METHODS AND MEDICAL COMPOSITIONS ADMINISTERED TO PROTECT MAMMALS TREATED USING AN EXTRACORPOREAL MEMBRANE OXYGENATION DEVICE

BACKGROUND

Related Applications

This application claims priority from U.S. Provisional Application No. 63/278,736, filed on November 12, 2021. The disclosures of that application are incorporated herein by reference.

Field

[0001] This disclosure relates to a methods and medical compositions administered to patients receiving treatment using an extracorporeal membrane oxygenation (ECMO) device to reduce mortality and morbidity adjunct to ECMO therapy. More particularly, the disclosure relates to gaseous pharmaceutical compositions administered in conjunction with ECMO therapy that include molecular hydrogen.

[0002] Sudden cardiac arrest (CA) is a major public health problem. This problem may be exacerbated during CA outside of a medical care facility. Restoring blood flow to vital organs may be delayed where a patient suffers CA outside of a medical facility. Such out-of-hospital cardiac arrests (OHCA) may lead to extensive ischemic injuries, damage a patient’s central nervous system (CNS) resulting in permanent cognitive disability or lead to death.

[0003] Extracorporeal cardiopulmonary resuscitation (ECPR) using an ECMO device may be indispensable in patients who lose their pulse for a long period. In some circumstances, ECPR can rescue selected patients who do not respond to conventional cardiopulmonary resuscitation (CPR). Recent systematic reviews and meta-analyses have indicated that ECPR improves survival rates in cohorts of selected patients with out-of-hospital cardiac arrest (OHCA).

However, the long-term survival of such patients with ECPR remains low due to the prolonged ischemia and severe organ damage. [0004] Much evidence has shown that ischemia reperfusion injury including excessive oxidant damage and systemic inflammatory reaction that may result from ECPR. This injury contributes to mortality and neurological impairment after CA in both humans and animals. Despite the benefits of ECPR in the quick restoration of oxygen supply and salvage of ischemic cell death, it is well known that ECMO, which includes an artificial oxygenation membrane and circuits, is associated with oxidative stress and systemic inflammatory reactions, resulting in coagulopathy and endothelial cell damage. Therefore, ECPR can be lifesaving but worsens the undesirable effects of post-CA physiology. It would be an improvement in the art to develop therapeutic targets to improve outcomes in patients with OHCA rescued by ECPR.

[0005] ECMO therapy is a procedure during which blood oxygenation and cardiac function are performed by a mechanical pump and membrane oxygenator outside the body. ECMO can provide short-term mechanical support to vital organs. It works by removing blood from the patient and pumping the blood through a membrane oxygenator. The oxygenator exchanges oxygen and carbon dioxide in patient’s blood, and the blood is then pumped to the patient. The pressure and flow created by the mechanical pump help circulate blood in the body, thereby supporting cardiac function.

[0006] Importantly, ECMO is not for treating diseases, rather it substitutes for the work of the heart and lungs, thus allowing them to “rest” until time or additional definitive treatment relieves the underlying medical condition. For example, in patients with refractory cardiac arrest despite standard cardiopulmonary resuscitation, ECMO can provide rapid circulatory support and the maintenance of circulation and perfusion of vital organs including brain and heart. Thus, extracorporeal circulatory support may be lifesaving, but also leads to excessive endothelial damage and systemic inflammatory reactions, which are potential therapeutic targets for improving survival outcomes in critically ill patients.

[0007] A number of adverse pathophysiological effects may be associated with ECMO therapy. These include ischemia-reperfusion injury and endothelial dysfunction, which are capable to inflicting serious morbidity. Because the ECMO device uses an artificial oxygenation membrane and circuits that contact the circulating blood, ECMO may induce excessive oxidative stress and inflammatory responses, resulting in coagulopathy and endothelial cell damage. Of note, the main mechanism responsible for death during and after ECMO remains unclear at this time. Established methods of administering ECMO do not provide pharmaceutical intervention for these and other ECMO-related adverse effects.

[0008] Hydrogen gas (H2) possesses antioxidant and antiapoptotic properties and has been shown to be beneficial after CA. It has been proposed that inhaled molecular hydrogen, H2, selectively reduces cytotoxic reactive oxygen species (ROS) such as hydroxyl radicals (*OH) and peroxynitrite (ONOO-). Inhaled H2 has been shown to attenuate myocardial and brain injury in a rat model of ventricular fibrillation (VF)-induced CA. Clinical pilot studies have shown beneficial effects of H2 in patients with acute myocardial infarction and in patients with OHCA who achieved successful return of spontaneous circulation (ROSC). However, whether H2 administration with ECPR can improve outcomes after CA and the precise mechanism responsible for the beneficial effects of H2 remains unknown.

[0009] Preclinical experiments suggest that inhaled hydrogen gas at a concentration of -4.0% protects against ischemia-reperfusion injury by selectively reducing cytotoxic reactive oxygen species, and by down-regulating endothelial damage. One of the present inventors has found that H2 administration via tracheal tube can improve brain and cardiac function and survival outcome after cardiopulmonary arrest resuscitation without ECMO in rats (Hayashida K, et al. Journal of the American Heart Association 2012; doi: 10.1611 / JAHA. 112.003459, and Hayashida K, et al. Circulation. 2014 Dec 9; 130 (24): 2173-80).

SUMMARY

[0010] The present disclosure relates to pharmaceutical gaseous pharmaceutical compositions and methods for administering such compositions that mitigate injuries in the setting of ECMO therapy. Administration of gaseous compositions including hydrogen gas via an ECMO device can unexpectedly improve the prognosis in critical ill or perioperative mammals receiving ECMO therapy, such as in human CA sufferers.

[0011] According to one aspect of the disclosure, a gaseous composition, including hydrogen gas is provided to a patient undergoing ECMO therapy. The hydrogen gas is administered as part of the composition of gasses used to oxygenate blood of the patient using the ECMO device. According to another aspect, instead of, or in addition to providing hydrogen gas via the ECMO device, hydrogen gas is also provided for inhalation, for example, by a ventilator device. [0012] According to another aspect of the disclosure, hydrogen gas is administered to a patient undergoing ECMO therapy in a pharmaceutically effective quantity and duration to protect brain function, improve survival time, improve myocardial contractility, and/or attenuate central venous congestion after a cardiac arrest.

[0013] According to another aspect of the disclosure, hydrogen gas is administered to a patient undergoing ECMO therapy in a pharmaceutically effective quantity and duration to alter the plasma concentrations of metabolites associated with endothelial damage and with vascular inflammation, including mitigation of syndecan-1 levels in plasma.

[0014] According to another aspect hydrogen gas is administered to a patient undergoing ECMO therapy in a pharmaceutically effective quantity and duration to increase brain oxygenation, to reduce reperfusion endothelial glycocalyx injury, to reduce endothelial inflammation, and/or to increase plasma concentrations of one or more of anti-inflammatory cytokines including, but not limited to Interleukin -10 (IL- 10), vascular endothelial growth factor (VEGF), and leptin.

[0015] According to another aspect of the disclosure, a gaseous pharmaceutical composition for administration to a mammal receiving ECMO therapy includes hydrogen and oxygen. According to a further aspect, the composition includes one or more inert gaseous components including, but not limited to, helium, nitrogen, or argon. According to one aspect, the concentration of hydrogen gas in the composition is between about 0.1% to about 4.0% of the composition. That is, the partial pressure of hydrogen is about 0.1% to about 4.0% of the total pressure. According to another aspect, the concentration of hydrogen gas is below the lower explosive limit. At standard atmospheric pressure, this limit is about 4.0% for hydrogen in pure oxygen (O2). Thus, according to another aspect, the composition comprises a gaseous mixture that is below the lower explosive limit of hydrogen under the ambient conditions where treatment using the composition is used.

[0016] According to yet another aspect, there is disclosed methods of treating mammals undergoing ECMO therapy by connecting the mammals circulatory system to the circuit of an ECMO device where the ECMO device includes a membrane in contact with the blood of the mammal, of providing a flow of a pharmaceutical gaseous composition to the ECMO device to a side of membrane opposite from the side in contact with the blood where the composition comprises hydrogen gas, of flowing the blood past the membrane so that gasses from the composition diffuse through the membrane and dissolve in the blood, and of flowing the blood to the mammal to restore blood flow to revive the mammal. According to a further aspect, in addition to providing the composition via the ECMO device, the composition is also administered via the lungs by inhalation, for example, using a medical ventilator.

[0017] According to another aspect, a pharmaceutical gaseous composition including hydrogen gas is administered before, at the same time, or after a mammal has achieved restoration of spontaneous circulation (ROSC), where ROSC has been facilitated using ECMO therapy.

According to one aspect, the composition is administered throughout ECMO therapy. According to another aspect, ECMO therapy is provided without the composition including hydrogen gas, for example, using pure oxygen or oxygen mixed with an inert gas, and the composition including hydrogen gas is administered via the lungs by inhalation and/or by a medical ventilator before or after ROSC.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] A more complete appreciation of the disclosure and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

[0019] Fig. 1 shows an exemplary apparatus for providing veno-arterial ECMO;

[0020] Fig. 2 shows an experimental timeline for an Example comparing groups of rats treated with ECMO therapy following CA with gaseous compositions including hydrogen gas (“H2 group”) and without hydrogen gas (“Placebo group”);

[0021] Fig. 3 shows tabulated values for baseline variables (body weight, MAP, heart rate, CVP, body temperature, arterial dp/dt(max), dp/dt(min), and EtCO2) before resuscitation for the H2 group and the Placebo group. [0022] Fig. 4 shows physiological and interventional variables during ECPR in the H2 group and the Placebo group;

[0023] Fig. 5A is a graph showing improved survival time after prolonged CA for the H2 group versus the Placebo group;

[0024] Fig. 5B shows a higher percentage of animals exhibiting responses to toe pinch and corneal reflex in the H2 group versus the Placebo group; and

[0025] Fig. 6 shows brain tissue oxygenation measured as PtCh during ECPR in the brains of animals subjected to prolonged CA and resuscitated by ECMO therapy for the H2 group and the Placebo group.

DETAILED DESCRIPTION

[0026] Illustrative embodiments of the present disclosure may be provided as improvements to the administration of ECMO therapy.

[0027] As discussed above, during CA, a patient’s circulation may be significantly diminished or may cease. Organs deprived of the flow of oxygenated blood sustain ischemic injury very soon after blood flow is diminished, resulting in permanent damage or death. Ischemic injury increases rapidly and unless circulation is restored quickly, a patient may suffer permanent injury or may die. Ischemic injury to the brain and CNS may leave the patient with permanent cognitive disabilities. Thus, it is vital that circulation be restored as soon as possible following CA. The risk of ischemic injury following CA is greatly increased when CA occurs outside of a hospital setting.

[0028] Under some circumstances ECMO provides a mechanism to rapidly and reliably restore circulation of oxygenated blood. ECMO devices drive blood through the patient’s circulatory system using a mechanical pump that can maintain adequate blood flow. Blood passes along a membrane oxygenator that allows oxygen to diffuse into the blood and allows carbon dioxide to diffuse out from the blood. A patient connected with an ECMO device is thus provided with a supply of oxygenated blood while medical treatment is provided to address the medical condition that caused the CA. [0029] While ECMO provides a method for reducing ischemic injury that may result from the loss of circulation, ECMO therapy itself may result in injury to the endothelium and to other organs. This injury may be the result of the introduction of reactive oxygen species because of interaction between the blood and surfaces within the ECMO device.

[0030] As discussed above, by introducing hydrogen gas according to embodiments of the disclosure as a component of the gas used to oxygenate blood during ECMO therapy, a surprising reduction in injury sustained because of ECMO therapy was achieved. The protective effects of hydrogen gas during ECMO are demonstrated to improved survival outcomes, improved survival times, and improved brain recovery after CA and ECPR.

[0031] As used in the present disclosure, the phrase “improved survival outcomes after CA” means that mammals treated using ECMO including hydrogen gas following CA had a reduced morbidity and/or reduced mortality compared with mammals treated with ECMO following CA without hydrogen gas. Reduced morbidity may include, but is not limited to improved brain recovery after CA.

[0032] As used in the present disclosure, the phrase “improved brain recovery after CA” means that mammals had a return of one or more cognitive, neurological, or sensory abilities following ROSC when treated with hydrogen gas during ECMO therapy as compared with ECMO treatment without hydrogen gas. The return of abilities may include recovery or improvement in functional status and/or an improvement in electrical signals indicating brain function. Such brain recovery may also include histological improvements to brain tissue including increased cerebral perfusion and/or reduced venous congestion.

[0033] Reduction in morbidity may also include a reduction in inflammation, for example, inflammation of the endothelium following CA and ECMO therapy. Reduction of inflammation may be evidenced by differences in the metabolome between mammals treated with ECMO after CA with and without hydrogen gas. Differences in the metabolome may include, but are not limited to a shift in D-glutamine and D-glutamate metabolism, a reduction in release of syndecan-1 into plasma after CA and ECPR, an increase in anti-inflammatory metabolites including IL- 10, VEGF, and leptin, and a reduction in pro-inflammatory cytokines/chemokines after ECMO after C A. [0034] Fig. 1 shows an exemplary system for providing veno-arterial ECMO. That is, blood flows from a major vein, in this example, the jugular vein, through the ECMO device, and is returned to the patient through a major artery, in this case, the femoral artery. The present disclosure is not limited to veno-arterial ECMO and can be used in combination with other arrangements to provide ECMO therapy including, but not limited to veno-venous ECMO, venoarterial ECMO, and in the setting of cardiopulmonary bypass.

[0035] In the arrangement 1 shown in Fig. 1, venous outflowing blood accumulates in a venous reservoir 10. A peristaltic pump 12 drives blood from the reservoir into an oxygenator 14. The oxygenator 14 includes a membrane in contact with venous blood. On the opposite side of the membrane, a flow of gas 16, including oxygen, passes along the membrane. Oxygen diffuses through the membrane and is absorbed by the blood. Carbon dioxide diffuses out from the venous blood through the membrane and flows out of the oxygenator along with the flow of gas. Oxygenated blood from the oxygenator is then returned to the patient through a cannula 18 inserted in the artery.

[0036] ECMO therapy is highly effective at maintaining a flow of oxygenated blood and may reduce ischemic organ damage if administered soon after the onset of CA. Thus, extracorporeal circulatory support may be lifesaving, but also leads to excessive endothelial damage and systemic inflammatory reactions, which are potential therapeutic targets for improving survival outcomes in critically ill patients. Resuscitation strategies using EE according to embodiments of the present disclosure may improve endothelial and cellular metabolism, in turn improving brain and other tissue viability and survival outcomes in patients with CA who require extracorporeal membrane oxygenation (ECMO) support.

[0037] According to one embodiment of the disclosure, the flow of gas supplied to the oxygenator 14 comprises oxygen combined with hydrogen gas. The gas composition may be provided by one or more gas tanks, by mechanical gas generators, by electrolytic gas generation, a hydrogen-absorbing alloy canister or by other means for providing medically suitable supplies of gas known in the field of the disclosure.

[0038] According to one embodiment, the gas composition provide to the oxygenator comprises between about 0.1% and about 4.0% EE mixed with pure oxygen. According to other embodiments, other gasses may be provided in the composition including, but not limited to nitrogen, helium, neon, or argon.

[0039] In practice, a patient being treated following CA is assessed to determine if CPR will likely result in ROSC. Once it is determined that ECPR is indicated, the patient is cannulized to provide access to a major vein and to a major artery. The patient may also be connected with a mechanical ventilator 20 to provide oxygen to the lungs. After purging the ECMO device of air, venous blood is provided to an ECMO device, as discussed above. A flow of oxygenated blood into the patient’s artery is provided by operation of the peristaltic pump 12. According to one embodiment of the disclosure, the membrane oxygenator 14 is provided with a gas composition 16 comprising hydrogen gas. The contents of the gas composition may be varied during ECMO treatment. According to one embodiment, as soon as ECMO therapy is started, a gas composition including hydrogen is provided. ECMO therapy may cease when the patient has ROSC. ECMO therapy using the gas composition including hydrogen may also be continued after ROSC is achieved.

[0040] According to another embodiment, ECMO therapy after CA may begin by providing pure oxygen to the oxygenator 14 to provide a maximum plasma oxygen partial pressure until ROSC is achieved. ECMO therapy may be continued after ROSC but with the composition of the gas including hydrogen gas to provide protection of the endothelium from inflammation and other damage during continued ECMO and/or to provide improved brain recovery after CA.

[0041] In addition to providing a gas composition via the ECMO device, the same gas composition or a different gas composition may be provided to the patient’s lungs via the mechanical ventilator 20. According to this embodiment, a composition of gas including hydrogen is provided to the patient both via the lungs and via ECMO therapy.

The methods herein disclosed are useful for improving prognosis in critical ill or perioperative mammals after the initiation of ECMO therapy. Examples of critical ill or perioperative medical conditions which may be connected an ECMO device include: refractory cardiac arrest (sudden heart attack); refractory shock due to acute myocardial infarction; acute heart failure; septic shock; anaphylactic shock; major trauma; hemorrhagic shock with hypotension; organ transplantation; organ transplant from a brain-dead donor; perioperative cardiac surgery or major surgery; cardiopulmonary bypass; acute respiratory distress syndrome (ARDS); acute respiratory failure in mammals.

Example 1

[0042] It has been shown that extracorporeal cardiopulmonary resuscitation (ECPR) improves survival rates in cohorts of selected patients with out-of-hospital cardiac arrest (CA). Despite the benefits of ECPR, extracorporeal membrane oxygenation (ECMO) includes an artificial oxygenation membrane and circuits that contact the circulating blood and induce excessive oxidative stress and inflammatory responses, resulting in coagulopathy and endothelial cell damage. It has also been shown that hydrogen gas (EE) possesses antioxidant and antiapoptotic properties and has been shown to be beneficial after CA in rats. An exemplary embodiment of the disclosure examined the administration of Eb combined with ECPR outcomes after prolonged CA in rats.

[0043] In this Example rats were subjected to 20 min of asphyxial CA and were resuscitated by ECPR. Mechanical ventilation (MV) was initiated at the beginning of ECPR. Animals were randomly assigned to the placebo or EE gas treatment groups. The supplement gas was administered with Ch through the ECPR membrane and MV. Survival time, electroencephalography (EEG), brain functional status, and brain tissue oxygenation were measured. Changes in the plasma levels of syndecan-1 (a marker of endothelial damage), multiple cytokines, chemokines, and metabolites were also evaluated.

[0044] In this exemplary embodiment, the survival rate at 4 h was 77.8% (7 out of 9) in the Eb group and 22.2 % (2 out of 9) in the placebo group. The Kaplan-Meier analysis showed that Eb significantly improved the 4 h-survival endpoint (log-rank P = 0.025 vs. placebo). All animals treated with Eb regained EEG activity, whereas no recovery was observed in animals treated with placebo. Eb therapy markedly improved intra-resuscitation brain tissue oxygenation and prevented an increase in central venous pressure after ECPR. Eb attenuated an increase in syndecan-1 levels and enhanced an increase in interleukin- 10, vascular endothelial growth factor, and leptin levels after ECPR. Metabolomics analysis identified significant changes at 2 h after CA/ECPR between the two groups, particularly in D-glutamine and D-glutamate metabolism. [0045] As discussed below, in this exemplary embodiment H2 gas therapy improved mortality in highly lethal CA rats rescued by ECPR and helped recover brain electrical activity. Without wishing to be bound by theory, it is hypothesized that H2 therapy may provide protective effects against endothelial damage.

[0046] METHODS

[0047] Animal Preparation

[0048] Male Sprague-Dawley rats (400-500 g, Charles River) were used in this study. The rats were housed in a rodent facility under a 12-h light/dark cycle and had free access to food and water. All experiments were performed in accordance with the National Institutes of Health guidelines for the use of experimental animals and were approved by the Institutional Animal Care and Use Committee of the Feinstein Institutes for Medical Research.

[0049] The rats were intubated, mechanically ventilated, and instrumented under anesthesia with 2% isoflurane as described previously. Fig. 1 shows the arrangement of apparatus 1 according to an embodiment of the disclosure. End-tidal carbon dioxide (EtCO2) was maintained at 40 ± 5 mmHg during the experiment. The left femoral artery was cannulated (PE-50, Becton Dickinson, Franklin Lakes, NJ) to monitor arterial pressure and the peak first derivative of arterial pressure (dP/dt), which are indicators of left ventricular function, and for blood sampling. The left femoral vein was cannulated (PE-50) to monitor central venous pressure (CVP) and for drug administration. The right external jugular vein and right femoral artery were respectively cannulated with a 14 G catheter for venous outflow and with a 20 G catheter for arterial inflow.

The esophageal temperature was maintained at 37.0°C ± 0.5°C using a thermostatically regulated heating pad and heating lamp throughout the procedure. Blood pressure and needle-probe electrocardiogram monitoring data were recorded and analyzed using a PC-based data acquisition system.

[0050] This study used a rat model of highly lethal prolonged asphyxia-induced CA, as reported previously. It has been previously shown that the survival rate was -20% at 4 h after CA/ECPR. Briefly, after injecting heparin (300 U) and vecuronium (2 mg/kg) via the left femoral vein, asphyxia was induced by stopping mechanical ventilation (MV), and isoflurane was discontinued. A mean arterial pressure (MAP) below 20 mmHg was defined as CA. After 20 min of asphyxia, ECPR was started with the initiation of veno-arterial ECMO flow and resumption of MV. The ECMO circuit consisted of a heat exchanger 22, an open venous reservoir 10, a membranous oxygenator 14 (Martin Humbs Engineering, Ingenieurbiiro fur Bauwesen, German), silicone tubing lines, and a roller pump 12 (MasterFlex, Barrington, IL, USA) primed with 10 mL of Normosol-R (Hospira, Lake Forest, IL), 10 mL of 6% Hespan (B. Braun Medical Inc., Bethlehem, PA, USA) and 0.3 mL of 8.4% sodium bicarbonate. As needed, an additional 5 mL of Normosol-R solution and 10 mL of donor blood were added to the venous reservoir 10 to maintain a constant circulating volume. The flow rates reached 130-150 mL/kg/min within 1 min, which approximated the normal cardiac output in the animal.

Following ROSC, the flow rate was gradually decreased to 40 mL/kg/min to prevent excessive volume administration. ECMO was performed for 30 min. All animals achieved successful ROSC, and the flow was continued for 30 min after ROSC in all cases. Once the flow was stopped, catheters inserted into the right external jugular vein and right femoral artery were removed, and the surgical wounds were sutured. After ROSC, the animals were ventilated with O2 supplemented with or without 2% H2 for the first 1 h after ROSC, followed by ventilation with 60% O2 for an additional 3 h. Survival time was monitored up to 4 h after CA/ECPR, according to a previous study. For sampling, 0.3 mL of blood was drawn from the left femoral artery catheter at the baseline and at designated times: 0.5, 1, and 2 h after ROSC. Death was defined as a MAP below 30 mmHg, lasting for 5 min.

[0051] Experimental Protocol

[0052] When ECPR was started and MV was resumed, animals were randomly assigned to two experimental groups: ECPR with 100% O2 (placebo group), and ECPR with 98% O2 supplemented with 2% H2 (H2 group). Fig. 2 shows the experimental timeline for the two experimental groups. The experimental gases were added to both the membrane oxygenator 14 and ventilator 20. Rats in both groups were ventilated with the experimental gas for the first 60 min after ROSC. As animals treated with ECPR using 100% or 98% O2 had similar survival rates in pilot experiments (data not shown), in this Example 100% O2 was used as a placebo. This approach was considered clinically relevant because ECPR for patients with OHCA is usually initiated with 100% O2 for the membrane oxygenator and ventilator. [0053] Assessment of Brain Function

[0054] It has been shown from previous studies with varying CA times, the toe pinch and corneal reflex were the only stimuli that animals responded to following severe CA. Thus, a lack of response to these stimuli evidenced complete loss of motor responses and a deep unresponsive coma. Therefore, following 20-min asphyxial CA and ECPR, brain function was assessed by the responses to toe pinch and corneal stimulation for 4 h after ROSC.

[0055] Brain Tissue Oxygen Monitoring

[0056] The animal’s head was stabilized in a stereotaxic instrument using ear bars. A small burr hole (2 mm) was drilled in the skull at 3 mm lateral and 3 mm posterior to the bregma. PtO2 was measured continuously using a Clark type tissue electrode (Integra® Licox® Brain Tissue Oxygen Monitoring, Integra LifeSciences Limited IDA Business and Technology Park, County Offaly, Ireland) inserted at 5 mm. There is no evidence in literature indicating that the Licox® oxygen monitoring analysis is affected by inhaled Hz.

[0057] Electroencephalogram (EEG) Monitoring

[0058] In a subgroup of rats subjected to CA and ECPR, EEG was measured for 4 h after ROSC, as reported previously. Briefly, before intubation and cannulation procedures, animals underwent implantation of EEG electrodes bilaterally under isoflurane using a stereotaxic apparatus (Stoelting, USA). Each animal had eight screw electrodes (Plastics One, Roanoke, VA) cortically implanted over the frontal (AP = +2, L = 2), parietal (AP = -4, L = 2), occipital (AP = -6, L = 2), and forelimb regions of the somatosensory cortex (n = 3 per group). The ground electrode was placed over the parasagittal right frontal lobe. The screws were held in place with dental cement. EEGs were recorded using an Intan RHS Stim/Recording 16 channel recording controller during the baseline, asphyxial CA, resuscitation, and after ROSC. Raw EEG signals were used to determine the EEG electrical activity.

[0059] Measurement of Plasma Syndecan-1

[0060] For quantitative determination of soluble syndecan-1 in plasma, a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Novus Biologicals, LLC, CO, USA) was used according to the manufacturer’s instructions. Duplicate measurements were performed for each sample by an investigator blinded to the experiment.

[0061] Multiplex Plasma Mediator Assay

[0062] Plasma eotaxin, epidermal growth factor (EGF), fractalkine, interferon (IFN)-y, interleukin (IL)-la, IL-lp, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, IL-17A, IL-18, IP- 10, growth-regulated oncogenes/keratinocyte chemoattractant (GRO/KC), tumor necrosis factor (TNF)-a, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colonystimulating factor (GM-CSF), monocyte chemotactic protein (MCP)-l, leptin, lipopolysaccharide-induced CXC chemokine (LIX), macrophage inflammatory protein (MIP)- la, MIP-2, regulated upon activation normal T cell express sequence (RANTES), and vascular endothelial growth factor (VEGF) levels were determined using the Rat Cytokine/Chemokine Array 27 Pl ex (Eve Technologies, Calgary, AB, Canada) according to the manufacturer’s protocol. The plasma samples were diluted 1 : 1 in PBS. Singlet measurements were performed for each sample by an investigator blinded to the experiment, and seven cytokines/chemokines (IL- la, GM-CSF, IL-2, EGF, IFN-y, GRO/KC, and MIP-2) were excluded from the analyses because they showed several out of range (OOR) measurements (defined as > 85%) for at least one time point. Values less than the OOR level were replaced with 0.001.

[0063] Untargeted Metabolomics with GC-MS

[0064] For metabolomics analysis, three groups of plasma were examined: samples obtained before CA induction (pre-CA, n = 8), samples at 2 h post-ROSC in the placebo group (placebo, n = 8), and at 2 h post-ROSC in the H2 group (H2, n = 8). An investigator blinded to the experiment derivatized the plasma samples for metabolic profiling for GC-MS using a two-step methoximation/silylation derivatization procedure. Plasma sample (50 pL) was mixed with 172 pL of sterile water, 555 pL of methanol, and 222 pL of chloroform by vortex for 20 s, centrifuged at 16,000 rpm at 4 °C for 3 min, and the supernatant was collected. Sterile water (400 pL) was added to the supernatant, and the mixture was centrifuged at 16,000 rpm at 4 °C for 3 min to collect the upper layer of the two-layer separation. The collected layer was evaporated by using a centrifugal concentrator and followed by lyophilization in a freeze dryer. Dried samples were kept drying before derivatization. Myristic acid d-27 in n-hexane (0.75 mg/mL, 10 pL) was added to the samples as the derivatization standard. First, the dried samples were methoximated with methoxyamine hydrochloride (40 mg/mL, 10 pL) in anhydrous pyridine at 30°C for 90 min. Next, the dried samples were silylated with N-methyl-N- trimethylsilyltrifluoroacetamide using 1 % trimethylchlorosilane (TMCS; 90 pL) at 37°C for 30 min. GC-MS analysis was performed on an Agilent 7890B gas chromatograph connected to an Agilent 5977B MSD (Agilent Technologies UK Ltd.). Samples were injected with an Agilent 7693 autosampler injector into deactivated splitless liners using the FiehnLib settings.

Compound identification was performed by comparing the retention time and the mass spectrum with a Fiehn metabolomics mass spectrum library. Peaks with a similarity index higher than 60% were assigned compound names, whereas those with less than 60% similarity were listed as unknown metabolites. The chromatograms were subjected to noise reduction prior to peak area integration. Any known artificial peaks, such as those due to noise, column bleeding, and the N- methyl-N-trimethylsilyltrifluoroacetamide derivatization procedure, were excluded from the data set. The integrated peak areas of multiple derivative peaks belonging to the same compound were summed and considered as a single compound. The relative peak area of each compound was calculated as the response after integrating the peak areas of the compounds.

[0065] Metabolomics Data Analysis

[0066] In total, 189 metabolites across the groups were detected using GC-MS. Of these, 76 metabolites in samples with a coefficient of variation (CV) value > 30% were rejected, which is acceptable for biomarker discovery. Of the 113 remaining metabolites, variables with missing data (> 50 % as threshold) were removed. Thereafter, the missing variables were replaced with the limit of detection (1/5 of the minimum positive value for each variable), as per the default setting of Metab o Analyst. Finally, a total of 53 metabolites were used for the study.

[0067] Principal component analysis (PCA) was used to gain an overview of all the samples and to identify the possible outliers. To visualize the changing patterns in metabolites for facilitate comparison among the three groups, a heatmap was generated by hierarchical clustering of plasma metabolites. Partial least squares-discriminant analysis (PLS-DA) identifies metabolites that carry the greatest group-separating information, as represented by the first latent variable. Using supervised machine learning, PLS-DA examines the discriminative capacity of high-dimensional and highly correlated data (metabolomics) and the relative importance of each feature within the dataset (i.e., each metabolite in the present study). PLS-DA was thus used to identify significantly changed metabolites among different groups (variable importance in the projection, VIP > 1 as a mean of all components). The models were refined by VIP selection to maximize Q2 (i.e., the cross-validated R2). A volcano plot was also generated to screen features with statistical significance (p < 0.05) and fold change (FC) > 1.2 or < 0.8. Finally, based on the VIP selection obtained using PLS-DA of the placebo and H2 groups, metabolite set enrichment analysis (MSEA) was conducted to evaluate the impact of individual metabolite alterations on different metabolic pathways. The MSEA was declared positive (i.e., differentially regulated) if it had a false discovery rate (FDR) < 0.05. The PCA, PLS-DA, volcano plots, heatmap, and MSEA were all performed with MetaboAnalyst (v5.0) using normalized data (auto-scaling feature and log transformation).

[0068] Statistical Analysis

[0069] Data for continuous variables are presented as mean ± standard error of the mean (SEM). Categorical data are presented as counts with frequencies. An unpaired two-tailed Student’ s t-test or Mann-Whitney U test was used to compare two independent groups, as appropriate for continuous variables. For normally distributed data, two-way analysis of variance (ANOVA) with or without repeated measures followed by Sidak’s correction for post- hoc comparisons was used as appropriate. For non-normally distributed data, the Friedman test followed by Dunn's multiple comparisons was used for post-hoc comparisons. Survival rates were estimated using the Kaplan-Meier method, and the log-rank test was used to compare the survival curves between groups. Statistical significance was considered at P < 0.05. GraphPad Prism 7.05 (GraphPad Software Inc., La Jolla, CA, USA) and SPSS (version 25.0; SPSS Inc., Chicago, IL, USA) were used for statistical analyses.

[0070] RESULTS

[0071] EE with ECPR Improved Survival Outcomes After CA

[0072] The baseline variables (body weight, MAP, heart rate, CVP, body temperature, arterial dp/dt(max), dp/dt(min), and EtCO2) were the same in each group. Fig. 3 shows tabulated values for these baseline variables before resuscitation. Fig. 4 shows the physiological and interventional variables during ECPR in rats of placebo and H2 group. There was no difference between each group in the time from ECPR initiation to ROSC, duration of pulseless electrical activity, or the parameters during ECPR except for the CVP at 10 min after ECPR initiation (CVP, placebo vs. H2: 5.7 ± 0.8 vs. 3.1 ± 0.8 mmHg, P = 0.036). There were no differences in the arterial lactate, pH, PaCh, PaCCh, base excess, or HCCh levels for the first 2 h after CA between the groups. The survival rate at 4 h after CA/ECPR was 77.8% (7 out of 9) in the H2 group and 22.2% (2 out of 9) in the placebo group. Kaplan-Meier analysis showed that H2 markedly improved the survival time after prolonged CA (log-rank P = 0.025 vs. placebo) as shown in Fig. 5A.

[0073] Following prolonged CA and ECPR, brain function was assessed based on the response to toe pinch and corneal stimulation. During the experiment, none of the animals showed a response to toe pinch, and one out of nine rats had corneal reflex in the placebo group. In the H2 group, two of nine animals responded to a toe pinch, and four rats showed corneal reflex. In total, the percentage of animals exhibiting either or both responses was significantly higher in the H2 group than in the placebo group (66.7% vs. 11.1%, P = 0.016 by Chi-square test) as shown in Fig. 5B. In all animals, EEG amplitudes disappeared at 51 ± 15 s after CA. After ROSC by ECPR, none of the animals treated with the placebo (0 out of 3) regained continuous EEG for the first 4 h after ROSC, whereas animals treated with H2 (3 out of 3) regained continuous EEG at 47 ± 9.9 min after ROSC, suggesting the beneficial effects of H2 on post-CA brain electrical recovery. These observations suggest that H2 administration along with ECPR improved the survival outcomes and brain recovery after CA and ECPR.

[0074] H2 Improved Brain Oxygenation During ECPR

[0075] To determine the impact of H2 on brain recovery after ECPR, tissue oxygenation was measure as PtO2 during ECPR in the brains of animals subjected to prolonged CA and resuscitated with placebo or H2. The results are shown in Fig. 6. After 20 min of asphyxial CA, the brain PtO2 of all animals markedly decreased to 28.6% ± 6.0 % of baseline. In the placebo group, ECPR with 100% O2 restored the PtCh levels at around the baseline level, which was ventilated with 30% O2. However, PtCh for the first 50 min after ECPR initiation did not differ from the baseline level in the placebo group. In contrast, PtCh in H2-treated animals increased strikingly to more than 200% of the baseline at 20-40 min after ECPR initiation. PtCh recovery at 20 min after ECPR initiation was significantly higher in the H2 group than in the placebo group (291% vs. 164% of baseline, P = 0.009).

[0076] H2 Attenuated the Elevated CVP Observed Early After CA and ECPR

[0077] H2 administration impacted hemodynamic parameters during the post-CA reperfusion period. In both groups, MAP increased within approximately 50 min after ROSC, and HR decreased approximately 1 h after ROSC. During the first 75 min after CA, there was no difference between the groups in body temperature, MAP, or HR. The H2 group exhibited markedly higher dp/dt(max) and lower dp/dt(min) early after ROSC compared with the baseline. Notably, there was a significant difference with respect to CVP. In the placebo group, CVP gradually increased after CA/ECPR. However, in the H2 group, CVP was maintained for the first 75 min, and H2-treated animals exhibited markedly lower CVP than the placebo group at 75 min after CA (P = 0.038). These observations suggest that H2 with ECPR improves myocardial contractility and thus attenuates central venous congestion early after ECPR.

[0078] H2 Attenuated Excessive Shedding of Endothelial Glycocalyx in Plasma and Enhanced Plasma IL- 10, Leptin, and VEGF Levels After CA/ECPR

[0079] To determine the mechanisms associated with the protective effects of H2, plasma syndecan-1 levels at the baseline and 120 min after CA with or without H2 were measured. Cardiac arrest and ECPR led to a considerable increase in plasma syndecan-1 levels at 120 min. H2 with ECPR abated the increase in plasma syndecan-1 levels, indicating that the protective effects of H2 on the outcomes are associated with an inhibitory effect on the release of endothelial glycocalyx shedding into the blood.

[0080] Plasma cytokine and chemokine levels at 2 h post-ROSC with and without H2 were measured. Cardiac arrest and ECPR markedly increased the plasma levels of IL- 10, VEGF, leptin. Likewise, CA and ECPR markedly increased the plasma levels of TNF-a, IL-ip, IL-6, fractalkine, IL-5, IL-18, IP-10, IL-4, eotaxin, MIP-la, IL-17a, IL-12, RANTES, G-CSF, LIX, and MCP-1. However, H2 gas administration at this time point did not affect the plasma levels of TNF-a, IL-lp, IL-6, fractalkine, IL-5, IL-18, IP-10, IL-4, eotaxin, MIP-la, IL-17a, IL-12, RANTES, G-CSF, LIX, and MCP-1, whereas the plasma levels of IL-10, VEGF, and leptin were increased by H2. [0081] DISCUSSION

[0082] This Example demonstrates the beneficial effects of EE when used in combination with ECPR on short-term outcomes in a highly lethal rat model of CA. Eb-treated animals showed improved survival time, with improvements in the functional status and electrical recovery of the brain after CA/ECPR. The beneficial effects of EE were associated with increased PtO2 in the brain during ECPR, and a decrease in plasma syndecan-1 levels, concurrent with the enhancement of plasma mediators after CA. Moreover, these findings provide the unique evidence of metabolic derangements, specifically a shift in D-glutamine and D-glutamate metabolism, in experimental CA/ECPR treated with EE.

[0083] Hypoxic-ischemia brain injury is common in patients who receive ECPR. Although the pathophysiology of brain injury during ECMO is not fully understood, and not intending to be bound by theory, inadequate cerebral oxygenation, cerebral blood flow (CBF) alterations, and abrupt PaCCh changes may play a critical role. In this example in a rat model of CA and ECPR, arterial lactate, PaCh, and pH, but not PaCCh, were markedly changed relative to the baseline upon initiating ECPR, but there was no difference in these parameters between post-CA animals with and without H2. During ECPR, H2-treated animals exhibited markedly higher O2 levels in the brain tissue compared to placebo-treated animals.

[0084] Previous studies have shown that a higher occurrence of cerebral oximetry desaturation during veno-arterial ECMO (V-A ECMO) was independently associated with mortality in patients who underwent ECMO. Together, these observations indicate that the salutary impact of H2 administration on survival and EEG recovery could be linked with improved brain tissue oxygenation during ECPR. In addition, as cerebral blood flow is dependent on constantly receiving a significant proportion of the cardiac output, improved mortality in the H2 group relative to the placebo group could be, at least partially, a consequence of better cerebral perfusion, because the parameters (dp/dt max and min, CVP) after ECPR tended to improve in the H2 group.

[0085] As shown in this exemplary embodiment, prolonged CA and subsequent ECPR led to a considerable increase in plasma syndecan-1 levels at 2 h after ROSC in rats. Previous experimental models have also demonstrated that ischemia reperfusion damages the endothelial glycocalyx, leading to the release of glycocalyx components such as syndecan-1 and heparan sulfate. Further, initiation of extracorporeal circulatory support is associated with excessive ROS and a complex innate immune response, leading to endothelial injury. Glycocalyx degradation is activated by ROS and pro-inflammatory cytokines, resulting in blood-brain barrier (BBB) leakage, brain edema, and poor neurologic outcomes after CA/CPR in rats. Preservation of the glycocalyx by hydrocortisone reduces BBB permeability and gene transcription-protein synthesis and inflammation, thus improving the neurologic outcomes after CA/CPR. A previous study has also shown that H2 suppresses the TNF-a release and endothelial glycocalyx degradation, thus preventing endothelial damage after hemorrhagic shock in rats. Based on the well-established anti-oxidant property of H2, it is hypothesized that H2 is sufficient to attenuate the endothelial glycocalyx degradation caused by CA/ECPR.

[0086] It was considered whether H2 exerts a therapeutic effect by suppressing the systemic inflammatory response after ECPR. In this Example, EE was found to abate the excessive release of syndecan-1 into plasma after CA and ECPR, consistent with the previous study. Unexpectedly, EE did not affect the plasma levels of most pro-inflammatory cytokines/chemokines after ECPR. However, the beneficial effects of H2 were associated with an exaggerated increase in the IL- 10, VEGF, and leptin levels.

[0087] Preclinical and clinical studies have demonstrated an association between cytokines and glycocalyx degradation biomarkers. IL- 10, a well-established anti-inflammatory cytokine, can block NF-KB activity, thereby decreasing the expression of cell adhesion molecules on the endothelial cell surface and thus inhibit leukocyte transmigration. This Example indicates that the beneficial effects of H2 with ECPR are associated with an enhanced anti-inflammatory response. The precise role of VEGF after CA and ECPR is not fully understood; however, VEGF is considered an endothelial survival factor that prevents microvascular apoptotic cell loss in vitro. Notably, the relationship between VEGF levels and mortality in critical illness is discordant in different studies. Leptin is generally considered a rapid stress mediator after injuries and has been found to exert neuroprotective effects in a mouse model of transient focal cerebral ischemia. Taken together, higher plasma IL-10, VEGF, and leptin concentrations shortly after reperfusion may provide beneficial effects on cerebrovascular damage after CA/ECPR. [0088] While illustrative embodiments of the disclosure have been described and illustrated above, it should be understood that these are exemplary of the disclosure and are not to be considered as limiting. Additions, deletions, substitutions, and other modifications can be made without departing from the spirit or scope of the disclosure. Accordingly, the disclosure is not to be considered as limited by the foregoing description.