EP3209128A1 - Nouvelle composition et solution ayant un niveau d'ions calcium contrôlé, procédé associé et son utilisation en reperfusion - Google Patents

Nouvelle composition et solution ayant un niveau d'ions calcium contrôlé, procédé associé et son utilisation en reperfusion

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Publication number
EP3209128A1
EP3209128A1 EP15853016.2A EP15853016A EP3209128A1 EP 3209128 A1 EP3209128 A1 EP 3209128A1 EP 15853016 A EP15853016 A EP 15853016A EP 3209128 A1 EP3209128 A1 EP 3209128A1
Authority
EP
European Patent Office
Prior art keywords
mmol
heart
solution
reperfusion
calcium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP15853016.2A
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German (de)
English (en)
Other versions
EP3209128A4 (fr
Inventor
Christopher White
Darren FREED
Larry Hryshko
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Tevosol Inc
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Individual
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Publication date
Priority claimed from PCT/CA2015/050297 external-priority patent/WO2015154193A1/fr
Application filed by Individual filed Critical Individual
Publication of EP3209128A1 publication Critical patent/EP3209128A1/fr
Publication of EP3209128A4 publication Critical patent/EP3209128A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol

Definitions

  • the present invention relates to novel compositions and solutions suitable for reperfusion and also relates to post-harvest preservation and protection of harvested donor hearts prior to their resuscitation and transplantation into recipient subjects.
  • Heart failure affects 10% of North Americans and is the leading hospital discharge diagnosis.
  • the diagnosis of heart failure is accompanied by a survival outlook that is comparable to a major cancer. There are limited
  • Cardiac transplantation remains the gold-standard therapeutic intervention for patients with end-stage heart failure, with an increasing number of individuals being added to the transplant waiting list every year.
  • wider application of this life-preserving intervention is limited by the availability of donors.
  • Data from the International Society of Heart and Lung Transplantation Registry shows that cardiac transplantation is in progressive decline in suitable donors (2007, Overall Heart and Adult Heart Transplantation Statistics). Two hundred and fifty eight Canadians have died during the last decade (2000 - 2010; Heart and Stroke Foundation of Canada) while waiting for heart transplantation. Similarly, in the United States, 304 patients died in 2010 alone while waiting for heart transplantation (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 it is being experienced across the globe.
  • Time is of the essence for removal of a heart from a donor and its successful transplantation into a recipient.
  • the following conventional principles generally apply for optimal donor heart preservation for the period of time between removal from the donor and transplantation: (i) minimization of cell swelling and edema, (ii) prevention of intracellular acidosis, (iii) prevention of injury caused by oxygen free radicals, and (iv) provision of substrate for regeneration of high- energy phosphate compounds, particularly adenosine triphosphate (ATP), during reperfusion.
  • ATP adenosine triphosphate
  • Such a patient is referred to as a "brainstem-dead” donor or a donor after brain death (“DBD”).
  • DBD brain death
  • Such a patient is referred to as a “non-heart- beating” donor, a “cardiac dead” donor, a donor after cardiac death, or a donor after circulatory death (DCD).
  • Brainstem-dead donors can be maintained under artificial respiration for extended periods of time to provide hemodynamic stability throughout their bodies until the point of organ retrieval. Cardiac perfusion is uncompromised and organ functionality is theoretically maintained. However, brainstem death itself can profoundly affect cardiac function. The humoral response to brainstem death is characterized by a marked rise 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
  • hypothermic apparatus, systems and methods have been recognized by those skilled in these arts, and alternative apparatus, systems and methods have been developed for preservation and maintenance of harvested organs at temperatures in the range of about 25 °C to about 35 °C (this can be referred to as "normothermic" temperatures, though normothermic more conventionally means a normal body temperature, i.e., an average of about 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 potentiate viability and cellular function;
  • pyruvate and adenosine for oxidative phosphorylation support transferrin as an attachment factor; insulin and sugars for metabolic support; glutathione to scavenge toxic free radicals as well as a source of impermeant; cyclodextrin as a source of impermeant, scavenger, and potentiator of cell attachment and growth factors; a high Mg 2+ concentration for microvessel metabolism support; mucopolysaccharides for growth factor potentiation and hemostasis; and endothelial growth factors.
  • Viaspan comprises potassium lactobionate, KH 2 PO 4 , MgS0 4 , 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). While harvested kidneys and livers can be maintained beyond twelve hours in normothermic systems, normothermic bathing and maintenance of harvested hearts by perfusion beyond 12 hours results in deterioration and irreversible debilitation of the hearts' functionality. Another disadvantage of using
  • normothermic, continuous-pulsed-perfusion systems for maintenance of harvested hearts is the time required to excise a heart from a donor, mount it into the normothermic perfusion system and then initiate and stabilize the perfusion process.
  • the excised donor heart After the excised donor heart has been stabilized, its physiological functionality is determined and, if transplantation criteria are met, the excised heart is transported as quickly as possible to a transplant facility.
  • the heart In the case of brainstem-dead donors, the heart generally is warm and beating when it is procured. It is then stopped, cooled, and put on ice until it is transplanted. Chilling the harvested heart reduces its metabolic activity and related demands by about 95%. However, some metabolic activity continues with the consequence that the heart muscle begins to die, and clinical data have shown that once the period of chilling of a harvested heart is prolonged beyond 4 hours, the risk of 1-year mortality post-transplant starts to rise.
  • risk of death at 1-year post-transplant for a recipient receiving a heart that has been preserved by chilling for six hours more than doubles compared to a recipient receiving a heart that has been chilled for less than 1 hour (Taylor et al., 2009, Registry of the International Society for Heart and Lung Transplantation: Twenty-sixth Official Adult Heart
  • Non-heart-beating donors have minimal brain function but do not meet the criteria for brainstem death, and therefore such donors cannot be legally declared brainstem dead. When it is clear that there is no hope for meaningful recovery of the patient, the physicians and family must be in
  • organs of non-heart-beating donors are necessarily exposed to variable periods of warm ischemia after cardiac arrest, which may result in varying degrees of organ damage.
  • duration of warm ischemia is not excessive, many types of organs, such as kidneys, livers, and lungs, can be harvested from non-heart-beating donors and are able to recover function after transplantation with success rates that approximate those for transplanted organs from brainstem-dead donors.
  • the present disclosure includes a novel solution comprising a preservation mixture comprising a calcium ion source; and a buffer for maintaining a pH of the solution, wherein the molar concentration of calcium ion (Ca 2+ ) in the solution is from 0.18 to 0.26 mmol/L, and the pH is lower than 7.4 and higher than 6.6.
  • the molar concentration of calcium ion (Ca 2+ ) may be 0.22 mmol/L.
  • the pH may be from 6.8 to 7.0, such as 6.9.
  • the preservation mixture may be a cardioplegia mixture comprising adenosine, lidocaine, and a magnesium ion source.
  • the solution may comprise 0.3 to 0.45 mmol/L of adenosine, 0.04 to 0.09 mmol/L of lidocaine, and 1 1 to 15 mmol/L of Mg 2+ .
  • the solution may comprise a sodium ion source and a potassium ion source.
  • the solution may comprise about 130 to about 160 mmol/L of Na + and 4 to 7 mmol/L of K + .
  • the solution may comprise chloride, an osmotic buffer and a reducing agent.
  • the solution may comprise 70 to 140 or 70 to 180 mmol/L of chloride, 8 to 12.5 mmol/L of glucose, 7.5 to 12.5 IU/L of insulin, 100 to 140 mmol/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 of adenosine; 0.04 to 0.09 mmol/L of lidocaine; 8 to 12.5 mmol/L of glucose; 110 to 130 mmol/L of NaCI; 4 to 7 mmol/L of KCI; 16 to 24 mmol/L of NaHC0 3 ; 0.9 to 1.4 mmol/L of NaH 2 P0 ; 0.18 to 0.26 mmol/L of CaCI 2 ; 1 1 to 15 mmol/L of MgCI 2 ; 7.5 to 12.5 IU/L of insulin; 100 to 140 mmol/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 0.4 mmol/L of adenosine; 0.05 mmol/L of lidocaine; 10 mmol/L of glucose; 123.8 mmol/L of NaCI; 5.9 mmol/L of KCI; 20 mmol/L of NaHC0 3 ; 1.2 mmol/L of NaH 2 P0 4 ; 0.22 mmol/L of CaCI 2 ; 13 mmol/L of MgCI 2 ; 10 IU/L of insulin; 120 mmol/L of D-mannitol; 1 mmol/L of pyruvate; and 3 mmol/L of reduced glutathione.
  • compositions for preparing the solution described in the preceding paragraph may comprise adenosine, lidocaine, and a calcium source, wherein the molar ratio of adenosine:calcium is from
  • the composition may further comprise a sodium source, a potassium source and a magnesium source, wherein the molar ratio of calcium:sodium is from 0.26:130 to 0.18:160, the molar ratio of
  • calcium:potassium is from 0.26:4 to 0.18 to 7, and the molar ratio of
  • calcium:magnesium is from 0.26:1 1 to 0.18:15.
  • the molar ratio of calcium:sodium may be 0.22:147, the molar ratio of calcium:potassium may be 0.22:5.9, and the molar ratio of calcium:magnesium may be 0.22:13.
  • the composition may also comprise chloride, glucose, insulin, D-mannitol, pyruvate, and reduced glutathione.
  • the solution as described herein may be used to reperfuse a donor heart and the present disclosure includes a method of reperfusion of a donor heart and use of the solution described herein for reperfusion of a donor heart.
  • the heart may be reperfused with the solution during removal of the heart from the donor.
  • the heart after removal from the donor may be reperfused in a reperfusion device.
  • the heart may be reperfused with the solution for at least 3 minutes immediately after removal of the heart from the donor.
  • the donor may be a donor after circulatory death.
  • the reperfusion may be at a temperature above about 25 °C and below about 37 °C.
  • the reperfusion may be at a temperature of about 35 °C during reperfusion.
  • selected embodiments of the present disclosure relate to solutions for immersion and bathing of a harvested heart while being concurrently flowed through the heart and its vasculature.
  • Some embodiments of the present disclosure pertain to use of solutions for ex vivo maintenance of harvested hearts to reduce and ameliorate post-harvest ischemic damage.
  • Some embodiments of the present disclosure pertain to methods for ex vivo maintenance of harvested hearts to minimize the occurrence and extent of post-harvest ischemic damage.
  • FIG. 1 is a schematic flowchart outlining the experimental protocols used in Example 1 ;
  • Fig. 2 is a chart showing the myocardial temperature achieved in harvested pig hearts after an initial 3-minute reperfusion period
  • Fig. 3 is a chart showing the effect of reperfusate temperature on the coronary blood flow through harvested pig hearts, measured after the initial 3- minute reperfusion period;
  • Fig. 4 is a chart showing the effect of reperfusate temperature on coronary vascular resistance to blood flow through harvested pig hearts, measured after the initial 3-minute reperfusion period;
  • Fig. 5 is a chart showing the effect of reperfusate temperature on coronary sinus lactate washout from harvested pig hearts, measured after the initial 3-minute reperfusion period;
  • Fig. 6 is a chart showing the effect of reperfusate temperature on the accumulation of Troponin I (a marker of myocardial injury) in the perfusate solution, measured 5 hours after harvest of the pig hearts;
  • Fig. 8 is a chart presenting the average extent of injury to endothelial cells and myocytes in harvested pig hearts, as observed in electron-microscopy micrographs and scored with a scoring system, as a function of reperfusion temperature;
  • Fig. 9 is a chart showing the effect of reperfusate temperature on the cardiac index of harvested pig hearts, measured 1 hour (“T1 "), 3 hours (“T3"), and 5 hours (“T5") after harvest of the pig hearts;
  • Fig. 10 is a chart showing the effect of reperfusate temperature on the systolic function of harvested pig hearts, measured 1 hour ("TV), 3 hours (“T3"), and 5 hours (“T5")after harvest of the pig hearts;
  • Fig. 11 is a chart showing the effect of reperfusate temperature on the diastolic function of harvested pig hearts, measured 1 hour (“T1 "), 3 hours (“T3"), and 5 hours (“T5") after harvest of the pig hearts;
  • Fig. 12 is a schematic chart outlining the temperatures and Ca 2+ ion concentrations of the cardioplegic solutions used in Example 2;
  • FIG. 13 is a schematic flowchart outlining the experimental protocols used in Example 2.
  • Fig. 14 is a chart showing the effect of Ca 2+ ion concentration in the reperfusate on weight gain in harvested pig hearts measured 1 hour after harvest;
  • Fig. 15 is a chart showing the effect of Ca 2+ ion concentration in the reperfusate on the cardiac output of harvested pig hearts measured 1 hour after harvest;
  • Fig. 16 is a chart showing the effect of Ca 2+ ion concentration on the contractility of the left ventricle during systole in harvested pig hearts, measured 1 hour after harvest;
  • Fig. 17 is a chart showing the effect of Ca 2+ ion concentration on relaxation of the left ventricle during diastole in harvested pig hearts, measured 1 hour after harvest;
  • Fig. 18 is a schematic chart outlining the temperatures, Ca 2+ ion concentrations, and pH values of the cardioplegic solutions used in Example 3;
  • Fig. 19 is a schematic flowchart outlining the experimental protocols used in Example 3.
  • Fig. 20 is a chart showing the effect of pH of the cardioplegic reperfusate solution on weight gain in harvested pig hearts, measured 1 hour after harvest;
  • Fig. 21 is a chart showing the effect of pH of the cardioplegic reperfusate solution on the cardiac output of harvested pig hearts, measured 1 hour after harvest;
  • Fig. 22 is a chart showing the effect of pH of the cardioplegic reperfusate solution on the contractility of the left ventricle during systole in harvested pig hearts, measured 1 hour after harvest;
  • Fig. 23 is a chart showing the effect of pH of the cardioplegic reperfusate solution on relaxation of the left ventricle during diastole in harvested pig hearts, measured 1 hour after harvest;
  • Fig. 24 is a schematic chart outlining the temperatures, Ca 2+ ion concentrations, and pH values of the cardioplegic reperfusate solutions, and the duration of reperfusion times used in Example 4;
  • Fig. 25 is a schematic flowchart outlining the experimental protocols used in Example 4, Part 1 ;
  • Fig. 26 is a chart showing the effect of duration of initial reperfusion on weight gain in harvested pig hearts
  • Fig. 27 is a chart showing the effects of duration of initial reperfusion on myocardial function of harvested pig hearts, measured 1 hour ("T1 "), 3 hours (“T3"), and 5 hours (“T5") after harvest;
  • Fig. 28 is a schematic flowchart outlining the experimental protocols used in Example 4, Part 2;
  • Fig. 29 is a chart showing the effect of an extended initial reperfusion with a cardioplegic reperfusate solution having a reduced concentration of anesthetic on weight gain in harvested pig hearts;
  • Fig. 30 is a chart showing the effect of extended initial reperfusion with a cardioplegic reperfusate solution having a reduced concentration of anesthetic on myocardial function of harvested pig hearts, measured 1 hour ("T1 "), 3 hours (“T3"), and 5 hours (“T5") after harvest; and
  • Fig. 31 is a chart showing the effect of anesthetic concentrations in cardioplegic reperfusate solutions on myocardial function of harvested pig hearts, measured 1 hour ("T1 "), 3 hours (“T3”), and 5 hours (“T5") after harvest.
  • Afterload means the mean tension produced by a chamber of the heart in order to contract. It can also be considered as the 'load' that the heart must eject blood against. Afterload is therefore a consequence of aortic large vessel compliance, wave reflection and small vessel resistance (left ventricular afterload) or similar pulmonary artery parameters (right ventricular afterload).
  • preload refers to the stretching of a single cardiac myocyte immediately prior to contraction and is therefore related to the sarcomere length. Since sarcomere length cannot be determined in the intact heart, other indices of preload such as ventricular end diastolic volume or pressure are used. As an example, preload increases when venous return is increased.
  • cardiac myocyte means a cardiac muscle cell.
  • stroke volume 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).
  • EDV end diastolic volume
  • ESV EDV - ESV.
  • the stroke volume is affected by changes in preload, afterload and inotropy (contractility). In normal hearts, the SV is not strongly influenced by afterload whereas in failing hearts, the SV is highly sensitive to afterload changes.
  • stroke work 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 enclosed by the pressure/volume loop is a measure of the ventricular stroke work, which is a product of the stroke volume and the mean aortic or pulmonary artery pressure (afterload), depending on whether one is considering the left or the right ventricle.
  • EF ejection fraction
  • EF SV/EDV. Healthy ventricles typically have ejection fractions greater than 0.55. Low EF usually indicates systolic dysfunction and severe heart failure can result in EF lower than 0.2.
  • EF is also used as a clinical indicator of the inotropy (contractility) of the heart. Increasing inotropy leads to an increase in EF, while decreasing inotropy decreases EF.
  • end systolic pressure volume relationship (ESPVR) describes the maximal pressure that can be developed by the left ventricle at any given left ventricular volume, or alternatively, by the right ventricle at any given right ventricular volume. This implies that the PV loop cannot cross over the line defining ESPVR for any given contractile state.
  • the slope of ESPVR (Ees) represents the end-systolic elastance, which provides an index of myocardial contractility.
  • the ESPVR is relatively insensitive to changes in preload, afterload and heart rate. This makes it an improved index of systolic function over other hemodynamic parameters like ejection fraction, cardiac output and stroke volume.
  • PRSW preload recruitable stroke work relationship
  • PVA pressure-volume area
  • dP/dt max is a measure of the global contractility of the left ventricle. The greater the contractile force exerted during systole, the greater the rate of increase in left ventricular pressure.
  • dP/dt min is a measure of the relaxation of the left ventricle during diastole.
  • DCD means donor after circulatory death, or donor after cardiac death.
  • DBD means donor after brain death.
  • the term "Langendorff perfusion” refers to a method of perfusing an excised heart with a nutrient-rich oxygenated solution in a reverse fashion via the aorta.
  • the backwards pressure causes the aortic valve to shut, thereby forcing the solution into the coronary vessels that supply the heart tissue with blood. This transports nutrients and oxygen to the cardiac muscle, allowing it to continue beating for several hours after its removal from the animal.
  • working heart refers to clinical ex vivo coronary perfusion throughout an excised heart by ventricular filling via the left atrium and ejection from the left ventricle via the aorta driven by the heart's contractile function and regular cardiac rhythm.
  • the excised heart is attached by cannulae to a perfusate reservoir and circulatory pumps in a Langendorff preparation.
  • the flow of perfusate through the excised heart in "working heart” mode is in the direction opposite to the flow of perfusate during Langendorff perfusion.
  • ischemia means a condition that occurs when blood flow and oxygen are kept from the heart.
  • the term "reperfusion” as used herein means passing a solution through a heart to re-establish supply of oxygen and provide protective or preservation materials to the heart, such as by pumping the solution through the heart in a perfusion device, and optionally immersing the heart in the solution.
  • the heart may be immersed in an oxygen-rich perfusate solution, which may be the same as the reperfusion solution or may be a different solution.
  • reperfusion injury refers to tissue damage in a harvested heart that occurs when a supply of oxygen via a perfusate solution is provided to the tissue after a period of ischemia or lack of oxygen. Depriving the heart of sufficient oxygen and nutrients during the ischemic period creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress, rather than restoration of normal function.
  • cardioplegia means an intentional and temporary cessation of, or maintenance of ceased or reduced, cardiac activities, such as by arresting or stopping the beating of the heart, for the purpose of preserving the health of the myocardium, including through a period of significantly reduced provision of oxygen and metabolic substrate. Cardioplegia can be imposed on a beating heart by chilling or by administration of a solution containing one or more chemicals that will cause paralysis of the heart muscle, or by both concurrently. In embodiments of the present disclosure, cardioplegia may also be achieved by providing limited oxygen and other supplies to the myocardium to preserve its health without fully restoring the cardiac activities of the heart.
  • cardioplegic solution as used herein means a solution containing chemical components that cause or maintain asystole (paralysis) of the heart in a mixture with components to preserve or protect heart cell functions.
  • homeostasis means the maintenance of a fairly stable metabolic equilibrium within and between the muscle cells of a harvested heart.
  • Normal serum potassium ion levels in human blood are in a range between 3.5 mEq/L and 5.0 mEq/L.
  • hypokalemic means having or
  • a hyperkalemic concentration includes any potassium ion concentration in excess of 6.0 mEq/L.
  • hypomic means a temperature that is less than about 20 °C.
  • Ischemia is accompanied by significant changes in ion-exchange patterns into and out of heart muscle cells as a consequence, primarily, of the loss of oxygen supply. As the availability of oxygen decreases and stops, the
  • Ischemic damage occurring during the procurement of a donor heart may be reduced by reperfusion of the harvested heart as soon as possible after its harvest in blood or a blood replacement product, as exemplified by Viaspan and CELSIOR ® (CELSIOR is a registered trademark of Genzyme Corp., Cambridge, Massachusetts, U.S.A.).
  • Reperfusion causes a prompt increase in the extracellular pH, which results in robust excretion of H + ions into the extracellular space.
  • H + ion movement into the extracellular space drives Na + ions into the cells.
  • intracellular pH levels results in the generation of reactive oxygen species that activate subcellular signals that in turn activate inflammatory cascades leading to apoptosis and cytokine release. Additionally, reactive oxygen species directly disrupt DNA structures and protein structures, thereby causing cell death.
  • Another problem associated with conventional reperfusion techniques is that it is very difficult in these techniques to modulate the intracellular levels of Ca 2+ ions during the reperfusion process, where reperfusion further increases the intracellular overload of Ca 2+ ions in heart muscle cells.
  • Contraction of a heart while the heart muscle cells are overloaded with intracellular Ca ions during reperfusion inevitably results in a disruptive type of necrosis, termed contraction band necrosis, as a result of massive myofibril contraction. Contraction band necrosis is considered to be the most severe form of reperfusion injury.
  • the rationale for chilling donor hearts immediately after their procurement and during reperfusion is to reduce metabolic activity within the heart muscle cells as quickly as possible to minimize the generation of reactive oxygen species during reperfusion and to minimize a subsequent intracellular overload of Ca 2+ ions during reperfusion.
  • the strategy comprises two components wherein the first component is an oxygenated cardioplegic composition for use as reperfusate solution during procurement of a harvested heart and for a period of time immediately after harvest during which the harvested heart is reperfused, preferably, for at least 3 minutes.
  • the reperfusate solution causes an immediate cessation of a donor heart's rhythmic beating upon reperfusion.
  • the at-least-3-minute reperfusion period starting immediately after the heart is harvested, is referred to as the immediate - early (“IE") period.
  • the second component of our strategy is to avoid chilling the heart during procurement process and during the post-harvest reperfusion period, and instead maintain normothermic conditions during harvest, during IE reperfusion, and during subsequent ex vivo maintenance of the harvested heart.
  • cardioplegic composition for reperfusion for example of a DCD heart for transplant, at temperatures from about 25 to about 37 °C, a number of factors may need to be considered.
  • a balanced approach in view of these factors may be required.
  • a source of potential complication is that the intracellular concentrations of a particular ion, especially the intracellular concentration of Ca 2+ or H + ions, which if not properly controlled could contribute to myocardial injury, can be sensitive to the extracellular concentrations of these ions as well as other ions.
  • the intracellular concentration of Ca 2+ in myocytes is expected to be affected not only by the extracellular concentration of Ca 2+ , but also, as a result of particular ion exchanges in the plasma membrane, by extracellular concentrations of other ions, such as H + and Na + .
  • the intracellular calcium ion concentration may be adjusted by changing extracellular concentration of one or more of Ca 2+ , Na + and H + .
  • changing the extracellular concentrations of H + and Na + may result in other changes which can affect other aspects of myocardial injury, in addition to optimizing intracellular Ca 2+ .
  • a solution for use as a reperfusion solution may include the following components:
  • a preservation mixture which may include adenosine to provide
  • concentration of Mg 2+ may also be included, as hypermagnesemia is also expected to assist in prevention of myocyte contraction during reperfusion.
  • the mixture may contain 0.3 to 0.45 mmol/L of adenosine, 0.04 to 0.09 mmol/L of lidocaine and 1 1 to 15 mmol/L of Mg 2+ .
  • a normakalemic concentration such as, 4 to 7 mmol/L.
  • the CI " concentration may be higher, such as up to about 180 mmol/L in the solution, it may be beneficial in some embodiments to have a lower CI " concentration such as for example, from 70 to 140 mmol/L, or up to about 140 mmol/L.
  • the pH-buffer may be provided by, for example, a combination of 16 to 24 mmol/L of HCO 3 1" and 0.9 to 1.4 mmol/L of H 2 P0 4 1" .
  • Substrates for energy metabolism such as a combination of 8 to 12.5 mmol/L of glucose and 0.75 to 1.25 mmol/L of pyruvate.
  • An osmotic agent in a concentration for obtaining an appropriate osmolarity such as, 100 to 140 mmol/L of D-mannitol.
  • An antioxidant or reducing agent in a concentration for obtaining an appropriate degree of protection from reactive oxygen species and physiological levels of reduction such as, 2.5 to 3.5 mmol/L of reduced glutathione.
  • one or more growth factors such as, 7.5 to 12.5 IU/L of insulin.
  • a pre-prepared cardioplegic composition may be titrated to the desired pH prior to use, such that the composition at the desired
  • a cardioplegic composition for causing an immediate cessation of a donor heart's rhythmic beating upon its contact with the cardioplegic composition may comprise an adenosine-lidocaine mixture, a normokalemic concentration of potassium ions, a concentration of Ca 2+ ions selected to maintain the intracellular level of Ca 2+ ions in the harvested heart's muscle cells at about 10 "4 mmol/L, and a pH of 6.9.
  • a suitable adenosine-lidocaine mixture may comprise 300 Mmol/L, 325
  • the cardioplegic composition may additionally comprise 8.012.5 mmol/L of glucose, 120-140 mmol/L of NaCI, 4.0-7.0 mmol/L of
  • a cardioplegic composition may include 400 pmol/L of adenosine, 50 Mmol/L of lidocaine, 10.0 mmol/L of glucose,
  • the cardioplegic composition may be oxygenated by bubbling a stream of 02 gas through the cardioplegic composition prior to and during its use for bathing and reperfusing a harvested donor.
  • Another selected embodiment of the present disclosure pertains to use of the selected oxygenated cardioplegic composition to reperfuse a harvested heart at a temperature of about 35 °C. Accordingly, the selected oxygenated cardioplegic composition is warmed to about 35 °C before contacting the heart during procurement and subsequent IE reperfusion for at least 3 minutes after procurement has been completed.
  • the harvested heart may be resuscitated by installation into a suitable apparatus for ex vivo maintenance of a functioning systolic harvested heart, by interconnection of conduit infrastructures provided within the apparatus with the hearts aorta, pulmonary artery, pulmonary vein, and vena cava, and bathing the excised heart in a constantly flowing perfusate solution comprising oxygenated blood and/or an oxygenated blood replacement solution. Additionally the constantly flowing perfusion solution is flowed through the heart's chambers while it is maintained in the apparatus.
  • Such apparatus are generally configured with the following: (i) a perfusate pumping system;(ii) flow sensors for monitoring the flow of perfusate to and from the installed heart s aorta, pulmonary artery, pulmonary vein, and vena cava; (iii) an ECG apparatus interconnectable with the excised heart; (v) probes interconnecting the installed heart with instruments for monitoring the excised heart's physiological functionality using load independent indices and load dependent indices; and optionally (vi) pacemakers for initiating or maintaining systolic function of the heart.
  • an example oxygenated cardioplegic composition disclosed herein to reperfuse a heart removed from a donor for transplant may provide a harvested heart with the ionic complement necessary for the ex Vo-maintained heart to continue generating ATP and pumping excess calcium out of the heart muscles cells while keeping the heart in a paralyzed condition i.e., a non-beating asystolic condition, thereby minimizing the potential for occurrence of contraction band necrosis.
  • cardioplegic composition for reperfusion of harvested hearts at temperatures from about 25 to about 35 °C can facilitate rapid restoration of calcium ion homeostasis and facilitate more rapid recovery and functional operation of the harvested heart after transplantation into a recipient subject.
  • such a solution may include a cardioplegia mixture.
  • the mixture contains a calcium ion source and a buffer for maintaining a pH of the solution.
  • the molar concentration of calcium ion (Ca 2+ ) in the solution is from 0.18 to 0.26 mmol/L and the pH is lower than 7.4 and higher than 6.6.
  • the molar concentration of calcium ion (Ca 2+ ) in the solution may be 0.22 mmol/L.
  • the pH may be from 6.8 to 7.0, such as 6.9.
  • the cardioplegia mixture 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 1 1 to 15 mmol/L of Mg 2+ .
  • the solution may also include a sodium ion source and a potassium ion source, such as about 130 to about 160 mmol/L of Na + and 4 to 7 mmol/L of K + .
  • the solution may further include chloride, an osmotic buffer and an antioxidant or reducing agent.
  • suitable osmotic buffers may include D-manitol, lactobionate, dextran, albumin, or the like.
  • Suitable antioxidants may include reduced glutathione, resveratrol, apelin analogs or the like.
  • the solution may contain, for example, 70 to 140 mmol/L chloride, 100 to 140 mmol/L of D- mannitol, and 2.5 to 3.5 mmol/L of reduced glutathione.
  • the solution may contain substrates for energy metabolism, such as one or more of glucose, pyruvate, free fatty acids (e.g. oleate or palmitate), triglycerides, or the like.
  • 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.
  • growth factors such as insulin, cardiotrophin-1 , erythropoietin, platelet-derived growth factors (PDGF), various forms of fibroblast growth factors (FGF), or the like.
  • the solution may contain 7.5 to 12.5 IU/L of insulin.
  • the solution may contain 0.3 to 0.45 mmol/L of adenosine; 0.04 to 0.09 mmol/L of lidocaine; 8 to 12.5 mmol/L of glucose; 110 to 130 mmol/L of NaCI; 4 to 7 mmol/L of KCI; 16 to 24 mmol/L of NaHC0 3 ; 0.9 to 1.4 mmol/L of NaH 2 P0 4 ; 0.18 to 0.26 mmol/L of CaCI 2 ;1 1 to 15 mmol/L of MgCI 2 ; 7.5 to 12.5 IU/L of insulin; 100 to 140 mmol/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 contain 0.4 mmol/L of adenosine; 0.05 mmol/L of lidocaine; 10 mmol/L of glucose; 123.8 mmol/L of NaCI; 5.9 mmol/L of KCI; 20 mmol/L of NaHC0 3 ; 1.2 mmol/L of NaH 2 PO ; 0.22 mmol/L of CaCI 2 ; 13 mmol/L of MgCI 2 ; 10 IU/L of insulin; 120 mmol/L of D-mannitol; 1 mmol/L of pyruvate; and 3 mmol/L of reduced glutathione.
  • a solution for reperfusion of an excised heart may include a cardioplegia mixture containing an anesthetic agent for paralyzing the heart and preventing myocyte contraction during reperfusion; and agents for protecting or restoring cardiac functions of the heart, the agents comprising a calcium source, a sodium source, and a potassium source, in amounts selected to restore and maintain calcium ion homeostasis in the heart at a temperature from about 25 to about 35 °C.
  • the solution may be at a temperature from about 25 to about 35 °C, such as about 35 °C.
  • a solution disclosed herein may be prepared and stored before use, or the solution may be prepared just before use by mixing pre-packaged compositions or materials, or by adding a solvent such as water or a buffer solution to a pre-formulation to form the desired solution.
  • a composition for preparing a reperfusion solution may include a mixture of adenosine, lidocaine, and a calcium source.
  • the molar ratio of adenosine:calcium may be from 0.3:0.26 to 0.45:0.18, such as 0.4:0.22, and the molar ratio of lidocaine alcium may be from 0.04:0.26 to 0.09:0. 8, such as 0.05:0.22.
  • the composition may also contain a sodium source, a potassium source and a magnesium source.
  • the molar ratio of calcium:sodium may be from 0.26: 130 to 0.18:160, such as 0.22:147.
  • the molar ratio of calcium:potassium may be from 0.26:4 to 0.18 to 7, such as 0.22:5.9.
  • calcium:magnesium may be from 0.26:1 1 to 0.18:15, such as 0.22:13.
  • the composition may also contain chloride, and one or more of glucose, insulin, D- mannitol, pyruvate, and reduced glutathione.
  • the composition may be mixed with a suitable pH buffer to prepare the desired reperfusion solution, such as a selected reperfusion solution described herein.
  • FIG. 1 For example, in a method for reperfusion of a heart for transplant, the heart may be reperfused with a reperfusion solution disclosed herein in a reperfusion device.
  • the reperfusion device may be similar to a conventional perfusion device and may be operated similarly except replacing the perfusion solution with a reperfusion solution described herein.
  • the Quest MPS®2 Myocardial Protection System provided by Quest Medical Inc., Allen, TX, USA, may be used as the reperfusion device.
  • a volume infusion pump may also be used to pump the reperfusion solution.
  • An infuser such as one that is typically used by a trauma patient, or a similar infuser, may be used for
  • BelmontTM rapid infuser RI-2 may be used in the reperfusion device.
  • the heart may be reperfused with the reperfusion solution for at least 3 minutes immediately after removal of the heart from the donor of the heart.
  • the donor may be a DCD donor, and the DCD heart may be maintained at a
  • FIG. 10 Further embodiments are related to methods of maintaining a heart for transplant.
  • the heart may be treated to maintain calcium ion homeostasis in the heart at a temperature from about 25 °C to about 37 °C, such as by use of a suitable solution or composition disclosed herein.
  • embodiments of solutions disclosed herein may be used for reperfusion of a donor heart, during removal of the heart, or immediately after removal of the heart from the donor, or both. Further, the solution may also be used as perfusion solution at other times or for other purposes as may be appropriate.
  • the heart may be removed from a donor after circulatory death (DCD) at a temperature from about 25 to 37 °C.
  • DCD circulatory death
  • a solution as described herein may also be used for reperfusion of other types of hearts such as a heart removed from a donor after brain death (DBD).
  • the solution may also be used at lower temperatures.
  • sample cardioplegic solutions used in these Examples were prepared at room temperature and their stated pH was measured at room temperature.
  • the lidocaine and D-mannitol solutions used to prepare the sample solutions were obtained from commercial sources,
  • sample solutions were prepared by adding the component ingredients to water.
  • the water was double-deionized and sterilized as known to those skilled in the art.
  • the sample solutions were oxygenated before use.
  • each heart was installed into a Quest MPS®2 Myocardial Protection System (MPS is a registered trademark of Quest Medical Inc., Allen, TX, USA) for precise control of the reperfusion pressure and temperature.
  • MPS is a registered trademark of Quest Medical Inc., Allen, TX, USA
  • the harvested hearts from first group of pigs were perfused for 3 minutes with a sample oxygenated cardioplegic composition (see TABLE I) that was chilled to 5 °C prior to commencing the reperfusion process.
  • the cardioplegic composition was initially prepared at room temperature and the pH of the composition was measured at room temperature.
  • the aortic perfusion pressure, coronary artery flow, and myocardial temperature were constantly monitored and recorded by the MPS ® 2 apparatus during the 3- minute initial reperfusion period.
  • each heart was removed from the Quest MPS ® 2 apparatus and transferred into an ex vivo heart perfusion (EVHP) apparatus where it was perfused with a constantly flowing supply of a blood-STEEN solution mixture (Hb 45 g/L; XVIVO Perfusion Inc., Englewood, CO, USA) wherein its systolic function was restored and maintained in a Langendorff mode at a normothermic temperature of 35 °C for 6 hours.
  • the aortic pressure and heart rate were constantly monitored and processed using the LABCHART ® software (LABCHART is a registered trademark of ADInstruments Pty. Ltd., Bella Vista, NSW, Australia).
  • each heart was transitioned from the Langendorff mode to a working mode by bringing the left atrial pressure from 0 to 8 mmHg and pacing the heart at 100 beats per minutes ("bpm").
  • bpm beats per minutes
  • 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 returned to the Langendorff mode.
  • composition as shown in TABLE I, which had been warmed to 35 °C prior to commencing the reperfusion process.
  • the data in Fig. 2 show that the myocardial temperatures recorded in the hearts receiving the IE reperfusion treatment with the sample oxygenated cardioplegic composition chilled to 5 °C dropped to about 10 °C by the end of the 3-minute IE reperfusion period.
  • the myocardial temperatures recorded in the hearts that received IE reperfusion with the sample oxygenated cardioplegic composition cooled to 25 °C were about 25 °C, while the myocardial temperatures recorded in the hearts that received reperfusion with the selected oxygenated cardioplegic composition were about 35 °C.
  • Fig. 3 shows that rates of coronary blood flow were reduced by about 15% in hearts that were reperfused with the sample oxygenated cardioplegic composition cooled to 25 °C compared to coronary blood flow in hearts that received reperfusion with the sample oxygenated cardioplegic composition.
  • Fig. 4 shows that the coronary vascular resistance in hearts reperfused with the cooled oxygenated cardioplegic composition dropped by about
  • Fig. 5 shows that the coronary sinus lactate dropped by more than 50% in hearts that received the chilled IE reperfusion treatment, and by about 25% in hearts that received the cooled IE reperfusion treatment, when compared to the coronary sinus lactate levels in the hearts receiving the normothermic IE reperfusion treatment.
  • Fig. 6 shows that levels of Troponin I (a marker for myocardial injury) increased as the temperature of the IE reperfusion temperature decreased, relative to the levels observed in hearts receiving the normothermic IE reperfusion treatment.
  • Fig. 7(A) is an electron micrograph showing a swollen endothelial cell in a capillary of a heart that received the chilled IE reperfusion treatment for 3 minutes
  • Fig. 7(B) is an electron micrograph showing a typical normal- appearing endothelial cell in a capillary of a heart that received the normothermic IE reperfusion treatment for 3 minutes.
  • Fig. 8 is a chart comparing the scores of endothelial injury and myocyte injury from hearts receiving chilled IE reperfusion for three minutes and from hearts receiving normothermic IE reperfusion for three minutes.
  • Fig. 9 is a chart showing the effects on cardiac indices of IE reperfusion with a cooled oxygenated cardioplegic composition and with a chilled oxygenated cardioplegic composition, with the effects of IP perfusion with a normothermic oxygenated cardioplegic composition.
  • Fig. 10 is a chart comparing the effects of the initial IE reperfusion temperatures on the subsequent systolic functioning of harvested hearts after 1 hour (“T1 "), 3 hours (“T3"), and 5 hours (“T5") of resuscitation and perfusion of the hearts with the blood-STEEN solution mixture.
  • Fig. 1 1 is a chart comparing the effects of the initial IE reperfusion temperatures on the subsequent diastolic functioning of harvested after 1 hour (“T1 "), 3 hours (“T3"), and 5 hours (“T5") of resuscitation and perfusion of the hearts with the blood-STEEN solution mixture.
  • the second study assessed the effects of reducing the Ca 2+ ion concentration in cardioplegic solutions to determine if lowering the Ca 2+ ion levels on the outside of myocytes would minimize the reverse mode functioning of the Na + /Ca 2+ pump thereby reducing the accumulation of Ca 2+ ions within the myocytes. Accordingly, this study assessed the effects of 50 pmol/L, 220 pmol/L, 500 pmol/L, and 1250 ⁇ /L of Ca 2+ ions in sample oxygenated cardioplegic solutions (Fig. 12). The components of these sample solutions are shown in TABLE II.
  • sample solutions were also prepared at room temperature and their stated pH values were measured at room temperature, as for sample solutions in Example I but with different calcium chloride concentrations at 0.05, 0.22, 0.5, or 1.25 mmol/L respectively. All reperfusions in this example were done at 35 °C.
  • pigs Twenty four pigs were separated into four groups and then euthanized following standard protocols and medical ethics procedures following the schematic flowchart shown in Fig. 13. Immediately after procurement of each heart was completed, each heart was installed into a Quest MPS ® 2 Myocardial Protection System. The harvested hearts from the first group of pigs were perfused for 3 minutes with a sample oxygenated cardioplegic composition containing 50 ⁇ / ⁇ _ Ca 2+ ions, which was warmed to 35 °C prior to commencing the reperfusion process.
  • the harvested hearts from the second group of pigs were perfused for 3 minutes with the sample oxygenated cardioplegic composition containing 220 ⁇ / ⁇ _ Ca 2+ ions, which was warmed to 35 °C prior to commencing the reperfusion process.
  • the harvested hearts from the third group of pigs were perfused for 3 minutes with the sample oxygenated cardioplegic composition containing 500 mol/L Ca 2+ ions, which was warmed to 35 °C prior to commencing the reperfusion process.
  • the harvested hearts from the fourth group of pigs were perfused for 3 minutes with the sample oxygenated cardioplegic composition containing 1 ,250 pmol/L Ca 2+ ions, which was warmed to 35 °C prior to commencing the reperfusion process.
  • the aortic perfusion pressure, coronary artery flow, and myocardial temperature were constantly monitored and recorded by the MPS ® 2 apparatus during the 3-minute initial reperfusion period. Blood gas samples were measured at 0, 30, 60, 120, and 80 seconds of the initial reperfusion period to collect data pertaining to changes occurring the partial pressure of O 2 (PaO 2 ), partial pressure of CO 2 (PaCOa), pH levels, electrolyte levels, lactate levels among others.
  • PaO 2 partial pressure of O 2
  • PaCOa partial pressure of CO 2
  • pH levels pH levels
  • electrolyte levels lactate levels among others.
  • each heart was transitioned from the Langendorff mode to a working mode by bringing the left atrial pressure from 0 to 8 mmHg and pacing the heart 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
  • Fig. 14 shows that the hearts initially reperfused at 35 °C with the sample oxygenated cardioplegic composition containing 220 pmol/L Ca 2+ ions developed significantly less myocardial edema than the hearts reperfused with oxygenated cardioplegic compositions containing one of the other three Ca 2+ ion concentrations.
  • Fig. 15 shows that the cardiac output (indexed for heart weight) of reperfused hearts improved as the Ca 2+ ion concentration in the oxygenated cardioplegic compositions was reduced from 1 ,250 ⁇ /L to 500 ⁇ /L to 220 ⁇ /L.
  • the cardiac output of hearts reperfused with an oxygenated cardioplegic composition containing 50 mol/L Ca 2+ ions was very poor.
  • Fig. 16 shows that the contractility of the left ventricle (as measured by dP/dt max) during systole in reperfused hearts improved as the Ca 2+ ion concentration in the oxygenated cardioplegic compositions was reduced from 1 ,250 ⁇ /L to 500 ⁇ /L to 220 ⁇ /L.
  • contractility of the left ventricle in hearts reperfused with the oxygenated cardioplegic composition containing 50 ⁇ /L Ca 2+ ions was very poor.
  • Fig. 17 shows that the relaxation of the left ventricle (as measured by dP/dt min) during diastole in reperfused hearts improved as the Ca 2+ ion concentration in the oxygenated cardioplegic compositions was reduced from 1 ,250 ⁇ / ⁇ _ to 500 ⁇ / ⁇ _ to 220 ⁇ /L.
  • relaxation of the left ventricle in hearts reperfused with the oxygenated cardioplegic composition containing 50 ⁇ / ⁇ _ Ca 2+ ions was very poor.
  • this study assessed the effects of adjusting the pH of sample hypocalcemic oxygenated cardioplegic compositions from 7.9 to 7.4, to 6.9, and to 6.4.
  • each heart was installed into a Quest MPS ® 2 Myocardial Protection System.
  • the harvested hearts from the first group of pigs were perfused for 3 minutes with the sample hypocalcemic oxygenated cardioplegic composition with a pH of 7.9, which was warmed to 35 °C prior to commencing the reperfusion process.
  • the harvested hearts from the second group of pigs were perfused for 3 minutes with a sample hypocalcemic oxygenated cardioplegic composition adjusted to a pH of 7.4, which was warmed to 35 °C prior to
  • the harvested hearts from the third group of pigs were perfused for 3 minutes with a sample hypocalcemic oxygenated cardioplegic composition adjusted to a pH of 6.9, which was warmed to 35 °C prior to commencing the reperfusion process.
  • the harvested hearts from the fourth group of pigs were perfused for 3 minutes with the sample hypocalcemic oxygenated cardioplegic composition adjusted to a pH of 6.4, which was warmed to 35 °C prior to commencing the reperfusion process.
  • the aortic perfusion pressure, coronary artery flow, and myocardial temperature were constantly monitored and recorded by the MPS ® 2 apparatus during the 3-minute initial reperfusion period. Blood gas samples were measured at 0, 30, 60, 120, and 180 seconds of the initial reperfusion period to collect data pertaining to changes occurring the partial pressure of 0 2 (Pa0 2 ), partial pressure of CO 2 (PaCO 2 ), pH levels, electrolyte levels, lactate levels among others.
  • each heart was removed from the Quest MPS ® 2 apparatus and transferred into an ex vivo heart perfusion (EVHP) apparatus where it was perfused with a constantly flowing supply of a blood-STEEN solution mixture (Hb 45 g/L; XVIVO Perfusion Inc., Englewood, CO, USA) wherein its systolic function was restored and maintained in a Langendorff mode at a normothermic temperature of 35 °C for 1 hour.
  • the aortic pressure and heart rate were constantly monitored and processed using the LABCHART ® software.
  • each heart was transitioned from the Langendorff mode to a working mode by bringing the left atrial pressure from 0 to 8 mmHg and pacing the heart 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 returned to the Langendorff mode.
  • Fig. 20 shows that the hearts initially reperfused at 35 °C with the sample hypocalcemic oxygenated cardioplegic compositions that was mildly acidic(i.e., pH 6.4) exhibited more myocardial edema than those that were reperfused with the more alkaline (i.e., pH of 7.9, 7.4, 6.9) hypocalcemic oxygenated cardioplegic compositions.
  • Fig. 21 shows that the cardiac outputs (indexed for heart weight) of reperfused hearts in a slightly acidic hypocalcemic oxygenated cardioplegic composition (i.e., pH 6.9) and a slightly alkaline hypocalcemic oxygenated cardioplegic composition (i.e., pH 7.4) were significantly better that the cardiac outputs of hearts reperfused in hypocalcemic oxygenated cardioplegic
  • compositions adjusted to pH 7.9 or 6.4 are adjusted to pH 7.9 or 6.4.
  • Fig. 22 shows that the contractility of the left ventricle (as measured by dP/dt max) during systole in reperfused hearts in a slightly acidic hypocalcemic oxygenated cardioplegic composition (i.e., pH 6.9) and a slightly alkaline hypocalcemic oxygenated cardioplegic composition (i.e., pH 7.4) were significantly better than the left ventricle contractility in hearts reperfused in hypocalcemic oxygenated cardioplegic compositions adjusted to pH 7.9 or 6.4.
  • a slightly acidic hypocalcemic oxygenated cardioplegic composition i.e., pH 6.9
  • a slightly alkaline hypocalcemic oxygenated cardioplegic composition i.e., pH 7.4
  • Fig. 23 shows that the relaxation of the left ventricle (as measured by dP/dt min) during diastole in reperfused hearts in a slightly acidic hypocalcemic oxygenated cardioplegic composition (i.e., pH 6.9) and a slightly alkaline
  • hypocalcemic oxygenated cardioplegic composition i.e., pH 7.4
  • hypocalcemic oxygenated cardioplegic compositions adjusted to pH 7.9 or 6.4.
  • Part 1 The next study assessed if there were potential incremental benefits to increasing the duration of reperfusion of harvested donor hearts with a mildly acidic hypocalcemic oxygenated cardioplegic composition.
  • this study assessed the effects of 3 minutes or 9 minutes reperfusion with a sample mildly acidic (pH 6.9) hypocalcemic (220 mol/L Ca 2+ ) oxygenated cardioplegic solution at 35 °C (Fig. 24).
  • the cardioplegic solution for Part 1 of this study contained 400 ⁇ /L adenosine and 500 ⁇ /L lidocaine.
  • the harvested hearts from the first group of pigs were perfused for 3 minutes with the sample mildly acidic hypocalcemic oxygenated cardioplegic composition warmed to 35 °C prior to commencing the reperfusion process for 3 minutes.
  • the harvested hearts from the second group of pigs were perfused for 9 minutes with the sample mildly acidic hypocalcemic oxygenated cardioplegic composition that was warmed to 35 °C prior to commencing the reperfusion process.
  • the aortic perfusion pressure, coronary artery flow, and myocardial temperature were constantly monitored and recorded by the MPS ® 2 apparatus during the 3-minute or 9-minute initial reperfusion period. Blood gas samples were measured at 0, 30, 60, 120, and 180 seconds of the initial reperfusion period to collect data pertaining to changes occurring the partial pressure of O 2 (Pa0 2 ), partial pressure of C0 2 (PaC0 2 ), pH levels, electrolyte levels, lactate levels among others.
  • each heart was removed from the Quest MPS ® 2 apparatus and transferred into an ex vivo heart perfusion (EVHP) apparatus where it was perfused with a constantly flowing supply of a blood-STEEN solution mixture (Hb 45 g/L; XVIVO Perfusion Inc., Englewood, CO, USA) wherein its systolic function was restored and maintained in a Langendorff mode at a normothermic temperature of 35 °C for 1 hour, 3 hours, and 5 hours.
  • aortic pressure and heart rate were constantly monitored and processed using the LABCHART ® software.
  • each heart was transitioned from the Langendorff mode to a working mode by bringing the left atrial pressure from 0 to 8 mmHg and pacing the heart 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 returned to the Langendorff mode for an additional 2 hours, after which the measurements were repeated (i.e., 3 hours after removal from reperfusion). After these measurements were completed, each heart was immediately returned to the Langendorff mode for an additional 2 hours, after which the measurements were repeated (i.e., 5 hours after removal from reperfusion).
  • Fig. 26 shows that the hearts initially reperfused for 9 minutes with the sample mildly acidic hypocalcemic oxygenated cardioplegic composition exhibited more myocardial edema than those that were reperfused for only 3 minutes.
  • Fig. 27 shows that the hearts initially reperfused for 9 minutes trended toward worsening function as ex vivo heart perfusion proceeded from 1 hour to 3 hours to 5 hours.
  • Part 2 The next study assessed the effects of reducing the lidocaine concentration in the sample mildly acidic hypocalcemic oxygenated cardioplegic composition. Accordingly, this study assessed the effects of 3 minutes or 9 minutes of reperfusion with a sample mildly acidic (pH 6.9) hypocalcemic (220 pmol/L Ca 2+ ) oxygenated cardioplegic solution at 35 °C containing 400 pmol/L adenosine and 50 pmol/L lidocaine (Fig. 28).
  • pigs were separated into two groups and then euthanized following standard protocols and medical ethics procedures following the schematic flowchart shown in Fig. 25.
  • each heart was installed into a Quest MPS ® 2 Myocardial Protection System.
  • the harvested hearts from the first group of pigs were perfused for 3 minutes with the sample mildly acidic hypocalcemic oxygenated cardioplegic composition warmed to 35 °C prior to commencing the reperfusion process for 3 minutes.
  • the harvested hearts from the second group of pigs were perfused for 9 minutes with the sample mildly acidic hypocalcemic oxygenated cardioplegic composition that was warmed to 35 °C prior to commencing the reperfusion process.
  • the aortic perfusion pressure, coronary artery flow, and myocardial temperature were constantly monitored and recorded by the MPS ® 2 apparatus during the 3-minute or 9-minute initial reperfusion period. Blood gas samples were measured at 0, 30, 60, 120, and 180 seconds of the initial reperfusion period to collect data pertaining to changes occurring the partial pressure of 0 2 (Pa0 2 ), partial pressure of CO 2 (PaC0 2 ), pH levels, electrolyte levels, lactate levels among others.
  • each heart was removed from the Quest MPS ® 2 apparatus and transferred into an ex vivo heart perfusion (EVHP) apparatus where it was perfused with a constantly flowing supply of a blood- STEEN solution mixture (Hb 45 g/L; XVIVO Perfusion Inc., Englewood, CO, USA) wherein its systolic function was restored and maintained in a Langendorff mode at a normothermic temperature of 35 °C for 1 hour, 3 hours, and 5 hours.
  • the aortic pressure and heart rate were constantly monitored and processed using the LABCHART ® software.
  • each heart was transitioned from the Langendorff mode to a working mode by bringing the left atrial pressure from 0 to 8 mmHg and pacing the heart 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 returned to the Langendorff mode for an additional 2 hours, after which the measurements were repeated (i.e., 3 hours after removal from reperfusion). After these measurements were completed, each heart was immediately returned to the Langendorff mode for an additional 2 hours, after which the measurements were repeated (i.e., 5 hours after removal from reperfusion).
  • Fig. 29 shows that there were not any significant differences in myocardial edema occurring in the hearts initially reperfused for 9 minutes compared with hearts perfused for 3 minutes in the sample mildly acidic hypocalcemic oxygenated cardioplegic composition containing 400 pmol/L adenosine and 50 ⁇ /L lidocaine.
  • Fig. 30 shows that prolonging the initial reperfusion period from 3 minutes to 9 minutes in the sample mildly acidic hypocalcemic oxygenated cardioplegic composition containing 400 ⁇ /L adenosine and 50 ⁇ /L lidocaine, did not have detrimental effects on the functional recovery of hearts perfused for 1 hour, 3 hours, and 5 hours after reperfusion.
  • Fig. 31 combines myocardial functional data from Part 1 (Fig. 27) and Part 2 (Fig. 30), wherein it is apparent that the 500 pmol/L concentration of lidocaine in the cardioplegic compositions used for initial ex vivo post-harvest reperfusion has debilitating effects of donor hearts. This data also demonstrates that prolonging the initial reperfusion period beyond 3 minutes is not beneficial for restoration of homeostasis and cardiac function in harvested donor hearts.

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Abstract

Cette invention concerne une solution comprenant un mélange de conservation comprenant une source d'ions calcium ; et un tampon pour maintenir le pH de la solution. La concentration molaire en ions calcium (Ca2+) dans la solution est de 0,18 à 0,26 mmol/L, et son pH est inférieur à 7,4 et supérieur à 6,6. Une composition pour la préparation de la solution peut comprendre de l'adénosine, de la lidocaïne, et une source de calcium, le rapport molaire adénosine:calcium étant de 0,3:0,26 à 0,45:0,18, et le rapport molaire lidocaïne:calcium étant de 0,04: 0,26 à 0,09:0.18. Un cœur de donneur peut être reperfusé à l'aide de cette solution. La solution peut être utilisée pour la reperfusion d'un cœur de donneur, p. ex. à une température d'environ 25 à environ 37°C, ledit donneur pouvant être un donneur après mort circulatoire.
EP15853016.2A 2014-10-24 2015-10-23 Nouvelle composition et solution ayant un niveau d'ions calcium contrôlé, procédé associé et son utilisation en reperfusion Withdrawn EP3209128A4 (fr)

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PCT/CA2015/051084 WO2016061700A1 (fr) 2014-10-24 2015-10-23 Nouvelle composition et solution ayant un niveau d'ions calcium contrôlé, procédé associé et son utilisation en reperfusion

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BR112017008076B1 (pt) 2021-06-29
CN107105640B (zh) 2021-10-12
AU2015336862A1 (en) 2017-06-01
BR112017008076A2 (pt) 2017-12-26
CA2965400A1 (fr) 2016-04-28
EP3209128A4 (fr) 2018-04-11
CN113841687A (zh) 2021-12-28
CA2965400C (fr) 2023-03-14
AU2015336862B2 (en) 2018-08-09
WO2016061700A1 (fr) 2016-04-28

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