Systems and methods for producing medical solutions with dissolved gas concentrations

Abstract

This disclosure relates to systems and methods for providing a perfusion solutions with dissolved hydrogen gas. Systems include a solution container having a fill tube, a gas transport tube, and an access tube; and a heating chamber sufficiently sized to house the solution container and apply heat sufficient to increase a temperature of a medical solution within the solution container to at least 100°C. The solution container comprises a headspace absent the medical solution and allows sufficient escape of dissolved gas from the medical solution upon introduction of the medical solution into the solution container. The gas transport tube comprises a hydrophobic filter or porous membrane in line with a valve configured to allow for control over gas egress from and gas ingress into the solution container. Perfusion solutions include a predetermined composition of dissolved gas having a pH2 greater than a pO2 and the pO2 greater than a pN2.

Description

[0001] Docket No. 220709-702601

[0002] SYSTEMS AND METHODS FOR PRODUCING MEDICAL SOLUTIONS WITH DISSOLVED GAS CONCENTRATIONS CROSS-REFERENCED APPLICATION

[0003] [1] This application claims the benefit of priority to U.S. Provisional Application No.

[0004] 63 / 761,711, filed on February 21, 2025, the entire contents of each of which are incorporated herein by reference.

[0005] BACKGROUND

[0006] [2] Ischemia Reperfusion Injury (IRI) is the primary driver of poor outcomes in organ transplant medicine. Organs donated for transplantation are susceptible to IRI due to oxidative stress arising from ischemia during organ recovery, transport, and grafting; and especially during the first 5 - 15 minutes of reperfusion in the organ recipient’s body.

[0007] [3] At present, most organ transplant protocols and technologies aim to reduce the effects IRI by minimizing one or more of the following factors: severity of hypoxia, total ischemic time, donor age, and donor risk factors such as smoking, diabetes, and other disease. Despite the best efforts, IRI remains a predominant factor in determining transplant outcomes.

[0008] [4] IRI can also be mitigated by antioxidant therapy, whereby antioxidants are administered to the organ prior to onset of acute oxidative stress, or during prolonged periods of mild-moderate oxidative stress. For organ transplant, the most effective antioxidant therapies involve pre-loading the organ tissue with a sufficient antioxidant concentration to combat the rapid production of damaging free radicals and reactive oxygen species (ROS), which peaks during the initial phases of reperfusion.

[0009] [5] Hydrogen gas (H2) is a known antioxidant that can be dissolved into donated organs to protect against IRI. Antioxidant effects of hydrogen have been reported in biological systems at concentration levels ranging from 15 ppb up to 1600 ppb (1% saturation up to 100% full saturation of H2 in water at one atmosphere). Nontoxicity, rapid transport through cellular barriers, and selective reactivity combine to make hydrogen a uniquely effective antioxidant in the IRI context.

[0010] [6] The beneficial effects of hydrogen follow a positive dose-response relationship; i.e., there is a positive correlation between hydrogen concentration in the tissue and the observed beneficial effect. Dose dependency is most pronounced in cases of acute oxidative stress wherein high ROS production rates are most effectively countered by a similarly high concentrations of dissolved hydrogen. In the context of organ transplant, effectiveness of hydrogen therapy is therefore maximized when the hydrogen concentration in the tissue is as high as is practically achievable,Docket No. 220709-702601

[0011] and the high hydrogen concentration is timed to coincide with the most acute phase of ROS production in the first 5 - 15 minutes of reperfusion.

[0012] BRIEF SUMMARY

[0013] [7] In view of the foregoing, there is a need for improved systems and methods of safe infusion of highly concentrated hydrogen gas into solutions for delivery to patients, or donated organs and tissues, to reduce or prevent the occurrence of oxidative stress, while simultaneously reducing or preventing a risk of dangerous levels of hydrogen subject to combustion. An additional problem to be solved is to avoid stripping the organ of dissolved oxygen during the infusion of hydrogen. Since the hydrogen infusion step is followed by a period of ischemia (an hours-long period of cold ischemia during storage, and / or a 0.5 - 1 hour long period of warm ischemia during surgical engraftment), depleted oxygen levels in the organ after the hydrogen infusion step may increase the severity of hypoxia and IRI in the organ.

[0014] [8] The systems and methods of the present disclosure address these needs and provide additional advantages as well. This section includes a summary of the claims in the commonly accepted definition of a comprehensive and usually brief recapitulation of the claims.

[0015] [9] In some aspects, the techniques described herein relate to a method for producing a hydrogen-rich medical perfusion solution, the method including, providing a medical perfusion solution in a container; degassing the medical perfusion solution to remove nitrogen and oxygen from the medical perfusion solution until a combination of a partial pressure of nitrogen and a partial pressure of oxygen in the medical perfusion solution is between 0 to 150 mm Hg; and adding hydrogen to the medical perfusion solution; and optionally, adding oxygen to the medical perfusion solution, after the degassing. In some aspects, the degassing includes using one of a vacuum, ultrasonic energy, heat, or a combination thereof to reduce a concentration of dissolved nitrogen in the medical perfusion solution. In some aspects, the adding oxygen includes adding dissolved oxygen to the medical perfusion solution to achieve a predetermined concentration of dissolved oxygen. In some aspects, the degassing replaces a portion of dissolved nitrogen with dissolved oxygen prior to the adding hydrogen. In some aspects, the degassing includes filling the container with the medical perfusion solution and purging at least one gas from a headspace of the container. In some aspects, the degassing further includes heating the container to above 100°C and purging the at least one gas from at least one valve in fluid communication with the headspace of the container. In some aspects, the purging is performed after the container has returned to a temperature below 100°C. In some aspects, the degassing further includes adding a predetermined amount of at least one of H2, 02, or a combination thereof to the headspace using the at least one valve. In some aspects, the adding is performed using at least one of: injection directly into theDocket No. 220709-702601

[0016] container, or a gas-filled and gas-permeable second container that is co-packaged with the medical perfusion solution inside a third gas-impermeable container. In some aspects, after the adding hydrogen, the medical perfusion solution has a hydrogen partial pressure (pH2) of at least 0.7 atm. In some aspects, after the adding oxygen, the medical perfusion solution has an oxygen partial pressure (pO2) of at least 0.7 atm. In some aspects, a ratio of the hydrogen to the oxygen is controlled to produce a combined solution with a predetermined partial pressure of each of the hydrogen and the oxygen. In some aspects, a ratio of the hydrogen to the oxygen in the medical perfusion solution ranges from 1 : 1 to 9: 1. In some aspects, the container is subsequently packaged in an outer gastight container. In some aspects, the medical perfusion solution is a denitrogenated solution.

[0017]

[0010] In some aspects, the techniques described herein relate to a system for producing medical solutions with dissolved hydrogen gas, the system including: at least one solution container having at least one fill tube, at least one gas transport tube, and at least one access tube; and a heating chamber sufficiently sized to house the at least one solution container and apply heat sufficient to increase a temperature of a medical solution within the at least one solution container to at least 100°C, wherein the at least one solution container includes a headspace absent the medical solution and allows sufficient escape of dissolved gas from the medical solution upon introduction of the medical solution into the at least one solution container, wherein the at least one gas transport tube includes a hydrophobic filter or porous membrane in line with at least one valve configured to allow for control over gas egress from the at least one solution container and gas ingress into the at least one solution container; and wherein, upon heating of the medical solution in the heating chamber, at least one dissolved gas is separated from the medical solution into the headspace such that the at least one dissolved gas is removable via the at least one gas transport tube upon opening of the at least one valve, thereby allowing for a sufficient amount of an additional gas composition to be introduced into the headspace so as to generate a predetermined composition of dissolved gas in the medical solution, the predetermined composition of dissolved gas having a partial pressure of dissolved hydrogen (pH2) greater than a partial pressure of dissolved oxygen (pO2) and the pO2 greater than a partial pressure of dissolved nitrogen (pN2). In some aspects, the at least one gas transport tube includes a hermetically sealable portion. In some aspects, the hydrophobic filter includes a pore size ranging from about 0.15 to about 0.3 micron. In some aspects, the at least one valve includes a bidirectional check valve. In some aspects, the at least one valve is closer to an external portion of the at least one gas transport tube than the hydrophobic filter. In some aspects, each of the at least one fill tube is separate from each of the at least one gas transport tube. In some aspects, the heating chamber is selected from an oven, an autoclave, a humidity chamber, a thermal shock chamber, a dry heat sterilizer, a heated ultrasonic bath, or aDocket No. 220709-702601

[0018] combination thereof. In some aspects, a system further includes a temperature controlling feature configured to maintain a temperature of the medical solution. In some aspects, a system further includes one or more gas sensors positioned to measure concentrations of dissolved oxygen or dissolved hydrogen in an egressing gas or a headspace of the medical solution, optionally wherein the one or more gas sensors comprise one or more dissolved gas sensors, one or more solution flow sensors, temperature sensors, optical sensors, or a combination thereof.. In some aspects, the concentrations of dissolved hydrogen and / or dissolved oxygen in the effluent medical solution are compared to a reference value so as to achieve a predetermined concentration of at least one of dissolved oxygen or dissolved hydrogen in the medical solution.

[0019]

[0011] In some aspects, the techniques described herein relate to a perfusion solution, the perfusion solution including, a fluid having a dissolved gas mixture of hydrogen, oxygen, and nitrogen, wherein a partial pressure of the dissolved hydrogen (pH2) is higher than 350 mm Hg, the partial pressure of the dissolved oxygen (pO2) ranges from 45 mm Hg to 300 mm Hg, and the partial pressure of dissolved nitrogen (pN2) is lower than a pO2 in the solution, wherein contact of the perfusion solution with an organ or tissue increases an amount of dissolved hydrogen and does not decrease an amount of dissolved oxygen within the organ or tissue as compared to the organ or tissue prior to contacting with the perfusion solution, wherein a predetermined concentration of dissolved oxygen in the organ or tissue is maintained over a course of hydrogen infusion so as to prevent a worsening of hypoxia or oxidative stress within the organ or tissue during the contact of organ or tissue with solution. In some aspects, the decrease in the amount of dissolved 02 results in a reduced occurrence of oxidative stress as compared to the organ or tissue prior to the contacting. In some aspects, the hypoxia or oxidative stress is represented by one or more of acute tubular necrosis (ATN), apoptotic cell death, Kidney Injury Marker 1 (KIM-1), Interleukin 6 (IL-6), CD68(+), Myeloperoxidase (MPO), Malondialdehyde (MDA), or a combination thereof. In some aspects, the partial pressure of the dissolved hydrogen is between about 450 mm Hg and about 500 mm Hg, and wherein the partial pressure of the dissolved oxygen is at least 1.5 times greater than the partial pressure of dissolved nitrogen. In some aspects, the fluid includes a University of Wisconsin (UW) solution or an organ or tissue preservation solution. In some aspects, a solution further includes one or more chelating agents selected from ethylenediaminetetraacetic acid (EDTA), Desferrioxamine (DFO), citric acid, Dimercaptosuccinic Acid (DMSA), or a combination thereof, wherein the one or more chelating agents are at concentrations ranging from 10-6 Molar to 10-4 Molar. In some aspects, the pN2 is less than 0.2 atm. In some aspects, the pN2 is less than 0.1 atm. In some aspects, a pO2 is greater than pN2 by a magnitude of at least 3x. In some aspects, a pH2 is greater than pO2 by a magnitude of at least 2x and greater than pN2 by a magnitude of at least 3x. In some aspects, when a concentration ofDocket No. 220709-702601

[0020] the hydrogen ranges from 40% to 80%, a pO2 is greater than a combined partial pressures of other gases, the other gases excluding the hydrogen and the oxygen. In some aspects, a solution further includes a bicarbonate buffer, wherein a partial pressure of carbon dioxide (pCO2) ranged from 0.04 atm - 0.06 atm, a pH2 ranges from 0.6 atm - 0.8 atm, and a pO2 ranges from 0.15 atm - 0.35 atm. In some aspects, a solution further includes one or more hydrogen sulfide donors selected from sodium thiosulfate (STS), sodium sulfide (Na2S), sodium hydrosulfide (NaHS), or a combination thereof, wherein a concentration of the one or more hydrogen sulfide donors ranges from 0.1 micromolar to 50 micromolar. In some aspects, the gas mixture in the fluid includes a binary mixture of hydrogen and oxygen.

[0021]

[0012] In some aspects, the techniques described herein relate to a method of producing medical solutions with dissolved hydrogen gas, the method including, providing an outer gas impermeable container and at least one inner gas permeable container; packaging a medical solution into the at least one inner gas permeable container, the medical solution being previously depleted of nitrogen having an amount of oxygen gas in a headspace of the at least one inner gas permeable container; and arranging the at least one inner gas permeable container and a predetermined amount of hydrogen gas within the outer gas impermeable container, wherein the predetermined amount of the hydrogen gas is sufficient to permeate into the at least one inner gas permeable container to equilibrate with the oxygen gas such that the medical solution has a predetermined dissolved gas composition, the predetermined dissolved gas composition having a partial pressure of dissolved hydrogen (pH2) higher than 350 mm Hg and a partial pressure of dissolved oxygen (pO2) ranging from 45 mm Hg to 300 mm Hg. In some aspects, the at least one inner gas permeable container includes a first inner gas permeable container and a second inner gas permeable container, wherein the medical solution is packaged into the first inner gas permeable container and the predetermined amount of hydrogen gas is packaged into the second inner gas permeable container. In some aspects, the at least one inner gas permeable container is transparent, flexible, sterile, or a combination thereof. In some aspects, a method further includes infusing the medical solution into a machine perfused organ or tissue. In some aspects, the infusing includes adjusting a concentration of dissolved nitrogen and dissolved oxygen in the machine perfused organ or tissue, the adjusting including administering a sweep gas prior to cessation of machine perfusion in preparation for hydrogen infusion, wherein the sweep gas is adjusted to an N2:O2 ratio of up to about 0.5. In some aspects, the sweep gas includes a mixture of N2 and 02.

[0022]

[0013] In some aspects, the techniques described herein relate to a method for producing a medical solution with substantially binary H2 / O2 dissolved gas mixture wherein an outer package contains a kit including: at least one container of oxygen- saturated or oxygen-rich solution (pO2 > 0.5 atm); and a gas-tight container of hydrogen gas to be combined with the oxygen-rich solution prior toDocket No. 220709-702601

[0023] using the medical solution. In some aspects, the outer package includes a gas-impermeable barrier. In some aspects, the at least one container includes a flexible material.

[0024]

[0014] In some aspects, the techniques described herein relate to a method for producing a medical solution with substantially binary H2 / O2 dissolved gas mixture wherein an outer package contains a kit including: at least one container of hydrogen-rich solution having a partial pressure of dissolved hydrogen (pH2) greater than 0.5 atm; and a gas-tight container of an oxygen gas, wherein an oxygen gas to be combined with the hydrogen-rich solution prior to using the hydrogen-rich solution. In some aspects, the outer package includes a gas-impermeable barrier. In some aspects, the at least one container includes a flexible material.

[0025]

[0015] In some aspects, the techniques described herein relate to a method for using binary dissolved gas mixtures of hydrogen and oxygen in a medical solution, the method including: combining a hydrogen-rich solution and an oxygen-rich solution within a container to form the binary dissolved gas mixture, wherein a volume ratio of hydrogen-rich solution to oxygen-rich solution ranges from 1:1 to 9:1. In some aspects, a concentration of glutathione is higher in the hydrogen-rich solution than in the oxygen-rich solution. In some aspects, the combining is performed at a time of use or immediately prior to the time of use. In some aspects, the combining includes heating the container to above 100°C and purging at least one gas from a headspace of the container. In some aspects, the purging is performed after the container has returned to a temperature below 100°C.

[0026]

[0016] In some aspects, a solution includes a fluid having a dissolved gas mixture of hydrogen, oxygen, and nitrogen, wherein a partial pressure of the hydrogen is higher than 350 mm Hg, the partial pressure of the oxygen ranges from 45 mm Hg to 300 mm Hg, and the partial pressure of dissolved nitrogen is lower than a concentration of the dissolved oxygen in the solution. In some aspects, the partial pressure of the hydrogen can be between about 450 mm Hg and about 500 mm Hg, and the partial pressure of the oxygen can be at least 1.5 times greater than the partial pressure of dissolved nitrogen.

[0027]

[0017] In some aspects, the techniques described herein relate to a method for producing a hydrogen-rich solution, the method including, providing a solution; degassing the solution to remove nitrogen and oxygen until the combination of PN2 and PO2 is between 0 to 150 mm Hg; adding hydrogen to the solution; and adding oxygen to the solution, after the degassing step. In some aspects, the degassing step can include using one of a vacuum, ultrasonic energy, heat, or a combination thereof to reduce the concentration of dissolved nitrogen in the solution. The adding oxygen step can include adding dissolved oxygen to the solution to achieve a physiologically appropriate concentration of dissolved oxygen. The degassing step can replace a portion of dissolved nitrogen with dissolved oxygen prior to the adding hydrogen step.Docket No. 220709-702601

[0028]

[0018] In some aspects, the techniques described herein relate to a method of producing medical solutions with dissolved hydrogen gas, the method including, injecting the dissolved hydrogen gas into a headspace of the container holding a degassed or denitrogenated solution. In some aspects, the container can be subsequently packaged in an outer gastight container.

[0029]

[0019] In some aspects, the techniques described herein relate to a method of producing medical solutions with dissolved hydrogen gas, the method including, packaging a nitrogen-depleted solution in an inner gas permeable container, arranging the inner gas permeable container within an outer gastight container, wherein the dissolved hydrogen is added to the medical solution via a second gas permeable container with hydrogen gas adjacent the gas permeable solution container and within the outer gastight container.

[0030]

[0020] In some aspects, the techniques described herein relate to a medical solution method of manufacturing, the method including, packaging a volume of nitrogen-depleted solution in a first gas permeable container; packaging a gas mixture containing hydrogen and oxygen in a second gas permeable container; and sealing the first and second gas permeable containers within an outer gas tight container.

[0031]

[0021] In some aspects, the techniques described herein relate to an organ perfusion system, the system including, a hydrogen infusion system, the hydrogen infusion system including a container of hydrogen infusion solution, a temperature controlling feature configured to maintain the temperature of the hydrogen infusion solution, and a shutoff valves to prevent flow of hydrogen infusion solution into the gas exchanger of the perfusion system. In some aspects, systems further include one or more dissolved gas sensors positioned to measure concentrations of dissolved oxygen and / or dissolved hydrogen in the effluent hydrogen infusion solution as it flows out of the organ or tissue. In some aspects, the measurement of dissolved hydrogen and / or dissolved oxygen in the effluent solution is compared to a reference value, to shut off or otherwise modify the flow of the hydrogen infusion solution to achieve the desired concentration of dissolved oxygen or dissolved hydrogen (or both) in the organ or tissue.

[0032]

[0022] In some aspects, the techniques described herein relate to a method for adjusting concentrations of dissolved nitrogen and dissolved oxygen in a machine perfused organ or tissue, the method including, administering a sweep gas prior to cessation of machine perfusion in preparation for hydrogen infusion, wherein the sweep gas is adjusted to an N2:O2 ratio of about 0.5.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034]

[0023] The novel features of the disclosure are set forth with particularity in the appended claims. The patent or application file contains at least one drawing executed in color. Copies of this patentDocket No. 220709-702601

[0035] or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0036]

[0024] FIGURE 1A illustrates exemplary scenarios of hydrogen outgassing rates from a static system as modeled by an exponential decay curve. FIGURE IB illustrates an experiment conducted by the inventor, where ischemic pig kidneys were exposed to standard flush solutions as well as two different hydrogen infusion solutions prior to hypothermic machine perfusion followed by warm oxygenated machine reperfusion. FIGURES 1C-1F illustrate prior art methods of infusing H2 into a fluid or organ.

[0037]

[0025] FIGURE 2 illustrates an embodiment of an HI system according to exemplary embodiments of this disclosure.

[0038]

[0026] FIGURE 3 illustrates an embodiment of N2 purging according to exemplary embodiments of this disclosure.

[0039]

[0027] FIGURES 4A-4B illustrate exemplary processes for manufacturing of a HI solution according to exemplary embodiments of this disclosure.

[0040]

[0028] FIGURES 5A-5B illustrate exemplary embodiments of packaging components for an HI solution according to exemplary embodiments of this disclosure.

[0041]

[0029] FIGURES 6A-6B illustrate exemplary process steps according to exemplary embodiments of this disclosure.

[0042]

[0030] FIGURES 7A, 7B, and 7C illustrate exemplary organ perfusion systems adapted to reduce the presence of dissolved nitrogen according to exemplary embodiments of this disclosure.

[0043]

[0031] FIGURE 8A: Above: Dissolved H2 in Solution 1 appears exert a moderate antioxidant effect based on molecular indicators Myeloperoxidase (MPO) and Malondialdeyde (MD A), which highlight cellular damage from reactive oxygen species (ROS). FIGURE 8B: Above, Dissolved H2 in Solution 1 does not appear to mitigate the early effects of acute hypoxic injury and resultant inflammation based on acute tubular necrosis (ATN) and interleukin-6 (IL-6).

[0044]

[0032] FIGURES 9A-9G: Above LEFT: Results for Experiment 1, Solution 1 versus Control Above RIGHT, Results for Experiment 2, Solution 2 versus Control.

[0045]

[0033] FIGURE 10A illustrates exemplary packaging devices for organ perfusion systems.

[0046] FIGURE 10B illustrates a seal at a distal end of the fill tube. FIGURE 10C illustrates the packaging device after initial filling of the inner space with a solution via the fill tube, which has been sealed with the seal. FIGURE 10D illustrates the packaging device after the gas in the headspace has been removed, e.g., via a positive pressure from pushing / squeezing of the packagingDocket No. 220709-702601

[0047] device or via a negative pressure relative to the inner space to force the gas in the headspace externally from the packaging device. FIGURE 10E further illustrates the packaging device inserted into a heating chamber for a degassing cycle. FIGURE 10F illustrates the packaging device that, using the valve, internal container pressure can be released and allows for the egress of dissolved gases from the headspace. FIGURE 10G illustrates the packaging device removed from the heating chamber (optional) and having a headspace of previously dissolved gases remaining after a thermal degassing cycle. FIGURE 10H illustrates that the composition in the headspace may be removed again, similar to what is performed as shown in FIG. 10D, with solution egress stopped by the filter. FIGURE 101 illustrates the packaging device further filled with a composition of H2, 02, or a combination thereof to fill the headspace as desired. FIGURE 10J illustrates the packaging device with the final gas composition, and with distal end of the gas transport tube sealed off, with the filter and the valve discarded.

[0048]

[0034] While the above-identified drawings set forth presently disclosed embodiments, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the presently disclosed embodiments.

[0049] DETAILED DESCRIPTION

[0050]

[0035] Described herein are systems and techniques for hydrogen gas and oxygen gas infusion into a solution with increased concentrations and effectiveness relative to those previously available. This disclosure relates generally to hydrogen gas infusion (HI) systems such that clinically-relevant concentrations of hydrogen and oxygen are safely infused into solutions and delivered to patients. In particular, the HI systems address the need for efficient control over levels of dissolved oxygen in the solution while maintaining levels of nitrogen below the oxygen levels. Further, the HI systems provide for consistent and predictable delivery of hydrogen and oxygen at a predetermined concentration with precision and within safe concentrations rather than existing methods that provide inconsistent or unpredictable delivery to treated areas. A key distinction of the instant HI system is its ability to eliminate nitrogen gas while optimizing oxygen and hydrogen concentration.

[0051]

[0036] After a period of ischemia, anoxia, or hypoxia, live organs or tissue can be subject to damage by reperfusion injury when blood supply returns. The absence of oxygen and other nutrients from the tissue during the period of ischemia can create conditions where restoration of circulation can result in inflammation and oxidative damage due, in part, to reactive oxygenDocket No. 220709-702601

[0052] species that come into existence during reperfusion. This can occur through induction of oxidative stress instead of, or in addition to, restoration to normal tissue function.

[0053]

[0037] The beneficial effects of hydrogen follow a positive dose-response relationship; i.e., there is a positive correlation between hydrogen concentration in the tissue and the observed beneficial effect. Dose dependency is most pronounced in cases of acute oxidative stress wherein high reactive oxygen species (ROS) production rates are most effectively countered by a similarly high concentrations of dissolved hydrogen. In the context of organ transplant, effectiveness of hydrogen is therefore maximized when the hydrogen concentration is as high as is practically achievable, and the high hydrogen concentration is timed to coincide with the most acute phase of ROS production in the first 5 - 15 minutes of reperfusion.

[0054]

[0038] Having a high concentration of hydrogen gas in the tissue matters the most at the period of time when the tissue is being reperfused, usually hours after the hydrogen was initially infused into the organ. Hydrogen gas is highly diffusive and able to rapidly permeate through organs, tissue, solutions, and through most container materials. When exposed to air, organs or solutions initially having a high concentration of hydrogen will be depleted by dissipation of hydrogen gas to the surroundings until the hydrogen concentration in the tissue falls below therapeutic levels. For many organ transplant procedures, several hours (4 to 36) may elapse between the initial infusion of hydrogen into the organ and the final reperfusion within the organ recipient’s body. Even with significant effort to minimize the rate of hydrogen outgassing from the organ storage system, it can be difficult to ensure that, by the time of reperfusion, the organ retains sufficient levels of dissolved hydrogen to combat the damaging free radicals produced by the cells during the initial few minutes of reperfusion.

[0055]

[0039] As illustrated in FIG. 1 A, the hydrogen outgassing rate from a static system can be modeled by an exponential decay curve, wherein the final hydrogen concentration is most affected by (a) the half-life of the hydrogen in the system; and (b) the initial concentration of hydrogen at time zero (tO) before dissipation commences. Maximizing the initial [H2] and / or maximizing the [H2] half-life of the system are useful methods to increase the final [H2] at the time of reperfusion.

[0056]

[0040] As shown in the example in FIG. 1 A, hydrogen concentration in the organ is shown over a six-hour period for four different hypothetical scenarios A, B, C, and D. The final concentration of hydrogen [H2] in the organ after a six-hour outgassing period is tabulated below for comparing scenarios. Scenario A maximizes neither the initial [H2] nor the [H2] half-life, resulting in a final [H2] at t6 = 100 ppb. Scenario B maximizes initial [H2] to achieve a final [H2] at t6 = 200 ppb. Scenario C increases the [H2] half-life to achieve a final [H2] at t6 = 283 ppb. Scenario D maximizes initial [H2] and increases the [H2] half-life achieve a final [H2] = 566 ppb.Docket No. 220709-702601

[0057]

[0041] The instant disclosure herein proposes to combat the problem of maximizing the residual amount of hydrogen in the tissue when it is reperfused with the organ recipient patient’s blood maximizing the initial concentration of hydrogen in the organ.

[0058]

[0042] An additional problem to be solved is to avoid stripping the organ of dissolved oxygen during the infusion of hydrogen. Since the hydrogen infusion step is followed by a period of ischemia (an hours-long period of cold ischemia during storage, and / or a 0.5 - 1 hour long period of warm ischemia during surgical engraftment), depleted oxygen levels in the organ after the hydrogen infusion step may increase the severity of hypoxia and ischemia reperfusion injury (IRI) in the organ.

[0059]

[0043] The ideal hydrogen infusion solution therefore has a high hydrogen concentration to maximize residual hydrogen concentration at the reperfusion step, and it should also have enough dissolved oxygen to match the dissolved oxygen levels already present in the tissues of the organ. Maximizing hydrogen while maintaining at least the minimum required oxygen level therefore requires the minimization of other dissolved gases such as Nitrogen or CO2 in the organ and in the solution. Any dissolved gases other than Hydrogen and Oxygen in the solution or the organ will tend to dilute the dissolved hydrogen and oxygen, thereby reducing their therapeutic effects.

[0060]

[0044] FIG. IB illustrates an experiment conducted by the inventor, where ischemic pig kidneys were exposed to standard flush solutions as well as two different hydrogen infusion solutions prior to hypothermic machine perfusion followed by warm oxygenated machine reperfusion as shown in the table in FIG. IB. Histopathology scoring for acute tubular necrosis (ATN) and immunostaining scores for reperfusion injury markers are tabulated below for all three arms. Arm 1 used a hydrogen infusion solution that maximized H2 at 96% of saturation and minimized 02 at less than 1% saturation. The Arm 1 solution had such a low oxygen concentration that it stripped the kidneys of their residual dissolved oxygen and made their ischemic state even worse than it would be for the control arm. The dissolved hydrogen in Arm 1 probably had a limited positive effect: Arm 1 scored better than the control arm in 4 out of 7 injury diagnostic markers. By contrast, Arm 2 used a hydrogen infusion solution that balanced H2 and 02 at 80% and 20% saturation respectively. Even though Arm 2 had a slightly lower hydrogen concentration than Arm 1, it had enough dissolved oxygen to match the oxygen levels of the control arm and prevent stripping the kidneys of their residual oxygen. Arm 2 scored better than both the control arm and Arm 1 in all 7 injury diagnostic markers. Results from the experiment of FIG. IB are shown below in TABLE 1.

[0061]

[0045] TABLE 1 : Results from experiment performed in FIG. IB.Docket No. 220709-702601

[0062]

[0063]

[0046] Comparing the dissolved gas profiles for Arm 2 and Control Arm, the best solution preparation process essentially replaced the dissolved nitrogen with dissolved hydrogen and left the dissolved oxygen levels roughly equivalent to normal atmospheric levels.

[0064]

[0047] Optimizing a hydrogen infusion solution with enough dissolved hydrogen to provide a therapeutic level of residual hydrogen at the time of reperfusion, while simultaneously having sufficient dissolved oxygen to prevent depletion the oxygen reserves of the organ tissue, requires a deliberate purging of nitrogen from the hydrogen infusion solution and / or the organ. Additionally, existing perfusion systems use a sweep gas that includes more nitrogen than oxygen; as the standard of care. The more of an increase made in the final oxygen mixture versus the baseline operational mode, the more effective is the claimed step of purging N2 from the organ as a way to facilitate the next step (hydrogen infusion). Nitrogen comprises about 80% of the ambient air and is thus ubiquitous in organs and solutions exposed to or in equilibrium with air. In addition, nitrogen is transported more slowly than oxygen through tissues and physiologic solutions, making high dissolved nitrogen concentrations a strong hindrance to hydrogen and oxygen infusion in solutions and organs. Unless specifically designed processes are used to eliminate the dissolved nitrogen and replace it with dissolved hydrogen, the dissolved nitrogen will dilute the dissolved hydrogen and / or the dissolved oxygen and reduce their therapeutic effectiveness. Per Dalton’s law, the sum of the partial pressures of different dissolved gases in a solution cannot exceed the barometric pressure (760 mm Hg at sea level), therefore any nitrogen gas remaining in solution is displacing hydrogen and / or oxygen.

[0065]

[0048] This instant disclosure proposes a safe and convenient way to optimize H2 and 02 levels in medical solutions and in organs while minimizing the presence of dissolved N2 and other diluent gases. For example, the more of an increase to the final oxygen mixture versus the baseline operational mode, the more effective the disclosed method can be at purging N2 from the organ, which can facilitate the next steps, e.g., hydrogen infusion. It is a process to prepare a hydrogen infusion solution that strips out dissolved nitrogen and replaces it with dissolved hydrogen, while leaving some or all of the oxygen levels unchanged. Dissolved oxygen can be added back to the solution after degassing if necessary, to achieve the desired final dissolved gas profile of H2 / O2Docket No. 220709-702601

[0066] in ratios ranging from 67 / 33 to 96 / 4 respectively, with the partial pressure of dissolved N2 in the solution or organ reduced to less than 150 mm Hg.

[0067]

[0049] Prior art disclosures acknowledge the potential benefits of dissolved hydrogen in medical solutions. The positive dose response for hydrogen is generally understood and high concentrations of dissolved hydrogen are sought after by the medical community. However, the prior art fails to address the simultaneous need to eliminate diluent gases, such as nitrogen, from a solution while retaining enough dissolved oxygen in the solution to prevent stripping native oxygen out of the organ, thereby potentially worsening the effects of prolonged hypoxia.

[0068]

[0050] Since prior art has not addressed the need for a basically binary gas mixture of H2 / O2 in solution (with all other gases minimized), no methods are proposed or implied in the prior art for producing such binary H2 / O2 solutions. Instead, the prior art emphasizes methods for infusing H2 into solutions without regard for any pretreatment steps that would be necessary to purge out dissolved N2 while providing enough dissolved 02 to address the basic oxygen needs of the organ tissues being treated by the solution. The final dissolved N2 levels can be important because it dilutes the dissolved hydrogen and therefore imposes an upper limit on hydrogen concentration. Given the initial 4:1 ratio for N2 / O2 in air and the lack of concern for infusing gases other than hydrogen, it is safe to assume that methods used in prior art have resulted in solutions where PN2 and PO2 were both proportionally reduced by the infusion of hydrogen gas, and the final PO2 in the solution would be low enough to either induce or worsen hypoxia in the tissue.

[0069]

[0051] As shown in FIG. 1C, one prior art method includes a medical solution that is bagged in a gas permeable material and then immersed for a period of time (e.g., hours) in a hydrogen-saturated water bath. Hydrogen diffuses from the water bath though the bag and into the medical solution. The medical solution is then used to infuse hydrogen into the target organ by immersion and / or perfusing the solution through the vasculature of the organ. Using the technique of FIG. 1C results in dissolved hydrogen levels reported at 1000-1200 ppb or more (PH2 about 450-530 mm Hg) within the solution. Hydrogen concentrations are measured and reported, but no discussion is made regarding final concentrations of dissolved oxygen or nitrogen in either the solution or the tissue. The technique in FIG. 1C results in an unpredictable mixture of H2, N2, and 02 in solution. It is likely that PH2 is limited by dilution / mixture with residual dissolved N2 or in bag headspace, and that P02 « PN2, in final solution, where final P02 in solution is lower than P02 in the organ tissues.

[0070]

[0052] As shown in FIG. ID, in an alternative prior art method, an organ, contained within a gas permeable container, is immersed in a hydrogen-saturated water bath similar to the process shown in FIG. 1C. Sufficient time (e.g., hours) is allowed for infusion of hydrogen to reach 1000 to 1200 ppb H2 concentration in the organ with no regard for maintaining enough dissolved 02 to supportDocket No. 220709-702601

[0071] the metabolism of the organ. In addition to the inability to maintain sufficient 02 levels, this method is limited to only very small organs or sections of tissue through which H2 can penetrate by diffusion within the allowed time.

[0072]

[0053] A third prior art method is illustrated in FIG. IE, in which, hydrogen is infused into the medical solution either by bubbling or by use of a membrane-type gas exchanger. The hydrogen feed gas flows through the solution as bubbles or through the gas exchanger as sweep gas. A pure H2 feed will strip out N2 and 02 together, such that a final solution having a high concentration of H2 will have a very low concentration of 02, which is not sufficient to support energy metabolism within the organ. In addition, this method has the significant disadvantage of producing large volumes of flammable / explosive waste gas containing H2 and 02.

[0073]

[0054] A fourth prior art method is shown in FIG. IF. As shown in FIG. IF, medical solutions are packaged within a gastight (impermeable) package. Also included within the confines of the gastight package is a time-delayed source of H2 gas intended to produce hydrogen gas after the outer gastight packaging layer has been sealed. The produced hydrogen gas is contained by the gas-tight outer packaging but diffuses through the gas-permeable inner solution packaging to produce a hydrogen-rich medical solution. Descriptions of this method estimate final H2 concentrations of 1000 ppb or more, but do not disclose or acknowledge any need to minimize dissolved nitrogen to prevent dilution of the hydrogen, or to balance dissolved oxygen within the solution. No solution pre-treatment steps are disclosed that would either reduce N2 levels or boost 02 levels in the solution as a preparation for H2 infusion.

[0074]

[0055] Therefore, the inventors have recognized and appreciated various unmet needs in the pursuit of high-concentration infusion of hydrogen gas with low level concentration of oxygen, with levels of nitrogen being undetectable or less than that of the concentration of oxygen, into medical solutions. For example, administration of high concentrations of molecular hydrogen via hydrogen-saturated aqueous solutions or blood in medical clinics for human treatment has been limited by the explosive danger posed by hydrogen gas. Consequently, although aqueous solutions with low concentrations of dissolved hydrogen are easy to produce, aqueous solutions having very high concentrations hydrogen concentrations at or near the saturation level, in combination with diluted 02, are difficult to produce in a healthcare setting without also producing a dangerous quantity of flammable or explosive gas. Further, administration of aqueous solutions with low concentrations of hydrogen have been known to result in heterogeneous microperfusion, in that not all targets, e.g., tissues or organs, receive the same amount of blood flow and thus hydrogen levels. In particular, to achieve a functional benefit in the impacted area, a minimum dose of hydrogen may be required, though remains unmet as a result of local underperfusion. This may lead to under delivery of hydrogen-saturated aqueous solutions. Timing of administering hydrogenDocket No. 220709-702601

[0075] gas to a patient has also been shown to be crucially important, as well as the duration of the hydrogen gas to be acting on the target, thus requiring high concentrations of hydrogen gas to be accessible on demand.

[0076]

[0056] Turning to the instant disclosure, the solutions provided herein overcome the challenges of the prior art methods while providing systems and methods for producing a solution containing a high concentration of hydrogen, a relatively lower concentration of oxygen, and a concentration of nitrogen that is clinically undetectable or less than the concentration of oxygen.

[0077]

[0057] Disclosed herein are methods for producing a hydrogen-rich medical perfusion solution. In some embodiments, methods include: providing a medical perfusion solution in a container; degassing the medical perfusion solution to remove nitrogen and oxygen from the medical perfusion solution until a combination of a partial pressure of nitrogen and a partial pressure of oxygen in the medical perfusion solution is between 0 to 150 mm Hg; and adding hydrogen to the medical perfusion solution; and optionally, adding oxygen to the medical perfusion solution, after the degassing.

[0078]

[0058] In some embodiments, the degassing includes using one of a vacuum, ultrasonic energy, heat, or a combination thereof to reduce a concentration of dissolved nitrogen in the medical perfusion solution. In some embodiments, the adding oxygen includes adding dissolved oxygen to the medical perfusion solution to achieve a predetermined concentration of dissolved oxygen. In some embodiments, the degassing replaces a portion of dissolved nitrogen with dissolved oxygen prior to the adding hydrogen. In some embodiments, the degassing includes filling the container with the medical perfusion solution and purging at least one gas from a headspace of the container. In some embodiments, the degassing further includes heating the container to above 100°C and purging the at least one gas from at least one valve in fluid communication with the headspace of the container. In some embodiments, the purging is performed after the container has returned to a temperature below 100°C. In some embodiments, the degassing further includes adding a predetermined amount of at least one of H2, 02, or a combination thereof to the headspace using the at least one valve.

[0079]

[0059] In some embodiments, the adding is performed using at least one of: injection directly into the container, or a gas-filled and gas-permeable second container that is co-packaged with the medical perfusion solution inside a third gas-impermeable container. In some embodiments, after the adding hydrogen, the medical perfusion solution has a hydrogen partial pressure (pH2) of at least 0.7 atm. In some embodiments, after the adding oxygen, the medical perfusion solution has an oxygen partial pressure (pO2) of at least 0.7 atm.

[0080]

[0060] In some embodiments, a ratio of the hydrogen to the oxygen is controlled to produce a combined solution with a predetermined partial pressure of each of the hydrogen and the oxygen.Docket No. 220709-702601

[0081] In some embodiments, a ratio of the hydrogen to the oxygen in the medical perfusion solution ranges from 1:1 to 9: 1.

[0082]

[0061] In some embodiments, the container is subsequently packaged in an outer gastight container.

[0083]

[0062] In some embodiments, the medical perfusion solution is a denitrogenated solution.

[0084]

[0063] Further disclosed herein are systems for producing medical solutions with dissolved hydrogen gas. In some embodiments, the system includes: at least one solution container having at least one fill tube, at least one gas transport tube, and at least one access tube; and a heating chamber sufficiently sized to house the at least one solution container and apply heat sufficient to increase a temperature of a medical solution within the at least one solution container to at least 100°C, wherein the at least one solution container includes a headspace absent the medical solution and allows sufficient escape of dissolved gas from the medical solution upon introduction of the medical solution into the at least one solution container, wherein the at least one gas transport tube includes a hydrophobic filter or porous membrane in line with at least one valve configured to allow for control over gas egress from the at least one solution container and gas ingress into the at least one solution container; and wherein, upon heating of the medical solution in the heating chamber, at least one dissolved gas is separated from the medical solution into the headspace such that the at least one dissolved gas is removable via the at least one gas transport tube upon opening of the at least one valve, thereby allowing for a sufficient amount of an additional gas composition to be introduced into the headspace so as to generate a predetermined composition of dissolved gas in the medical solution, the predetermined composition of dissolved gas having a partial pressure of dissolved hydrogen (pH2) greater than a partial pressure of dissolved oxygen (pO2) and the pO2 greater than a partial pressure of dissolved nitrogen (pN2).

[0085]

[0064] In some embodiments, the at least one gas transport tube includes a hermetically sealable portion. In some embodiments, the hydrophobic filter includes a pore size ranging from about 0.15 to about 0.3 micron. In some embodiments, the at least one valve includes a bidirectional check valve. In some embodiments, the at least one valve is closer to an external portion of the at least one gas transport tube than the hydrophobic filter. In some embodiments, each of the at least one fill tube is separate from each of the at least one gas transport tube.

[0086]

[0065] In some embodiments, the heating chamber is selected from an oven, an autoclave, a humidity chamber, a thermal shock chamber, a dry heat sterilizer, a heated ultrasonic bath, or a combination thereof.

[0087]

[0066] In some embodiments, the system further includes a temperature controlling feature configured to maintain a temperature of the medical solution. In some embodiments, the system further includes one or more gas sensors positioned to measure concentrations of dissolved oxygenDocket No. 220709-702601

[0088] or dissolved hydrogen in an egressing gas or a headspace of the medical solution, optionally wherein the one or more gas sensors comprise one or more dissolved gas sensors, one or more solution flow sensors, temperature sensors, optical sensors, or a combination thereof..

[0089]

[0067] In some embodiments, the concentrations of dissolved hydrogen and / or dissolved oxygen in the effluent medical solution are compared to a reference value so as to achieve a predetermined concentration of at least one of dissolved oxygen or dissolved hydrogen in the medical solution.

[0090]

[0068] Further disclosed herein are perfusion solutions, including: a fluid having a dissolved gas mixture of hydrogen, oxygen, and nitrogen, wherein a partial pressure of the dissolved hydrogen (pH2) is higher than 350 mm Hg, the partial pressure of the dissolved oxygen (pO2) ranges from 45 mm Hg to 300 mm Hg, and the partial pressure of dissolved nitrogen (pN2) is lower than a pO2 in the solution, wherein contact of the perfusion solution with an organ or tissue increases an amount of dissolved hydrogen and does not decrease an amount of dissolved oxygen within the organ or tissue as compared to the organ or tissue prior to contacting with the perfusion solution, wherein a predetermined concentration of dissolved oxygen in the organ or tissue is maintained over a course of hydrogen infusion so as to prevent a worsening of hypoxia or oxidative stress within the organ or tissue during the contact of organ or tissue with solution. The maintenance of a stable concentration of dissolved oxygen in the organ over the course of hydrogen infusion should be tailored to prevent a worsening of hypoxia or oxidative stress within the organ or tissue during the contact of organ or tissue with solution.

[0091]

[0069] In some embodiments, the decrease in the amount of dissolved 02 results in a reduced occurrence of oxidative stress as compared to the organ or tissue prior to the contacting. In some embodiments, the hypoxia or oxidative stress is represented by one or more of acute tubular necrosis (ATN), apoptotic cell death, Kidney Injury Marker 1 (KIM-1), Interleukin 6 (IL-6), CD68(+), Myeloperoxidase (MPO), Malondialdehyde (MDA), or a combination thereof.

[0092]

[0070] In some embodiments, the partial pressure of the dissolved hydrogen is between about 450 mm Hg and about 500 mm Hg, and wherein the partial pressure of the dissolved oxygen is at least 1.5 times greater than the partial pressure of dissolved nitrogen.

[0093]

[0071] In some embodiments, the fluid includes a University of Wisconsin (UW) solution or an organ or tissue preservation solution. In some embodiments, a fluid further includes one or more chelating agents selected from ethylenediaminetetraacetic acid (EDTA), Desferrioxamine (DFO), citric acid, Dimercaptosuccinic Acid (DMSA), or a combination thereof, wherein the one or more chelating agents are at concentrations ranging from 10-6 Molar to 10-4 Molar. In some embodiments, a solution further includes a bicarbonate buffer, wherein a partial pressure of carbon dioxide (pCO2) ranged from 0.04 atm - 0.06 atm, a pH2 ranges from 0.6 atm - 0.8 atm, and a pO2 ranges from 0.15 atm - 0.35 atm. In some embodiments, a solution further includes one or moreDocket No. 220709-702601

[0094] hydrogen sulfide donors selected from sodium thiosulfate (STS), sodium sulfide (Na2S), sodium hydrosulfide (NaHS), or a combination thereof, wherein a concentration of the one or more hydrogen sulfide donors ranges from 0.1 micromolar to 50 micromolar.

[0095]

[0072] In some embodiments, the pN2 is less than 0.2 atm. In some embodiments, the pN2 is less than 0.1 atm. In some embodiments, a pO2 is greater than pN2 by a magnitude of at least 3x. In some embodiments, a pH2 is greater than pO2 by a magnitude of at least 2x and greater than pN2 by a magnitude of at least 3x. In some embodiments, when a concentration of the hydrogen ranges from 40% to 80%, a pO2 is greater than a combined partial pressures of other gases, the other gases excluding the hydrogen and the oxygen. In some embodiments, the gas mixture in the fluid includes a binary mixture of hydrogen and oxygen.

[0096]

[0073] Further disclosed herein are methods of producing medical solutions with dissolved hydrogen gas, including: providing an outer gas impermeable container and at least one inner gas permeable container; packaging a medical solution into the at least one inner gas permeable container, the medical solution being previously depleted of nitrogen having an amount of oxygen gas in a headspace of the at least one inner gas permeable container; and arranging the at least one inner gas permeable container and a predetermined amount of hydrogen gas within the outer gas impermeable container, wherein the predetermined amount of the hydrogen gas is sufficient to permeate into the at least one inner gas permeable container to equilibrate with the oxygen gas such that the medical solution has a predetermined dissolved gas composition, the predetermined dissolved gas composition having a partial pressure of dissolved hydrogen (pH2) higher than 350 mm Hg and a partial pressure of dissolved oxygen (pO2) ranging from 45 mm Hg to 300 mm Hg.

[0097]

[0074] In some embodiments, the at least one inner gas permeable container includes a first inner gas permeable container and a second inner gas permeable container, wherein the medical solution is packaged into the first inner gas permeable container and the predetermined amount of hydrogen gas is packaged into the second inner gas permeable container. In some embodiments, the at least one inner gas permeable container is transparent, flexible, sterile, or a combination thereof.

[0098]

[0075] In some embodiments, a method further includes infusing the medical solution into a machine perfused organ or tissue. In some embodiments, the infusing includes adjusting a concentration of dissolved nitrogen and dissolved oxygen in the machine perfused organ or tissue, the adjusting including administering a sweep gas prior to cessation of machine perfusion in preparation for hydrogen infusion, wherein the sweep gas is adjusted to an N2:O2 ratio of up to about 0.5. In some embodiments, the sweep gas includes a mixture of N2 and 02.

[0099]

[0076] Further disclosed herein are methods for producing a medical solution with substantially binary H2 / O2 dissolved gas mixture wherein an outer package contains a kit including: at least one container of oxygen- saturated or oxygen-rich solution (pO2 > 0.5 atm); and a gas-tightDocket No. 220709-702601

[0100] container of hydrogen gas to be combined with the oxygen-rich solution prior to using the medical solution. In some embodiments, the outer package includes a gas-impermeable barrier. In some embodiments, the at least one container includes a flexible material.

[0101]

[0077] Further disclosed herein are methods for producing a medical solution with substantially binary H2 / O2 dissolved gas mixture wherein an outer package contains a kit including: at least one container of hydrogen-rich solution having a partial pressure of dissolved hydrogen (pH2) greater than 0.5 atm; and a gas-tight container of an oxygen gas, wherein an oxygen gas to be combined with the hydrogen-rich solution prior to using the hydrogen-rich solution. In some embodiments, the outer package includes a gas-impermeable barrier. In some embodiments, the at least one container includes a flexible material.

[0102]

[0078] Further disclosed herein are methods for using binary dissolved gas mixtures of hydrogen and oxygen in a medical solution, the method including: combining a hydrogen-rich solution and an oxygen-rich solution within a container to form the binary dissolved gas mixture, wherein a volume ratio of hydrogen-rich solution to oxygen-rich solution ranges from 1:1 to 9:1. In some embodiments, a concentration of glutathione is higher in the hydrogen-rich solution than in the oxygen-rich solution. In some embodiments, the combining is performed at a time of use or immediately prior to the time of use. In some embodiments, the combining includes heating the container to above 100°C and purging at least one gas from a headspace of the container. In some embodiments, the purging is performed after the container has returned to a temperature below 100°C.

[0103]

[0079] As shown in FIG. 2, a process block diagram is illustrated that describes an embodiment which can being with a stock medical solution 101. In some embodiments, the stock medical solution can be saline solution or other biocompatible solutions known, or unknown. Initially, the process can begin with a stock solution 101 that was previously equilibrated with air. The stock solution 101 can, therefore, have a high concentration of dissolved N2 and an approximately biologic concentration of 02 (approaching 80% N2 and 20% 02 by dissolved gas partial pressure). The solution 101 can be treated by a degassing step 102 in which the majority of the dissolved N2 and 02 are removed from the solution. Typical degassing processes can include heating, ultrasonic degassing, vacuum degassing, inert gas purging, or any combination of the above. As described above, the prior art methods do not provide for a degassing step 102 for production of hydrogenrich medical solutions. As a corollary, the use of degassed solution as a feedstock for hydrogenrich medical solutions has not been disclosed or implied in the prior art.

[0104]

[0080] Any combination of the aforementioned degassing methods can remove both N2 and 02 from the solution, leaving the resulting degassed solution 103 having an 02 concentrations lower than those typically required by organ tissues for maintaining normal aerobic metabolism (PO2Docket No. 220709-702601

[0105] from 50 to 160 mm Hg). Thus, the resultant solution can therefore increase the risk of hypoxic stress in the organ. The dissolved oxygen in degassed solution 103, being too low to support aerobic metabolism, can be boosted after the degassing step 102 by the oxygen addition step 104 to bring oxygen levels back to an appropriate target percentage (such as 10%, 20%, or 30% by dissolved gas partial pressure) based on the desired effect on the target organ. As noted above, currently available methods do not provide for or disclose the addition of oxygen once the oxygen has been degassed. In the instant disclosure, in an embodiment, an oxygen addition step 104 may occur before solution packaging by using oxygen as the sweep gas in a gas exchanger, or by bubbling oxygen gas through the degassed solution 103. In some embodiments, the oxygen can be infused into the solution after packaging. After packaging, the oxygen gas may be injected as a gas headspace in the solution package, or it may occur as the result of oxygen gas diffusion through a gas-permeable solution container after the gastight outer packaging layer is sealed.

[0106]

[0081] The quantity of oxygen gas added to the system, either as dissolved oxygen gas in the solution or as a gas phase added to the packaging, can be calculated to provide the solution with the target oxygen concentration at its expected use temperature. In an embodiment, an organ preservation solution can be expected to be used at 5 deg. C., and the desired oxygen partial pressure can be 150 mm Hg (1 / 5 of an atmosphere). In such an embodiment, the minimum volume of oxygen in the system can be approximately 1.8 cc of oxygen gas per 1 liter of solution (from the known oxygen solubility in water) plus 20% of any headspace within the gastight packaging. For this scenario, the “system” is defined as the entire volume of space contained within the final, gastight packaging of the product.

[0107]

[0082] The hydrogen addition step 105 may occur either before or after the oxygen addition step 104. It can be advantageous to maintain separate steps for 104 and 105 in order to avoid mixing large quantities of H2 and 02 in the production environment. As noted above, the mixing of large quantities of H2 and 02 can result in an explosion, therefore the H2 supply and the 02 supply gases should not be mixed. At the scale of 1 to 4 liter solution packaging, the volume of H2 and 02 within the package is small enough to minimize the fire hazard of H2 / O2 gas mixtures. Aqueous diffusions of H2 and 02 are nonreactive, and small mixtures of H2 and 02 gas in the headspace of the solution container are well contained within a humid environment and away from ignition sources. In an example, the H2 can be injected directly into the headspace of the gas-permeable solution container. In other embodiments the H2 can be injected into the dead space between the gas permeable solution container and the gastight layer of outer packaging. The advantage of performing step 105 in the packaging can be that, unlike bubbling or gas exchanger methods, it produces no H2-laden waste gas stream and minimizes the total volume of H2 needed to infuse the solution with the desired concentration of hydrogen.Docket No. 220709-702601

[0108]

[0083] Over time, for example a number of days on the storage shelf, diffusion of both oxygen and hydrogen through the gas permeable solution container can ensure equilibration of the H2 and 02 gases between the solution and any remaining headspace within the gastight packaging.

[0109]

[0084] The instant disclosure provides for an H2 / O2 ratio in a binary gas mixture in the solution which can be controlled by adding a calculated ratio in the total volumes of H2 and 02 to the system, where:

[0110]

[0085] Final molar ratio of H2 to 02 in solution = (VH2(in aqueous solution at predicted use temperature) + VH2(in packaging headspace)) / (V02(in aqueous solution at predicted use temperature) + V02(in packaging headspace))

[0111]

[0086] In general it is desirable to minimize the total volume of hydrogen and oxygen required, which can be achieved by using enough gas to saturate the solutions while leaving a minimum of headspace within the sealed gastight package.

[0112]

[0087] Figure 3 depicts an alternative process wherein the N2 gas can be purged from the stock solution 201 by using oxygen gas to displace all of the dissolved nitrogen in an oxygen substitution step 202. The resulting solution at step 203 can be high in dissolved 02 and low in dissolved N2. In an example, the oxygen gas can be bubbled through the solution to achieve a solution at step 203 having oxygen at 95% saturation and nitrogen at 5% saturation. The subsequent hydrogen addition step 204 adds enough hydrogen to the solution or space within the gastight packaging (using any of the same methods described for Figure 2, oxygen addition step 104) to dilute the oxygen down to the desired target of 10%, 20% or 30% 02 by volume with the balance being a high concentration of H2 such as 70%, 80%, or 90% by volume (minus a low residue of whatever N2 remains). In another example, the process depicted in Figure 3 may also include a moderate degassing step between steps 203 and 204 to decrease the dissolved oxygen levels to a desired value by vacuum degassing or ultrasonic degassing for a short period. The added oxygen reduction step may lower the total volume of the H2 / O2 gas mixture contained within the final solution package.

[0113]

[0088] Figure 4A and 4B depict example values for dissolved gas concentrations at sequential steps of the process described by Figure 2. For example, in Figure 4A, the oxygen addition step can happen before the hydrogen addition step. In Figure 4B, in an embodiment, the order can be reversed for the separate gas additions. Either sequence can be used as long as the resultant solution has most of the nitrogen removed from the initial stock solution.

[0114]

[0089] Figures 5A and 5B depict exemplary embodiments of packaging components wherein gas addition may occur concurrent with the packaging process, and gas equilibration may occur after the gastight (or gas impermeable) packaging has been sealed. Figure 5A depicts an embodiment wherein solution 401 can be contained within a gas-permeable, sterile, flexible, and transparentDocket No. 220709-702601

[0115] container 403 similar or equivalent to a medical IV bag. The inner headspace 402 within the container 403 may contain the added 02, H2, or both. The flexible container 403 can be packaged within a gastight container 404. The headspace 405 between the inner gas permeable container 403 and the outer gastight container 404 may contain the added 02, H2, or both. In an example, the gastight container 404 can be a flexible laminated film containing aluminum foil as the impermeable barrier. In some embodiments, the gastight container 404 comprises a transparent gas-impermeable material, such as, for example, a polymer film, a submicron layer of a glass, oxide, or ceramic that has been deposited on the polymer film, or the like. The 02 and H2 can be added to the inner headspace 402 of container 403, and the outer headspace 405 can be evacuated by vacuum before sealing the flexible outer gastight container 404. Atmospheric pressure outside the flexible barrier of outer gastight container 404 can collapse the barrier of outer gastight container 404 around the inner container 403 to minimize the headspace 405 between inner container 403 and outer gastight container 404.

[0116]

[0090] Figure 5B depicts an alternative embodiment wherein an additional gas-permeable container 407 can be added to the outer gastight container 404. The additional gas-permeable container 407 can contain the hydrogen gas for the hydrogen gas addition step 105 from the example in FIG. 2, or step 204 from the example in FIG. 3. In this example, the solution 401 can be contained in gas-permeable container 403 with inner headspace 402 containing the added oxygen. To further prevent mixing of H2 and 02 gases in large quantities outside the gastight package, the hydrogen gas 406 can be added to the system in its own gas permeable container 407. The outer headspace 405 can be evacuated by vacuum prior to sealing the outer gastight container 404. The sealed outer gastight container 404 can collapse against containers 407 and 403 to minimize the volume of outer headspace 405. Over a period of hours to days, a volume of the hydrogen gas 406 and the oxygen gas volume in inner headspace 402 can diffuse through the gas permeable containers 403 and 407 to equilibrate at the desired concentration in the solution 401. In some examples, some residual H2 / O2 mixture can remain in container 407 for later use in the medical setting, after opening the outer gastight container 404 to use the solution 401.

[0117]

[0091] Figures 6A and 6B depict additional examples of processes where some residual nitrogen may remain in the system after degassing step 102 from Figure 2, or step 202 from Figure 3. In an example shown in Figure 6A, the stock solution degassing process can remove about 80% of the dissolved nitrogen and oxygen, leaving a residue of dissolved N2 and 02 gases in solution. The mostly degassed solution can be packaged in a flexible, gas permeable container with no headspace. A volume of H2 gas (predetermined to infuse the solution with the desired concentration of H2) can be injected into the gas permeable container to form a H2 gas headspace in contact with the solution. A volume of 02 gas (predetermined to infuse the solution with theDocket No. 220709-702601

[0118] desired concentration of 02) can additionally be injected into the gas permeable container to mix with the H2 gas headspace in contact with the solution. The flexible container holding the solution, the residual dissolved N2 and 02, and the mixed H2 / O2 headspace can be vacuum sealed within a flexible gastight container. Diffusion and equilibration of all gases within the gastight container occurs within a few days of packaging. The residual N2 left over from the partial degassing step can displace some H2 and can reduces the final concentration somewhat compared to what it could have been in the absence of residual N2.

[0119]

[0092] In an example depicted in Figure 6B, the stock solution can be partially denitrogenated by bubbling oxygen gas through it. The solution can be packaged with an excess of dissolved 02. Hydrogen can be injected into the solution package in sufficient volume to eventually dilute the oxygen down to the desired concentration and infuse the solution with dissolved hydrogen. A vacuum-sealed, flexible, and gastight outer packaging layer can contain the solution and all gases, which equilibrate to provide a solution with a high H2 level and a biologically compatible 02 level.

[0120]

[0093] In cases of machine perfusion of donated organs, systems and methods may be desired to purge or at least reduce the amount of dissolved nitrogen from a machine-perfused organ prior to, or as part of, a hydrogen infusion step. For transplanted organs, the end of a machine perfusion step marks an opportunity to optimize the hydrogen and oxygen gas concentrations within the organs to prepare them for the warm ischemic period during which the organ is surgically grafted into the organ recipient’s body. Removal or at least reduction of the dissolved N2 in the organ can prepare the organ to receive an optimized mixture of dissolved H2 and 02 in preparation for grafting. Figures 7A, 7B, and 7C depict an organ perfusion system that can be adapted to reduce the presence of dissolved nitrogen so that an optimal binary mixture of dissolved H2 and 02 can be infused to protect the organ from hypoxia and reperfusion injury during the surgical grafting and reperfusion steps, respectively.

[0121]

[0094] Figure 7A depicts an embodiment of an adapted organ perfusion system in the mode of oxygenated machine perfusion. A donated organ 601 can be maintained in an oxygenated state during transport or storage by a perfusion circuit 612 comprising the organ 601, a fluid recirculation pump 603, and a gas exchanger 602 all in a closed fluid pathway. A source of oxygen gas 604 can be provided to feed the gas exchanger 602 with a sweep gas 609. In most examples, the oxygen sweep gas 609 feeding the gas exchanger 602 can include a mixture of 02 and N2 gas. Due to the presence of N2 in the sweep gas 609, organ 601 will have a significant concentration of dissolved nitrogen gas after even a short period of time in the active perfusion circuit 612.

[0122]

[0095] In a mode of oxygenated machine perfusion, shutoff valves 610 can separate the perfusion circuit 612 from the hydrogen infusion components 613. The hydrogen infusion components canDocket No. 220709-702601

[0123] include a hydrogen infusion solution 605 that can be in thermal contact with a temperature regulator 608, an effluent analyzer 606, and an effluent container 607.

[0124]

[0096] Figure 7B depicts a transition point between oxygenated machine perfusion mode, as shown in FIG. 7A, and a nitrogen reducing mode in preparation for hydrogen infusion. The sweep gas 609 from FIG. 7A (which typically includes a nitrogen-rich mixture of N2 and 02) can be replaced by a sweep gas 615 containing mostly 02 with little or no N2. The reduced N2 levels and elevated 02 levels in sweep gas 615 can cause a corresponding drop in dissolved N2 and rise in dissolved 02 in organ 601. Having replaced most of the dissolved N2 in organ 601 with dissolved 02, organ 601 can be ready for hydrogen infusion. In cases where dissolved N2 still predominates in the tissue, the high fraction and slow bulk transport of N2 through tissue are a hinderance to rapid and efficient uptake of hydrogen gas, as well as to adjusting dissolved oxygen levels.

[0125]

[0097] Figure 7C depicts an embodiment of the hydrogen infusion step carried out in the adapted organ perfusion machine. Valves 610 can isolate organ 601 from the perfusion circuit 612 and stop all flow of perfusate solution through the gas exchanger 602. Optionally, additional isolation valves (not shown) can stop all flow of sweep gas 615 through gas exchanger 602. A hydrogen infusion solution 605 can flow through organ 601 to infuse H2 into the organ. The infusion solution flowing out of the organ can flow through an effluent analyzer 606 and into an effluent container 607. The effluent analyzer 606 can include 1) dissolved gas sensors, which can measure dissolved oxygen or hydrogen, 2) solution flow sensors, and 3) electronics to compare the dissolved oxygen or dissolved hydrogen in the effluent solution to a reference value. Other sensors can be disposed within the analyzer 606 to sense other parameters of the infusion solution flowing out of the organ, including, but not limited to, temperature sensors, optical sensors, and others. The effluent analyzer 606 can also function to display measured values or send commands to pumps or actuators controlling the flow of hydrogen infusion solution 605.

[0126]

[0098] In an example, the hydrogen infusion solution 605 can have a high H2 concentration with PH2 =730 mm Hg, and a low 02 concentration with PO2 < 30 mm Hg. Having this dissolved gas composition, the hydrogen infusion solution can quickly infuse hydrogen into the organ 601, and can simultaneously reduce the dissolved oxygen in the organ. An effluent analyzer 606 can measure the dissolved oxygen concentration in the effluent and stops the flow of hydrogen infusion solution 605 when the dissolved oxygen has reached the lowest acceptable concentration.

[0127]

[0099] In another example, the hydrogen infusion solution 605 can be prepared by one previously described processes for nitrogen removal and oxygen balancing depicted in FIGS. 2 through 6B. The solution 605 therefore can have pH2 = 600 mm Hg and pO2 = 150 mm Hg, with pN2 less than 10 mm Hg. As the infusion solution 605 flows through organ 601, the dissolved gases in theDocket No. 220709-702601

[0128] organ (initially a mix of N2 and 02) can converge on a mixture matching the low nitrogen, binary H2 / O2 gas profile of the infusion solution 605. The very low nitrogen content can make room for a high concentration of hydrogen and a physiologically acceptable concentration of oxygen to provide optimal condition for the organ to be removed from the machine and undergo a period of ischemia while the organ is surgically grafted into the organ recipient’s body.

[0129]

[0100] In some embodiments, a hydrogen infusion solution is adapted to be administered to a subject during a normothermic regional perfusion (NRP) procedure. In some embodiments, a hydrogen infusion step is performed on an organ donor during an NRP procedure. In the NRP context, a hydrogen-rich solution is used to prime the lines of the perfusion system and to perfuse the donor organs prior to introducing warm, oxygenated perfusate or blood back to the organs. In some embodiments the hydrogen-rich solution is co-administered to the donor vasculature in parallel with the oxygen-rich perfusate or blood in a manner calculated to maintain a therapeutic concentration of hydrogen during the normothermic regional reperfusion process. In some embodiments, the volume flow ratio of the hydrogen-rich solution to oxygen-rich perfusate (or blood) is 1:10. In another embodiment, the ratio may be as high as 1:1. In another embodiment, the NRP system continuously increases the flow of oxygenated perfusate while continuously decreasing the flow of hydrogen-rich solution to effect a complete transition from the initial state of hydrogen-rich perfusion to the final state of oxygen-rich perfusion.

[0130]

[0101] In another embodiment, the NRP equipment may be configured to first perform normothermic regional perfusion for a predetermined time period (30 minutes for example) and then begin to cool the organs off for transport by pumping or channeling a cool, hydrogen-rich solution through the organs to protect them during ischemic transport and handling.

[0131]

[0102] Further described herein are techniques for organ perfusion. In some embodiments, such techniques include organ perfusion systems that perfuse all or part of the organ donor’s body, or just an organ by itself.

[0132]

[0103] The perfusion system and perfusion protocol is configured to deliver both hydrogen-rich solutions and oxygen-rich solutions in a stepwise fashion: First, administer hydrogen-rich solution by use of a pre-packaged container of hydrogen-rich medical solution. The hydrogenrich solution can be hydrogen-rich saline used to prime the perfusion circuit. In some embodiments, the hydrogen-rich solution is non normothermic or below normal body temperature. Second, administer a mixture of hydrogen-rich solution and oxygen-rich perfusate in parallel, where the flow rates of hydrogen-rich solutions and oxygen rich solutions are adjusted to favor hydrogen-rich solution in the beginning and oxygen-rich solution in the end. Third, administer mostly oxygen-rich, normothermic perfusate solution with the option toDocket No. 220709-702601

[0133] continue administering up to 10% Hydrogen-rich solution by volume via parallel injection into the arterial flow path.

[0134]

[0104] In some embodiments, when the adding oxygen step includes adding dissolved oxygen to the solution to achieve a physiologically appropriate concentration of dissolved oxygen, the solution is heated in thermal cycle prior to hydrogen infusion.

[0135]

[0105] In some embodiments, the heating cycle (time, temperature, and internal atmosphere) is optimized to accomplish both sterilization and degassing. In some embodiments, the heating cycle comprises a duration of heat cycle in excess of the minimum necessary to achieve sterilization, (longer heat cycles favor degassing). In some embodiments, the heating cycle comprises a steam purge through the heating chamber at rates sufficient to minimize 02 and N2 partial pressure within the chamber (to favor 02 and N2 degassing).

[0136]

[0106] In some embodiments, when the adding oxygen step includes adding dissolved oxygen to the solution to achieve a physiologically appropriate concentration of dissolved oxygen, the system is further configured to the following sequence of steps to optimize the manufacturing process:

[0137]

[0107] Step 1 : Fill container with solution;

[0138]

[0108] Step 2: Purge air / 02 / N2 from container headspace;

[0139]

[0109] Step 3: Degassing by heating above 100°C;

[0140]

[0110] Step 4: Purge gas from container headspace after heating cycle has cooled (e.g., below 100°C; and

[0141]

[0111] Step 5: Add packaging gas comprising hydrogen, oxygen, or a mixture thereof to the solution using any of the previously described methods (injection directly into the primary container or inclusion of H2 and 02 gases within the outer, gas impermeable container or addition via a gas-filled, gas permeable container co-packaged with the medical solution inside a common gas-impermeable container.

[0142]

[0112] Further described herein are packaging techniques configured to optimize the above manufacturing process.

[0143]

[0113] In some embodiments, a package includes a solution container having a fill tube being separate from a gas transport tube. In some embodiments, the gas transport tube comprises a hydrophobic filter and a sealable or closable portion whereby the gas transport tube can be hermetically sealed after all gas transport steps are complete. In some embodiments, the hydrophobic filter has a nominal pore size for sterile filtering. In some embodiments, the gas transport tube comprises a bidirectional check valve in line with the filter.

[0144]

[0114] In some embodiments, such organ perfusion systems include packaging devices such as the exemplary packaging device 1000 illustrated in FIGS. 10A-10J. As illustrated in FIG. 10A,Docket No. 220709-702601

[0145] packaging device 1000 comprising at least one container 1001, each of the at least one container 1001 comprising an inner space 1002. The packaging device 1000 further includes at least one fill tube 1003, at least one access tube 1004, and at least one gas transport tube 1005. The fill tube 1003 allows for filling the inner space 1002 of the container 1001 with a solution 1006. In some embodiments, the solution 1006 comprises any solution described herein, such as, for example, a medical solution, a hydrogen-rich solution, or the like. In some embodiments, the gas transport tube 1005 comprises at least one valve 1007 and at least one filter 1008 in line with each other. The at least one valve 1007 is configured to allow for control over gas egress from the container 1001 and gas ingress into the container 1001. For example, the at least one valve 1007 is configured to allow for control over gas (e.g., oxygen, hydrogen, nitrogen) egress from the container and gas ingress (e.g., air) into the container 1001. In some embodiments, the valve 1007 comprises a bi-directional check valve. In some embodiments, the container 1001 further comprises a headspace 1009 after it has been filled, wherein the headspace 1009 comprises at least one gas. In some embodiments, the filter 1008 is sterile. In some embodiments, the filter 1008 is hydrophobic. In some embodiments, the filter 1008 has a predetermined pore size ranging from between about 0.01 to about 0.5 micron, between about 0.1 to about 0.3 micron, between about 0.2 to about 0.4 micron, of about 0.15 micron, of about 0.2 micron, of about 0.21 micron, of about 0.22 micron, of about 0.23 micron, of about 0.24 micron, of about 0.25 micron, or the like.

[0146]

[0115] FIG. 10B illustrates a seal 1010 at a distal end of the fill tube 1003. The seal 1010 may be a sealable or closable portion of the fill tube 1003. The seal 1010 may be a hermetic seal. The seal 1010 may be applied once one or more gas transport steps described herein are completed within the packaging device 1000.

[0147]

[0116] The package design can facilitate the degassing process as follows: the first headspace purge from Step 2 in the above section can be accomplished by either pulling vacuum on the gas transport tube or by pressurizing (squeezing) the container to push gas out through the hydrophobic filter of the gas transport tube. In some embodiments, the flow automatically stops when the gas is gone and the solution comes in contact with the hydrophobic filter. The bidirectional check valve with controlled cracking pressure prevents air ingress into the container. The container of solution (now without headspace) is placed into the heating chamber with gas transport tube oriented towards the top of the container so that gases evolving from the solution can exit through the gas transport tube during heating. The solution is thermally degassed (heated to drive out dissolved gases). In some embodiments, any headspace remaining in the container (from gases recently driven out of solution) are removed either via vacuum or by applying external pressure to the solution container. In some embodiments, a hydrophobic filter canDocket No. 220709-702601

[0148] prevent egress of liquid phase so only gas phase can be forced out of the solution container. In some embodiments, packaging gas (hydrogen, oxygen, or mixture thereof) can be pushed through the hydrophobic filter and bi-directional check valve to create a headspace of the intended H2 / O2 mixture within the solution container.

[0149]

[0117] In some embodiments, the gas transport tube is hermetically sealed to prevent further fluid motion. In some embodiments, gas transport tube seal is made between the hydrophobic filter and the main solution reservoir so the filter can be discarded after the gas transport tube is sealed.

[0150]

[0118] FIG. IOC illustrates the packaging device 1000 after initial filling of the inner space 1002 with a solution 1006 via the fill tube 1003, which has been sealed with the seal 1010. The headspace 1009 at this point comprises an initial gas composition. FIG. 10D illustrates the packaging device 1000 after the gas in the headspace 1009 has been removed, e.g., via a positive pressure from pushing / squeezing of the packaging device 1000 or via a negative pressure relative to the inner space 1002 to force the gas in the headspace 1009 externally from the packaging device 1000. The filter 1008 is shown to prevent egress of the solution 1006.

[0151]

[0119] FIG. 10E further illustrates the packaging device 1000 inserted into a heating chamber 1100 for a degassing cycle. The container 1001 may be oriented upright during heating within the heating chamber. In some embodiments, the heating chamber 1100 comprises an over, an autoclave, a humidity chamber, a thermal shock chamber, a dry heat sterilizer, a heated ultrasonic bath, or the like. FIG. 10F illustrates the packaging device 1000 that, using the valve 1007, internal container pressure can be released and allows for the egress of dissolved gases from the headspace 1009. At this point, a new composition of gases in the headspace 1009 is formed from 02, N2, or other dissolved gases coming out of the solution 1006.

[0152]

[0120] FIG. 10G illustrates the packaging device 1000 removed from the heating chamber 1100 (optional) and having a headspace 1009 of previously dissolved gases remaining after a thermal degassing cycle. FIG. 10H illustrates that the composition in the headspace 1009 may be removed again, similar to what is performed as shown in FIG. 10D, with solution egress stopped by the filter 1008.

[0153]

[0121] FIG. 101 illustrates the packaging device 1000 further filled with a composition of H2, 02, or a combination thereof to fill the headspace 1009 as desired. As discussed above, these steps may be repeated as necessary until a final gas composition as desired is obtained within the headspace 1009. FIG. 10J illustrates the packaging device 1000 with the final gas composition, and with distal end of the gas transport tube 1005 sealed off, with the filter 1008 and the valve 1007 discarded. The final gas composition in the headspace 1009 can then be mixed with theDocket No. 220709-702601

[0154] solution 1006 to obtain the dissolved gas solution for infusion, perfusion, or any other process described herein.

[0155]

[0122] While reference to the human body, tissue, and organs is made herein with regard to the use of the instant disclosure, it will be appreciated that this disclosure can be applicable for other living organism and other non-biological systems.

[0156] Illustrative Embodiments

[0157]

[0123] Embodiment 1. A method for producing a hydrogen-rich medical perfusion solution, the method comprising,

[0158]

[0124] a. providing a medical perfusion solution in a container;

[0159]

[0125] b. degassing the medical perfusion solution to remove nitrogen and oxygen from the medical perfusion solution until a combination of a partial pressure of nitrogen and a partial pressure of oxygen in the medical perfusion solution is between 0 to 150 mm Hg; and

[0160]

[0126] c. adding hydrogen to the medical perfusion solution; and

[0161]

[0127] d. optionally, adding oxygen to the medical perfusion solution, after the degassing.

[0162]

[0128] Embodiment 2. The method of Embodiment 1, wherein the degassing includes using one of a vacuum, ultrasonic energy, heat, or a combination thereof to reduce a concentration of dissolved nitrogen in the medical perfusion solution.

[0163]

[0129] Embodiment 3. The method of any one of Embodiment 1 or Embodiment 2, wherein the adding oxygen includes adding dissolved oxygen to the medical perfusion solution to achieve a predetermined concentration of dissolved oxygen.

[0164]

[0130] Embodiment 4. The method of any one of Embodiment 1-Embodiment 3, wherein the degassing replaces a portion of dissolved nitrogen with dissolved oxygen prior to the adding hydrogen.

[0165]

[0131] Embodiment 5. The method of any one of Embodiment 1-Embodiment 4, wherein the degassing comprises filling the container with the medical perfusion solution and purging at least one gas from a headspace of the container.

[0166]

[0132] Embodiment 6. The method of Embodiment 5, wherein the degassing further comprises heating the container to above 100°C and purging the at least one gas from at least one valve in fluid communication with the headspace of the container.

[0167]

[0133] Embodiment 7. The method of any one of Embodiment 5 or Embodiment 6, wherein the purging is performed after the container has returned to a temperature below 100°C.

[0168]

[0134] Embodiment 8. The method of any one of Embodiment 6-Embodiment 7, wherein the degassing further comprises adding a predetermined amount of at least one of H2, 02, or a combination thereof to the headspace using the at least one valve.Docket No. 220709-702601

[0169]

[0135] Embodiment 9. The method of Embodiment 8, wherein the adding is performed using at least one of: injection directly into the container, or a gas-filled and gas-permeable second container that is co-packaged with the medical perfusion solution inside a third gas-impermeable container.

[0170]

[0136] Embodiment 10. The method of any one of Embodiment 1 -Embodiment 9, wherein, after the adding hydrogen, the medical perfusion solution has a hydrogen partial pressure (pH2) of at least 0.7 atm.

[0171]

[0137] Embodiment 11. The method of any one of Embodiment 1 -Embodiment 10, wherein, after the adding oxygen, the medical perfusion solution has an oxygen partial pressure (pO2) of at least 0.7 atm.

[0172]

[0138] Embodiment 12. The method of any one of Embodiment 1 -Embodiment 11, wherein a ratio of the hydrogen to the oxygen is controlled to produce a combined solution with a predetermined partial pressure of each of the hydrogen and the oxygen.

[0173]

[0139] Embodiment 13. The method of any one of Embodiment 1 -Embodiment 12, wherein a ratio of the hydrogen to the oxygen in the medical perfusion solution ranges from 1 : 1 to 9:1.

[0174]

[0140] Embodiment 14. The method of any one of Embodiment 1-Embodiment 13, wherein the container is subsequently packaged in an outer gastight container.

[0175]

[0141] Embodiment 15. The method of any one of Embodiment 1-Embodiment 14, wherein the medical perfusion solution is a denitrogenated solution.

[0176]

[0142] Embodiment 16. A system for producing medical solutions with dissolved hydrogen gas, the system comprising:

[0177]

[0143] a. at least one solution container having at least one fill tube, at least one gas transport tube, and at least one access tube; and

[0178]

[0144] b. a heating chamber sufficiently sized to house the at least one solution container and apply heat sufficient to increase a temperature of a medical solution within the at least one solution container to at least 100°C,

[0179]

[0145] c wherein the at least one solution container comprises a headspace absent the medical solution and allows sufficient escape of dissolved gas from the medical solution upon introduction of the medical solution into the at least one solution container,

[0180]

[0146] d. wherein the at least one gas transport tube comprises a hydrophobic filter or porous membrane in line with at least one valve configured to allow for control over gas egress from the at least one solution container and gas ingress into the at least one solution container; andDocket No. 220709-702601

[0181]

[0147] e wherein, upon heating of the medical solution in the heating chamber, at least one dissolved gas is separated from the medical solution into the headspace such that the at least one dissolved gas is removable via the at least one gas transport tube upon opening of the at least one valve, thereby allowing for a sufficient amount of an additional gas composition to be introduced into the headspace so as to generate a predetermined composition of dissolved gas in the medical solution, the predetermined composition of dissolved gas having a partial pressure of dissolved hydrogen (pH2) greater than a partial pressure of dissolved oxygen (pO2) and the pO2 greater than a partial pressure of dissolved nitrogen (pN2).

[0182]

[0148] Embodiment 17. The system of Embodiment 16, wherein the at least one gas transport tube comprises a hermetically sealable portion.

[0183]

[0149] Embodiment 18. The system of any one of Embodiment 16 or Embodiment 17, wherein the hydrophobic filter comprises a pore size ranging from about 0.15 to about 0.3 micron.

[0184]

[0150] Embodiment 19. The system of any one of Embodiment 16-Embodiment 18, wherein the at least one valve comprises a bidirectional check valve.

[0185]

[0151] Embodiment 20. The system of any one of Embodiment 16-Embodiment 19, wherein the at least one valve is closer to an external portion of the at least one gas transport tube than the hydrophobic filter.

[0186]

[0152] Embodiment 21. The system of any one of Embodiment 16-Embodiment 20, wherein each of the at least one fill tube is separate from each of the at least one gas transport tube.

[0187]

[0153] Embodiment 22. The system of any one of Embodiment 16-Embodiment 21, wherein the heating chamber is selected from an oven, an autoclave, a humidity chamber, a thermal shock chamber, a dry heat sterilizer, a heated ultrasonic bath, or a combination thereof.

[0188]

[0154] Embodiment 23. The system of any one of Embodiment 16-Embodiment 22, further comprising a temperature controlling feature configured to maintain a temperature of the medical solution.

[0189]

[0155] Embodiment 24. The system of any one of Embodiment 16-Embodiment 23, further comprising one or more gas sensors positioned to measure concentrations of dissolved oxygen or dissolved hydrogen in an egressing gas or a headspace of the medical solution, optionally wherein the one or more gas sensors comprise one or more dissolved gas sensors, one or more solution flow sensors, temperature sensors, optical sensors, or a combination thereof..

[0190]

[0156] Embodiment 25. The system of Embodiment 24, wherein the concentrations of dissolved hydrogen and / or dissolved oxygen in the effluent medical solution are compared to aDocket No. 220709-702601

[0191] reference value so as to achieve a predetermined concentration of at least one of dissolved oxygen or dissolved hydrogen in the medical solution.

[0192]

[0157] Embodiment 26. A perfusion solution, the perfusion solution comprising,

[0193]

[0158] a. a fluid having a dissolved gas mixture of hydrogen, oxygen, and nitrogen,

[0194]

[0159] b. wherein a partial pressure of the dissolved hydrogen (pH2) is higher than 350 mm Hg, the partial pressure of the dissolved oxygen (pO2) ranges from 45 mm Hg to 300 mm Hg, and the partial pressure of dissolved nitrogen (pN2) is lower than a pO2 in the solution,

[0195]

[0160] c wherein contact of the perfusion solution with an organ or tissue increases an amount of dissolved hydrogen and does not decrease an amount of dissolved oxygen within the organ or tissue as compared to the organ or tissue prior to contacting with the perfusion solution, wherein a predetermined concentration of dissolved oxygen in the organ or tissue is maintained over a course of hydrogen infusion so as to prevent a worsening of hypoxia or oxidative stress within the organ or tissue during the contact of organ or tissue with solution; optionally wherein the decrease in the amount of dissolved 02 results in a reduced occurrence of oxidative stress as compared to the organ or tissue prior to the contacting.

[0196]

[0161] Embodiment 27. The solution of Embodiment 26, wherein the oxidative stress is represented by one or more of acute tubular necrosis (ATN), apoptotic cell death, Kidney Injury Marker 1 (KIM-1), Interleukin 6 (IL-6), CD68(+), Myeloperoxidase (MPO), Malondialdehyde (MDA), or a combination thereof.

[0197]

[0162] Embodiment 28. The solution of Embodiment 26 or Embodiment 27, wherein the partial pressure of the dissolved hydrogen is between about 450 mm Hg and about 500 mm Hg, and wherein the partial pressure of the dissolved oxygen is at least 1.5 times greater than the partial pressure of dissolved nitrogen.

[0198]

[0163] Embodiment 29. The solution of any one of Embodiment 26-Embodiment 28, wherein the fluid comprises a University of Wisconsin (UW) solution or an organ or tissue preservation solution.

[0199]

[0164] Embodiment 30. The solution of any one of Embodiment 26-Embodiment 29, further comprising one or more chelating agents selected from ethylenediaminetetraacetic acid (EDTA), Desferrioxamine (DFO), citric acid, Dimercaptosuccinic Acid (DMSA), or a combination thereof, wherein the one or more chelating agents are at concentrations ranging from 10-6 Molar to 10-4 Molar.

[0200]

[0165] Embodiment 31. The solution of any one of Embodiment 26-Embodiment 30, wherein the pN2 is less than 0.2 atm.

[0201]

[0166] Embodiment 32. The solution of any one of Embodiment 26-Embodiment 31, wherein the pN2 is less than 0.1 atm.Docket No. 220709-702601

[0202]

[0167] Embodiment 33. The solution of any one of Embodiment 26-Embodiment 32, wherein a pO2 is greater than pN2 by a magnitude of at least 3x.

[0203]

[0168] Embodiment 34. The solution of any one of Embodiment 26-Embodiment 33, wherein a pH2 is greater than pO2 by a magnitude of at least 2x and greater than pN2 by a magnitude of at least 3x.

[0204]

[0169] Embodiment 35. The solution of any one of Embodiment 26-Embodiment 34, wherein, when a concentration of the hydrogen ranges from 40% to 80%, a pO2 is greater than a combined partial pressures of other gases, the other gases excluding the hydrogen and the oxygen.

[0205]

[0170] Embodiment 36. The solution of any one of Embodiment 26-Embodiment 35, further comprising a bicarbonate buffer, wherein a partial pressure of carbon dioxide (pCO2) ranged from 0.04 atm - 0.06 atm, a pH2 ranges from 0.6 atm - 0.8 atm, and a pO2 ranges from 0.15 atm - 0.35 atm.

[0206]

[0171] Embodiment 37. The solution of any one of Embodiment 26-Embodiment 36, further comprising one or more hydrogen sulfide donors selected from sodium thiosulfate (STS), sodium sulfide (Na2S), sodium hydrosulfide (NaHS), or a combination thereof, wherein a concentration of the one or more hydrogen sulfide donors ranges from 0.1 micromolar to 50 micromolar.

[0207]

[0172] Embodiment 38. The solution of any one of Embodiment 26-Embodiment 37, wherein the gas mixture dissolved in the fluid comprises a binary mixture of hydrogen and oxygen.

[0208]

[0173] Embodiment 39. A method of producing medical solutions with dissolved hydrogen gas, the method comprising,

[0209]

[0174] a. providing an outer gas impermeable container and at least one inner gas permeable container;

[0210]

[0175] b. packaging a medical solution into the at least one inner gas permeable container, the medical solution being previously depleted of nitrogen having an amount of oxygen gas in a headspace of the at least one inner gas permeable container; and

[0211]

[0176] c arranging the at least one inner gas permeable container and a predetermined amount of hydrogen gas within the outer gas impermeable container,

[0212]

[0177] d. wherein the predetermined amount of the hydrogen gas is sufficient to permeate into the at least one inner gas permeable container to equilibrate with the oxygen gas such that the medical solution has a predetermined dissolved gas composition, the predetermined dissolved gas composition having a partial pressure of dissolved hydrogen (pH2) higher than 350Docket No. 220709-702601

[0213] mm Hg and a partial pressure of dissolved oxygen (p02) ranging from 45 mm Hg to 300 mm Hg

[0214]

[0178] Embodiment 40. The method of Embodiment 39, wherein the at least one inner gas permeable container comprises a first inner gas permeable container and a second inner gas permeable container, wherein the medical solution is packaged into the first inner gas permeable container and the predetermined amount of hydrogen gas is packaged into the second inner gas permeable container.

[0215]

[0179] Embodiment 41. The method of any one of Embodiment 39 or Embodiment 40, wherein the at least one inner gas permeable container is transparent, flexible, sterile, or a combination thereof.

[0216]

[0180] Embodiment 42. The method of any one of Embodiment 39-Embodiment 41, further comprising infusing the medical solution into a machine perfused organ or tissue.

[0217]

[0181] Embodiment 43. The method of Embodiment 42, wherein the infusing comprises adjusting a concentration of dissolved nitrogen and dissolved oxygen in the machine perfused organ or tissue, the adjusting comprising administering a sweep gas prior to cessation of machine perfusion in preparation for hydrogen infusion, wherein the sweep gas is adjusted to an N2:O2 ratio of up to about 0.5.

[0218]

[0182] Embodiment 44. The method of Embodiment 43, wherein the sweep gas comprises a mixture of N2 and 02.

[0219]

[0183] Embodiment 45. A method for producing a medical solution with substantially binary H2 / O2 dissolved gas mixture wherein an outer package contains a kit comprising:

[0220]

[0184] a. at least one container of oxygen- saturated or oxygen-rich solution (pO2 > 0.5 atm); and

[0221]

[0185] b. a gas-tight container of hydrogen gas to be combined with the oxygen-rich solution prior to using the medical solution.

[0222]

[0186] Embodiment 46. The method of Embodiment 45, wherein the outer package comprises a gas-impermeable barrier.

[0223]

[0187] Embodiment 47. The method of any one of Embodiment 45 or Embodiment 46, wherein the at least one container comprises a flexible material.

[0224]

[0188] Embodiment 48. A method for producing a medical solution with substantially binary H2 / O2 dissolved gas mixture wherein an outer package contains a kit comprising:

[0225]

[0189] a. at least one container of hydrogen-rich solution having a partial pressure of dissolved hydrogen (pH2) greater than 0.5 atm; and

[0226]

[0190] b. a gas-tight container of an oxygen gas,Docket No. 220709-702601

[0227]

[0191] c wherein an oxygen gas to be combined with the hydrogen-rich solution prior to using the hydrogen-rich solution.

[0228]

[0192] Embodiment 49. The method of Embodiment 48, wherein the outer package comprises a gas-impermeable barrier.

[0229]

[0193] Embodiment 50. The method of any one of Embodiment 48 or Embodiment 49, wherein the at least one container comprises a flexible material.

[0230]

[0194] Embodiment 51. A method for using binary dissolved gas mixtures of hydrogen and oxygen in a medical solution, the method comprising:

[0231]

[0195] a. combining a hydrogen-rich solution and an oxygen-rich solution within a container to form the binary dissolved gas mixture, wherein a volume ratio of hydrogen-rich solution to oxygen-rich solution ranges from 1 : 1 to 9: 1.

[0232]

[0196] Embodiment 52. The method of Embodiment 51, wherein a concentration of glutathione is higher in the hydrogen-rich solution than in the oxygen-rich solution.

[0233]

[0197] Embodiment 53. The method of any one of Embodiment 51 or Embodiment 52, wherein the combining is performed at a time of use or immediately prior to the time of use.

[0234]

[0198] Embodiment 54. The method of any one of Embodiment 51-Embodiment 53, wherein the combining comprises heating the container to above 100°C and purging at least one gas from a headspace of the container.

[0235]

[0199] Embodiment 55. The method of Embodiment 54, wherein the purging is performed after the container has returned to a temperature below 100°C.

[0236]

[0200] Embodiment 56. A solution, the solution comprising,

[0237]

[0201] Embodiment 57. a fluid having a dissolved gas mixture of hydrogen, oxygen, and nitrogen,

[0238]

[0202] Embodiment 58. wherein a partial pressure of the hydrogen is higher than 350 mm Hg, the partial pressure of the oxygen ranges from 45 mm Hg to 300 mm Hg, and the partial pressure of dissolved nitrogen is lower than a concentration of the dissolved oxygen in the solution.

[0239]

[0203] Embodiment 59. The solution of Embodiment 56,

[0240]

[0204] Embodiment 60. wherein the partial pressure of the hydrogen is between about 450 mm Hg and about 500 mm Hg, and

[0241]

[0205] Embodiment 61. wherein the partial pressure of the oxygen is at least 1.5 times greater than the partial pressure of dissolved nitrogen.

[0242]

[0206] Embodiment 62. A method for producing a hydrogen-rich solution, the method comprising,

[0243]

[0207] Embodiment 63. providing a solution;Docket No. 220709-702601

[0244]

[0208] Embodiment 64. degassing the solution to remove nitrogen and oxygen until the combination of PN2 and PO2 is between 0 to 150 mm Hg;

[0245]

[0209] Embodiment 65. adding hydrogen to the solution; and

[0246]

[0210] Embodiment 66. adding oxygen to the solution, after the degassing step.

[0247]

[0211] Embodiment 67. The method of Embodiment 62, wherein the degassing step includes using one of a vacuum, ultrasonic energy, heat, or a combination thereof to reduce the concentration of dissolved nitrogen in the solution.

[0248]

[0212] Embodiment 68. The method of any one of Embodiment 62-Embodiment 67, wherein the adding oxygen step includes adding dissolved oxygen to the solution to achieve a physiologically appropriate concentration of dissolved oxygen.

[0249]

[0213] Embodiment 69. The method of any one of Embodiment 62-Embodiment 68, wherein degassing step replaces a portion of dissolved nitrogen with dissolved oxygen prior to the adding hydrogen step.

[0250]

[0214] Embodiment 70. A method of producing medical solutions with dissolved hydrogen gas, the method comprising,

[0251]

[0215] Embodiment 71. injecting the dissolved hydrogen gas into a headspace of the container holding a degassed or denitrogenated solution.

[0252]

[0216] Embodiment 72. The method of Embodiment 70, wherein the container is subsequently packaged in an outer gastight container.

[0253]

[0217] Embodiment 73. A method of producing medical solutions with dissolved hydrogen gas, the method comprising,

[0254]

[0218] Embodiment 74. packaging a nitrogen-depleted solution in an inner gas permeable container,

[0255]

[0219] Embodiment 75. arranging the inner gas permeable container within an outer gastight container,

[0256]

[0220] Embodiment 76. wherein the dissolved hydrogen is added to the medical solution via a second gas permeable container with hydrogen gas adjacent the gas permeable solution container and within the outer gastight container.

[0257]

[0221] Embodiment 77. A medical solution method of manufacturing, the method comprising,

[0258]

[0222] Embodiment 78. packaging a volume of nitrogen-depleted solution in a first gas permeable container;

[0259]

[0223] Embodiment 79. packaging a gas mixture containing hydrogen and oxygen in a second gas permeable container; andDocket No. 220709-702601

[0260]

[0224] Embodiment 80. sealing the first and second gas permeable containers within an outer gas tight container.

[0261]

[0225] Embodiment 81. An organ perfusion system, the system comprising,

[0262]

[0226] Embodiment 82. a hydrogen infusion system, the hydrogen infusion system comprising a container of hydrogen infusion solution, a temperature controlling feature configured to maintain the temperature of the hydrogen infusion solution, and a shutoff valves to prevent flow of hydrogen infusion solution into the gas exchanger of the perfusion system.

[0263]

[0227] Embodiment 83. The system of Embodiment 81, further comprising one or more dissolved gas sensors positioned to measure concentrations of dissolved oxygen and / or dissolved hydrogen in the effluent hydrogen infusion solution as it flows out of the organ.

[0264]

[0228] Embodiment 84. The system of any one of Embodiment 81-Embodiment 83, wherein the measurement of dissolved hydrogen and / or dissolved oxygen in the effluent solution is compared to a reference value, to shut off or otherwise modify the flow of the hydrogen infusion solution to achieve the desired concentration of dissolved oxygen or dissolved hydrogen (or both) in the organ.

[0265]

[0229] Embodiment 85. A method for adjusting concentrations of dissolved nitrogen and dissolved oxygen in a machine perfused organ, the method comprising,

[0266]

[0230] Embodiment 86. administering a sweep gas prior to cessation of machine perfusion in preparation for hydrogen infusion,

[0267]

[0231] Embodiment 87. wherein the sweep gas is adjusted to an N2:O2 ratio of about 0.5.

[0268]

[0232] Embodiment 88. A solution, the solution comprising:

[0269]

[0233] Embodiment 89. a fluid having a dissolved gas mixture of hydrogen and oxygen, wherein the dissolved gas mixture is a substantially binary mixture of hydrogen and oxygen.

[0270]

[0234] Embodiment 90. The solution of Embodiment 88, wherein the fluid comprises UW solution or an organ preservation solution.

[0271]

[0235] Embodiment 91. The solution of any one of Embodiment 88-Embodiment 90, further comprising one or more chelating agents selected from ethylenediaminetetraacetic acid (EDTA), Desferrioxamine (DFO), citric acid, Dimercaptosuccinic Acid (DMSA), or a combination thereof, wherein the one or more chelating agents are at concentrations ranging from 10-6 Molar to 10-4 Molar.

[0272]

[0236] Embodiment 92. The solution of any one of Embodiment 88-Embodiment 91, further comprising a partial pressure of nitrogen (pN2) less than 0.2 atm.

[0273]

[0237] Embodiment 93. The solution of any one of Embodiment 88-Embodiment 92, wherein the pN2 is less than 0.1 atmDocket No. 220709-702601

[0274]

[0238] Embodiment 94. The solution of any one of Embodiment 88-Embodiment 93, wherein a partial pressure of oxygen (pO2) is greater than a pN2.

[0275]

[0239] Embodiment 95. The solution of any one of Embodiment 88-Embodiment 94, wherein a pO2 is greater than pN2 by a magnitude of at least 3x.

[0276]

[0240] Embodiment 96. The solution of any one of Embodiment 88-Embodiment 95, wherein a partial pressure of hydrogen (pH2) is at greater than a pN2.

[0277]

[0241] Embodiment 97. The solution of any one of Embodiment 88-Embodiment 90, wherein a pH2 is greater than pO2 by a magnitude of at least 2x and greater than pN2 by a magnitude of at least 3x.

[0278]

[0242] Embodiment 98. The solution of any one of Embodiment 88-Embodiment 90, wherein, when a concentration of the hydrogen ranges from 40% to 80%, a pO2 is greater than a combined partial pressures of other gases, the other gases excluding the hydrogen and the oxygen.

[0279]

[0243] Embodiment 99. The solution of any one of Embodiment 88-Embodiment 90, further comprising a bicarbonate buffer, wherein a partial pressure of carbon dioxide (pCO2) ranged from 0.04 atm - 0.06 atm, a pH2 ranges from 0.6 atm - 0.8 atm, and a pO2 ranges from 0.15 atm - 0.35 atm.

[0280]

[0244] Embodiment 100. The solution of any one of Embodiment 88-Embodiment 90, further comprising one or more hydrogen sulfide donors selected from sodium thiosulfate (STS), sodium sulfide (Na2S), sodium hydrosulfide (NaHS), or a combination thereof, wherein a concentration of the one or more hydrogen sulfide donors ranges from 0.1 micromolar to 50 micromolar.

[0281]

[0245] Embodiment 101. A method for producing medical solutions with substantially binary (H2 / O2) dissolved gas mixture, the method comprising:

[0282]

[0246] Embodiment 102. packaging two or more packages of solution within an outer package or container, wherein at least one of the inner containers comprises a gas-tight container to maintain a gas-impermeable separation between hydrogen-rich and oxygen-rich solutions until the time of use.

[0283]

[0247] Embodiment 103. The method of Embodiment 101, wherein the hydrogen-rich solution has hydrogen partial pressure of at least 0.7 atm

[0284]

[0248] Embodiment 104. The method of any one of Embodiment 101-Embodiment 103, wherein the oxygen-rich solution has an oxygen partial pressure of at least 0.7 atm.

[0285]

[0249] Embodiment 105. The method of any one of Embodiment 101-Embodiment 104, wherein the volume ratios of hydrogen-rich solution to oxygen rich solution is calculated to produce a combined solution with the desired partial pressures of hydrogen and oxygen.Docket No. 220709-702601

[0286]

[0250] Embodiment 106. A method for producing a medical solution with substantially binary H2 / O2 dissolved gas mixture wherein an outer package contains a kit comprising:

[0287]

[0251] Embodiment 107. a container of degassed medical solution, optionally wherein the container is flexible;

[0288]

[0252] Embodiment 108. at least one volume of gas or gas mixture in a gas-impermeable container to be combined with the degassed solution prior to using the solution.

[0289]

[0253] Embodiment 109. The method of Embodiment 106, wherein a single gas container comprises a gas mixture of H2 and 02 in a ratio ranging from 1 :9 to 9: 1

[0290]

[0254] Embodiment 110. The method of any one of Embodiment 106-Embodiment 109, wherein two separate gas containers are used, each containing either substantially pure H2 or substantially pure 02 gas to be combined with the degassed solution prior to using the solution

[0255] Embodiment 111. A method for producing a medical solution with substantially binary H2 / O2 dissolved gas mixture wherein an outer package contains a kit comprising:

[0291]

[0256] a. a container of oxygen-saturated or oxygen-rich solution (pO2 > 0.5 atm), optionally wherein the container is flexible; and

[0292]

[0257] b. a gas-tight container of hydrogen gas to be combined with the oxygen-rich solution prior to using the solution, optionally with the outer packaging comprising a gas-impermeable barrier.

[0293]

[0258] Embodiment 112. A method for producing a medical solution with substantially binary H2 / O2 dissolved gas mixture wherein an outer package contains a kit comprising:

[0294]

[0259] a. a container of hydrogen-saturated or hydrogen-rich solution (pH2 > 0.5 atm), optionally wherein the container is flexible;

[0295]

[0260] b. a gas-tight container of oxygen gas to be combined with the hydrogen-rich solution prior to using the solution, optionally with the outer packaging comprising a gas-impermeable barrier.

[0296]

[0261] Embodiment 113. A method for using binary dissolved gas mixtures of hydrogen and oxygen in a medical solution, in which a hydrogen-rich solution and a separate oxygen-rich solution are combined within a single package or container at the time of use (or immediately prior) to form the binary gas mixture .

[0297]

[0262] Embodiment 114. The method of Embodiment 113, wherein the volume ratio of H2-rich solution to 02-rich solution ranges from 1 : 1 to 9: 1.

[0298]

[0263] Embodiment 115. The method of any one of Embodiment 113 -Embodiment 114, wherein the concentration of glutathione is higher in the H2-rich solution than in the 02-rich solution.Docket No. 220709-702601

[0299] EXAMPLES

[0300]

[0264] Described below are examples of ways in which techniques described herein may be implemented. It should be appreciated that these examples are merely illustrative, that embodiments are not limited to operating in accordance with the specific examples shown in the figures and discussed below, and that other embodiments are possible.

[0301] Example 1. Exemplary dissolved gas compositions for organ preservation.

[0302]

[0265] The purpose of this example is to provide exemplary results of dissolved gas mixtures as described above in kidney organs.

[0303]

[0266] In organ preservation solutions, dissolved hydrogen and dissolved oxygen play critical roles and their levels should be optimized for best effect. An insufficient concentration of hydrogen reduces the antioxidant effects of hydrogen, which are known to follow a positive dose-response relationship. An insufficient concentration of oxygen in the organ preservation solution can strip residual oxygen out of an already hypoxic organ, worsening the effects of hypoxic injury.

[0304]

[0267] In the context of hydrogen-rich organ preservation solutions, the optimized dissolved gas mixture is a binary mixture of H2 and 02, with a ratio of H2:O2 ranging from 2: 1 to 9: 1.

[0305] Tertiary gases like nitrogen and carbon dioxide may be present as residue from processing or storage conditions, but due to competition between dissolved gases (Dalton’s Law of Partial Pressures) the presence of tertiary gases can only result in a reduction in the partial pressure of one or more of the beneficial gases H2 and 02. Reducing the concentration of H2 in solution may reduce its antioxidant efficacy. Reducing the concentration of 02 in solution below the residual 02 concentrations in the ischemic organ tissues may irreversibly increase the negative effects of hypoxia on cell death and organ disfunction.

[0306]

[0268] Nomenclature Note: For convenience and ease of interpretation, percentages are used to describe how much of a given gas is dissolved in an aqueous solution. The percentage quantifies the amount of a gas dissolved in solution as a percentage of the solubility limit for said gas. For example, 80% H2 in solution can be interpreted as a concentration of hydrogen that is 80% of the saturation limit for H2 in solution. Conveniently these solubility percentages also correspond to (a) the volume fraction of said gas in a gas phase mixture in equilibrium with the solution; and (b) the partial pressure of the dissolved gas as a percentage of the total system pressure (per Dalton’s Law).

[0307]

[0269] Example: an aqueous solution having “80% H2 and 20% 02” has a dissolved hydrogen concentration [H2] that is 80% of the H2 solubility limit at 1 atm, and a hydrogen partial pressure (pH2) that is 80% of the total system pressure. The solution also has a dissolvedDocket No. 220709-702601

[0308] oxygen concentration

[0002] that is 20% of the 02 solubility limit at 1 atm, and oxygen partial pressure (pO2) that is 20% of the total system pressure.

[0309]

[0270] The max solubilities of H2, 02, and N2 in water at 25°C, 1 atm are given below for reference, (solubility limits change for temperature and solvent conditions)

[0310]

[0271] H2 solubility limit at pH2 = 1 atm: 0.00078 mol / L = 0.78 mM

[0311]

[0272] 02 solubility limit atpO2 = 1 atm: 0.0013 mol / L = 1.3 mM

[0312]

[0273] N2 solubility limit at pN2 = 1 atm: 0.00061 mol / L = 0.61 mM

[0313]

[0274] The partial pressures of all gases in the solution must sum to the total system pressure per Dalton’s Law. For convenience, system pressure is assumed to be sea level atmospheric pressure 1 atm = 760 mm Hg.

[0314]

[0275] Conventionally, hydrogen-rich organ preservation solutions recognize the importance of maximizing hydrogen concentrations in order to maximize antioxidant effect. However, the inventors have recognized and appreciated the role or necessity of dissolved oxygen as an ingredient of hydrogen-rich organ preservation solutions. Where oxygen is mentioned, it is treated as a gas to be displaced or removed as much as possible from solution or ignored as a minor impurity, not a gas to be retained for its beneficial impact on residual oxygen stores within the organ tissue. In some previous systems, dissolved oxygen concentration is intentionally eliminated from solution to prevent catalyzed reaction between dissolved hydrogen and dissolved oxygen.

[0315]

[0276] In some previous systems, processes and methods aim to maximize hydrogen concentration in solution by displacing other gases already present in the solution to make “room” for dissolved hydrogen, without any means of selectively retaining important gases like 02 or of selectively removing nonbeneficial gases like N2 (which competes with both H2 and 02 based on the solubility limits (Dalton’s Law of Partial Pressures)).

[0316]

[0277] Supporting Evidence: Two experiments were performed that highlight the difference between solution production methods and their resulting gas compositions, and the ultimate effect of the gas compositions on efficacy as an organ preservation solution.

[0317]

[0278] Experimental model and background: The porcine kidney DCD model imposes a 1-hour period of warm ischemia on both kidneys (no blood flow through renal arteries) prior to removing the kidneys from the donor animal’s body. The warm ischemic period in the porcine DCD model kidney is designed to simulate the warm ischemic period in clinical organ recovery between the time of cardiac death of the human donor and the subsequent time of surgical recovery of the organ for transplant. In the porcine DCD model, immediately upon removal from the warm ischemic conditions inside the donor body, the kidney is flushed by renal artery perfusion with cold University of Wisconsin (UW) solution. After flushing with coldDocket No. 220709-702601

[0318] preservation solution, kidneys are maintained at 4 °C for 4 hours and then reperfused with warm, oxygenated blood for 4 hours at physiologic temperature (about 39°C). Urine output, renal vascular resistance, and creatinine clearance are monitored throughout reperfusion to assess initial organ function. After the 4-hour reperfusion period the kidneys are sectioned and samples preserved for biochemical and immunohistochemical analysis. For each kidney treated with a hydrogen-rich UW solution, the contralateral kidney from the same donor animal was flushed with unmodified UW solution to serve as a paired control and to represent the current standard of care.

[0319]

[0279] Experiment 1, Solution 1 (UW with 95% H2, 1%O2) versus Control (UW solution with 80% N2, 20% 02).

[0320]

[0280] Solution 1 production method: Stock UW solution was bubbled with pure H2 for one hour at atmospheric pressure. Gas composition for Solution 1 at the time of use was 95% H2, 1% 02, 4% N2 and other trace gases.

[0321]

[0281] Control solution : Stock UW solution, equilibrated with air (80% N2, 20% 02) at time of use.

[0322]

[0282] Results of Experiment 1: Hydrogen-rich Solution 1 did not improve the overall viability or function of the kidneys. A modest reduction in oxidative stress indicators Malondialdehyde (MDA) and myeloperoxidase (MPO) suggest a weak antioxidant effect in the treated kidneys versus their controls. However, indicators of acute hypoxic injury (acute tubular necrosis (ATN) and IL-6) appeared worse in the treated kidneys, indicating that the hydrogen-treated kidneys may have suffered a more significant hypoxic injury than their control kidneys despite the high concentration of hydrogen in Solution 1

[0323]

[0283] FIG. 8A: LEFT: Dissolved H2 in Solution 1 appears exert a moderate antioxidant effect based on molecular indicators Myeloperoxidase (MPO) and Malondialdeyde (MDA), which highlight cellular damage from reactive oxygen species (ROS)

[0324]

[0284] FIG. 8B: LEFT, Dissolved H2 in Solution 1 does not appear to mitigate the early effects of acute hypoxic injury and resultant inflammation based on acute tubular necrosis (ATN) and interleukin-6 (IL-6). The apparent increase in ATN and IL-6 markers with Solution 1 may indicate a more profound hypoxic injury in the treatment arm versus the control arm despite the high concentration of the antioxidant H2.

[0325]

[0285] Experiment 2, Solution 2 (UW with 80% H2, 20% 02) versus Control (UW with 80% N2, 20% 02)

[0326]

[0286] Solution 2 production method:

[0327]

[0287] Stock UW solution vacuum degassed for 24 hours

[0328]

[0288] Degassed solution vacuum packaged in a flexible, gas-tight package.Docket No. 220709-702601

[0329]

[0289] Injected pure 02 gas into the gas-tight packaging.

[0330]

[0290] Injected pure H2 gas into the gas-tight packaging. (Volume of injected H2 was roughly 4X the volume of injected 02 to maintain equilibrium between solution and a gas volume having 80%H2, 20%O2 by volume)

[0331]

[0291] Sealed the gas-tight packaging with a final seal for stable shelf storage and shipping.

[0332]

[0292] H2 and 02 gases cannot escape the gas-tight outer packaging, but they quickly penetrate the plastic solution container within the gas-tight packaging to reach equilibrium between gases dissolved in solution and the residual gas phase in the package (80%H2, 20%O2).

[0333]

[0293] Solution 2 gas composition was measured at time of use, 3 weeks after production and packaging: 80% H2, 20% 02.

[0334]

[0294] Control Solution gas composition at time of use: 80% N2, 20% 02 (equilibrated with air)

[0335]

[0295] Results of Experiment 2: Hydrogen-rich Solution 2 outperformed the control arm by every measurement method used in the study. Key Terms for data labels:

[0336]

[0296] ATN score - average prevalence of acute tubular necrosis (ATN) as observed by pathologist on scale of 1 to 5 where 1 = no necrotic tubular cells per field, 5 = all necrotic tubular cells per field.

[0337]

[0297] TUNEL - average fraction of the tissue area that stained positive for DNA fragmentation, indicative of apoptotic cell death.

[0338]

[0298] KIM-1 - average fraction of the tissue area that stained positive for Kidney Injury Marker 1, an early indicator of hypoxic and oxidative injury in kidneys

[0339]

[0299] IL-6 - average fraction of the tissue area that stained positive for Interleukin 6. IL-6 contributes to tubular cell damage by enhancing inflammation, which can increase apoptosis (as detected by TUNEL staining) and necrosis in the proximal tubules

[0340]

[0300] CD68(+) - average fraction of tissue area staining positive for CD68+ early pro-inflammatory marker in kidney injury tending to recruit and polarize immune cells towards formation of scarring as opposed to regeneration.

[0341]

[0301] MPO - Myeloperoxidase produced by neutrophils in response to renal tubular cell injury; MPO is a key mediator of oxidative stress and tissue damage due to its role in generating reactive oxygen species (ROS).

[0342]

[0302] MDA - Malondialdehyde (MDA) is a reactive aldehyde and a well-established marker of oxidative stress and lipid peroxidation, playing a significant role in the early stages of kidney ischemia-reperfusion injury (IRI).

[0343]

[0303] FIGS. 9A-9G: LEFT: Results for Experiment 1, Solution 1 versus Control RIGHT, Results for Experiment 2, Solution 2 versus Control

[0344]

[0304] ConclusionsDocket No. 220709-702601

[0345]

[0305] Although unrecognized in prior art, dissolved oxygen plays an important role in hydrogen-rich organ preservation solutions. Solutions having extremely high hydrogen concentrations (Solution 1, with H2 at 95% of saturation limit) can deliver a high antioxidant dose and still underperform if their near absence of dissolved oxygen causes a net reduction of dissolved oxygen within the tissue.

[0346]

[0306] Owing to the need to maximize the concentration of dissolved H2 while still allowing enough “space” for dissolved 02, the optimum dissolved gas mixture is a binary H2 / O2 combination. To match the dissolved oxygen levels that are flushed through organs using everyday stock solution that are equilibrated with air, a practical upper bound of 20% was placed on the dissolved oxygen allotment and reserve the remaining 80% for dissolved hydrogen.

[0347] Producing binary H2 / O2 dissolved gas compositions from stock solutions requires unique production methods, as disclosed in the patent application.

[0348]

[0307] The production methods reported in prior art may produce solutions with various hydrogen saturation levels ranging from 30% to 90% or more, but the mixture of other gases comprising the balance will inevitably comprise more nitrogen than oxygen due to the initial ratio of 4: 1 N2 to 02 in the air-equilibrated state.

[0349]

[0308] Example 1 : By prior art methods, a solution targeting 10% dissolved 02 (to prevent stripping residual 02 from organ tissues) would likely have 40% dissolved N2 as well, leaving room for only 50% H2. Such a solution would deliver a relatively low dose of H2 to the organ, resulting in a lesser antioxidant benefit than could have been delivered otherwise.

[0350]

[0309] Example 2: By prior art methods, a solution targeting 80% H2 by diluting or displacing the other gases in solution would displace dissolved nitrogen and oxygen from solution in equal proportions. Having started in a 4: 1 ratio of nitrogen to oxygen due to equilibration with air, the resulting dissolved gas profile would be approximately 80% H2, 16% N2, and 4% 02. The 4% 02 may be insufficient to prevent stripping residual 02 from the organ tissues, and the 16% N2 takes up “space” in the allotment that could otherwise be used for more dissolved H2 or 02.

[0351] DEFINITIONS

[0352]

[0310] As used herein, “about” and its grammatical equivalents in relation to a reference numerical value and its grammatical equivalents as used herein can include a range of values plus or minus 10% from that value. For example, the amount “about 10” encompasses amounts from 9 to 11. The term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

[0353] [3H] As used herein, a “cell” refers to a biological cell. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaea cell, a cell of a single-cell eukaryoticDocket No. 220709-702601

[0354] organism, a protozoa cell, a cell from a plant, an algal cell, a fungal cell, a fungal protoplast cell, an animal cell, and the like. Sometimes a cell is not originating from a natural organism, e.g., a cell can be a synthetically made, sometimes termed an artificial cell.

[0355]

[0312] Although various features of the disclosure may be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the disclosure may be described herein in the context of separate embodiments for clarity, various aspects and embodiments can be implemented in a single embodiment.

[0356]

[0313] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0357]

[0314] Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0358]

[0315] The word “exemplary” is used herein to mean serving as an example, instance, or illustration. Any embodiment, implementation, process, feature, etc. described herein as exemplary should therefore be understood to be an illustrative example and should not be understood to be a preferred or advantageous example unless otherwise indicated.

[0359]

[0316] While exemplary embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments described herein, or combinations of one or more of these embodiments or aspects described therein may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Docket No. 220709-702601CLAIMS1. A method for producing a hydrogen-rich medical perfusion solution, the method comprising, providing a medical perfusion solution in a container;degassing the medical perfusion solution to remove nitrogen and oxygen from the medical perfusion solution until a combination of a partial pressure of nitrogen and a partial pressure of oxygen in the medical perfusion solution is between 0 to 150 mm Hg; andadding hydrogen to the medical perfusion solution; andoptionally, adding oxygen to the medical perfusion solution, after the degassing.

2. The method of claim 1, wherein the degassing includes using one of a vacuum, ultrasonic energy, heat, or a combination thereof to reduce a concentration of dissolved nitrogen in the medical perfusion solution.

3. The method of any one of claim 1 or claim 2, wherein the adding oxygen includes adding dissolved oxygen to the medical perfusion solution to achieve a predetermined concentration of dissolved oxygen.

4. The method of any one of claims 1-3, wherein the degassing replaces a portion of dissolved nitrogen with dissolved oxygen prior to the adding hydrogen.

5. The method of any one of claims 1 -4, wherein the degassing comprises filling the container with the medical perfusion solution and purging at least one gas from a headspace of the container.

6. The method of claim 5, wherein the degassing further comprises heating the container to above 100°C and purging the at least one gas from at least one valve in fluid communication with the headspace of the container.

7. The method of any one of claim 5 or claim 6, wherein the purging is performed after the container has returned to a temperature below 100°C.

8. The method of any one of claims 6-7, wherein the degassing further comprises adding a predetermined amount of at least one of H2, 02, or a combination thereof to the headspace using the at least one valve.

9. The method of claim 8, wherein the adding is performed using at least one of: injection directly into the container, or a gas-filled and gas-permeable second container that is co-packaged with the medical perfusion solution inside a third gas-impermeable container.

10. The method of any one of claims 1 -9, wherein, after the adding hydrogen, the medical perfusion solution has a hydrogen partial pressure (pH2) of at least 0.7 atm.

11. The method of any one of claims 1-10, wherein, after the adding oxygen, the medical perfusion solution has an oxygen partial pressure (pO2) of at least 0.7 atm.Docket No. 220709-70260112. The method of any one of claims 1-11, wherein a ratio of the hydrogen to the oxygen is controlled to produce a combined solution with a predetermined partial pressure of each of the hydrogen and the oxygen.

13. The method of any one of claims 1-12, wherein a ratio of the hydrogen to the oxygen in the medical perfusion solution ranges from 1:1 to 9:1.

14. The method of any one of claims 1-13, wherein the container is subsequently packaged in an outer gastight container.

15. The method of any one of claims 1-14, wherein the medical perfusion solution is a denitrogenated solution.

16. A system for producing medical solutions with dissolved hydrogen gas, the system comprising:at least one solution container having at least one fill tube, at least one gas transport tube, and at least one access tube; anda heating chamber sufficiently sized to house the at least one solution container and apply heat sufficient to increase a temperature of a medical solution within the at least one solution container to at least 100°C,wherein the at least one solution container comprises a headspace absent the medical solution and allows sufficient escape of dissolved gas from the medical solution upon introduction of the medical solution into the at least one solution container,wherein the at least one gas transport tube comprises a hydrophobic filter or porous membrane in line with at least one valve configured to allow for control over gas egress from the at least one solution container and gas ingress into the at least one solution container; and wherein, upon heating of the medical solution in the heating chamber, at least one dissolved gas is separated from the medical solution into the headspace such that the at least one dissolved gas is removable via the at least one gas transport tube upon opening of the at least one valve, thereby allowing for a sufficient amount of an additional gas composition to be introduced into the headspace so as to generate a predetermined composition of dissolved gas in the medical solution, the predetermined composition of dissolved gas having a partial pressure of dissolved hydrogen (pH2) greater than a partial pressure of dissolved oxygen (pO2) and the pO2 greater than a partial pressure of dissolved nitrogen (pN2).

17. The system of claim 16, wherein the at least one gas transport tube comprises a hermetically sealable portion.

18. The system of any one of claim 16 or claim 17, wherein the hydrophobic filter comprises a pore size ranging from about 0.15 to about 0.3 micron.

19. The system of any one of claims 16-18, wherein the at least one valve comprises a bidirectional check valve.Docket No. 220709-70260120. The system of any one of claims 16-19, wherein the at least one valve is closer to an external portion of the at least one gas transport tube than the hydrophobic filter.

21. The system of any one of claims 16-20, wherein each of the at least one fill tube is separate from each of the at least one gas transport tube.

22. The system of any one of claims 16-21, wherein the heating chamber is selected from an oven, an autoclave, a humidity chamber, a thermal shock chamber, a dry heat sterilizer, a heated ultrasonic bath, or a combination thereof.

23. The system of any one of claims 16-22, further comprising a temperature controlling feature configured to maintain a temperature of the medical solution.

24. The system of any one of claims 16-23, further comprising one or more gas sensors positioned to measure concentrations of dissolved oxygen or dissolved hydrogen in an egressing gas or a headspace of the medical solution, optionally wherein the one or more gas sensors comprise one or more dissolved gas sensors, one or more solution flow sensors, temperature sensors, optical sensors, or a combination thereof.

25. The system of claim 24, wherein the concentrations of dissolved hydrogen and / or dissolved oxygen in the effluent medical solution are compared to a reference value so as to achieve a predetermined concentration of at least one of dissolved oxygen or dissolved hydrogen in the medical solution.

26. A perfusion solution, the perfusion solution comprising,a fluid having a dissolved gas mixture of hydrogen, oxygen, and nitrogen, wherein a partial pressure of the dissolved hydrogen (pH2) is higher than 350 mm Hg, the partial pressure of the dissolved oxygen (pO2) ranges from 45 mm Hg to 300 mm Hg, and the partial pressure of dissolved nitrogen (pN2) is lower than a pO2 in the solution,wherein contact of the perfusion solution with an organ or tissue increases an amount of dissolved hydrogen and does not decrease an amount of dissolved oxygen within the organ or tissue as compared to the organ or tissue prior to contacting with the perfusion solution, wherein a predetermined concentration of dissolved oxygen in the organ or tissue is maintained over a course of hydrogen infusion so as to prevent a worsening of hypoxia or oxidative stress within the organ or tissue during the contact of organ or tissue with solution.

27. The solution of claim 26, wherein the oxidative stress is represented by one or more of acute tubular necrosis (ATN), apoptotic cell death, Kidney Injury Marker 1 (KIM-1), Interleukin 6 (IL-6), CD68(+), Myeloperoxidase (MPO), Malondialdehyde (MDA), or a combination thereof.

28. The solution of any one of claim 26 or claim 27, wherein the partial pressure of the dissolved hydrogen is between about 450 mm Hg and about 500 mm Hg, and wherein the partial pressureDocket No. 220709-702601of the dissolved oxygen is at least 1.5 times greater than the partial pressure of dissolved nitrogen.

29. The solution of any one of claim 26-28, wherein the fluid comprises a University of Wisconsin (UW) solution or an organ or tissue preservation solution.

30. The solution of any one of claims 26-29, further comprising one or more chelating agents selected from ethylenediaminetetraacetic acid (EDTA), Desferrioxamine (DFO), citric acid, Dimercaptosuccinic Acid (DMSA), or a combination thereof, wherein the one or more chelating agents are at concentrations ranging from 10-6 Molar to 10-4 Molar.

31. The solution of any one of claims 26-30, wherein the pN2 is less than 0.2 atm.

32. The solution of any one of claims 26-31, wherein the pN2 is less than 0.1 atm.

33. The solution of any one of claims 26-32, wherein a pO2 is greater than pN2 by a magnitude of at least 3x.

34. The solution of any one of claims 26-33, wherein a pH2 is greater than pO2 by a magnitude of at least 2x and greater than pN2 by a magnitude of at least 3x.

35. The solution of any one of claims 26-34, wherein, when a concentration of the hydrogen ranges from 40% to 80%, a pO2 is greater than a combined partial pressures of other gases, the other gases excluding the hydrogen and the oxygen.

36. The solution of any one of claims 26-35, further comprising a bicarbonate buffer, wherein a partial pressure of carbon dioxide (pCO2) ranged from 0.04 atm - 0.06 atm, a pH2 ranges from 0.6 atm - 0.8 atm, and a pO2 ranges from 0.15 atm - 0.35 atm.

37. The solution of any one of claims 26-36, further comprising one or more hydrogen sulfide donors selected from sodium thiosulfate (STS), sodium sulfide (Na2S), sodium hydrosulfide (NaHS), or a combination thereof, wherein a concentration of the one or more hydrogen sulfide donors ranges from 0.1 micromolar to 50 micromolar.

38. The solution of any one of claims 26-37, wherein the gas mixture dissolved in the fluid comprises a binary mixture of hydrogen and oxygen.

39. A method of producing medical solutions with dissolved hydrogen gas, the method comprising, providing an outer gas impermeable container and at least one inner gas permeable container;packaging a medical solution into the at least one inner gas permeable container, the medical solution being previously depleted of nitrogen having an amount of oxygen gas in a headspace of the at least one inner gas permeable container; andarranging the at least one inner gas permeable container and a predetermined amount of hydrogen gas within the outer gas impermeable container,Docket No. 220709-702601wherein the predetermined amount of the hydrogen gas is sufficient to permeate into the at least one inner gas permeable container to equilibrate with the oxygen gas such that the medical solution has a predetermined dissolved gas composition, the predetermined dissolved gas composition having a partial pressure of dissolved hydrogen (pH2) higher than 350 mm Hg and a partial pressure of dissolved oxygen (pO2) ranging from 45 mm Hg to 300 mm Hg.

40. The method of claim 39, wherein the at least one inner gas permeable container comprises a first inner gas permeable container and a second inner gas permeable container, wherein the medical solution is packaged into the first inner gas permeable container and the predetermined amount of hydrogen gas is packaged into the second inner gas permeable container.

41. The method of any one of claim 39 or claim 40, wherein the at least one inner gas permeable container is transparent, flexible, sterile, or a combination thereof.

42. The method of any one of claims 39-41, further comprising infusing the medical solution into a machine perfused organ or tissue.

43. The method of claim 42, wherein the infusing comprises adjusting a concentration of dissolved nitrogen and dissolved oxygen in the machine perfused organ or tissue, the adjusting comprising administering a sweep gas prior to cessation of machine perfusion in preparation for hydrogen infusion, wherein the sweep gas is adjusted to an N2:O2 ratio of up to about 0.5.

44. The method of claim 43, wherein the sweep gas comprises a mixture of N2 and 02.

45. A method for producing a medical solution with substantially binary H2 / O2 dissolved gas mixture wherein an outer package contains a kit comprising:at least one container of oxygen-saturated or oxygen-rich solution (pO2 > 0.5 atm); and a gas-tight container of hydrogen gas to be combined with the oxygen-rich solution prior to using the medical solution.

46. The method of claim 45, wherein the outer package comprises a gas-impermeable barrier.

47. The method of any one of claim 45 or claim 46, wherein the at least one container comprises a flexible material.

48. A method for producing a medical solution with substantially binary H2 / O2 dissolved gas mixture wherein an outer package contains a kit comprising:at least one container of hydrogen-rich solution having a partial pressure of dissolved hydrogen (pH2) greater than 0.5 atm; anda gas-tight container of an oxygen gas,wherein an oxygen gas to be combined with the hydrogen-rich solution prior to using the hydrogen-rich solution.

49. The method of claim 48, wherein the outer package comprises a gas-impermeable barrier.Docket No. 220709-70260150. The method of any one of claim 48 or claim 49, wherein the at least one container comprises a flexible material.

51. A method for using binary dissolved gas mixtures of hydrogen and oxygen in a medical solution, the method comprising:combining a hydrogen-rich solution and an oxygen-rich solution within a container to form the binary dissolved gas mixture, wherein a volume ratio of hydrogen-rich solution to oxygen-rich solution ranges from 1 : 1 to 9: 1.

52. The method of claim 51, wherein a concentration of glutathione is higher in the hydrogen-rich solution than in the oxygen-rich solution.

53. The method of any one of claim 51 or claim 52, wherein the combining is performed at a time of use or immediately prior to the time of use.

54. The method of any one of claims 51-53, wherein the combining comprises heating the container to above 100°C and purging at least one gas from a headspace of the container.

55. The method of claim 54, wherein the purging is performed after the container has returned to a temperature below 100°C.

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