KR100304594B1 - Composition for the Preservation of Organs and Blood Cells - Google Patents

Abstract

The present invention relates to the composition of an organ to be transplanted and a liquid for preserving blood, and in particular, an organ containing asparagine, aspartic acid, pyruvate, and polyamines of putrescine, spermidine, spermine, etc., in a basic salt solution. And a composition of red blood cell preservatives.

During organ transplantation, transplant organs not only receive ischemic damage during storage and transplantation but also have serious sequelae such as reperfusion injury during reperfusion after transplantation. According to the present invention, by adding asparagine, aspartic acid, pyruvate, and polyamines putrescine, spermidine, spermine, etc., the basic salt solution is stored for 90 hours for the pancreas, 60 hours for the kidneys and at least 30 hours for the liver. The retention period can be provided, and it is possible to control the deterioration of erythrocyte function by applying the same to a known erythrocyte preservation solution by reducing cell damage of erythrocytes.

Description

Composition for the Preservation of Organs and Blood Cells

The present invention relates to a composition containing asparagine, aspartic acid, pyruvate, and polyamines such as putrescine, spermidine, spermine and their salts or mixtures thereof as an active ingredient, which prevents tissue damage of the transplant organ and protects its function. will be. More specifically, the present invention is a liquid or powder composition containing asparagine, aspartic acid, pyruvate and polyamines of putrescine, spermidine, spermine and salts thereof, for ischemic and organ perfusion after transplantation. The present invention relates to a composition for preserving organ tissues, which is used to prevent and recover damage to organs to be transplanted.

Long-term preservatives are widely used, such as Euro-Collins (EU), Sacks, and UW fluid developed by the University of Wisconsin, including Collins. In addition to kidneys, lungs, brains, bones, cartilage, skin, etc. are applied to transplants, and erythrocyte preservatives are widely used CPDA-1 and AS-3 preservatives. Conventionally, in order to develop effective preservatives, cold preservation methods at 2 to 3 ° C and prevention of cell expansion by using hydroxyethyl starch have been added to existing preservatives. Still, the following problems are not solved.

First, it does not solve the pH change in the tissue. The preservation of organs is by cold preservation, in which case anaerobic glycolysis is mainly activated, resulting in a low pH situation with increased lactate. Low pH situations metabolically limit several systems, which is beneficial for tissue preservation, but many tissue damage occurs because the pH must be restored to normal during transplantation.

Second, oxidative damage of tissues due to ischemia-reperfusion should be addressed. When organs are transplanted and reperfused, various mechanisms of oxidative damage trigger tissue destruction. Currently, long-term preservation methods are not used to effectively control such tissue damage.

The inventors of the present invention prevent oxygen radical dependent tissue damage by controlling the NADH / NAD ratio using aspartic acid malate shuttle metabolism, and in particular, pyruvate, which has a biochemical mechanism similar to that of aspartate, can damage heart tissue. It has been shown that it can effectively prevent cardiac tissue damage caused by ischemic myocardial reperfusion injury and myocardial toxicity drug and prevent myocardial function deterioration (Korean Patent Application No. 98-16349, 98-016349). It is not known whether putrescine, spermidine, spermine and their salts prevent radical damage to tissues by removing lipid peroxides by oxygen radicals. Oxygen radicals, which are highly reactive, react with unsaturated fatty acids in biofilms to produce various types of lipid peroxides. At this time, various kinds of aldehydes such as 4-hydroxynonenal, malondialdehyde, acrolein, etc. are produced. These aldehydes react with macromolecules such as proteins and nucleic acids in vivo to damage living tissues.

The present inventors have been studying ways to solve the problems of the prior art, focusing on the fact that asparagine can react with acetaldehyde and spermidine with malondialdehyde, respectively, to make a complex, heart, liver, By preventing the ischemia and reperfusion injury of transplanted organ tissues such as lungs, it is possible to prevent tissue damage of organs of preservation. In addition, the present invention is added to known erythrocyte preservatives to reduce cellular damage of erythrocytes, thereby controlling the degradation of erythrocyte function It was found that this can be done to complete the present invention.

Therefore, the present invention is a liquid or powder composition containing asparagine, aspartic acid, pyruvate and polyamines of putrescine, spermidine, spermine and salts thereof, and the organ to be transplanted by ischemia damage and reperfusion injury after transplantation. Its purpose is to prevent and recover from the damage of people.

In another aspect, the present invention is to provide a composition for preserving erythrocytes used to reduce the cell damage of erythrocytes by controlling the degradation of erythrocyte function by adding the composition to a known erythrocyte preservation solution. .

The present invention relates to the composition of a liquid for preserving the organs and red blood cells to be transplanted, characterized in that it contains asparagine, aspartic acid, pyruvate, and polyamine putrescine, spermidine, spermine, etc., in a known preservative. It relates to a composition of organ and erythrocyte preservation liquid.

Long-term preservation of carcasses for transplantation is commonly done in hospitals, and the most common method for organ preservation involves cooling the organs immediately after transferring them from the carcass donor. Cooling is usually done by combining external cooling and rapid engine perfusion to lower the temperature as soon as possible. The organ is then immersed in a flush-out solution in a simple plastic container and then immersed in ice again to keep the extractor under 0-4 ° C. The composition of the flush-out solution to provide such an optimal preservation environment is currently under extensive research.

Organ transplantation is divided into (1) organ extraction (2) cold storage of organs (3) ischemic period during transplantation (4) reperfusion process. Since transplantation can result in ischemic damage during storage and transplantation, the transplantation must be completed as soon as possible to ensure the function or high survival rate. However, when organ storage or transplantation time is delayed, tissue ischemia progresses and ischemic damage is caused, and reperfusion damage occurs after transplantation. Reperfusion injury refers to a phenomenon in which tissue damage is accelerated when re-perfusion of the ischemic tissue is progressed to a certain degree. Therefore, if you can overcome the ischemia and reperfusion injury of the transplant organ, you can store the organs harvested longer than now, so you can prepare for transplantation freely and improve the success rate of organ transplantation.

The mechanisms of tissue ischemia and reperfusion injury are not fully understood, but important causes include oxygen radical response, calcium influx, increased prostaglandin, inflammatory response due to increased leukocyte activity, and decreased blood flow due to vascular endothelial injury. have. Considering these mechanisms as a major factor, efforts to prevent ischemia and reperfusion injury have been actively conducted, but the mechanisms of cellular metabolism in transplantation organs, especially the mechanism of reperfusion injury after transplantation, have not been clearly identified. Oxygen radicals, which are highly reactive, react with unsaturated fatty acids in biological membranes to produce various kinds of lipid peroxides. At this time, various kinds of aldehydes, 4-hydroxynonenal, malondialdehyde, and acrolein, are produced. It reacts with macromolecules such as proteins and nucleic acids in vivo and damages tissues.

The long-term preservative for transplantation of the present invention is an impermeable to the cells, as shown in Table 1 below, anionic lactobionate and raffinose, asparagine, asparagine, pyruvate and polyamines such as putrescine, spermidine, spermine and the like. It consists of a solution osmolality of about 320 Mosm / L and 140 mM K ⁺ and 30 mM Na ⁺ . In addition, it shows as Table 2 about the composition for red blood cell preservatives.

Table 1 Compositions of Stock Solution and Perfusate

matter Content per liter K ⁺ -lactobionate 100mmol KH ₂ PO ₄ 20mmol MgSO ₄ 5mmol Raffinos 30mmol Adenosine 5mmol Glutathione (reduction) 5mmol insulin 100 U Allopurinol 1mmol Dexamethasone 10mg Hydroxyethyl starch 45 g Pyruvate 3mmol Asparagine 5mmol Aspartate 5mmol Putrescine 0.5mmol Spermidine 50 µm Spermine 50 µm Glutamine 3mmol Tryptophan 1mmol pH 7.4 Osmotic pressure 320 mOsm / Liter

Table 2 Erythrocyte Preservative Composition

matter Content per liter Dextrose 120mmol NaCl 150mmol Mannitol 45mmol Adenine 2mmol Phosphate (Na ₂ HPO ₄ ) 10mmol citrate 10mmol Hydroxyethyl starch 10mmol Pyruvate 3mmol Asparagine 5mmol Aspartate 5mmol Putrescine 0.2mmol Spermidine 20 μmol Spermine 50 µm Glutamine 5mmol pH 7.4 Osmotic pressure 450 mOsm / Liter

Use within the range of 0.01-2 mM putrescine, spermidine 1-500 μM and spermine 1-500 μM as polyamine preservatives to preserve organs and blood is sufficient to maintain the efficacy of the present invention. . Preferably, the composition is added and used in the range of 0.1 to 10 mM of pyruvate, 0.1 to 10 mM of asparagine, and 0.1 to 10 mM of aspartic acid.

Asparagine, aspartic acid, pyruvate, and polyamines putrescine, spermidine, and spermine in the compositions of Tables 1 and 2 prevent rapid changes in pH of cells in organs including the liver and pancreas, and also asparagine and spurs. Midines form complexes by reacting acetaldehyde with malondialdehyde, respectively, and remove aldehydes that cause tissue damage caused by oxygen radical reactions to prevent ischemia and reperfusion injury in transplanted organ tissues such as heart, liver and lung. It can also reduce the cell damage of erythrocytes, thereby controlling the degradation of erythrocyte function.

Important considerations for successful cold storage include, first, the prevention of intracellular acid poisoning. Even during cooling, ischemia stimulates glycolysis and glycogen breakdown, resulting in increased lactic acid production and hydrogen ion concentrations. Tissue acid poisoning is lethal to cells and can induce lysosomal instability, lysosomal enzyme activity, and alteration of mitochondrial properties. Therefore, the prevention of intracellular acid poisoning is an important prerequisite for excellent preservation of organs, and according to the present invention, it is possible to prevent intracellular acid poisoning since it has a buffering capacity capable of preventing a sudden pH change in the cell.

Second, ischemia metabolic changes such as energy depletion and anaerobic glycolysis progress slowly because cold organ preservation is perfused and contained in the fluid (cold ischemia state), but prolonged exposure to artificial perfusion and normal cells Cellular damage occurs due to metabolic inhibition. Antioxidants and adenosine that prevent tissue damage have been shown to prevent tissue damage caused by cold ischemia, suggesting that oxygen radical reactions and energy depletion are major causes of tissue damage. Therefore, when the antioxidants glutathione, allopurinol and adenosine are added to the composition, a more synergistic effect can be expected in long-term storage efficiency.

Hereinafter, the present invention will be described in more detail with reference to examples and test examples, but the following examples are only possible embodiments of the present invention and should not be interpreted in a limiting sense. Those skilled in the art to which the present invention pertains can modify and use some components or proportions of the composition according to the above examples, so that the scope of the present invention extends to these equivalent areas.

Example 1 Preparation of Long-Term Preservation Solution

100 g of hydroxyethyl starch having a molecular weight of 200,000 to 300,000 Daltons and a degree of substitution of 0.4 to 0.5 was dissolved in distilled-deionized water to obtain a 10% W / W solution. The 10% W / W starch solution was stirred well with a mixer, and the compositions shown in Tables 1 and 2 were slowly added to dissolve to prepare a long-term preservation solution and a red blood cell preservation solution, respectively.

Experimental Example 1 Preservation Experiment of Dog Pancreas

Female mongrel dogs weighing 15-25 kg, 5 animals in each experimental group, were anesthetized with pentatol and obtained through the midline incision. Immediately after flushing out (control) or 48 and 90 hours after cold storage, the transplanted tissue with attached spleen was implanted into the iliac vessels. The pancreatic duct was then opened to the left to freely drain pancreatic fluid into the abdominal cavity.

All subject dogs were injected with 0.5G Mandol I.V (intravenous injection) prior to transplantation and during the first three days after transplantation. The dogs were then fed a standard dog food diet containing biocase. Animals were divided into three groups as follows, and Euro Collins (EU) solution and UW solution and the present invention preservative (MB) were used as control preservatives. Preservation of the transplanted organ was placed in a double plastic bag and covered with each stock solution and placed in an ice-water bath.

Group I (control): transplant transplant organ immediately, group 2 (48-hour cold storage), group 3 (90-hour cold storage).

Blood glucose levels were measured daily for the first week after transplantation and once every two weeks thereafter.

Table 3 Mean Glucose Levels (mg% ± SD) for 1 week after transplantation of the preserved pancreas

Experimental group Euro Collins (EU) UW Preservation solution (MB) of the present invention 1 group (control) 90.9 ± 7.3 90.9 ± 7.3 90.9 ± 7.3 Group 2 (48 hours) 132.9 ± 11.0 115.4 ± 9.3 107.2 ± 5.5 Group 3 (90 hours) 211.4 ± 16.2 178.9 ± 10.5 137.2 ± 6.9

Results of blood glucose levels 1 week after transplantation for each animal are shown in Table 3. During the first week after transplantation, the mean blood glucose level of the present invention was lowest compared to the known stock solution compared to the control group. All the dogs became hyperglycemia (over 200 mg /% blood glucose) after the removal of the pancreas, whereas in the preservation solution of the present invention, five animals in group 3 were observed for long-term survival. Three were sacrificed after two to three months and one remained for four months.

Test Example 2 Preservation Experiment of Rabbit Liver

The quality of preservation was evaluated by storing the rabbit liver separated by a known surgical procedure in a Euro- Collins solution, a UW solution and a solution of the present invention (MB). Bile formation during normal heat perfusion during cold storage of liver is the most useful parameter of viability, and is shown in Table 4 by measuring the rate of bile formation between rabbits stored for 30 hours in cold storage for known storage solutions and the present invention.

Table 4 Measurement of bile production rate (ml / 100gm / hr ± SD) of liver by each preservative

contrast Euro Collins UW Preservation solution of the present invention (MB) 5.9 ± 1.5 1.7 ± 0.9 3.4 ± 0.7 4.1 ± 0.5

In addition, the preservation solution was tested for efficacy as an orthotopic liver transplant model in dogs. After simple flush perfusion with the test stock solution, five series of dog livers were stored for 30 hours and then transplanted using combined 'cuff' and suture techniques. All livers immediately revealed a satisfactory shape, all five dogs awoke immediately, and platelet counts were normal 6 hours after surgery. The biliary tract and GOT and alkaline phosphatase enzyme values at 7 days after the procedure are shown in Table 5.

Table 5 Comparison of Biliary Blot, Geothy, and Alkaline Phosphorase Enzyme Values by Preservatives

Euro Collins UW Preservation solution of the present invention Biopsies mg% 0.6 ± 0.3 0.5 ± 0.6 0.4 ± 0.7 Blood GOT (SGOT) 62 ± 12 51 ± 23 48 ± 17 Alkaline Phosphotase 245 ± 14 289 ± 40 240 ± 45

After 30 hours of cold storage (2-4 ° C.) and normal heat reperfusion, the described preservative solution of the present invention was found to be superior to other well-known cold storage solutions based on bile production and enzyme value.

Test Example 3 Kidney Preservation Experiment

The preservation of kidney with the cold stock solution and the effect on renal function after reperfusion were investigated.

1) The kidneys of dogs were stored for 60 hours in Euro Collins (EC), UW and the present stock solution (MB). Renal function was measured during reperfusion into the IPK model using albumin containing craps-henselate solution at 37 ° C. over 90 minutes. Urine samples are collected every 30 minutes to produce GFR (creatinine clearance), urine / plasma protein (U / P), and sodium

Resorption (% Na) was measured and the results are shown in Table 6.

During reperfusion, both cold storage kidney groups reduced kidney function. Compared to the kidneys stored with EuroCollins and UW stocks, the kidneys with stocks of the present invention significantly improved GFR and sodium resorption during IPK (Table 6). This improvement in function means that the kidneys preserved in the preservative of the present invention can treat cold ischemic damage faster than the kidneys stored in known solutions.

Table 6 Comparison of GFR and Sodium Resorption Rates of Kidneys Stored in Each Preservative

30 minutes 60 minutes 90 minutes GFRuL / min.g contrast 230.5 ± 31.7 210.6 ± 47.5 125.4 ± 25.6 EC 29.6 ± 13.0 36.2 ± 18.3 45.0 ± 11.8 UW 37.3 ± 17.3 48.0 ± 20.1 67.5 ± 23.5 MB 59.3 ± 15.3 77.3 ± 23.3 87.3 ± 33.3 U / P contrast 0.17 ± 0.01 0.12 ± 0.02 0.18 ± 0.02 EC 0.41 ± 0.1 0.35 ± 0.1 0.31 ± 0.1 UW 0.58 ± 0.1 0.41 ± 0.2 0.33 ± 0.2 MB 0.38 ± 0.1 0.27 ± 0.1 0.29 ± 0.1 % Na contrast 101.5 ± 0.7 98.0 ± 0.4 99.1 ± 0.5 EC 65.3 ± 8.9 56.2 ± 0.1 49.4 ± 10.5 UW 85.7 ± 7.7 86.0 ± 6.9 89.8 ± 10.2 MB 88.1 ± 5.7 85.1 ± 12.7 92.7 ± 5.6

2) A series of five kidneys preserved in the preservation solution of the present invention for 60 hours were autografted. Serum creatine (mean ± SD) after transplantation is shown in Table 7.

Table 7 Serum creatinine content after transplantation (mean ± SD)

Work 0 One 2 3 4 5 6 7 8 9 10 Serum creatine mg% 1.3 ± 0.2 3.1 ± 0.7 2.5 ± 1.1 2.4 ± 1.0 2.2 ± 0.9 2.0 ± 0.8 1.6 ± 0.5 1.6 ± 0.4 1.4 ± 0.4 1.4 ± 0.3 1.2 ± 0.3

This experiment confirmed that the preservation solution of the present invention can excellently preserve the renal function even after 60 hours kidney storage.

Test Example 4 Preservation Experiment of Rabbit Heart

Two groups of rabbits weighing about 2.0kg without gender are divided into four groups, the first group is the Euro Collins solution as the control group, the second group is the UW solution, and the third group is the present invention preservation solution (MB). Used as.

The laboratory instrument was equipped with a constant-pressure Langendorff perfusion device, and the column containing Krebs-Henseleit (KH) buffer was maintained at a constant pressure of 150 cmH ₂ O, and 95% O ₂ and 5% CO ₂ The buffer solution was bubbled through the bubble tube with the mixed gas, and the pH was maintained in the range of 7.35 to 7.45.

The rabbit's surgery was performed by opening the abdominal cavity, elevating the scoliosis, cutting the rib cage along both sternums, and exposing the heart. The pericardium was removed and immediately placed in 4 ° C K-H solution to cool. The aorta of the rabbit heart was connected to the aortic tube of the Langendorf apparatus to perfuse K-H buffer at 37 ° C. to recover the heartbeat. Two pacemaker electrodes are placed on the front wall of the right ventricle, and a thin conduit with a latex balloon at the end of the pacemaker is inserted through the left atrium into the left ventricle to inflate the diastolic terminal pressure to 10 mmHg and connect to a pressure transducer. Left ventricular developing pressure (LVDP) was observed and recorded through a physiograph, and a pressure differential (dp / dt) was also recorded using a differential calculator.

After the heart of the extracted rabbit was in equilibrium state for 20 minutes, perfusion solution was shut off immediately, and the cardiac arrest solution at 4 ° C. was injected at a pressure of 50 cmH 2 O. As soon as the heartbeat was stopped, it was immediately removed from the Langendorf apparatus and stored in the planned experimental cardiac stock by simple cold deposition. After storage for 10 hours, the rabbit aorta was reattached to the aortic tube of the Langendorf apparatus, a pacemaker electrode, a late ventricle balloon of the left ventricle, and the like were re-perfused for 20 minutes while the myocardium was gradually warmed with K-H buffer at 37 ° C. If there were ventricular fibrillation, defibrillation was performed and the same heart rate as the equilibrium state was maintained to exclude errors due to differences in heart rate.

Cardiac function tests were performed to measure left ventricular pressure, perfusion volume, and pressure differential at 10 and 20 minutes of equilibrium and reperfusion periods, respectively. Pressure differential was analyzed by total pressure differential (total max dp / dt), maximum systolic pressure differential (+ max dp / dt), and maximum diastolic pressure differential (-max dp / dt).

The preservative solution of the present invention showed better recovery rate of left ventricular pressure, perfusion amount, and diastolic pressure differential value than the known cardiac preservative solution, and thus, it was confirmed that the preservative solution was beneficial for myocardial protection.

(Table 8) Recovery rate of cardiac function by cardiac preservation of left ventricular pressure (LVDP) and pressure differential dp / dt

Preservative Perfusion amount (ml / min) LVDP (mmHg) Total dp / dt + dp / dt -dp / dt EC 65.6 ± 14.1 67 ± 16.5 66 ± 17.5 77 ± 16.1 56 ± 20.6 UW 83.5 ± 11.9 71 ± 18.3 72 ± 12.5 75 ± 29.6 67 ± 11.8 MB 114.1 ± 21.4 92 ± 15.3 92 ± 14.5 96 ± 24.0 89 ± 12.5

From the above results, it was confirmed that the preservation solution of the present invention is excellent in preservation of kidney, pancreas, liver and heart and can be used for simple cold storage or continuous perfusion.

Test Example 5 Red Blood Cell Conservation Experiment

After collecting about 100ml of blood in a bag containing CPDA-1, AS-1 and the preservative of the present invention in a German shepherd (5 animals, average weight 30Kg), the preparation of red blood cells of AABB's guidelines (AABB) Red blood cells were prepared and stored at 4 ° C. according to standard guidelines. After 2 months, stored blood was thawed and transfused into the same dog to assess the presence of transfusion side effects and transfusion effects.

1) 100ml of blood was collected from each dog in each blood preservation solution, and then left in the refrigerator for 3 days to separate plasma and prepare concentrated red blood cells.

2) After 2 months, the preserved blood was taken out and thawed by shaking for 20 minutes in a 37 ° C water bath.

Wipe off the blood bag, and then slowly inject 25 ml of 12% NaCl solution within 30 seconds, shake by hand, and leave at room temperature for 2 minutes. Then, 25 ml of 0.9% saline added with 0.2% dextrose was slowly injected for 1 minute, mixed, and then left for 2 minutes.

3) The cell washer was washed two more times with 1000 ml of 0.9% saline added with 0.2% dextrose. The washed red blood cells were collected in a PVC bag and stored in a blood refrigerator.

4) The thawing erythrocytes were transfused for 2 hours to two dogs to be transfused over 4 hours. 24 hours after the transfusion, the transfusion dogs were checked for side effects, and blood was collected to evaluate whether there was a real transfusion effect.

(Table 9) Ratio of RBC status according to each preservative before and after refrigeration

CDPA-1 AS-1 Invention RBC / unit (mil) before refrigeration 455.2 456.5 452.2 RBC / unit (mil) after refrigeration 331.6 361.8 388.1 RBC recovery rate (%) 72.8% 79.2% 85.8% Osmotic pressure (mOsmol) 289 312 360 LDH (IU / L) 850 798 708 K ⁺ concentration (mEq / L) 8.8 8.5 10.2 plasma Hb (mg / dL) 680 760 834

Table 10. Effect of Red Blood Cell Transfusion on Each Preservative

CDPA-1 AS-1 Invention Blood transfusion (ml) 400 400 400 Hb (blood transfusion) 13.0 12.8 13.1 Hb (after blood transfusion) 11.5 11.2 13.5 Side Effect Loss of appetite Loss of appetite normal

Currently, red blood cell preparations are stored using CPDA-1 blood preservatives worldwide, in which case the maximum shelf life of blood is only 35 days, and up to 42 days using blood preservatives such as AS-1. The use of hydroxyethyl starch, various amino acids and polyamines as a protective agent used in the preparation of refrigerated red blood cells showed that it was superior to conventional preservatives by transferring intracellular moisture of red blood cells out of cells to prevent intracellular crystal formation and destruction of cell membranes. .

Criteria for evaluating safety in the manufacture of red blood cells are based on the standards set by the American Blood Bank Association. Red blood cell recovery should exceed 80% of red blood cells, and the osmotic pressure in the supernatant of thawed blood should be kept below 500 mOsmol / L. In the two-month cold storage experiment with the red blood cell preservative of the present invention, the red blood cell recovery rate and osmotic pressure were satisfied, and the state of the dog after blood transfusion was also excellent compared to the known preservative.

The preservative for preserving organs and blood according to the present invention provides 90 hours of preservation for the pancreas, 60 hours of preservation of the kidneys and at least 30 hours of preservation of the liver. Reduction of damage can control the deterioration of erythrocyte function.

Claims (2)

A preservative for preserving organs and blood, the polyamine comprises 0.01-2 mM putrescine, 1-500 μM spermidine, 1-500 μM spermine and a pharmaceutically acceptable carrier as an active ingredient. Composition for preservation and storage of organs or blood. The organ or blood composition for preservation and storage according to claim 1, which contains 0.1 to 10 mM of pyruvate, 0.1 to 10 mM of asparagine, and 0.1 to 10 mM of aspartic acid.