ORGAN AND BIOLOGICAL TISSUE PRESERVATION COLD STORAGE SOLUTION

Abstract: Cold storage solutions for the preservation of organs and biological tissues prior to implantation, including a cellular energy production stimulator under anaerobic conditions, an anti-inflammatory agent, and an oxygen free radical scavenger.

Description: ORGAN AND BIOLOGICAL TISSUE PRESERVATION COLD STORAGE SOLUTION Reference to Related Application This application claims the benefit of U.S. Provisional Application No. 60/240,023 filed on October 13, 2000, entitled "Organ and Biological Tissue Preservation Cold Storage Solution," which is incorporated herein by reference. Field of Invention The invention relates to the field of organ and biological tissue preservation. In particular, the invention relates to cold storage solutions for the preservation of organs and biological tissues for implant. Background of Invention It is believed that the ability to preserve human organs for a few days by cold storage after initial flushing with an intracellular electrolyte solution or by pulsatile perfusion with an electrolyte-protein solution has allowed sufficient time for histo-compatibility testing of donor and recipient. It is also believed that preservation by solution or perfusion has also allowed for organ sharing among transplant centers, careful preoperative preparation of the recipient, time for preliminary donor culture results to become available, and vascular repairs of the organ prior to implantation. It is believed that the 1990's has been a decade characterized by increasing waiting times for cadaveric organs. In renal transplantation, the growing disparity between available donors and patients on the waiting list has stimulated efforts to maximize utilization of cadaveric organs. An obstacle that may arise in the effort to increase utilization is that maximal utilization may require transplantation of all available organs, including extended criteria donor organs. However, by extending the criteria for suitability of donor organs, transplant clinicians may risk a penalty with respect to graft function, diminishing the efficiency of organ utilization if transplanted organs exhibit inferior graft survival. Consequently, interventions that both improve graft function and improve the ability of clinicians to assess the donor organ may be crucial to achieving the goal of maximizing the efficiency of cadaveric transplantation. The mechanisms of injuries sustained by the cadaveric renal allograft during pre-preservation, cold ischemic preservation and reperfusion are believed to be complex and not fully understood. However, it is believed that there exists ample evidence to suggest that many of the injurious mechanisms occur as a result of the combination of prolonged cold ischemia and reperfusion (I/R). Reperfusion alone may not be deleterious to the graft, since reperfusion after short periods of cold ischemia may be well-tolerated, but reperfusion may be necessary for the manifestation of injuries that originate during deep and prolonged hypothermia. It is suggested that four major components of I/R injury that affect the preserved renal allograft begin during cold ischemia and are expressed during reperfusion. These include endothelial injury, leukocyte sequestration, platelet adhesion and increased coagulation. Hypothermically-induced injury to the endothelium during preservation may lead to drastic alterations in cytoskeletal and organelle structures. During ischemic stress, profound changes in endothelial cell calcium metabolism may occur. These changes may be marked by the release of calcium from intracellular depots and by the pathological influx of calcium through the plasma membrane. Hypothermic preservation may disrupt the membrane electrical potential gradient, resulting in ion redistribution and uncontrolled circulation of Ca++. The depletion of ATP stored during I/R may compromise ATP-dependent pumps that extrude Ca++ from the cell and the energy intensive shuttle of organelle membranes, causing a dramatic elevation of intracellular free Ca++. Alterations in cytosolic Ca++ concentration may disrupt several intracellular functions, many of which may result in damaging effects. Unregulated calcium homeostasis has been implicated in the development of endothelial and parenchymal injury and is believed to be a fundamental step in the sequelae of steps leading to lethal cell injury. Among the most significant damaging effects of increased cytosolic Ca++ are believed to be the activation of phosphohpase Al, 2 and C, the cytotoxic production of reactive oxygen species by macrophages, the activation of proteases that enhance the conversion of xanthine dehydrogenase to xanthine oxidase, and mitochondrial derangements. Solutions for preserving organs are described in U.S. Patent Nos. 4,798,824 and 4,879,283, the disclosures of which are incorporated herein in their entirety. One such solution is Viaspan ® cold storage solution, which may be used for hypothermic flushing and storage of organs. Despite such solutions, it is believed that there remains a need for organ and tissue preserving solutions that allow for static storage and preservation, while demonstrating superior quality preservation of organ and tissue viability and function. Summary of the Invention The invention provides an organ and tissue preserving solution for static storage preservation that demonstrates superior quality preservation when compared to existing preserving media, in terms of organ and tissue viability and function. The organ and biological tissue preservation aqueous cold storage solution includes a cellular energy production stimulator under anaerobic conditions, an anti-inflammatory agent, and an oxygen free radical scavenger. The invention also provides a preserved organ or biological tissue, including a cadaveric organ or tissue within a cold storage solution of the invention in a deep hypothermic condition or physiological condition. The invention also provides a method for preserving an organ or biological tissue. The method includes flushing a cadaveric organ or tissue with a cold storage solution of the invention, allowing the flushed cadaveric organ or tissue to be enveloped in the cold storage solution, and storing the cadaveric organ or tissue in the cold storage solution in a deep hypothermic condition or physiological condition. The invention further provides a method of preparing an organ or biological tissue preservation cold storage solution. The method includes providing a solution with distilled water or deionized water, adding potassium lactobionate, potassium phosphate, raffmose, adenosine, allopurinol, pentastarch, insulin and dexamethasone to the solution, and mixing superoxide dismutase into the solution. Detailed Description of the Invention In accordance with the invention, the organ and biological tissue preservation cold storage solution includes a cellular energy production stimulator under anaerobic conditions, an anti-inflammatory agent, and an oxygen free radical scavenger. The organ and biological tissue preservation cold storage solution is intended for infusion into the vasculature of cadaveric and living donor organs for transplantation. Once infused, the donor organs are exsanguinated and blood is replaced by the solution in the native vasculature of the organs to return the organs to a normothermic condition. The solution may be used under deep hypothermic conditions or physiological conditions. The solution remains in the vasculature of the organ, as well as envelopes the entire organ during the period of cold ischemia. This method of preservation allows for the extended storage of organs, tissues, and all biological substances. When the organ or tissue is returned to normothermic conditions, the solution is replaced with blood or other physiologic media. Variations of this solution may also be used for machine perfusion preservation. The cold storage solution of the invention may be used in the same manner and for the same tissues and organs as known storage solutions. A cold storage solution of the invention includes a cellular energy production stimulator under anaerobic conditions. Insulin, which is a polypeptide hormone, is the primary hormone responsible for controlling the uptake, utilization, and storage of cellular nutrients. Insulin stimulates transport of substrates and ions into cells, promotes translocation of proteins between cellular compartments and activates and inactivates specific enzymes. A cold storage solution of the invention also includes an anti-inflammatory agent. Dexamethasone, which is a steroid analogue, is used for its anti-inflammatory properties. Dexamethasone has a prolonged plasma half-life and pronounced growth-suppressing properties. A cold storage solution of the invention also contains an oxygen free radical scavenger. One example, superoxide dismutase, is known for its potent free oxygen radical scavenging properties. Preferably, the superoxide dismutase is conjugated to polyethylene glycol so that its half-life is extended by a factor of about 100 times. Superoxide dismutase is a potent scavenger of several classes of free oxygen radicals during cold ischemia and upon reperfusion. When conjugated to polyethylene glycol, superoxide dismutase can remain active for several hours in the cold storage solution of the invention. According to a preferred embodiment of the invention, an organ and biological tissue preservation cold storage solution containing superoxide dismutase in the preserving solution significantly improves vascular resistance, vascular flow, and calcium efflux during the organ preservation period. The inhibition of calcium efflux over time in kidneys preserved by the proposed solution suggests that, in addition to vasoactive effects, an additional cytoprotective and cryoprotective effect may also be important in ameliorating ischemic injury. These improvements are substantiated ultrastructurally by improved appearance of mitochondria in proximal tubular cells compared to mitochondria from kidneys not exposed to the proposed solution. A cold storage solution of the invention may also contain components that are typically used in known cold storage solutions. See, U. S. Patent Nos. 4,798,824 and 4,879,283. For example, other components that may be utilized in the solution include: potassium lactobionate, which is an impermeant anion that reduces cell swelling, KH 2 PO , which provides acid-base buffering and maintains the pH of the solution, MgSO , which stabilizes cellular and organelle membranes, and raffmose, which is a complex sugar that reduces cell swelling and provides energy stores for metabolically stressed cells. In addition, adenosine, which is a precursor to ATP synthesis, allopurinol, which is also a free oxygen radical scavenger, and pentastarch, which is an oncotic supporter, may be added to the solution. NaCl and KOH may also be used for acid-base buffering and maintenance of the pH of the solution. In a preferred embodiment, the organ or biological tissue preservation cold storage solution includes, but is not limited to: Table 1 In a more preferred embodiment, the organ or biological tissue preservation cold storage solution includes, but is not limited to: Table 2 In a most preferred embodiment, the organ or biological tissue preservation cold storage solution includes, but is not limited to: Table 3 A cold storage solution of the invention may be prepared by combining the components described above with sterile water, such as distilled and/or deionized water. For example, to prepare the organ and biological tissue preservation cold storage solution, approximately 700- 900mL, or preferably about 800 mL, of sterile water is poured into a one liter beaker at approximately room temperature. Although a one liter beaker is used in this example, any other container of any size may be used to prepare the solution, where the component amounts would be adjusted accordingly. In the most preferred embodiment, the following are added, in any order, to the solution and each is mixed until dissolved in the solution: approximately 100 mol/L of potassium lactobionate, approximately 25 mol L of potassium phosphate, approximately 30 mol/L of raffmose, approximately 5 mol/L of adenosine, approximately 1 mol/L of allopurinol and approximately 50 g of modified pentastarch. The modified pentastarch is a fractionated colloid mixture of 40-60 kDaltons in diameter and is modified by infusing the pentastarch under 3 arm of pressure through a dialyzing filter with a bore size of about 40-60 kDaltons. Approximately 40 U of insulin and approximately 8mg/L dexamethasone are also added to the solution. Then, in a second step, approximately 25,000 U of superoxide dismutase, which is conjugated to polyethylene glycol, is added to the solution. The first and second step may also be reversed. The invention also provides a method for preserving an organ or biological tissue. The method flushes a cadaveric organ or tissue with a cold storage solution of the invention, allows the cadaveric organ or tissue to be enveloped in the cold storage solution, and then stores the cadaveric organ or tissue in the cold storage solution in a deep hypothermic condition or physiological condition. Additional cold storage solution may be added to ensure adequate preservation of the organ or tissue. Preferred temperatures range from about 2-10°C in the deep hypothermic condition and are about 37°C, or room temperature, in the physiological condition. In one embodiment, the cold storage solution is first cooled to below 10°C using an ice bath or other cooling means known in the art. It is typical to inspect the cooled solution for any precipitates which may be removed by filfration prior to use. Alternatively, the organ or tissue to be preserved may be placed in the solution and then cooled. The invention further provides a preserved organ or biological tissue comprising a cadaveric organ or tissue within a cold storage solution of the invention in a deep hypothermic condition or physiological condition. As discussed above, preferred temperatures range from about 2-10°C in the deep hypothermic condition and are about 37°C, or room temperature, in the physiological condition. The invention is further explained by the following examples of the invention as well as comparison examples. In all of the examples, kidneys were procured from heart-beating donors and preserved in a laboratory by cold storage preservation. Although kidneys were used in the examples, any organ or biological tissue may be preserved in the cold storage solution. Randomization was accomplished as an open labeled, sequential analysis. All agents were added immediately prior to vascular flush. Data Collected The following donor, preservation, and postoperative recipient outcome data were collected for either Example 1 or 2: donor age (D age, years), final donor creatinine (D Cr, mg/dL), donor infraoperative urine output (U/O, mL), cold ischemic time (CIT, hours), perfusion time (PT, hours), perfusate [Na+] (mM/lOOg), perfusate [C1-] (mM/lOOg), perfusate [K+] (mM/lOOg), perfusate [Ca++] (mM/lOOg), perfusate pH, renal flow during MP (FL, mL/min lOOg), renal resistance during MP (RES, mmHg/(mL/min/100g), recipient age (R age, years), recipient discharge creatinine (R Cr, mg/dL), initial length of recipient hospital stay (LOS, days), immediate graft function (IF, %) defined as urine production exceeding 2000mL during the first 24 post-operative hours, delayed renal allograft graft function (DGF, %) defined as the need for dialysis within the first 7 days post-transplant, and present function (3 Mo or 1 Yr, %) defined as 3 month or one year post-operative graft status. Method of Preservation All cold stored kidneys subject to the above criteria were flushed and cold stored at 4°C in IL of either University of Wisconsin (UW) solution (Niaspan ® cold storage solution, Dupont Pharma, Wilmington, DE) or the Cold Organ Storage (Perfusion) solution (OPS) of the present invention. The University of Wisconsin solution, which is also the Control-UW solution, is described in U.S. Patent Νos. 4,798,824 and 4,879,283. Statistical analysis All data are reported as mean values ± SEM unless otherwise noted. Paired and unpaired student's t-tests were used where appropriate. All statistical analyses were performed by Statview 4.5 software (Abacus Concepts, Berkeley, CA). EXAMPLE 1 Comparison of selected donor, preservation, and outcome variables by method and type of organ preservation solution (mean +/-SEM) n = number of recipients ns = not significant OPS University of p value (unpaired (n=152) Wisconsin Student's t-test) (n=160) Donor characteristics Donor age (y) 44.1 42.1 ns Final serum creatinine(mg/dl) 1.1 0.9 ns Preservation characteristics Cold ischemic time (h) 23 24 ns Outcome characteristics Delayed graft function (%) 17 32 0.03 1 yr. graft survival (%) 98 91 0.04 EXAMPLE 2 Comparison of selected donor, preservation, and outcome characteristics by type of organ preservation solution - cold storage formulation (mean +/-SEM) SOD=superoxide dismutase-polyethylene glycol (25,000 units L) n = number of recipients SOD Control-UW p value (unpaired (n=48) solution (n=86) student's t-test) (Embodiment of Table 3) Donor Characteristics Donor age (y) 39.2+/-10 44.0+/-12 0.8 Final serum creatinine 1.1+/-0.6 0.9+/-0.5 0.65 ( g/dl) Intraoperative urine 250+/-80 200+/-80 0.45 output(ml) Preservation characteristics Cold ischemic time (h) 26+/-4 23+/-4 0.59 Outcome characteristics Immediate function 79+/-5 77+/-5 Delayed grant function 20+/-4 22+/-5 O 3 month function (%) 91+/-4 87+/-5 While the invention has been disclosed with reference to certain preferred embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the sphere and scope of the invention, as defined in the appended claims and their equivalents thereof. For example, although the detailed description may refer, at times, to only organs, the terms "organs" and "organ" encompass all organs, tissues and body parts that may be transplanted. Accordingly, it is intended that the invention not be limited to the described embodiments, but that it have the full scope defined by the language of the following claims.

Claims: What we claim is: 1. An organ or biological tissue preservation aqueous cold storage solution comprising: a cellular energy production stimulator under anaerobic conditions; an anti-inflammatory agent; and an oxygen free radical scavenger. 2. The cold storage solution of claim 1 wherein the cellular energy production stimulator comprises insulin. 3. The cold storage solution of claim 1 wherein the anti-inflammatory agent comprises dexamethasone. 4. The cold storage solution of claim 1 wherein the oxygen free radical scavenger comprises superoxide dismutase. 5. The cold storage solution of claim 4 wherein the superoxide dismutase conjugates to polyethylene glycol. 6. The cold storage solution of claim 1 further comprising potassium lactobionate, KH 2 PO , MgSO 4 , and raffmose. 7. The cold storage solution of claim 1 further comprising adenosine, allopurinol and pentastarch. 8. The cold storage solution of claim 1 further comprising NaCl and KOH. 9. The cold storage solution of claim 1 wherein the cellular energy production stimulator comprises about 4-100U/L insulin, the anti-inflammatory agent comprises about 4-24mg/L dexamethasone, and the oxygen free radical scavenger comprises about 1,000-100,000 U/L superoxide dismutase, further comprising: about 50-150mM potassium lactobionate; about 10-40mM KH 2 PO 4 ; about 2-8mM MgSO 4 ; about 10-50mM raffmose; about l-20mM adenosine; about 1-1 OmM allopurinol; and about 40-60g/L pentastarch. 10. The cold storage solution of claim 1 wherein the cellular energy production stimulator comprises about 20-60U/L insulin, the anti-inflammatory agent comprises about 6-16mg/L dexamethasone, and the oxygen free radical scavenger comprises about 5,000-50,000 U/L superoxide dismutase, further comprising: about 75-125mM potassium lactobionate; about 20-30mM KH 2 PO 4 ; about 3-7mM MgSO ; about 20-40mM raffmose; about 2-1 OmM adenosine; about l-5mM allopurinol; and about 45-55g/L pentastarch. 11. The cold storage solution of claim 1 wherein the cellular energy production stimulator comprises about 40U/L insulin, the anti-inflammatory agent comprises about 8mg/L dexamethasone, and the oxygen free radical scavenger comprises about 25,000 U/L superoxide dismutase, further comprising: about lOOmM potassium lactobionate; about 25mM KH 2 PO 4 ; about 5mM MgSO 4 ; about 30mM raffmose; about 5mM adenosine; about ImM allopurinol; and about 50g/L pentastarch. 12. The cold storage solution of claim 1 further comprising sterile water. 13. A preserved organ or biological tissue comprising at least one of a cadaveric organ and tissue within the cold storage solution of claim 1 in at least one of a deep hypothermic condition and physiological condition. 14. The preserved organ or biological tissue of claim 13 wherein the cold storage solution is infused into vasculature of at least one of a cadaveric organ, living donor organ, and tissue. 15. The preserved organ or biological tissue of claim 13 wherein the deep hypothermic condition comprises a temperature of about 2-10°C. 16. The preserved organ or biological tissue of claim 13 wherein the physiological condition comprises a temperature of about 37°C. 17. The preserved organ or biological tissue of claim 13 wherein the cold storage solution is cooled to below 10°C. 18. The preserved organ or biological tissue of claim 13 wherein any precipitates in the cold storage solution are removed prior to use. 19. An organ or biological tissue preservation aqueous cold storage solution comprising: about 1,000-100,000U/L superoxide dismutase; about 50-150mM potassium lactobionate; about 10-40mM KH 2 PO 4 ; about 2-8mM MgSO 4 ; about 10-50mM raffmose; about l-20mM adenosine; about 1-1 OmM allopurinol; about 40-60g/L pentastarch; about 4-100U/L insulin; about 4-24mg/L dexamethasone; and about 700-900mL sterile water. 20. A method for preserving an organ or biological tissue comprising: flushing at least one of a cadaveric organ and tissue with a cold storage solution, having a cellular energy production stimulator under anaerobic conditions, an anti-inflammatory agent, and an oxygen free radical scavenger; allowing the flushed at least one of a cadaveric organ and tissue to be enveloped in the cold storage solution; and storing the at least one of a cadaveric organ and tissue in the cold storage solution in at least one of a deep hypothermic condition and physiological condition. 21. The method of claim 20 wherein the flushing comprises: infusing the solution into vasculature of the at least one of a cadaveric organ and tissue; and exsanguinating the at least one of a cadaveric organ and tissue. 22. The method of claim 20 wherein the storing comprises: replacing blood in vasculature of the at least one of a cadaveric organ and tissue with the solution. 23. The method of claim 20 further comprising: replacing the solution with at least blood to return the at least one of a cadaveric organ and tissue to a normothermic condition. 24. The method of claim 20 further comprising: cooling the solution to below 10°C; inspecting the cooled solution for precipitates; and removing any precipitates by filtration. 25. A method of preparing an organ or biological tissue preservation cold storage solution comprising: providing a solution with sterile water; adding potassium lactobionate, potassium phosphate, raffinose, adenosine, allopurinol, pentastarch, insulin and dexamethasone to the solution; and mixing superoxide dismutase into the solution. 26. The method of claim 25 further comprising: mixing the solution until all components are dissolved. 27. The method of claim 25 further comprising: infusing the pentastarch under pressure through a dialyzing filter; and conjugating the superoxide dismutase to polyethylene glycol.

Inventors: ARRINGTON BEN O'MAR, POLYAK MAXIMILIAN, ARRINGTON, BEN, O'MAR, POLYAK, MAXIMILIAN
Applicants: PIKE LAB INC, PIKE LABORATORIES, INC.
Application year: 2001
Main classification: A61K38
IPC codes: A01N1