Methods to improve organ survival rates

Abstract

To provide methods of improving the viability of an organ.SOLUTION: The present disclosure provides methods of improving the viability of one or more organs by continuously administering a composition comprising NOx gas directly to the organ(s). A method of improving the viability of an organ intended for transplant comprises continuously administering a composition comprising NOx gas directly to the organ via an organ perfusion system or ventilation. The composition is administered for at least one hour but not more than 12 hours. The composition is administered after the organ has been harvested from a donor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 550,463, filed on 25 August 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] This disclosure is NO x The present invention provides a method for improving the survival rate of one or more organs by directly and continuously administering a gas-containing composition to the organ(s)(s). [Background technology]

[0003] Cells, tissues, organs, and organisms deprived of adequate blood flow suffer ischemic damage due to oxidative stress, ultimately leading to death. Conventional methods to mitigate ischemic damage include perfusing the affected tissue with oxygen, but this procedure can cause significant tissue damage and may result in serious and / or permanent injury, such as brain injury during stroke or cardiac arrest.

[0004] Attempts have been made to reduce ischemia and reperfusion injury by inducing a metabolically reduced state in tissues and organs. For example, in situations where living tissue is preserved for transplantation (or grafting), one common method to reduce metabolic activity is to immerse the tissue or organ in a physiological fluid such as saline and place it in a cold environment. However, such methods cannot be relied upon for long periods, and the success of organ transplantation and limb reattachment remains inversely proportional to the amount of time the organ or limb has not been in contact with an intact organism.

[0005] Therefore, in this field, there remains a need to improve organ survival before ischemia and / or reperfusion injury occurs. [Overview of the project] [Means for solving the problem]

[0006] In one embodiment, the present disclosure relates to a method for improving the viability of an organ intended for transplantation, wherein NO is delivered to the organ via an organ perfusion system or ventilation. x This includes methods that involve directly and continuously administering a gas-containing composition.

[0007] In another aspect, the disclosure relates to a method for improving the survival rate of an organ damaged by ischemia-reperfusion, wherein the organ is perfused through an organ perfusion system or ventilation to the organ at a concentration of NO less than or equal to about 20 ppm. x The method includes administering a gas-containing composition directly and continuously. In various embodiments, the organ requiring treatment is an organ damaged by trauma, surgery, respiratory arrest, or cardiac arrest. In specific embodiments, the organ requiring treatment is an organ intended for transplantation. In exemplary embodiments, the organ requiring treatment is an organ extracted from a donor and intended for transplantation.

[0008] In another embodiment, the present disclosure relates to a method for improving the survival rate of an organ damaged by ischemia-reperfusion, comprising (a) about 20 ppm to about 40 ppm of NO x (b) administering a composition containing gas ("loading dose") to an organ for up to approximately 1 hour ("loading period"), and then (b) delivering NO to an organ via an organ perfusion system or ventilation at a concentration of approximately 20 ppm or less. x The method comprises administering a gas-containing composition directly and continuously to an organ. In various embodiments, the organ requiring treatment is an organ damaged by trauma, surgery, respiratory arrest, or cardiac arrest. In specific embodiments, the organ requiring treatment is an organ intended for transplantation. In exemplary embodiments, the organ requiring treatment is an organ extracted from a donor and intended for transplantation.

[0009] In another embodiment, the present disclosure relates to a method for improving the viability of an organ intended for transplantation, wherein the organ is perfused with 20 ppm or less of NO through an organ perfusion system or ventilation. x The method comprises administering a gas-containing composition directly and continuously. In some embodiments, the organ is present in a brain-dead donor. In other embodiments, the organ is NO xIt has been removed from the donor prior to administration of the composition containing the gas. In an exemplary embodiment, the organ is a lung, a kidney, or a heart.

[0010] In another aspect, the present disclosure is a method of improving the survival rate of an organ for transplantation, comprising: (a) administering to the organ a composition containing NO gas at about 20 ppm to about 40 ppm (a "loading dose") for up to about 1 hour (a "loading period"), and then (b) continuously administering to the organ, via an organ perfusion system or ventilation, a composition containing NO gas at 20 ppm or less. x In some embodiments, the organ is from a brain-dead donor. In other embodiments, the organ has been removed from the donor prior to administration of the composition containing the NO gas. In an exemplary embodiment, the organ is a lung, a kidney, or a heart. x In some embodiments, the organ is from a brain-dead donor. In other embodiments, the organ has been removed from the donor prior to administration of the composition containing the NO gas. In an exemplary embodiment, the organ is a lung, a kidney, or a heart. x It has been removed from the donor prior to administration of the composition containing the gas. In an exemplary embodiment, the organ is a lung, a kidney, or a heart.

[0011] In another aspect, the present disclosure is a method of treating ischemia-reperfusion injury of an organ in need thereof, comprising continuously administering, directly to the organ via an organ perfusion system or ventilation, a composition containing NO gas at 20 ppm or less. x In various embodiments, the organ in need of treatment is an organ damaged by trauma, surgery, respiratory arrest, or cardiac arrest. In certain embodiments, the organ in need of treatment is an organ for transplantation. In an exemplary embodiment, the organ in need of treatment is an organ removed from a donor for transplantation.

[0012] In another aspect, the present disclosure is a method of treating ischemia-reperfusion injury of an organ in need thereof, comprising: (a) administering to the organ a composition containing NO gas at about 20 ppm to about 40 ppm (a "loading dose") for up to about 1 hour (a "loading period"), and then (b) continuously administering to the organ, via an organ perfusion system or ventilation, a composition containing NO gas at 20 ppm or less. x ガス(「負荷用量」)を含む組成物を最長約1時間(「負荷期間」)器官に投与すること、次いで(b)器官灌流システムまたは換気を介して、20ppm以下のNO xThe method comprises administering a gas-containing composition directly and continuously to an organ. In various embodiments, the organ requiring treatment is an organ damaged by trauma, surgery, respiratory arrest, or cardiac arrest. In specific embodiments, the organ requiring treatment is an organ intended for transplantation. In exemplary embodiments, the organ requiring treatment is an organ extracted from a donor and intended for transplantation.

[0013] In another embodiment, the present disclosure relates to a method for transplantation, comprising (a) NO 20 ppm or less. x The present invention provides a method comprising (b) administering a gas-containing composition directly to an organ intended for transplantation for up to 12 consecutive hours, and (b) transplanting the organ into a recipient. In some embodiments, the organ is located in a brain-dead donor. In other embodiments, the organ is located in NO x The organ is removed from the donor before administration of the gas-containing composition. In exemplary embodiments, the organ is the lung, kidney, or heart.

[0014] In another embodiment, the present disclosure relates to a method for transplantation, comprising (a) about 20 ppm to about 40 ppm of NO x (b) Administer a composition containing gas ("loading dose") to the organs for up to approximately 1 hour ("loading period"), followed by (b) NO 20 ppm or less. x The present invention provides a method comprising (c) administering a gas-containing composition directly to an organ intended for transplantation for up to 12 consecutive hours, and then transplanting the organ into a recipient. In some embodiments, the organ is located in a brain-dead donor. In other embodiments, the organ is located in NO x The organ is removed from the donor before administration of the gas-containing composition. In exemplary embodiments, the organ is the lung, kidney, or heart.

[0015] Other aspects and variations of this disclosure are described in more detail below. This specification also provides, for example, the following items: (Item 1) A method for improving the survival rate of an organ intended for transplantation, comprising delivering 20 ppm or less of NO to the organ via an organ perfusion system or ventilation.x The method comprising directly and continuously administering a composition containing a gas. (Item 2) The method according to item 1, wherein the composition is administered for at least 1 hour, but not for more than 12 hours. (Item 3) The method according to item 1 or item 2, wherein the composition is administered after the organ has been collected from the donor. (Item 4) The method according to item 1 or item 2, wherein the composition is administered while the organ is in the body of a brain-dead donor. (Item 5) The method according to any one of items 1 to 3, wherein the organ is selected from the group consisting of the lungs, heart, liver, kidneys, pancreas, intestines, thymus, and cornea. (Item 6) NO x The method described in any one of items 1 to 5, wherein the amount is approximately 10 ppm to approximately 15 ppm. (Item 7) NO x The method described in any one of items 1 to 5, wherein the amount is approximately 5 ppm to approximately 10 ppm. (Item 8) NO x The method described in any one of items 1 to 5, wherein the amount is approximately 1 ppm to approximately 5 ppm. (Item 9) The method according to any one of items 1 to 8, wherein the composition is an organ perfusion fluid and optionally further comprises one or more additional compounds. (Item 10) The method according to item 9, wherein the perfusion fluid is a cell-free perfusion fluid. (Item 11) The above method involves approximately 20 ppm to approximately 40 ppm of NO x A composition containing gas ("loading dose") with NO content of 20 ppm or less x The method according to any one of items 1 to 10, further comprising administering the gas-containing composition to the organ for up to about one hour ("loading period") immediately before continuous administration. (Item 12) The method according to item 11, wherein the load dose composition is a perfusion solution. (Item 13) The method according to item 12, wherein the perfusion fluid is a cell-free perfusion fluid. (Item 14) NO x The method described in any one of items 1 to 13, wherein the total gas administration time does not exceed 12 hours. (Item 15) A method described in any one of items 1 to 14, which significantly maintains mitochondrial function. (Item 16) The method according to any one of items 1 to 15, wherein mitochondrial reactive oxygen species (mtROS) are reduced in the organ compared to an untreated control. (Item 17) A method for improving the survival rate of an organ damaged by ischemia-reperfusion, wherein the organ is perfused with 20 ppm or less of NO through an organ perfusion system. x The method comprising directly and continuously administering a composition containing a gas. (Item 18) The method according to item 17, wherein the composition is administered for at least 1 hour, but not for more than 12 hours. (Item 19) The method according to item 17 or item 18, wherein the organ is intended for transplantation, and the composition is administered after the organ has been harvested from a donor. (Item 20) The method according to item 17 or item 18, wherein the organ is intended for transplantation, and the composition is administered while the organ is in the body of a brain-dead donor. (Item 21) The method according to any one of items 17 to 20, wherein the organ is selected from the group consisting of the lungs, heart, liver, kidneys, pancreas, intestines, thymus, and cornea. (Item 22) NO x The method described in any one of items 17 to 21, wherein the amount is approximately 10 ppm to approximately 15 ppm. (Item 23) NO xThe method described in any one of items 17 to 21, wherein the amount is approximately 5 ppm to approximately 10 ppm. (Item 24) NO x The method described in any one of items 17 to 21, wherein the amount is approximately 1 ppm to approximately 5 ppm. (Item 25) The method according to any one of items 17 to 23, wherein the composition is an organ perfusion fluid and optionally further comprises one or more additional compounds. (Item 26) The method according to item 25, wherein the perfusion fluid is a cell-free perfusion fluid. (Item 27) The above method involves approximately 20 ppm to approximately 40 ppm of NO x A composition containing gas ("loading dose") with NO content of 20 ppm or less x The method according to any one of items 17 to 26, further comprising administering the gas-containing composition to the organ for up to about one hour ("loading period") immediately before continuous administration. (Item 28) The method according to item 27, wherein the load dose composition is a perfusion solution. (Item 29) The method according to item 28, wherein the perfusion fluid is a cell-free perfusion fluid. (Item 30) NO x The method described in any one of items 17 to 29, wherein the total gas administration time does not exceed 12 hours. (Item 31) The method described in any one of items 17-30, which significantly maintains mitochondrial function. (Item 32) The method according to any one of items 17 to 31, wherein mitochondrial reactive oxygen species (mtROS) are reduced in the organ compared to an untreated control. (Item 33) It is a transplantation method, (a) NO below 20 ppm via the organ perfusion system x The gas-containing composition is administered directly and continuously to the organ intended for transplantation, and (b) Transplanting the organ to the recipient. The method, including the method described above. (Item 34) The method according to item 33, wherein the composition is administered for at least 1 hour, but not exceeding 12 hours. (Item 35) The method according to item 33 or item 34, wherein the composition is administered after the organ has been collected from the donor. (Item 36) The method according to item 33 or item 34, wherein the composition is administered while the organ is in the body of a brain-dead donor. (Item 37) The method according to any one of items 33 to 36, wherein the organ is selected from the group consisting of the lungs, heart, liver, kidneys, pancreas, intestines, thymus, and cornea. (Item 38) NO x The method described in any one of items 33 to 37, wherein the amount is approximately 10 ppm to approximately 15 ppm. (Item 39) NO x The method described in any one of items 33 to 37, wherein the amount is approximately 5 ppm to approximately 10 ppm. (Item 40) NO x The method described in any one of items 33 to 37, wherein the amount is approximately 1 ppm to approximately 5 ppm. (Item 41) The method according to any one of items 33 to 40, wherein the composition is an organ perfusion fluid and optionally further comprises one or more additional compounds. (Item 42) The method according to item 41, wherein the perfusion fluid is a cell-free perfusion fluid. (Item 43) The method described above involves applying NO at a concentration of approximately 20 ppm to 40 ppm for a maximum of approximately 1 hour immediately preceding step (a) ("loading period"). x The method according to any one of items 33 to 42, further comprising administering a composition containing a gas ("loading dose") to the organ. (Item 44) The method according to item 43, wherein the load dose composition is a perfusion solution. (Item 45) The method according to item 44, wherein the perfusion fluid is a cell-free perfusion fluid. (Item 46) NO x The method described in any one of items 33 to 45, wherein the total gas administration time does not exceed 12 hours. (Item 47) The method described in any one of items 33-46, which significantly maintains mitochondrial function. (Item 48) The method according to any one of items 33 to 47, wherein mitochondrial reactive oxygen species (mtROS) are reduced in the organ compared to an untreated control.

[0016] The application file includes at least one color photograph. A copy of this patent application publication containing the color photograph is available from the Ministry upon request and payment of the required fees. [Brief explanation of the drawing]

[0017] [Figure 1] This is a diagram illustrating an embodiment of an EVLP circuit. [Figure 2] This is a diagram illustrating an embodiment of an EVLP circuit. [Modes for carrying out the invention]

[0018] This disclosure is NO x This disclosure provides a method for improving the survival rate of one or more organs by directly and sequentially administering a gas-containing composition to the organ(s)(s). This disclosure encompasses methods for improving the survival rate of organs intended for transplantation, as well as organs suffering from ischemia-reperfusion injury due to other causes. Furthermore, this disclosure provides methods for improving the capacity of transplanted organs and methods for transplantation. When used herein, "NO x The term "gas" refers to gaseous nitrogen oxides. In preferred embodiments, NO xThe gas is gaseous nitric oxide (gNO). Non-limiting examples of further components of the composition may include inert diluent gases (e.g., helium, neon), human albumin, sodium caprylate, N-acetyl-DL-tryptophan, and oxygen gas (O2). x Administering a gas-containing composition directly and continuously to an organ means that the organ will continuously produce NO without interruption during the administration period. x This means direct contact with the gas. This disclosure is not limited by the type of organ. Non-limiting examples of suitable organs include the liver, kidney, pancreas, heart, lung, intestine, thymus, cornea, angiogenic composite allograft (e.g., face, hand, etc.), or any combination thereof. Organ survival rates are NO x The results will be improved compared to organs obtained by methods that do not involve direct, continuous administration of gas-containing compositions to the organ. As used herein, the term “survival rate” indicates the suitability of an organ for its intended purpose. Measurements of organ survival rate may vary depending on the type of organ and are known in the art.

[0019] Hereinafter, we present some definitions that apply throughout this disclosure. As used herein, “approximately” refers to a numerical value, including integers, fractions, and percentages, whether expressly indicated or not. The term “approximately” generally refers to a range of numerical values, such as ±0.5 to 1%, ±1 to 5%, or ±5 to 10%, of the enumerated value, and is considered equivalent to, for example, having the same function or result as, the cited value.

[0020] The term “comprising” means “including, but not necessarily limited to.” This is especially true for open-ended inclusion or membership in the aforementioned combinations, groups, series, etc. As used herein, the terms “comprising” and “including” are comprehensive and / or open-ended and do not exclude additional, unlisted elements or method processes. The term “essentially consisting of” is more restrictive than “comprising” but not as restrictive as “consisting of.” Specifically, the term “essentially consisting of” limits membership to specific materials or steps and those that do not substantially affect the essential characteristics of the claimed invention.

[0021] As used herein, the term "ischemia-reperfusion injury" refers to injury resulting from ischemia, reperfusion, or both.

[0022] As used herein, the terms “to treat,” “to treat,” or “treatment” refer to both therapeutic actions and preventive or preventive measures, the purpose of which is to prevent or slow (reduce) undesirable physiological changes or disease / disorder. Beneficial or desirable clinical outcomes include, but are not limited to, relief of symptoms, reduction of the extent of the disease, stable (i.e., non-worsening) state of the disease, delayed or slowed progression of the disease, remission or mitigation of the disease state, and remission (partial or complete remission), whether detectable or undetectable. “Treatment” may also mean extending survival compared to the survival expected without treatment. Persons requiring treatment include those already suffering from the disease or disorder, those prone to developing the disease or disorder, or those in a condition where prevention of the disease or disorder is necessary.

[0023] (a) NO x gas-containing composition According to this disclosure, NO xThe gas-containing composition is administered directly to the organ in a continuous manner. The composition of this disclosure may be a gas or a liquid. If the composition of this disclosure is a liquid, NO x The gas is solubilized in the liquid. In other words, "NO is a liquid composition." x "Composition containing gas" is solubilized NO x It is a liquid containing gas. Similarly, "20 ppm NO, which is a liquid composition." x The "composition containing gas" is a liquid containing 20 ppm nitric oxide, and the 20 ppm nitric oxide is solubilized in the liquid NO x This is the amount of gas. Furthermore, in embodiments where the composition is a liquid, NO is administered directly to the organs. x The amount of gas is NO dissolved in the liquid. x It is the amount of gas. NO x The gas can be produced and provided by any method known in the art.

[0024] In some embodiments, the composition is a gas. x In addition to the gas, the composition may further include one or more additional components, including but not limited to inert diluent gases (multiple, e.g., helium, neon), nitrogen, oxygen, and water. In exemplary embodiments, NO x The gas is gNO. If the composition is a gas, the composition can be administered directly to the organs via a ventilator or any other method known in the art.

[0025] In other embodiments, the composition is a perfusion fluid. The term “perfusion fluid” refers to any fluid used for preservation, perfusion, or reperfusion of tissue or organ. Perfusion fluids are often sterile and isotonic. Solubilized NO xIn addition to gas, the perfusion solution may further contain one or more additional components, including but not limited to sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. The composition of the perfusion solution may also vary between organs. In preferred embodiments, the perfusion solution is a cell-free perfusion solution. Such solutions may include, but are not limited to, Celsior solution, Krebs-Henseleit solution, normal saline solution, University of Wisconsin solution, St. Thomas II solution, Collins solution, Stanford solution, Perfidex®, Steen Solution®, or combinations thereof. In exemplary embodiments, the composition is a cell-free perfusion solution, NO x The gas is gNO. In further embodiments, the cell-free perfusion fluid is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. Methods suitable for directly administering perfusion fluid to organs are known in the art and include, but are not limited to, organ perfusion systems. This disclosure is not limited to any particular organ perfusion system. Generally speaking, an organ perfusion system may comprise a pump for the movement and control of the perfusion fluid, means for controlling the temperature of the system, a cannula, and means for measuring physiological parameters. Non-limiting examples of organ perfusion systems are disclosed in U.S. Patent No. 9,629,358, which is incorporated herein by reference. Non-limiting examples of organ perfusion systems are also disclosed in Figures 1 and 2.

[0026] To improve organ survival rates, a therapeutically effective dose of NO x The gas is administered directly to the organs. Depending on one or more types of the administered composition, NO is delivered to the organs. xDirect administration of gas may occur via an organ perfusion system, a ventilator, or any combination thereof. In embodiments using an organ perfusion system and a ventilator in combination, the organ perfusion system and ventilator are used simultaneously to administer NO x A gas-containing composition may be administered directly to the organ. Alternatively, or in addition, an organ perfusion system and a ventilator may be used in succession to administer NO x The gas-containing composition can be administered directly to the organs, with varying amounts overlapping between two administration methods (e.g., no overlap, overlap of a few seconds, a few minutes, or a few hours). For example, it can be administered first by a ventilator and then by a perfusion system, or vice versa.

[0027] "NO x A "therapeutic dose of gas" is defined as a sufficient amount of NO to improve the survival rate of an organ as defined herein, when administered directly to an organ. x This indicates the amount of gas. NO is a component of the "therapeutic effective dose". x The amount of NO can be determined by those skilled in the art, although it varies depending on various factors. As will be discussed in more detail below, a therapeutically effective amount of NO for the treatment of organs with ischemia-reperfusion injury. x The gas concentration is less than 20 ppm. This is a therapeutically effective dose of NO. x The gas can be used alone or in load doses of NO x It can be used after gas administration. NO x The loading dose of the gas can be used to increase vasodilation in ischemic organs, and may be particularly suitable when the composition of this disclosure first comes into contact with the organ after it has been removed from the donor. However, NO provided in loading doses x The amount of gas typically used is NO, which can be safely used to treat ischemia-reperfusion injury for extended periods (e.g., more than one hour). x It exceeds the amount of gas.

[0028] In one or more embodiments, NO xThe gas is administered at an initial concentration and optionally increased as needed to achieve the desired effect (e.g., increased organ viability). For example, the initial nitric oxide concentration is approximately 0.05 ppm to approximately 50 ppm or approximately 1 ppm to approximately 50 ppm and may be optionally gradually increased until the desired effect is achieved or the nitric oxide threshold is met. An exemplary nitric oxide administration would begin with an initial concentration of approximately 1 ppm, and then increase to the desired NO x The NO concentration can be increased in increments of approximately 0.1 ppm to 5 ppm until an effect is achieved. x Ensure that the concentration does not exceed 50 ppm and / or that the methemoglobin level is less than or greater than approximately 5%. Alternatively, exemplary nitric oxide administration may begin at an initial concentration of 5 ppm, and then the desired NO x The concentration can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO x Ensure that the concentration does not exceed 50 ppm and / or that the methemoglobin level is less than or greater than approximately 5%. In another exemplary embodiment, nitric oxide administration is started at an initial concentration of 10 ppm, and then the desired NO x The concentration can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO x Ensure that the concentration does not exceed 50 ppm and / or that the methemoglobin level is less than or greater than approximately 5%. In yet another exemplary embodiment, administration of nitric oxide is started at an initial concentration of 15 ppm, and then the desired NO x The concentration can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO x Ensure that the concentration does not exceed 50 ppm and / or that the methemoglobin level is less than or greater than approximately 5%. In yet another exemplary embodiment, administration of nitric oxide is started at an initial concentration of 20 ppm, and then the desired NO x The concentration can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO xEnsure that the concentration does not exceed 50 ppm and / or the level of methemoglobin is less than or does not exceed about 5%. In each of the above embodiments, the administration can be for 5 minutes, 10 minutes, 15 minutes, 30 minutes or 60 minutes. Alternatively, the administration can be for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0029] In one or more embodiments, NO x gas is administered at an initial concentration and optionally increased as needed to obtain the desired effect (e.g., increased survival rate of an organ). For example, the initial nitric oxide concentration is from about 0.05 ppm to about 35 ppm or from about 1 ppm to about 35 ppm and can be incrementally increased as desired until the desired effect is obtained or the nitric oxide threshold is met. An exemplary nitric oxide administration starts at an initial concentration of about 1 ppm and then can be increased in increments of about 0.1 ppm to about 5 ppm until the desired NO x effect is obtained, provided that the NO x concentration does not exceed 35 ppm and / or the level of methemoglobin is less than or does not exceed about 5%. Alternatively, an exemplary nitric oxide administration starts at an initial concentration of 5 ppm and then can be increased in increments of 0.1 ppm to 5 ppm until the desired NO x effect is obtained, provided that the NO x concentration does not exceed 35 ppm and / or the level of methemoglobin is less than or does not exceed about 5%. In another exemplary embodiment, the nitric oxide administration starts at an initial concentration of 10 ppm and then can be increased in increments of 0.1 ppm to 5 ppm until the desired NO x effect is obtained, provided that the NO x concentration does not exceed 35 ppm and / or the level of methemoglobin is less than or does not exceed about 5%. In yet another exemplary embodiment, the nitric oxide administration starts at an initial concentration of 15 ppm and then can be increased in increments of 0.1 ppm to 5 ppm until the desired NO xIt can be increased in increments of 0.1 ppm to 5 ppm until the effect is obtained, but NO x Ensure that the concentration does not exceed 35 ppm and / or the level of methemoglobin is less than or does not exceed about 5%. In yet another exemplary embodiment, the administration of nitric oxide starts at an initial concentration of 20 ppm and then, until the desired NO x It can be increased in increments of 0.1 ppm to 5 ppm until the effect is obtained, but NO x Ensure that the concentration does not exceed 35 ppm and / or the level of methemoglobin is less than or does not exceed about 5%. In each of the above embodiments, the administration can be for 5 minutes, 10 minutes, 15 minutes, 30 minutes or 60 minutes. Alternatively, the administration can be for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0030] In one or more embodiments, NO x The gas is administered at an initial concentration and optionally increased as needed to obtain the desired effect (e.g., increased survival rate of an organ). For example, the initial nitric oxide concentration is from about 0.05 ppm to about 20 ppm or from about 1 ppm to about 20 ppm and can be incrementally increased as desired until the desired effect is obtained or the nitric oxide threshold is met. An exemplary administration of nitric oxide starts at an initial concentration of about 1 ppm and then, until the desired NO x It can be increased in increments of about 0.1 ppm to about 5 ppm until the effect is obtained, but NO x Ensure that the concentration does not exceed 20 ppm and / or the level of methemoglobin is less than or does not exceed about 5%. Alternatively, an exemplary administration of nitric oxide starts at an initial concentration of 5 ppm and then, until the desired NO x It can be increased in increments of 0.1 ppm to 5 ppm until the effect is obtained, but NO xEnsure that the concentration does not exceed 20 ppm and / or that the methemoglobin level is less than or greater than approximately 5%. In another exemplary embodiment, nitric oxide administration is started at an initial concentration of 10 ppm, and then the desired NO x The concentration can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO x Ensure that the concentration does not exceed 35 ppm and / or that the methemoglobin level is less than or greater than approximately 5%. In yet another exemplary embodiment, administration of nitric oxide is started at an initial concentration of 15 ppm, and then the desired NO x The concentration can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO x Ensure that the concentration does not exceed 20 ppm and / or that the methemoglobin level is less than or greater than approximately 5%. In yet another exemplary embodiment, administration of nitric oxide is started at an initial concentration of 20 ppm, and then the desired NO x The concentration can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO x Ensure that the concentration does not exceed 20 ppm and / or that the methemoglobin level is less than or greater than approximately 5%. In each of the above embodiments, the administration may be for 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 60 minutes. Alternatively, the administration may be for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0031] In one or more embodiments, NO x The gas is administered at an initial concentration for an initial period of time, and then at a lower second concentration for a second period of time to achieve the desired effect (e.g., increased organ viability). For example, NO xThe gas may be administered at an initial concentration of approximately 20 ppm to 40 ppm for up to approximately 1 hour, after which it may be reduced to below 20 ppm to improve organ survival. An exemplary nitric oxide administration may begin at an initial concentration of approximately 20 ppm to 40 ppm, and then the increment may be reduced over an initial period until the nitric oxide concentration reaches below 20 ppm. The rate of reduction may be constant or variable. The nitric oxide concentration may be further adjusted as needed, for example, based on monitoring of a nitric oxide marker. Alternatively, an exemplary nitric oxide administration may begin at an initial concentration of approximately 20 ppm to 40 ppm, maintain a constant concentration over an initial period, and then reduce to below 20 ppm. The nitric oxide concentration may be further adjusted as needed, for example, based on monitoring of a nitric oxide marker. In each of the above embodiments, the total time for nitric oxide administration (the sum of the time at the initial concentration and the time for the concentration to decrease) may be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0032] In each of the embodiments described above, the initial nitric oxide concentration, the increase or decrease in the nitric oxide increment, and the maximum nitric oxide concentration and / or threshold of the nitric oxide or nitric oxide marker may vary depending on the application and / or the specific organ being treated. The increment may vary throughout the adjustment of the nitric oxide supply. If monitoring indicates that the nitric oxide or nitric oxide marker is meeting or exceeding the nitric oxide threshold, the nitric oxide may also be gradually reduced.

[0033] In each of the above embodiments, NO x The concentration is the last NO x It is also possible to incrementally adjust the concentration by a specific percentage. Such incremental percentages are NO xThis may include changes in concentration of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 125%, 150%, 175%, and 200%.

[0034] NO x Instead of adjusting the concentration, or in addition to that, NO x NO is supplied directly to organs, such as by regulating the flow rate of gas. x NO x The gas dosage can be adjusted.

[0035] In further embodiments, nitric oxide administration is adjusted based on monitoring of nitric oxide or nitric oxide markers. As used herein, “nitric oxide marker” indicates a direct or indirect indicator of the nitric oxide concentration in a fluid. For example, nitric oxide markers include, among others, methemoglobin and NO x (That is, NO, nitrite ion (NO2) - ), nitrate ion (NO3 - ) etc.) are included. Such adjustments are done manually or NO x This may be performed automatically by the supply device. The NO supply device may also issue alarms based on monitoring. If the monitoring device is a separate component from the NO supply device, the monitoring device may transmit monitoring information to the NO supply device via any suitable wired or wireless connection. For example, if the nitric oxide or nitric oxide marker in the fluid falls below a certain threshold, the NO supply may be increased until the nitric oxide or nitric oxide marker in the fluid meets the threshold. Similarly, if the nitric oxide or nitric oxide marker in the fluid exceeds a certain threshold, the amount of NO administered may be reduced. The nitric oxide or nitric oxide marker can be monitored continuously or intermittently, for example, at regular intervals.

[0036] In one or more embodiments, such monitoring may include monitoring methemoglobin and / or NO. These nitric oxide markers can be measured directly by techniques such as pulse oximetry or optical measurements, or by any other means that directly or indirectly measure or correlate NO or the NO marker. For example, in another measurement technique, a probe is placed in the perfusion fluid to measure the NO in the fluid. x It can measure levels and provide real-time analysis of the perfusion fluid.

[0037] In one or more embodiments, nitric oxide or a nitric oxide marker is monitored by comparing a measured value of nitric oxide or a nitric oxide marker with a nitric oxide threshold. The nitric oxide threshold may be a safety limit to ensure that methemoglobinemia does not occur. For example, the nitric oxide threshold may be a methemoglobin level, such as the percentage of methemoglobin relative to red blood cells. In exemplary embodiments, the nitric oxide threshold is in the range of about 1% to about 15% methemoglobin, or about 3% to about 10% methemoglobin. Therefore, if the methemoglobin level meets or exceeds an acceptable range such as ≤3%, ≤4%, ≤5%, ≤6%, ≤7%, ≤8%, ≤9%, ≤10%, ≤11%, or ≤12%, the nitric oxide dose can be adjusted.

[0038] The level of NO2 in the perfusion fluid can also be monitored. Recirculation of the fluid may cause NO2 to accumulate in the fluid. If the NO2 concentration rises and exceeds a certain threshold, the NO supply device can adjust the NO dosage and / or issue an alarm. NO2 can also be removed using reducing agents, scrubbers, bases, or other appropriate means.

[0039] (b) Ischemia-reperfusion injury The term "ischemia-reperfusion injury" refers to injury resulting from ischemia, reperfusion, or both. Ischemia refers to insufficient blood supply to an organ, and ischemic injury occurs when blood supply to a tissue or area of ​​an organ is cut off. The act of restoring blood flow to an organ or tissue is called reperfusion, and reperfusion injury occurs as a result of restoring blood flow to a tissue or organ after ischemia. Ischemia can be the result of injury or disease suffered by an organism. Examples of certain diseases that can induce ischemia or hypoxia include, but are not limited to, trauma or surgery, respiratory or cardiac arrest, tumors, heart disease, and neurological disorders. Examples of certain injuries that can cause an ischemic or hypoxic state include, but are not limited to, trauma such as burns, cuts, amputations, gunshot wounds, or surgical trauma. Furthermore, injuries can also include internal injuries such as stroke or heart attack, which cause a sudden decrease in circulation. Other injuries include reduced circulation due to non-invasive stress such as exposure to cold or radiation, or planned reductions in circulation such as during cardiac surgery or organ donor procedures prior to the extraction of organs for transport, and subsequent transplantation to recipients.

[0040] One aspect of this disclosure comprises a method for treating ischemia-reperfusion injury in an organ requiring such treatment. This method involves NO levels below 20 ppm. x The method involves administering a gas-containing composition directly and continuously to an organ. In some embodiments, the composition contains 1 ppm to 20 ppm of nitric oxide. In other embodiments, the composition contains about 1 ppm to about 10 ppm of nitric oxide, about 5 ppm to about 15 ppm of nitric oxide, or about 10 ppm to 20 ppm. In other embodiments, the composition contains about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 11 ppm, about 12 ppm, about 13 ppm, about 14 ppm, about 15 ppm, about 16 ppm, about 17 ppm, about 18 ppm, about 19 ppm, or about 20 ppm of nitric oxide. xSuitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, and more preferably a cell-free perfusion solution. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. In various embodiments, the organ requiring treatment is an organ damaged by external injury, surgery, respiratory arrest, or cardiac arrest. In specific embodiments, the organ requiring treatment is an organ intended for transplantation. In exemplary embodiments, the organ requiring treatment is an organ extracted from a donor and intended for transplantation.

[0041] The administration may be 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 60 minutes. Alternatively, the administration may be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer. In embodiments where an organ is the target of transplantation, NO x Gas administration is preferably not to exceed 12 hours in total. In certain embodiments, administration begins simultaneously with ischemia. In other embodiments, administration begins some time after ischemia has started, but preferentially as close as possible to the onset of ischemia. For example, administration may begin about 5, 10, 15, 20, 25, or 30 minutes after the onset of ischemia. Administration may also begin during reperfusion, or alternatively, continue after reperfusion has started. In some cases, administration may continue for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours, or longer than 12 hours after reperfusion has started.

[0042] Effective treatment of ischemia-reperfusion injury can be evaluated by any method known in the art, including but not limited to measuring cellular function (e.g., metabolic capacity, ATP content, etc.), measuring cellular damage (e.g., histological evaluation, protein oxidation, morphological changes, etc.), measuring inflammation, and / or measuring organ function.

[0043] In a further embodiment, a method for treating ischemia-reperfusion injury in an organ requiring it involves approximately 20 ppm to 40 ppm of NO x A composition containing gas ("loading dose") has NO content of 20 ppm or less. x The procedure may include an additional step in which the gas-containing composition is administered for up to approximately one hour immediately prior to administration ("loading period"). For example, the loading dose may be administered over approximately 10 minutes, 15 minutes, 20 minutes, 30 minutes, or between approximately 10 minutes and 30 minutes. In another example, the loading dose may be administered over approximately 30 minutes, 35 minutes, 40 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, or between 30 minutes and 60 minutes. The two compositions may be the same except for the nitric oxide concentration. Alternatively, the two compositions may be different. The nitric oxide concentration in the loading dose may be gradually reduced over the loading period until the nitric oxide concentration is 20 ppm or less. The rate of reduction may be constant or not. Alternatively, the nitric oxide concentration in the loading dose may be kept constant over the loading period and then reduced to a nitric oxide concentration of 20 ppm or less. In preferred embodiments, the two compositions are identical, and the composition is a perfusion solution, preferably a cell-free perfusion solution. In exemplary embodiments, the organ is a heart, lung, or kidney, and the organ is intended for transplantation.

[0044] Another aspect of this disclosure comprises a method for improving the survival rate of organs damaged by ischemia-reperfusion. This method involves NO levels below 20 ppm. x The method involves administering a gas-containing composition directly and continuously to an organ. In some embodiments, the composition contains 1 ppm to 20 ppm of nitric oxide. In other embodiments, the composition contains about 1 ppm to about 10 ppm of nitric oxide, about 5 ppm to about 15 ppm of nitric oxide, or about 10 ppm to 20 ppm. In other embodiments, the composition contains about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 11 ppm, about 12 ppm, about 13 ppm, about 14 ppm, about 15 ppm, about 16 ppm, about 17 ppm, about 18 ppm, about 19 ppm, or about 20 ppm of nitric oxide. xSuitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, and more preferably a cell-free perfusion solution. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. Improving the viability of organs damaged by ischemia and / or reperfusion may include maintaining mitochondrial function or reducing oxidative damage. Other means known in the art for assessing organ viability may also be used, which include, but are not limited to, measuring cellular function (e.g., metabolic capacity, ATP content, etc.), measuring cellular damage (e.g., histological evaluation, morphological changes, etc.), measuring inflammation, and / or measuring organ function.

[0045] In one embodiment, the disclosure encompasses a method for maintaining mitochondrial function in an organ with ischemia-reperfusion injury. As used herein, mitochondrial function may be measured by the respiratory control ratio (RCR), an indicator of mitochondrial binding state. Generally speaking, the RCR represents the ratio of the oxidation rate (state 3) in the presence of excess substrate and adenosine diphosphate to the oxidation rate after ADP has been phosphorylated to a steady-state concentration (state 4). In some embodiments, NO is present at 20 ppm or less. x Direct administration of a gas-containing composition to an organ significantly maintains mitochondrial function in organs with ischemia-reperfusion injury. As used herein, "significantly maintained" means as described herein. x This refers to a difference of less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% in mitochondrial function between the gas-treated organ and the control organ that did not undergo ischemia-reperfusion. In other words, significantly maintained mitochondrial function means that the organ underwent similar ischemia-reperfusion injury but did not undergo direct and continuous NO x This may indicate improved mitochondrial function compared to similar organs that were not administered the gas. xSuitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0046] In other embodiments, the disclosure includes methods for reducing oxidative damage to organs with ischemia-reperfusion injury. Generally speaking, these methods involve reducing NO levels below 20 ppm. x This involves administering a gas-containing composition directly and continuously to an organ. Where used herein, “reduction of oxidative damage” or “reduction of oxidative damage” may be measured compared to an organ treated under similar conditions, but it is directly and continuously NO x No gas is administered. For example, oxidative damage may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to organs treated under similar conditions, but this is directly and sequentially due to NO x No gas is administered. In certain embodiments, mitochondrial reactive oxygen species (mtROS) are directly and continuously reduced compared to untreated controls. x NO decreases within the organs of ischemia-reperfusion injury after administration. x Suitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0047] In some embodiments, the present disclosure includes methods for increasing the activity of superoxide dismutase 2 (SOD2 or manganese-dependent superoxide dismutase (MnSOD)) in organs with ischemia-reperfusion injury. These methods involve NO levels below 20 ppm. x The present invention involves administering a gas-containing composition directly and sequentially to an organ, thereby increasing the activity of MnSOD in the organ compared to a control organ that has not been in contact with the composition of the present invention. For example, the activity of MnSOD may increase by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to an organ treated under similar conditions, but directly and sequentially. x No gas is administered. Methods for measuring MnSOD activity are well known in the art. NO x Suitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0048] In other embodiments, the disclosure includes a method for inhibiting nitrotyrosine formation in organs with ischemia-reperfusion injury. This method involves NO levels below 20 ppm. x The present invention involves directly and continuously administering a gas-containing composition to an organ, thereby inhibiting the formation of nitrotyrosine adducts in the organ compared to a control organ that has not been in contact with the composition of the present invention. For example, nitrotyrosine formation may be inhibited by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to an organ treated under similar conditions, but directly and continuously. x No gas is administered. Methods for measuring the formation of nitrotyrosine adducts are known in the art. NO xSuitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0049] In certain embodiments, the disclosure includes a method for preventing the inactivation of mitochondrial complex I activity, complex II activity, complex III activity, complex IV activity, or combination thereof in organs with ischemia-reperfusion injury. This method involves NO content of 20 ppm or less. x The method involves continuously administering a gas-containing composition directly to an organ, with NO content of 20 ppm or less. x Administering the gas prevents inactivation of the activity of mitochondrial complexes I, II, III, IV, or combinations thereof, compared to a control organ. In one embodiment, NO is administered at concentrations of 20 ppm or less. x By directly and continuously administering the gas-containing composition to the organs, inactivation of mitochondrial complex I activity is prevented. In another embodiment, NO is 20 ppm or less. x By directly and continuously administering the gas-containing composition to the organs, inactivation of mitochondrial complex II activity is prevented. In yet another embodiment, NO is 20 ppm or less. x By directly and continuously administering the gas-containing composition to the organs, inactivation of mitochondrial complex III activity is prevented. In another embodiment, NO is 20 ppm or less. x By directly and continuously administering the gas-containing composition to the organs, inactivation of mitochondrial complex IV activity is prevented. In preferred embodiments, NO is 20 ppm or less. x By directly and continuously administering the gas-containing composition to the organs, inactivation of mitochondrial complex I and mitochondrial complex II activity is prevented. In another preferred embodiment, NO is 20 ppm or less. xBy directly and continuously administering a gas-containing composition to an organ, inactivation of mitochondrial complex II and mitochondrial complex III activity is prevented. For example, the inactivation of activity in each of the above embodiments may be inhibited by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to organs treated under similar conditions, but it is directly and continuously NO x No gas is administered. Methods for measuring mitochondrial complex I activity, complex II activity, complex III activity, or complex IV activity are known in the art. NO x Suitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0050] In each of the above embodiments, NO is 20 ppm or less. x The administration of the gas-containing composition may be for the duration necessary to improve organ viability. For example, in some embodiments, the administration may be 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 60 minutes. In other embodiments, the administration may be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer. In embodiments where the organ is intended for transplantation, NO xGas administration is preferably not to exceed 12 hours in total. In certain embodiments, administration begins simultaneously with ischemia. In other embodiments, administration begins some time after ischemia has started, but preferentially as close to the onset of ischemia as possible. In some embodiments, administration begins approximately 5, 10, 15, 20, 25, or 30 minutes after the onset of ischemia. Administration may also begin during reperfusion, or alternatively, continue after reperfusion has started. In some cases, administration may continue for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours, or longer than 12 hours after reperfusion has started.

[0051] In a further embodiment, the above method involves using approximately 20 ppm to approximately 40 ppm of NO. x A composition containing gas ("loading dose") has NO content of 20 ppm or less. x The procedure may include an additional step administered up to approximately one hour immediately prior to administering the gas-containing composition. For example, the loading dose may be administered over approximately 10 minutes, 15 minutes, 20 minutes, 30 minutes, or between approximately 10 and 30 minutes. In another example, the loading dose may be administered over approximately 30 minutes, 35 minutes, 40 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, or between 30 and 60 minutes. The two compositions may be the same except for the nitric oxide concentration. Alternatively, the two compositions may be different. The nitric oxide concentration in the loading dose may be gradually reduced over a period of up to approximately one hour until the nitric oxide concentration is 20 ppm or less. The rate of reduction may be constant or not. Alternatively, the nitric oxide concentration in the loading dose may be kept constant over a period of up to approximately one hour, and then reduced to a nitric oxide concentration of 20 ppm or less. In preferred embodiments, the two compositions are identical, and the composition is a perfusion solution, preferably a cell-free perfusion solution. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. In exemplary embodiments, the organ is the heart, lung, or kidney.

[0052] (c) Methods to improve the survival rate of organs intended for transplantation Organs with ischemia-reperfusion injury include organs intended for transplantation. Therefore, this disclosure includes methods for improving the survival rate of organs intended for transplantation. Such methods are NO x This involves administering a gas-containing composition directly to an organ via an organ perfusion system or ventilation for a continuous period of up to 12 hours. In other words, from the time the organ is procured until it is transplanted to the recipient, the organ is NO x There is no direct contact with the gas and no interruption. As used herein, procurement refers to both the identification of an organ donor and the extraction of the organ, and can be used interchangeably with either term. In some embodiments, the composition is administered after the organ has been taken from the donor. In other embodiments, the composition is administered while the organ is still in the donor's body. In these embodiments, the donor may be a brain-dead donor or a cardiac-arrest donor. In some examples, administration may be 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 60 minutes. In other examples, administration may be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer. NO xSuitable compositions containing gas are described in Section (a). In preferred embodiments, the composition is a perfusion solution, and more preferably a cell-free perfusion solution. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. While we do not wish to be bound by theory, it is thought that the methods of the present disclosure can increase the number of organs available for transplantation by improving the viability of organs that previously did not meet the criteria for transplantation, thereby allowing the use of more organs obtained from more donors (e.g., brain-dead related donors, cardiac arrest donors, etc.). Improving the viability of organs intended for transplantation may, in part, include maintaining mitochondrial function or reducing oxidative damage to the organ. Other means known in the art for assessing organ viability may also be used, which include, but are not limited to, measuring cellular function (e.g., metabolic capacity, ATP content, etc.), measuring cellular damage (e.g., histological evaluation, morphological changes, etc.), measuring inflammation, and / or measuring organ function. In exemplary embodiments, the organ is the heart, lung, or kidney.

[0053] In one embodiment, this disclosure encompasses a method for maintaining mitochondrial function in an organ intended for transplantation. As used herein, mitochondrial function may be measured by the respiratory control ratio (RCR), an indicator of mitochondrial binding state. Generally speaking, the RCR represents the ratio of the oxidation rate (state 3) in the presence of excess substrate and adenosine diphosphate to the oxidation rate after ADP has been phosphorylated to a steady-state concentration (state 4). In some embodiments, NO is present at concentrations of 20 ppm or less. x By directly administering a gas-containing composition to an organ, mitochondrial function is significantly maintained in the organ intended for transplantation. As used herein, "significantly maintained" means as described herein. x Organs treated with gas, NO xThis indicates a difference of less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% in mitochondrial function compared to a control organ not treated with gas. In other words, significantly maintained mitochondrial function means that mitochondrial function has undergone similar ischemia-reperfusion injury but has not been directly and continuously maintained. x This may indicate improved mitochondrial function compared to similar organs intended for transplantation that have not been administered gas. x Suitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0054] In certain embodiments, the disclosure includes methods for reducing oxidative damage to organs intended for transplantation. For example, mitochondrial reactive oxygen species (mtROS) can be reduced within organs intended for transplantation. Such methods include reducing NO to 20 ppm or less. x The method involves administering a gas-containing composition directly and continuously to an organ. Direct and continuous administration may occur before the organ is removed from the donor, during transport / storage, during transplantation to the recipient, after transplantation to the recipient, or any combination thereof. In a preferred embodiment, the organ is NO from the time of organ removal until transplantation to the recipient. x It does not come into direct contact with the gas and is not interrupted. When used herein, “reduction of oxidative damage” or “reduction of oxidative damage” may be measured compared to organs treated under similar conditions, but it is not directly and continuously NO x No gas is administered. For example, oxidative damage may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to organs treated under similar conditions, but this is directly and sequentially due to NO xNo gas is administered. In certain embodiments, mitochondrial reactive oxygen species (mtROS) are directly and continuously reduced compared to a non-treated control. x NO decreases within the organs of ischemia-reperfusion injury after administration. x Suitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0055] In some embodiments, the present disclosure includes methods for increasing the activity of superoxide dismutase 2 (SOD2 or manganese-dependent superoxide dismutase (MnSOD)) in organs intended for transplantation. This method involves NO levels below 20 ppm. x The present invention involves administering a gas-containing composition directly and sequentially to an organ, thereby increasing the activity of MnSOD in the organ compared to a control organ that has not been in contact with the composition of the present invention. For example, the activity of MnSOD may increase by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to an organ treated under similar conditions, but directly and sequentially. x The gas is not administered. Direct, continuous administration can occur before the organ is removed from the donor, during transport / storage, during transplantation to the recipient, after transplantation to the recipient, or any combination thereof. In a preferred embodiment, the organ is NO from the time of organ removal until transplantation to the recipient. x It comes into direct contact with the gas and is not interrupted. Methods for measuring MnSOD activity are known in the art. NO xSuitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0056] In other embodiments, the disclosure includes a method for inhibiting nitrotyrosine formation in an organ intended for transplantation. This method involves NO below 20 ppm. x The present invention involves administering a gas-containing composition directly and sequentially to an organ, thereby inhibiting the formation of nitrotyrosine adducts in the organ compared to a control organ that has not been in contact with the composition of the present invention. For example, the formation of nitrotyrosine may be inhibited by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to an organ treated under similar conditions, but directly and sequentially. x The gas is not administered. Direct, continuous administration can occur before the organ is removed from the donor, during transport / storage, during transplantation to the recipient, after transplantation to the recipient, or any combination thereof. In a preferred embodiment, the organ is NO from the time of organ removal until transplantation to the recipient. x It is in direct contact with the gas and will not be interrupted. Methods for measuring the formation of nitrotyrosine adducts are known in the art. NO x Suitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0057] In certain embodiments, the disclosure includes a method for preventing the inactivation of mitochondrial complex I activity, complex II activity, complex III activity, complex IV activity, or combination thereof in an organ intended for transplantation. This method involves NO content of 20 ppm or less. x The method involves continuously administering a gas-containing composition directly to an organ, with NO content of 20 ppm or less. x Administering the gas prevents inactivation of the activity of mitochondrial complexes I, II, III, IV, or combinations thereof, compared to a control organ. In one embodiment, NO is administered at concentrations of 20 ppm or less. x By directly and continuously administering the gas-containing composition to the organs, inactivation of mitochondrial complex I activity is prevented. In another embodiment, NO is 20 ppm or less. x By directly and continuously administering a gas-containing composition to organs, inactivation of mitochondrial complex II activity is prevented. In yet another embodiment, NO content of 20 ppm or less is used. x By directly and continuously administering the gas-containing composition to the organs, inactivation of mitochondrial complex III activity is prevented. In another embodiment, NO is 20 ppm or less. x By directly and continuously administering the gas-containing composition to the organs, inactivation of mitochondrial complex IV activity is prevented. In preferred embodiments, NO is 20 ppm or less. x By directly and continuously administering the gas-containing composition to the organs, inactivation of mitochondrial complex I and mitochondrial complex II activity is prevented. In another preferred embodiment, NO is 20 ppm or less. x By directly and continuously administering a gas-containing composition to an organ, inactivation of mitochondrial complex II and mitochondrial complex III activity is prevented. For example, the inactivation of activity in each of the above embodiments may be inhibited by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to organs treated under similar conditions, but it is directly and continuously NO xThe gas is not administered. Direct, continuous administration can occur before the organ is removed from the donor, during transport / storage, during transplantation to the recipient, after transplantation to the recipient, or any combination thereof. In a preferred embodiment, the organ is NO from the time of organ removal until transplantation to the recipient. x Direct contact with the gas is not interrupted. Methods for measuring mitochondrial complex I activity, complex II activity, complex III activity, or complex IV activity are known in the art. NO x Suitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0058] In each of the above methods, NO is supplied to the organs via the organ perfusion system. x Direct administration of gas-containing compositions may occur over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours via an organ perfusion system, a ventilator, or any combination thereof. In embodiments using an organ perfusion system and a ventilator in combination, NO may be administered simultaneously using the organ perfusion system and ventilator. x A gas-containing composition may be administered directly to the organ. Alternatively, or in addition, an organ perfusion system and a ventilator may be used in succession to administer NO x The gas-containing composition can be administered directly to the organs, with varying amounts overlapping between two administration methods (e.g., no overlap, overlap of a few seconds, a few minutes, or a few hours). For example, it can be administered first by a ventilator and then by a perfusion system, or vice versa.

[0059] In a further embodiment, the above method involves using approximately 20 ppm to approximately 40 ppm of NO. x A composition containing gas ("loading dose") has NO content of 20 ppm or less. xThe gas-containing composition is administered up to approximately one hour immediately before administration, NO x Gas administration may include additional steps, not exceeding a total of 12 hours. For example, the loading dose can be administered over approximately 10 minutes, 15 minutes, 20 minutes, 30 minutes, or between 10 and 30 minutes, in which case the NO concentration is less than 20 ppm. x The gas-containing composition is administered over a period of approximately 11.8 hours or less. In another example, the loading dose may be administered over approximately 30 minutes, 35 minutes, 40 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, or between 30 and 60 minutes, in which case the NO concentration is 20 ppm or less. x The gas-containing composition is administered for 11.5 hours or less. The two compositions may be the same except for the nitric oxide concentration, or they may be different. The nitric oxide concentration in the loading dose can be gradually reduced over a period of up to about 1 hour until the nitric oxide concentration is 20 ppm or less. The rate of reduction may be constant or not. Alternatively, the nitric oxide concentration in the loading dose may be kept constant over a period of up to about 1 hour, and then reduced to a nitric oxide concentration of 20 ppm or less. In a preferred embodiment, the two compositions are the same, and the composition is a perfusion solution, preferably a cell-free perfusion solution. In a further embodiment, the cell-free perfusion solution is Steen Solution®, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. In an exemplary embodiment, the organ is the heart, lung, or kidney.

[0060] (d) Methods for improving the post-transplant function of organs intended for transplantation. Another aspect of this disclosure comprises a method for improving the post-transplant capacity of an organ intended for transplantation. This method involves NO 20 ppm or less. x The method involves administering a gas-containing composition directly to an organ via an organ perfusion system for up to 12 hours continuously. In some examples, the administration may be for 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 60 minutes. In other examples, the administration may be for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours. NO xSuitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, and more preferably a cell-free perfusion fluid. Improvement of the post-transplant capacity of an organ intended for transplantation may include maintaining mitochondrial function or reducing oxidative damage. Other means known in the art for assessing organ viability may also be used, including but not limited to measuring cellular function (e.g., metabolic capacity, ATP content, etc.), measuring cellular damage (e.g., histological evaluation, morphological changes, etc.), measuring inflammation, and / or measuring organ function. In exemplary embodiments, the organ is the heart, lung, or kidney.

[0061] In one embodiment, this disclosure encompasses a method for maintaining mitochondrial function in a transplanted organ. As used herein, mitochondrial function may be measured by the respiratory control ratio (RCR), an indicator of mitochondrial binding state. Generally speaking, the RCR represents the ratio of the oxidation rate (state 3) in the presence of excess substrate and adenosine diphosphate to the oxidation rate after ADP has been phosphorylated to a steady-state concentration (state 4). In some embodiments, NO is present at concentrations of 20 ppm or less. x By directly administering a gas-containing composition to an organ, mitochondrial function is significantly maintained in the transplanted organ. As used herein, "significantly maintained" means as described herein. x Organs treated with gas, NO x This indicates a difference of less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% in mitochondrial function compared to a control organ not directly and continuously treated with gas. In other words, significantly maintained mitochondrial function is defined as having undergone similar ischemia-reperfusion injury but not directly and continuously treated with NO. x This may indicate improved mitochondrial function compared to similar organs that were not administered the gas. xSuitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0062] In other embodiments, the disclosure includes methods for reducing oxidative damage to transplanted organs. Generally speaking, these methods involve reducing NO to 20 ppm or less. x This involves administering a gas-containing composition directly and continuously to an organ. Where used herein, “reduction of oxidative damage” or “reduction of oxidative damage” may be measured compared to an organ treated under similar conditions, but it is directly and continuously NO x No gas is administered. For example, oxidative damage may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to organs treated under similar conditions, but this is directly and sequentially due to NO x No gas is administered. In certain embodiments, mitochondrial reactive oxygen species (mtROS) are directly and continuously reduced compared to untreated controls. x NO decreases in the transplanted organ after administration. x Suitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0063] In a further embodiment, the above method involves using approximately 20 ppm to approximately 40 ppm of NO. x A composition containing gas ("loading dose") has NO content of 20 ppm or less. x The gas-containing composition is administered up to approximately one hour immediately before administration, NOx Gas administration may include additional steps, not exceeding a total of 12 hours. For example, the loading dose can be administered over approximately 10 minutes, 15 minutes, 20 minutes, 30 minutes, or between 10 and 30 minutes, in which case the NO concentration is less than 20 ppm. x The gas-containing composition is administered over a period of approximately 11.8 hours or less. In another example, the loading dose may be administered over approximately 30 minutes, 35 minutes, 40 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, or between 30 and 60 minutes, in which case the NO concentration is 20 ppm or less. x The gas-containing composition is administered for 11.5 hours or less. The two compositions may be the same except for the nitric oxide concentration, or they may be different. The nitric oxide concentration in the loading dose can be gradually reduced over a period of up to about 1 hour until the nitric oxide concentration is 20 ppm or less. The rate of reduction may be constant or not. Alternatively, the nitric oxide concentration in the loading dose may be kept constant over a period of up to about 1 hour, and then reduced to a nitric oxide concentration of 20 ppm or less. In preferred embodiments, the two compositions are the same, and the composition is a perfusion solution, preferably a cell-free perfusion solution. In exemplary embodiments, the organ is the heart, lung, or kidney. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0064] (e) Transplant method In another embodiment, the present disclosure provides a transplantation method, which involves (a) NO 20 ppm or less. xThe method comprises (b) administering a gas-containing composition directly to an organ intended for transplantation for up to 12 consecutive hours, and (b) transplanting the organ into the recipient. The administration may be for 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 60 minutes. Alternatively, the administration may be for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours. In some embodiments, the composition contains 1 ppm to 20 ppm of nitric oxide. In other embodiments, the composition contains about 1 to about 10 ppm of nitric oxide, about 5 ppm to about 15 ppm of nitric oxide, or about 10 ppm to 20 ppm. In other embodiments, the composition contains about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 11 ppm, about 12 ppm, about 13 ppm, about 14 ppm, about 15 ppm, about 16 ppm, about 17 ppm, about 18 ppm, about 19 ppm, or about 20 ppm of nitric oxide. x Suitable compositions containing gas are described in section (a). In preferred embodiments, the composition is a perfusion solution, and more preferably a cell-free perfusion solution. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. In exemplary embodiments, the organ is the heart, lung, or kidney.

[0065] In certain embodiments, the organ intended for transplantation is removed from the donor before step (a) above. In these embodiments, administration begins some time after the onset of ischemia, but preferentially as close as possible to the onset of ischemia. For example, administration may begin approximately 5, 10, 15, 20, 25, or 30 minutes after the onset of ischemia. The timing of administration may or may not correspond to the onset of reperfusion.

[0066] In a further embodiment, this method uses NO at a concentration of approximately 20 ppm to 40 ppm. x A composition containing gas ("loading dose") has NO content of 20 ppm or less. xThe procedure may include an additional step in which the gas-containing composition is administered for up to approximately one hour immediately prior to administration ("loading period"). For example, the loading dose may be administered over approximately 10 minutes, 15 minutes, 20 minutes, 30 minutes, or between approximately 10 minutes and 30 minutes. In another example, the loading dose may be administered over approximately 30 minutes, 35 minutes, 40 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, or between 30 minutes and 60 minutes. The two compositions may be the same except for the nitric oxide concentration. Alternatively, the two compositions may be different. The nitric oxide concentration in the loading dose may be gradually reduced over the loading period until the nitric oxide concentration is 20 ppm or less. The rate of reduction may be constant or not. Alternatively, the nitric oxide concentration in the loading dose may be kept constant over the loading period and then reduced to a nitric oxide concentration of 20 ppm or less. In preferred embodiments, the two compositions are identical, and the composition is a perfusion solution, preferably a cell-free perfusion solution. In further embodiments, the cell-free perfusion solution is Steen Solution®, which optionally comprises sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. In exemplary embodiments, the organ is the heart, lung, or kidney.

[0067] Examples The following examples illustrate various iterations of the present invention.

[0068] Example 1: Lung transplant protocol This study includes a total of 20 lungs (8 lungs with gNO and perfusion, 8 lungs with perfusion only, and 4 lungs with gNO ventilation and perfusion) according to the modified standard lung donor selection criteria. The three-group study includes gNO added to perfusion, perfusion only, and gNO ventilation and perfusion (pilot study). The XVivo perfusion device system with cell-free perfusion (Steen solution) is used. The maximum cryo-ischemic time for lungs is 8–10 hours. The duration of Ex-Vivo lung perfusion is up to 12 hours. Lung health is assessed by evaluation systems, biomarker evaluation, and histopathological evaluation.

[0069] Evaluation system The evaluation system includes a 0-10 rating (overall score) using a composite scale of three variables. The three variables are: 1) Delta PaO2 weighted as 0-4 using four categories: 0 = <350 mmHg; 1 = ≥350-<400 mmHg; 2 = ≥400-<450 mmHg; 3 = ≥450-<500 mmHg; 4 = ≥500 mmHg; 2) Static lung compliance weighted as 0-4 (change from baseline): 0 = no improvement or deterioration in compliance; 1 = 1-3% improvement; 2 = 4-7% improvement; 3 = 8-11% improvement; and 4 = 12-15% improvement in compliance; and 3) Pulmonary vascular resistance (PVR) weighted as 0-2: 0 = no change or increase in PVR; 1 = 1-7% decrease in PVR; 2 = 8-15% decrease in PVR.

[0070] Biomarker evaluation The biomarkers evaluated are damage assessment molecular protein (DAMP), high-mobility group box-1 (HMGB1), S100A8 (MRP8, cargranulin A), S100A9 (MRP14, cargranulin B), and serum amyloid A (SAA). The target cytokines are TNFα-1, IL1-β, IL-6, NLRP3, IL-10, and donor cell-free DNA.

[0071] Histopathological evaluation The histopathological parameters evaluated are interstitial and alveolar edema, hyaline membrane formation, and evidence of vascular integrity / damage (CD31 staining).

[0072] Procurement of donor lungs Current clinical practice after organ retrieval from a donor involves static, low-temperature preservation until transplantation to the recipient. During retrieval, the lungs undergo a low-temperature lung flush using a low-potassium dextran preservation solution, combined with local cooling and lung ventilation. Subsequently, the lungs are transported at 4°C in a statically inflated state. Hypothermia, in the face of ischemia, reduces metabolic activity while maintaining cell viability, essentially slowing down the process of cell death (5% of the metabolic rate at 37°C). Therefore, low-temperature preservation is the mainstay of lung preservation in previous technologies. However, a significant reduction in the metabolic function of the organ remains, thus hindering meaningful lung evaluation and recovery.

[0073] The donor lung procurement technique for this trial is as follows: Bronchoscopy and median sternotomy are performed. The pericardium and multiple cavities are opened. Both lungs are replaced and PO2 is assessed with a 100% FiO2 challenge. The whole body is heparinized. A purse-string suture is made in the major pulmonary aorta (PA). A cannula is inserted into the PA through the purse-string. The cannula is degassed and connected to a deflated Perfadex tube (the Perfadex bag should be ejected only by gravity (unpressurized), and the bag should not be more than 1 meter above the lung). 500 mcg of alprostidil is administered directly to the major PA. The superior vena cava (SVC), the opened left and right atria, and the cross-clamp aorta are ligated.

[0074] Administer 4 liters of antegrade Perfadex. Ensure rapid and thorough drainage of fluid from both the right and left atria. Place ice locally in both lungs (but use only one lung per cycle). Maintain ventilation at room air FiO2 with a tidal volume of 4-6 ml / kg / min and a respiratory rate of 10 breaths per minute. Once infusion is complete, resection the heart. If a heart has been transplanted, incise the PA at the PA bifurcation. Do not divide the PA bifurcation. If a heart has not been transplanted, incise the PA at the RVOT just outside the pulmonary valve. Ensure the left atrial cuff remains intact. Infuse cryo-Perfadex (2 liters total) into the retrograde pulmonary veins. Incise the pericardium bilaterally at the level of the diaphragm and completely separate the pericardium from the diaphragm by crossing it. Incise the inferior pulmonary ligaments bilaterally. Dissect the posterior pericardium from the cranial posterior medialis to the tracheal bifurcation. Dissect the cranial side of the trachea down to the level of the cricoid cartilage. Pull the endotracheal tube up to the level of the larynx. Inflate the lung to 50% of its total lung volume at 50% FiO2. Divide the trachea above the cricoid cartilage with two staple loads. Dissect the posterior trachea from the tail of the posterior mediastinum (esophagus) toward the diaphragm. Dissect the left pulmonary artery (PA) from the aortic arch and divide the ligamentum arteriosus. Remove the lung and preserve it in ice-cold Perfadex solution. If a heart has been procured for transplantation, resect 10 cm of the donor's descending aorta and place it in the same cold Perfadex solution bag as the lung. This will later be used for pulmonary artery reconstruction.

[0075] Ex-Vivo Lung Perfusion (EVLP) The main principle of extracorporeal lung (EVLP) is to perfuse and ventilate the donor organ outside the body in a sealed container that maintains temperature, moisture, and sterility. The EVLP circuit consists of a centrifugal pump that circulates the perfusion fluid, passing through a membrane gas exchanger and a leukocyte-removing filter before entering the pulmonary artery (PA). This system is very similar to those used in extracorporeal circuits (ECCs) during cardiac surgery. Acellular Steen solution is used as the perfusion fluid in the EVLP process.

[0076] In short, the EVLP process is as follows: The lungs are placed in a specially covered plastic chamber to keep them in a stable position during ventilation and to provide a warm, humid environment. Perfusion fluid (propelled by a centrifugal pump) enters the lungs through a cannula placed in the pulmonary artery (PA). The return flow from the lungs is passive, by gravity, through the pulmonary veins (PV), and the perfusion fluid is collected in a reservoir before being recirculated via a pump and membrane oxygenator. The gas oxygenator is connected to a tank containing a special gas mixture of oxygen (6%), carbon dioxide (8%), and nitrogen (86%). A ventilator provides an airway to the lungs and is also connected to a tracheoporsal tube.

[0077] The lung graft (cooled during organ harvesting and then subjected to static cryopreservation) is gradually rewarmed over 45 minutes. Pulmonary perfusion is initiated at a low flow rate (0.10–0.15 l / min) and then gradually increased in parallel with rewarming to a pulmonary artery supply of 40–50% of the estimated cardiac output (at 70 ml / kg / min). The PA pressure should be kept low (<15–20 mmHg) if the capillary-alveolar barrier is weakened by IRI after organ harvesting, as ischemia-reperfusion injury (IRI) can impair integrity, increase permeability of the alveolar capillary membrane, and lead to the formation of pulmonary edema.

[0078] During the rewarming phase, oxygen is supplied to the graft via a membrane oxygenator, followed by pCO2 and pH levels close to those normally measured in the PA. A catheter is placed in situ to continuously measure PA and left atrium (LA) pressure throughout the EVLP process. Mechanical ventilation of the lungs is initiated when the perfusion fluid temperature reaches 32 degrees Celsius (typically about 30 minutes after the start of perfusion).

[0079] The following outlines the 19 steps of the EVLP process. This is a quick reference guide, not a comprehensive protocol.

[0080] Step 1. Transport the lungs to XPS. Ensure compliance with UNOS standards.

[0081] Step 2. Insertion of the lung cannula. Trim the LA cannula (green) to fit the appropriate atrial opening and suture with a continuous polypropylene suture. For the PA cannula (yellow), open the PA lumen and insert the XVIVO cannula. If pulmonary artery reconstruction is required, suture a segment of the donor's descending aorta to the pulmonary artery, and then insert the cannula as described above. Secure the cannula in place in the glove using umbilical tape or silk tie and return the tie to the cannula below the pressure line. During intubation, clamp the trachea to prevent deflation. Insert the ET tube into the trachea and secure it with umbilical tape or silk tie.

[0082] Step 3. Backwash the back table. Backwash with 1 liter of cold Perfadex and check for leaks around the cannula insertion point. Leave the ET tubing clamped.

[0083] Step 4. System Setup. Set up the XPS perfusion circuit with the PGM. Open the dome by removing the plastic, then open the first blue layer wrapping on the back table. Place the clear U-shaped drape on the top bar of the XPS on the blue table and secure the XPS w-clip covering the blue table. Position the "U" opening of the U-shaped drape to the right to allow the tubing to pass through. Aseptically open the second blue layer of the dome on the blue table of the Xvivo machine. Attach the red 3 / 8-inch drain line to the back of the dome, and the quarter-inch line around the outside of the pole and through the red roller pump. Release your hand from the drain bag and attach the orange and red lines to the bag. Add a return line from the top of the reservoir to the two-way valve. Close the two-way valve for the reservoir on the red line and open it for the drain bag. Set up the transducer, rinse with sterile saline, add the blue tubing to the heater / cooler, then add the venous gas line to the back of the Quadrox. Ensure there are sufficient amounts of O2 and trigas.

[0084] Step 5. Purge the system. Add 1500 ml of STEEN to the reservoir, add the medications (10,000 units of heparin, 500 mg of methylprednisolone, 1 gm of ceftazidine), turn on the Cardiohelp and heater / cooler (set to 23) first, followed by the UPS, touchscreen, and ventilator. Purge the perfusion circuit by removing the yellow cap on the back of the Quadrox, the clear cap on either of the reservoirs, and the blue cap on the leukocyte filter. Increase Cardiohelp to 1000 RPM to check for leaks in the circuit, and after 1 minute increase Cardiohelp to 3000 RPM. After a few minutes, increase Cardiohelp to 5000 RPM and run for 1 minute to ensure all air is coming out of the lines, then reduce the RPM to below 250.

[0085] Step 6. Logging data settings. Enter the required information on the touchscreen setup page and change the PH to an acceptable level up to the alarm (6.8). On the service page, press the green PGM calibration button and enter the PH and PO2 based on the calibration number on the PGM package. Set the timer on the main screen (1 and 2 count up, #3 counts down (#3 is set to 10 minutes)). Press Settings for ventilator and press Confirm with Mode (S) CMV+. Enter the parameters in the spreadsheet and press Confirm. Also, set alarms for each parameter in the flow sheet. On the I / O panel on the back of the XPS, position the inspiratory port on the right side of the lung circuit and the expiratory port (blue tip) on the left side. Connect the flow sensor tubing with white for transparent and blue for blue. Connect the venous gas mixing line. Connect the high-pressure O2 line. The humidifier filter is placed at the lung-side end of the circuit between the flow sensor and ET-T. Perform preoperative checks, press System, and press Test / Calibrate. Select one calibration test at a time. Airtightness - Press the following steps, Flow sensor - Press the following steps, O2 cell does not require testing.

[0086] Step 7. Retrograde flushing. Using a sterile tube clamp, the direction of flow in the perfusion circuit is reversed, so that the flow proceeds through the LA and exits the PA. Starting at 750 RPM, slowly increase the RPM until there is enough flow to fill the PA cannula, flushing the lung until approximately 250cc of blood is removed from the STEEN solution. Use a recycle pump to direct this STEEN solution into a dump bag. Once the blood has been removed, attach the cannula and circuit to the LA side and ensure the lung is filled with STEEN solution. There should be enough STEEN to pass through the LA cannula and fill the PA cannula, then reset the direction of the recycle pump to enter the reservoir. Reposition the sterile clamp on top of the PA cannula and attach it to the circuit. Clamp the bridge (this should be the only clamp).

[0087] Step 8. Calibration. Calibrate the perfusion flow sensor and pressure sensor. (The flow sensor can be calibrated before the lung is placed in the circuit for retrograde flow). The pressure sensor should be calibrated immediately after retrograde flow and the lung is in the circuit (at the top of the fluid level – where the cannula meets the lung and the pressure two-way stopcock, pull the syringe from the pressure sensor line to make everything the same level, fill with STEEN and make sure to move the two-way stopcock to shut it off from saline).

[0088] Step 9. Proceed. Start Timer 1 (Perfusion Timer) and follow the EVLP Workup Sheet for the initial 1-hour settings.

[0089] Step 10. Initial setup for the first hour. Every 10 minutes, the Cardiohelp and heater / cooler settings will be adjusted until they reach their maximum values. Ventilation cannot be started until the temperature reaches 32°C. Bronchi may be affected at 32°C. Trigas sweeping should be started simultaneously with the ventilator startup.

[0090] Step 11. Lung replacement. To avoid confusion with the O2 challenge, lung replacement involves holding the exhalation key on the ventilator for 15 seconds. To hold exhalation, this key should be pressed after inhalation.

[0091] Step 12. O2 Challenge. In the O2 Challenge, only change the ventilator settings and follow the replenishment settings on the EVLP workup sheet. (Multiply Vt by IBW x 10, increase FiO2 to 100%, BPM to a maximum of 10)

[0092] Step 13. Gas release. At the final moment of the O2 challenge, arterial and venous perfusion fluid samples should be taken from the transducer. Press the gray PH button on the main screen to lock the gas values. (When results are returned from the lab, perform pinpoint calibration to PH on both LA and PA.) Record the appropriate information and then reset the ventilator to its normal settings (leave alarms and settings high at this point).

[0093] Step 14. X-ray. After completing the O2 challenge, an X-ray should be performed to make a baseline comparison for future X-rays.

[0094] Step 15. Dilution of STEEN. Dilution of STEEN is required after (or during) the replenishment, challenge, gas aspiration, and X-ray. Open the + / - pump control window on the touchscreen. Touch the [Remove] button and slide your finger down until the remove button is locked (if a clamp system is used, remove the clamp instead). Monitor the reservoir level drop. Once sufficient STEEN has been removed, touch the [Remove] button to shut off (if a clamp system is used, replace the clamp). Add 3 or 4 fresh STEEN. You may also add another dose of the drug.

[0095] Step 16. Maintain and assess the lungs for the next 3–5 hours. Repeat steps 12 and 13 (subtract pinpoint calibration) at the 50-minute mark of each time period.

[0096] Step 17. Repeat Step 14 about one hour before removing the lung for transplantation. Comparing the first and second X-rays can help determine if the lung is suitable for transplantation.

[0097] Step 18. Repeat Step 15 after 6 hours of perfusion.

[0098] Step 19. Rapid Cooling. Once the lung transplant is accepted, set the heater / cooler to 15°C, clamp the ET tube when the lung is 50% inflated at 32°C, remove the lung from the perfusion circuit, rinse with 2 liters of cold Perfadex, and place in a sterile bag with the Perfadex. (Similar to standard donor procurement protocols).

[0099] Step 20. Clean and store the XPS. Wipe the surface of the XPS with disinfectant, store it with the toggle switch facing upwards, and plug it into an outlet for charging. Gas inhalation should be performed at the end of each 10-minute challenge.

[0100] For reference, please see Figures 1 and 2, which show schematic diagrams of potential systems.

[0101] Example 2: Inhaled nitric oxide improves brain mitochondrial function in a blinded, randomized, controlled piglet asphyxia model of cardiac arrest.

[0102] Introduction Neurological injury following cardiac arrest (CA) in children remains common. Inhaled nitric oxide (iNO) may alleviate cerebral mitochondrial dysfunction, a critical convergence point for CA-induced secondary brain injury. We hypothesized that animals treated with 20 ppm iNO during CPR and 4 hours after return to spontaneous circulation (ROSC) following asphyxia and cardiac arrest would improve cerebral blood flow (CBF) and mitochondrial function, as defined by increased respiratory control ratio (RCR) and decreased mitochondrial reactive oxygen species (mtROS) compared to placebo.

[0103] method Four-week-old pigs were suffocated for seven minutes, followed by ventricular fibrillation. Guideline CPR was performed with a compression depth (CD) of at least one-third of the chest diameter, and standard epinephrine was administered for 10 minutes or until ROSC in protocolized care after ROSC. Participants were randomized in a blinded manner (starting with 20 ppm iNO and entering CPR at 1 minute, n=10, or placebo, n=10). Sham pigs (n=4) did not receive CA or CPR. Baseline and serial CBF measurements were performed using invasive clinical and non-invasive optical instruments. Cortical and hippocampal tissues were analyzed by high-resolution respiratory measurements to assess mitochondrial function. T-tests and ANOVA were used where applicable. Longitudinal hemodynamic variables were compared using generalized estimating equations to control for within-subject correlations.

[0104] result Seven out of ten animals in the placebo group and 10 out of ten in the iNO group (p=0.21) survived. There were no significant differences in invasive or non-invasive cardiopulmonary bypass (CBF) between the treatment groups during CPR and after ROSC. Cortical and hippocampal RCR was significantly higher (p=0.04, 0.007), and mtROS production was significantly lower in iNO-treated animals (p<0.001, p=0.03). There were no differences in systemic or pulmonary hemodynamics between the iNO and placebo groups, but mean pulmonary artery pressure tended to be lower in iNO animals during CPR (28.1±9.8v, 42.6±6.0, p=0.14). iNO preserves mitochondrial function in the brain (increased RCR) and limits mtROS production in a porcine model of pediatric cardiac arrest. Further research is needed to evaluate this potential neuroprotective effect of iNO in ischemia-reperfusion injury and cardiac arrest.

Claims

1. A system for increasing the activity of superoxide dismutase 2 (SOD2 or manganese-dependent superoxide dismutase (MnSOD)) in organs with ischemia-reperfusion injury, wherein the system is Administering a loading dose containing 20 ppm to 40 ppm NO gas to the aforementioned organ, The method involves directly and continuously administering a composition containing NO gas at a concentration of 0.05 ppm to 20 ppm to the aforementioned organs. It is configured to do the following: The activity of MnSOD was increased in the organ compared to a control organ that had not been in contact with the composition. The aforementioned loading dose is administered immediately before the composition, in a system.

2. The system according to claim 1, wherein the activity of MnSOD can be increased by 5% to 95% compared to organs treated under similar conditions but not directly and sequentially administered with NO gas.

3. The system according to claim 1, further configured to monitor the level of methemoglobin and reduce the dose of NO gas when the level of methemoglobin exceeds 5%.

4. The system according to claim 1, wherein the composition further comprises a cell-free perfusion solution, the cell-free perfusion solution comprising sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

5. The system according to claim 1, wherein the organ is the heart, lungs, or kidneys.

6. The system according to claim 1, wherein the administration of the composition is carried out for a period of time necessary to improve the survival rate of the organ.

7. The system according to claim 6, wherein the administration of the composition is carried out over a period of 5 to 60 minutes, or over a period of 1 to 12 hours.

8. The system according to claim 1, wherein administration begins simultaneously with the onset of ischemia, or administration begins within 5 to 30 minutes after the onset of ischemia.

9. The system according to claim 1, wherein administration begins during reperfusion, or administration continues for 1 to 12 hours after reperfusion has begun.

10. A system for inhibiting nitrotyrosine formation in organs with ischemia-reperfusion injury, wherein the system is Administering a loading dose containing 20 ppm to 40 ppm NO gas to the aforementioned organ, The method involves directly and continuously administering a composition containing NO gas at a concentration of 0.05 ppm to 20 ppm to the aforementioned organs. It is configured to do the following: The formation of nitrotyrosine adducts was inhibited in the organ compared to a control organ that was not in contact with the composition. The aforementioned loading dose is administered immediately before the composition, in a system.

11. The system according to claim 10, wherein nitrotyrosine formation is inhibited by 5% to 95% compared to organs treated under similar conditions but not directly and sequentially administered with NO gas.

12. The system according to claim 10, further configured to monitor the level of methemoglobin and reduce the dose of NO gas when the level of methemoglobin exceeds 5%.

13. The system according to claim 10, wherein the composition further comprises a cell-free perfusion solution, the cell-free perfusion solution comprising sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

14. The system according to claim 10, wherein the organ is the heart, lungs, or kidneys.

15. The system according to claim 10, wherein the administration of the composition is carried out for a period of time necessary to improve the survival rate of the organ.

16. The system according to claim 15, wherein the administration of the composition is carried out over a period of 5 to 60 minutes, or over a period of 1 to 12 hours.

17. The system according to claim 10, wherein administration begins simultaneously with the onset of ischemia, or administration begins within 5 to 30 minutes after the onset of ischemia.

18. The system according to claim 10, wherein administration begins during reperfusion, or administration continues for 1 to 12 hours after reperfusion has begun.

19. A system for preventing the inactivation of mitochondrial complex I activity, complex II activity, complex III activity, complex IV activity, or combination thereof in an organ with ischemia-reperfusion injury, wherein the system is Administering a loading dose containing 20 ppm to 40 ppm NO gas to the aforementioned organ, The method involves directly and continuously administering a composition containing NO gas at a concentration of 0.05 ppm to 20 ppm to the aforementioned organs. It is configured to do the following: Administering NO gas at 20 ppm or less prevents inactivation of mitochondrial complexes I, II, III, IV, or combinations thereof compared to a control organ. The aforementioned loading dose is administered immediately before the composition, in a system.

20. The system according to claim 19, wherein inactivation of mitochondrial complex I activity, complex II activity, complex III activity, complex IV activity, or combination thereof is inhibited by 5% to 95% compared to organs treated under similar conditions but not directly and sequentially administered with NO gas.

21. The system according to claim 19, further configured to monitor the level of methemoglobin and reduce the dose of NO gas when the level of methemoglobin exceeds 5%.

22. The system according to claim 19, wherein the composition further comprises a cell-free perfusion solution, the cell-free perfusion solution comprising sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

23. The system according to claim 19, wherein the organ is the heart, lungs, or kidneys.

24. The system according to claim 19, wherein the administration of the composition is carried out for a period of time necessary to improve the survival rate of the organ.

25. The system according to claim 24, wherein the administration of the composition is carried out over a period of 5 to 60 minutes or over a period of 1 to 12 hours.

26. The system according to claim 19, wherein the administration begins simultaneously with the onset of ischemia, or the administration begins within 5 to 30 minutes after the onset of ischemia.

27. The system according to claim 19, wherein administration begins during reperfusion, or administration continues for 1 to 12 hours after reperfusion has begun.

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