BR112017008076B1 - SOLUTION WITH CONTROLLED CALCIUM ION LEVEL, COMPOSITION FOR SOLUTION PREPARATION AND RELATED PROCESS AND USE FOR REPERFUSION - Google Patents

Abstract

The present invention relates to a solution comprising a preservative mixture comprising a source of calcium ion; and a buffer for maintaining a pH of the solution. the molar concentration of calcium ion (ca2+) in the solution is from 0.18 to 0.26 mmol/l and the ph is less than 7.4 and greater than 6.6. a composition for preparing the solution may comprise adenosine, lidocaine and a source of calcium, wherein the molar ratio of adenosine:calcium is from 0.3:0.26 to 0.45:0.18 and the molar ratio of lidocaine :calcium is from 0.04:0.26 to 0.09:0.18. a donor heart may be reperfused with the solution. the solution can be used for reperfusion of a donor heart, such as at a temperature of approximately 25 to approximately 37°C. the donor may be a donor after circulatory death.

Description

REFERENCE TO RELATED PATENTS APPLICATIONS

[0001] This patent application is a continuation in part of the patent application of PCT International Patent Application No. PCT/CA2015/050297, filed April 10, 2015, which claims the benefit and priority of U.S. Provisional Patent Application No. 61/978132, filed April 10, 2014, the entire contents of each of which are incorporated herein by reference.

[0002] This patent application claims the benefit and priority of U.S. Provisional Patent Application No. 62/068,524, filed October 24, 2014, the entire contents of which are incorporated herein by reference.

FIELD

[0003] The present invention relates to new compositions and solutions suitable for reperfusion and also refers to post-collection preservation and protection of donor hearts collected before their resuscitation and transplantation in recipients.

BACKGROUND

[0004] Heart failure affects 10% of Americans and is the main hospital discharge diagnosis. Diagnosis of heart failure is accompanied by a probability of survival that is comparable to that of major cancer. There are limited rehabilitation options available to patients who are suffering from heart failure, and few strategies actually rehabilitate the heart. Heart transplantation remains the other standard therapeutic intervention for patients with end-stage heart failure, with an increasing number of individuals being added to the transplant waiting list each year. However, wider application of this life-saving intervention is limited by the availability of donors. Data from the International Society of Heart and Lung Transplantation Registry show that heart transplantation is in progressive decline and suitable donors (2007, Overall Heart and Adult Heart Transplantation Statistics). Two hundred fifty-eight Canadians have died over the past decade (2000 - 2010; Heart and Stroke Foundation of Canada) while awaiting heart transplants. Similarly, in the United States, 304 patients died in 2010 just while waiting for a heart transplant (Organ Procurement and Transplantation Network, U.S. Dept. of Health & Human Services). This phenomenon is primarily due to a shortage of suitable organ donors and is being felt across the globe.

[0005] Time is of the essence for the removal of a heart from a donor and its successful transplantation into a recipient. The following conventional principles generally apply for optimal preservation of the donor heart during the time period between donor removal and transplantation: (i) minimization of cell swelling and edema, (ii) prevention of intracellular acidosis, (iii ) prevention of damage caused by oxygen free radicals and (iv) provision of substrate for the regeneration of high energy phosphate compounds, particularly adenosine triphosphate (ATP), during reperfusion. There are two main sources of donor hearts for transplantation. First, breathing patients who have suffered irreversible loss of brain function as a result of non-penetrating head trauma or intracerebral hemorrhage. Such a patient is referred to as a "brain dead" donor or a post-brain death ("DBD") donor. Second, patients who suffered circulatory death. Such a patient is referred to as a "no heartbeat" donor, a "cardiac death" donor, a cardiac postmortem donor, or a circulatory postmortem donor (DCD).

[0006] Brain-dead donors can be maintained with artificial respiration for extended periods of time to provide hemodynamic stability throughout their entire bodies to the point of organ removal. Cardiac perfusion is not flexible and organ functionality is theoretically maintained. However, brain death itself can profoundly affect cardiac function. The humoral response to brain death is characterized by a remarkable increase in circulating catecholamines. Physiological responses to this "catecholamine storm" include vasoconstriction, hypertension, and tachycardia, all of which increase myocardial oxygen demand. Increased levels of catecholamine circulating throughout the vascular system induce vasoconstriction, which, in turn, compromises myocardial oxygen supply and can lead to subendocardial ischemia. This imbalance between myocardial oxygen supply and demand is a factor implicated in decreased cardiac function after brain death (Halejcio-Delophont et al., 1998, Increase in myocardial interstitial adenosine and net lactate production in brain-dead pigs: an in vivo microdialysis study, Transplantation 66(10):1278-1284; Halejcio-Delophont et al., 1998, Consequences of brain death on coronary blood flow and myocardial metabolism. Transplant Proc. 30(6):2840-2841. Structural myocardial damage that occurs after brain death is characterized by myocytolysis, contraction band necrosis, subendocardial hemorrhage, edema, and interstitial mononuclear cell infiltration (Baroldi et al., 1997, Type and extent of myocardial injury related to brain damage and its significance in heart transplantation: a morphometric study. J. Heart Lung Transplant 16(10):9941000). - well reduced cardiac function and the current understanding is that only 40% of hearts can be recovered from this population of transplant donors.

[0007] Various equipment, systems and perfusion methods have been developed for the ex vivo maintenance and transport of collected organs. Most employ hypothermic conditions to reduce organ metabolism, decrease organ energy requirements, delay the elimination of high-energy phosphate stores, delay lactic acid buildup, and delay morphological and functional deterioration associated with interruption of oxygenated blood supply . The collected organs are usually perfused in these systems with solutions comprising antioxidants and pyruvate at low temperatures to maintain their physiological functionality.

[0008] The disadvantages of hypothermic equipment, systems and methods have been recognized by experts in these arts and equipment, alternative systems and methods have been developed for the preservation and maintenance of collected organs at temperatures ranging from approximately 25°C to approximately 35°C (these may be referred to as "normothermic" temperatures, although normothermic more conventionally means a normal body temperature, ie an average of approximately 37°C). Normothermic systems typically use perfusates based on the ViaspanTM formulation (also known as the University of Wisconsin solution or UW solution) supplemented with one or more of the following: serum albumin as a source of protein and colloid; trace elements to enhance pyruvate and adenosine cell viability and function to support oxidative phosphorylation; transferrin as a binding factor; insulin and sugars for metabolic support; glutathione to scavenge toxic free radicals as well as a source of waterproofing agent; cyclodextrin as a source of waterproofing, scavenging and cell binding enhancer and growth factors; a high concentration of Mg2+ to support microvessel metabolism; mucopolysaccharides for the potentiation and hemostasis of growth factors; and endothelial growth factors. For example, Viaspan comprises potassium lactobionate, KH2PO4, MgSO4, raffinose, adenosine, glutathione, allopurinol and hydroxyethyl starch. Other normothermic perfusion solutions have been developed and used (Muhlbacher et al., 1999, Preservation solutions for transplantation. Transplant Proc. 31(5):2069-2070). Although collected kidneys and livers can be maintained for more than twelve hours in normothermic systems, bathing and normothermic maintenance of hearts collected by perfusion for more than 12 hours results in deterioration and irreversible impairment of the functionality of the hearts. Another disadvantage of using normothermic continuous pulsed perfusion systems for the maintenance of collected hearts is the time required to excise a heart from a donor, mount it within the normothermic perfusion system, and then initiate and stabilize the perfusion process.

[0009] After the excised donor heart has been stabilized, its physiological functionality is determined and, if the transplantation criteria are met, the excised heart is transported as quickly as possible to a transplant unit.

[0010] In the case of brain-dead donors, the heart is usually warm and is beating when acquired. This is then stopped, cooled and placed on ice until transplanted. Cooling the collected heart reduces its metabolic activity and related demands by approximately 95%. However, some metabolic activity continues with the consequence that the heart muscle begins to die and clinical data have shown that since the cooling period of a collected heart is prolonged more than 4 hours, the risk of mortality within 1 year after the transplantation starts to increase. For example, the risk of death within 1 year after transplantation for a recipient who receives a heart that has been preserved by cooling for six hours more than doubles compared to a recipient who receives a heart that has been cooled for less than an hour ( Taylor et al., 2009, Registry of the International Society for Heart and Lung Transplantation: Twenty-sixth Official Adult Heart Transplant Report - 2009. JHLT 28(10):1007-1022).

[0011] Well-defined criteria have been developed for harvesting organs for transplantation from donors in which the heart is not beating (Kootstra et al., 1995, Categories of non-heart-beating donors. Transplant Proc. 27(5):2893- 2894; Bos, 2005, Ethical and legal issues in non-heart-beating organ donation. Transplantation, 2005. 79(9): p. 1143-1147). Donors whose heart is not beating have minimal brain function but do not meet the criteria for brain death and therefore such donors cannot legally be declared brain dead. When it is evident that there is no hope for the patient's meaningful recovery, physicians and the family must agree to withdraw support measures. Up to this point in care, patients whose heart is not beating are often supported with mechanical ventilation as well as intravenous inotropic or vasopressor medication. However, only those patients with single-system organ failure, namely neurological system failure, can be considered for organ donation. Withdrawal of life support, but commonly interruption of mechanical ventilation, is followed by anoxic cardiac arrest, after which the patient must remain asystolic for five minutes before organ acquisition is allowed. Consequently, donor organs in which the heart is not beating are necessarily exposed to variable periods of warm ischemia after cardiac arrest, which can result in varying degrees of organ damage. However, as long as the duration of warm ischemia is not excessive, many types of organs, such as kidneys, livers and lungs, can be collected from donors whose heart is not beating and are able to regain function after transplantation. with success rates approaching those for organs transplanted from brain-dead donors. Although hearts collected from brain-dead donors are exposed to an ischemic period limited to the time from organ acquisition to transplantation, hearts collected from donors after cardiac death are exposed to much larger ischemic injury events, including an arrest event hypoxemic, warm ischemic injury that occurs during the mandatory five-minute waiting period before organ harvesting can be started, and additional ischemic injury that occurs during reperfusion of the heart after it is harvested. Due to the ischemic damage that occurs before organ harvesting begins, hearts from donors where the heart is not beating are not used for transplantation.

SUMMARY

[0012] The present disclosure includes a new solution comprising a preservation mixture comprising a source of calcium ion; and a buffer for maintaining a solution pH, in which the molar concentration of calcium ion (Ca2+) in the solution is from 0.18 to 0.26 mmol/L and the pH is less than 7.4 and greater than 6 .6. The molar concentration of calcium ion (Ca2+) can be 0.22 mmol/L. The pH can be from 6.8 to 7.0, such as 6.9. The preservation mixture can be a cardioplegia mixture comprising adenosine, lidocaine and a magnesium ion source. The solution may comprise 0.3 to 0.45 mmol/L adenosine, 0.04 to 0.09 mmol/L lidocaine and 11 to 15 mmol/L Mg2+. The solution may comprise a sodium ion source and a potassium ion source. The solution may comprise approximately 130 to approximately 160 mmoles/L of Na+ and 4 to 7 mmoles/L of K+. The solution can comprise chloride, an osmotic buffer and a reducing agent. The solution can comprise 70 to 140 or 70 to 180 mmoles/L of chloride, 8 to 12.5 mmoles/L of glucose, 7.5 to 12.5 IU/L of insulin, 100 to 140 mmoles/L of D- mannitol, 0.75 to 1.25 mmol/L of pyruvate and 2.5 to 3.5 mmol/L of reduced glutathione. The solution may comprise 0.3 to 0.45 mmol/L adenosine; 0.04 to 0.09 mmol/L lidocaine; 8 to 12.5 mmoles/L of glucose; 110 to 130 mmoles/L of NaCl; 4 to 7 mmoles/L KCl; 16 to 24 mmoles/L of NaHCO3; 0.9 to 1.4 mmol/L NaH2PO4; 0.18 to 0.26 mmol/L of CaCl2; 11 to 15 mmoles/L of MgCl2; 7.5 to 12.5 IU/L of insulin; 100 to 140 mmoles/L of D-mannitol; 0.75 to 1.25 mmol/L of pyruvate; and 2.5 to 3.5 mmoles/L of reduced glutathione. The solution may comprise 0.4 mmol/L adenosine; 0.05 mmol/L lidocaine; 10 mmoles/L glucose; 123.8 mmoles/L of NaCl; 5.9 mmoles/L KCl; 20 mmoles/L of NaHCO3; 1.2 mmol/L NaH2PO4; 0.22 mmol/L of CaCl2; 13 mmoles/L of MgCl2; 10 IU/L of insulin; 120 mmoles/L of D-mannitol; 1 mmol/L of pyruvate; and 3 mmoles/L of reduced glutathione.

[0013] A composition for preparing the solution described in the previous paragraph is also provided. The composition may comprise adenosine, lidocaine and a source of calcium, wherein the molar ratio of adenosine:calcium is from 0.3:0.26 to 0.45:0.18 and the molar ratio of lidocaine:calcium is 0 .04:0.26 to 0.09:0.18. The molar ratio of adenosine:calcium can be 0.4:0.22 and the molar ratio of lidocaine:calcium can be 0.05:0.22. The composition may further comprise a sodium source, a potassium source and a magnesium source, wherein the calcium:sodium molar ratio is from 0.26:130 to 0.18:160, the calcium:potassium molar ratio is from 0.26:4 to 0.18 to 7 and the calcium:magnesium molar ratio is from 0.26:11 to 0.18:15. The calcium:sodium molar ratio can be 0.22:147, the calcium:potassium molar ratio can be 0.22:5.9, and the calcium:magnesium molar ratio can be 0.22:13. The composition may further comprise chloride, glucose, insulin, D-mannitol, pyruvate and reduced glutathione.

[0014] The solution that is described herein can be used for reperfusion of a donor heart and the present disclosure includes a method of reperfusion of a donor heart and of using the solution described herein for reperfusion of a donor heart . The heart may be reperfused with the solution during removal of the donor heart. The heart after removal from the donor may undergo reperfusion in a reperfusion device. The heart can be reperfused with the solution for at least 3 minutes immediately after removing the donor heart. The donor can be a donor after circulatory death. Reperfusion can occur at a temperature above approximately 25°C and below approximately 37°C. Reperfusion can take place at a temperature of approximately 35°C during reperfusion.

[0015] In such a method or use, selected embodiments of the present disclosure pertain to solutions for dipping and bathing a collected heart while it is concurrently drained through the heart and its vasculature.

[0016] Some modalities of the present disclosure refer to the use of solutions for the ex vivo maintenance of collected hearts to reduce and to improve post-harvest ischemic lesions.

[0017] Some modalities of the present disclosure refer to methods for ex vivo maintenance of collected hearts to minimize the occurrence and extent of post-harvest ischemic lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In the Figures, which illustrate, for the purpose of example only, modalities of this invention:

[0019] Figure 1 ("Figure 1) is a schematic flowchart describing the experimental protocols used in Example 1;

[0020] Figure 2 is a graph showing the myocardial temperature reached in pig hearts collected after an initial 3-minute reperfusion period;

[0021] Figure 3 is a graph showing the effect of reperfusion temperature on coronary blood flow through collected pig hearts, measured after the initial 3-minute reperfusion period;

[0022] Figure 4 is a graph showing the effect of reperfusion temperature on coronary vascular resistance to blood flow through collected pig hearts, measured after the initial 3-minute reperfusion period;

[0023] Figure 5 is a graph showing the effect of reperfusion temperature on the elimination of lactate from the coronary sinus of collected pig hearts, measured after the initial 3-minute reperfusion period;

[0024] Figure 6 is a graph showing the effect of reperfusate temperature on the accumulation of Troponin I (a marker of myocardial damage) in the perfusate solution, measured 5 hours after collection from pig hearts;

[0025] Figure 7(A) is a representative micrograph of a section along a collected pig heart subjected to reperfusion at 5°C that shows swollen endothelial cells lining a capillary, while Figure 7( B) is a representative micrograph of a section through a collected pig heart reperfused at 35°C showing normal endothelial cells lining a capillary;

[0026] Figure 8 is a graph showing the mean extent of lesion in endothelial cells and myocytes in collected pig hearts, which is observed in electron microscopy micrographs and classified with a classification system as a function of the temperature of reperfusion;

[0027] Figure 9 is a graph showing the effect of reperfusion temperature on the cardiac index of pig hearts collected, measured at one hour ("T1"), 3 hours ("T3") and 5 hours ("T5" ) after collecting the pig hearts;

[0028] Figure 10 is a graph showing the effect of reperfusion temperature on the systolic function of collected pig hearts, measured at one hour ("T1"), 3 hours ("T3") and 5 hours ("T5" ) after collecting the pig hearts;

[0029] Figure 11 is a graph showing the effect of reperfusion temperature on the diastolic function of collected pig hearts, measured at one hour ("T1"), 3 hours ("T3") and 5 hours ("T5" ) after collecting the pig hearts;

[0030] Figure 12 is a schematic graph describing the temperatures and Ca2+ ion concentrations of the cardioplegic solutions used in Example 2;

[0031] Figure 13 is a schematic flowchart describing the experimental protocols used in Example 2;

[0032] Figure 14 is a graph showing the effect of the concentration of Ca2+ ion in the reperfusate on the weight gain in collected pig hearts measured one hour after collection;

[0033] Figure 15 is a graph showing the effect of Ca2+ ion concentration on cardiac output reperfusate from collected pig hearts measured one hour after collection;

[0034] Figure 16 is a graph showing the effect of Ca2+ ion concentration on the left ventricular contraction capacity during systole in collected pig hearts, measured one hour after collection;

[0035] Figure 17 is a graph showing the effect of Ca2+ ion concentration on left ventricular relaxation during diastole in collected pig hearts, measured one hour after collection;

[0036] Figure 18 is a schematic graph describing the temperatures, the Ca2+ ion concentrations and the pH values of the cardioplegic solutions used in Example 3;

[0037] Figure 19 is a schematic flowchart describing the experimental protocols used in Example 3;

[0038] Figure 20 is a graph showing the effect of the pH of the cardioplegic reperfusate solution on the weight gain in collected pig hearts, measured one hour after collection;

[0039] Figure 21 is a graph showing the effect of the pH of the cardioplegic reperfusate solution on the cardiac output of collected pig hearts, measured one hour after collection;

[0040] Figure 22 is a graph showing the effect of the pH of the cardioplegic reperfusate solution on the left ventricular contraction capacity during systole in collected pig hearts, measured one hour after collection;

[0041] Figure 23 is a graph showing the effect of the pH of the cardioplegic reperfusate solution on the relaxation of the left ventricle during diastole in collected pig hearts, measured one hour after collection;

[0042] Figure 24 is a schematic graph depicting the temperatures, Ca2+ ion concentrations and pH values of cardioplegic reperfusate solutions and duration of reperfusion times used in Example 4;

[0043] Figure 25 is a schematic flowchart describing the experimental protocols used in Example 4, Part 1;

[0044] Figure 26 is a graph showing the effect of duration of initial reperfusion on weight gain in collected pig hearts;

[0045] Figure 27 is a graph showing the effects of initial reperfusion duration on myocardial function from collected pig hearts, measured at one hour ("T1"), 3 hours ("T3") and 5 hours (" T5") after collection;

[0046] Figure 28 is a schematic flowchart describing the experimental protocols used in Example 4, Part 2;

[0047] Figure 29 is a graph showing the effect of an initial extended reperfusion with a solution of cardioplegic reperfusate that has a reduced concentration of anesthetic on weight gain in collected pig hearts;

[0048] Figure 30 is a graph showing the effect of extended initial reperfusion with a solution of cardioplegic reperfusate that has a reduced concentration of anesthetic on the myocardial function of collected pig hearts, measured at one hour ("T1"), 3 hours ("T3") and 5 hours ("T5") after collection; and

[0049] Figure 31 is a graph showing the effect of anesthetic concentrations in cardioplegic reperfusate solutions on the myocardial function of collected pig hearts, measured at one hour ("T1"), 3 hours ("T3") and 5 hours ("T5") after collection.

DETAILED DESCRIPTION

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In order that the invention described herein may be fully understood, the following terms and definitions are provided herein.

[0051] The word "comprise" or variations such as "comprises" or "comprising" shall be understood to imply the inclusion of a quoted whole number or groups of whole numbers, but not the exclusion of any other whole number or group of whole numbers .

[0052] The term "around" or "approximately" means within 20%, preferably within 10% and more preferably within 5% of a given value or range.

[0053] The term "afterload" means the average tension produced by a chamber of the heart for the purpose of contraction. It can also be thought of as the 'load' against which the heart has to eject blood. Afterload is therefore a consequence of large aortic vessel distension, wave reflection, and small vessel resistance (left ventricular afterload) or similar pulmonary artery parameters (right ventricular afterload).

[0054] The term "preload" refers to the extent of a single cardiac myocyte immediately before contraction and is therefore related to the length of the sarcomere. Since the sarcomere length cannot be determined in the intact heart, other preload indices such as ventricular end-diastolic volume or pressure are used. As an example, preload increases when venous return is increased.

[0055] The term "cardiac myocyte" means a cardiac muscle cell.

[0056] The term "systolic volume" (SV) means the volume of blood ejected by the right/left ventricle in a single contraction. It is the difference between the end-diastolic volume (EDV) and the end-systolic volume (ESV). Mathematically, SV = EDV - ESV. Stroke volume is affected by changes in preload, afterload, and inotropy (contraction capacity). In normal hearts, SV is not strongly influenced by afterload whereas in failing hearts, SV is highly sensitive to changes in afterload.

[0057] The term "systolic work" (SW) refers to the work performed by the left or right ventricle to eject the stroke volume into the aorta or pulmonary artery, respectively. The area delimited by the pressure/volume loop is a measure of the ventricular systolic work, which is a product of the systolic volume and the main aortic or pulmonary artery pressure (afterload), depending on whether the left or right ventricle is considered. .

[0058] The term "ejection fraction" (EF) means the fraction of end-diastolic volume that is ejected out of the ventricle during each contraction. Mathematically, EF = SV/EDV. Healthy ventricles typically have ejection fractions greater than 0.55. Low EF usually indicates systolic dysfunction and severe heart failure may result from an EF less than 0.2. EF is also used as a clinical indicator of the inotropy (contracting capacity) of the heart. Increased inotropy leads to an increase in EF, whereas a decrease in inotropy decreases EF.

[0059] The term "end systolic pressure-volume ratio" (ESPVR) describes the maximum pressure that can be produced by the left ventricle in any supplied left ventricular volume or alternatively, by the right ventricle in any supplied right ventricular volume. This implies that the PV loop cannot cross the line that sets the ESPVR to any given contractile state. The slope of ESPVR (Ees) represents the end-systolic elastance, which provides an index of the capacity to contract the myocardium. ESPVR is relatively insensitive to changes in preload, afterload, and heart rate. This makes it an improved index of systolic function relative to other hemodynamic parameters such as ejection fraction, cardiac output, and stroke volume. The ESPVR becomes steeper and shifts to the left as inotropy (contraction capacity) increases. ESPVR becomes flatter and shifts to the right as inotropy decreases.

[0060] The term "recruitable systolic work to preload ratio" (PRSW) means a measure of cardiac contraction capacity and is the linear relationship between SW and EDV.

[0061] The term "volume pressure area" (PVA) means the total mechanical energy generated by the ventricular contraction. This equates to the sum of the systolic work (SW) covered within the PV loop and the elastic potential energy (PE). Mathematically, PVA = PE + SW.

[0062] The term "dP/dt max" is a measure of the global capacity for contraction of the left ventricle. The greater the contractile force exerted during systole, the greater the rate of increase in left ventricular pressure.

[0063] The term "dP/dt min" is a measure of left ventricular relaxation during diastole.

[0064] As used herein, the term "DCD" means post-death circulatory donor or post-death cardiac donor. As used herein, the term "DBD" means post-brain death donor.

[0065] The term "Langendorff perfusion" refers to a method of perfusing an excised heart with an oxygenated nutrient-rich solution in an inverted manner through the aorta. Retrograde pressure causes the aortic valve to close, thus forcing the solution into the coronary vessels that supply blood to the heart tissue. This transports nutrients and oxygen to the heart muscle, allowing it to continue beating for several hours after it is removed from the animal.

[0066] The term "beating heart" as used herein, refers to clinical ex vivo coronary perfusion throughout the heart excised by ventricular filling through the left atrium and left ventricular ejection through the aorta controlled by the contractile function of the heart and regular heart rate. The excised heart is connected through a cannula to a reservoir of perfusate and circulatory pumps in a Langendorff preparation. Perfusate flow through the excised heart in the "beating heart" mode occurs in the opposite direction to perfusate flow during Langendorff perfusion.

[0067] The term "ischemia" means a condition that occurs when blood flow and oxygen are not supplied to the heart.

[0068] The term "reperfusion" as used herein means passing a solution through a heart to re-establish the oxygen supply and provide protective or preservation materials to the heart, such as by pumping the solution through the heart in a perfusion device and optionally by immersing the heart in the solution. Optionally, during reperfusion the heart can be immersed in an oxygen-rich perfusate solution, which can be the same as a reperfusion solution or can be a different solution.

[0069] The term "reperfusion injury" as used herein refers to tissue damage in a collected heart that occurs when an oxygen supply through a perfusate solution is supplied to the tissue after a period of ischemia or lack of oxygen. Deprivation of the heart of sufficient oxygen and nutrients during the ischemic period creates a condition in which the restoration of circulation results in inflammation or oxidative damage through the induction of oxidative stress, rather than the restoration of normal function.

[0070] The term "cardioplegia" as used herein means an intentional and temporary interruption or maintenance of ceased or reduced cardiac activities, such as through the interruption or suspension of the heartbeat, for the purpose of preserving the health of the myocardium, including the over a period of significantly reduced provision of oxygen and metabolic substrate. Cardioplegia can be imposed on a beating heart by cooling or by administering a solution that contains one or more chemical reagents that will cause the heart muscle to paralyze, or by both concurrently. In embodiments of the present disclosure, cardioplegia can also be achieved by providing oxygen and other limited supplies to the myocardium to preserve its health without completely restoring heart activity.

[0071] The term "cardioplegic solution" as used herein means a solution that contains chemical components that cause or maintain asystole (paralysis) of the heart in a mixture with components to preserve or protect the functions of cardiac cells.

[0072] The term "homeostasis" as used herein means the maintenance of a completely stable metabolic balance within and between the muscle cells of a collected heart.

[0073] The term "normokalemic" as used herein means that it possesses or is characterized by a normal concentration of potassium ion in the blood. Normal serum potassium ion levels in human blood range between 3.5 mEq/L and 5.0 mEq/L.

[0074] The term "hyperkalemic" as used herein means that it possesses or is characterized by a potassium ion concentration in the blood that is significantly elevated relative to a normokalemic concentration. A hyperkalemic concentration includes any potassium ion concentration in excess of 6.0 mEq/L.

[0075] The term "hypothermic" as used herein means a temperature that is less than approximately 20°C.

[0076] The medically and legally prescribed events that have to occur for the ethical acquisition of hearts that can be transplanted from post-death circulatory donors (DCD) inevitably cause an occurrence of cardiac arrest and a sequence of ischemic events that result in damage to the muscles of the heart. These prescribed events cannot be modified.

[0077] Ischemia is accompanied by significant changes in ion exchange patterns into and out of cardiac muscle cells as a consequence primarily of the loss of oxygen supply. As oxygen availability decreases and is interrupted, the metabolism of cardiac muscle cells changes from aerobic to anaerobic with an immediate consequence of rapidly decreasing intracellular pH levels. Low intracellular pH results in increasing amounts of H+ ions that are excreted from muscle cells within the extracellular spaces. At the same time, the ionic potential across cell membranes decreases due to significantly reduced Na+/Ca2+ ion exchange as a result of lower intracellular ATP levels. The end result is an increased overload in intracellular Ca2+ levels. Increased intracellular Ca2+ levels activate Ca2+-dependent proteases, which disrupt cell structure resulting in cell death. The severity of such an injury increases with the duration of the ischemic conditions.

[0078] The ischemic injury that occurs during the acquisition of a donor heart can be reduced by reperfusion of the collected heart as soon as possible after its collection in blood or a blood substitute product, which is exemplified by Viaspan and CELSIOR® (CELSIOR is a registered trademark of Genzyme Corp., Cambridge, Massachusetts, USA). Reperfusion causes a rapid increase in extracellular pH, which results in robust excretion of H+ ions into the extracellular space. The movement of H+ ions within the extracellular space drives Na+ ions into cells. Higher intracellular Na+ ion concentrations reverse the exchange of Na+/Ca2+ ions across myocyte cell membranes, resulting in "reverse mode" excretion of accumulated intracellular Na+ ions accompanied by an influx of Ca2+ ions, by recovery of ATP synthesis and a further subsequent excretion of Ca2+ ions. However, although reperfusion can restore aerobic respiration and metabolism in collected hearts, reperfusion commonly results in additional damage (known as reperfusion injury) to cardiac muscle cells. For example, the immediate rise in intracellular pH levels results in the production of reactive oxygen species that activate subcellular signals which in turn activate inflammatory cascades that lead to apoptosis and cytokine release. Additionally, reactive oxygen species directly disrupt DNA structures and protein structures, thus causing cell death. Another problem associated with conventional reperfusion techniques is that it is very difficult in these techniques to modulate intracellular levels of Ca2+ ions during a reperfusion process, in which reperfusion additionally increases the intracellular overload of Ca2+ ions in cardiac muscle cells;

Contraction of a heart while cardiac muscle cells are overloaded with intracellular Ca2+ ions during reperfusion inevitably results in a destructive type of necrosis, termed contraction band necrosis, as a result of massive contraction of the myofibrils. Contraction band necrosis is considered the most severe form of reperfusion injury.

[0080] Consequently, the rationale of cooling donor hearts immediately after their acquisition and during reperfusion is to reduce metabolic activity within cardiac muscle cells as quickly as possible to minimize the production of reactive oxygen species during reperfusion and to minimize a subsequent intracellular overload of Ca2+ ions during reperfusion.

[0081] The present inventors have found that myocardial damage in donor hearts can be minimized by a strategy focused on maintaining calcium ion homeostasis within and around the heart during collection and reperfusion processes. The present inventors' strategy comprises two components in which the first component is an oxygenated cardioplegic composition for use as a reperfusate solution during the acquisition of a collected heart and for a period of time immediately after collection during which the collected heart is subjected to reperfusion, preferably, for at least 3 minutes. Reperfusate solution causes an immediate interruption of the rhythmic beat of a donor heart after reperfusion. The reperfusion period of at least 3 minutes, which begins immediately after the heart is collected, is referred to as the immediate-initial ("EI") period. The second component of the present inventors' strategy is to avoid heart cooling during the acquisition process and during the post-collection reperfusion period and instead maintain normothermic conditions during collection, during IE reperfusion and during ex vivo maintenance of the collected heart.

[0082] It has been recognized that it would be beneficial to prevent myocyte contraction before intracellular calcium overload has been produced in a donor heart and before ATP stores in the heart can be replenished. It is expected that after a period of reperfusion or perfusion, as oxygen and energy substrates are supplied to the heart, the heart can begin to beat again. However, if the heart starts to beat again when there is intracellular calcium overload, this can result in contracture. Thus, it is expected that the reduction of intracellular calcium ion concentration will eliminate or prevent intracellular calcium overload before restarting myocyte contraction or that the complete restoration of cardiac activities may reduce reperfusion injuries. The present inventors' results indicate that intracellular calcium concentrations and consequently reperfusion injuries can be reduced by controlling, at least in part, the calcium content in a reperfusion solution.

[0083] When selecting the components and their concentrations in a cardioplegic composition for reperfusion, for example, from a DCD heart for transplantation, at temperatures of approximately 25 to approximately 37°C, a number of factors may need to be considered. To reduce or to minimize myocardial damage in such a donor heart during reperfusion, a balanced approach in view of these factors may be necessary. For example, one source of potential complication is that intracellular concentrations of a particular ion, especially the intracellular concentration of Ca2+ or H+ ions, which if not properly controlled could contribute to myocardial damage, may be sensitive to extracellular concentrations of these ions as well as of other ions. For example, the intracellular concentration of Ca2+ in myocytes is expected to be affected not only by the extracellular concentration of Ca2+, but also, as a result of particular ionic exchanges in the plasma membrane, by the extracellular concentrations of other ions, such as H+ and Na+. Thus, the intracellular calcium ion concentration can be adjusted by changing the extracellular concentration of one or more of Ca2+, Na+ and H+. However, changing extracellular concentrations of H+ and Na+ can result in other changes that can affect other aspects of myocardial damage, in addition to optimizing intracellular Ca2+. Another factor that must be considered is providing enough calcium ions in a reperfusion solution to avoid a phenomenon known as the "calcium paradox" - in which the hypocalcemic heart muscles are again exposed to the normal level of Ca2+, the cells can become overloaded with Ca2+, which can cause significant cell damage or injury. To achieve optimal results, these different effects must be considered in a balanced approach when selecting the components and their respective concentrations.

[0084] In one embodiment, a solution for use as a reperfusion solution may include the following components: - A preservation mixture which may include adenosine to provide oxidative phosphorylation support and lidocaine to prevent myocyte contraction during reperfusion. Additionally, a relatively high concentration of Mg2+ can also be included, as hypermagnesemia is expected to help prevent myocyte contraction during reperfusion. For example, the mixture can contain 0.3 to 0.45 mmol/L of adenosine, 0.04 to 0.09 mmol/L of lidocaine, and 11 to 15 mmol/L of Mg2+. - Ca2+ at a concentration of 0.18 mmol/L to 0.26 mmol/L, to provide a lower than physiological concentration of extracellular calcium ions in a normal heart. - Na+, such as at a concentration of 130 mmoles/L to 160 mmoles/L, to provide an appropriate concentration of extracellular sodium ion. - K+ at a normokalemic concentration, such as 4 to 7 mmoles/L. - Cl- in a concentration ranging, for example, from 70 to 180 mmoles/L. Although in some modalities the Cl- concentration may be higher, such as up to approximately 180 mmoles/L in solution, this may be beneficial in some modalities for obtaining a lower Cl- concentration such as, for example, 70 up to 140 mmoles/L or up to approximately 140 mmoles/L. - A pH buffer for maintaining the pH of a reperfusion solution that is greater than 6.7 and less than 7.4 at the desired operating temperature for reperfusion. The pH buffer can be provided, for example, by a combination of 16 to 24 mmoles/L of HCO31- and 0.9 to 1.4 mmol/L of H2PO41-. - Substrates for energy metabolism, such as a combination of 8 to 12.5 mmoles/L glucose and 0.75 to 1.25 mmol/L pyruvate. - An osmotic agent in a concentration to obtain an appropriate osmolarity, such as 100 to 140 mmoles/L of D-mannitol. - An antioxidant or reducing agent in a concentration to achieve an appropriate degree of protection from reactive oxygen species and physiologically reducing levels, such as 2.5 to 3.5 mmoles/L of reduced glutathione. - Optionally, one or more growth factors, such as 7.5 to 12.5 IU/L insulin.

[0085] During use, a pre-prepared cardioplegic composition can be titrated to the desired pH before use, so that the composition at the desired temperature for reperfusion is at the desired pH at the time of reperfusion.

[0086] A cardioplegic composition to cause an immediate interruption of the rhythmic beat of a donor heart after its contact with the cardioplegic composition may comprise a mixture of adenosine-lidocaine, a normokalemic composition of potassium ions, a concentration of Ca2+ ions selected to maintain the intracellular level of Ca2+ ions in heart muscle cells collected at approximately 10-4 mmoles/L and a pH of 6.9. A suitable adenosine-lidocaine mixture may comprise 300 μmol/L, 325 μmol/L, 350 μmol/L, 375 μmol/L, 400 μmol/L, 425 μmol/L, 450 μmol/L adenosine and 40 μmol/L , 45 μmol/L, 50 μmol/L, 55 μmol/L, 60 μmol/L, 70 μmol/L, 80 μmol/L, 90 μmol/L of lidocaine. The cardioplegic composition may further comprise 8.012.5 mmoles/L of glucose, 120-140 mmoles/L of NaCl, 4.07.0 mmoles/L of KCl, 12.0-16.0 mmoles/L of NaHCO3, 0, 9-1.4 mmol/L of NaH2PO4, 0.18-0.26 mmol/L of CaCl2, 11.0-15.0 mmol/L of MgCl2, 7.5-12.5 IU/L of insulin, 100.0-140.0 mmoles/L of D-mannitol, 0.75-1.25 mmol/L of pyruvate and 2.5-3.5 mmoles/L of reduced glutathione. In a particular modality, a cardioplegic composition may include 400 μmol/L adenosine, 50 μmol/L lidocaine, 10.0 mmol/L glucose, 123.8 mmol/L NaCl, 5.9 mmol/L KCl , 20 mmol/L of NaHCO3, 1.2 mmol/L of NaH2PO4, 0.22 mmol/L of CaCl2, 13.0 mmol/L of MgCl2, 10.0 IU/L of insulin, 120.0 mmol/L of D-mannitol, 1.0 mmol/L of pyruvate and 3.0 mmol/L of reduced glutathione.

[0087] The cardioplegic composition can be oxygenated by bubbling a stream of O2 gas through the cardioplegic composition before and during its use to bathe and reperfuse a collected donor.

[0088] Another selected modality of the present disclosure refers to the use of the oxygenated cardioplegic composition selected for the reperfusion of a collected heart at a temperature of approximately 35°C. Consequently, the selected oxygenated cardioplegic composition is heated to approximately 35°C prior to contact with the heart during acquisition and subsequent IE reperfusion over at least 3 minutes after the acquisition is completed. After the initial IE reperfusion period on the selected oxygenated cardioplegic composition under normothermic conditions, the collected heart can be resuscitated by placing it within an appropriate equipment for ex vivo maintenance of a collected functioning systolic heart through the infra-red interconnection. conductive structures provided within the equipment with the heart aorta, pulmonary artery, pulmonary vein and vena cava and bathing the excised heart in a constant flow perfusate solution comprising oxygenated blood and/or an oxygenated blood substitute solution. Additionally, the constant flow perfusion solution is drained through the chambers of the heart while the heart is kept in the instrument. Such equipment is generally configured with the following (i) a perfusate pumping system; (ii) flow sensors for monitoring the flow of perfusate to and from the placed aorta of the heart, pulmonary artery, pulmonary vein and vena cava; (iii) an ECG device that can be interconnected to the excised heart; (v) probes that interconnect the placed heart with instruments for monitoring the physiological functionality of the excised heart using load-independent indices and load-dependent indices; and optionally (vi) pacemakers to initiate or maintain systolic heart function.

[0089] It is expected that the use of an example oxygenated cardioplegic composition disclosed herein for the reperfusion of a heart removed from a donor for transplantation may provide a collected heart with the ionic complement necessary for the heart to be maintained ex vivo continue to produce ATP and pump calcium out of the heart muscle cells while keeping the heart in a paralyzed condition, that is, an asystolic condition without beating, thus minimizing the potential for contraction band necrosis to occur. While not wishing to be limited to any particular theory, it is likely that the use of such a cardioplegic composition for reperfusion of hearts collected at temperatures of approximately 25 to approximately 35°C could facilitate the rapid restoration of calcium ion homeostasis and facilitate further recovery. rapid and functional operation of the heart collected after transplantation in a recipient individual.

[0090] Without wishing to be limited to any particular theory, it is also expected that when a heart removed from a DCD donor is reperfused immediately after its removal from the donor with an adequate cardioplegic solution with calcium ion concentration and pH controlled throughout of a sufficient period of time, it is possible to avoid excessive reperfusion injuries, such as those caused by intracellular calcium overload, in the heart, without cooling the DCD heart to below approximately 25°C before, during and after reperfusion and for provide a suitable heart for transplantation.

[0091] In one modality, such a solution may include a mixture of cardioplegia. The mixture contains a source of calcium ion and a buffer to maintain a pH of the solution. The molar concentration of calcium ion (Ca2+) in the solution is from 0.18 to 0.26 mmol/L and the pH is less than 7.4 and greater than 6.6. The molar concentration of calcium ion (Ca2+) in the solution can be 0.22 mmol/L. The pH can be from 6.8 to 7.0, such as 6.9. In specific modalities, the cardioplegia mix may include adenosine, lidocaine, and a magnesium ion source, such as 0.3 to 0.45 mmol/L of adenosine, 0.04 to 0.09 mmol/L of lidocaine, and 11 to 15 mmoles/L of Mg2+. The solution may also include a sodium ion source and a potassium ion source, such as approximately 130 to approximately 160 mmoles/L of Na+ and 4 to 7 mmoles/L of K+. The solution can also include chloride, an osmotic buffer and an antioxidant or reducing agent. For example, suitable osmotic buffers can include D-mannitol, lactobionate, dextran, albumin or the like. Suitable antioxidants can include reduced glutathione, resveratrol, apelin analogues or the like. The solution can contain, for example, 70 to 140 mmoles/L of chloride, 100 to 140 mmoles/L of D-mannitol and 2.5 to 3.5 mmoles/L of reduced glutathione. The solution may contain substrates for energy metabolism, such as one or more of glucose, pyruvate, free fatty acids (for example, oleate or palmitate), triglycerides or the like. For example, in some modalities, the solution may contain 8 to 12.5 mmol/L of glucose and 0.75 to 1.25 mmol/L of pyruvate. The solution may contain one or more growth factors, such as insulin, cardiotrophin-1, erythropoietin, platelet-derived growth factors (PDGF), various forms of fibroblast growth factors (FGF) or the like. For example, the solution may contain 7.5 to 12.5 IU/L of insulin. Thus, depending on the application, the solution may contain 0.3 to 0.45 mmol/L of adenosine; 0.04 to 0.09 mmol/L lidocaine; 8 to 12.5 mmoles/L of glucose; 110 to 130 mmoles/L of NaCl; 4 to 7 mmoles/L KCl; 16 to 24 mmoles/L of NaHCO3; 0.9 to 1.4 mmol/L NaH2PO4; 0.18 to 0.26 mmol/L of CaCl2; 11 to 15 mmol/L of MgCl2; 7.5 to 12.5 IU/L of insulin; 100 to 140 mmoles/L of D-mannitol; 0.75 to 1.25 mmol/L of pyruvate; and 2.5 to 3.5 mmoles/L of reduced glutathione. More specifically, the solution may contain 0.4 mmol/L of adenosine; 0.05 mmol/L lidocaine; 10 mmoles/L glucose; 123.8 mmoles/L of NaCl; 5.9 mmoles/L KCl; 20 mmoles/L of NaHCO3; 1.2 mmol/L NaH2PO4; 0.22 mmol/L of CaCl2; 13 mmol les/L MgCl2; 10 IU/L of insulin; 120 mmoles/L of D-mannitol; 1 mmol/L of pyruvate; and 3 mmoles/L of reduced glutathione.

[0092] In different embodiments, the solution for reperfusion of an excised heart may include the cardioplegia mixture containing an anesthetic agent to paralyze the heart and prevent contraction of myocytes during reperfusion; and agents for protecting or restoring cardiac functions, agents comprising a source of calcium, a source of sodium and a source of potassium, in selected amounts to restore and maintain calcium ion homeostasis in the heart at a temperature of approximately 25 to approximately 35°C. The solution can be at a temperature of approximately 25 to approximately 35°C, such as approximately 35°C.

[0093] As can be appreciated by those skilled in the art, a solution disclosed herein can be prepared and stored prior to use or the solution can be prepared just before use by mixing pre-packaged compositions or materials or by adding a solvent such as water or a buffer solution in a preformulation to form the desired solution. For example, a composition for preparing a reperfusion solution can include a mixture of adenosine, lidocaine and a source of calcium. The adenosine:calcium molar ratio can be from 0.3:0.26 to 0.45:0.18, such as 0.4:0.22 and the lidocaine:calcium molar ratio can be 0.04: 0.26 to 0.09:0.18, such as 0.05:0.22. The composition may also contain a source of sodium, a source of potassium and a source of magnesium. The calcium:sodium molar ratio can be from 0.26:130 to 0.18:160, such as 0.22:147. The calcium:potassium molar ratio can be from 0.26:4 to 0.18 to 7, such as 0.22:5.9. The calcium:magnesium molar ratio can be from 0.26:11 to 0.18:15, such as 0.22:13. The composition can also contain chloride and one or more of glucose, insulin, D-mannitol, pyruvate and reduced glutathione. The composition can be mixed with a suitable pH buffer to prepare the desired reperfusion solution, such as a selected reperfusion solution described herein.

[0094] Additional modalities refer to methods of preservation and preparation of hearts for transplantation. For example, in a method for reperfusion of a heart for transplantation, the heart may be reperfused with a reperfusion solution disclosed herein in a reperfusion apparatus. The reperfusion device may be similar to a conventional perfusion device and may be operated similarly except for replacing the perfusion solution with a reperfusion solution described here. For example, the Quest MPS®2 Myocardial Protection System, provided by Quest Medical Inc., Allen, TX, USA, can be used as a reperfusion device. A volume infusion pump can also be used to pump a reperfusion solution. An infuser, such as one that is typically used by a trauma patient or a similar infuser, can be used for reperfusion. For example, the BelmontTM RI-2 Rapid Infuser can be used in a reperfusion device.

[0095] The heart can be reperfused with a reperfusion solution for at least 3 minutes immediately after removing the donor heart from the heart. The donor can be a DCD donor and the DCD heart can be maintained at a temperature above approximately 25°C and below approximately 37°C, such as at approximately 35°C at any stage of the acquisition, reperfusion, procedures, perfusion, storage and transplantation.

[0096] Additional modalities are related to methods of maintaining a heart for transplantation. For example, the heart can be treated to maintain calcium ion homeostasis in the heart at a temperature of approximately 25°C to approximately 37°C, such as through the use of a suitable solution or composition disclosed herein. .

[0097] As can now be considered, the modalities of solutions disclosed herein can be used for reperfusion of a donor heart, during heart removal or immediately after removal of the donor heart, or both. Additionally, the solution can also be used as a perfusion solution at other times or for other purposes that may be appropriate. Conveniently, the heart can be removed from a donor after circulatory death (DCD) at a temperature of approximately 25 to 37°C. In different embodiments, a solution that is described here can also be used for reperfusion of other types of hearts such as a heart removed from a donor after brain death (DBD). In some embodiments, the solution can also be used at lower temperatures.

[0098] Although some modalities have been described herein with reference to reperfusion or cardioplegic solutions or compositions or cardioplegia mixtures, it can be understood that these are preservation compositions, solutions or mixtures, which can preserve or protect the functions cells and therefore the health of the cell in the organ that will be transplanted.

[0099] The following examples are provided to more fully describe the disclosure and are presented for non-limiting illustrative purposes. EXAMPLES

[00100] The cardioplegic sample solutions used in these Examples were prepared at room temperature and their quoted pH was measured at room temperature. The lidocaine and D-mannitol solutions used to prepare the sample solutions were obtained from commercial suppliers.

[00101] All sample solutions were prepared by adding the component ingredients to the water. The water was double deionized and sterilized as is known to those skilled in the art. Sample solutions were oxygenated prior to use. Example 1:

[00102] It is evident that strategies to minimize trauma and ex vivo injury after collection in donor hearts require an understanding of the ionic exchanges that occur in the heart during ischemia and during/after reperfusion.

[00103] During ischemia, the heart's metabolism is changed from aerobic to anaerobic with a subsequent production of protons within cardiac myocytes. The efflux of excess protons through myocyte cell walls in exchange for Na+ ions that enter via the Na+/K+ pump. Once ATP stores within myocytes are depleted, myocytes become unable to pump Na+ ions back out through the Na+/K+ pump. As a result, as the duration of ischemia increases, there is an accumulation of: (i) Na+ ions inside the myocytes and (ii) Na+ ions and H+ ions inside and outside the myocytes.

[00104] During reperfusion, the H+ ions on the outside of the myocytes are washed away resulting in the occurrence of a high Na+/H+ gradient along the myocyte walls resulting in a high influx of Na+ ions into the myocytes . The increased concentration of Na+ ions causes the Na+/Ca2+ pump to work in a reverse mode resulting in an influx of Ca2+ ions into the myocytes as the Na+/Ca2+ pump tries to balance the levels of Na+ ions in and out of myocytes. If a myocyte overloaded with Ca2+ is allowed to contract, a fatal hypercontract can occur (hypercontract is also commonly referred to as "contraction band necrosis"). Consequently, a primary goal of resuscitation of a DCD heart is to mitigate an overload of Ca2+ ions in myocytes.

[00105] Consequently, the objectives of the present inventors were to prevent a collected DCD heart from contracting by reperfusion with a cardioplegic solution containing anesthetic while providing the necessary substrates to generate ATP so that the reperfused heart could restore its homeostasis by pumping Na+ and Ca2+ ions and thereby minimizing trauma and reperfusion injury from ischemia. Due to the production of ATP to provide the energy needed to exchange the ions through the Na+/K+ pumps and the Na+/Ca2+ pumps, it was the present inventors' idea that reperfusion of collected donor hearts would facilitate faster restoration of homeostasis of ions and the recovery of cardiac function. Consequently, the first study evaluated the effects of reperfusion temperature on collected donor hearts.

[00106] Eighteen pigs were separated into three groups and then euthanized following standard protocols and medical ethics procedures following the schematic flowchart shown in Figure 1.

[00107] Six pigs were assigned to the first group ("cold" group). Immediately after each heart acquisition was completed, each heart was placed into a Quest MPS®2 Myocardial Protection System (MPS is a registered trademark of Quest Medical Inc., Allen, TX, USA) for accurate pressure and blood pressure control. reperfusion temperature. Hearts collected from the first group of pigs were perfused for 3 minutes with an oxygenated cardioplegic sample composition (see TABLE I) which was cooled to 5°C before starting the reperfusion process. The cardioplegic composition was initially prepared at room temperature and the pH of the composition was measured at room temperature. Aortic perfusion pressure, coronary artery flow and myocardial temperature were constantly monitored and recorded by the MPS®2 equipment during the initial 3-minute reperfusion period. Blood gas samples were measured at 0, 30, 60, 120 and 180 seconds of the initial reperfusion period to collect data regarding changes that occur in partial pressure of O2 (PaO2), partial pressure of CO2 (PaCO2), levels of pH, electrolyte levels, lactate levels among others. TABLE I Sample I - Cardioplegic Solution (pH = 7.35)

[00108] After the initial 3-minute reperfusion period was completed, each heart was removed from the Quest MPS®2 instrument and transferred into an ex vivo cardiac perfusion instrument (EVHP) where it was perfused with a flow supply constant of a blood-STEEN solution mixture (Hb 45 g/L; XVIVO Perfusion Inc., Englewood, CO, USA) in which its systolic function was restored and maintained in a Langendorff mode at a normothermic temperature of 35° C for 6 hours. Aortic pressure and heart rate were constantly monitored and processed using LABCHART® software (LABCHART is a registered trademark of ADInstruments Pty. Ltd., Bella Vista, NSW, Australia). After 1 hour, 3 hours and 5 hours of perfusion with the blood-STEEN solution mixture in the EVHP equipment, each heart underwent a transition from Langendorff mode to a functioning mode, taking left atrial pressure from 0 to 8 mmHg and regulating the heart rate at 100 beats per minute ("bpm"). Cardiac output, coronary blood flow, aortic root and coronary sinus blood gases were measured and cardiac function was assessed with a pressure-volume loop catheter. After these measurements were completed, each heart was immediately taken back to Langendorff mode.

[00109] Five pigs were assigned to the second group ("refrigerated" group) and were processed as previously described for the first group with the only exception that IE reperfusion was performed with an oxygenated cardioplegic sample composition as shown in TABLE I, which was refrigerated at 25°C before starting the reperfusion process.

[00110] Seven pigs were assigned to the third group ("normothermic" group) and were processed as previously described for the first group with the only exception that IE reperfusion was performed with an oxygenated cardioplegic sample composition as shown in the TABLE I, which was heated to 35°C before starting the reperfusion process.

[00111] The data in Figure 2 show that the myocardial temperatures recorded in the hearts that received the IE reperfusion treatment with the oxygenated cardioplegic composition of the sample cooled to 5°C fell to approximately 10°C at the end of the IE reperfusion period of 3 minutes. The myocardial temperatures recorded in hearts that received IE reperfusion with the oxygenated cardioplegic composition of a sample refrigerated at 25°C were approximately 25°C, while the myocardial temperatures recorded in the hearts that received reperfusion with the selected oxygenated cardioplegic composition were of approximately 35°C.

[00112] Figure 3 shows that coronary blood flow rates were reduced by approximately 15% in hearts that underwent reperfusion with the oxygenated cardioplegic composition of sample refrigerated to 25°C compared with coronary blood flow in hearts that received reperfusion with the oxygenated cardioplegic composition of the sample. However, coronary blood flow rates were reduced by approximately 50% in hearts that were reperfused with the oxygenated cardioplegic composition from a sample cooled to 5°C compared to coronary blood flow in hearts that were reperfused with the cardioplegic composition sample oxygenate.

[00113] Figure 4 shows that coronary vascular resistance in hearts that underwent reperfusion with the refrigerated oxygenated cardioplegic composition fell by approximately 40% compared with hearts that underwent reperfusion with the oxygenated cardioplegic composition, while the oxygenated cardioplegic composition. nothing cold caused a reduction of more than 50% in coronary vascular resistance.

[00114] Figure 5 shows that the coronary sinus lactate dropped by more than 50% in hearts that received the cooled IE reperfusion treatment and by approximately 25% in the hearts that received the refrigerated IE reperfusion treatment, when compared to the levels of coronary sinus lactate in hearts that received normothermic IE reperfusion treatment.

[00115] Figure 6 shows that Troponin I levels (a marker for myocardial injury) increased as the temperature of the IE reperfusion decreased, compared to the levels observed in the hearts that received the normothermal IE reperfusion treatment.

[00116] Figure 7(A) is an electron micrograph showing a swollen endothelial cell in a capillary of a heart that received the IE reperfusion treatment cooled for 3 minutes, while Figure 7(B) is an electron micrograph that shows a typical normal-appearing endothelial cell in a capillary from a heart that has received normothermic IE reperfusion treatment for 3 minutes.

[00117] Figure 8 is a graph comparing the endothelial injury and myocyte injury scores of hearts that received IE reperfusion cooled for three minutes and hearts that received IE reperfusion normothermic for three minutes.

[00118] Figure 9 is a graph showing the effects on cardiac indices of IE reperfusion with a cooled oxygenated cardioplegic composition and with a cooled oxygenated cardioplegic composition, with the effects of IP perfusion with a normothermic oxygenated cardioplegic composition.

[00119] Figure 10 is a graph comparing the effects of initial IE reperfusion temperatures on subsequent systolic functioning of hearts collected after one hour ("T1"), 3 hours ("T3") and 5 hours (" T5") of resuscitation and perfusion of the hearts with the mixture of blood-STEEN solution.

[00120] Figure 11 is a graph comparing the effects of initial IE reperfusion temperatures on the subsequent diastolic functioning of hearts collected after one hour ("T1"), 3 hours ("T3") and 5 hours ("T5" ) of resuscitation and perfusion of the hearts with the mixture of blood-STEEN solution.

[00121] The data collected in this study demonstrate that the initial reperfusion conditions, which last only 3 minutes, have a significant impact on the severity of post-harvest trauma and the functional recovery of hearts collected from porcine DCD donors. Example 2:

[00122] The second study evaluated the effects of reducing the concentration of Ca2+ ions in cardioplegic solutions to determine whether decreasing the levels of Ca2+ ions on the outside of myocytes would minimize the reverse-mode operation of the Na+/Ca2+ pump, thereby reducing accumulation of Ca2+ ions within myocytes. Consequently, this study evaluated the effects of 50 μmol/L, 220 μmol/L, 500 μmol/L, and 1250 μmol/L of Ca2+ ions in oxygenated cardioplegic sample solutions (Figure 12). The components of these sample solutions are shown in TABLE II. Sample solutions were also prepared at room temperature and their quoted pH values were measured at room temperature, just as for sample solutions in Example I, but with calcium chloride concentrations different from 0.05, 0.22, 0 .5 or 1.25 mmol/L respectively. All reperfusions in this example were performed at 35°C. TABLE II Sample II - Cardioplegic solutions (pH = 7.35)

[00123] Twenty-four pigs were separated into four groups and then euthanized following standard protocols and medical ethics procedures following the schematic flowchart shown in Figure 13. Immediately after the acquisition of each heart was completed, each heart was placed on a Quest MPS®2 Myocardial Protection System. The hearts collected from the first group of pigs were perfused for 3 minutes with an oxygenated cardioplegic sample composition containing 50 μmol/L of Ca2+ ions, which was heated to 35°C before starting the reperfusion process. The hearts collected from the second group of pigs were perfused for 3 minutes with the oxygenated cardioplegic composition of a sample containing 220 μmol/L of Ca2+ ions, which was heated to 35°C before starting the reperfusion process. The hearts collected from the third group of pigs were perfused for 3 minutes with the oxygenated cardioplegic composition of a sample containing 500 μmol/L of Ca2+ ions, which was heated to 35°C before starting the reperfusion process. The hearts collected from the fourth group of pigs were perfused for 3 minutes with the oxygenated cardioplegic sample composition containing 1,250 μmol/L of Ca2+ ions, which was heated to 35°C before starting the reperfusion process.

[00124] Aortic perfusion pressure, coronary artery flow and myocardial temperature were constantly monitored and recorded by the MPS®2 equipment during the initial 3-minute reperfusion period. Blood gas samples were measured at 0, 30, 60, 120 and 180 seconds of the initial reperfusion period to collect data regarding changes that occur in the partial pressure of O2 (PaO2), in the partial pressure of CO2 (PaCO2), in pH levels, in electrolyte levels, in lactate levels among others.

[00125] After the initial 3-minute reperfusion period was completed, each heart was removed from the Quest MPS®2 instrument and transferred into an ex vivo cardiac perfusion instrument (EVHP) where it was perfused with a supply of constant flow of a blood-STEEN solution mixture (Hb 45 g/L; XVIVO Perfusion Inc., Englewood, CO, USA) in which its systolic function was restored and maintained in a Langendorff mode at a normothermic temperature of 35 °C for one hour. Aortic pressure and heart rate were constantly monitored and processed using LABCHART® software. In one hour of perfusion with the blood-STEEN solution mixture in the EVHP equipment, each heart underwent a transition from Langendorff mode to a functioning mode, taking left atrial pressure from 0 to 8 mmHg and regulating the heart rhythm at 100 bpm . Cardiac output, coronary blood flow, aortic root and coronary sinus blood gases were measured, and cardiac function was assessed with a pressure-volume loop catheter. After these measurements were completed, each heart was immediately taken back to Langendorff mode.

[00126] Figure 14 shows that hearts initially reperfused at 35°C with oxygenated cardioplegic sample composition containing 220 μmol/L of Ca2+ ions developed significantly less myocardial edema than hearts that underwent reperfusion with oxygenated cardioplegic compositions containing one of the other three concentrations of Ca2+ ions.

[00127] Figure 15 shows that cardiac output (indexed for heart weight) of hearts that underwent reperfusion improved as the concentration of Ca2+ ions in oxygenated cardioplegic compositions was reduced from 1,250 μmol/L to 500 μmol/ L to 220 µmoles/L. However, the cardiac output of hearts that underwent reperfusion with an oxygenated cardioplegic composition containing 50 μmol/L of Ca2+ ions was very weak.

[00128] Figure 16 shows that the contraction capacity of the left ventricle (which is measured by dP/dt max) during systole in reperfused hearts improved as the concentration of Ca2+ ions in oxygenated cardioplegic compositions was reduced by 1,250 μmol/L up to 500 μmol/L to 220 μmol/L. However, the contracting capacity of the left ventricle in hearts that underwent reperfusion with the oxygenated cardioplegic composition containing 50 μmol/L of Ca2+ ions was very weak.

[00129] Figure 17 shows that the relaxation of the left ventricle (which is measured by dP/dt min) during diastole in hearts that underwent reperfusion improved as the concentration of Ca2+ ions in the oxygenated cardioplegic compositions was reduced by 1,250 μmol/ L up to 500 μmol/L to 220 μmol/L. However, the relaxation of the left ventricle in hearts that underwent reperfusion with the oxygenated cardioplegic composition containing 50 μmol/L of Ca2+ ions was very weak.

[00130] The data collected during this study demonstrate that hypocalcemic oxygenated cardioplegic compositions at 35°C significantly enhanced the functional recovery of the myocardium. The best performance in this study was with a Ca2+ ion concentration of 220 µmoles/L. However, it appears that reducing the Ca2+ ion concentration too low, for example to 50 μmol/L, can have detrimental effects, a phenomenon previously described as the "calcium paradox". Example 3:

[00131] The following study assessed whether there were potential additional benefits to acidification of a hypocalcemic oxygenated cardioplegic composition. Consequently, this study evaluated the effects of pH adjustment of hypocalcemic oxygenated cardioplegic sample compositions from 7.9 to 7.4, to 6.9 and to 6.4.

TABELA IIIB Amostra IIIB - Solução cardioplégica (pH = 7,35)

TABELA IIIC Amostra IIIC - Solução cardioplégica (pH = 6,9)

TABELA IIID Amostra IIID - Solução cardioplégica (pH = 6,4)

[00132] The components of these sample solutions IIIA through IIID are shown in TABLES IIIA through IIID respectively. TABLE IIIA Sample IIIA - Cardioplegic Solution (pH = 7.9)

TABLE IIIB Sample IIIB - Cardioplegic Solution (pH = 7.35)

TABLE IIIC Sample IIIC - Cardioplegic Solution (pH = 6.9)

TABLE IIID Sample IIID - Cardioplegic Solution (pH = 6.4)

[00133] The cardioplegic sample solutions contained 220 μmol/L of Ca2+ ions and all reperfusions were performed at 35°C (Figure 18).

[00134] Twenty-four pigs were separated into four groups and were then euthanized following standard protocols and medical ethics procedures following the schematic flowchart shown in Figure 19. Immediately after the acquisition of each heart was completed, each heart was placed on a Quest MPS®2 Myocardial Protection System. The hearts collected from the first group of pigs were perfused for 3 minutes with the hypocalcemic oxygenated cardioplegic sample composition with a pH of 7.9, which was heated to 35°C before starting the reperfusion process. The hearts collected from the second group of pigs were perfused for 3 minutes with a hypocalcemic oxygenated cardioplegic sample composition adjusted to a pH of 7.4, which was warmed to 35°C before starting the reperfusion process. The hearts collected from the third group of pigs were perfused for 3 minutes with a hypocalcemic oxygenated cardioplegic sample composition adjusted to a pH of 6.9, which was heated to 35°C before starting the reperfusion process. The hearts collected from the fourth group of pigs were perfused for 3 minutes with the hypocalcemic oxygenated cardioplegic sample composition adjusted to a pH of 6.4, which was warmed to 35°C before starting the reperfusion process.

[00135] Aortic perfusion pressure, coronary artery flow and myocardial temperature were constantly monitored and recorded by the MPS®2 equipment during the initial 3-minute reperfusion period. Blood gas samples were measured at 0, 30, 60, 120 and 180 seconds of the initial reperfusion period to collect data regarding changes that occur in the partial pressure of O2 (PaO2), in the partial pressure of CO2 (PaCO2 ), in the pH levels, in the levels of electrolytes, in the levels of lactate, among others.

[00136] After the initial 3-minute reperfusion period was completed, each heart was removed from the Quest MPS®2 instrument and transferred into an ex vivo cardiac perfusion instrument (EVHP) where it was perfused with a supply of constant flow of a blood-STEEN solution mixture (Hb 45 g/L; XVIVO Perfusion Inc., Englewood, CO, USA) in which its systolic function was restored and maintained in a Langendorff mode at a normothermic temperature of 35 °C for one hour. Aortic pressure and heart rate were constantly monitored and processed using LABCHART® software. After one hour of perfusion with the blood-STEEN solution mixture in the EVHP equipment, each heart underwent a transition from Langendorff mode to a functioning mode, taking left atrial pressure from 0 to 8 mmHg and regulating the heart rhythm at 100 bpm . Cardiac output, coronary blood flow, aortic root and coronary sinus blood gases were measured and cardiac function was assessed with a pressure-volume loop catheter. After these measurements were completed, each heart was immediately taken back to Langendorff mode.

[00137] Figure 20 shows that hearts initially reperfused at 35°C with hypocalcemic oxygenated cardioplegic compositions from a sample that was mildly acidic (ie, pH 6.4) exhibited more myocardial edema than those that underwent reperfusion. - perfusion with the more alkaline hypocalcemic oxygenated cardioplegic compositions (ie, pH 7.9, 7.4, 6.9).

[00138] Figure 21 shows the cardiac outputs (indexed for heart weight) of hearts that underwent reperfusion in a slightly acidic hypocalcemic oxygenated cardioplegic composition (ie, pH 6.9) and a slightly hypocalcemic oxygenated cardioplegic composition alkaline (ie, pH 7.4) were significantly better than cardiac outputs from hearts that underwent reperfusion in hypocalcemic oxygenated cardioplegic compositions adjusted to pH 7.9 or 6.4.

[00139] Figure 22 shows the left ventricular contraction capacity (which is measured by dP/dt max) during systole in reperfused hearts in a slightly acidic hypocalcemic oxygenated cardioplegic composition (ie, pH 6.9) and a slightly alkaline hypocalcemic oxygenated cardioplegic composition (ie, pH 7.4) was significantly better than the left ventricular contraction capacity in reperfused hearts in hypocalcemic oxygenated cardioplegic compositions adjusted to pH 7.9 or 6.4 .

[00140] Figure 23 shows that left ventricular relaxation (which is measured by dP/dt min) during diastole in reperfused hearts in a slightly acidic hypocalcemic oxygenated cardioplegic composition (ie, pH 6.9) and a Slightly alkaline hypocalcemic oxygenated cardioplegic composition (ie, pH 7.4) was significantly better than left ventricular relaxation in hearts that underwent reperfusion in hypocalcemic oxygenated cardioplegic compositions adjusted to pH 7.9 or 6.4.

[00141] The data collected during this study demonstrate that initial alkaline reperfusion is detrimental and that significant acidity (eg pH 6.4) is detrimental as well. However, it appears that mild acidosis (eg pH 6.6 to 6.9) is beneficial. Example 4:

[00142] Part 1: The following study assessed whether there were potential additional benefits to increasing the duration of reperfusion of donor hearts collected with a mildly acidic hypocalcemic oxygenated cardioplegic composition.

[00143] The sample solutions used for these tests were the same as the IIIC Sample Solutions described above.

[00144] Consequently, this study evaluated the effects of reperfusion for 3 minutes or 9 minutes with an oxygenated cardioplegic sample solution mildly acidic (pH 6.9) hypocalcemic (220 μmol/L Ca2+) at 35°C ( Figure 24). The cardioplegic solution for Part 1 of this study contained 400 μmol/L of adenosine and 500 μmol/L of lidocaine.

[00145] Twelve pigs were separated into two groups and then euthanized following standard protocols and medical ethics procedures following the schematic flowchart shown in Figure 25. Immediately after the acquisition of each heart was completed, each heart was placed in a Quest MPS®2 Myocardial Protection System. The hearts collected from the first group of pigs were perfused for 3 minutes with the hypocalcemic oxygenated cardioplegic composition of a mildly acidic sample heated to 35°C before starting the reperfusion process for 3 minutes. The hearts collected from the second group of pigs were perfused for 9 minutes with the hypocalcemic oxygenated cardioplegic composition of a mildly acidic sample that was heated to 35°C before starting the reperfusion process.

[00146] Aortic perfusion pressure, coronary artery flow and myocardial temperature were constantly monitored and recorded by the MPS®2 equipment during the initial reperfusion period of 3 minutes or 9 minutes. Blood gas samples were measured at 0, 30, 60, 120 and 180 seconds of the initial reperfusion period to collect data regarding changes that occur in the partial pressure of O2 (PaO2), in the partial pressure of CO2 (Pa - CO2), in pH levels, in electrolyte levels, in lactate levels, among others.

[00147] After the initial 3-minute reperfusion period or the initial 9-minute reperfusion period had been completed, each heart was removed from the Quest MPS®2 instrument and transferred into an ex vivo cardiac perfusion instrument (EVHP) where he was perfused with a constant flow supply of a blood-STEEN solution mixture (Hb 45 g/L; XVIVO Perfusion Inc., Englewood, CO, USA) in which his systolic function was restored and maintained in a Langendorff mode at a normothermic temperature of 35°C for one hour, 3 hours and 5 hours. Aortic pressure and heart rate were constantly monitored and processed using LABCHART® software. After one hour of perfusion with the blood-STEEN solution mixture in the EVHP equipment, each heart underwent a transition from Langendorff mode to a functioning mode, taking left atrial pressure from 0 to 8 mmHg and regulating the heart rhythm at 100 bpm . Cardiac output, coronary blood flow, aortic root and coronary sinus blood gases were measured and cardiac function was assessed with a pressure-volume loop catheter. After these measurements were completed, each heart was immediately taken back to Langendorff mode for an additional two hours, after which the measurements were repeated (ie, 3 hours after removal of reperfusion). After these measurements were completed, each heart was immediately taken back to Langendorff mode for an additional two hours, after which measurements were repeated (ie, 5 hours after removal of reperfusion).

[00148] Figure 26 shows that hearts that initially underwent reperfusion for 9 minutes with the hypocalcemic oxygenated cardioplegic composition of mildly acidic sample exhibited more myocardial edema than those that underwent reperfusion for only 3 minutes.

[00149] Figure 27 shows that hearts that initially underwent reperfusion for 9 minutes tended towards worsening function since ex vivo perfusion of the heart occurred from one hour to 3 hours to 5 hours.

[00150] These data suggest that the high concentration (500 μmoles/L) of lidocaine could be toxic.

[00151] Part 2: The following study evaluated the effects of reducing the concentration of lidocaine on the hypocalcemic oxygenated cardioplegic composition of a mildly acidic sample. Consequently, this study evaluated the effects of 3 minutes or 9 minutes of reperfusion with a mildly acidic (pH 6.9) hypocalcemic (220 μmol/L Ca2+) oxygenated cardioplegic sample solution at 35°C containing 400 μmol/L of adenosine and 50 μmol/L of lidocaine (Figure 28).

[00152] The components of this sample solution are shown in TABLE IV. TABLE IV Sample IV - Cardioplegic Solution (pH = 6.9)

[00153] Twelve pigs were separated into two groups and then euthanized following standard protocols and medical ethics procedures following the schematic flowchart shown in Figure 25. Immediately after the acquisition of each heart was completed, each heart was placed in a Quest MPS®2 Myocardial Protection System. The hearts collected from the first group of pigs were perfused for 3 minutes with the hypocalcemic oxygenated cardioplegic composition of a mildly acidic sample heated to 35°C before starting the reperfusion process for 3 minutes. The hearts collected from the second group of pigs were perfused for 9 minutes with the hypocalcemic oxygenated cardioplegic composition of a mildly acidic sample that was heated to 35°C before starting the reperfusion process.

[00154] Aortic perfusion pressure, coronary artery flow and myocardial temperature were constantly monitored and recorded by the MPS®2 equipment during the initial reperfusion period of 3 minutes or 9 minutes. Blood gas samples were measured at 0, 30, 60, 120 and 180 seconds of the initial reperfusion period to collect data regarding changes that occur in the partial pressure of O2 (PaO2), in the partial pressure of CO2 (PaCO2), in pH levels, in electrolyte levels, in lactate levels among others.

[00155] After the initial 3-minute reperfusion period or the initial 9-minute reperfusion period had been completed, each heart was removed from the Quest MPS®2 instrument and transferred into an ex vivo cardiac perfusion instrument (EVHP) where he was perfused with a constant flow supply of a blood-STEEN solution mixture (Hb 45 g/L; XVIVO Perfusion Inc., Englewood, CO, USA) in which his systolic function was restored and maintained in a Langendorff at a normothermic temperature of 35°C for one hour, 3 hours and 5 hours. Aortic pressure and heart rate were constantly monitored and processed using LABCHART® software. In one hour of perfusion with the blood-STEEN solution mixture in the EVHP equipment, each heart underwent a transition from Langendorff mode to a functioning mode, taking left atrial pressure from 0 to 8 mmHg and regulating the heart rhythm at 100 bpm . Cardiac output, coronary blood flow, aortic root and coronary sinus blood gases were measured, and cardiac function was assessed with a pressure-volume loop catheter. After these measurements were completed, each heart was immediately taken back to Langendorff mode for an additional two hours, after which the measurements were repeated (ie, 3 hours after removal of reperfusion). After these measurements were completed, each heart was immediately taken back to Langendorff mode for an additional two hours, after which measurements were repeated (ie, 5 hours after removal of reperfusion).

[00156] Figure 29 shows that there were no significant differences in myocardial edema occurring in hearts that initially underwent reperfusion for 9 minutes compared with hearts that were perfused for 3 minutes in the hypocalcemic oxygenated cardioplegic composition of a mildly acidic sample containing 400 μmol/ L of adenosine and 50 μmol/L of lidocaine.

[00157] Figure 30 shows that the extension of the initial reperfusion period from 3 minutes to 9 minutes in the hypocalcemic oxygenated cardioplegic composition of a mildly acidic sample containing 400 μmol/L of adenosine and 50 μmol/L of lidocaine, did not had detrimental effects on the functional recovery of hearts that were perfused for one hour, 3 hours and 5 hours after reperfusion.

[00158] Figure 31 combines myocardial functional data from Part 1 (Figure 27) and Part 2 (Figure 30), in which it is evident that the 500 μmol/L lidocaine concentration in the cardioplegic compositions used for reperfusion Initial ex vivo post-collection has debilitating effects on donor hearts. These data also demonstrate that extending the initial reperfusion period beyond 3 minutes is not beneficial for the restoration of homeostasis and cardiac function in collected donor hearts.

[00159] The data presented here indicate that a potentially efficient composition for a cardioplegic solution for initial reperfusion of donor hearts is shown in TABLE IV.

[00160] It will be understood that any range of values disclosed herein is intended to specifically include any intermediate value or sub-range within the range provided and all intermediate values and sub-ranges are individually and specifically disclosed.

[00161] It will also be understood that the word "a" or "an" is intended to mean "one or more" or "at least one" and any singular form is intended to include the plurals herein.

[00162] It will be further understood that the term "comprises", which includes any variation thereof, is intended to be unlimited and means "includes, but is not limited to" , unless specifically stated otherwise.

[00163] When a list of terms is provided here with an "or" before the last item, any one of the listed items or any suitable combination of two or more of the listed items can be selected and used.

[00164] Other modifications in the modalities described above are possible. The invention is therefore defined by the claims, which are to be given a broad interpretation consistent with the description as a whole.

Claims (20)

1. A solution comprising: a mixture of cardioplegia comprising a source of calcium ion and lidocaine; and a buffer for maintaining a pH of the solution, characterized in that the solution comprises 0.04 to 0.09 mmol/L of lidocaine and 0.18 to 0.26 mmol/L of calcium ion (Ca2+) and pH is less than 7.4 and greater than 6.6. 2. Solution according to claim 1, characterized in that the pH is from 6.8 to 7.0. 3. Solution according to claim 1, characterized in that the pH is 6.9. 4. Solution according to any one of claims 1 to 3, characterized in that it comprises: 0.3 to 0.45 mmol/L of adenosine; 0.04 to 0.09 mmol/L lidocaine; 8 to 12.5 mmoles/L of glucose; 120 to 130 mmoles/L of NaCl; 4 to 7 mmoles/L KCl; more than 16 mmoles/L and less than or equal to 24 mmoles/L of NaHCO3; 0.9 to 1.4 mmol/L NaH2PO4; 0.18 to 0.26 mmol/L of CaCl2; 11 to 15 mmoles/L of MgCl2; 7.5 to 12.5 IU/L of insulin; 100 to 140 mmoles/L of D-mannitol; 0.75 to 1.25 mmol/L of pyruvate; and 2.5 to 3.5 mmoles/L of reduced glutathione. 5. Solution according to any one of claims 1 to 3, characterized in that it comprises: 0.3 to 0.45 mmol/L of adenosine; 0.04 to 0.09 mmol/L lidocaine; 8 to 12.5 mmoles/L of glucose; 110 to less than 120 mmoles/L NaCl; 4 to 7 mmoles/L KCl; 16 to 24 mmoles/L of NaHCO3; 0.9 to 1.4 mmol/L NaH2PO4; 0.18 to 0.26 mmol/L of CaCl2; 11 to 15 mmoles/L of MgCl2; 7.5 to 12.5 IU/L of insulin; 100 to 140 mmoles/L of D-mannitol; 0.75 to 1.25 mmol/L of pyruvate; and 2.5 to 3.5 mmoles/L of reduced glutathione. 6. Solution according to claim 1, characterized in that it comprises: 0.3 to 0.45 mmol/L of adenosine; 0.04 to 0.09 mmol/L lidocaine; 8 to 12.5 mmoles/L of glucose; 120 to 140 mmoles/L of NaCl; 4 to 7 mmoles/L KCl; 12 to 16 mmoles/L of NaHCO3; 0.9 to 1.4 mmol/L NaH2PO4; 0.18 to 0.26 mmol/L of CaCl2; 11 to 15 mmoles/L of MgCl2; 7.5 to 12.5 IU/L of insulin; 100 to 140 mmoles/L of D-mannitol; 0.75 to 1.25 mmol/L of pyruvate; and 2.5 to 3.5 mmoles/L of reduced glutathione, where the pH is 6.6 to 6.9. 7. Solution according to any one of claims 1 to 3, characterized in that it comprises: 0.4 mmol/L of adenosine; 0.05 mmol/L lidocaine; 10. mmoles/L of glucose; 11.8 mmoles/L NaCl; 12. mmoles/L KCl; 20. mmoles/L of NaHCO3; 21. mmol/L of NaH2PO4; 0.22 mmol/L of CaCl2; 22. mmoles/L MgCl2; 23. IU/L of insulin; 120. mmoles/L of D-mannitol; 121. mmol/L of pyruvate; and 122. mmoles/L of reduced glutathione. 8. Solution according to any one of claims 1 to 7, characterized in that the solution is oxygenated. 9. Composition for preparing the solution as defined in any one of claims 1 to 8, characterized in that it comprises adenosine, lidocaine and a source of calcium, in which the molar ratio of adenosine:calcium is 0.3:0, 26 to 0.45:0.18 and the molar ratio of lidocaine:calcium is 0.04:0.26 to 0.09:0.18. 10. Composition according to claim 9, characterized in that the molar ratio of adenosine:calcium is 0.4:0.22 and the molar ratio of lidocaine:calcium is 0.05:0.22. 11. Composition according to any one of claims 9 to 10, characterized in that it further comprises chloride, glucose, insulin, D-mannitol, pyruvate and reduced glutathione. 12. Process characterized by the fact that it comprises the reperfusion of a donor heart with the solution as defined in any one of claims 1 to 8. 13. Process according to claim 12, characterized in that the heart is reperfused with the solution during the removal of the heart from a heart donor. 14. Process according to claim 12, characterized in that the heart is reperfused with the solution in a reperfusion device. 15. Process according to claim 14, characterized in that the heart is reperfused with the solution for at least 3 minutes immediately after removal of the heart from a heart donor. 16. Process according to any one of claims 12 to 15, characterized in that the heart is at a temperature above approximately 25°C and below approximately 37°C. 17. Use of the solution as defined in any one of claims 1 to 8, characterized in that it is for reperfusion of a donor's heart. 18. Use according to claim 17, characterized in that the solution is used for reperfusion of the heart during removal of the heart from a donor. 19. Use according to claim 17, characterized in that the solution is used for reperfusion of the heart for at least 3 minutes immediately after removal of the heart from a donor. 20. Use according to any one of claims 17 to 19, characterized in that the heart is at a temperature above approximately 25°C and below approximately 37°C.

Images