Use of methylene blue to treat ischemia-reperfusion injury

Abstract

This method provides in situ quantification of the overall mitochondrial redox state, the redox state of individual cytochromes such as complex III and IV, and methods for treatment should the redox state be identified as hyperoxidized. Mitochondrial rescue from IRI-mediated injury can be achieved using methylene blue, which acts as an alternate electron donor to bypass complex III.

Description

Attorney Docket No. 030258-000102 WOPTUSE OF METHYLENE BLUE TO TREAT ISCHEMIA-REPERFUSION INJURYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application 63 / 687,821 filed on August 28, 2024, the content of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The technology described herein relates to methods for preventing and treating ischemia-reperfusion injury.GOVERNMENT SUPPORT

[0003] This invention was made with government support under Grant. No. 5R01DK134590-02, awarded by The National Institute of Health. The government has certain rights in the invention.BACKGROUND

[0004] Mitochondria are highly studied in cell physiology and pathology (Brand, M.D., et al., Br J Dermatol (2013)) because they stand at a critical intersection between life and death, comprising the complex pathways that direct metabolism as well as those key to injury. The central element of mitochondrial function is the electron transport chain (ETC), which consists of four complexes (cl, ell, cIII, cIV) in the inner mitochondrial membrane that use electron flow to create an electrochemical gradient and power ATP generation at complex V (eV). Under physiological conditions, much of the oxygen consumed during oxidative phosphorylation is reduced to water; however, a portion of electrons leak along the ETC and covalently reduce oxygen to form the highly reactive superoxide (Jastroch, M., et al., Essays Biochem (2010); Mazat, J.P, et al., Cell Mol Life Sci (2020)). While some ROS is essential for normal cell signaling, significant changes may lead to metabolic dysfunction, oxidative cell damage, and programmed cell death, which are defining features of countless disease states (Bardaweel, S.K., et al., Eurasian J Med (2018)). Although extensive research has investigated the role of mitochondria in various tissues and disease states (Fromenty, B. and Roden, M. J. Hepatol (2023); Hiran, A. et al., Oxidative Stress (2020); Schatten, H et al., Reprod Biol Endocrinol (2014); Li, J.L. et al., Front Mol Neurosci (2021)), the precise sequence of events and underlying mechanisms of mitochondrial injury remain poorly understood.14929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0005] Several techniques are currently employed to assess mitochondrial injury, including both direct and indirect methods, each with distinct advantages and limitations. For instance, indirect assessments may involve measuring coenzymes such as NADH and FADFL, which transfer electrons within the electron transport chain (ETC); ATP, the primary energy output of mitochondria; or mitochondrial breakdown products like cell-free flavin mononucleotide (FMN), as others have done in liver transplantation (Bruinsma, B.G., et al., Transplantation (2017); Eden, J., et al., J Hepatol (2025)). While these biomarkers offer valuable insight into mitochondrial function and injury, they do so through indirect means. Direct assessments offer more specific evaluations but present additional challenges. For example, mitochondrial membrane potential (MMP), typically measured using cationic dyes, serves as a widely used indicator of mitochondrial dysfunction. However, it does not reveal the precise site or mechanism of injury (Logan, A., et al., Cell Metab (2016); Sivandzade, F.A., et al., Bio Protoc (2019)). Other direct analytical techniques, such as electron microscopy and mitochondrial respirometry, can yield detailed structural and functional data but require tissue sampling and specialized processing. While these methods have been applied in liver transplantation contexts (Meszaros, A.T., et al., EBioMedicine (2022)), the need for tissue samples can pose practical limitations, particularly in clinical or time-sensitive settings (Kuznetsov, A.V, et al., Anal Biochem (2002); Koliaki, C., et al., Cell Metab (2015)). There remains a significant need for in situ methods for determining mitochondrial injury.SUMMARY

[0006] The technology described herein relates to a method for preventing or treating ischemia-reperfusion injury (IRI) using methylene blue or a functional variant thereof. Using non-destructive resonance Raman Spectroscopy (RRS), dysfunction of whole mitochondria and individual complexes was monitored in real time. Analysis of RRS allows in situ quantification of the overall mitochondrial redox state, as well as the redox state of individual cytochromes such as complex III and IV. A model for warm IRI in organ transplantation is disclosed herein, whereby dysfunction at complex III is characterized by hyperoxidation during ex vivo machine perfusion, due to electron leakage. Mitochondria were rescued from IRI-mediated injury using methylene blue, which acts as an alternate electron donor to bypass complex III.

[0007] One aspect of any of the embodiments is a method for preventing or treating ischemia-reperfusion injury in an organ or a tissue, the method comprising administering an24929-4870-6393 6Attorney Docket No. 030258-000102 WOPT effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0008] In one embodiment of any of the aspects, wherein the organ is a solid organ or a tissue thereof. In one embodiment of any of the aspects, the organ or tissue is vascularized. For example, the tissue is of a liver, a heart, a kidney, a brain, a retina, or skin. In another embodiment, the organ is a liver, a heart, a kidney, a brain, a retina, or skin.

[0009] In one embodiment of any of the aspects, prior to treatment, the tissue or organ has been subjected to ischemia followed by initiation of reperfusion, and the tissue or organ is at risk of or has suffered an ischemia-reperfusion injury.

[0010] In some embodiments, the method further comprises administering an effective amount of an oxygenation support to the tissue or organ, in addition to the administering of the methylene blue or functional variant thereof.

[0011] One aspect of any of the embodiments is method for preventing or treating ischemia-reperfusion injury in a tissue or an organ, the method comprising: (a) determining if an entire mitochondria and / or at least one electron transport chain (ETC) complex of the tissue or organ is hyperoxidized; and (b) if the entire mitochondria and / or the at least one ETC complex of the tissue or organ is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0012] When referring to redox states and / or 3RMR values, the terms “mitochondria” and “entire mitochondria” and “whole mitochondria” are used interchangeably throughout.

[0013] Some embodiments of any of the aspects further comprise determining if the mitochondria and / or at least one electron transport chain (ETC) complex is hyperoxidized prior to administering the effective amount of methylene blue or the functional variant thereof. For example, the method further comprises quantifying a Resonance Raman Reduced Mitochondrial Ratio (3RMR) value for the redox state of the mitochondria or the at least one ETC complex of the tissue or organ using a Raman spectroscopy method and determining if the 3RMR value indicates that the entire mitochondria and / or the at least one ETC complexes is hyperoxidized.

[0014] One aspect of any of the embodiments is a method for preventing or treating ischemia-reperfusion injury in a tissue or an organ, the method comprising: (a) quantifying a Resonance Raman Reduced Mitochondrial Ratio (3RMR) value of a mitochondrial complex of the tissue or organ using a Raman spectroscopy method; (b) determining if the 3RMR value indicates that the mitochondrial complex is hyperoxidized; and (c) if the 3RMR value34929-4870-6393 6Attorney Docket No. 030258-000102 WOPT indicates that the mitochondrial complex is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGs. 1A-1B depict a summary of the RRS data processing and computational pipeline. FIG. 1A: Flow chart of data analysis using NI Lab VIEW software. The raw spectrum from a representative rodent liver is adjusted to remove cosmic rays and dark current, and the fluorescence baseline is subtracted, leaving the Raman spectrum (ym). A regression analysis is performed against the library of known spectra to determine the best fit, which represents the relative concentrations of chromophores in the tissue within the Raman shift range of 700 to 1500 cm'1(yr), and together achieves random residual spectrum of unexplained signal accounting for less than 4% of the measured spectrum (ym- yr= residual; Formula VI) FIG. IB: Libraries of known spectra are created from the purified, isolated analytes, including reduced and oxidized whole mitochondria (x17x2), reduced and oxidized complex III (x3, x4), reduced and oxidized complex IV (x5, x6), and reduced and oxidized hemoglobin (x7, x8). Since mitochondrial complexes have resonance maxima of the reduced form closer to the excitation wavelength of 441 nm than that of oxidized form, a unique enhancement factor pair was assigned to each analyte to counterbalance their difference in Soret absorption strength■■■)• Finally, shown are their mathematical relationship that yields 3RMR values(Formulae I, II, III, IV). yrrepresents the relative concentrations of chromophores in the tissue within the Raman shift range of 700 to 1500 cm'1(Formula V).

[0016] FIGs. 2A-2L depict RRS detects mitochondrial hyperoxidation during subnormothermic machine perfusion (SNMP) of rodent livers with extended warm ischemia (WI). Study designed with n=6 per group with data presented as median ± interquartile range (IQR). Statistical significance at each time point was assessed using an unpaired two-tailed Student’s t-test with Welch’s correction, where the following symbols denote p < 0.05 for the corresponding comparisons: (#) Fresh vs Ih WI; (*) Ih WI vs 3h WI; (+) Fresh vs 3h WI. FIG. 2A: Schematic of experimental design. Rat livers are cannulated and explanted, then exposed to either 0, 1, or 3 hours of warm ischemia prior to SNMP. FIG. 2B: The range of possible 3RMR_mito values from 0% (fully oxidized) to 100% (fully reduced) with corresponding biological interpretations. FIG. 2C: Schematic of the hypothesized mechanism of the hyperoxidized mitochondrial state, which can be attributed to electron leakiness at complex III. FIG. 2D: Images of fresh (left) and 3h WI (right) rodent livers after44929-4870-6393 6Attorney Docket No. 030258-000102 WOPTSNMP, which show evidence of IRI (weight gain, pale color) in 3h WI livers. From RRS measures, FIG. 2E: 3RMR_mito, FIG. 2F: 3RMR CIII and FIG. 2G: 3RMR_complex IV indicated increasingly over-oxidized mitochondria and hyperoxidized complex III as a vulnerable site of injury in 3h WI livers. During SNMP, FIG. 2H: flow rate, FIG. 21: oxygen consumption, and FIG. 2J: outflow lactate level collectively showed poor aerobic respiration and viability in 3h WI liver. After SNMP, FIG. 2K: mitochondrial energy charge, and FIG. 2L: TUNEL images, with dead cells (dark dots) and relative edema (white space) in images, further confirmed IRI effects in 3h livers.

[0017] FIGs. 3A-3D demonstrate Resonance Raman Spectroscopy (RRS) oxygen stress test interrogates the mechanism of mitochondrial hyperoxidation in 3h WI rodent livers. Mechanistically, if hyperoxidation occurs due to electron leakiness, ETC complexes would not be able to accumulate electrons and could not become reduced as quickly in response to ischemia. FIG. 3A: Schematic of the oxygen stress test method. FIG. 3B: Representative RRS measure spectrum during reperfusion (with O2) and ischemia (without O2). FIG. 3C: 3RMR_mito values after 3 mins of ischemia induced by the oxygen stress test. Study designed with n=3 per group with data presented as median ± interquartile range (IQR). Statistical significance at each time point was assessed using an unpaired two-tailed Student’s t-test with Welch’s correction, where the following symbols denote p < 0.05 for the corresponding comparisons: (#) Fresh vs Ih WI; (*) Ih WI vs 3h WI; (+) Fresh vs 3h WI. FIG. 3D: Representative trendline of 3RMR_mito values as a function of the oxygen stress test. After the depletion of oxygen during the RRS oxygen stress test, mitochondria of 3h WI livers become reduced more slowly than that of fresh and Ih WI livers.

[0018] FIGs. 4A-4K demonstrate Methylene Blue (MB) mitigates IRI-mediated mitochondrial injury of 3h WI rodent livers. FIG. 4A: Schematic of MB therapeutic mechanism via bypassing complex III, and the proposed benefit of Hemoglobin Based Oxygen Carriers (HBOC) via increasing oxygen delivery. FIG. 4B: Doses and administration of MB and HBOC during machine perfusion. From RRS measures, FIG. 4C: 3RMR_mito, FIG. 4D: 3RMR CIII, FIG. 4E: 3RMR_complex IV confirmed the MB effects, evident with fully oxidized complex III due to electron transfer bypass and reduced complex IV due to enhanced electron deposition without changes to perfusate flow rate for oxygen supply. Viability biomarkers FIG. 4F: flow rate, FIG. 4G: oxygen consumption, FIG. 4H: outflow lactate level. After SNMP, FIG. 41: mitochondrial energy charge, FIG. 4 J: weight gain, and FIG. 4K: Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) images showed non-significant differences in cell death (dark dots) and relative tissue edema (white54929-4870-6393 6Attorney Docket No. 030258-000102 WOPT space) between the 3h WI+MB and 3h WI+HBOC livers. Study designed with n=3-6 per group with data presented as median ± interquartile range (IQR). Statistical significance at each time point was assessed using an unpaired two-tailed Student’s t-test with Welch’s correction, where the following symbols denote p < 0.05 for the corresponding comparisons: (#) 3h WI vs 3h WI+MB; (*) 3h WI+MB vs 3h WI+MB+HBOC; (+) 3h WI vs 3h WI+MB+HBOC. HBOC used in the present study significantly lowered the mitochondrial signal strength, thereby the 3RMR values were not able to be quantified for these groups.

[0019] FIGs. 5A-5H demonstrate Methylene Blue improves hemodynamics and aerobic metabolism of Donor after cardiac death (DCD) pig livers. FIG. 5A: Schematic showing timeline of procurement, treatment, and measurements. After SNMP, FIG. 5B: images of DCD livers representative of each study group. During SNMP, FIG. 5C: 3RMR_mito, FIG. 5D: portal flow rate, FIG. 5E: portal vascular resistance, FIG. 5F: oxygen consumption, FIG. 5G: outflow lactate level, and FIG. 5H: outflow potassium level. Study designed with n=4 per group with data presented as median ± interquartile range (IQR). Statistical significance at each time point was assessed using an unpaired two-tailed Student’s t-test with Welch’s correction, where the following symbols denote p < 0.05 for the corresponding comparisons: (*) 30 min WI vs 30 min WI+MB+HBOC; (#) 30 min WI vs 45 min WI+MB; (+) 30 min WI+MB+HBOC vs 45 min WI+MB; (f) 30 min WI vs Fresh DBD; (Q) 30 min WI+MB+HBOC vs Fresh DBD; (0) 45 mins WI+MB vs Fresh DBD. HBOC used in the present study significantly lowered the mitochondrial signal strength; thereby the 3RMR values were not able to be quantified for these groups.

[0020] FIGs. 6A-6H depict spectral differences in peak positions and intensities of RRS measurements obtained from isolated mitochondrial Complex III (CIII) and Complex IV (CIV). For highlighting redox differences, FIG. 6A: comparison of oxidized and reduced CIII with the FIG. 6B: associated computed difference, and FIG. 6C: comparison of oxidized and reduced CIV with the FIG. 6D: associated computed difference. For highlighting complex-specific spectral differences, FIG. 6E: comparison of CIII and CIV in the oxidized state with the FIG. 6F: associated computed difference, and FIG. 6G: comparison of CIII and CIV in the reduced state with the FIG. 6H: associated computed difference.

[0021] FIGs. 7A-7C depict the fit of whole mitochondria library with individual complexes. The 441nm excitation source used is close to the Soret absorption maximum of reduced Complex IV, which results in maximal resonant enhancement of this chromophore. A legend below each panel denotes which trace is assigned to each library and complex. FIG.64929-4870-6393 6Attorney Docket No. 030258-000102 WOPT7A: A regression using the Complex III and Complex IV libraries fully explain the measured spectrum of reduced mitochondria, resulting in minimal unexplained residual (shown in dotted line). Using only FIG. 7B: Complex III or FIG. 7C: Complex IV does not fully explain the spectrum and results in a non-random structure in the residual that deviates (as denoted by an asterisk).

[0022] FIG. 8 : RRS spectral analysis showed non-significant resonance from methylene blue. Left panel: shows measured spectrum (top trace), fit (middle trace), and residue (bottom trace) from a rat liver perfused without methylene blue. Right panel: shows measured spectrum, fit, and residue from a rat liver perfused with methylene blue. There are no additional peaks in the spectrum from the liver with methylene blue, indicating nonsignificant resonance from methylene blue.

[0023] FIG. 9 : Soret absorption maxima of hemoglobin and key mitochondrial ETC complexes (Complex III and IV). Absorption peaks for oxidized (open circles) and reduced (filled circles) forms are shown across the Soret band (400-450 nm). The selected RRS excitation wavelength ( = 441 nm), indicated by a gray arrow, closely aligns with reduced- form maxima, enhancing their sensitivity for redox assessment.

[0024] FIGs. 10A-10J depict additional parameters for the confirmation of IRI in 3h WI rodent livers during SNMP. Study designed with n=6 per group with data presented as median ± interquartile range (IQR). Statistical significance at each time point was assessed using an unpaired two-tailed Student’s t-test with Welch’s correction, where the following symbols denote p < 0.05 for the corresponding comparisons: (#) Fresh vs Ih WI; (*) Ih WI vs 3h WI; (+) Fresh vs 3h WI. FIG. 10A: vascular resistance; hepatic injury makers: FIG. 10B: AST, FIG. 10C: ALT, and FIG. 10D: potassium outflow concentrations, FIG. 10E: cytochrome c release into the perfusate (as a proxy for apoptosis), FIG. 10F: NAD / NADH ratio (inversely proportionate to oxidative stress), FIG. 10G: bile production, FIG. 10H: weight gain, FIG. 101: ATP concentration, and FIG. 10J: H&E images of representative livers, showing tissue edema (white space between stained cells) at 20X magnification.

[0025] FIGs. 11A-11G depict rodent liver experiments using antimycin A (AA) and oxygen stress test (OST) to validate the reliability of RRS in detecting well-characterized mitochondrial phenomena. Study design with n=3 per group with data presented as median ± interquartile range (IQR). Statistical significance at each time point was assessed using an unpaired two-tailed Student’s t-test with Welch’s correction, where the following symbols denote p < 0.05 for the corresponding comparisons: (#) Fresh vs IhWI; (+) Fresh vs 3h WI; ($) Fresh vs Fresh+AA; (*) IhWI vs 3h WI; (%) IhWI vs Fresh+AA; (&) 3h WI vs74929-4870-6393 6Attorney Docket No. 030258-000102 WOPTFresh+AA. FIG. 11A: Experimental design for the Fresh+AA liver group compared to main WI groups and fresh controls. FIG. 11B: Oxygen consumption in Fresh+AA livers during well-oxygenated SNMP confirmed cessation of mitochondrial respiration following AA administration. (* indicates p<0.05) FIG. 11C: Time-course plots of 3RMR_mito during OST from a representative Fresh+AA liver. FIG. 11D: the 3RMR_mito values at 3 minutes of OST demonstrated distinct mitochondrial redox responses to acute ischemia, differentiating healthy liver groups (fresh and Ih WI livers) from damaged liver groups (3h WI and fresh+AA livers). FIG. HE: 3RMR_mito, FIG. HF: 3RMR_CIII, and FIG. HG: 3RMR CIV values showed a trend toward differences between healthy and damaged liver groups. However, fresh+AA livers showed no detectable 3RMR CIV response to the acute ischemia of OST with below threshold signal levels. This is hypothesized to be due to a complete shutdown of Complex IV activity due to AA, whereas 3h WI livers had a diminished, yet partially rescuable, Complex IV function.

[0026] FIGs. 12A-12J depict additional parameters for the assessment of MB effects in rodent livers. Study designed with n=3-6 per group, with data presented as median ± interquartile range (IQR). Statistical significance at each time point was assessed using an unpaired two-tailed Student’s t-test with Welch’s correction, where the following symbols denote p < 0.05 for the corresponding comparisons: (#) 3h WI vs 3h WI+MB; (+) 3h WI vs 3h WI+MB+HBOC; (*) 3h WI+MB vs 3h WI+MB+HBOC. For the cytochromes c results, the values for 3hWI+MB+HBOC is not reported since HBOC interfered with the enzyme- linked immunosorbent assay (ELISA) assay. FIG. 12A: vascular resistance; hepatic injury makers: FIG. 12B: AST, FIG. 12C: ALT and FIG. 12D: potassium outflow concentrations, FIG. 12E: concentration of cytochrome c release in perfusate (as a proxy for apoptosis), FIG. 12F: NAD / NADH ratio (inversely proportionate to oxidative stress), FIG. 12G: bile production, FIG. 12H: ATP concentration, FIG. 121: H&E images of representative livers of 3h WI+MB (left) and 3h WI+MB+HBOC (right) at 20X magnification, and FIG. 12J: images of a 3h WI+MB rodent liver at 30, 90, and 150 mins that show the progression of MB stains over SNMP time.

[0027] FIGs. 13A-13H depict additional parameters for viability assessment of DCD pig livers during SNMP. Study design with n=4-5 per group with data presented as median ± interquartile range (IQR). Statistical significance at each time point was assessed using an unpaired two-tailed Student’s t-test with Welch’s correction, where the following symbols denote p < 0.05 for the corresponding comparisons: (*) 30 min WI vs 30 min WI+MB+HBOC; (#) 30 min WI vs 45 min WI+MB; (+) 30 min WI+MB+HBOC vs 45 min 84929-4870-6393 6Attorney Docket No. 030258-000102 WOPTWI+MB; (f) 30 min WI vs Fresh DBD; (Q) 30 min WI+MB+HBOC vs Fresh DBD; (0) 45 mins WI+MB vs Fresh DBD. Hepatic injury markers: FIG. 13A: AST and FIG. 13B: ALT outflow concentrations; FIG. 13C: cytochrome c release / concentration in perfusate; Biopsy based energetic measurements: FIG. 13D: NAD / NADH ratio (inversely proportionate to oxidative stress); FIG. 13E: mitochondrial energy charge; and FIG. 13F: ATP Concentration; FIG. 13G: TUNEL staining (Scale bar: 100 pm) and FIG. 13H: H&E staining of representative livers with 30 mins WI (left) and 30 mins WI+MB+HBOC (right) (Scale bar: 100 pm).

[0028] FIGs. 14A-14E depict rodent liver RR analysis of 3h WI, Fresh and 3h WI+MB treated groups. *p<0.05, 3h WI vs fresh; +p<0.05, 3h WI vs 3h WI+MB. FIG. 1A: Resonance Raman (RR) spectroscopy as a tool that can quantitatively assess the oxygenation state of mitochondria (RR reduced mitochondrial ratio or 3RMR) and that of individual cytochromes via surface readings. FIG. 14B: IRI mechanism in extended warm ischemic, non-transplantable livers. FIG. 14C: whole mitochondria (3RMR); FIG. 14D: Complex III; FIG. 14E: Complex IV redox state.

[0029] FIGs. 15A-15H depict DCD pig liber MB+HEMOPURE treatment compared with no treatment during SNMP. FIG 15A: DCD pig liver perfused with MB+HEMOPURE. FIG. 15B: The effect of MB and HEMOPURE on electron transport chain. FIG. 15C: portal resistance; FIG. 15D: portal flow rate; FIG. 15E: potassium level; FIG. 15F: oxygen consumption; FIG. 15G: lactate level; FIG. 15H: glucose level.

[0030] FIG. 16 depicts (top) images of fresh (left) and 3h WI (right) rodent livers after SNMP, which show evidence of IRI (weight gain, pale color) in 3h WI livers; (bottom) the potential mechanism of the hyperoxidized mitochondrial state, which can be attributed to electron leakiness at complex III.

[0031] FIG. 17 depicts the range of possible 3RMR_mito values from 0% (fully oxidized) to 100% (fully reduced) with corresponding biological interpretations; Raman spectra of oxidized / reduced mitochondrial, complex III and complex IV; and mathematical determination of the Resonance Raman Reduced Mitochondrial Ratio (3RMR).

[0032] FIG. 18 depicts results in IRI rodent liver models: detection of mitochondrial leakiness in IRI livers during 3h SNMP (n = 6).

[0033] FIG. 19 depicts results in IRI rodent liver models: assessment of MB effects in overcoming mitochondrial leakiness (n = 3).94929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0034] FIG. 20 depicts results in DCD pig livers: assessment of MB+ HBOC treatment in rescuing DCD pig livers from IRI progression during SNMP (n=4 control livers, n=5 treated livers).DETAILED DESCRIPTION

[0035] One aspect of any of the embodiments is a method for preventing or treating ischemia-reperfusion injury in an organ or a tissue, the method comprising administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0036] Methylene blue (MB) has a chemical formula of CieHisCINsS, as shown in Formula VIII below. Methylene blue is FDA approved to treat a blood disorder called methemoglobinemia, a condition in which hemoglobin has decreased ability to carry oxygen. Methylene blue shuttles electrons from complex I (CI) to cytochrome c (which are then transferred to complex IV (CIV)), therefore bypassing complex III (CIII). As such a functional variant of methylene blue shares function(s) with methylene blue, including: shuttling electrons from complex I to cytochrome c and / or bypassing CIII. A functional variant of methylene blue can include a structural variant (e.g., an analog) of methylene blue that maintains the function(s) of methylene blue, including: shuttling electrons from complex I to cytochrome c and / or bypassing CIII. Possible structural variants include variants of methylene blue wherein one or more functional group on the molecule (— N(CH3)2, — H) are replaced with other R groups, such as alkyl, alkenyl, alkynyl, phenyl, amino, hydroxy, alkoxy, aryl, heteroaryl, cyclyl, heterocyclyl. Additional possible structural variants include variants of methylene blue wherein one or more or heteroatoms (N, S) are replaced with different atoms, such as N, S, O, or C. Structural variants may comprise alternative counterions to CF, such as F, Br , F, OH, NO3’, NO2’, HCOf, C1O4, C1O3, CH3COO;H2PO4 ', or another anion with a -1 charge.

[0037] Formula VIII: Methylene blue104929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0038] Non-limiting examples of functional variants of methylene blue include mitoquinone, azure A, diaminophenolthiazines, phenazine methosulfate, or 2,6-indophenol, or any combination thereof. Mitoquinone, as shown in Formula IX below, is a synthetic analogue of coenzyme Q 10. It is a mitochondria-targeted antioxidant capable of reducing mitochondrial overproduction of reactive oxygen species.

[0039] Formula IX: Mitoquinone

[0040] Azure A is an organic dye with structural similarities to methylene blue, as shown in Formula X below. Azure A is commonly used as a biological stain. As it is a photosensitizer, it is capable of disrupting mitochondrial function through intrinsic and photodynamic effects.

[0041] Formula X: Azure A114929-4870-6393 6Attorney Docket No. 030258-000102 WOPTstructure shown in Formula XI below, wherein two R groups are amine groups. Thiazines are a class of heterocyclic compounds with a six membered ring comprising four carbons, a nitrogen, and a sulfur. These molecules often absorb and emit visible light, making them useful as dyes. Diaminophenolthiazines interact with the ETC by altering electron flow, either inhibiting respiration or inducing photooxidation.

[0043] Formula XI: phenolthiazine

[0044] Phenazine methosulfate, as shown in Formula XII below, is commonly used in cell proliferation assays. Since the reduced phenazine methosulfate is easily oxidized by oxygen, it is used in assays as an electron carrier between enzymes and oxygen, cytochrome c, indophenols, or tetrazolium salts. The reduced phenazine methosulfate can be used as an electron donor to reduce cytochrome c or in photosynthetic experiments.

[0045] Formula XII: Phenazine methosulfate124929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0046] 2,6-indophenol, also referred to as 2,6-Dichlorophenolindophenol (DCPIP or DPIP), is a chemical compound used as a redox dye. The oxidized form is blue in color, with maximal absorption at 600 nm. The photosynthetic electron transport chain can reduce DCPIP as a substitute for NADP+. Reduction of DCPIP, with color change from blue to colorless, can be used as an exogenous acceptor of electrons in the mitochondrial ETC, resulting from succinate oxidation to fumarate promoted by Complex II. 2,6-indophenol is shown as shown in Formula XIII below.

[0047] Formula XIII: 2,6-indophenol

[0048] In some embodiments, the methylene blue, or functional variant thereof, is administered to the organ or subject intravenously. In some embodiments, the methylene blue, or functional variant thereof, is administered to the organ or subject over a period of time of about 5 minutes to 30 minutes, such as over a 5 minute, 10 minute, 15 minute, 20 minute, 25 minute, or 30 minute period.134929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0049] As used herein the term “therapeutic effect” refers to a consequence of treatment, the results of which are judged to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation (e.g., ischemia-reperfusion injury). A therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.

[0050] As used herein, the terms “therapeutically effective amount” and “effective amount” are used interchangeably to refer to an amount of a compound or a composition (e.g., methylene blue or a functional variant thereof) that is sufficient to provide the intended benefit (e.g. prevention, prophylaxis, delay of onset of symptoms, or amelioration of symptoms of a disease, such as ischemia-reperfusion injury). In prophylactic or preventative applications, an effective amount can be administered to a subject susceptible to, or otherwise at risk of developing a disease, disorder or condition to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including a biochemical, histologic and / or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes.

[0051] Depending on the route of administration, effective doses (e.g., of methylene blue or a functional variant thereof) can be calculated according to the body weight, body surface area, or organ size of the subject to be treated. Optimization of the appropriate dosages can readily be made by one skilled in the art in light of pharmacokinetic data observed in human clinical trials. Alternatively, or additionally, the dosage to be administered can be determined from studies using animal models for the particular type of condition to be treated (e.g., ischemia-reperfusion injury), and / or from animal or human data obtained from agents which are known to exhibit similar pharmacological activities. The final dosage regimen will be determined by the attending surgeon or physician, considering various factors which modify the action of active agent, e.g., the agent’s specific activity, the agent’s specific half-life in vivo, the severity of the condition and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any present infection, time of administration, the use (or not) of other concomitant therapies, and other clinical factors.

[0052] Determination of an effective amount (e.g., of methylene blue or a functional variant thereof) is well within the capability of those skilled in the art. Generally, the actual effective amount can vary with the specific compound, the use or application technique, the desired effect, the duration of the effect and side effects, the subject’s history, age, condition, 144929-4870-6393 6Attorney Docket No. 030258-000102 WOPT sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents. Accordingly, an effective dose of compound described herein is an amount sufficient to produce at least some desired therapeutic effect in a subject.

[0053] In some embodiments of any of the aspects, an effective amount of methylene blue or a functional variant thereof is from 0.02 mg / ml - 0.1 mg / ml, e.g., in perfusate in machine perfusion (e.g., where the total volume of perfusate can be 500 mL for rat livers or 2 L for pig livers and human livers). For example, an effective amount of methylene blue or a functional variant thereof is 0.020 mg / ml, 0.021 mg / ml, 0.022 mg / ml, 0.023 mg / ml, 0.024 mg / ml, 0.025 mg / ml, 0.026 mg / ml, 0.027 mg / ml, 0.028 mg / ml, 0.029 mg / ml, 0.030 mg / ml, 0.031 mg / ml, 0.032 mg / ml, 0.033 mg / ml, 0.034 mg / ml, 0.035 mg / ml, 0.036 mg / ml, 0.037 mg / ml, 0.038 mg / ml, 0.039 mg / ml, 0.040 mg / ml, 0.041 mg / ml, 0.042 mg / ml, 0.043 mg / ml, 0.044 mg / ml, 0.045 mg / ml, 0.046 mg / ml, 0.047 mg / ml, 0.048 mg / ml, 0.049 mg / ml, 0.050 mg / ml, 0.051 mg / ml, 0.052 mg / ml, 0.053 mg / ml, 0.054 mg / ml, 0.055 mg / ml, 0.056 mg / ml, 0.057 mg / ml, 0.058 mg / ml, 0.059 mg / ml, 0.060 mg / ml, 0.061 mg / ml, 0.062 mg / ml, 0.063 mg / ml, 0.064 mg / ml, 0.065 mg / ml, 0.066 mg / ml, 0.067 mg / ml, 0.068 mg / ml, 0.069 mg / ml, 0.070 mg / ml, 0.071 mg / ml, 0.072 mg / ml, 0.073 mg / ml, 0.074 mg / ml, 0.075 mg / ml, 0.076 mg / ml, 0.077 mg / ml, 0.078 mg / ml, 0.079 mg / ml, 0.080 mg / ml, 0.081 mg / ml, 0.082 mg / ml, 0.083 mg / ml, 0.084 mg / ml, 0.085 mg / ml, 0.086 mg / ml, 0.087 mg / ml, 0.088 mg / ml, 0.089 mg / ml, 0.090 mg / ml, 0.091 mg / ml, 0.092 mg / ml, 0.093 mg / ml, 0.094 mg / ml, 0.095 mg / ml, 0.096 mg / ml, 0.097 mg / ml, 0.098 mg / ml, 0.099 mg / ml, 0.100 mg / ml.

[0054] In some embodiments of any of the aspects, an effective amount of methylene blue or a functional variant thereof is from about 0.02 mg / ml - about 0.1 mg / ml, e.g., in perfusate in machine perfusion. For example, an effective amount of methylene blue or a functional variant thereof is about 0.020 mg / ml, about 0.021 mg / ml, about 0.022 mg / ml, about 0.023 mg / ml, about 0.024 mg / ml, about 0.025 mg / ml, about 0.026 mg / ml, about 0.027 mg / ml, about 0.028 mg / ml, about 0.029 mg / ml, about 0.030 mg / ml, about 0.031 mg / ml, about 0.032 mg / ml, about 0.033 mg / ml, about 0.034 mg / ml, about 0.035 mg / ml, about 0.036 mg / ml, about 0.037 mg / ml, about 0.038 mg / ml, about 0.039 mg / ml, about 0.040 mg / ml, about 0.041 mg / ml, about 0.042 mg / ml, about 0.043 mg / ml, about 0.044 mg / ml, about 0.045 mg / ml, about 0.046 mg / ml, about 0.047 mg / ml, about 0.048 mg / ml, about 0.049 mg / ml, about 0.050 mg / ml, about 0.051 mg / ml, about 0.052 mg / ml, about 0.053 mg / ml, about 0.054 mg / ml, about 0.055 mg / ml, about 0.056 mg / ml, about 0.057 mg / ml, about 0.058 mg / ml, about 0.059 mg / ml, about 0.060 mg / ml, about 0.061 mg / ml, about 0.062 mg / ml, about 0.063 154929-4870-6393 6Attorney Docket No. 030258-000102 WOPT mg / ml, about 0.064 mg / ml, about 0.065 mg / ml, about 0.066 mg / ml, about 0.067 mg / ml, about 0.068 mg / ml, about 0.069 mg / ml, about 0.070 mg / ml, about 0.071 mg / ml, about 0.072 mg / ml, about 0.073 mg / ml, about 0.074 mg / ml, about 0.075 mg / ml, about 0.076 mg / ml, about 0.077 mg / ml, about 0.078 mg / ml, about 0.079 mg / ml, about 0.080 mg / ml, about 0.081 mg / ml, about 0.082 mg / ml, about 0.083 mg / ml, about 0.084 mg / ml, about 0.085 mg / ml, about 0.086 mg / ml, about 0.087 mg / ml, about 0.088 mg / ml, about 0.089 mg / ml, about 0.090 mg / ml, about 0.091 mg / ml, about 0.092 mg / ml, about 0.093 mg / ml, about 0.094 mg / ml, about 0.095 mg / ml, about 0.096 mg / ml, about 0.097 mg / ml, about 0.098 mg / ml, about 0.099 mg / ml, or about 0.100 mg / ml.

[0055] In some embodiments of any of the aspects, an effective amount is from about 0.02 mg / ml - 0.1 mg / ml of methylene blue or a functional variant thereof, e.g., in perfusate in machine perfusion. For example, an effective amount of methylene blue or a functional variant thereof is from about 0.02 mg / ml - 0.1 mg / ml, 0.02 mg / ml - 0.08 mg / ml, 0.02 mg / ml - 0.06 mg / ml, 0.02 mg / ml - 0.04 mg / ml, 0.04 mg / ml - 0.1 mg / ml, 0.04 mg / ml - 0.09 mg / ml, 0.04 mg / ml - 0.08 mg / ml, 0.04 mg / ml - 0.06 mg / ml, 0.05 mg / ml - 0.1 mg / ml, 0.05 mg / ml - 0.09 mg / ml, 0.05 mg / ml - 0.08 mg / ml, 0.05 mg / ml - 0.07 mg / ml, 0.05 mg / ml - 0.06 mg / ml, 0.06 mg / ml - 0.1 mg / ml, 0.06 mg / ml - 0.095 mg / ml, 0.06 mg / ml - 0.09 mg / ml, 0.06 mg / ml - 0.085 mg / ml, 0.06 mg / ml - 0.08 mg / ml, 0.06 mg / ml - 0.075 mg / ml, 0.06 mg / ml - 0.07 mg. ml, 0.06 mg / ml - 0.065 mg / ml, 0.07 mg / ml - 0.1 mg / ml, 0.07 mg / ml - 0.095 mg / ml, 0.07 mg / ml - 0.09 mg / ml, 0.07 mg / ml - 0.085 mg / ml, 0.07 mg / ml - 0.08 mg / ml, 0.08 mg / ml - 0.1 mg / ml, 0.08 mg / ml - 0.095 mg / ml, or 0.09 mg / ml - 0.1 mg / ml.

[0056] In some embodiments of any of the aspects, an effective amount of methylene blue or a functional variant thereof is from 1.25 mg / kg - 6.25 mg / kg of a subject’s body weight (e.g., using an 80 kg male with 5L of blood as reference). For example, an effective amount is 1.25 mg / kg, 1.30 mg / kg, 1.35 mg / kg, 1.40 mg / kg, 1.45 mg / kg, 1.50 mg / kg, 1.55 mg / kg, 1.60 mg / kg, 1.65 mg / kg, 1.70 mg / kg, 1.75 mg / kg, 1.80 mg / kg, 1.85 mg / kg, 1.90 mg / kg, 1.95 mg / kg, 2.00 mg / kg, 2.05 mg / kg, 2.10 mg / kg, 2.15 mg / kg, 2.20 mg / kg, 2.25 mg / kg, 2.30 mg / kg, 2.35 mg / kg, 2.40 mg / kg, 2.45 mg / kg, 2.50 mg / kg, 2.55 mg / kg, 2.60 mg / kg, 2.65 mg / kg, 2.70 mg / kg, 2.75 mg / kg, 2.80 mg / kg, 2.85 mg / kg, 2.90 mg / kg, 2.95 mg / kg, 3.00 mg / kg, 3.05 mg / kg, 3.10 mg / kg, 3.15 mg / kg, 3.20 mg / kg, 3.25 mg / kg, 3.30 mg / kg, 3.35 mg / kg, 3.40 mg / kg, 3.45 mg / kg, 3.50 mg / kg, 3.55 mg / kg, 3.60 mg / kg, 3.65 mg / kg, 3.70 mg / kg, 3.75 mg / kg, 3.80 mg / kg, 3.85 mg / kg, 3.90 mg / kg, 3.95 mg / kg, 4.00 mg / kg, 4.05 mg / kg, 4.10 mg / kg, 4.15 mg / kg, 4.20 mg / kg, 4.25 mg / kg, 4.30 mg / kg, 4.35 mg / kg, 4.40 mg / kg, 4.45 mg / kg, 4.50 mg / kg, 4.55 mg / kg, 4.60 mg / kg, 4.65 mg / kg, 4.70164929-4870-6393 6Attorney Docket No. 030258-000102 WOPT mg / kg, 4.75 mg / kg, 4.80 mg / kg, 4.85 mg / kg, 4.90 mg / kg, 4.95 mg / kg, 5.00 mg / kg, 5.05 mg / kg, 5.10 mg / kg, 5.15 mg / kg, 5.20 mg / kg, 5.25 mg / kg, 5.30 mg / kg, 5.35 mg / kg, 5.40 mg / kg, 5.45 mg / kg, 5.50 mg / kg, 5.55 mg / kg, 5.60 mg / kg, 5.65 mg / kg, 5.70 mg / kg, 5.75 mg / kg, 5.80 mg / kg, 5.85 mg / kg, 5.90 mg / kg, 5.95 mg / kg, 6.00 mg / kg, 6.05 mg / kg, 6.10 mg / kg, 6.15 mg / kg, 6.20 mg / kg, or 6.25 mg / kg.

[0057] In some embodiments of any of the aspects, an effective amount of methylene blue or a functional variant thereof is from about 1.25 mg / kg - about 6.25 mg / kg of a subject’s body weight. For example, an effective amount of methylene blue or a functional variant thereof is about 1.25 mg / kg, about 1.30 mg / kg, about 1.35 mg / kg, about 1.40 mg / kg, about 1.45 mg / kg, about 1.50 mg / kg, about 1.55 mg / kg, about 1.60 mg / kg, about 1.65 mg / kg, about 1.70 mg / kg, about 1.75 mg / kg, about 1.80 mg / kg, about 1.85 mg / kg, about 1.90 mg / kg, about 1.95 mg / kg, about 2.00 mg / kg, about 2.05 mg / kg, about 2.10 mg / kg, about 2.15 mg / kg, about 2.20 mg / kg, about 2.25 mg / kg, about 2.30 mg / kg, about 2.35 mg / kg, about 2.40 mg / kg, about 2.45 mg / kg, about 2.50 mg / kg, about 2.55 mg / kg, about 2.60 mg / kg, about 2.65 mg / kg, about 2.70 mg / kg, about 2.75 mg / kg, about 2.80 mg / kg, about 2.85 mg / kg, about 2.90 mg / kg, about 2.95 mg / kg, about 3.00 mg / kg, about 3.05 mg / kg, about 3.10 mg / kg, about 3.15 mg / kg, about 3.20 mg / kg, about 3.25 mg / kg, about 3.30 mg / kg, about 3.35 mg / kg, about 3.40 mg / kg, about 3.45 mg / kg, about 3.50 mg / kg, about 3.55 mg / kg, about 3.60 mg / kg, about 3.65 mg / kg, about 3.70 mg / kg, about 3.75 mg / kg, about 3.80 mg / kg, about 3.85 mg / kg, about 3.90 mg / kg, about 3.95 mg / kg, about 4.00 mg / kg, about 4.05 mg / kg, about 4.10 mg / kg, about 4.15 mg / kg, about 4.20 mg / kg, about 4.25 mg / kg, about 4.30 mg / kg, about 4.35 mg / kg, about 4.40 mg / kg, about 4.45 mg / kg, about 4.50 mg / kg, about 4.55 mg / kg, about 4.60 mg / kg, about 4.65 mg / kg, about 4.70 mg / kg, about 4.75 mg / kg, about 4.80 mg / kg, about 4.85 mg / kg, about 4.90 mg / kg, about 4.95 mg / kg, about 5.00 mg / kg, about 5.05 mg / kg, about 5.10 mg / kg, about 5.15 mg / kg, about 5.20 mg / kg, about 5.25 mg / kg, about 5.30 mg / kg, about 5.35 mg / kg, about 5.40 mg / kg, about 5.45 mg / kg, about 5.50 mg / kg, about 5.55 mg / kg, about 5.60 mg / kg, about 5.65 mg / kg, about 5.70 mg / kg, about 5.75 mg / kg, about 5.80 mg / kg, about 5.85 mg / kg, about 5.90 mg / kg, about 5.95 mg / kg, about 6.00 mg / kg, about 6.05 mg / kg, about 6.10 mg / kg, about 6.15 mg / kg, about 6.20 mg / kg, or about 6.25 mg / kg.

[0058] In some embodiments, the effective amount of methylene blue or a functional variant thereof is comprised by a pharmaceutical composition. In some embodiments, the effective amount of methylene blue or a functional variant thereof is in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is employed herein to refer to those174929-4870-6393 6Attorney Docket No. 030258-000102 WOPT compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient (e.g., methylene blue or a functional variant thereof) would not be found to occur in or within nature. In some embodiments, the pharmaceutically acceptable carrier for methylene blue or a functional variant thereof is an aqueous dextrose solution (e.g., 5% Dextrose solution in water).

[0059] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having an ischemia-reperfusion injury with an effective amount of methylene blue or a functional variant thereof. Subjects having an ischemia-reperfusion injury can be identified by a physician using the methods described herein (e.g., a Raman spectroscopy method). Symptoms and / or complications of an ischemia-reperfusion injury, which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to organ or tissue failure (e.g., heart failure, cerebral dysfunction), inflammation (e.g., systemic inflammation), and / or cell death (including apoptosis, autophagy, necrosis, and / or necroptosis). Exposure to risk factors for ischemia-reperfusion injury (e.g. a tissue or organ transplantation, a complex surgery, a myocardial infarction, or a stroke) can also aid in determining if a subject is likely to have an ischemia-reperfusion injury or in making a diagnosis of an ischemia-reperfusion injury.

[0060] The compositions described herein can be administered to a subject having or diagnosed as having an ischemia-reperfusion injury. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. methylene blue or a functional variant thereof to a subject in order to alleviate a symptom of an ischemia-reperfusion injury. As used herein, "alleviating a symptom of an ischemia-reperfusion injury" is ameliorating any condition or symptom associated with the ischemia-reperfusion injury . As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.184929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0061] As described herein, levels of hyperoxidization in mitochondria and / or electron transport chain (ETC) complex(es) can be increased in an ischemia-reperfusion injury and / or in a tissue, organ, or subject with an ischemia-reperfusion injury. Accordingly, in one aspect of any of the embodiments, described herein is a method of treating or preventing an ischemia-reperfusion injury in a tissue, organ, or subject in need thereof, the method comprising administering methylene blue or a functional variant thereof to a tissue, organ, or subject determined to have a level of mitochondrial and / or ETC complex hyperoxidation that is increased relative to a reference (e.g., a non-ischemic, healthy tissue, organ, or subject). In one aspect of any of the embodiments, described herein is a method of treating or preventing an ischemia-reperfusion injury in a tissue, organ, or subject in need thereof, the method comprising: a) determining the level of mitochondrial and / or ETC complex hyperoxidation in the tissue, organ, or subject; and b) administering methylene blue or a functional variant thereof to the tissue, organ, or subject if the level of mitochondrial and / or ETC complex hyperoxidation is increased relative to a reference.

[0062] In some embodiments of any of the aspects, the method comprises administering methylene blue or a functional variant thereof to a tissue, organ, or subject previously determined to have a level of mitochondrial and / or ETC complex hyperoxidation that is increased relative to a reference. In some embodiments of any of the aspects, described herein is a method of treating or preventing an ischemia-reperfusion injury in a tissue, organ, or subject in need thereof, the method comprising: a) first determining the level of mitochondrial and / or ETC complex hyperoxidation in a tissue, organ, or subject; and b) then administering methylene blue or a functional variant thereof to the tissue, organ, or subject if the level of mitochondrial and / or ETC complex hyperoxidation is increased relative to a reference.

[0063] In one aspect of any of the embodiments, described herein is a method of treating or preventing an ischemia-reperfusion injury in a tissue, organ, or subject in need thereof, the method comprising: a) determining if the tissue, organ, or subject has an increased level of mitochondrial and / or ETC complex hyperoxidation; and b) administering methylene blue or a functional variant thereof to the tissue, organ, or subject if the level of mitochondrial and / or ETC complex hyperoxidation is increased relative to a reference. In some embodiments of any of the aspects, the step of determining if the tissue, organ, or subject has an increased level of mitochondrial and / or ETC complex hyperoxidation can comprise performing or having performed an assay on an tissue, organ, or subject (e.g., ex vivo or in vivo) to determine / measure the level of mitochondrial and / or ETC complex194929-4870-6393 6Attorney Docket No. 030258-000102 WOPT hyperoxidation in the tissue, organ, or subject. In some embodiments of any of the aspects, the step of determining if the tissue, organ, or subject has an increased level of mitochondrial and / or ETC complex hyperoxidation can comprise performing or having performed an assay on a tissue, organ, or subject (e.g., ex vivo or in vivo) to determine / measure the level of mitochondrial and / or ETC complex hyperoxidation in the tissue, organ, or subject. In some embodiments of any of the aspects, the step of determining if the tissue, organ, or subject has an increased level of mitochondrial and / or ETC complex hyperoxidation can comprise ordering or requesting an assay on the tissue, organ, or subject to determine / measure the level of mitochondrial and / or ETC complex hyperoxidation in the tissue, organ, or subject. In some embodiments of any of the aspects, the step of determining if the tissue, organ, or subject has an increased level of mitochondrial and / or ETC complex hyperoxidation can comprise receiving the results of an assay on the tissue, organ, or subject to determine / measure the level of mitochondrial and / or ETC complex hyperoxidation in the tissue, organ, or subject. In some embodiments of any of the aspects, the step of determining if the tissue, organ, or subject has an increased level of mitochondrial and / or ETC complex hyperoxidation can comprise receiving a report, results, or other means of identifying the tissue, organ, or subject as a subject with an increased level of mitochondrial and / or ETC complex hyperoxidation.

[0064] In one aspect of any of the embodiments, described herein is a method of treating or preventing an ischemia-reperfusion injury in a tissue, organ, or subject in need thereof, the method comprising: a) determining if the tissue, organ, or subject has an increased level of mitochondrial and / or ETC complex hyperoxidation; and b) instructing or directing that the tissue, organ, or subject be administered methylene blue or a functional variant thereof if the level of mitochondrial and / or ETC complex hyperoxidation is increased relative to a reference. In some embodiments of any of the aspects, the step of determining if the tissue, organ, or subject has an increased level of mitochondrial and / or ETC complex hyperoxidation can comprise performing or having performed an assay on the tissue, organ, or subject to determine / measure the level of mitochondrial and / or ETC complex hyperoxidation in the tissue, organ, or subject. In some embodiments of any of the aspects, the step of determining if the tissue, organ, or subject has an increased level of mitochondrial and / or ETC complex hyperoxidation can comprise performing or having performed an assay on the tissue, organ, or subject to determine / measure the level of mitochondrial and / or ETC complex hyperoxidation in the tissue, organ, or subject. In some embodiments of any of the aspects, the step of determining if the tissue, organ, or subject has an increased level of204929-4870-6393 6Attorney Docket No. 030258-000102 WOPT mitochondrial and / or ETC complex hyperoxidation can comprise ordering or requesting an assay on the tissue, organ, or subject to determine / measure the level of mitochondrial and / or ETC complex hyperoxidation in the tissue, organ, or subject. In some embodiments of any of the aspects, the step of instructing or directing that the tissue, organ, or subject be administered a particular treatment can comprise providing a report of the assay results. In some embodiments of any of the aspects, the step of instructing or directing that the tissue, organ, or subject be administered a particular treatment can comprise providing a report of the assay results and / or treatment recommendations in view of the assay results.

[0065] One aspect of any of the embodiments is a method for preventing or treating ischemia-reperfusion injury in a tissue or an organ, the method comprising: (a) determining if an entire mitochondria and / or at least one electron transport chain (ETC) complex of the tissue or organ is hyperoxidized; and (b) if the entire mitochondria and / or the at least one ETC complex of the tissue or organ is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0066] In one embodiment of any of the aspects, the organ is a solid organ or a tissue thereof. In one embodiment of any of the aspects, the organ or tissue is vascularized (i.e., comprises blood vessels of the cardiovascular system. For example, the tissue is of a liver, a heart, a kidney, a brain, a retina, or skin. In another embodiment, the organ is a liver, a heart, a kidney, a brain, a retina, or skin.

[0067] For example, the organ is a heart, a lung, a liver, a kidney, a brain, a spleen, a pancreas, a small intestine, a large intestine, skin, a skeletal muscle, a cardiac muscle, a smooth muscle, an adrenal gland, a thyroid gland, a parathyroid gland, bone marrow, a placenta, a retina, a pituitary gland, a thymus, a lymph node, an ovary, a teste, a uterus, a periosteum, a synovial membrane, or a mucous membrane.

[0068] For example, the tissue is of a heart, a lung, a liver, a kidney, a brain, a spleen, a pancreas, a small intestine, a large intestine, skin, a skeletal muscle, a cardiac muscle, a smooth muscle, an adrenal gland, a thyroid gland, a parathyroid gland, bone marrow, a placenta, a retina, a pituitary gland, a thymus, a lymph node, an ovary, a teste, a uterus, a periosteum, a synovial membrane, a mucous membrane, or a subcutaneous tissue.

[0069] In one embodiment of any of the aspects, the tissue or organ is on or in a subject, and / or the method occurs in vivo. In another embodiment of any of the aspects, the tissue or organ has been removed from a subject, and / or the method occurs ex vivo.214929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0070] In one embodiment of any of the aspects, prior to treatment, the tissue or organ has been subjected to ischemia followed by initiation of reperfusion, and / or the tissue or organ is at risk of or has suffered an ischemia-reperfusion injury. In one embodiment of any of the aspects, the ischemia and / or reperfusion is from: a tissue or organ transplantation, a surgery (e.g., a complex surgery), a myocardial infarction, or a stroke. In one embodiment of any of the aspects, the surgery (e.g., complex surgery) is a cardiac surgery (e.g., a complex cardiac surgery). For example, the cardiac surgery (e.g., complex cardiac surgery) comprises cardioplegia, which is the deliberate temporary stopping of the heart. In one embodiment of any of the aspects, the surgery (e.g., complex surgery) is a surgery of any vascularized organ, which involves ceasing or decreasing blood flow to the organ during the surgery. The term “complex surgery” can refer to a surgical procedure that is high-risk, involves multiple steps, and / or includes specialized techniques such as cardioplegia or other forms of organ ischemia.

[0071] In one embodiment of any of the aspects, the organ or tissue is a Donor after Circulatory Death (DCD) organ or tissue. Donation after Circulatory Death (DCD) refers to the recovery of organ(s) for transplantation from a deceased donor after irreversible cessation of circulatory and respiratory functions, which can occur when life-sustaining treatment is withdrawn, and the heart stops beating, leading to the declaration of death by a physician.

[0072] In some embodiments of any of the aspects, the ischemia is warm ischemia. In one embodiment of any of the aspects, the reperfusion is machine perfusion. For example, the machine perfusion is subnormothermic machine perfusion.

[0073] In some embodiments of any of the aspects, the ischemia occurs for at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 120 minutes, or at least 180 minutes. For example, the ischemia occurs for 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, 40 minutes, 42 minutes, 44 minutes, 46 minutes, 48 minutes, 50 minutes, 52 minutes, 54 minutes, 56 minutes, 58 minutes, 60 minutes, 62 minutes, 64 minutes, 66 minutes, 68 minutes, 70 minutes, 72 minutes, 74 minutes, 76 minutes, 78 minutes, 80 minutes, 82 minutes, 84 minutes, 86 minutes, 88 minutes, 90 minutes, 92 minutes, 94 minutes, 96 minutes, 98 minutes, 100 minutes, 102 minutes, 104 minutes, 106 minutes, 108 minutes, 110 minutes, 112 minutes, 114 minutes, 116 minutes, 118 minutes, 120 minutes, 122 minutes, 124 minutes, 126 minutes, 128 minutes, 130 minutes, 132 minutes, 134 minutes, 136 minutes, 138 minutes, 140 minutes, 142 minutes, 144 minutes, 146 minutes, 148 minutes, 150 minutes, 152 minutes, 154 minutes, 156 minutes, 158 minutes, 160 minutes, 162 minutes, 164 minutes, 166 minutes, 168 minutes, 170 minutes, 172 minutes, 174224929-4870-6393 6Attorney Docket No. 030258-000102 WOPT minutes, 176 minutes, 178 minutes, 180 minutes, 30-60 minutes, 60-120 minutes, 120-180 minutes, 30-120 minutes, 60-180 minutes, or 30-180 minutes.

[0074] In some embodiments, the ischemia occurs for about 30 minutes, about 32 minutes, about 34 minutes, about 36 minutes, about 38 minutes, about 40 minutes, about 42 minutes, about 44 minutes, about 46 minutes, about 48 minutes, about 50 minutes, about 52 minutes, about 54 minutes, about 56 minutes, about 58 minutes, about 60 minutes, about 62 minutes, about 64 minutes, about 66 minutes, about 68 minutes, about 70 minutes, about 72 minutes, about 74 minutes, about 76 minutes, about 78 minutes, about 80 minutes, about 82 minutes, about 84 minutes, about 86 minutes, about 88 minutes, about 90 minutes, about 92 minutes, about 94 minutes, about 96 minutes, about 98 minutes, about 100 minutes, about 102 minutes, about 104 minutes, about 106 minutes, about 108 minutes, about 110 minutes, about 112 minutes, about 114 minutes, about 116 minutes, about 118 minutes, about 120 minutes, about 122 minutes, about 124 minutes, about 126 minutes, about 128 minutes, about 130 minutes, about 132 minutes, about 134 minutes, about 136 minutes, about 138 minutes, about 140 minutes, about 142 minutes, about 144 minutes, about 146 minutes, about 148 minutes, about 150 minutes, about 152 minutes, about 154 minutes, about 156 minutes, about158 minutes, about 160 minutes, about 162 minutes, about 164 minutes, about 166 minutes, about 168 minutes, about 170 minutes, about 172 minutes, about 174 minutes, about 176 minutes, about 178 minutes, or about 180 minutes.

[0075] In some embodiments of any of the aspects, the reperfusion occurs for at least30 minutes, at least 45 minutes, at least 60 minutes, at least 120 minutes, or at least 180 minutes. iFor example, the reperfusion occurs for 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, 40 minutes, 42 minutes, 44 minutes, 46 minutes, 48 minutes, 50 minutes, 52 minutes, 54 minutes, 56 minutes, 58 minutes, 60 minutes, 62 minutes, 64 minutes, 66 minutes, 68 minutes, 70 minutes, 72 minutes, 74 minutes, 76 minutes, 78 minutes, 80 minutes, 82 minutes, 84 minutes, 86 minutes, 88 minutes, 90 minutes, 92 minutes, 94 minutes, 96 minutes, 98 minutes, 100 minutes, 102 minutes, 104 minutes, 106 minutes, 108 minutes, 110 minutes, 112 minutes, 114 minutes, 116 minutes, 118 minutes, 120 minutes, 122 minutes, 124 minutes, 126 minutes, 128 minutes, 130 minutes, 132 minutes, 134 minutes, 136 minutes, 138 minutes, 140 minutes, 142 minutes, 144 minutes, 146 minutes, 148 minutes, 150 minutes, 152 minutes, 154 minutes, 156 minutes, 158 minutes, 160 minutes, 162 minutes, 164 minutes, 166 minutes, 168 minutes, 170 minutes, 172 minutes, 174 minutes, 176 minutes, 178 minutes, 180 minutes, 30-60 minutes, 60-120 minutes, 120-180 minutes, 30-120 minutes, 60-180 minutes, or 30-180 minutes.234929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0076] In some embodiments, the reperfusion occurs for about 30 minutes, about 32 minutes, about 34 minutes, about 36 minutes, about 38 minutes, about 40 minutes, about 42 minutes, about 44 minutes, about 46 minutes, about 48 minutes, about 50 minutes, about 52 minutes, about 54 minutes, about 56 minutes, about 58 minutes, about 60 minutes, about 62 minutes, about 64 minutes, about 66 minutes, about 68 minutes, about 70 minutes, about 72 minutes, about 74 minutes, about 76 minutes, about 78 minutes, about 80 minutes, about 82 minutes, about 84 minutes, about 86 minutes, about 88 minutes, about 90 minutes, about 92 minutes, about 94 minutes, about 96 minutes, about 98 minutes, about 100 minutes, about 102 minutes, about 104 minutes, about 106 minutes, about 108 minutes, about 110 minutes, about 112 minutes, about 114 minutes, about 116 minutes, about 118 minutes, about 120 minutes, about 122 minutes, about 124 minutes, about 126 minutes, about 128 minutes, about 130 minutes, about 132 minutes, about 134 minutes, about 136 minutes, about 138 minutes, about 140 minutes, about 142 minutes, about 144 minutes, about 146 minutes, about 148 minutes, about 150 minutes, about 152 minutes, about 154 minutes, about 156 minutes, about 158 minutes, about 160 minutes, about 162 minutes, about 164 minutes, about 166 minutes, about 168 minutes, about 170 minutes, about 172 minutes, about 174 minutes, about 176 minutes, about 178 minutes, or about 180 minutes.

[0077] In some embodiments of any of the aspects, the reperfusion occurs for up to 24 hours. For example, the reperfusion occurs for 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, 40 minutes, 42 minutes, 44 minutes, 46 minutes, 48 minutes, 50 minutes, 52 minutes, 54 minutes, 56 minutes, 58 minutes, 60 minutes, 62 minutes, 64 minutes, 66 minutes, 68 minutes, 70 minutes, 72 minutes, 74 minutes, 76 minutes, 78 minutes, 80 minutes, 82 minutes, 84 minutes, 86 minutes, 88 minutes, 90 minutes, 92 minutes, 94 minutes, 96 minutes, 98 minutes, 100 minutes, 102 minutes, 104 minutes, 106 minutes, 108 minutes, 110 minutes, 112 minutes, 114 minutes, 116 minutes, 118 minutes, 120 minutes, 122 minutes, 124 minutes, 126 minutes, 128 minutes, 130 minutes, 132 minutes, 134 minutes, 136 minutes, 138 minutes, 140 minutes, 142 minutes, 144 minutes, 146 minutes, 148 minutes, 150 minutes, 152 minutes, 154 minutes, 156 minutes, 158 minutes, 160 minutes, 162 minutes, 164 minutes, 166 minutes, 168 minutes, 170 minutes, 172 minutes, 174 minutes, 176 minutes, 178 minutes, or 180 minutes, 2 hours, 2.25 hours, 2.5 hours, 2.75 hours, 3 hours, 3.25 hours, 3.5 hours, 3.75 hours, 4 hours, 4.25 hours, 4.5 hours, 4.75 hours, 5 hours, 5.25 hours, 5.5 hours, 5.75 hours, 6 hours, 6.25 hours, 6.5 hours, 6.75 hours, 7 hours, 7.25 hours, 7.5 hours, 7.75 hours, 8 hours, 8.25 hours, 8.5 hours, 8.75 hours, 9 hours, 9.25 hours, 9.5 hours, 9.75 hours, 10 hours, 10.25 hours, 10.5 hours, 10.75 hours, 11 244929-4870-6393 6Attorney Docket No. 030258-000102 WOPT hours, 11.25 hours, 11.5 hours, 11.75 hours, 12 hours, 12.25 hours, 12.5 hours, 12.75 hours, 13 hours, 13.25 hours, 13.5 hours, 13.75 hours, 14 hours, 14.25 hours, 14.5 hours, 14.75 hours, 15 hours, 15.25 hours, 15.5 hours, 15.75 hours, 16 hours, 16.25 hours, 16.5 hours,16.75 hours, 17 hours, 17.25 hours, 17.5 hours, 17.75 hours, 18 hours, 18.25 hours, 18.5 hours, 18.75 hours, 19 hours, 19.25 hours, 19.5 hours, 19.75 hours, 20 hours, 20.25 hours,20.5 hours, 20.75 hours, 21 hours, 21.25 hours, 21.5 hours, 21.75 hours, 22 hours, 22.25 hours, 22.5 hours, 22.75 hours, 23 hours, 23.25 hours, 23.5 hours, 23.75 hours, 24 hours, 30 minutes to 24 hours, 1-24 hours, 6-24 hours, 18-24 hours, 1-6 hours, 6-12 hours, 12-18 hours, 6-24 hours, or 12-24 hours.

[0078] In some embodiments of any of the aspects, the reperfusion occurs for up to about 24 hours. For example, the reperfusion occurs for about 30 minutes, about 32 minutes, about 34 minutes, about 36 minutes, about 38 minutes, about 40 minutes, about 42 minutes, about 44 minutes, about 46 minutes, about 48 minutes, about 50 minutes, about 52 minutes, about 54 minutes, about 56 minutes, about 58 minutes, about 60 minutes, about 62 minutes, about 64 minutes, about 66 minutes, about 68 minutes, about 70 minutes, about 72 minutes, about 74 minutes, about 76 minutes, about 78 minutes, about 80 minutes, about 82 minutes, about 84 minutes, about 86 minutes, about 88 minutes, about 90 minutes, about 92 minutes, about 94 minutes, about 96 minutes, about 98 minutes, about 100 minutes, about 102 minutes, about 104 minutes, about 106 minutes, about 108 minutes, about 110 minutes, about 112 minutes, about 114 minutes, about 116 minutes, about 118 minutes, about 120 minutes, about 122 minutes, about 124 minutes, about 126 minutes, about 128 minutes, about 130 minutes, about 132 minutes, about 134 minutes, about 136 minutes, about 138 minutes, about 140 minutes, about 142 minutes, about 144 minutes, about 146 minutes, about 148 minutes, about 150 minutes, about 152 minutes, about 154 minutes, about 156 minutes, about 158 minutes, about 160 minutes, about 162 minutes, about 164 minutes, about 166 minutes, about 168 minutes, about 170 minutes, about 172 minutes, about 174 minutes, about 176 minutes, about 178 minutes, or about 180 minutes, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75 hours, about 3 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, about 4 hours, about 4.25 hours, about 4.5 hours, about 4.75 hours, about 5 hours, about 5.25 hours, about 5.5 hours, about 5.75 hours, about 6 hours, about 6.25 hours, about 6.5 hours, about6.75 hours, about 7 hours, about 7.25 hours, about 7.5 hours, about 7.75 hours, about 8 hours, about 8.25 hours, about 8.5 hours, about 8.75 hours, about 9 hours, about 9.25 hours, about9.5 hours, about 9.75 hours, about 10 hours, about 10.25 hours, about 10.5 hours, about 10.75 hours, about 11 hours, about 11.25 hours, about 11.5 hours, about 11.75 hours, about 12254929-4870-6393 6Attorney Docket No. 030258-000102 WOPT hours, about 12.25 hours, about 12.5 hours, about 12.75 hours, about 13 hours, about 13.25 hours, about 13.5 hours, about 13.75 hours, about 14 hours, about 14.25 hours, about 14.5 hours, about 14.75 hours, about 15 hours, about 15.25 hours, about 15.5 hours, about 15.75 hours, about 16 hours, about 16.25 hours, about 16.5 hours, about 16.75 hours, about 17 hours, about 17.25 hours, about 17.5 hours, about 17.75 hours, about 18 hours, about 18.25 hours, about 18.5 hours, about 18.75 hours, about 19 hours, about 19.25 hours, about 19.5 hours, about 19.75 hours, about 20 hours, about 20.25 hours, about 20.5 hours, about 20.75 hours, about 21 hours, about 21.25 hours, about 21.5 hours, about 21.75 hours, about 22 hours, about 22.25 hours, about 22.5 hours, about 22.75 hours, about 23 hours, about 23.25 hours, about 23.5 hours, about 23.75 hours, or about 24 hours.

[0079] In some embodiments of any of the aspects, the method is performed during or after the reperfusion. In some embodiments of any of the aspects, the methylene blue or functional variant thereof is administered during the reperfusion. In some embodiments of any of the aspects, the methylene blue or functional variant thereof is administered after the reperfusion. In some embodiments of any of the aspects, the methylene blue or functional variant thereof is administered during the reperfusion and after the reperfusion. In some embodiments of any of the aspects, the methylene blue or functional variant thereof is administered before skin closure during a tissue or organ transplantation or a complex surgery. In some embodiments of any of the aspects, the methylene blue or functional variant thereof is administered immediately after transplantation of the tissue or organ into a subject. For example, the methylene blue or a variant thereof is administered about 0.5 minutes, 0.75 minutes, 1 minute, 1.25 minutes, 1.5 minutes, 1.75 minutes, 2 minutes, 2.25 minutes, 2.5 minutes, 2.75 minutes, 3 minutes, 3.25 minutes, 3.5 minutes, 3.75 minutes, 4 minutes, 4.25 minutes, 4.5 minutes, 4.75 minutes, 5 minutes, 5.25 minutes, 5.5 minutes, 5.75 minutes, 6 minutes, 6.25 minutes, 6.5 minutes, 6.75 minutes, 7 minutes, 7.25 minutes, 7.5 minutes, 7.75 minutes, 8 minutes, 8.25 minutes, 8.5 minutes, 8.75 minutes, 9 minutes, 9.25 minutes, 9.5 minutes, 9.75 minutes, 10 minutes, 10.25 minutes, 10.5 minutes, 10.75 minutes, 11 minutes, 11.25 minutes, 11.5 minutes, 11.75 minutes, 12 minutes, 12.25 minutes, 12.5 minutes, 12.75 minutes, 13 minutes, 13.25 minutes, 13.5 minutes, 13.75 minutes, 14 minutes, 14.25 minutes, 14.5 minutes, 14.75 minutes, or 15 minutes after transplantation of the tissue or organ into a subject.

[0080] In some embodiments of any of the aspects, the method is not performed during the ischemia. In some embodiments of any of the aspects, the methylene blue or functional variant thereof is not administered during the ischemia.264929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0081] In some embodiments, the method further comprises administering an effective amount of an oxygenation support to the tissue or organ, in addition to the administering of the methylene blue or functional variant thereof.

[0082] The term “oxygenation support” as used herein refers to a composition used for oxygen therapy, also referred to as supplemental oxygen, which is the use of oxygen as medical treatment.

[0083] For example, the oxygenation support is whole blood, packed Red Blood Cells (RBCs), artificial Hemoglobin Based Oxygen Carriers (HBOCs), Supplemental Oxygen Therapy, Mechanical Ventilation, Hyperbaric Oxygen Therapy (HBOT), Extracorporeal Membrane Oxygenation (ECMO), cardiopulmonary bypass (CPB), or Hypothermic Oxygenated Machine Perfusion (HOPE). In some embodiments of any of the aspects, the oxygenation support is HBOCs.

[0084] Some embodiments of any of the aspects further comprise determining if the mitochondria and / or at least one electron transport chain (ETC) complex is hyperoxidized prior to administering the effective amount of methylene blue or the functional variant thereof. For example, the method further comprises quantifying a Resonance Raman Reduced Mitochondrial Ratio (3RMR) value for the redox state of the mitochondria or the at least one ETC complex of the tissue or organ using a Raman spectroscopy method and determining if the 3RMR value indicates that the whole mitochondria and / or the at least one ETC complexes is hyperoxidized. In some embodiments of any of the aspects, the excitation wavelength used in the Raman spectroscopy method is 441 nm. In some embodiments of any of the aspects, the excitation wavelength used in the Raman spectroscopy method is within the Soret absorption band (400 nm-450 nm).

[0085] A resonance Raman spectroscopy (RRS) system permits clinicians to assess organ fitness safely and confidently prior to transplantation. This approach is compatible with current clinical standards of organ preservation and produces results rapidly in real-time. The RRS system can be used to measure resonance Raman reduced mitochondrial ratios (3RMR) in biological tissue. Measuring 3RMR can be used to quantify the amount of damage to a tissue for transplant caused by ischemia. This measurement can be used to determine the viability of the tissue for transplantation and predict transplant success.

[0086] In Raman spectroscopy, the wavelength of light from a narrowband laser is shifted to lower energy by a precise quantity determined by the frequency of the vibrational mode of the molecules it encounters. The wavelength shift (also called a Stokes shift) of inelastically scattered light can be separated from fluorescence to measure a redox state-274929-4870-6393 6Attorney Docket No. 030258-000102 WOPT specific spectral signature of a molecule. In the special case of RRS, the optically excited state overlaps a strong electronic absorption line, resulting in orders of magnitude enhancement of the Raman cross-section.

[0087] Relevant to cellular energetics, the resonance Raman profiles of porphyrin structures (present in hemoglobin, myoglobin, and mitochondrial cytochromes) are amplified by 4 to 6 orders of magnitude (called an enhancement factor) when excited near the Soret absorption band (400 nm-450 nm). This enhancement makes the in vivo quantification of small quantities of such structures possible, even in a complex environment. Using this approach, the redox state of mitochondrial cytochromes in isolated mitochondria, in myocytes, and in bloodless tissues can be determined. In addition to determining mitochondrial cytochrome redox state, RRS can also be used to determine other mitochondrial breakdown products, as well as other tissue health indicators, such as tissue oxyhemoglobin saturation. The RRS system offers continuous multi-point monitoring of organs during machine perfusion.

[0088] An RRS technique for quantification of 3RMR values is further described in US Patent 11,717,167 B2 and US Patent Publication 2023 / 0109459 Al, the contents of each of which are incorporated herein in their entireties.

[0089] In some embodiments of any of the aspects, the at least one ETC complex is Complex I (CI), Complex II (CII), or Complex III (CIII). For example, the at least one ETC complex is Complex III (CIII).

[0090] In some embodiments of any of the aspects, the 3RMR value for the mitochondria in a liver being below 10% (e.g., below about: 5%, 6%, 7%, 8%, 9%, 10%) or the 3RMR value for CIII being in a liver below about 20% (e.g., below about: 15%, 16%, 17%, 18%, 19%, 20%) indicate that the entire mitochondria or the CIII are hyperoxidized. In other embodiments, the 3RMR value for the entire mitochondria in a liver being above 10% (e.g., above about: 5%, 6%, 7%, 8%, 9%, 10%) or the 3RMR value for CIII in a liver being above about 20% (e.g., above about: 15%, 16%, 17%, 18%, 19%, 20%), indicate that the entire mitochondria or CIII are not hyperoxidized. Without wishing to be bound by theory, it is expected that the 3RMR values for the mitochondria and / or the CIII in the liver will be similar (e.g., within 1%, 5%, or 10% points) to the 3RMR values in non-liver organs.

[0091] One aspect of any of the embodiments is a method for preventing or treating ischemia-reperfusion injury in a tissue or an organ, the method comprising: (a) quantifying a Resonance Raman Reduced Mitochondrial Ratio (3RMR) value of a mitochondrial complex of the tissue or organ using a Raman spectroscopy method; (b) determining if the 3RMR284929-4870-6393 6Attorney Docket No. 030258-000102 WOPT value indicates that the mitochondrial complex is hyperoxidized; and (c) if the 3RMR value indicates that the mitochondrial complex is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0092] In some embodiments of any of the aspects, the at least one ETC complex is Complex I (CI), Complex II (CII), or Complex III (CIII). In some embodiments of any of the aspects, the at least one ETC complex is CIII. In some embodiments, if the mitochondria and / or the at least one ETC complex are physiologically oxidized, no treatment is administered. In some embodiments, if the mitochondria and / or the at least one ETC complex are hyper-reduced, the treatment comprises oxygen and / or an oxygenation support.

[0093] For whole mitochondria in the liver, a 3RMR value of about 10% to about 45% is representative of physiologically oxidized mitochondria. A 3RMR value of above about 45% in whole mitochondria in the liver is hyper-reduced. For CIII, a 3RMR value of about 20% to about 45% is considered physiologically oxidized in the liver , and a 3RMR value above about 45% is considered hyper-reduced in the liver. Without wishing to be bound by theory, it is expected that the 3RMR values for the mitochondria and / or the CIII in the liver will be similar (e.g., within 1%, 5%, or 10% points) to the 3RMR values in non-liver organs.

[0094] In some embodiments, after step (c), the method further comprises: (d) quantifying a 3RMR value of a second mitochondrial ETC complex of the tissue or organ using the Raman spectroscopy method; and (e) determining if the 3RMR value indicates that the second mitochondrial ETC complex is reduced.

[0095] In some embodiments, after step (e), the method further comprises: (f) if the 3RMR value indicates that the second mitochondrial ETC complex is not reduced, continuing to administer the methylene blue or the functional variant thereof to the tissue or organ; or (g) if the 3RMR value indicates that the second mitochondrial ETC complex is reduced, ceasing to administer the methylene blue or the functional variant thereof to the tissue or organ and / or increasing the oxygen delivery to the tissue or organ.

[0096] In some embodiments of any of the aspects, the second mitochondrial ETC complex is Complex IV (CIV). In some embodiments, the 3RMR value being at or above about 1% (e.g., above about: 0.5%, 0.9% 1.0%, 1.1%, 1.5%) in a liver indicates that the CIV is reduced; and wherein the 3RMR value being below about 1% (e.g., below about: 0.5%, 0.9% 1.0%, 1.1%, 1.5%) in a liver indicates that the CIV is not reduced. Without wishing to294929-4870-6393 6Attorney Docket No. 030258-000102 WOPT be bound by theory, it is expected that the 3RMR values for the CIV in the liver will be similar (e.g., within 1%, 5%, or 10% points) to the 3RMR values in non-liver organs.

[0097] In some embodiments, the 3RMR value of the mitochondria, the CIII, or the CIV is calculated by: (i) obtaining a raw spectrum using the Raman spectroscopy method; (ii) performing baseline subtraction of the raw spectrum to produce a resonance Raman spectrum; (iii) performing a linear regression analysis of the resonance Raman spectrum against a library of known analytes selected from oxidized and reduced resonance Raman spectra of the mitochondria, CIII, or CIV, to determine the relative concentration of: oxidized entire mitochondria and reduced entire mitochondria, or oxidized CIII and reduced CIII, or oxidized CIV and reduced CIV, in the tissue or organ; wherein, during the linear regression analysis, each known analyte is assigned a specific enhancement factor (fn), and (iv) wherein the 3RMR value of the mitochondria, the CIII, or the CIV is calculated as the ratio of their corresponding reduced concentration divided by the sum of the oxidized and reduced concentrations.

[0098] In the 3RMR calculation, the f values are fixed for all measurements at a specific excitation wavelength. For an excitation wavelength of 441 nm, the f values are 0.15 for reduced entire mitochondria (fi) and 1 for oxidized entire mitochondria (fz). P values are coefficient values. The P value calculated from the analytical algorithm reflects the relative contribution of each form (reduced or oxidized) to the total measure spectrum (ym). P values can change between measurements while f values are fixed and do not. While P already gives the relative ratio, P alone only shows the ratio under a specific wavelength (e.g., 441 nm). To report the true biological ratio, the 3RMR calculation also factors in f, which accounts for wavelength-specific differences in signal. For example, if the wavelength is changed (e.g., from 441 nm to 420 nm), the f values will be different, and the P values will also change. The product of P and f (here written as fnpnin the 3RMR calculations) remains the same across wavelengths (e.g., 441 nm, 420 nm, etc.).

[0099] In some embodiments, the 3RMR value of the entire mitochondria is calculated by: Formula I: 3RMR_mito = i / ?i / ( / i / ?i + f ftz)- In some embodiments, the 3RMR value of the CIII is calculated by: Formula II: 3RMR_CIII =+ A / ^)- In some embodiments, the 3RMR value of the CIV is calculated by Formula III: 3RMR_CIV =

[0100] One aspect of any of the embodiments is methylene blue or a functional variant thereof for use in a method of treating or preventing ischemia-reperfusion injury.304929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0101] One aspect of any of the embodiments is methylene blue or a functional variant thereof for use in a method of treating or preventing ischemia-reperfusion injury, the method comprising: administering an effective amount of methylene blue or a functional variant thereof to an tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0102] One aspect of any of the embodiments is methylene blue or a functional variant thereof for use in a method of treating or preventing ischemia-reperfusion injury, the method comprising: (a) determining if an entire mitochondria or at least one electron transport chain (ETC) complex of the tissue or organ is hyperoxidized; and (b) if the entire mitochondria or the at least one ETC complex of the tissue or organ is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0103] One aspect of any of the embodiments is methylene blue or a functional variant thereof for use in a method of treating or preventing ischemia-reperfusion injury, the method comprising: (a) quantifying a Resonance Raman Reduced Mitochondrial Ratio (3RMR) value of a mitochondrial complex of the tissue or organ using a Raman spectroscopy method; (b) determining if the 3RMR value indicates that the mitochondrial complex is hyperoxidized; and (c) if the 3RMR value indicates that the mitochondrial complex is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0104] One aspect of any of the embodiments is methylene blue or a functional variant thereof for use in the manufacture of a medicament for treating or preventing ischemia-reperfusion injury.Definitions

[0105] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected herein. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.314929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0106] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0107] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%. In some embodiments of the various aspects described herein, the term “about” when used in connection with percentages can mean ±5%. The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0108] As used herein, the use of “or” means “and / or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

[0109] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0110] The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0111] As used herein the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characterise cfs) of that embodiment of the invention.324929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0112] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0113] The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[0114] As used herein, the term “analog” refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance.

[0115] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference or a p- value of less than 0.05.

[0116] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0117] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as334929-4870-6393 6Attorney Docket No. 030258-000102 WOPT compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[0118] The terms “prevent,” “preventing” and “prevention” (and grammatical variations thereof) refer to prevention and / or delay of the onset of a disease, disorder and / or a clinical symptom(s) in a subject and / or a reduction in the severity of the onset of the disease, disorder and / or clinical symptom(s) relative to what would occur in the absence of treatment. The prevention can be complete, e.g., the total absence of the disease, disorder and / or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and / or clinical symptom(s) in the subject and / or the severity of onset is less than what would occur in the absence of the treatment.

[0119] As used herein, the terms “treat,” “treating,” and / or “treatment” include abrogating, substantially inhibiting, slowing or reversing the progression of a disorder, disease or condition, substantially ameliorating clinical symptoms of a disorder, disease or condition, or substantially preventing the appearance of clinical symptoms of a disorder, disease or condition, obtaining beneficial or desired clinical results. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder, disease or condition); (b) limiting development of symptoms characteristic of the disorder, disease or condition(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder, disease or condition(s) being treated; (d) limiting recurrence of the disorder, disease or condition(s) in subjects that have previously had the disorder, disease or condition(s); and (e) limiting recurrence of symptoms in subjects that were previously asymptomatic for the disorder, disease or condition(s). Beneficial or desired clinical results, such as pharmacologic and / or physiologic effects include, but are not limited to, preventing the disease, disorder or condition from occurring in a subject predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (e.g., not worsening) of the disease, disorder or condition, preventing spread of the disease, disorder or condition, delaying or slowing of the344929-4870-6393 6Attorney Docket No. 030258-000102 WOPT disease, disorder or condition progression, amelioration or palliation of the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0120] As used herein, the term “subject” or “patient” refers to any organism to which a compound or composition disclosed herein can be administered, e.g., for experimental, diagnostic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient” and “subject” are used interchangeably herein. A subject can be male or female.

[0121] Preferably, the subject is a mammal. The mammal can be a primate (e.g., human or non-human primate), mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of human diseases and disorders. In addition, compounds, compositions and methods described herein can be used with domesticated animals and / or pets.

[0122] In some embodiments, the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. Examples of subjects include humans, dogs, cats, cows, goats, and mice. The term subject is further intended to include transgenic species. In some embodiments, the subject can be of European ancestry. In some embodiments, the subject can be of African American ancestry. In some embodiments, the subject can be of Asian ancestry.

[0123] In one embodiment, a patient is a human, such as a human infant (e.g. less than 1 years old), child (e.g. between 1 and 12 years old), adolescent (e.g. between 12 and 18 years old), adult (e.g. 18 to 65 years), or elderly (e.g. older than 65).354929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0124] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. ischemia-reperfusion injury) or one or more complications related to such a condition, and optionally, have already undergone treatment for ischemia-reperfusion injury or the one or more complications related to ischemia-reperfusion injury. Alternatively, a subject can also be one who has not been previously diagnosed as having ischemia-reperfusion injury or one or more complications related to ischemia-reperfusion injury. For example, a subject can be one who exhibits one or more risk factors for ischemia-reperfusion injury or one or more complications related to ischemia-reperfusion injury or a subject who does not exhibit risk factors.

[0125] A “subject in need” of treatment for a particular condition (e.g., ischemiareperfusion injury) can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0126] As used herein, “administration,” “administering,” and variants thereof refers to introducing a compound or a composition into a subject and includes concurrent and sequential introduction of a compound or a composition. “Administration” can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. “Administration” also encompasses in vitro and ex vivo treatments. The introduction of a compound or a composition into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, or topically. Administration includes selfadministration and the administration by another. Administration can be carried out by any suitable route. A suitable route of administration allows the compound or the composition to perform its intended function. Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion. In some embodiments, administration will generally be local rather than systemic.

[0127] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will self-administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining364929-4870-6393 6Attorney Docket No. 030258-000102 WOPT patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” of compositions includes both methods practiced on the human body and also the foregoing activities. It is noted that administering can be in vitro, ex vivo or in vivo.

[0128] The treatments described herein can be administered at once or can be divided into a number of smaller doses to be administered at intervals of time. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to methylene blue or functional variant thereof. The desired dose or amount can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be one or more doses and / or treatments daily over a period of weeks or months. Examples of dosing and / or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. It is understood that the precise dosage and duration of treatment will be a function of the location of where the composition is parenterally administered, the carrier and other variables that can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values can also vary with the age of the individual treated. It is to be further understood that for any subject, specific dosage regimens can need to be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations. Hence, the concentration ranges set forth herein are intended to be exemplary and are not intended to limit the scope or practice of the claimed formulations.

[0129] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in cell biology, immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology 374929-4870-6393 6Attorney Docket No. 030258-000102 WOPT and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADAM Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0130] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:

[0131] Paragraph 1 : A method for preventing or treating ischemia-reperfusion injury in an organ or a tissue, the method comprising administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0132] Paragraph 2: A method for preventing or treating ischemia-reperfusion injury in a tissue or an organ, the method comprising: (a) determining if a mitochondria and / or at least one electron transport chain (ETC) complex of the tissue or organ is hyperoxidized; and (b) if the mitochondria and / or the at least one ETC complex of the tissue or organ is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0133] Paragraph 3: The method of any of paragraphs 1-2, wherein the organ is a solid organ or a tissue thereof.

[0134] Paragraph 4: The method of any of paragraphs 1-3, wherein the organ or tissue is vascularized.384929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0135] Paragraph 5: The method of any of paragraphs 1-4, wherein the tissue is of a liver, a heart, a kidney, a brain, a retina, or skin.

[0136] Paragraph 6: The method of any of paragraphs 1-5, wherein the organ is a liver, a heart, a kidney, a brain, a retina, or skin.

[0137] Paragraph 7: The method of any one of paragraphs 1-6, wherein the tissue or organ is on or in a subject, and the method occurs in vivo.

[0138] Paragraph 8: The method of any one of paragraphs 1-7, wherein the tissue or organ has been removed from a subject, and the method occurs ex vivo.

[0139] Paragraph 9: The method of any one of paragraphs 1-8, wherein prior to treatment, the tissue or organ has been subjected to ischemia followed by initiation of reperfusion, and the tissue or organ is at risk of or has suffered an ischemia-reperfusion injury.

[0140] Paragraph 10: The method of any of paragraphs 1-9, wherein the ischemia and / or reperfusion is from: a tissue or organ transplantation, a complex surgery, a myocardial infarction, or a stroke.

[0141] Paragraph 11 : The method of paragraph 10, wherein the complex surgery is a complex cardiac surgery.

[0142] Paragraph 12: The method of paragraph 11, wherein the complex cardiac surgery comprises cardioplegia.

[0143] Paragraph 13: The method of paragraphs 1 or 2, wherein the organ or tissue is a Donor after Circulatory Death (DCD) organ or tissue.

[0144] Paragraph 14: The method of any of paragraphs 1-13, wherein the ischemia is warm ischemia.

[0145] Paragraph 15: The method of any of paragraphs 1-13, wherein the reperfusion is machine perfusion.

[0146] Paragraph 16: The method of paragraph 15, wherein the machine perfusion is subnormothermic machine perfusion.

[0147] Paragraph 17: The method of any one of paragraphs 1-16, wherein the ischemia occurs for at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 120 minutes, or at least 180 minutes.

[0148] Paragraph 18: The method of any of paragraphs 1-17, wherein the reperfusion occurs for at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 120 minutes, or at least 180 minutes.394929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0149] Paragraph 19: The method of any of paragraphs 1-18, wherein the reperfusion occurs for up to about 24 hours.

[0150] Paragraph 20: The method of any one of paragraphs 1-19, wherein the method is performed during or after the reperfusion.

[0151] Paragraph 21 : The method of any one of paragraphs 1-20, wherein the methylene blue or functional variant thereof is administered during or after the reperfusion.

[0152] Paragraph 22: The method of any of paragraphs 1-21, wherein the methylene blue or functional variant thereof is administered before skin closure during a tissue or organ transplantation or a complex surgery.

[0153] Paragraph 23: The method of any of paragraphs 1-22, wherein the methylene blue or functional variant thereof is administered immediately after transplantation of the tissue or organ into a subject.

[0154] Paragraph 24: The method of any one of paragraphs 1-23, wherein the method is not performed during the ischemia.

[0155] Paragraph 25: The method of any one of paragraphs 1-24, wherein the methylene blue or functional variant thereof is not administered during the ischemia.

[0156] Paragraph 26: The method of any one of paragraphs 1-25, further comprising administering an effective amount of an oxygenation support to the tissue or organ, in addition to the administering of the methylene blue or functional variant thereof.

[0157] Paragraph 27: The method of paragraph 26, wherein the oxygenation support is whole blood, packed Red Blood Cells (RBCs), artificial Hemoglobin Based Oxygen Carriers (HBOCs), Supplemental Oxygen Therapy, Mechanical Ventilation, Hyperbaric Oxygen Therapy (HBOT), Extracorporeal Membrane Oxygenation (ECMO), cardiopulmonary bypass (CPB), or Hypothermic Oxygenated Machine Perfusion (HOPE).

[0158] Paragraph 28: The method of paragraphs 26 or 27, wherein the oxygenation support is HBOCs.

[0159] Paragraph 29: The method of paragraph 1, further comprising determining if the mitochondria and / or at least one electron transport chain (ETC) complex is hyperoxidized prior to administering the effective amount of methylene blue or the functional variant thereof.

[0160] Paragraph 30: The method of paragraphs 2 or 29, further comprising quantifying a Resonance Raman Reduced Mitochondrial Ratio (3RMR) value for the redox state of the mitochondria or the at least one ETC complex of the tissue or organ using a404929-4870-6393 6Attorney Docket No. 030258-000102 WOPTRaman spectroscopy method, and determining if the 3RMR value indicates that the mitochondria and / or the at least one ETC complexes is hyperoxidized.

[0161] Paragraph 31 : The method of paragraphs 2, 29 or 30, wherein the at least one ETC complex is Complex I (CI), Complex II (CII), or Complex III (CIII).

[0162] Paragraph 32: The method of any of paragraphs 2 and 29-31, wherein the at least one ETC complex is Complex III (CIII).

[0163] Paragraph 33 : The method of paragraph 32, wherein the 3RMR value for the mitochondria in a liver being below 10% or the 3RMR value for CIII being below 20% indicate that the mitochondria or the CIII are hyperoxidized.

[0164] Paragraph 34: The method of paragraph 32, wherein the 3RMR value for the mitochondria in a liver being above 10% or the 3RMR value for CIII being above 20%, indicate that the mitochondria or CIII are not hyperoxidized.

[0165] Paragraph 35: Amethod for preventing or treating ischemia-reperfusion injury in a tissue or an organ, the method comprising: (a) quantifying a Resonance Raman Reduced Mitochondrial Ratio (3RMR) value of a mitochondrial complex of the tissue or organ using a Raman spectroscopy method; (b) determining if the 3RMR value indicates that the mitochondrial complex is hyperoxidized; and (c) if the 3RMR value indicates that the mitochondrial complex is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0166] Paragraph 36: The method of paragraph 35, wherein the at least one ETC complex is Complex I (CI), Complex II (CII), or Complex III (CIII).

[0167] Paragraph 37: The method of paragraph 35, wherein if the mitochondria and / or the at least one ETC complex are physiologically oxidized, no treatment is administered.

[0168] Paragraph 38: The method of paragraph 35, wherein if the mitochondria and / or the at least one ETC complex are hyper-reduced, the treatment comprises oxygen and / or an oxygenation support.

[0169] Paragraph 39: The method of paragraph 35, wherein after step (c), the method further comprises: (d) quantifying a 3RMR value of a second mitochondrial ETC complex of the tissue or organ using the Raman spectroscopy method; and(e) determining if the 3RMR value indicates that the second mitochondrial ETC complex is reduced.

[0170] Paragraph 40: The method of paragraph 39, wherein after step (e), the method further comprises: (f) if the 3RMR value indicates that the second mitochondrial ETC414929-4870-6393 6Attorney Docket No. 030258-000102 WOPT complex is not reduced, continuing to administer the methylene blue or the functional variant thereof to the tissue or organ; or (g) if the 3RMR value indicates that the second mitochondrial ETC complex is reduced, ceasing to administer the methylene blue or the functional variant thereof to the tissue or organ and / or increasing the oxygen delivery to the tissue or organ.

[0171] Paragraph 41 : The method of paragraphs 39 or 40, wherein the second mitochondrial ETC complex is Complex IV (CIV).

[0172] Paragraph 42: The method of paragraph 41, wherein the 3RMR value being at or above 1% in a liver indicates that the CIV is reduced; and wherein the 3RMR value being below 1% in a liver indicates that the CIV is not reduced.

[0173] Paragraph 43: The method of any one of paragraphs 30-42, wherein the 3RMR value of the mitochondria, the CIII, or the CIV is calculated by: (i) obtaining a raw spectrum using the Raman spectroscopy method; (ii) performing baseline subtraction of the raw spectrum to produce a resonance Raman spectrum; (iii) performing a linear regression analysis of the resonance Raman spectrum against a library of known analytes selected from oxidized and reduced resonance Raman spectra of the mitochondria, CIII, or CIV, to determine the relative concentration of: oxidized mitochondria and reduced mitochondria, or oxidized CIII and reduced CIII, or oxidized CIV and reduced CIV, in the tissue or organ; wherein, during the linear regression analysis, each known analyte is assigned a specific enhancement factor (fn), and (iv) wherein the 3RMR value of the mitochondria, the CIII, or the CIV is calculated as the ratio of their corresponding reduced concentration divided by the sum of the oxidized and reduced concentrations.

[0174] Paragraph 44: The method of any one of paragraphs 30-43, wherein the 3RMR value of the mitochondria is calculated by: Formula I: 3RMR_mito =+ ^)-

[0175] Paragraph 45: The method of any one of paragraphs 30-44, wherein the 3RMR value of the CIII is calculated by: Formula II:+ J-

[0176] Paragraph 46: The method of any one of paragraphs 30-45, wherein the 3RMR value of the CIV is calculated by Formula III:+6?6).

[0177] Paragraph 47: Methylene blue or a functional variant thereof for use in a method of treating or preventing ischemia-reperfusion injury.

[0178] Paragraph 48: Methylene blue or a functional variant thereof for use in a method of treating or preventing ischemia-reperfusion injury, the method comprising:424929-4870-6393 6Attorney Docket No. 030258-000102 WOPT administering an effective amount of methylene blue or a functional variant thereof to an tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0179] Paragraph 49: Methylene blue or a functional variant thereof for use in a method of treating or preventing ischemia-reperfusion injury, the method comprising: (a) determining if a mitochondria or at least one electron transport chain (ETC) complex of the tissue or organ is hyperoxidized; and (b) if the mitochondria or the at least one ETC complex of the tissue or organ is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemiareperfusion injury.

[0180] Paragraph 50: Methylene blue or a functional variant thereof for use in a method of treating or preventing ischemia-reperfusion injury, the method comprising: (a) quantifying a Resonance Raman Reduced Mitochondrial Ratio (3RMR) value of a mitochondrial complex of the tissue or organ using a Raman spectroscopy method; (b) determining if the 3RMR value indicates that the mitochondrial complex is hyperoxidized; and (c) if the 3RMR value indicates that the mitochondrial complex is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

[0181] Paragraph 51 : Methylene blue or a functional variant thereof for use in the manufacture of a medicament for treating or preventing ischemia-reperfusion injury.

[0182] Paragraph 52: The method of any of the preceding paragraphs, wherein the functional variant of methylene blue is selected from the group consisting of: mitoquinone, azure A, a diaminophenolthiazine, phenazine methosulfate, and 2,6-indophenol.

[0183]

[0184] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0185] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

[0186] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and 434929-4870-6393 6Attorney Docket No. 030258-000102 WOPT examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0187] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0188] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLES

[0189] Example 1: Detection of IRI in liver mitochondrial respiratory chain complexes during machine perfusion using non-contact resonance Raman SpectroscopyMitochondrial dysfunction is a critical factor in several diseases, but current in situ assessment methods are severely limited. Introduced herein is a non-destructive approach to monitor mitochondrial dysfunction in real-time using resonance Raman Spectroscopy (RRS). This method provides in situ quantification of the overall mitochondrial redox state, as well as the redox state of individual cytochromes such as complex III and IV. Demonstrated herein is RRS utility with a rodent liver model of warm ischemia-reperfusion injury (IRI) in organ transplantation, whereby dysfunction at complex III is characterized by hyperoxidation during ex vivo machine perfusion, due to electron leakage. Mitochondria were rescued from IRI- mediated injury using methylene blue, which acts as an alternate electron donor to bypass complex III. RRS-guided treatment was further tested on porcine marginal livers with extended444929-4870-6393 6Attorney Docket No. 030258-000102 WOPT warm ischemia (WI; 30-45 mins), showing recovery of hemodynamics and oxygen / lactate values that approached controls without WI.

[0190] The embodiments disclosed herein pertain to technology for rapidly and dynamically measuring the redox states of whole mitochondria from the surface of whole organs without the need for tissue processing. The distinct heme structures present in mitochondrial cytochromes generate resonance Raman scattering events that are captured and quantified with a Resonance Raman Spectroscopy (RRS) device. Disclosed herein are methods to selectively measure the redox states of individual complexes (cIII and cIV) in the ETC non- invasively from the surface of mammalian livers, thereby facilitating mechanistic insights into mitochondrial injury during ischemia-reperfusion injury.

[0191] To demonstrate the advantage of RRS to better understand mitochondrial dysfunction from a dynamic perspective, the mitochondrial redox state as a function of ischemia-reperfusion injury (IRI) in the context of liver transplantation was studied. Mitochondrial injury significantly contributes to the IRI development since the organelle’s integrity is highly sensitive to oxygen supply changes as seen in ischemia and reperfusion events

[0019] , Exposure to Warm Ischemia (WI), whereby the organ remains in situ at body temperature without circulatory support, poses a significant risk for organ viability as it leads to decreased patient and graft survival rates [20-22], In light of this, limits on WI time as well as other criteria (e.g. steatosis, fibrosis, perfusate chemistry, etc.) have adversely impacted the utilization of Donation after Circulatory Death (DCD) liver, which remained low at 20-25% in the USA over the last 10 years

[0023] , As DCD is increasingly considered for the purpose of alleviating severe shortages of viable organs [24-26], there is a critical need to safely increase the utilization of DCD livers. Leveraging RRS technology, these studies elucidate mechanisms of Wl-induced mitochondrial injury, specifically those that can be used in IRI prevention / recovery strategies aimed at improving / preserving the viability of DCD livers for transplantation.

[0192] In this study, the overall mitochondrial redox state, defined as the ratio of reduced to total mitochondria (or Resonance Raman Reduced Mitochondrial Ratio: 3RMR), was determined in rodent DCD liver models with incremental WI time and a subnormothermic machine perfusion (SNMP) protocol. Optimization of the regression library for specific measurement of individual complexes of the ETC, including cIII and cIV, allows for identification of the precise location of injury leading to mitochondrial dysfunction (i.e., 3RMR CIII and 3RMR CIV). Livers exposed to severe ischemic events exhibit hyperoxidation at complex III during reperfusion. Methylene blue (MB) is an FDA-approved 454929-4870-6393 6Attorney Docket No. 030258-000102 WOPT drug that shuttles electrons from complex I to cytochrome c (which are then transferred to CIV), therefore bypassing CIII

[0028] , When added to the perfusate, MB resulted in the complete oxidation of CIII and increased the reduced state of CIV, supporting its expected mode of action. This rescued rodent liver mitochondria from IRI-mediated injury during machine perfusion, both with and without hemoglobin-based oxygen carrier (HBOC) to enhance oxygen delivery. Translational potential of the RRS-guided treatment is demonstrated through the MB- supplemented perfusion on DCD porcine livers with extended WI time (i.e., 45 mins) that had been expected to be non -transplantable, showing recovery of hemodynamics and oxygen / lactate values, with no statistical differences with transplantable DBD (Donation after Brain Death) controls. Overall, bioengineering advances can be used to enhance mechanistic understanding of mitochondrial dysfunction, and these insights are leveraged herein to therapeutically intervene and rescue DCD livers from IRI. Ultimately, these insights and strategies can used to make more DCD livers available for transplantation as well as better understand IRI across broad pathological conditions.

[0193] Results. 1. Spectroscopic quantification of mitochondrial redox states. The RRS system disclosed herein was used to quantify mitochondrial redox state with expanded features to include complexes III and IV, as shown in FIGs. 1A-1B (and FIGs. 6A-6H and 7A-7C). CIII and CIV are both rich in heme groups with resonant Soret band absorption in the ~430- 450 nm range [29, 30], which overlaps with the 441 nm excitation wavelength. These properties result in their strong and spectrally distinct Raman signals, as shown in FIGs. 6A- 6H. In contrast, Complex I (CI) is not Raman-active under these conditions due to the absence of a heme group [31, 32], While complex II (CII) may be Raman active with a Soret peak of 416 / 426

[0033] , it is present at relatively lower concentrations, as compared to CIII

[0034] , and hence was not captured in the present study.

[0194] The RRS system uses a non-contact optical laser probe with an excitation wavelength of 441 nm on the organ surface and records the raw RR spectrum, which is subjected to non-specific background subtraction followed by spectral analysis (FIG. 1A). A regression algorithm is utilized to explain the measured RR spectrum (ym) as a weighted sum (yrequation) of pre-recorded library RR spectra from isolated, purified analytes such as entire mitochondria, ETC CIII and CIV, as well as hemoglobin (or hemoglobin-based oxygen carrier, HBOC) in both reduced and oxidized states (FIG. IB). Methylene Blue (MB) was confirmed to have non-significant spectroscopic Raman resonance with 441 nm excitation wavelength (FIG. 8). Leveraging spectral differences, the 3RMR values are spectroscopically quantified464929-4870-6393 6Attorney Docket No. 030258-000102 WOPT using regression coefficients of corresponding relevant components in the weighted sum yrequation. Specifically, the 3RMR_mito is calculated by+ f2^2) (Formula I) ratio, of which is the coefficient of reduced mitochondria spectrum (x- and f2 / 32is the coefficient of oxidized mitochondria spectrum (x2). The 3RMR CIII (3RMR_CIII = / 33 / ( / 3 + A ); Formula II) and 3RMR_CIV (3RMR_CIV = f5p5 / (f5p5+ / 6£>);Formula III) are also determined using this mathematical approach (FIG. IB). FIG. 9 illustrates the justification for the use of a 441 nm wavelength, which was based on the Soret absorption maxima of key mitochondrial components (CIII and CIV).

[0195] 2. Detection of hyperoxidation in rodent livers with IRI during machine perfusion. During SNMP, the 3RMR_mito values range from 0% (completely oxidized) to 100% (completely reduced) in correspondence with various physiological and pathophysiological conditions (FIG. 2B). During the optimal reperfusion of healthy rodent organs, 3RMR_mito measures have been shown to be consistently stable within a range of low values (10-30%), reflecting a predominantly oxidized state for the entire mitochondria during optimal reperfusion protocol [16-18], The 3RMR_mito values that exceed this range, therefore, typically indicate various degrees of deoxygenation or ischemia driven by electron accumulation within the ETC complexes. In contrast, the 3RMR_mito values below the optimal range indicate hyperoxidation, with one mechanism due to electron leaks out of the ETC complexes, as illustrated in FIG. 2C.

[0196] Based on validated DCD rodent livers

[0027] , the experiment included three study groups (n=6 per group): fresh livers without WI time, transplantable livers with short Ih WI time, and non-viable livers with prolonged 3h WI time (FIG. 2A). Through visual inspection alone, the 3h WI livers exhibited evident signs of IRI after 3 hours of SNMP, characterized by a notable pale appearance (FIG. 2D). This was confirmed by other perfusion-based parameters, such as increased vascular resistance and weight gain, elevated levels of aspartate transaminase (AST) and alanine aminotransferase (ALT), and absent bile production during SNMP (FIGs. 10A-10J) Further, NAD:NADH ratios and ATP levels in 3h WI were not recoverable after SNMP (FIGs. 10F, 101), demonstrating the severity of injury to these livers, as compared to previous work, whereby SNMP with human livers lead to some improvements in energetic cofactors albeit with less total ischemic time

[0035] , Nonetheless, since SNMP alone cannot recover 3h WI livers, this provides an opportunity to leverage this condition to determine therapeutic effectiveness. Like metabolic disruption in livers with cold ischemia

[0016] , electrons accumulate during warm ischemia (high 3RMR) for Ih and 3h WI livers, which was also474929-4870-6393 6Attorney Docket No. 030258-000102 WOPT shown here at T=0 mins in FIG. 2E. Immediately after reperfusion, the 3RMR_mito values rapidly dropped to less than 20% after 10 mins of SNMP, indicating the effects of reoxygenation. With more reperfusion time, however, the 3RMR_mito values of 3h WI livers were getting significantly lower than those in fresh and Ih WI livers, which was statistically significant after 30 mins in both cases, indicating a progressive hyperoxidized redox state.

[0197] Furthermore, insights about the exact site of IRI-mediated hyperoxidation were achievable with this technology. First, the 3RMR CIII values were significantly lower in 3h WI livers than in fresh and Ih WI livers (p<0.05 at all time-points) (FIG. 2F), and the 3RMR CIII values in 3h WI livers of 3h WI livers decreased from 20 (20-23.75)% at the beginning (10 mins of SNMP) to 8 (3.75-12.25)% at 180 minutes (n=6 per time-point, p<0.05). This result showed that hyperoxidization of complex III (almost 20% lower than fresh liver baseline at 180 mins) is much more severe than that of the overall mitochondria (roughly 9% difference from fresh liver baseline at 180 mins), indicating that complex III is a particularly vulnerable site of injury during reperfusion time. Second, there was no difference in the redox states of complex IV among the three liver groups, and the 3RMR CIV values were zero (completely oxidized) during SNMP (FIG. 2G). This 3RMR CIV result, when considered alone, indicates sufficient local oxygen supply to complex IV in non-viable 3h WI livers, which contradicts the overall interpretation of other perfusion parameters, that is the 3h WI livers had very restricted oxygen delivery and poor aerobic metabolism. Compared to fresh livers, the 3h WI livers had 50% slower flow rate of perfusate delivering oxygen (FIG. 2H), decreased oxygen consumption (FIG. 21), elevated lactate level (FIG. 2 J), reduced mitochondrial energy charge (FIG. 2K), and excessive cell death evident in TUNEL imaging (FIG. 2L). However, when considering that 3h WI livers had hyperoxidized complex III and overall hyper-oxidized mitochondria, the corresponding 3RMR CIV measures become more understandable. Demonstrated herein is that in 3h WI livers, this hyperoxidation is driven by premature electron leaks from complex III, thereby significantly reducing electron transfer to complex IV, allowing the limited oxygen supply (from 50% perfusate flow rate) to suffice for electron transfer out of complex IV (oxidation). This prevents electron accumulation and reduction in complex IV, which would otherwise increase 3RMR CIV values under similar oxygen-limited conditions.

[0198] To further interrogate the mechanism underlying this hyper-oxidized state, which was hypothesized to be due to electron leakiness at complex III, an oxygen stress test (OST) was developed. During the OST, livers are transitioned from continuous oxygenated perfusion (i.e., SNMP) to acute ischemia by stopping both perfusate flow and oxygen delivery, leading to an additional period of ischemia (FIGs. 3A-3D). This stress test is based on the484929-4870-6393 6Attorney Docket No. 030258-000102 WOPT principle that if electrons are leaking from the mitochondria, then they would display a slower rise in 3RMR (and hence less electron accumulation) with additional ischemia time. Indeed, after the same 3 mins of ischemia introduced at 90 mins of SNMP, the 3RMR_mito of 3h WI livers only reached 18.64 (17.07 - 22.41) % while 3RMR_mito of fresh and Ih WI livers were 57.07 (45.7 - 65.56) % and 63.59 (56.68 - 66.7) % respectively (n=3 per group, p<0.05 fresh vs 3h WI livers & Ih WI vs 3h WI livers). In other words, the average rate of change in 3RMR_mito values was less than 4 % / min in 3h WI livers but more than 15% / min in fresh and Ih WI livers.

[0199] To further characterize and validate this pathological oxidation within the ETC, the effects of antimycin A (AA), a well-characterized Complex III inhibitor known to increase ROS production at this site, were investigated

[0036] , Shown in FIG. 11A is the study group called Fresh + Antimycin A (Fresh+AA). These rodent livers did not undergo warm ischemia (WI) but instead received a dose of AA at a concentration of -0.02 mM during SNMP. Following AA administration, oxygen consumption in the liver declined significantly and approached zero by 90 minutes, despite uninterrupted delivery of well-oxygenated perfusate (FIG. 11B) Then, livers underwent an oxygen stress test (OST), and the RRS device was used to measure corresponding shifts in mitochondrial redox states (FIGs. 11C-11G). During the OST, both fresh and Ih WI livers (>90% transplantable

[0037] ) demonstrated rapid redox transitions, reaching approximately 60% for 3RMR_mito values within just 3 minutes of ischemia onset (FIG. 3D). In contrast, 3h WI livers reached only -20% over the same period (FIG. 3D) The Fresh+AA livers exhibited a redox response during OST that resembled the impaired pattern seen in 3h WI livers (FIGs. 11C-11D). Finally, FIGs. 11E-11G show the 3RMR_mito, 3RMR CIII, and 3RMR CIV throughout the full duration of the OST. Only the 3RMR of CIV trended differently between 3h WI and Fresh+AA livers, whereby livers exposed to AA remained completely oxidized throughout the oxygen stress test. This indicates complete blockage of electron transfer during AA exposure, which is consistent with its mechanisms of action. Taken together, the combination of OST and AA supports the central finding that mitochondrial dysfunction, specifically characterized by pathological oxidation within the ETC, can be readily detected and quantified by the RRS device.

[0200] 3. Intervention with methylene blue to rescue mitochondria from Wl-induced IRI development. With the understanding of progressive IRI-mediated hyperoxidation at CIII in 3h WI livers, the utility of MB, which shuttles electrons directly from complex I to cytochrome c, was investigated

[0028] , It was hypothesized that by using MB to bypass cIII during the mitochondrial respiration process, cIV would receive more electrons for consumption of494929-4870-6393 6Attorney Docket No. 030258-000102 WOPT molecular oxygen and livers could be rescued from IRI-mediated mitochondrial injury (FIGs. 4A-4B). Besides treatment with MB alone, the effects of MB with artificial hemoglobin-based oxygen carriers (HBOC) were also tested, which is expected to enhance oxygen delivery

[0038] ,

[0201] The RRS assessment supported the hypothesis, with promising outcomes from MB intervention. First, 3RMR_mito values trended higher with MB treatment, although statistically non-significantly compared to non-treated 3h WI livers (FIG. 4C). Second, 3h WI+MB livers had 3RMR CIII values drop to almost zero (fully oxidized) throughout SNMP (FIG. 4D), while their corresponding 3RMR_CIV values increased up to 40% (FIG. 4E). These results are consistent with the hypothesis that MB has effectively altered the electron transfer process to bypass complex III, leaving it without electron deposition from upstream complexes and thus fully oxidized, while improving electron deposition to complex IV. The reduced state of complex IV in 3 h WI+MB livers, also indicated that the new demand from more electron deposition exceeded oxygen supply from the current SNMP protocol, whose perfusate flow rate did not improve compared to that of 3h WI livers without treatment (FIG. 4F), thereby justifying the purpose of the hemoglobin-based oxygen carriers (HBOC).However, while previous work has shown mitochondrial measurements by RRS in the presence of packed Red Blood Cells (RBCs) and whole blood are feasible [17, 18], the HBOC used in the present study significantly lowered the mitochondrial signal strength. As a result, the 3RMR values were not able to be quantified for these groups.

[0202] The positive effects of MB in preventing IRI-mediated injury were also observed with other organ viability biomarkers. Oxygen consumption trended higher in 3h WI+MB+HBOC liver groups than in non-treated 3h WI livers, and statistical significance was observed at T= 90 mins (p=0.0054) and T=150 mins (p=0.0468) (FIG. 4G). Lactate outflow concentration trended lower in 3h WI+MB livers (with p=0.0384 at T=120 mins) and were statistically lower in 3h WI+MB+HBOC livers than in non-treated 3h WI livers at all timepoints except T=10 mins (FIG. 4H). Compared to fresh liver group, lactate level was non- significantly different (p>0.05) at T=10, 30 and 180 mins for 3h WI+MB livers; and at all time points except T=60 mins for 3h WI+MB+HBOC. The observation that lactate level of non- viable 3h WI livers was lowered to physiological range highlighted that MB was able to enhance aerobic respiration. The additional boost of improvement for oxygen consumption, outflow lactate level and mitochondrial energy charge (FIGs. 4G-4I) in 3h WI+MB+HBOC livers also indicates that HBOC provided some synergistic benefits to the therapeutic cocktail. However, the persistent challenge of suboptimal oxygen delivery due to low perfusate flow rate504929-4870-6393 6Attorney Docket No. 030258-000102 WOPT has hindered many other potential avenues of recovery including weight gain (FIG. 4J), cell death (FIG. 4K), and other aspects of liver IRI (FIGs. 12A-12J).

[0203] 4. Translating perfusion-based MB intervention to pig model of DCD livers.Following the demonstration of improved outcomes using MB -supplemented machine perfusion for rodent liver models, the same perfusion-based intervention was tested with pig livers (FIG. 5A-5B), comparing fresh control DBD (donation after brain death) livers without warm ischemia (Fresh DBD control, n=4), DCD (donation after circulatory death) livers with 30 minutes of warm ischemia without treatment (DCD 30min WI, n=4), 30 min WI livers treated with MB+HBOC (30min WI+MB+HB0C=5), and 45 min WI livers treated with MB alone (45 min WI+MB, n=4). These groups were selected since DBD livers are the primary source of livers for transplantation and serve as a reference for optimal graft quality in the absence of warm ischemia. Further, the clinical cut-off for warm ischemia time in liver transplantation is < 30 mins, whereby ischemic times above this are considered non- transplantable. As such, an untreated 30-minute WI group was included to define the marginal viability threshold, while the 30-minute WI group treated with MB+HBOC was included to define the combined therapeutic effectiveness of both methylene blue and the hemoglobinbased oxygen carrier. Finally, a 45 min WI+MB group was included since the ability to rescue livers with greater than 30 min warm ischemia time would significantly increase the donor pool of organs. Further, these livers were treated with MB alone since it specifically targets the hyperoxidation of CIII and hence was hypothesized to have the main therapeutic advantage.

[0204] The schematic of the experimental design and representative livers from each study group are shown in FIG. 5A-5B. Images show that fresh DBD livers displayed a uniform brownish surface, consistent with healthy tissue. In contrast, 30 min WI livers exhibited patchy areas of paler color, indicating mild injury. The 30 min WI+MB+HBOC group appeared red in color, as the HBOC component dominated the visual appearance and masked the blue hue of MB. The 45 min WI+MB livers showed a distinct blue coloration due to MB staining. Consistent with these images, Fresh DBD controls showed values in the healthy 3RMR_mito range (FIG. 5C), as observed with rodent livers herein and in other studies [16, 17], Within the initial 10 mins of SNMP, RRS assessment showed that both 30min WI livers and 45min WI+MB liver groups had a marked decrease in 3RMR_mito values, reflecting the shift from ischemia to oxygenated reperfusion as seen with rodent livers (FIG. 5C). For the rest of SNMP time, 3RMR_mito values were predominantly less than 10% in 30min WI livers, indicating mitochondrial hyperoxidization. The 3RMR_mito values of 45min WI+MB livers also trended consistently low between zero to 5%, but this is an expected result considering MB therapeutic 514929-4870-6393 6Attorney Docket No. 030258-000102 WOPT effects. In specific, as shown in rodent livers, MB made complex III fully oxidized (FIG. 4D) instead of restoring its regular 30-35% reduced state (FIG. 2F), and if oxygen delivery is improved for sufficient electron transfer at complex IV, this complex would also become fully oxidized (FIG. 2G), which together can lead to an artificially hyperoxidized state of the entire mitochondria under MB treatment.

[0205] Indeed, hemodynamic results indicated that MB-treated livers were likely to benefit from higher oxygen delivery. Compared to 30mins WI livers, portal flow rate increased 2-fold in 30mins WI+MB+HBOC livers and almost 4-fold in 45mins WI+MB livers (FIG. 5D, p<0.05 at all time-points for both treated liver groups in comparison to 30min WI livers, except T=180 min for 45mins WI+MB). Portal vascular resistance trended lower in 30mins WI+MB+HBOC livers (although p>0.05, except at T=10 min) and significantly decreased in 45mins WI+MB livers in comparison to non-treated WI livers (FIG. 5E, p<0.05 at all timepoints except T=30 min). Further, MB improved aerobic respiration during SNMP. The 30mins WI+MB+HBOC livers exhibited a 2-fold higher oxygen consumption (FIG. 5F, p<0.05 at T=30, 60, 90, 150, 180 mins) and a trend of lower lactate outflow concentration although nonsignificant statistically (FIG. 5G) than non-treated WI livers. In addition, the 45mins WI+MB livers consumed as much oxygen as the 30mins WI+MB+HBOC livers (FIG. 5F), with even a lower trend of lactate outflow concentration (FIG. 5G, p<0.05 at all time-points except T=10min, 45mins WI+MB vs 30mins WI livers). Potassium outflow concentration, a liver injury biomarker, continuously decreased throughout reperfusion time for all three liver groups (FIG. 5H), thus showing the overall benefits of SNMP for DCD liver functional recovery. Perhaps the most important outcome was the striking similarities between Fresh DBD and 45 min WI+MB livers, whereby there were no significant differences in hemodynamics and oxygen / lactate values, demonstrating the ability of methylene blue to recover the function of livers exposed to severe warm ischemia. 3RMR_mito at T=60 (FIG. 5C) and select injury markers at select time points were significantly different comparing Fresh DBD and 45 min WI+MB livers, including potassium at T=30 min (FIG. 5H), AST at T=180 min (FIG. 13A), and cytochrome c at T=120 min (FIG. 13C). 45 min WI livers treated with MB had lower total ATP by the end of perfusion, as compared to untreated 30 min WI controls (FIG. 13F), suggesting additional metabolic supports may be required for complete metabolic recovery.

[0206] Taken together, these results indicate ability for the clinical translation of this perfusion-based MB intervention to improve the recovery of human DCD livers that are currently considered marginal with extended warm ischemia durations. They also showcase the usefulness of the RRS technology for fundamental scientific endeavors that can help develop 524929-4870-6393 6Attorney Docket No. 030258-000102 WOPT effective targeted therapeutic intervention for the perfusion / recovery of DCD livers with extended WI time.

[0207] Herein is presented a RRS technique to detect IRI-mediated mitochondrial injury in livers during machine perfusion, and to evaluate mitochondria-targeted therapeutics such as MB. As compared to other methods that require complicated and / or time-consuming tissue sampling and mitochondrial isolations, the RRS system can provide non-destructive label-free measurements, while collecting the redox states of whole mitochondria and individual complexes in the ETC, including CIII and CIV (FIGs. 1A-1B, 6A-6H, 7A-7C). The RRS system can be used both for individual time-point evaluation during machine perfusion time (FIGs. 2A-2L), and for continuous monitoring of dynamic changes during an oxygen stress test (FIGs. 3A-3D). Taken together, this illustrates the versatility of the RRS approach that can be easily adapted to assess IRI-mediated mitochondrial injury in other tissues or organs.

[0208] The ability of the RRS technology in solving complex biological problems was demonstrated by using the RRS assessment to elucidate Wl-induced mitochondrial injury pathway in liver IRI development, a multi-factorial problem that is detrimental to clinical outcomes of DCD liver transplants [39, 40], The initial observation centered on a decrease in the 3RMR CIII as a function of perfusion from rodent livers exposed to 3 hours of warm ischemia (3h WI; FIG. 2F, triangle markers), indicating a progression towards an oxidized state. This was matched with biochemical results to confirm increased stress in the 3h WI liver group, including reduced oxygen consumption (FIG. 21), elevated lactate levels (FIG. 2J), decreased energy charge (FIG. 2K), visible cell death (as shown by TUNEL staining; FIG. 2L), elevated AST and ALT levels (FIGs. 10B-10C), and release of cytochrome c into the perfusate (FIG. 10E). To interrogate the mechanism underlying hyperoxidation at CIII, several experimental approaches were employed, including the oxygen stress test (OST) and therapeutic interventions (with MB and antimycin A, AA). Firstly, an OST was developed, wherein perfused livers were exposed to an additional period of ischemia (defined by a compete pause of both perfusate fluid flow and oxygen air supply for 10 min; FIGs. 3A-3D). This stress test is based on the principle that if electrons leak from mitochondria, then they would display a slower rise in 3RMR with additional ischemia time since they cannot retain electrons required for a higher (more reduced) 3RMR. Indeed, after the same 3 mins of ischemia introduced at 90 mins of SNMP, the 3RMR_mito of 3h WI livers only reached 18.64 (17.07 - 22.41) % while 3RMR_mito of fresh and Ih WI livers were 57.07 (45.7 - 65.56) % and 63.59 (56.68 - 66.7) % respectively (n=3 per group, p<0.05 fresh vs 3h WI livers & Ih WI534929-4870-6393 6Attorney Docket No. 030258-000102 WOPT vs 3h WI livers). In other words, the average rate of change in 3RMR_mito values was less than 4 % / min in 3h WI livers but more than 15% / min in fresh and Ih WI livers.

[0209] Secondly the known mode of action of two therapeutics was leveraged, including MB and AA. While MB shuttles electrons from complex I to cytochrome c (which then transfers to CIV, thereby bypassing CIII), AA is a well-characterized Complex III inhibitor known to increase ROS production at this site

[0036] , Specifically, when 3h WI livers were treated with MB (±HBOC), a decrease in 3RMR CIII and an increase in 3RMR CIV was observed (see FIGs 4D-4E). Although 3RMR values outside of the normal zone (i.e. 12-20%; FIG. 2B) are typically associated with mitochondrial stress, in this case, the values reflect the therapeutic action of MB. As noted above, since MB shuttles electrons from complex I to cytochrome c, the decrease in 3RMR CIII (i.e. hyperoxidation) in the presence of MB is evidence that electrons are effectively bypassing CIII. In contrast, the increase in 3RMR CIV indicates that MB is shuttling electrons from CI and depositing them to CIV (via cytochrome c), resulting in the rise in 3RMR CIV (or relatively high reduced state). This relatively high reduced state at CIV could have also been matched with not enough oxygen molecules for electron transfer (FIG. 4E). This is hypothesized to be due to suboptimal re-introduction of oxygenated perfusate in current SNMP protocol. Gradual re-warming [41, 42] or hypothermic [43, 44] machine perfusion protocols could change this dynamic.

[0210] Since MB shuttle electrons from upstream CI to cytochrome c and therefore CIV afterwards, under MB treatment, it is expected that all three CI, CII, and CIII to be hyperoxidized or even completely oxidized because they no longer received any electrons to be reduced. CIV, however, is expected to either stay fully oxidized (3RMR<1%) if oxygen delivery is high enough to match the increased electron deposition by MB or to be reduced under insufficient oxygen delivery condition.

[0211] Finally, the effects of AA were investigated, whereby livers from the “Fresh + Antimycin A” (Fresh+AA) group exhibited a redox response during the oxygen stress test (OST) that closely mirrored the impaired redox dynamics observed in 3h WI livers (FIGs. 11A-11G). The striking similarity in RRS-derived redox shift behavior between the Fresh+AA and 3h WI groups during the OST supports the central finding that mitochondrial dysfunction, specifically characterized by pathological oxidation at complex III within the ETC, plays a key role in driving liver IRI following prolonged warm ischemia. One mechanism for this hyperoxidation can be due to electron leakage.

[0212] While these therapeutic interventions provided a means to interrogate the underlying mechanism of hyperoxidation at CIII, methylene blue in particular has high544929-4870-6393 6Attorney Docket No. 030258-000102 WOPT translational potential to overcome hyperoxidation at CIII, including in the liver transplant setting. Indeed, in testing MB for recovering IRI in rodent livers, the results demonstrated improved aerobic metabolism, evident by significantly improved lactate level and increased oxygen consumption (FIGs. 4G-4H), thereby motivating the translation of MB to a pre-clinical large animal model such as the pig. In pursuit of this goal, outcomes included similarities between Fresh DBD and 45 min WI+MB pig livers, whereby there were almost no differences in hemodynamics and oxygen / lactate values, demonstrating the ability of methylene blue to recover the function of livers exposed to severe warm ischemia (FIGs. 5A-5H and 13A-13H). The 45min WI+MB group outperformed the 30min WI+MB+HBOC group in portal flow, oxygen consumption, and lactate levels, indicating the HBOC can even be counterproductive in the swine model. HBOC has been reported to scavenge nitric oxide, leading to vasoconstriction and impaired microcirculation [45-47], In this capacity, these unintended effects of HBOC can reduce effective tissue perfusion and oxygen delivery at the microvascular level, which is critical for MB to reach and act on injured mitochondria, thus blunting the therapeutic impact of MB. Irrespective of the underlying reason for the counterproductive effects of HBOC, a comparison of the MB+HBOC versus MB alone group further supports the central hypothesis that pathological oxidation within the ETC is a key driver of liver IRI following prolonged warm ischemia. The MB only treatment included lower ATP levels at the end of perfusion, as compared to untreated WI controls.

[0213] Overall, the RRS assessment presented here permitted the detection of a IRI- mediated mitochondrial injury pathway and guided an effective mitochondria-targeted therapeutic approach that is scalable from small to large animal models of DCD livers. While demonstrated herein is the capability of RRS to track mitochondrial injury during SNMP with acellular perfusates, previous work has also shown its ability to measure mitochondrial function during normothermic machine perfusion with RBC-based perfusates

[0017] , thereby demonstrating its translational relevance. However, the present study did observe a decrease in mitochondrial signal strength in the presence of HBOC, thereby prohibiting the tracking of 3RMR in these groups. The impact of this technology includes an increase in organ availability. Implementations can be used for developing therapeutic cocktails for organ mitochondrial preservation, improving machine perfusion protocols to mitigate IRI during DCD organ recovery, and establishing an extended clinical criterion to include RRS-derived biomarkers for assessment of other marginal DCD organs with extended WI time prior to transplant.Altogether, by leveraging the principles presented here, solutions to solve ischemia-reperfusion injury far beyond transplantation are achievable and described herein.554929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0214] Experimental Design. Rodent: For the initial step, the effect of different durations of warm ischemia on the liver viability was investigated. 18 rat livers were divided into 3 experimental groups (n=6 per group): 1-hour warm ischemia (Ih WI), 3 hours warm ischemia (3h WI), and fresh. The livers underwent subnormothermic machine perfusion (SNMP) at room temperature (21°C) for 3 hours with an acellular solution. Supplementarily, 12 rat livers (n=3 per group: fresh, Ih WI, 3h WI, and fresh+AA) were used separately to perform oxygen stress tests (FIGs. 3A-3D, 11A-11G) For these livers, SNMP was performed for 90 mins with acellular solution and then livers were subjected to a compete pause of both perfusate fluid flow and oxygen air supply for a dynamic RRS evaluation of mitochondrial response to acute ischemic stress.

[0215] Next, the effects of MB and HBOC were investigated in the 3h WI group. For this step, a total number of 6 livers were divided into 2 experimental groups (n=3 per group): 3h WI treated with MB (3h WI+MB) and 3hWI treated with both MB and HBOC (3h WI+MB+HBOC). The livers were subjected to SNMP for 3 hours using acellular solution combined with the treatments.

[0216] Swine: 17 DCD pig livers were allocated into four groups for study. The first group (30min WI, n=4) underwent 3 -hour SNMP without treatment after a 30-minute warm ischemic period. The second group (30minWI+MB+HBOC, n=5) received a combined treatment of MB and HBOC during SNMP following the same warm ischemic period. The third group (45min WI+MB, n=4) was subjected to an extended 45-minute warm ischemic period, followed by treatment with MB alone during 3-hour SNMP, to assess the effects of MB on liver with extended warm ischemia during SNMP. The fourth group (Fresh DBD, n=4) was procured without any WI time and immediately put on an SNMP system.

[0217] Liver Procurement. Rodent: Livers from healthy, adult female Lewis rats (10-12 weeks old, weighing 175-200 g) (CHARLES RIVER LABORATORIES, Wilmington, MA, USA) were used for all experiments to ensure the consistency between groups. The animals were housed socially in a temperature and humidity-controlled room and provided unrestricted food and water. The liver was procured as previously described

[0048] , The liver was weighted, and the perfusion was started immediately after procurement for fresh group. For WI groups, following procurement, the liver was placed in a temperature-controlled chamber with saline and maintained at 36 ± 0.1°C for either 1 hour or 3 hours.

[0218] Swine: Yorkshire pigs (around 40 kg, mix sex) were obtained 1 week before surgery to allow acclimatization. The liver was procured as previously described [49, 50], Briefly, the pigs were sedated with Atropine (0.04mg / kg), TELAZOL (4.4 mg / kg), and xylazine 564929-4870-6393 6Attorney Docket No. 030258-000102 WOPT(2.2 mg / kg), and Abolus ofPropofol (0.16-0.33 mg / kg) was administered through the intravenous (IV) line. General anesthesia was maintained via continuous isoflurane (3-5%) inhalation and intravenous fentanyl (5-20 ug / kg / h) as needed. Heart rate, blood pressure, oxygen saturation and ventilation were monitored. Abolus of heparin (100 U / kg) was administered. After a midstemal incision and sternotomy, the descending aorta was cannulated

[0051] , Laparotomy was performed. Next, the animal was switched to intravenous anesthesia, and the ventilator was turned off, allowing the animal to undergo cardiac arrest. Once the mean arterial pressure fell below 30 mmHg, a 30-minute timer was started to track the warm ischemic time. For extended warm ischemia, liver was left unflushed for an extra 15 minutes (45 minutes total) in the abdominal cavity. Immediately after cross-clamp and flush initiation through the aorta, the portal vein was cannulated and secured with 2.0 Prolene suture. Once the liver has been flushed with IL of cold (4 °C) Lactated Ringer followed by IL of University of Wisconsin Solution (UW), the surrounding structures were carefully cut with a wide incision around the hilum, and the liver was removed from abdominal cavity. Livers were prepared on the back table by cannulating the portal vein, hepatic artery, and bile duct, and by removing the gallbladder. This was followed by flushing out the UW with 2L of cold (4 °C) Lactated Ringer solution through portal vein (1.5 L) and hepatic artery (0.5 L) shortly before initiating the perfusion. The liver was connected to the perfusion machine after the vessels were prepared on the back table.

[0219] Machine Perfusion. Rodent: The perfusion system setup and operation are previously described

[0052] , The flow rates were manually adjusted for the maximum pressure of 5 mmHg and temperatures were maintained at subnorm othermic temperatures (SNMP, ~21°C). The oxygen stress test (OST) involved transitioning from continuous oxygenated perfusion (i.e., SNMP) to acute ischemia by stopping both perfusate flow and oxygen delivery.

[0220] Swine: SNMP was initiated using a device that permits dual perfusion through the portal vein and hepatic artery in a closed circuit (Liver Assist Organ Assist, Groningen, Netherlands). The pressures were set to 5 mmHg for the portal vein and 55 mmHg for the hepatic artery, the flow rates were automatically adjusted, as previously described [49, 50], The temperature of the system was set to 21°C.

[0221] Perfusion Solution. Rodent: The perfusion solution consisted of 500 mL phenol red-free Williams’ Medium E (SIGMA- ALDRICH, St Louis, MO, USA) supplemented with 5 g bovine serum albumin (SIGMA- ALDRICH, USA), 12 mg water-soluble dexamethasone (SIGMA-ALDRICH, USA), 5 mL penicillin-streptomycin (THERMO FISHER SCIENTIFIC, Waltham MA, USA), 5 ml L-glutamine (SIGMA- ALDRICH, China), 2.5 uL insulin574929-4870-6393 6Attorney Docket No. 030258-000102 WOPT(Pharmacy), 1000 U heparin (Pharmacy), as previously described [49, 50], As illustrated in FIG. 11A, for the experimental groups containing antimycin A (AA), these livers received a direct bolus injection of AA (10 mL of 1 mM) during SNMP. This dose was then recirculated within SNMP system containing 500 mL of perfusate, yielding a final AA concentration of -0.02 mM. As illustrated in FIG. 4B, for the experimental groups containing methylene blue (provided by BEANTOWN CHEMICAL, Hudson, NH, USA), a total dose of 50 mg of MB drug for 500 mL of perfusate was administered in the following fashion: initial 25 mg MB (0.05 mg / mL) shortly prior to SNMP to allow uniform distribution of MB within the perfusion system, followed by an additional 5 mg MB (0.001 mg / mL) at T= 30, 60, 90, 120, and 150 minutes. MB doses were adapted from previous study in rodent models

[0053] , For 3hWI+MB+HBOC group 25% HBOC-201 v / v (provided by HEMOGLOBIN OXYGEN THERAPEUTICS, Souderton, PA) was added to the solution.

[0222] Swine: The same acellular Williams’ E based solution was used with a volume of 2L for each pig liver perfusion. The 30minWI group was perfused only with acellular solution. The 30minWI+MB+HBOC group was supplemented with MB and HBOC as described above.

[0223] Mitochondrial Redox State Assessment by RRS device. Device Overview and Components. The RRS system by PEND AR TECHNOLOGIES comprises several components: a 441 nm laser pump source, a fiber optic probe with coaxial light emission and collection, and a high-resolution spectrometer. The laser delivers 8mW of single-line excitation light, which is focused into a 1.5 mm spot located 9 mm from the probe tip. The RRS measurements were taken on the liver surface at the center of the left lateral lobe, as recommended previously

[0016] , which identified this as an optimal site for assessing mitochondrial redox state (3RMR_mito) during ex vivo machine perfusion of rodent livers. The same location was used for pig livers based on this guidance. The scattered light from the sample is collected, passed through a filter to remove the excitation signal, and then directed to the spectrometer. The spectrometer has a resolution of 8 cm'1(FWHM) and includes an internal acetaminophen calibration reference to ensure accuracy. The system uses a temperature- controlled image sensor to maintain consistent dark current and cosmic rays are eliminated from the Charge-Coupled Device (CCD) signal before processing. Spectra are captured every second and integrated over a 180-second period for analysis.

[0224] Library Creation. Whole mitochondria isolation procedure '. Chromophores for the mitochondrial library were developed by PEND AR in partnership with the National Institutes of Health Cardiac Energetics Laboratory and based on previous experiments [18, 54], 584929-4870-6393 6Attorney Docket No. 030258-000102 WOPTFresh whole mitochondria were isolated from rodent and rabbit hearts, as described previously

[0054] , Briefly, hearts were perfused in situ with Krebs-Hanseleit buffer to remove blood and supply calcium before excision. After removing fat and connective tissues, the left ventricles and septa were dissected, weighed, and minced with scissors in an isotonic ice-cold buffer A (0.28M sucrose, 10 mM HEPES, 1 mM EDTA, 1 mM EGTA, and 5 mM K2HPO4and KOH to adjust pH to 7.1). The minced tissue was diluted with buffer A to a 10% w / v suspension and disrupted in a tissue homogenizer over ice. It was then treated with 2.5 mg / 5 g tissue wet weight of trypsin at 4°C for 15 min. This was followed by stopping the trypsin activity by adding 13 mg / 5 g tissue wet weight of trypsin inhibitor and 100 mg / 10 mL of bovine serum albumin. The homogenate was then centrifuged at 600 g for 10 min at 4°C to collect the supernatant containing mitochondria. The pellet was resuspended in buffer A and homogenized before centrifuged again at 600 g for 10 min at 4°C. The supernatant was collected and pooled with the previous supernatant. The pellet was resuspended in 40 mL buffer A and recentrifuged at 600 g for a total of three times, where the subsequent supernatants are combined. The final pooled supernatant containing the mitochondria was then centrifuged at 8000 g for 10 min at 4°C to pellet the isolated mitochondria. The pelleted mitochondria were resuspended in buffer A, followed by another 8000 g centrifugation. The pelleted mitochondria were washed and resuspended in buffer B (125 mM KC1, 15 mM NaCl, 20 mM Hepes, 1 mM EGTA, 5 mM MgCh, 5 mM potassium phosphate pH 7.1). This was followed by another centrifugation at 8000 g and resuspension in 1 mL buffer B. This mitochondrial suspension is further purified using PERCOLL gradient centrifugation, as described previously

[0055] , to remove broken mitochondria and cell debris. The final mitochondrial suspension in buffer B was quantified spectroscopically for its Cytochrome a content as a reference, as described previously

[0056] ,

[0225] Whole mitochondria library generation: The purified mitochondrial samples were then exposed to oxidizing versus reducing conditions. Briefly, a 350 mL sample of 4 nmol cyt a / mL solution in Fiskum Buffer was placed in a 1 mL cuvette and exposed to environmental oxygen to produce the oxidized form. To fully reduce the sample, a small amount of hydrogen sulfide (HS) was added, and nitrogen was blown over the cuvette before the cap was replaced. These preparations were contained in a glass vial and placed 9 mm from the RRS probe while shielding from room light to collect the spectra for a period of 600 seconds. The spectra were averaged, and the baseline fluorescence background was subtracted to produce a pure spectrum for the oxidized / reduced state of the whole mitochondria, which was added to the library. A clear difference in RRS was observed because of exposure to oxidizing versus reducing594929-4870-6393 6Attorney Docket No. 030258-000102 WOPT conditions, which can be visualized as differences in peak positions and intensities in the Raman spectrum, as shown in FIG. IB and FIGs. 6A-6H.

[0226] Complex III & IV isolation procedure '. In addition to the spectra for whole mitochondria, individual complexes III and IV of the electron transport chain were also purified, as previously described

[0057] , CIII and CIV were focused on since both are rich in heme groups with resonant Soret band absorption in the -430-450 nm range [29, 30], which overlaps with the 441 nm excitation wavelength. These properties result in their strong and spectrally distinct Raman signals, as shown in FIGs. 6A-6H. In contrast, Complex I (CI) is not Raman-active under our conditions due to the absence of a heme group [31, 32], While complex II (CII) may be Raman active with a Soret peak of 416 / 426

[0033] , it is present at relatively lower concentrations, as compared to CIII

[0034] , and hence was not captured in the present study (confirmation of this is provided in FIGs. 7A-7C and described further under Complex III & IV library generation below).

[0227] Complex III and IV are isolated using a procedure described previously [54, 57], Briefly, a stock sample of isolated mitochondria (corresponding to 200 nmoles of cytochrome a) is used as the starting material. The mitochondria are diluted to 10 nmol cytochrome a / mL in ice-cold buffer C (50 mM Tris, ImM MgSCU, pH 8.45). Then, 10% w / v n- P-D-dodecyl maltoside (DM) is added dropwise to a final concentration of 1% with gentle mixing on ice. The extract was centrifuged at 40,000 g for 40 min at 4°C, and the supernatant was applied to a 2.5 x 20 cm glass column (BIO RAD), previously packed withDi ethyl aminoethyl (DEAE) Sepharose Fast Flow anion exchange resin (GE HEALTHCARE) equilibrated with 5 column volumes of buffer C (containing 0.02% w / v DM). After washing with 2 column volumes of buffer C, elution of mitochondrial complexes was achieved by applying a linear 0-400 mM NaCl gradient in the same buffer and collecting the elute in 4 mL fractions. Green fractions containing Complex IV eluted close to the middle of the gradient (~180mLNaCl), before the reddish fractions containing Complex III. The pooled fractions containing each complex were concentrated to 20-30 nmoles cytochrome a / mL.

[0228] Complex III & IV library generation: Once the individual complexes were isolated, each complex was then exposed to oxidizing and reducing conditions. An 11 mM sample of Complex III in Tris buffer was reduced using 2 mL of ascorbate (1 M) to reduce cytochrome cl and 2mL of HS to fully reduce cytochromes bi. and bu. An 18.67 mM sample of Complex IV in Tris buffer was reduced in multiple steps as Complex IV undergoes a catalytic oxidation process with multiple states

[0058] , To fully reduce Complex IV, two additions of 2 mL of H2O2 (10 mM) were made followed by 2 mL of HS to produce the fully reduced form.604929-4870-6393 6Attorney Docket No. 030258-000102 WOPTLibrary spectra were collected after each addition to capture each step of the cycle. Fully oxidized, O, and reduced, R, spectra were added to the final reference library to represent the steady states of complex IV. All samples were prepared in a 1 mL glass vial which was held in a fixed position at 9 mm from the probe lens and enclosed by a black box to prevent light contamination. RRS spectra were collected for 600 seconds for each complex and oxidation state.

[0229] To visually illustrate the spectral differences between CIII versus CIV as well as whole mitochondria versus individual complexes, FIGs. 6A-6H and FIGs. 7A-7C, are presented respectively. FIGs. 6A-6H shows the spectral differences (in peak positions and intensities) between redox states of the same complex (FIGs. 6A-6D) versus differences between complexes (FIGs. 6E-6H) at 441 nm excitation wavelength. For example, in FIG. 6E, the oxidized state of CIII and CIV are shown as a solid and dotted line, respectively, while the corresponding computed differences (generated by subtracting one spectra from the other) are shown in the panel below as a black line (i.e., FIG. 6F). For example, the (“nu-four“) band, centered around 1370-1380 cm ', differs between complex III and IV, with complex III exhibiting sharper, lower-frequency peaks, while complex IV shows broader, upshifted bands. While this one example is highlighted at the nu-four band (1370-1380 cmthe regression analysis determines the best fit of the experimental spectra against the library of known spectra across the full range (700 to 1500 cm'1).

[0230] To show the relationship between spectra from whole mitochondria versus individual complexes, included are FIGs. 7A-7C. The 441nm excitation source used is close to the Soret absorption maximum of reduced Complex IV, which results in maximal resonant enhancement of this chromophore. A regression using the reduced Complex III and Complex IV libraries (FIG. 7A) fully explains the measured spectrum of reduced mitochondria, resulting in minimal unexplained residual (shown as a black line). Using only the Complex III (FIG. 7B) or Complex IV (FIG. 7C) reduced library does not fully explain the spectrum and results in a non-random structure in the residual especially near the v4 band around 1356 cm'1. In short, the sum of the individual spectrum of complex III and complex IV are differentiated and together fully explain the spectrum of the whole mitochondria.

[0231] Generation of other libraries'. Libraries were also created for hemoglobin and included in the regression algorithm when HBOC was present during perfusion. Fresh blood was collected via exsanguination of a rodent and oxygenated in a cuvette using 100% oxygen (fully oxygenated) or deoxygenated using sodium dithionate (fully deoxygenated). Resonance Raman spectra were created by imaging through the glass cuvette wall. A small stirring magnet 614929-4870-6393 6Attorney Docket No. 030258-000102 WOPT at the base of the cuvette was used to mix the solution. To produce oxygenated state, a pure oxygen headspace was established inside the cuvette. To produce deoxy state, sodium dithionate was added, and a pure nitrogen headspace was established. Spectra in the oxy and deoxy states were measured for 10 minutes. The resonance Raman spectra show unique markers, especially at the nu 4 band, where the peak at 1356 cm'1is the deoxyHb peak and 1376 cm'1is the oxyHb peak that were used to confirm fully oxygenated and deoxygenated states. Finally, additional spectral profiles of methylene blue and the base perfusate were also investigated; however, since there were no Raman-active components (FIG. 8) these were not incorporated in the regression.

[0232] Wavelength selection: As shown in FIG. 9, 441 nm closely aligns with the absorption peaks of the reduced forms of Complex III, Complex IV, and hemoglobin, thereby enhancing sensitivity to their reduced states. Although the study focuses on the reperfusion phase, when mitochondria and ETC components are predominantly oxidized, accurate detection of their reduced states remains critical for calculating redox status using the 3RMR ratio: reduced / (reduced + oxidized). Because reduced forms are less abundant during oxygenated reperfusion phase, selecting a wavelength that preferentially excites them ensures sufficient signal strength to detect even subtle redox shifts in mitochondrial and ETC complexes.

[0233] Data Analysis. During a measurement, scattered light is collected by the probe, where a band pass filter removes the laser wavelength leaving the inelastically scattered photons to be analyzed by the spectrometer. The CCD collects a 2D image of the signal, which is read once per second, with one region of the CCD capturing signal from the liver tissue and another from an internal acetaminophen reference. The location of the acetaminophen peaks is used to shift the measured spectrum to account for changes due to temperature or instrument variations over time. Hot pixels and cosmic rays are subtracted from the image based on a large deviation from the surrounding pixels. The 2D images collapsed to ID spectra, which are continually averaged together for 180 seconds.

[0234] Over the range of baseline analysis (550 cm' SOcm'1) a slow varying function (5thorder polynomial) is fit to the spectrum to approximate the signal fluorescence. This background is subtracted to form the initial RRS spectrum (ym) shown in FIG. 1A. To refine the baseline estimate during the regression, a baseline refinement is iteratively performed to isolate the sharp RRS peaks from the slower varying fluorescence background. After each iteration of the regression, a cubic spline function is fit to the residual with the resulting new baseline estimate subtracted from the RRS spectrum before the next iteration.624929-4870-6393 6Attorney Docket No. 030258-000102 WOPT

[0235] T linear regression is performed over a range of 700 cm'1- 1500 cm'1to generate a best fit curve (yr) which represents the sum of the regression coefficients for the library chromophores times the library spectra. For the liver tissue, a library consisting of previously obtained spectra of complex III / complex IV or whole mitochondria in both oxidized and reduced states was used, as described above. Since mitochondrial complexes have resonance maxima of reduced form closer to the excitation wavelength of 441 nm than that of oxidized form (FIG. 9), a unique enhancement factor pair was assigned to each analyte to counterbalance their difference in Soret absorption strength (also shown as fxin FIG. IB). In specific, reduced / oxidized enhancement factors of the library spectra are 0.15 (fi) / 1 (f2) for whole mitochondria, 0.28 (fs) / 1.4 (ft) for complex III, 0.15 (fs) / I (fe) for complex IV, and 0.17 (f?) / I (fs) for hemoglobin. Each coefficient (Bn) is multiplied by an enhancement factor (fn) to account for their relative resonance enhancement. The resulting values of coefficients times factors are used to represent the relative concentration of each chromophore in the tissue. The redox state of each cytochrome complex can be calculated as the ratio of the reduced concentration divided by the sum of the oxidized and reduced concentrations.

[0236] As illustrated in FIGs. 1A-1B, the 3RMR values (%) are calculated as follows: 3RMR_mito = / ?i / ( i / ?i + f P ), FormulaFormulaII; 3RMR_CIV = fsPs / f sPs + A / A)> Formula III. In addition, the RRS device also provides oxygen saturation (%) values calculated as StO2=Formula IV. Additionally, the residual spectrum, calculated as ym- yr= residual (Formula VI), is minimized to achieve a high quality of the fitting libraries. The root mean square of the residual spectrum (RMSerror) is calculated and compared to the estimated shot noise from the CCD, where an RMSerror close to the shot noise indicates that the residual is random noise rather than systematic. This ensures that the unexplained spectrum is not from unknown chromophores

[0018] , Finally, at least 10% of the total signal is mitochondrial specific, thereby ensuring there is enough signal strength to detect the most subtle spectral features to promote accurate i dentifi cati on / quantifi cati on .

[0237] Liver Viability Assessment. Inflow and outflow samples were analyzed every 30 minutes with SIEMENS RAPID-POINT 500 (SIEMENS, Munich, Germany) to primarily monitor pH, pOy (mmHg), lactate concentration (mmol / mL), and other ion levels. Concurrently, temperature, flow rate and pressure were recorded. Vascular resistance was calculated as pressure (mmHg) / flow rate (mL / min) / liver weight (g). Outflow samples were also used to measure hepatic injury markers (AST) and ALT) using Activity Assay Kit634929-4870-6393 6Attorney Docket No. 030258-000102 WOPT(CAYMAN CHEMICAL, Ann Arbor, MI, USA) and cellular apoptosis marker (Cytochrome C) using ELISA Kit (ABCAM, USA). The produced bile was collected in EPPENDORF tubes and was measured in milliliter (mL) at the end of perfusion. Liver weight was recorded before and after perfusion to determine the weight chance. Oxygen consumption (mLCh / min per g) was calculated as following: Formula VII: [O2 solubility coefficient (mL O2 / mL per mmHg) ] x [portal vein partial oxygen pressure (mmHg) - vena cava partial oxygen pressure (mmHg)] x portal vein flow (mL / min) / liver weight (g) + [HBOC concentration (g / dL) / 100] x HBOC oxygen binding capacity (mL 02 / g) x [(portal vein oxygen saturation - vena cava oxygen saturation) / ! 00] x portal vein flow (mL / min) / liver weight (g). HBOC concentration for 25% v / v was determined to be 3.25 g / dL and HBOC oxygen binding capacity is 1.39 mL 02 / g (provided by HEMOGLOBIN OXYGEN THERAPEUTICS, Souderton, PA).

[0238] Histological Imaging. Wedge biopsies were taken at the end of perfusion, fixed in 10% formalin and moved to 70% ethanol after 24 hours. Samples were paraffin embedded, cut, stained and analyzed as previously described

[0048] , In brief, H&E (hematoxylin and eosin) staining was used for vasculature and tissue edema visualization. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining was used to visualize cellular level of apoptosis.

[0239] Evaluation of Liver Energy Charge. The tissue samples for measurement of bioenergetic molecules were taken at the end of perfusion and immediately flash frozen in liquid nitrogen. Prior to tissue homogenization, samples were stored at -80°C. Liquid Chromatography-Mass Spectrometry (LC-MS) was performed by a Mass Spectrometry Core Facility (Boston MA, USA) as previously described

[0035] ,

[0240] Statistical Study. GRAPHPAD PRISM 9 software and MICROSOFT EXCEL were used to perform graphing and statistical analysis. In presenting figures, data are represented as median (Interquartile range). Statistical significance at each time point was assessed using an unpaired two-tailed Student’s T-test with Welch’s correction during machine perfusion, where significantly different comparisons between the two study groups are as indicated in the figure legends. Two-tailed statistical significance was set at < 0.05, with (*) denoting p < 0.05 unless other denotations for groups are indicated.

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Cucullo, Analysis of the Mitochondrial Membrane Potential Using the Cationic JC-1 Dye as a Sensitive Fluorescent Probe. Bi o Protoc, 2019.9(1).13. Meszaros, A. T. , et al . , Mitochondrial respiration during normothermic liver machine perfusion predicts clinical outcome. EBioMedicine, 2022. 85: p. 104311.14. Kuznetsov, A. V, et al., Evaluation of mitochondrial respiratory function in small biopsies of liver. Anal Biochem, 2002. 305(2): p. 186-94.15. Koliaki, C., et al., Adaptation of hepatic mitochondrial function in humans with nonalcoholic fatty liver is lost in steatohepatitis. Cell Metab, 2015. 21(5): p. 739-46.16. de Vries, R.J., et al., Non-invasive quantification of the mitochondrial redox state in livers during machine perfusion. PLoS One, 2021. 16(10): p. e0258833.654929-4870-6393 6Attorney Docket No. 030258-000102 WOPT17. Jain, R., et al., Real-time monitoring of mitochondrial oxygenation during machine perfusion using resonance Raman spectroscopy predicts organ function. 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Nemeth, N., et al., Hemorheological and Microcirculatory Factors in Liver Ischemia- Reperfusion Injury-An Update on Pathophysiology, Molecular Mechanisms and Protective Strategies. Int J Mol Sci, 2021. 22(4).41. Mahboub, P, et al., Gradual rewarming with a hemoglobin-based oxygen carrier improves viability of donation after circulatory death in rat livers. Front Transplant, 2024. 3: p. 1353124.42. Minor, T., et al., Controlled oxygenated rewarming as novel end-ischemic therapy for cold stored liver grafts. A randomized controlled trial. Clin Transl Sci, 2022. 15(12): p. 2918- 2927.43. van Rij n, R. , et al . , Hypothermic Machine Perfusion in Liver Transplantation - A Randomized Trial. N Engl J Med, 2021. 384(15): p. 1391-1401.44. McGiflfin, D.C., et al., Hypothermic oxygenated perfusion (HOPE) safely and effectively extends acceptable donor heart preservation times: Results of the Australian and New Zealand trial. J Heart Lung Transplant, 2024. 43(3): p. 485-495.674929-4870-6393 6Attorney Docket No. 030258-000102 WOPT45. Yu, B., K.D. Bloch, and W.M. Zapol, Hemoglobin-based red blood cell substitutes and nitric oxide. Trends Cardiovasc Med, 2009. 19(3): p. 103-7.46. Cabrales, P. and J.M. Friedman, HBOC vasoactivity: interplay between nitric oxide scavenging and capacity to generate bioactive nitric oxide species. Antioxid Redox Signal, 2013. 18(17): p. 2284-97.47. Song, B.K., et al., Effects of a hemoglobin-based oxygen carrier (HBOC-201) and derivatives with altered oxygen affinity and viscosity on systemic and microcirculatory variables in a top-load rat model. Microvasc Res, 2014. 95: p. 124-30.48. Tessier, S.N., et al., Partial freezing of rat livers extends preservation time by 5-fold. Nat Commun, 2022. 13(1): p. 4008.49. Chen, M., et al., Contrast-Enhanced Ultrasound to Quantifyc Perfusion in a Machine- Perfused Pig Liver. Annu Int Conf IEEE Eng Med Biol Soc, 2018. 2018: p. 3128-3131.50. Bruinsma, B.G., et al., Subnormothermic machine perfusion for ex vivo preservation and recovery of the human liver for transplantation. Am J Transplant, 2014. 14(6): p. 1400-9.51. Olverson, G.t., et al., Cardiac Loading using Passive Left Atrial Pressurization and Passive Afterload for Graft Assessment. J Vis Exp, 2024(210).52. Bruinsma, B.G., et al., Supercooling preservation and transplantation of the rat liver. Nat Protoc, 2015. 10(3): p. 484-94.53. Shi, Z.F., et al., Methylene blue ameliorates brain edema in rats with experimental ischemic stroke via inhibiting aquaporin 4 expression. Acta Pharmacol Sin, 2021. 42(3): p. 382- 392.54. Covian, R., et al., Spectroscopic identification of the catalytic intermediates of cytochrome c oxidase in respiring heart mitochondria. Biochimica et Biophysica Acta (BB A) - Bioenergetics, 2023. 1864(2): p. 148934.55. Glancy, B . and R. S . Balaban, Protein composition and function of red and white skeletal muscle mitochondria. Am J Physiol Cell Physiol, 2011. 300(6): p. C 1280-90.56. Balaban, R.S., VK. Mootha, and A. Arai, Spectroscopic determination of cytochrome c oxidase content in tissues containing myoglobin or hemoglobin. Anal Biochem, 1996. 237(2): p. 274-8.57. Ljungdahl, P.O., J.D. Pennoyer, and B.L. Trumpower,

[0018] Purification of cytochrome bcl complexes from phylogenically diverse species by a single method, in Methods in Enzymology. 1986, Academic Press, p. 181-191.684929-4870-6393 6Attorney Docket No. 030258-000102 WOPT58. Siletsky, S.A. and A. A. Konstantinov, Cytochrome c oxidase: Charge translocation coupled to single -electron partial steps of the catalytic cycle. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012. 1817(4): p. 476-488.

[0242] Example 2: Mitochondrial Recovery From Ischemia-reperfusion Injury In DCD Pig Livers Detected By Resonance Raman Spectroscopy

[0243] Purpose: Donor after cardiac death (DCD) livers have the potential to extend the donor pool but ischemia-reperfusion injury (IRI) remains a critical problem that can limit their utilization. Mitochondria play a critical role in liver IRI by energy stress and overproduction of reactive oxygen species. In this study, it was first aimed to induce mitochondrial stress by extended warm ischemia (WI) and detect reperfusion injury using Resonance Raman (RR) Spectroscopy during machine perfusion in rodent models. Next, methylene blue (MB) was introduced to the perfusate to rescue mitochondria from electron leaks. Finally, the effects were translated to DCD pig livers, with added MB and the hemoglobin-based oxygen carriers (e.g., HEMOPURE, also known as HBOC-201, a bovine hemoglobin-based oxygen carrier) for optimal oxygen delivery.

[0244] Methods: Rodent (Lewis) livers (n=6) were procured and subjected to 3h WI followed by 3h acellular subnormothermic perfusion (SNMP), RR analysis compared with freshly procured (n=6) livers. Then, 0.01% MB was added to the perfusate, and RR analysis was assessed to investigate the drug’s effectiveness on 3h WI livers (n=3). Next, livers were harvested from Yorkshire pigs 30 mins after cardiac death. 0.01% MB and 25% HEMOPURE were added to perfusate (n=3). Results were compared to control livers (n=4) with no treatment during 3h SNMP.

[0245] Results: Rodent 3h WI livers showed lower reduced mitochondrial cytochromes (defined as “3RMR”, FIG. 14C) and significant decrease in reduced complex III compared to fresh, indicating a leak in electron chain. With MB treatment, RR analysis confirmed electron bypass as complex III turned fully oxidized (FIG. 14D) while complex IV became reduced (FIG. 14E) due to the extra electron deposit. DCD pig livers treated with MB+HEMOPURE showed improved vascular resistance (FIG. 15C) with higher flow rate (FIG. 15D) than control group. There were also increased oxygen consumption (FIG. 15F), and reduced lactate level (FIG. 15G).

[0246] Conclusions: one of the mitochondrial pathways of IRI was detected with RR analysis. Treatment with MB+HEMOPURE in DCD pig livers increased electron and oxygen 694929-4870-6393 6Attorney Docket No. 030258-000102 WOPT delivery to complex IV, leading to higher oxygen consumption and lower lactate level, indicating the enhancement of aerobic metabolism via recovery of mitochondrial function.

[0247] Example 3: MITOCHONDRIAL RECOVERY FROM ISCHEMIAREPERFUSION INJURY IN DCD PIG LIVERS DETECTED BY RESONANCE RAMAN SPECTROSCOPY

[0248] Disclosed herein is a biomedical technology to discern the redox states of mitochondria and electron transport chain (ETC) complexes in a quantitative, nondestructive, and label-free approach. To highlight the significant abilities of the technology, this investigation was aimed at assessing mitochondrial damage as part of ischemiareperfusion injury (IRI) during machine perfusion, a critical determinant in prediction of the success / outcomes of liver transplantation.

[0249] The technology disclosed herein utilizes Resonance Raman Spectroscopy (RRS) system with a non-contact measurement probe and a 1.5-meter fiber optic cable to transmit laser light to and collect RRS scattering from the organ surface, and a 441 nm absorption excitation wavelength laser, and a high-resolution spectrometer in a small footprint device. The mitochondrial redox state (3RMR) values are computed by regression algorithm using RRS spectral libraries of isolated whole mitochondria and ETC complexes previously recorded, and are evaluated with classic biometrics to distinguish various physiological states. Demonstrated herein are methods to induce mitochondrial stress by extended warm ischemia (WI) followed by detection of mitochondrial leakiness by the described RRS technology during sub-normothermic machine perfusion (SNMP) of rodent IRI models, as well as assessment of the effects of methylene blue (MB) in overcoming mitochondrial leakiness. The methods are applied to rescue of Donor after Circulatory Death (DCD) pig livers from IRI mitochondrial leakiness using MB with artificial hemoglobinbased oxygen carrier (HBOC) during SNMP. Results in IRI rodent liver models are shown in FIGs. 18-19. FIG. 18 depicts experiment 1, detection of mitochondrial leakiness in IRI livers during 3h SNMP (n = 6). FIG. 19 depicts experiment 2, assessment of MB effects in overcoming mitochondrial leakiness (n = 3). FIG. 20 depicts results in DCD pig livers: Experiment 3, assessment of MB+ HBOC treatment in rescuing DCD pig livers from IRI progression during SNMP (n=4 control livers, n=5 treated livers).

[0250] Rodent 3h WI livers showed lower 3RMR mito values (p<0.05) and significant decrease in reduced complex III (p<0.05) compared to fresh, indicated an overoxidized mitochondrial state with an electron leak particularly at complex III. With MB treatment, RRS analysis confirmed electron bypass as complex III turned fully oxidized while 704929-4870-6393 6Attorney Docket No. 030258-000102 WOPT complex IV became reduced due to the extra electron deposit. Treatment with MB+HBOC in DCD pig livers increased electron and oxygen delivery to complex IV, leading to higher portal flow rate (p<0.05), oxygen consumption (p<0.05) and lower decreasing trends of lactate and potassium levels, indicating the enhancement of aerobic metabolism via recovery of mitochondrial function.References for Example 31. DA Perry, JW Salvin, et al. Responsive monitoring of mitochondrial redox states in heart muscle predicts impending cardiac arrest. Science Translational Medicine. 2017.2. HAPrag, et al. Mitochondrial ROS production during ischemia-reperfusion injury. Oxidative Stress, Chapter 26. 2020.

[0251] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.714929-4870-6393 6

Claims

1. Attorney Docket No. 030258-000102 WOPTCLAIMSWhat is claimed is:

1. A method for preventing or treating ischemia-reperfusion injury in an organ or a tissue, the method comprising administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

2. A method for preventing or treating ischemia-reperfusion injury in a tissue or an organ, the method comprising:(a) determining if a mitochondria and / or at least one electron transport chain (ETC) complex of the tissue or organ is hyperoxidized; and(b) if the mitochondria and / or the at least one ETC complex of the tissue or organ is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

3. The method of any of claims 1-2, wherein the organ is a solid organ or a tissue thereof.

4. The method of any of claims 1-3, wherein the organ or tissue is vascularized.

5. The method of any of claims 1-4, wherein the tissue is of a liver, a heart, a kidney, a brain, a retina, or skin.

6. The method of any of claims 1-5, wherein the organ is a liver, a heart, a kidney, a brain, a retina, or skin.

7. The method of any one of claims 1-6, wherein the tissue or organ is on or in a subject, and the method occurs in vivo.

8. The method of any one of claims 1-7, wherein the tissue or organ has been removed from a subject, and the method occurs ex vivo.724929-4870-6393 6Attorney Docket No. 030258-000102 WOPT9. The method of any one of claims 1-8, wherein prior to treatment, the tissue or organ has been subjected to ischemia followed by initiation of reperfusion, and the tissue or organ is at risk of or has suffered an ischemia-reperfusion injury.

10. The method of any of claims 1-9, wherein the ischemia and / or reperfusion is from: a tissue or organ transplantation, a complex surgery, a myocardial infarction, or a stroke.

11. The method of claim 10, wherein the complex surgery is a complex cardiac surgery.

12. The method of claim 11, wherein the complex cardiac surgery comprises cardioplegia.

13. The method of claims 1 or 2, wherein the organ or tissue is a Donor after Circulatory Death (DCD) organ or tissue.

14. The method of any of claims 1-13, wherein the ischemia is warm ischemia.

15. The method of any of claims 1-13, wherein the reperfusion is machine perfusion.

16. The method of claim 15, wherein the machine perfusion is subnormothermic machine perfusion.

17. The method of any one of claims 1-16, wherein the ischemia occurs for at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 120 minutes, or at least 180 minutes.

18. The method of any of claims 1-17, wherein the reperfusion occurs for at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 120 minutes, or at least 180 minutes.

19. The method of any of claims 1-18, wherein the reperfusion occurs for up to about 24 hours.734929-4870-6393 6Attorney Docket No. 030258-000102 WOPT20. The method of any one of claims 1-19, wherein the method is performed during or after the reperfusion.

21. The method of any one of claims 1-20, wherein the methylene blue or functional variant thereof is administered during or after the reperfusion.

22. The method of any of claims 1-21, wherein the methylene blue or functional variant thereof is administered before skin closure during a tissue or organ transplantation or a complex surgery.

23. The method of any of claims 1-22, wherein the methylene blue or functional variant thereof is administered immediately after transplantation of the tissue or organ into a subject.

24. The method of any one of claims 1-23, wherein the method is not performed during the ischemia.

25. The method of any one of claims 1-24, wherein the methylene blue or functional variant thereof is not administered during the ischemia.

26. The method of any one of claims 1-25, further comprising administering an effective amount of an oxygenation support to the tissue or organ, in addition to the administering of the methylene blue or functional variant thereof.

27. The method of claim 26, wherein the oxygenation support is whole blood, packed Red Blood Cells (RBCs), artificial Hemoglobin Based Oxygen Carriers (HBOCs), Supplemental Oxygen Therapy, Mechanical Ventilation, Hyperbaric Oxygen Therapy (HBOT), Extracorporeal Membrane Oxygenation (ECMO), cardiopulmonary bypass (CPB), or Hypothermic Oxygenated Machine Perfusion (HOPE).

28. The method of claims 26 or 27, wherein the oxygenation support is HBOCs.744929-4870-6393 6Attorney Docket No. 030258-000102 WOPT29. The method of claim 1, further comprising determining if the mitochondria and / or at least one electron transport chain (ETC) complex is hyperoxidized prior to administering the effective amount of methylene blue or the functional variant thereof.

30. The method of claims 2 or 29, further comprising quantifying a Resonance Raman Reduced Mitochondrial Ratio (3RMR) value for the redox state of the mitochondria or the at least one ETC complex of the tissue or organ using a Raman spectroscopy method, and determining if the 3RMR value indicates that the mitochondria and / or the at least one ETC complex is hyperoxidized.

31. The method of claims 2, 29 or 30, wherein the at least one ETC complex is Complex I (CI), Complex II (CII), or Complex III (CIII).

32. The method of any of claims 2 and 29-31, wherein the at least one ETC complex is Complex III (CIII).

33. The method of claim 32, wherein the 3RMR value for the mitochondria in a liver being below 10% or the 3RMR value for CIII being below 20% indicate that the mitochondria or the CIII are hyperoxidized.

34. The method of claim 32, wherein the 3RMR value for the mitochondria in a liver being above 10% or the 3RMR value for CIII being above 20%, indicate that the mitochondria or CIII are not hyperoxidized.

35. A method for preventing or treating ischemia-reperfusion injury in a tissue or an organ, the method comprising:(a) quantifying a Resonance Raman Reduced Mitochondrial Ratio (3RMR) value of a mitochondrial complex of the tissue or organ using a Raman spectroscopy method;(b) determining if the 3RMR value indicates that the mitochondrial complex is hyperoxidized; and(c) if the 3RMR value indicates that the mitochondrial complex is hyperoxidized, administering an effective amount of methylene blue or a754929-4870-6393 6Attorney Docket No. 030258-000102 WOPT functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

36. The method of claim 35, wherein the at least one ETC complex is Complex I (CI), Complex II (CII), or Complex III (CIII).

37. The method of claim 35, wherein if the mitochondria and / or the at least one ETC complex are physiologically oxidized, no treatment is administered.

38. The method of claim 35, wherein if the mitochondria and / or the at least one ETC complex are hyper-reduced, the treatment comprises oxygen and / or an oxygenation support.

39. The method of claim 35, wherein after step (c), the method further comprises:(d) quantifying a 3RMR value of a second mitochondrial ETC complex of the tissue or organ using the Raman spectroscopy method; and(e) determining if the 3RMR value indicates that the second mitochondrial ETC complex is reduced.

40. The method of claim 39, wherein after step (e), the method further comprises:(f) if the 3RMR value indicates that the second mitochondrial ETC complex is not reduced, continuing to administer the methylene blue or the functional variant thereof to the tissue or organ; or(g) if the 3RMR value indicates that the second mitochondrial ETC complex is reduced, ceasing to administer the methylene blue or the functional variant thereof to the tissue or organ and / or increasing the oxygen delivery to the tissue or organ.

41. The method of claim 39 or 40, wherein the second mitochondrial ETC complex is Complex IV (CIV).

42. The method of claim 41, wherein the 3RMR value being at or above 1% in a liver indicates that the CIV is reduced; and wherein the 3RMR value being below 1% in a liver indicates that the CIV is not reduced.764929-4870-6393 6Attorney Docket No. 030258-000102 WOPT43. The method of any one of claims 30-42, wherein the 3RMR value of the mitochondria, the CIII, or the CIV is calculated by:(i) obtaining a raw spectrum using the Raman spectroscopy method;(ii) performing baseline subtraction of the raw spectrum to produce a resonance Raman spectrum;(iii) performing a linear regression analysis of the resonance Raman spectrum against a library of known analytes selected from oxidized and reduced resonance Raman spectra of the mitochondria, CIII, or CIV, to determine the relative concentration of: oxidized mitochondria and reduced mitochondria, or oxidized CIII and reduced CIII, or oxidized CIV and reduced CIV, in the tissue or organ; wherein, during the linear regression analysis, each known analyte is assigned a specific enhancement factor (fn), and(iv) wherein the 3RMR value of the mitochondria, the CIII, or the CIV is calculated as the ratio of their corresponding reduced concentration divided by the sum of the oxidized and reduced concentrations.

44. The method of any one of claims 30-43, wherein the 3RMR value of the mitochondria is calculated by: Formula I: 3RMR_mito =+ fzftz)-45. The method of any one of claims 30-44, wherein the 3RMR value of the CIII is calculated by: Formula II: 3RMR_CIII = fsPs / fsPs46. The method of any one of claims 30-45, wherein the 3RMR value of the CIV is calculated by Formula III: 3RMR_CIV = fsfts / fsfts + f6p6~).

47. Methylene blue or a functional variant thereof for use in a method of treating or preventing ischemia-reperfusion injury.

48. Methylene blue or a functional variant thereof for use in a method of treating or preventing ischemia-reperfusion injury, the method comprising: administering an effective amount of methylene blue or a functional variant thereof to an tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.774929-4870-6393 6Attorney Docket No. 030258-000102 WOPT49. Methylene blue or a functional variant thereof for use in a method of treating or preventing ischemia-reperfusion injury, the method comprising:(a) determining if a mitochondrial or at least one electron transport chain (ETC) complex of the tissue or organ is hyperoxidized; and(b) if the mitochondrial or the at least one ETC complex of the tissue or organ is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

50. Methylene blue or a functional variant thereof for use in a method of treating or preventing ischemia-reperfusion injury, the method comprising:(a) quantifying a Resonance Raman Reduced Mitochondrial Ratio (3RMR) value of a mitochondrial complex of the tissue or organ using a Raman spectroscopy method;(b) determining if the 3RMR value indicates that the mitochondrial complex is hyperoxidized; and(c) if the 3RMR value indicates that the mitochondrial complex is hyperoxidized, administering an effective amount of methylene blue or a functional variant thereof to the tissue or organ to thereby prevent or treat the ischemia-reperfusion injury.

51. Methylene blue or a functional variant thereof for use in the manufacture of a medicament for treating or preventing ischemia-reperfusion injury.

52. The method of any of the preceding claims, wherein the functional variant of methylene blue is selected from the group consisting of: mitoquinone, azure A, a diaminophenolthiazine, phenazine methosulfate, and 2,6-indophenol.784929-4870-6393 6

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