Compositions and method for tissue preservation   

Abstract: Methods and compositions for resuscitating, storing, and preserving functional integrity of organs and tissues. Metabolic function is maintained by sustaining ATP levels, mitochondrial function, cardiomyocyte contractility, prevention of acidosis, inhibition of induction of apoptosis, maintaining ionontrophy and lusiotrophy by regulating calcium, sodium, potassium and chloride ions.

This application is a continuation of U.S. application Ser. No. 12/527,119, filed Aug. 13, 2009, which claims priority to International Application No. PCT/US2008/002170, filed Feb. 19, 2008, and which claims priority to U.S. Provisional Application No. 60/901,844 filed Feb. 17, 2007, U.S. Provisional Application No. 60/902,587, filed Feb. 20, 2007, and U.S. Provisional Application No. 60/966,511, filed Aug. 27, 2007, each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to tissue preservation.

BACKGROUND OF THE INVENTION

The major obstacles in cardiac transplantation are the limited availability of donor hearts and the poor quality of donor hearts due to deterioration during storage. Using current practices and preservation solutions, the limit of preservation time is 4-6 hours, and in the United States, all cardiac allografts are presently obtained from brain-dead, beating heart donors maintained on life support systems. Moreover, current practices result in a significant incidence of accelerated vasculopathy in transplanted hearts. As such, there is a pressing need for long-term storage solutions that preserve the structural and physiological integrity of donor hearts.

SUMMARY

The invention provides a solution for preserving a human or human-compatible harvested organ in need of preservation or resuscitation during a preservation or evaluation period prior to implantation, including transplantation or reimplantation. The solution of the invention also allows the organ to be transported to alternate geographic locations during the preservation period. The invention provides improved compositions, methods, and devices for organ storage, which preserve the functional integrity of the organ as well as restore the function to a non-functioning or deteriorated organ. Increasing the use of non-beating heart (NBH) donors and storage of donor hearts for a longer period of time would increase the size of the donor pool substantially and allow transport of donor hearts over longer distances to increase availability to recipients. Transplantation of hearts with intact, functioning coronary endothelium as a result of storage in the improved solution minimizes vasculopathy that occurs after cardiac transplantation using current technology.

The invention provides for compositions for preserving or resuscitating a biological tissue. The composition contains a physiological salt solution and at least one, at least two, at least three, at least four, or at least five of the following compositions: a substrate for the production of adenosine tri phosphate (ATP), a substrate for the consumption of ammonia, a reagent that buffers intracellular acidity, a reagent that quenches reactive oxygen species, and/or a reagent that balances tissue edema/dehydration.

In one aspect, the composition contains a physiological salt solution and a substrate for the production of ATP. Optionally, the substrate for the production of ATP is phosphocreatine, creatine ethyl ester, dicreatine malate, creatine gluconate, fructose, sucrose, ribose, hexose or pentose. Alternatively, the substrate for the production of ATP is creatine orotate, creatine monohydrate, adenosine, or dextrose/glucose.

The composition for preserving or resuscitating a biological tissue contains a physiological salt solution and a compound for the consumption of ammonia. Optionally, the compound for the consumption of ammonia is ornithine or carbomyl phosphate. Alternatively, the compound for the consumption of ammonia is L-citrulline malate.

In another aspect, the composition for preserving or resuscitating a biological tissue contains a physiological salt solution and a reagent that buffers intracellular acidity. In one aspect, the reagent that buffers intracellular acidity is Histidine, Glutamine, Tryptophan, Lysine, or Taurine. Alternatively, the reagent that buffers intracellular acidity is sodium bicarbonate, THAM, or L-carnosine.

Optionally, the composition for preserving or resuscitating a biological tissue contains a physiological salt and a reagent that quenches reactive oxygen species. In one aspect, the reagent that quenches reactive oxygen species is dithiothreitol (DTT), beta-Mercaptoethanol, Acetylcysteine, Alpha lipoic acid, Taurine, Reserveratrol, Lutein, Selenium, Methionine, or Tocopherols/Vitamin E.

In yet another aspect, the composition for preserving or resuscitating a biological tissue contains a physiological salt and a reagent that balances tissue water content (edema/dehydration). Reagents that balance tissue water content include Mannitol, urea, glycerine, isosorbide, or raffinose pentahydrate. Optionally, the reagent that balances tissue water content is the penta fraction of raffinose pentahydrate.

The solution is used to resuscitate a living donor heart, a temporally stored living donor heart for transplantation, as well as cadaveric donor heart and permits extended temporal storage of living, stored, or cadaveric organs such as the heart. Cardiomyocyte function in living donor and cadaveric hearts are preserved as well as endothelial function in coronary vasculature and chambers of the living donor and the cadaveric heart. The solution also mediates reversal of metabolic and degenerative changes and inhibition of cell death and progression to cell viability in the hearts (and other organs) when the organ is contacted with and stored in the solution of the invention shortly after death. Metabolic function is maintained by sustaining ATP levels, mitochondrial function, cardiomyocyte contractility, prevention of acidosis, inhibition of induction of apoptosis. Ionontrophy and lusiotrophy are maintained by regulating calcium, sodium, potassium and chloride ions. Buffering capacity of the solution prevent acidosis. The solution preserves calcium mobilization, nitric oxide generation in the organ as well as maintains both endothelium-dependent and independent vasomotor function in the coronary vasculature. Dehydration and subsequent over-hydration (edema) is prevented upon reperfusion by manipulating ionic concentrations and aquaporin channels, and ischemia-reperfusion injury is prevented. The solution and storage system is a self-sustaining regenerative system for production of substrates for ATP and nitric oxide.

The compositions prevent ischemia-reperfusion injury. This function is mediated by ascorbic acid and glutathione, carnitine (by preventing accumulation of long chain acyl-CoA that leads to generation of free radicals-ischemi-reperfusion injury), carnosine and alpha lipoic acid which are free radical, (hydroxyl radical, singlet oxygen, peroxyl radical and superoxide) scavenger. The composition contains calcium chloride, potassium chloride, potassium phosphate, magnesium sulfate, sodium bicarbonate, D-glucose, adenosine, glutathione, insulin, and a reagent to prevent dehydration and/or edema of an organ and/or tissue. The solution also contains other salts such as magnesium chloride, sodium chloride, and/or sodium phosphate dibasic.

Dehydration of the heart (during storage) and over-hydration or edema after reperfusion is controlled by manipulating sodium (less) and potassium ions (more) (i.e., osmolarity). The solution is slightly hypotonic and hyposmolar, hence driving the water into the heart during storage and balancing out during reperfusion. The increase in external KCl concentration counterbalances the K current (close to Nerst potential) and prevents potassium from moving out along with water (shell of hydration of K ion). Dehydration/edema is also controlled by manipulating the aquaporin channels via the ionic currents. Also, external K may delay/decrease the movement of Ca ions into the cell, thus preventing dehydration via Ca activated K channel For example, the solution contains 125 mM sodium and 7 mM potassium. In one aspect, the composition contains calcium chloride, potassium chloride, potassium phosphate, magnesium sulfate, sodium bicarbonate, D-glucose, adenosine, glutathione, insulin, and a substrate for the production of ATP. For example, the substrate for the production of ATP is creatine orotate, creatine monohydrate, adenosine, or dextrose/glucose. Dichloroacetate increases ATP production by inhibiting the kinase enzyme that phosphorylates PDH enzyme making it inactive. Dichloroacetate induces ATP synthesis by facilitating the TCA cycle.

In the citrulline malate-arginine cycle, malate (cleaved from citrulline) enters the TCA cycle to generate more ATP. Also, citrulline malate is converted to arginine and fumarate; fumarate enters the TCA cycle to facilitate more ATP production. Both malate and fumarate in TCA cycle leads to more ATP production.

The composition contains calcium chloride, potassium chloride, potassium phosphate, magnesium sulfate, sodium bicarbonate, D-glucose, adenosine, glutathione, insulin, and a reagent that buffers acidity. A reagent that buffers intracellular acidity is creatine orotate via facilitated synthesis of carnosine. Creatine monohydrate buffers acidity by increasing energy production and decreased lactate accumulaton. Acidity is also buffered by sodium bicarbonate, Tris-hydroxymethyl aminomethane (THAM), and L-carnosine (intracellular acidity). Dichloroacetate controls acidity by lowering lactate levels in the preserved organ, and thus the solution. L-carnitine facilitates a decrease in myocardial lactate production, hence reducing acidity.

The composition contains calcium chloride, potassium chloride, potassium phosphate, magnesium sulfate, sodium bicarbonate, D-glucose, adenosine, glutathione, insulin, and a substrate for the consumption of ammonia. For example, the substrate for the consumption of ammonia is L-citrulline malate Ammonia combines with carbamoyl phosphate to form citrulline malate, which forms a substrate for nitric oxide and ATP.

The composition contains calcium chloride, potassium chloride, potassium phosphate, magnesium sulfate, sodium bicarbonate, D-glucose, adenosine, glutathione, insulin, a reagent to prevent dehydration and/or edema of an organ and/or tissue, a substrate for the production of ATP, a reagent that buffers acidity, and a substrate for the consumption of ammonia. Preferably, the composition includes the following compounds and concentrations:

about 0.147 g/L calcium chloride (1 mM)

about 0.52 g/L potassium chloride (7 mM)

about 0.06 g/L potassium phosphate (monobasic) (0.44 mM)

about 0.11 g/L magnesium chloride (hexahydrate) (0.50 mM)

about 0.125 g/L magnesium sulfate (heptahydrate) (0.50 mM)

about 7.30 g/L sodium chloride (125 mM)

about 0.35 g/L sodium bicarbonate (4.2 mM)

about 0.05 g/L sodium phosphate (dibasic; heptahydrate) (0.19 mM)

about 1.98 g/L dextrose/D-glucose (11 mM)

about 0.27 g/L adenosine (1 mM)

about 0.46 g/L glutathione (reduced; 1.5 mM)

about 0.18 g/L ascorbic acid (1 mM)

about 0.21 g/L L-arginine (1 mM)

about 0.27 g/L creatine orotate (0.5 mM)

about 0.30 g/L creatine monohydrate (2 mM)

about 0.15 g/L L-citrulline malate (1 mM)

about 0.08 g/L dichloroacetate (0.5 mM)

about 2.3 g/L-carnosine (10 mM)

about 2.0 g/L-carnitine (10 mM)

about 0.021 g/LAlpha-Lipoic acid (0.1 mM)

about 0.50 ml/L insulin (10 mg/ml)

THAM (Tris-Hydroxymethyl Aminomethane; 100 mM stock @12.1 gm/L) to adjust

pH (about 1 L distilled water).

The ingredients are mixed together to form a solution. Insulin is optionally added after the other ingredients are mixed. For example, insulin is added minutes, e.g., 0.5, 1, 2, 5, minutes to hours, e.g., 0.5, 1, 2, 3, 4, or 5 hours prior to immersing an organ in the solution.

A preferred composition includes amounts of the compounds in the following ranges to achieve a desired ratio of compositions:

0.1-0.5 g/L calcium chloride

0.25-0.75 g/L potassium chloride (7 mM)

0.01-0.5 g/L potassium phosphate (monobasic)

0.01-0.5 g/L magnesium chloride (hexahydrate)

0.01-0.5 g/L magnesium sulfate (heptahydrate)

5.0-10.0 g/L sodium chloride (125 mM)

0.01-0.5 g/L sodium bicarbonate

0.01-0.5 g/L sodium phosphate (dibasic; heptahydrate)

1.0-5.0 g/L D-dextrose/glucose (11 mM)

0.01-0.5 g/L adenosine (1 mM)

0.01-0.75 g/L glutathione (reduced; 1.5 mM)

0.01-0.5 g/L ascorbic acid (1 mM)

0.01-0.5 g/L L-arginine (1 mM)

0.01-0.5 g/L creatine orotate (0.5 mM).

0.01-0.5 g/L creatine monohydrate (2 mM)

0.01-0.5 g/L L-citrulline malate (1 mM)

0.01-0.5 g/L dichloroacetate (0.5 mM)

0.23-2.3 g/L-carnosine (1-10 mM)

0.20-2.0 g/L-carnitine (1-10 mM)

0.0021-0.21 g/LAlpha-Lipoic acid (0.01-1.0 mM)

0.25-0.75 ml/L insulin (10 mg/ml)

THAM to adjust pH (0.01-3.0 L distilled water).

For example, the solution contains:

about 0.14 g/L calcium chloride

about 0.52 g/L potassium chloride (7 mM)

about 0.06 g/L potassium phosphate (monobasic)

about 0.10 g/L magnesium chloride (hexahydrate)

about 0.10 g/L magnesium sulfate (heptahydrate)

about 7.31 g/L sodium chloride (125 mM)

about 0.35 g/L sodium bicarbonate

about 0.05 g/L sodium phosphate (dibasic; heptahydrate)

about 1.98 g/L dextrose/D-glucose (11 mM)

about 0.27 g/L adenosine (1 mM)

about 0.46 g/L glutathione (reduced; 1.5 mM)

about 0.18 g/L ascorbic acid (1 mM)

about 0.21 g/L L-arginine (1 mM)

about 0.27 g/L creatine orotate (0.5 mM)

about 0.30 g/L creatine monohydrate (2 mM)

about 0.15 g/L L-citrulline malate (1 mM)

about 0.08 g/L dichloroacetate (0.5 mM)

about 2.3 g/L-carnosine (10 mM)

about 2.0 g/L-carnitine (10 mM)

about 0.021 g/LAlpha-Lipoic acid (0.1 mM)

about 0.50 ml/L insulin (10 mg/ml)

THAM to adjust pH (about 1 L distilled water).

The solutions described above are used to maintain the physiological integrity of an organ. The organ or tissue, which is preferably isolated from a human body or the body of an animal, is contacted for a duration of at least 1 hour. Alternatively, the organ or tissue is perfused in situ, i.e., prior to being removed from the body. For example, the organ or tissue is contacted in situ in a cadaver or in a living patient. The organ or tissue is contacted with the solution from a period of about 1-5 hours, e.g., 4 hours up to several days, e.g., 1, 2, 5, 8, days or more.

A variety of different organs and tissue types are favorably stored and resuscitated using the solutions. For example, the organ or tissue is a heart. Other suitable tissues/organs include kidney, liver, stomach, spleen, pancreas, lung, brain, eye, intestines, bladder, skin or dermal tissue, blood vessels such as veins or arteries, heart valves, sperm, and oocyte(s).

The method of maintaining or restoring the physiological integrity of an organ or tissue is carried out by contacting the organ or tissue with the solution(s) described above for a duration of time such that esterase activity is in the range of >150 arbitrary fluorescence photon units, intracellular NO concentration is in the range of >1 nM, and mitochondrial membrane potential ratio of the cells of the organ is >1.00. The solution preserves intact cardiac myocyte contractile proteins (myosin heavy chain (HC), myosin light chain (LC), acitinin, actin, troponinc C); eNOS, caveolin, and vWF, e.g., as assessed with Western and Immunoblots, whereas storage of tissues/organs such as heart in previous solutions led to degradation of such proteins such as myosin heavy chain thereby compromising the integrity and function of the transplanted organ. Absence or fragmentation of these compostions indicate deterioration of the organ. Clinical parameters include Left Ventricular End Diastolic Pressure (LVEDP) 0-30 mm Hg, BP 100-140/60-100, pH>6.8. The solution also inhibits stenosis, plaques, clot formation due to damaged endothelium, as well formation of an atheroma (arterial sclerosis), which is a significant problem in transplant cases.

Also within the invention is a method of evaluating the physiological integrity of an organ or tissue. The organ or tissue is contacted with solution(s) described above (or another solution) and a level of concentration or activity of one or more of the following parameters is detected or evaluated. Biological indices to be evaluated include esterase, nitric oxide, or cellular membrane potential. Esterase activity is in the range of >150 arbitrary (fluorescence photon) units, intracellular NO concentration is in the range of >1 nM, and mitochondrial membrane potential of the cells of said organ is in the range of >1.00 (ratio of polarized versus depolarized, where >1 indicates that the mitochondrial membrane is polarized and the tissue is healthy. The foregoing values indicate that the organ or tissue, e.g., a heart, is suitable for transplantation into a recipient living mammal.

The compositions for making the storage/resuscitation solution are optionally packaged in a kit with the ingredients/amounts listed below or multiples thereof, i.e., scaled up to make 2, 3, 5, 10, 20 times the amount of solution. An exemplary kit contains

0.1-0.5 g/L calcium chloride

0.25-0.75 g/L potassium chloride (7 mM)

0.01-0.5 g/L potassium phosphate (monobasic)

0.01-0.5 g/L magnesium chloride (hexahydrate)

0.01-0.5 g/L magnesium sulfate (heptahydrate)

5.0-10.0 g/L sodium chloride (125 mM)

0.01-0.5 g/L sodium bicarbonate

0.01-0.5 g/L sodium phosphate (dibasic; heptahydrate)

1.0-5.0 g/L D-glucose (11 mM)

0.01-0.5 g/L adenosine (1 mM)

0.01-0.75 g/L glutathione (reduced; 1.5 mM)

0.01-0.5 g/L ascorbic acid (1 mM)

0.01-0.5 g/L L-arginine (1 mM)

0.01-0.5 g/L creatine orotate (0.5 mM)

0.01-0.5 g/L creatine monohydrate (2 mM)

0.01-0.5 g/L L-citrulline malate (1 mM)

0.01-0.5 g/L dichloroacetate (0.5 mM)

0.23-2.3 g/L-carnosine (1-10 mM)

0.20-2.0 g/L-carnitine (1-10 mM)

0.0021-0.21 g/LAlpha-Lipoic acid (0.01-1.0 mM)

0.25-0.75 ml/L insulin (10 mg/ml)

THAM to adjust pH

These ingredients packaged together with instructions for use and are mixed in 0.01-2.0 L of distilled water. As is described above, insulin is optionally added shortly before use, i.e., shortly before an organ is added to the solution. The kit is packaged or sold without the sterile water component. For example, the kit contains

about 0.14 g/L calcium chloride

about 0.52 g/L potassium chloride (7 mM)

about 0.06 g/L potassium phosphate (monobasic)

about 0.10 g/L magnesium chloride (hexahydrate)

about 0.10 g/L magnesium sulfate (heptahydrate)

about 7.31 g/L sodium chloride (125 mM)

about 0.35 g/L sodium bicarbonate

about 0.05 g/L sodium phosphate (dibasic; heptahydrate)

about 1.98 g/L dextrose/D-glucose (11 mM)

about 0.27 g/L adenosine (1 mM)

about 0.46 g/L glutathione (reduced; 1.5 mM)

about 0.18 g/L ascorbic acid (1 mM)

about 0.21 g/L L-arginine (1 mM)

about 0.27 g/L creatine orotate (0.5 mM)

about 0.30 g/L creatine monohydrate (2 mM)

about 0.15 g/L L-citrulline malate (1 mM)

about 0.08 g/L dichloroacetate (0.5 mM)

about 2.3 g/L-carnosine (10 mM)

about 2.0 g/L-carnitine (10 mM)

about 0.021 g/LAlpha-Lipoic acid (0.1 mM)

about 0.50 ml/L insulin (10 mg/ml)

THAM to adjust pH

Optionally, the solution is nano-sized to increase the efficiency of traversing the cellular membrane. Nano-sizing refers to the reduction of the particle size to the sub-micron range, with the final particle size typically being 1-10 ηm. The reduction of particle size leads to a significant increase in the efficiency of the solution in traversing the cellular membrane. In one aspect, the efficiency is increased such that at least 20%, at least 25%, at least 50%, at least 75%, or at least 100% of the solution traverses the cellular membrane.

The invention provides for nano-sizing for the solution of the invention prior to use in the methods described herein. Alternatively, the invention provides for nano-sizing the water prior to adding the other compounds/reagents of the solution. In yet another aspect, the invention provides for nano-sizing the water and nano-sizing each compound/reagent of the solution separately prior to mixing in solution.

In one aspect, the composition comprises water packets or water clusters in a nanometer range of size. Optionally, the water packets or water clusters are 1-10 ηm, 1-25 ηm, 25-50 ηm, 50-75 ηm, 75-100 ηm, 100-200 ηm, 200-500 ηm, or 500-999 ηm.

The invention also provides methods of measuring the pH of an excised heart comprising contacting the excised heart with the solution of the invention, and determining the pH of the excised heart, wherein a pH between 6.8 and 7.0 indicates the heart is suitable for transplantation.

The invention also provides methods of measuring the pH of the solution of the invention comprising contacting an excised heart with the solution of the invention, and determining the pH of the solution of the invention, wherein a pH between 6.8 and 7.0 indicates said heart is suitable for transplantation.

In addition to solutions and methods of storing/preserving organs and tissues, the invention includes a perfusion device. Elements of the device include a chamber assembly, a perfusion circuit including a first conduit for providing the solution to the organ, and a temperature control unit. The device includes an immobilized phosphoenolpyruvate (PEP) carboxykinase and lactate dehydrogenase (LDH) columns to make it a self sustaining system. For example, the solution coming out of the heart chamber may contain some lactic acid, which is then continuously converted to pyruvate by the LDH as the solution is pumped back into the chamber. Pyruvate then enters the TCA cycle for continuous generation of ATP. Similarly, oxaloacetate that is formed in the TCA cycle is converted to Phosphoenol pyruvate by PEP carboxykinase. PEP is converted to pyruvate producing ATP. Also, pyruvate enters the TCA cycle to generate more ATP. Thus, the device permits the cycle to continue regenerating substrates. Energy metabolism is sustained in the system until all the glucose and/or fatty acids are consumed only upon prolonged storage, e.g., greater than about 2 weeks or 10 days. For example, the device is suitable for vascular tissue such as heart and blood vessels, as well as kidney, liver, stomach, spleen, pancreas, lung, brain, eye, intestines, and bladder.

A system for maintenance or resuscitation of a mammalian organ includes a container for keeping the organ in contact with the composition or solution described above, a delivery means for delivering the composition to at least one vascular vessel of the organ, a removal means for transport of the composition away from the organ, a temperature control means, an oxygenation means, a filtering means, and a flow control means. The delivery means, removal means, temperature means, oxygenation means, filtering means, and flow control means provide or restore a physiological acceptable mammalian environment. A self-sustaining system includes the following elements: continuous production of nitric oxide, continuous generation of ATP, buffering of acidosis and hydrogen ions, and quenching of ammonia. The elements are preferably interconnected.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. All references cited herein are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of photomicrographs depicting the esterase activity (green) and cellular necrosis (red) of ventricular cardiomyocytes in the anterior and posterior left ventricular (LV) wall at 0 minutes, 15 minutes and 120 minutes post-harvest.

FIG. 2 is a bar chart demonstrating the esterase activity of ventricular cardiomyocytes in the anterior and posterior LV wall at 0 minutes, 15 minutes and 120 minutes post-harvest.

FIG. 3 is a series of photomicrographs showing the ratio of polarized (red) to depolarized (green) ventricular cardiac myocyte mitochondria in the anterior and posterior LV wall at 0 minutes, 15 minutes and 120 minutes post-harvest.

FIG. 4 is a bar graph representing the ratio of polarized to depolarized ventricular cardiac myocyte mitochondria in the anterior and posterior LV wall at 0 minutes, 15 minutes, 30 minutes, and 120 minutes post-harvest.

FIG. 5 is a series of photomicrographs indicating the necrosis (red) and esterase activity (green) of the posterior LV wall of porcine hearts that were incubated in either the solution of the invention, referred to as “Lazarus”, or Celsior for 4 hours.

FIG. 6 is a graphical representation of the esterase activity in the anterior and posterior LV myocardium in each experimental group, i.e., 4 hour storage in Celsior; 4 hour storage in Lazarus; NBHD 4 hour storage in Lazarus; and NBHD 10 day storage in Lazarus.

FIG. 7 is a bar graph identifying the esterase activity in the anterior and posterior LV wall of the NBHD model after 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours of storage in Lazarus solution.

FIG. 8 is a series of images demonstrating the ratio of red (polarized) to green (depolarized) cardiac myocyte mitochondria in the anterior LV wall after 0 minutes, 60 minutes, 120 minutes, 180 minutes, and 240 minutes of storage in either Celsior or Lazarus solution.

FIG. 9 is a series of photomicrographs showing necrosis (red) and esterase activity (green) of porcine heart left anterior descending artery that was incubated in either Lazarus or Celsior for 4 hours.

FIG. 10 is a series of images depicting calcium mobilization (red) and NO production (green) of Celsior or Lazarus-treated porcine heart left anterior descending artery before and after stimulation with bradykinin.

FIG. 11 is a graphical representation of calcium mobilization and NO production of Celsior-treated porcine heart left anterior descending artery after stimulation with bradykinin.

FIG. 12 is a bar graph demonstrating calcium mobilization and NO production of 4 hour Lazarus-treated porcine heart (1 hour post-mortem) left anterior descending artery after stimulation with bradykinin.

FIG. 13 is a series of images depicting immunofluorescence labeling of Myosin and Actin in the myocardium after 4 hours incubation in either Celsior or Lazarus.

FIG. 14 is a series of photomicrographs demonstrating immunofluorescence labeling of Troponin C and Actinin in the myocardium after 4 hours of incubation in Celsior or Lazarus.

FIG. 15 is a photograph of a Western blot showing the presence of Myosin light chain protein in anterior (A) and posterior (P) biopsies of porcine post-mortem heart stored in Lazarus or Celsior for 240 minutes.

FIG. 16 is a photograph of a Western blot showing the presence of Actinin in anterior (A) and posterior (P) biopsies of porcine post-mortem heart stored in Lazarus or Celsior for 240 minutes.

FIG. 17 is a series of photomicrographs showing immunofluorescence labeling of eNOS in the endothelium after 4 hours of incubation in Celsior or Lazarus.

FIG. 18 is a series of images depicting necrosis (red) and esterase activity (green) in the myocardium and left main coronary artery (1 hour post-mortem) after 10 days of storage in Lazarus solution.

FIG. 19 is a bar chart demonstrating esterase activity in the anterior and posterior myocardium (1 hour post-mortem) after 10 days of storage in Lazarus solution.

FIG. 20 is a series of photomicrographs showing calcium mobilization (red) and NO production (green) of 10 day Lazarus-treated porcine heart left anterior descending artery after stimulation with bradykinin.

FIG. 21 is a graphical representation of the esterase activity in a liver after 10 days of storage in Lazarus solution.

FIG. 22 is a bar graph showing the percent necrosis in a liver after 10 days of storage in Lazarus solution.

FIG. 23 is a diagram showing the energy metabolism in the heart during storage.

FIG. 24 is a diagram illustrating the ammonia production in the heart during storage.

FIG. 25 is a diagram demonstrating the role of creatine in the production of ATP and the maintenance of metabolic mechanisms in the heart during storage.

FIG. 26 is a line drawing of an ex vivo resuscitation and preservation device for a heart.

FIG. 27 is a line drawing of an ex vivo preservation device of a heart in non-working state.

FIG. 28 is a diagram depicting an experimental design.

FIG. 29 is a drawing illustrating a Gardos channel.

FIG. 30 is a drawing of an aquaporin (water channel).

DETAILED DESCRIPTION

The present invention provides a novel storage solution for donor hearts taking into account various biochemical processes that occur during storage of an organ for transplantation. The solution permits a substantially longer preservation period, the use of non-beating heart (NBH) donors, and improved clinical outcomes for those receiving cardiac transplants. The solution includes compositions the address various damaging processes (listed below) that occur during storage of an organ.

Techniques for the preservation of the donor heart for transplantation have changed very little despite 30 years of extensive research in the field. In cardiac transplantation, major obstacles are the limited availability and poor quality of donor hearts after some amount of storage time. Using current practices and preservation solutions, the limit of preservation time is 4-6 hours (Ferrera Ret al., 1994 Ann Thorac Surg, 57:1233-1239; Oshima K et al., 1999 J of Heart and Lung Trans, 18(9): 852-861), and in the United States, all cardiac allografts are presently obtained from brain-dead, beating heart donors maintained on life support systems. As a result of this severely limited organ pool and time restraints on preservation, 10%-40% of all cardiac transplant candidates die awaiting a new organ. In addition, currently there is a significant incidence of accelerated vasculopathy in transplanted hearts presumably due, in part, to the poor quality of donor hearts after storage.

The ability to store donor hearts for a longer period of time would increase the size of the donor pool and allow for transport of donor hearts longer distances to suitable recipients. Moreover, superior preservation might allow for the use of non-beating heart donors (NBHD) and further increase the donor pool. Finally, improvement of the condition of donor hearts after storage would improve the clinical results for the recipient.

The preservation of a harvested donor human heart prior to transplant entails protection of endothelial cells, cardiac myocyte function, and mechano-contractile coupling, and is related to the long-term survival of a transplanted heart. Despite advances in science and technology of prolonging organ viability and storage, few changes have been made in the clinical practice of donor heart preservation during the past 20 years. The currently available preservation protocols use hypothermic arrest and simple storage using a variety of crystalloid-based cardioplegic and preservation solutions, and are limited in terms of the length of storage of 4-6 hours. In addition, these techniques invariably subject donor hearts to periods of global ischemia that may result in functional and metabolic stunning leading to ischemia-reperfusion injury. In addition, the two major limitations of successful long term cardiac transplantation today are the lack of enough donor hearts to meet the demand of the large number of patients who potentially could benefit from transplantation, and the relatively high rate of long-term transplanted heart failures, which are primarily due to accelerated atherosclerotic disease.

Surgical techniques for harvesting and transplantation of the human heart are very well established. What happens to the explanted heart during storage generally dictates the successful outcome of the transplant. Therefore, there is a pressing need for storage solutions and conditions. The invention provides storage solutions, methods and devices that maintain the structural and functional integrity of explanted donor hearts and thus prolongs ex-vivo storage prior to surgical re-implantation. Application of this newly developed technology should enable the resuscitation of an arrested heart within 30 minutes of the arrest. As such, patients dying in the emergency room due to non-cardiac trauma can become potential heart donors for transplantation.

This invention provides a system for preserving harvested human heart (or other organs) in need of preservation or resuscitation during preservation and evaluation period prior to implantation and/or transplantation. This system allows for organs to be preserved at normothermic temperatures in viable state (and be transported to alternate geographic locations for transplantation if required), thus potentially increasing the extremely limited availability of these much-needed organs. In order to preserve the heart in a viable state, the invention considers both the anaerobic and aerobic modes of metabolism, the requirements for endogenous vasodialators and an energy source. The preservation solution described herein maintains the viability of the organ for an extended period, e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 8 hours, about 10 hours, about 15 hours, about 20 hours, or about 1 day. The solution of the invention provides nutrients suitable for cardiac myocyte and endothelial cell energy metabolism. In another aspect, the solutions provide protective ingredients that can help the tissues/cells resist the damaging effects of prolonged storage. Moreover, the solutions provide protection against desiccation and rehydration edema upon transplantation.

The invention also provides a perfusion system that allows for circulation of the solution not only through the coronary vasculature, but also all the compartments of the static heart. This ensures that the nutrient rich solution bathes the whole organ inside and outside.

The invention also provides a method to gently prime the heart with nutrients and intermediate metabolites prior to transplantation. Such priming of the heart physiologically converts the static heart into a restored sinus rhythm without detrimental contractures and ischemia-reperfusion injury.

Thus, the compositions, methods, systems/devices and media of the present invention can preserve, resuscitate and maintain the donor heart in either the static or beating state during the time of preservation to insure homogenous distribution of the preservation medium. Maintaining the heart in the beating state further serves to sustain normal metabolic, contractile, and endothelial functions beyond the current 4-6 hour window of hypothermic arrest and storage for donor hearts.

The invention provides a novel storage solution for donor hearts taking into account various biochemical processes that occur during storage of an organ for transplantation. The solution of the invention will allow a substantially longer preservation period, the use of NBH donors, and improved clinical outcomes for those receiving cardiac transplants.

Following are some potentially damaging processes that occur during storage of an organ and how they are addressed by the solution of the invention. Reactive oxygen species are generated during storage; however, ascorbic acid and reduced glutathione (i.e. reducing agents) present in the solution consume oxygen free radicals during storage. Hydrogen ion production during storage can lead to acidosis; however, the solution contains agents with buffering activity. ATP is diminished during storage; however, the solution contains substrates for the production of ATP and maintenance of metabolic mechanisms in the heart. Ammonia is produced as a result of the breakdown of amino acids during storage; however, the solution contains substrates for the consumption of ammonia and production of substrate for anaerobic/aerobic metabolism. Storage can also result in dehydration/edema of the organ; however, the ionic composition of the solution is manipulated in order to maintain normal water content in the cell to prevent edema in the reperfused myocyte. This also provides an ideal environment for myocyte contraction.

FIGS. 23-25 illustrate some metabolic pathways in the heart during storage and how these processes are addressed by the solutions of the invention. FIG. 23 illustrates energy metabolism pathways in the heart during storage, while FIG. 24 depicts pathways pertaining to ammonia production in the heart. FIG. 25 depicts energy metabolism in the heart relating to ATP stores and production of hydrogen ions.

Myocardial Dehydration during storage

A significant problem in organ storage is dehydration. For example, in heart tissue, myocardial dehydration occurs via the following metabolic processes. A decrease in ATP leads to an increase in Na+ in the cell, secondary to Na+ leak and decrease in Na—K ATPase activity. The Na—Ca exchanger reverses causing Ca+ to build up in the cell. Subsequently, a further increase in Ca+ results in Ca+-induced Ca+ release from the sarcoplasmic reticulum (SR). Ca+ also induces the GARDOS Channel, which leads to the loss of K+ and water (FIG. 29). Aquaporins are a route for water out of the cell, and upon reperfusion, back into the cell (FIG. 30.)

In order to counteract the above-mentioned sequence of events, the solution contains a relatively low Na+ concentration (about 125 mM) and a slightly higher K+ concentration (7 mM). By preventing the dehydration of the myocardium, the solutions of the invention prevent the rapid uptake of water into the cells once perfusion is reestablished.

Prior to the invention described herein, none of the currently available organ storage solutions had adequately addressed the potential role of activated hemichannels and/or aquaporins in induction of edema and ionic imbalance in the stored organs, especially the heart. Connexin 43 containing hemichannels, that are also the components of gap junctions, and aquaporins (AQP1) have been demonstrated in cardiomyocytes and the endothelial cells in the heart. Activation of these channels may play an important role in development of ionic disproportion, edema, and stiffness of the organs during storage. Increased cell volume (edema) and stiffness can lead to the failure of the organ upon transplant, or may require significant pharmacological intervention to reverse this trend before the organ can function. Problems associated with these channels being activated is addressed by the invention to improve viable preservation of stored organs.

Optimizing the solution of the invention would prevent activation of hemichannels, thus avoiding edema, ionic imbalance and energy depletion during organ storage. These constituents provide a favorable environment and cellular support during ex-vivo storage. As shown in the Examples below, the protection of structure and function of cardiomyocytes and the endothelium in BHD and NBHD hearts, stored over a prolonged period in the solution of the invention, supports this hypothesis.

A decrease in physiological concentration of external calcium (<1-1.3 mM), a reduction in glutathione (altered redox potential), energy state (<ATP), phosphorylation, or in activation of MAP kinase, or any increase in reactive oxygen species and osmolarity leads to opening of hemichannels. All of these parameters, which are encountered by the heart or organ during storage, can operate independently of one another in activating the hemichannels. Opening of hemichannels, because of nonselectivity, allows free flow of external Na+, Cl−, and Ca2+ into, and K+ out of the cytoplasm. Due to the build up of excess intracellular negative charges (negatively charged macromolecules), the increase in internal Cl− concentration would require water uptake via AQP1 and/or hemichannels to maintain isosmotic condition. Subsequent closing of the channels would establish a higher steady state cell volume. However, to counterbalance the increased volume and to avoid rupture, the cell compensates by rearranging the cytoskeleton leading to cell stiffness. Increased cell volume (edema) and stiffness leads to the failure of the organ upon transplant, or may require significant pharmacological intervention to reverse this trend before the organ can function. Activation of hemichannels leads to water and ion accumulation and edema in the heart during storage. Components of the solution of the invention, including physiological concentration of calcium, will prevent any of the above-mentioned detrimental changes from taking place in the stored organs.

Optionally, the solution is nano-sized to increase the efficiency of the solution traversing the cellular membrane by any method known in the art, including the method described in U.S. Pat. Nos. 6,521,248 and 7,198,254, which are incorporated herein by reference in their entireties. Nano-sizing refers to the reduction of the particle size to the sub-micron range, with the final particle size typically being 1-10 ηm. The reduction of particle size leads to a significant increase in the efficiency of the solution in traversing the cellular membrane. In one aspect, the efficiency is increased such that at least 20%, at least 25%, at least 50%, at least 75%, or at least 100% of the solution traverses the cellular membrane.

The invention provides for nano-sizing for the solution of the invention prior to use in the methods described herein. Alternatively, the invention provides for nano-sizing the water prior to adding the other compounds/reagents of the solution. In yet another aspect, the invention provides for nano-sizing the water and nano-sizing each compound/reagent of the solution separately prior to mixing in solution.

In one aspect, the composition comprises water packets or water clusters in a nanometer range of size. Optionally, the water packets or water clusters are 1-10 ηm, 1-25 ηm, 25-50 ηm, 50-75 ηm, 75-100 ηm, 100-200 ηm, 200-500 ηm, or 500-999 ηm.

Increasing storage time significantly, improving condition of donor hearts/organs after storage, and allowing for the use of NBHD will have a profound impact on the current landscape of transplant surgery Immediate impact could be achieved if the solution is used in place of current preservation solutions.

Assessment of myocardial and endothelial structure and function at the cellular level indicates that solution can accomplish each one of those goals. Structure and function of the myocardium is significantly improved after storage in the solution of the invention, referred to as “Lazarus”, if compared to Celsior (a commonly used storage solution for cardiac transplants) when the conditions are exactly the same. Furthermore, these parameters are still preserved after one hour of death (NBHD) and 10 days or two weeks of storage.

Experiments were performed to compare Lazarus to Celsior (a presently used storage solution in cardiac transplantation) with respect to their effects on the myocardium and coronary endothelium as assessed using various microscopic and biochemical analyses. Comparisons were made using beating heart and NBHD models.

The Lazarus solution contained the following:

Calcium chloride 0.14 gm/L Potassium chloride (7 mM) 0.52 gm/L Potassium phosphate (monobasic) 0.06 gm/L

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