Methods of improving viability of organ

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 TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 550,463, filed August 25, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] This disclosure is a x A method is provided for improving the viability of one or more organs by continuously administering a gas-containing composition directly to the organ(s). [Background technology]

[0003] Cells, tissues, organs, and organisms deprived of adequate blood flow suffer ischemic injury due to oxidative stress and ultimately death. Traditional methods of alleviating ischemic injury involve perfusing the affected tissue with oxygen, but this treatment can cause significant tissue damage, potentially resulting in serious and / or permanent injuries, such as brain damage during stroke or cardiac arrest.

[0004] Attempts have been made to reduce ischemia and reperfusion injury by inducing a hypometabolic state in tissues and organs. For example, in situations where living tissue is being preserved for transplantation (transplant or graft), one common method for reducing metabolic activity is by immersing the tissue or organ in a physiological fluid, such as saline, and placing it in a cold environment. However, such methods cannot be relied upon for long periods of time, and the success of organ transplantation and limb reattachment remains inversely related to the time the organ or limb is out of contact with the intact organism.

[0005] Thus, there remains a need in the art to improve organ viability prior to ischemia and / or reperfusion injury. Summary of the Invention [Means for solving the problem]

[0006] In one aspect, the present disclosure provides a method for improving the viability of an organ intended for transplantation, comprising administering NO to the organ via an organ perfusion system or ventilation. x The present invention encompasses methods that involve direct, continuous administration of a gas-containing composition.

[0007] In another aspect, the present disclosure provides a method for improving the viability of an organ injured by ischemia-reperfusion, comprising administering about 20 ppm or less of NO to the organ via an organ perfusion system or ventilation. x The present invention encompasses methods comprising the direct continuous administration of a composition comprising a gas. 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 intended for transplantation. In an exemplary embodiment, the organ in need of treatment is an organ removed from a donor and intended for transplantation.

[0008] In another aspect, the present disclosure provides a method for improving the viability of an organ damaged by ischemia-reperfusion, comprising: (a) administering to an organ a concentration of about 20 ppm to about 40 ppm NO x (b) administering a composition containing the gas ("loading dose") to the organ for up to about 1 hour ("loading period"); and (c) administering to the organ via an organ perfusion system or ventilation a concentration of about 20 ppm or less of NO. x The present invention encompasses methods comprising continuously administering a gas-containing composition directly to an organ. 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 intended for transplantation. In an exemplary embodiment, the organ in need of treatment is an organ removed from a donor and intended for transplantation.

[0009] In another aspect, the present disclosure provides a method for improving the viability of an organ intended for transplantation, comprising administering 20 ppm or less NO to the organ via an organ perfusion system or ventilation. x In some embodiments, the organ is present in a brain-dead donor. In other embodiments, the organ is present in a brain-dead donor. xIt has been removed from the donor prior to administration of the gas-containing composition. In an exemplary embodiment, the organ is a lung, kidney, or heart.

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

[0011] In another aspect, the present disclosure provides a method of treating ischemia-reperfusion injury in an organ in need thereof, comprising administering 20 ppm or less NO directly to the organ via an organ perfusion system or ventilation. x The present invention encompasses methods comprising continuously administering a composition comprising a gas. 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 intended for transplantation. In an exemplary embodiment, the organ in need of treatment is an organ removed from a donor and intended for transplantation.

[0012] In another aspect, the present disclosure provides a method of treating ischemia-reperfusion injury in an organ in need thereof, comprising: (a) administering to an organ a concentration of about 20 ppm to about 40 ppm NO x (b) administering a composition containing the gas ("loading dose") to the organ for up to about 1 hour ("loading period"), and then (c) administering 20 ppm or less of NO via an organ perfusion system or ventilation. xThe present invention encompasses methods comprising continuously administering a gas-containing composition directly to an organ. 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 intended for transplantation. In an exemplary embodiment, the organ in need of treatment is an organ removed from a donor and intended for transplantation.

[0013] In another aspect, the present disclosure provides a method for transplantation, comprising: (a) administering to a patient a dose of 20 ppm or less of NO x (b) administering a composition comprising a gas 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 present in a brain-dead donor. In other embodiments, the organ is administered in a manner that utilizes NO gas. x It has been removed from the donor prior to administration of the gas-containing composition. In an exemplary embodiment, the organ is a lung, kidney, or heart.

[0014] In another aspect, the disclosure provides a method for transplantation, comprising: (a) administering from about 20 ppm to about 40 ppm NO x (b) administering to the organ a composition containing 20 ppm or less NO gas (a "loading dose") for up to about 1 hour (a "loading period"); x (c) administering a composition comprising a gas directly to an organ intended for transplantation for up to 12 consecutive hours; and (d) transplanting the organ into a recipient. In some embodiments, the organ is present in a brain-dead donor. In other embodiments, the organ is administered in a manner that utilizes NO gas. x It has been removed from the donor prior to administration of the gas-containing composition. In an exemplary embodiment, the organ is a lung, kidney, or heart.

[0015] Other aspects and iterations of the present disclosure are described more thoroughly below. The present specification also provides, for example, the following items: (Item 1) A method for improving the viability of an organ intended for transplantation, comprising administering to said organ via an organ perfusion system or ventilation a concentration of 20 ppm or less of NO.x The method comprises directly and continuously administering a composition comprising a gas. (Item 2) 10. The method of claim 1, wherein the composition is administered for at least 1 hour but not more than 12 hours. (Item 3) 3. The method of claim 1 or claim 2, wherein the composition is administered after the organ is harvested from the donor. (Item 4) 3. The method of claim 1 or 2, wherein the composition is administered while the organ is in the body of a brain-dead donor. (Item 5) 4. The method according to any one of items 1 to 3, wherein the organ is selected from the group consisting of lung, heart, liver, kidney, pancreas, intestine, thymus, and cornea. (Item 6) NO x The method according to any one of items 1 to 5, wherein the amount of is about 10 ppm to about 15 ppm. (Item 7) NO x 6. The method according to any one of items 1 to 5, wherein the amount of is about 5 ppm to about 10 ppm. (Item 8) NO x The method according to any one of items 1 to 5, wherein the amount of is about 1 ppm to about 5 ppm. (Item 9) 9. The method of any one of items 1 to 8, wherein the composition is an organ perfusate and optionally further comprises one or more additional compound(s). (Item 10) 10. The method of claim 9, wherein the perfusion fluid is a cell-free perfusion fluid. (Item 11) The method comprises: x Compositions containing gas ("loading dose") containing 20 ppm or less of NO x 11. The method of any one of items 1 to 10, further comprising administering the composition comprising a gas to the organ for up to about 1 hour immediately prior to sequential administration (the "loading period"). (Item 12) 12. The method of claim 11, wherein the loading dose composition is a perfusate. (Item 13) Item 13. The method of item 12, wherein the perfusion fluid is a cell-free perfusion fluid. (Item 14) NO x 14. The method according to any one of items 1 to 13, wherein the total administration time of the gas does not exceed 12 hours. (Item 15) 15. The method according to any one of items 1 to 14, wherein mitochondrial function is significantly maintained. (Item 16) 16. The method of 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) 1. A method for improving the survival rate of an organ damaged by ischemia-reperfusion, comprising administering 20 ppm or less of NO to said organ via an organ perfusion system. x The method comprises directly and continuously administering a composition comprising a gas. (Item 18) 18. The method of claim 17, wherein the composition is administered for at least 1 hour but not more than 12 hours. (Item 19) 19. The method of claim 17 or 18, wherein the organ is intended for transplantation and the composition is administered after the organ is harvested from the donor. (Item 20) 19. The method of claim 17 or 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) 21. The method according to any one of items 17 to 20, wherein the organ is selected from the group consisting of lung, heart, liver, kidney, pancreas, intestine, thymus, and cornea. (Item 22) NO x 22. The method according to any one of items 17 to 21, wherein the amount of is about 10 ppm to about 15 ppm. (Item 23) NO x22. The method according to any one of items 17 to 21, wherein the amount of is about 5 ppm to about 10 ppm. (Item 24) NO x 22. The method according to any one of items 17 to 21, wherein the amount of is about 1 ppm to about 5 ppm. (Item 25) 24. The method of any one of items 17 to 23, wherein the composition is an organ perfusate and optionally further comprises one or more additional compound(s). (Item 26) 26. The method of claim 25, wherein the perfusion fluid is a cell-free perfusion fluid. (Item 27) The method comprises: x Compositions containing gas ("loading dose") containing 20 ppm or less of NO x 27. The method of any one of items 17 to 26, further comprising administering the composition comprising a gas to the organ for up to about 1 hour immediately prior to sequential administration (the "loading period"). (Item 28) 28. The method of claim 27, wherein the loading dose composition is a perfusate. (Item 29) 29. The method of claim 28, wherein the perfusion fluid is a cell-free perfusion fluid. (Item 30) NO x 30. The method according to any one of items 17 to 29, wherein the total administration time of the gas does not exceed 12 hours. (Item 31) 31. The method according to any one of items 17 to 30, wherein mitochondrial function is significantly maintained. (Item 32) 32. The method of 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) A transplantation method comprising: (a) NOx levels below 20 ppm via an organ perfusion system x Continuously administering a gas-containing composition directly to the organ intended for transplantation; and (b) transplanting said organ into a recipient. The method comprising: (Item 34) 34. The method of claim 33, wherein the composition is administered for at least 1 hour but not more than 12 hours. (Item 35) 35. The method of claim 33 or 34, wherein the composition is administered after the organ is harvested from the donor. (Item 36) 35. The method of claim 33 or 34, wherein the composition is administered while the organ is in the body of a brain-dead donor. (Item 37) 37. The method according to any one of items 33 to 36, wherein the organ is selected from the group consisting of lung, heart, liver, kidney, pancreas, intestine, thymus, and cornea. (Item 38) NO x 38. The method according to any one of items 33 to 37, wherein the amount of is about 10 ppm to about 15 ppm. (Item 39) NO x 38. The method according to any one of items 33 to 37, wherein the amount of is about 5 ppm to about 10 ppm. (Item 40) NO x 38. The method according to any one of items 33 to 37, wherein the amount of is about 1 ppm to about 5 ppm. (Item 41) 41. The method of any one of items 33 to 40, wherein the composition is an organ perfusate and optionally further comprises one or more additional compound(s). (Item 42) 42. The method of claim 41, wherein the perfusion fluid is a cell-free perfusion fluid. (Item 43) The method further comprises administering about 20 ppm to about 40 ppm NO for up to about 1 hour (the "loading period") immediately prior to step (a). x 43. The method of any one of items 33 to 42, further comprising administering to said organ a composition comprising a gas (a "loading dose"). (Item 44) 44. The method of claim 43, wherein the loading dose composition is a perfusate. (Item 45) 45. The method of claim 44, wherein the perfusion fluid is a cell-free perfusion fluid. (Item 46) NO x 46. ​​The method according to any one of items 33 to 45, wherein the total administration time of the gas does not exceed 12 hours. (Item 47) 47. The method according to any one of items 33 to 46, wherein mitochondrial function is significantly maintained. (Item 48) 48. The method of 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 will contain at least one photograph executed in color. Copies of this patent application publication including color photographs will be provided by the Agency upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is an illustration of an embodiment of an EVLP circuit. [Figure 2] 1 is an illustration of an embodiment of an EVLP circuit. DETAILED DESCRIPTION OF THE INVENTION

[0018] This disclosure is a x Provided are methods for improving the viability of one or more organs by continuously administering a composition comprising a gas directly to the organ(s). The present disclosure encompasses methods for improving the viability of organs intended for transplantation, as well as organs with ischemia-reperfusion injury due to other causes. The present disclosure also provides methods for improving transplant organ performance and methods for transplantation. As used herein, "NO" refers to a gas-containing composition. x The term "gas" refers to gaseous nitrogen oxides. In a preferred embodiment, NO xThe gas is gaseous nitric oxide (gNO). Non-limiting examples of additional components of the composition may include an inert diluent gas (e.g., helium, neon, etc.), human albumin, sodium caprylate, N-acetyl-DL-tryptophan, and oxygen gas (O). x Continuous administration of a gas-containing composition directly to an organ means that the organ receives NO without interruption during the administration period. x This means that the organ is in direct contact with the gas. The present 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, vascularized composite allografts (e.g., face, hand, etc.), or any combination thereof. Organ viability is determined by the NO x The viability of an organ may be improved compared to organs obtained by methods that do not involve direct, continuous administration of a gas-containing composition to the organ. As used herein, the term "viability" refers to the suitability of an organ for its intended purpose. Measurements of organ viability may vary by organ type and are known in the art.

[0019] Several definitions that apply throughout this disclosure are now provided. As used herein, "about" refers to numerical values, including integers, fractions, percentages, etc., whether explicitly stated or not. The term "about" generally refers to a range of numerical values, such as ±0.5-1%, ±1-5%, or ±5-10% of the recited value, that are considered equivalent to the recited value, e.g., have the same function or result.

[0020] The term "comprising" means "including, but not necessarily limited to." This indicates open-ended inclusion or membership, particularly in the aforementioned combinations, groups, series, etc. As used herein, the terms "comprising" and "including" are inclusive and / or open-ended and do not exclude additional, unrecited elements or method steps. The term "consisting essentially of" is more restrictive than "comprising," but not as restrictive as "consisting of." Specifically, the term "consisting essentially of" limits membership to certain materials or steps, and those that do not materially affect the essential characteristics of the claimed invention.

[0021] As used herein, the term "ischemia-reperfusion injury" refers to damage caused by ischemia, reperfusion, or both.

[0022] As used herein, the terms "treat," "treating," or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, where the goal is to prevent or slow (alleviate) an undesirable physiological change or disease / disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stable (i.e., not worsening) state of disease, delay or slowing of disease progression, remission or palliation of the disease state, and remission (partial or complete remission), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or disorder, as well as those prone to have the disease or disorder or those in whom the disease or disorder is to be prevented.

[0023] (a)NO x Gas-containing composition According to this disclosure, NO xThe gas-containing composition is continuously administered directly to the organ. The composition of the present disclosure can be a gas or a liquid. When the composition of the present disclosure is a liquid, NO x The gas is solubilized in the liquid. In other words, "NO x The "gas-containing composition" is a solubilized NO x Similarly, "20 ppm NOx, where the composition is a liquid, x The "gas-containing composition" is a liquid containing 20 ppm nitric oxide, and the 20 ppm nitric oxide is NO solubilized in the liquid. x Additionally, in embodiments where the composition is a liquid, NO administered directly to the organ x The amount of gas is determined by the amount of NO dissolved in the liquid. x The amount of gas. NO x The gas may be generated 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, an inert diluent gas(es), nitrogen, oxygen, and water. In an exemplary embodiment, NO x The gas is gNO. When the composition is a gas, it can be administered directly to the organ via a ventilator or any other method known in the art.

[0025] In other embodiments, the composition is a perfusion solution. The term "perfusion solution" refers to any fluid used in the preservation, perfusion, or reperfusion of tissues or organs. Perfusion solutions are frequently sterile and isotonic. Solubilized NO xIn addition to gas, the perfusion solution may further comprise 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 a preferred embodiment, 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 an exemplary embodiment, the composition is a cell-free perfusion solution, and NO x The gas is gNO. In a further embodiment, the cell-free perfusion solution is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. Suitable methods for administering perfusion solution directly to an organ are known in the art and include, but are not limited to, organ perfusion systems. The present disclosure is not limited to any particular organ perfusion system. Generally speaking, an organ perfusion system may include a pump for moving and controlling the perfusion solution, a means for controlling the temperature of the system, a cannula, and a means for measuring physiological parameters. A non-limiting example of an organ perfusion system is disclosed in U.S. Pat. No. 9,629,358, which is incorporated herein by reference. Non-limiting examples of organ perfusion systems are also disclosed in FIGS. 1 and 2.

[0026] Therapeutic doses of NO to improve organ viability x The gas is administered directly to the organ. Depending on the type or types of compositions administered, NO is delivered to the organ. xDirect administration of gas can occur via an organ perfusion system, a ventilator, or any combination thereof. In embodiments using a combined organ perfusion system and ventilator, the organ perfusion system and ventilator are used simultaneously to administer NO x Alternatively, or in addition, 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 series to deliver NO. x The gas-containing composition can be administered directly to the organ, e.g., with varying amounts of overlap between the two administration methods (e.g., no overlap, overlap of a few seconds, minutes, or hours), e.g., first via a ventilator and then via a perfusion system, or vice versa.

[0027] "NO x A "therapeutically effective amount of NO gas" is a gas that, when administered directly to an organ, is sufficient to improve the viability of the organ as defined herein. x The amount of NO gas that constitutes a "therapeutically effective amount" is shown. x The amount of NO will vary depending on a variety of factors and can be determined by one skilled in the art. As described in further detail below, a therapeutically effective amount of NO for the treatment of organs with ischemia-reperfusion injury is x The therapeutically effective amount of NO gas is less than 20 ppm. x The gas can be administered alone or with a loading dose of NO x Can be used after gas administration. x A loading dose of gas can be used to increase vasodilation of an ischemic organ and may be particularly appropriate when the composition of the present disclosure first contacts the organ after removal from the donor. However, NO provided in the loading dose x The amount of gas is typically sufficient to safely treat ischemia-reperfusion injury for extended periods (e.g., periods greater than 1 hour). x Exceeding the amount of gas.

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

[0029] In one or more embodiments, NO x The gas is administered at an initial concentration, optionally increased as needed to achieve the desired effect (e.g., increased organ viability). For example, the initial nitric oxide concentration may be about 0.05 ppm to about 35 ppm, or about 1 ppm to about 35 ppm, optionally increased gradually until the desired effect is achieved or the nitric oxide threshold is met. An exemplary nitric oxide administration begins with an initial concentration of about 1 ppm, followed by a desired NO concentration. x It can be increased in increments of about 0.1 ppm to about 5 ppm until an effect is achieved, but NO x Ensure that the concentration does not exceed 35 ppm and / or that methemoglobin levels are less than or equal to about 5%. Alternatively, an exemplary nitric oxide administration may begin with an initial concentration of 5 ppm, followed by a desired NO concentration. x It 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 methemoglobin levels are less than or equal to about 5%. In another exemplary embodiment, nitric oxide administration begins at an initial concentration of 10 ppm, followed by a desired NO concentration. x It can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO x In yet another exemplary embodiment, nitric oxide administration begins at an initial concentration of 15 ppm, followed by a desired NO concentration of 15 ppm. xIt can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO x In yet another exemplary embodiment, nitric oxide administration begins at an initial concentration of 20 ppm, followed by a desired NO concentration of 10 ppm. x It can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO x Ensure concentrations do not exceed 35 ppm and / or methemoglobin levels are less than or equal to about 5%. In each of the above embodiments, administration can be for 5, 10, 15, 30, or 60 minutes. Alternatively, administration can be for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours.

[0030] In one or more embodiments, NO x The gas is administered at an initial concentration, optionally increased as needed to achieve the desired effect (e.g., increased organ viability). For example, the initial nitric oxide concentration may be about 0.05 ppm to about 20 ppm, or about 1 ppm to about 20 ppm, and optionally increased gradually until the desired effect is achieved or the nitric oxide threshold is met. An exemplary nitric oxide administration begins with an initial concentration of about 1 ppm, followed by a desired NO concentration. x It can be increased in increments of about 0.1 ppm to about 5 ppm until an effect is achieved, but NO x Ensure that concentrations do not exceed 20 ppm and / or that methemoglobin levels are less than or equal to about 5%. Alternatively, an exemplary nitric oxide administration may begin with an initial concentration of 5 ppm, followed by a desired NO concentration. x It 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 20 ppm and / or that methemoglobin levels are less than or equal to about 5%. In another exemplary embodiment, nitric oxide administration begins at an initial concentration of 10 ppm, followed by a desired NO concentration. x It can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO x In yet another exemplary embodiment, nitric oxide administration begins at an initial concentration of 15 ppm, followed by a desired NO concentration of 15 ppm. x It can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO x In yet another exemplary embodiment, nitric oxide administration begins at an initial concentration of 20 ppm, followed by a desired NO concentration. x It can be increased in increments of 0.1 ppm to 5 ppm until an effect is achieved, but NO x Ensure concentrations do not exceed 20 ppm and / or methemoglobin levels are less than or equal to about 5%. In each of the above embodiments, administration can be for 5, 10, 15, 30, or 60 minutes. Alternatively, administration can be for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 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 second, lower concentration for a second period of time to achieve the desired effect (e.g., increased organ viability). xThe gas may be administered at an initial concentration of about 20 ppm to about 40 ppm for up to about one hour, and then decreased to 20 ppm or less to improve organ viability. Exemplary nitric oxide administration can begin at an initial concentration of about 20 ppm to about 40 ppm, and then incrementally decrease over an initial period of time until a nitric oxide concentration of 20 ppm or less is reached. The rate of decrease may or may not be constant. The nitric oxide concentration can be further adjusted as needed, for example, based on monitoring of nitric oxide markers. Alternatively, exemplary nitric oxide administration can begin at an initial concentration of about 20 ppm to about 40 ppm, remain constant over an initial period of time, and then decrease to a nitric oxide concentration of 20 ppm or less. The nitric oxide concentration can be further adjusted as needed, for example, based on monitoring of nitric oxide markers. In each of the above embodiments, the total time of nitric oxide administration (the sum of the time of initial concentration and the time of concentration decline) 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 above embodiments, the initial nitric oxide concentration, the incremental increase or decrease in nitric oxide, the maximum nitric oxide concentration and / or threshold of nitric oxide or nitric oxide markers may vary depending on the application and / or based on the particular organ being treated. The increments may vary throughout the adjustment of nitric oxide delivery. Nitric oxide may also be gradually decreased if monitoring indicates that nitric oxide or nitric oxide markers meet or exceed the nitric oxide threshold.

[0033] In each of the above embodiments, NO x The final NO concentration x The concentration can also be adjusted incrementally by a specific percentage. xThese may include 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% changes in concentration.

[0034] NO x Instead of or in addition to adjusting the concentration, NO x NO delivered directly to organs, such as regulating gas flow x By any means to adjust the amount of gas, NO x The gas dosage can be adjusted.

[0035] In further embodiments, administration of nitric oxide is adjusted based on monitoring of nitric oxide or a nitric oxide marker. As used herein, "nitric oxide marker" refers to a direct or indirect indicator of nitric oxide concentration in a fluid. For example, nitric oxide markers include, among others, methemoglobin and NO. x (i.e., NO, nitrite ions (NO2 - ), nitrate ions (NO3 - Such adjustments may be made manually or x This monitoring may be performed automatically by the delivery device. The NO delivery device may also issue an alarm based on the monitoring. If the monitoring device is a separate component from the NO delivery device, the monitoring device can transmit monitoring information to the NO delivery 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 delivery of NO 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 decreased. Nitric oxide or nitric oxide markers can be monitored continuously or intermittently, for example, at regular intervals.

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

[0037] In one or more embodiments, nitric oxide or a nitric oxide marker is monitored by comparing the measured value of the nitric oxide or a nitric oxide marker to a nitric oxide threshold. The nitric oxide threshold can be a safety limit to ensure that methemoglobinemia does not occur. For example, the nitric oxide threshold can be a methemoglobin level, such as the ratio of methemoglobin 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. Thus, if the methemoglobin level meets or exceeds an acceptable range, such as ≦3%, ≦4%, ≦5%, ≦6%, ≦7%, ≦8%, ≦9%, ≦10%, ≦11%, or ≦12%, administration of nitric oxide can be adjusted.

[0038] The NO2 level in the perfusate may also be monitored. Fluid recirculation can cause NO2 to accumulate in the fluid. If the NO2 concentration rises above a certain threshold, the NO delivery device can adjust the administration of NO and / or sound an alarm. NO2 can also be removed using reducing agents, scrubbers, bases, or other suitable means.

[0039] (b) Ischemia-reperfusion injury The term "ischemia-reperfusion injury" refers to damage caused by ischemia, reperfusion, or both. Ischemia refers to insufficient blood supply to an organ; ischemic injury occurs when blood supply to a tissue or organ region 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 specific 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 specific injuries that can cause ischemic or hypoxic conditions include, but are not limited to, trauma such as burns, cuts, amputations, gunshot wounds, or surgical trauma. Injuries can also include internal injuries, such as strokes or heart attacks, which result in a sudden decrease in circulation. Other insults include reduced circulation due to non-invasive stresses such as exposure to cold or radiation, or deliberate reduction of circulation such as during cardiac surgery or treatment of organ donors prior to harvesting the donor organ for transport and transplantation into a recipient.

[0040] One aspect of the present disclosure includes a method for treating ischemia-reperfusion injury in an organ in need thereof, the method comprising administering 20 ppm or less NO x This involves continuously administering a composition containing the gas directly to the 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 comprising the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, and even more preferably, a cell-free perfusion solution. In a further embodiment, the cell-free perfusion solution is Steen Solution™, optionally comprising sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. 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 intended for transplantation. In an exemplary embodiment, the organ in need of treatment is an organ removed from a donor and intended for transplantation.

[0041] Administration can be for 5, 10, 15, 30, or 60 minutes. Alternatively, administration can be for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more hours. In embodiments where the organ is intended for transplantation, NO x Preferably, gas administration does not exceed a total of 12 hours. In certain embodiments, administration begins simultaneously with ischemia. In other embodiments, administration begins some time after the onset of ischemia, but preferentially begins 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. Administration can also begin during reperfusion or, alternatively, continue after reperfusion has begun. In some cases, administration can continue for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more hours after reperfusion has begun.

[0042] Effective treatment of ischemia-reperfusion injury can be assessed 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, the method for treating ischemia-reperfusion injury in an organ in need thereof comprises administering about 20 ppm to about 40 ppm NO x The composition containing the gas ("loading dose") contains no more than 20 ppm NO x The method may include an additional step of administering a gas-containing composition for up to about 1 hour (the "loading period") immediately prior to administration. For example, the loading dose may be administered for about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or between about 10 and about 30 minutes. In another example, the loading dose may be administered for about 30 minutes, about 35 minutes, about 40 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or between 30 and 60 minutes. The two compositions may be identical except for the nitric oxide concentration. Alternatively, the two compositions may be different. The nitric oxide concentration in the loading dose may be gradually decreased over the loading period until the nitric oxide concentration is 20 ppm or less. The rate of decrease may or may not be constant. Alternatively, the nitric oxide concentration in the loading dose may be held constant over the loading period and then decreased 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 perfusate, preferably a cell-free perfusate. In an exemplary embodiment, the organ is a heart, lung, or kidney, and the organ is intended for transplantation.

[0044] Another aspect of the present disclosure includes a method for improving the survival rate of an organ damaged by ischemia-reperfusion, the method comprising administering 20 ppm or less NO x This involves continuously administering a composition containing the gas directly to the 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 gas-containing compositions are described in section (a). In a preferred embodiment, the composition is a perfusion solution, and even more preferably, a cell-free perfusion solution. In a further embodiment, the cell-free perfusion solution is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. Improving the viability of an organ 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, 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.

[0045] In one embodiment, the present disclosure encompasses a method for maintaining mitochondrial function in an organ with ischemia-reperfusion injury. As used herein, mitochondrial function can be measured by the respiratory control ratio (RCR), which is an index of mitochondrial connectivity. Generally speaking, the RCR represents the ratio of the oxidation rate in the presence of excess substrate and adenosine diphosphate (State 3) to the oxidation rate after ADP phosphorylation to a steady-state concentration (State 4). In some embodiments, the RCR is measured by administering 20 ppm or less of NO. x By administering a composition containing NO gas directly to an organ, mitochondrial function is significantly maintained in an organ that has been subjected to ischemia-reperfusion injury. As used herein, "significantly maintained" refers to the NO gas composition described herein. x A significant maintenance indicates less than a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% difference in mitochondrial function between organs treated with NO gas and control organs that did not undergo ischemia-reperfusion. In other words, a significant maintenance indicates a difference between organs that underwent similar ischemia-reperfusion injury but without direct continuous NO gas administration. x This may show improved mitochondrial function compared to similar organs not receiving the gas. xSuitable compositions comprising the gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, even more preferably an acellular perfusion fluid, and the organ is the heart, lung, or kidney. In further embodiments, the acellular perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0046] In other embodiments, the present disclosure encompasses a method for reducing oxidative damage to an organ suffering from ischemia-reperfusion injury. Generally speaking, the method comprises administering 20 ppm or less of NO x As used herein, "reduction of oxidative damage" or "reduction of oxidative damage" may be measured relative to an organ treated under similar conditions, but it may be measured by administering directly and continuously NO gas to the organ. x 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, which may be directly and continuously administered NO. x In certain embodiments, mitochondrial reactive oxygen species (mtROS) are increased by direct continuous NO gas administration compared to untreated controls. x is reduced in organs with ischemia-reperfusion injury to which NO is administered. x Suitable compositions comprising the gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, even more preferably an acellular perfusion fluid, and the organ is the heart, lung, or kidney. In further embodiments, the acellular perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0047] In some embodiments, the present disclosure encompasses a method of increasing the activity of superoxide dismutase 2 (SOD2 or manganese-dependent superoxide dismutase (MnSOD)) in an organ suffering from ischemia-reperfusion injury. The method comprises administering 20 ppm or less of NO x The present invention relates to a method for treating MnSOD by directly and continuously administering a composition containing NO gas to an organ, wherein the activity of MnSOD is increased in the organ compared to a control organ not contacted with the composition of the present disclosure. For example, the activity of MnSOD may be increased 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, which is directly and continuously administered with NO gas. x No gas is administered. Methods for measuring MnSOD activity are known in the art. x Suitable compositions comprising the gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, even more preferably an acellular perfusion fluid, and the organ is the heart, lung, or kidney. In further embodiments, the acellular perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0048] In another embodiment, the present disclosure encompasses a method for inhibiting the formation of nitrotyrosine in an organ having ischemia-reperfusion injury, the method comprising administering 20 ppm or less of NO x The present invention relates to a method for treating an organ by directly and continuously administering a composition containing NO gas to the organ, wherein the formation of nitrotyrosine adducts in the organ is inhibited compared to a control organ not contacted with the composition of the present invention. For example, the formation of nitrotyrosine can 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, which is achieved by directly and continuously administering NO gas to the organ. x No gas is administered. Methods for measuring the formation of nitrotyrosine adducts are known in the art. xSuitable compositions comprising the gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, even more preferably an acellular perfusion fluid, and the organ is the heart, lung, or kidney. In further embodiments, the acellular perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0049] In certain embodiments, the present disclosure encompasses a method for preventing the inactivation of mitochondrial complex I activity, complex II activity, complex III activity, complex IV activity, or a combination thereof, in an organ suffering from ischemia-reperfusion injury. The method comprises administering 20 ppm or less of NO. x The method comprises administering continuously to an organ a composition containing a gas containing 20 ppm or less of NO. x The administration of the gas prevents the inactivation of mitochondrial complex I, complex II, complex III, complex IV, or a combination thereof, compared to a control organ. In one embodiment, the administration of the gas at 20 ppm or less NO x Continuous administration of a gas-containing composition directly to the organ prevents inactivation of mitochondrial complex I activity. In another embodiment, 20 ppm or less of NO x Continuous administration of a gas-containing composition directly to the organ prevents inactivation of mitochondrial complex II activity. In yet another embodiment, 20 ppm or less of NO x Continuous administration of a gas-containing composition directly to the organ prevents inactivation of mitochondrial complex III activity. In another embodiment, 20 ppm or less of NO x Continuous administration of a gas-containing composition directly into the organ prevents the inactivation of mitochondrial complex IV activity. In a preferred embodiment, 20 ppm or less of NO x Continuous administration of a gas-containing composition directly to the organ prevents the inactivation of mitochondrial complex I and mitochondrial complex II activity. In another preferred embodiment, 20 ppm or less of NO xDirect and continuous administration of a composition containing NO gas to an organ prevents inactivation of mitochondrial complex II and mitochondrial complex III activity. For example, 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 an organ treated under similar conditions, which is achieved by direct and continuous administration of NO gas. 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. x Suitable compositions comprising the gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, even more preferably an acellular perfusion fluid, and the organ is the heart, lung, or kidney. In further embodiments, the acellular perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

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

[0051] In a further embodiment, the method comprises administering from about 20 ppm to about 40 ppm of NO x The composition containing the gas ("loading dose") contains no more than 20 ppm NO x The method may include an additional step of administering the gas-containing composition immediately before the administration of the loading dose for up to about one hour. For example, the loading dose may be administered for about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or between about 10 and about 30 minutes. In another example, the loading dose may be administered for about 30 minutes, about 35 minutes, about 40 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or between 30 and 60 minutes. The two compositions may be identical except for the nitric oxide concentration. Alternatively, the two compositions may be different. The nitric oxide concentration in the loading dose may be gradually decreased over a period of up to about one hour until the nitric oxide concentration is 20 ppm or less. The rate of decrease may or may not be constant. Alternatively, the nitric oxide concentration in the loading dose may be held constant for a period of up to about one hour, and then decreased 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 fluid, preferably a cell-free perfusion fluid. In a further embodiment, the cell-free perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. In an exemplary embodiment, the organ is a heart, lung, or kidney.

[0052] (c) Methods for improving the viability of organs intended for transplantation. Organs with ischemia-reperfusion injury include organs intended for transplantation. Accordingly, the present disclosure encompasses methods for improving the survival rate of organs intended for transplantation. Such methods include administering NO x The method involves administering a composition containing the gas directly to the organ via an organ perfusion system or ventilation for up to 12 hours continuously. In other words, the organ is maintained in a state where it is free of NO from the time of organ procurement until the time of transplantation into the recipient. x The gas is in direct contact with the organ and is not interrupted. As used herein, procurement refers to both organ donor identification and organ removal, and either term can be used interchangeably. In some embodiments, the composition is administered after the organ is harvested from the donor. In other embodiments, the composition is administered while the organ is in the donor's body. In these embodiments, the donor may be a brain-dead or cardiac-beating donor. In some instances, administration may be for 5, 10, 15, 30, or 60 minutes. In other instances, administration may be for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more hours. xSuitable compositions containing gases are described in section (a). In a preferred embodiment, the composition is a perfusion solution, and even more preferably, a cell-free perfusion solution. In a further embodiment, the cell-free perfusion solution is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. Without wishing to be bound by theory, it is believed that the disclosed methods can increase the number of organs available for transplantation by improving the viability of organs that previously did not meet transplantation criteria, thereby allowing the use of more organs from more donors (e.g., brain-dead related donors, cardiac-beating donors, etc.). Improving organ viability for transplantation purposes may include, in part, maintaining mitochondrial function or reducing oxidative damage to the organ. 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 an exemplary embodiment, the organ is a heart, lung, or kidney.

[0053] In one embodiment, the present disclosure encompasses a method for maintaining mitochondrial function in an organ intended for transplantation. As used herein, mitochondrial function can be measured by the respiratory control ratio (RCR), which is an index of mitochondrial connectivity. Generally speaking, the RCR represents the ratio of the oxidation rate in the presence of excess substrate and adenosine diphosphate (State 3) to the oxidation rate after ADP phosphorylation to a steady-state concentration (State 4). In some embodiments, the RCR is measured by administering 20 ppm or less of NO. x By administering a composition containing the gas directly to the organ, mitochondrial function is significantly preserved in the organ intended for transplantation. As used herein, "significantly preserved" refers to the NO gas described herein. x Gas-treated organs and NO xmitochondrial function compared to control organs not treated with NO gas. In other words, significantly maintained indicates less than a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% difference in ... x It may show improved mitochondrial function compared to similar organs intended for transplant that are not gassed. x Suitable compositions comprising the gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, even more preferably an acellular perfusion fluid, and the organ is the heart, lung, or kidney. In further embodiments, the acellular perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0054] In certain embodiments, the present disclosure encompasses methods for reducing oxidative damage to organs intended for transplantation. For example, mitochondrial reactive oxygen species (mtROS) can be reduced in organs intended for transplantation. Such methods include administering 20 ppm or less of NO x The direct and continuous administration of a composition comprising a gas to the organ can occur before the organ is removed from the donor, during transport / storage, during transplantation into the recipient, after transplantation into the recipient, or any combination thereof. In a preferred embodiment, the organ is not exposed to NO gas from the time of removal until transplantation into the recipient. x As used herein, "reduction of oxidative damage" or "reduction of oxidative damage" may be measured relative to an organ treated under similar conditions, but it does not include direct and continuous NO gas contact. x 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, which may be directly and continuously administered NO. xIn certain embodiments, mitochondrial reactive oxygen species (mtROS) are increased by direct, continuous NO gas administration compared to untreated controls. x is reduced in organs with ischemia-reperfusion injury to which NO is administered. x Suitable compositions comprising the gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, even more preferably an acellular perfusion fluid, and the organ is the heart, lung, or kidney. In further embodiments, the acellular perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0055] In some embodiments, the present disclosure encompasses a method for increasing the activity of superoxide dismutase 2 (SOD2 or manganese-dependent superoxide dismutase (MnSOD)) in an organ intended for transplantation. The method comprises administering 20 ppm or less of NO x The present invention relates to a method for treating MnSOD by directly and continuously administering a composition containing NO gas to an organ, wherein the activity of MnSOD is increased in the organ compared to a control organ not contacted with the composition of the present disclosure. For example, the activity of MnSOD may be increased 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, which is directly and continuously administered with NO gas. x The direct and continuous administration can occur before the organ is removed from the donor, during transport / storage, during transplantation into the recipient, after transplantation into the recipient, or any combination thereof. In a preferred embodiment, the organ is not administered NO gas from the time of removal until the time of transplantation into the recipient. x Direct contact with the gas is maintained without interruption. Methods for measuring MnSOD activity are known in the art. xSuitable compositions comprising the gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, even more preferably an acellular perfusion fluid, and the organ is the heart, lung, or kidney. In further embodiments, the acellular perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0056] In another embodiment, the present disclosure encompasses a method for inhibiting the formation of nitrotyrosine in an organ intended for transplantation, the method comprising administering 20 ppm or less of NO x The present invention relates to a method for treating an organ comprising administering directly and continuously to an organ a composition comprising a NO gas, wherein the formation of nitrotyrosine adducts in the organ is inhibited compared to a control organ not contacted with the composition of the present disclosure. 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, which is achieved by directly and continuously administering NO gas to the organ. x The direct and continuous administration can occur before the organ is removed from the donor, during transport / storage, during transplantation into the recipient, after transplantation into the recipient, or any combination thereof. In a preferred embodiment, the organ is not administered NO gas from the time of removal until the time of transplantation into the recipient. x The gas is in direct contact with the gas without interruption. Methods for measuring the formation of nitrotyrosine adducts are known in the art. x Suitable compositions comprising the gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, even more preferably an acellular perfusion fluid, and the organ is the heart, lung, or kidney. In further embodiments, the acellular perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0057] In certain embodiments, the present disclosure encompasses a method for preventing the inactivation of mitochondrial complex I activity, complex II activity, complex III activity, complex IV activity, or a combination thereof, in an organ intended for transplantation. The method comprises administering 20 ppm or less of NO x The method comprises administering continuously to an organ a composition containing a gas containing 20 ppm or less of NO. x The administration of the gas prevents the inactivation of mitochondrial complex I, complex II, complex III, complex IV, or a combination thereof, compared to a control organ. In one embodiment, the administration of the gas at 20 ppm or less NO x Continuous administration of a gas-containing composition directly to the organ prevents inactivation of mitochondrial complex I activity. In another embodiment, 20 ppm or less of NO x Continuous administration of a gas-containing composition directly to the organ prevents inactivation of mitochondrial complex II activity. In yet another embodiment, 20 ppm or less of NO x Continuous administration of a gas-containing composition directly to the organ prevents inactivation of mitochondrial complex III activity. In another embodiment, 20 ppm or less of NO x Continuous administration of a gas-containing composition directly into the organ prevents the inactivation of mitochondrial complex IV activity. In a preferred embodiment, 20 ppm or less of NO x Continuous administration of a gas-containing composition directly to the organ prevents the inactivation of mitochondrial complex I and mitochondrial complex II activity. In another preferred embodiment, 20 ppm or less of NO x Direct and continuous administration of a composition containing NO gas to an organ prevents inactivation of mitochondrial complex II and mitochondrial complex III activity. For example, 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 an organ treated under similar conditions, which is achieved by direct and continuous administration of NO gas. xThe direct and continuous administration can occur before the organ is removed from the donor, during transport / storage, during transplantation into the recipient, after transplantation into the recipient, or any combination thereof. In a preferred embodiment, the organ is not administered NO gas from the time of removal until the time of transplantation into the recipient. x The NO gas is directly contacted with the mitochondrial complex I, complex II, complex III, or complex IV activity. Methods for measuring NO activity are known in the art. x Suitable compositions comprising the gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, even more preferably an acellular perfusion fluid, and the organ is the heart, lung, or kidney. In further embodiments, the acellular perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0058] In each of the above methods, NO is delivered to the organ via the organ perfusion system. x Direct administration of the gas-containing composition can occur over 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 via an organ perfusion system, a ventilator, or any combination thereof. In embodiments using a combined organ perfusion system and a ventilator, the organ perfusion system and the ventilator can be used simultaneously to administer NO. x Alternatively, or in addition, 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 series to deliver NO. x The gas-containing composition can be administered directly to the organ, e.g., with varying amounts of overlap between the two administration methods (e.g., no overlap, overlap of a few seconds, minutes, or hours), e.g., first via a ventilator and then via a perfusion system, or vice versa.

[0059] In a further embodiment, the method comprises administering from about 20 ppm to about 40 ppm of NO x The composition containing the gas ("loading dose") contains no more than 20 ppm NO xThe gas-containing composition is administered up to approximately 1 hour before the administration of NO x The administration of gas can include additional steps for a total of no more than 12 hours. For example, the loading dose can be administered for about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or between about 10 and about 30 minutes, in which case no more than 20 ppm NO is administered. x The gas-containing composition is administered for about 11.8 hours or less. In another example, the loading dose may be administered over about 30 minutes, about 35 minutes, about 40 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or between 30 and 60 minutes, where the gas-containing composition contains 20 ppm or less NO. 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. Alternatively, the two compositions may be different. The nitric oxide concentration in the loading dose may be gradually decreased over a period of up to about 1 hour until the nitric oxide concentration is 20 ppm or less. The rate of decrease may or may not be constant. Alternatively, the nitric oxide concentration in the loading dose may be held constant for a period of up to about 1 hour, and then decreased 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 fluid, preferably a cell-free perfusion fluid. In a further embodiment, the cell-free perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. In an exemplary embodiment, the organ is a heart, lung, or kidney.

[0060] (d) A method for improving the post-transplant performance of an organ intended for transplantation. Another aspect of the present disclosure includes a method for improving the post-transplant performance of an organ intended for transplantation, the method comprising administering 20 ppm or less of NO x The method includes administering a gas-containing composition directly to an organ via an organ perfusion system for up to 12 consecutive hours. In some instances, administration can be for 5, 10, 15, 30, or 60 minutes. In other instances, administration can be for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. xSuitable compositions containing gases are described in section (a). In a preferred embodiment, the composition is a perfusate, and even more preferably a cell-free perfusate. Improving the post-transplant performance 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 an exemplary embodiment, the organ is a heart, lung, or kidney.

[0061] In one embodiment, the present disclosure encompasses a method for maintaining mitochondrial function in a transplanted organ. As used herein, mitochondrial function can be measured by the respiratory control ratio (RCR), which is an index of mitochondrial connectivity. Generally speaking, the RCR represents the ratio of the oxidation rate in the presence of excess substrate and adenosine diphosphate (State 3) to the oxidation rate after ADP phosphorylation to a steady-state concentration (State 4). In some embodiments, the RCR is measured by administering 20 ppm or less of NO. x By administering a composition containing NO gas directly to the organ, mitochondrial function is significantly maintained in the organ after transplantation. As used herein, "significantly maintained" refers to the NO gas composition described herein. x Gas-treated organs and NO x mitochondrial function compared to control organs that had undergone similar ischemia-reperfusion injury but had not been directly or continuously treated with NO gas. x This may show improved mitochondrial function compared to similar organs not receiving the gas. xSuitable compositions comprising the gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, even more preferably an acellular perfusion fluid, and the organ is the heart, lung, or kidney. In further embodiments, the acellular perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0062] In other embodiments, the present disclosure encompasses a method for reducing oxidative damage in a transplanted organ. Generally speaking, the method comprises administering 20 ppm or less of NO x As used herein, "reduction of oxidative damage" or "reduction of oxidative damage" may be measured relative to an organ treated under similar conditions, but it may be measured by administering directly and continuously NO gas to the organ. x 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, which may be directly and continuously administered NO. x In certain embodiments, mitochondrial reactive oxygen species (mtROS) are increased by direct continuous NO gas administration compared to untreated controls. x is reduced in transplanted organs to which NO is administered. x Suitable compositions comprising the gas are described in section (a). In preferred embodiments, the composition is a perfusion fluid, even more preferably an acellular perfusion fluid, and the organ is the heart, lung, or kidney. In further embodiments, the acellular perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0063] In a further embodiment, the method comprises administering from about 20 ppm to about 40 ppm of NO x The composition containing the gas ("loading dose") contains no more than 20 ppm NO x The gas-containing composition is administered up to approximately 1 hour before the administration of NOx The administration of gas can include additional steps for a total of no more than 12 hours. For example, the loading dose can be administered for about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or between about 10 and about 30 minutes, in which case no more than 20 ppm NO is administered. x The gas-containing composition is administered for about 11.8 hours or less. In another example, the loading dose may be administered over about 30 minutes, about 35 minutes, about 40 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or between 30 and 60 minutes, where the gas-containing composition contains 20 ppm or less NO. 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. Alternatively, the two compositions may be different. The nitric oxide concentration in the loading dose may be gradually decreased over a period of up to about 1 hour until the nitric oxide concentration is 20 ppm or less. The rate of decrease may or may not be constant. Alternatively, the nitric oxide concentration in the loading dose may be held constant for a period of up to about 1 hour, and then decreased 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 fluid, preferably a cell-free perfusion fluid. In an exemplary embodiment, the organ is a heart, lung, or kidney. In a further embodiment, the cell-free perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin.

[0064] (e) Transplant method In another aspect, the present disclosure provides a method of implantation, the method comprising: (a) administering 20 ppm or less NO xThe method includes (a) administering a gas-containing composition directly to an organ intended for transplantation continuously for up to 12 hours, and (b) transplanting the organ into a recipient. Administration can be for 5, 10, 15, 30, or 60 minutes. Alternatively, administration can be for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 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 comprises 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 comprising the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, and even more preferably, an acellular perfusion solution. In a further embodiment, the acellular perfusion solution is Steen Solution™, optionally comprising sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. In an exemplary embodiment, the organ is a heart, lung, or kidney.

[0065] In certain embodiments, the organ intended for transplantation has been removed from the donor prior to step (a) above. In these embodiments, administration begins some time after the onset of ischemia, but preferentially begins 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, the method comprises adding about 20 ppm to about 40 ppm NO x The composition containing the gas ("loading dose") contains no more than 20 ppm NO xThe method may include an additional step of administering a gas-containing composition for up to about 1 hour (the "loading period") immediately prior to administration. For example, the loading dose may be administered for about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or between about 10 and about 30 minutes. In another example, the loading dose may be administered for about 30 minutes, about 35 minutes, about 40 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or between 30 and 60 minutes. The two compositions may be identical except for the nitric oxide concentration. Alternatively, the two compositions may be different. The nitric oxide concentration in the loading dose may be gradually decreased over the loading period until the nitric oxide concentration is 20 ppm or less. The rate of decrease may or may not be constant. Alternatively, the nitric oxide concentration in the loading dose may be held constant over the loading period and then decreased 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 fluid, preferably a cell-free perfusion fluid. In a further embodiment, the cell-free perfusion fluid is Steen Solution™, optionally containing sodium caprylate, N-acetyl-DL-tryptophan, and human albumin. In an exemplary embodiment, the organ is a heart, lung, or kidney.

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

[0068] Example 1: Lung Transplant Protocol This study will include a total of 20 lungs (8 lungs with gNO and perfusate, 8 lungs with perfusate only, and 4 lungs with gNO ventilation and perfusate) according to modified standard lung donor selection criteria. A three-arm study will include gNO added to the perfusate, perfusate only, and gNO ventilation and perfusate (pilot study). The XVivo perfusion device system with acellular perfusate (Steen solution) will be used. The maximum cold ischemia time for the lungs will be 8-10 hours. The duration of ex-vivo lung perfusion will be up to 12 hours. Lung health will be assessed by a scoring system, biomarker evaluation, and histopathological assessment.

[0069] Rating System The rating system includes a 0-10 rating (total score) using a composite scale of three variables: 1) delta PaO2 weighted from 0 to 4 using four categories: 0 = <350 mmHg; 1 = ≥350 to <400 mmHg; 2 = ≥400 to <450 mmHg; 3 = ≥450 to <500 mmHg; and 4 = ≥500 mmHg; 2) pulmonary static compliance weighted from 0 to 4 (change from baseline): 0 = no improvement or worsening 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 from 0 to 2: 0 = no change or increase in PVR; 1 = 1-7% decrease in PVR; and 2 = 8-15% decrease in PVR.

[0070] Biomarker evaluation Biomarkers evaluated include damage-assessing molecules (DAMPs), high-mobility group box-1 (HMGB1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), and serum amyloid A (SAA). Cytokines of interest include TNFα-1, IL1-β, IL-6, NLRP3, IL-10, and donor cell-free DNA.

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

[0072] Donor lung procurement Current clinical practice after organ retrieval from a donor is for static storage at low temperature until transplantation into a recipient. During retrieval, the lung undergoes a hypothermic lung washout using a low-potassium dextran preservation solution in combination with topical cooling and lung ventilation. The lung is then transported at 4°C in a statically inflated state. Hypothermia, in the face of ischemia, reduces metabolic activity while preserving cellular viability, essentially slowing the process of cell death (5% of metabolic rate at 37°C). Therefore, hypothermic storage is the mainstay of prior art lung preservation. However, there is a significant reduction in organ metabolic function, thereby preventing meaningful lung evaluation and recovery.

[0073] The donor lung procurement technique for this study was as follows: Bronchoscopy and median sternotomy were performed. The pericardium and multiple cavities were opened. Both lungs were recruited and PO2 was assessed with a 100% FiO2 challenge. The system was heparinized. A purse string suture was placed in the main pulmonary artery (PA). The PA was cannulated through the purse string. The cannula was deflated and connected to deflated Perfadex tubing (the Perfadex bag should only drain by gravity (non-pressurized), and the bag should not be higher than 1 meter above the lungs). 500 mcg of Alprostidil was administered directly into the main PA. The superior vena cava (SVC), patented left and right atria, and cross-clamped aorta were ligated.

[0074] Deliver 4 liters of antegrade Perfadex. Ensure rapid and consistent drainage of both the right and left atria. Apply topical ice to both lungs (but use only one lung per cycle). Maintain ventilation with room air FiO2 at a tidal volume of 4-6 ml / kg / min and a respiratory rate of 10 breaths per minute. Once the infusion is complete, excise the heart. If the heart is transplanted, incise the PA at the PA bifurcation. Do not divide the PA bifurcation. If the heart is not transplanted, incise the PA at the RVOT just proximal to the pulmonary valve. Ensure the left atrial cuff is still intact. Inject cold Perfadex (2 liters total) into the retrograde pulmonary veins. Incise the pericardium bilaterally at the level of the diaphragm and transect it to completely separate it from the diaphragm. Incise the inferior pulmonary ligaments on both sides. Dissect the posterior pericardium from the posterior longitudinal membrane cranial to the tracheal carina. Dissect the trachea cranially down to the level of the cricoid cartilage. Retract the endotracheal tube to the level of the larynx. Inflate the lungs to 50% of total lung capacity at 50% FiO2. Divide the trachea above the cricoid cartilage with two staple loads. Dissect the posterior trachea caudal to the posterior mediastinum (esophagus) towards the diaphragm. Dissect the left PA from the aortic arch and divide the ligamentum arteriosum. Remove the lungs and store them in ice-cold Perfadex solution. If a heart is being procured for transplant, resect 10 cm of the donor's descending aorta and place it in the same cold Perfadex solution bag as the lungs. This will later be used to reconstruct the pulmonary artery.

[0075] Ex-vivo lung perfusion (EVLP) The main principle of EVLP consists of perfusing and ventilating the lung outside the body in a sealed container that maintains the temperature, moisture, and sterility of the donor organ. The EVLP circuit consists of a centrifugal pump that circulates the perfusate through a membrane gas exchanger and a leukocyte-removal filter before entering the pulmonary artery (PA). This system is very similar to that used in extracorporeal circuits (ECC) in cardiac surgery. Acellular Steen's solution is used as the perfusate in the EVLP process.

[0076] Briefly, the EVLP process is as follows: The lungs are placed in a specially lined plastic chamber to hold them in a stable position during ventilation and to provide a warm, moist environment. Perfusate (propelled by a centrifugal pump) enters the lungs through a cannula placed in the pulmonary artery (PA). Return flow from the lungs is passive via the pulmonary veins (PV) by gravity, and the perfusate is collected in a reservoir before being recirculated through the 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%). An airway to the lungs is established by a ventilator and is connected to a tracheal chest tube.

[0077] Lung grafts (cooled during organ harvest and undergo static hypothermic storage) are gradually rewarmed over 45 minutes. Pulmonary perfusion is initiated at a low flow rate (0.10–0.15 L / min) and then gradually increased to a pulmonary arterial supply of 40–50% of estimated cardiac output (at 70 mL / kg / min) in parallel with rewarming. PA pressures should be kept low (<15–20 mmHg) once the capillary-alveolar barrier is weakened by ischemia-reperfusion injury (IRI) after organ procurement, as IRI can compromise the integrity and increase the permeability of the alveolar-capillary membrane, leading to the formation of pulmonary edema.

[0078] During the rewarming phase, O2 supply to the graft is provided by a membrane oxygenator, which then provides pCO2 and pH close to normal measurements in the PA. Catheters are placed in situ to continuously measure PA and left atrial (LA) pressure throughout the EVLP process. Once the perfusate temperature reaches 32 degrees Celsius (usually about 30 minutes after the start of perfusion), mechanical ventilation of the lungs is initiated.

[0079] Below is a 19-step progression of the EVLP process. This is not a comprehensive protocol, but rather a quick reference guide.

[0080] Step 1. Transporting the lungs to XPS. Compliant with UNOS standards.

[0081] Step 2. Pulmonary cannulation. Trim the LA cannula (green) to fit the appropriate atrial opening and suture it with a continuous polypropylene suture. The PA cannula (yellow) opens the PA lumen for insertion of the XVIVO cannula. If pulmonary artery reconstruction is required, suture a segment of the donor's descending aorta to the pulmonary artery, then cannulate as described above. Use umbilical tape or a silk tie to secure the cannula in place on the globe and return the tie to the cannula below the pressure line. During intubation, clamp the trachea to prevent constriction. Insert an ET tube into the trachea and secure it with umbilical tape or a silk tie.

[0082] Step 3. Flush the back table with 1 liter of cold Perfadex and check for leaks around the cannulation. Keep the ET tube clamped.

[0083] Step 4. System Setup. Set up the XPS perfusion circuit on the PGM. Open the dome by removing the plastic, then open the first blue layer wrapping on the back table. Place a clear U-shaped drape on the top bar of the XPS on the blue table and secure it with the XPS w-clips covering the blue table. Place the "U" opening of the U-shaped drape on the right side 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 thread the quarter line around the outside of the pole and into the red roller pump. Release 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 stopcock. Close the two-way stopcock to the reservoir at the red line and open it to the drain bag. Set up the transducer, flush with sterile saline, add the blue tubing to the heater / cooler, and then add the intravenous gas line to the back of the Quadrox. Ensure there is sufficient O2 and trigger gas.

[0084] Step 5. Purge the system. Add 1500 ml of STEEN to the reservoir and add medications (heparin 10,000 units, methylprednisolone 500 mg, ceftazidime 1 gm). Turn on Cardiohelp and the 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 one 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 one minute, increase Cardiohelp to 3000 RPM. After a few minutes, increase Cardiohelp to 5000 RPM and run it for one minute to ensure all air is 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 (set #3 to 10 minutes)). Press Set for Ventilator, Mode (S) CMV+, and Confirm. Enter the spreadsheet parameters and confirm. Also, set alarms for each flowsheet parameter. On the I / O panel on the back of the XPS, place 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: white to clear and blue to blue. Connect the venous gas blend line. Connect the high-pressure O2 line. Place the humidification filter at the lung end of the circuit between the flow sensor and the ET-T. Perform pre-op checks, press System, and then press Test & Calibrate. Select each calibration test one at a time. Airtightness: Press the following steps. Flow Sensor: Press the following steps. O2 Cell: No need to test.

[0086] Step 7. Retrograde flush. Using a sterile tubing clamp, change the flow direction in the perfusion circuit so that flow is forward in the LA and out the PA. Starting at 750 RPM, slowly increase the RPM until there is enough flow to fill the PA cannula and flush the lungs until there is approximately 250 cc of STEEN solution, excluding blood. Use the recycle pump to direct this STEEN solution into the dump bag. Once the blood has been removed, attach the cannula and circuit to the LA side and ensure the lungs are filled with STEEN solution. There should be enough STEEN through the LA cannula to fill the PA cannula, then reset the recycle pump direction 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 and pressure sensors. (The flow sensor can be done before the lung is placed on the circuit for retrograde flow.) The pressure sensor should be done immediately after retrograde flow and the lung is on the circuit (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 sure they are all at the same height, fill with STEEN, and move the two-way stopcock to ensure it is shut off from saline).

[0088] Step 9. Antegrade. Start Timer 1 (perfusion timer) and follow the EVLP workup sheet for settings for the first hour.

[0089] Step 10. First hour settings. Every 10 minutes, Cardiohelp and the heater / cooler are changed until they reach their maximum values. Ventilation cannot begin until the temperature reaches 32°C. At 32°C, the bronchi may be treated. The trigger gas sweep must begin simultaneously with the start of the ventilator.

[0090] Step 11. Lung Refill. To avoid confusion with the O2 challenge, lung refill is performed by manually holding the exhalation key on the ventilator for 15 seconds. To hold exhalation, this key should be pressed after inspiration.

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

[0092] Step 13. Venting. At the final moment of the O2 challenge, arterial and venous perfusate samples should be taken from the transducers. Press the gray PH button on the main screen to lock the gas values. (When results are returned from the lab, perform pinpoint calibrations for PH in both the LA and PA.) Record the appropriate information, then reset to normal settings on the ventilator (keep alarms and settings high at this point).

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

[0094] Step 15. Diluting STEEN. After refilling, challenge, gas aspiration, and x-ray are complete (or between x-rays), diluting STEEN is required. Open the + / - Pump Control window on the touchscreen. Touch the Remove button and slide your finger down until the Remove button locks (if the clamp method is used, remove the clamp instead). Watch the reservoir level drop. Once enough STEEN has been removed, touch the Remove button to shut off (replace the clamp if the clamp method is used). Add three or four fresh vials of STEEN. You can also add another dose of medication.

[0095] Step 16. Maintain and assess lungs for the next 3-5 hours. At the 50 minute mark of each hour, repeat steps 12 and 13 (subtract pinpoint calibration).

[0096] Step 17. Repeat step 14 approximately 1 hour before harvesting the lung for transplant. Comparing the first and second x-rays will help determine if the lung is suitable for transplant.

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

[0098] Step 19. Rapid cooling. Once the lung is accepted for transplant, set the heater / cooler to 15°C. When the lung is 50% inflated at 32°C, clamp the ET tube, disconnect the lung from the perfusion circuit, flush it with 2 L of cold Perfadex, and place it in a sterile bag with the Perfadex (as per standard donor procurement protocol).

[0099] Step 20. Clean and store the XPS. Wipe down the surface of the XPS with disinfectant, store it with the toggle switch in the up position, and plug it in to charge. Gas should be drawn at the end of each 10-minute challenge.

[0100] For reference, 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 study.

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

[0103] method Four-week-old pigs were asphyxiated for 7 minutes and then induced into 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, with protocolized care following ROSC. Subjects were randomized in a blinded fashion (initiating a 1-minute CPR period with 20 ppm iNO, n = 10, or placebo, n = 10). Sham controls (n = 4) did not undergo CA or CPR. Baseline and continuous CBF measurements were performed using invasive clinical and noninvasive optical devices. Cortical and hippocampal tissues were analyzed by high-resolution respirometry to assess mitochondrial function. T-tests and ANOVAs were used where appropriate. Longitudinal hemodynamic variables were compared using generalized estimating equations to control for within-subject correlations.

[0104] result Seven out of 10 placebo and 10 out of 10 iNO patients (p=0.21) survived. There were no significant differences in invasive or noninvasive CBF between treatment groups during CPR or after ROSC. Cortical and hippocampal RCRs were 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 during CPR tended to be lower in iNO-treated animals (28.1±9.8 vs. 42.6±6.0, p=0.14). iNO preserves cerebral mitochondrial function (increased RCR) and limits mtROS production in a porcine model of pediatric CA. Further studies are needed to evaluate this potential neuroprotective effect of iNO during ischemia-reperfusion injury and cardiac arrest.

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[Claim 1] Systems, devices, methods, etc.

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