CA1282342C - Perfusate for the preservation of organs - Google Patents

Abstract

Abstract The present invention relates to new compositions for the preservation of organs prior to implantation comprising hydroxyethyl starch substantially free of ethylene glycol, ethylene chlorohydrin and acetone in a pharmaceutically acceptable organ perfusate.

Description

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Perfusate for the Preservation of Organs ackground of the Invention P~enal preservation 9 the ex v~vo storage of cadaveric kidneys, is a relatively new field~ Preservat;on of cadaveric 5 kîdneys ~or transplantation is common practice in hospitals;
however~ advances have been limited to trial and error expelimentation, l~though this approach has been partially successful from a clinîcal standpoint, the actual principles behind these ~uccesses are not well understood.
As renal transplantation has evolved from a strictlyr research procedure to an es~ablished clirucal therapy for end-stage renal disease, renal preservation has progressed from the laboratory research stage to an established clinical method. At present, the two most commonly use~ methods for 15 renal preservation are simple hypothermic storage and continuous perfusion. With simple hypothermic storage, the most common method of clinical renal preservation, the organs are removed from the cadaver donor and are cooled rapidly~
This is usually achieved by a combination of external cooling ~ and a short period of per~usion to drop the core temperature as quickl~ as possible. The kidneys are then stored, immersed in a flush-out solution in a simple plastic container, anc3 kept at a temperature of O to 4 by immer~;ing the container in ice. The advantages of this method are its 25 simplicity, its low cost, and the ease OI transportation of the organs. The composition of the flush-out solution to provide optimum preselvation ha~ been extensively studieù.

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The second method of renal preservation which has undergone extensive laboratory investigation, as wel] as clinical testing, is continuous pulsatile perfusiorl. 'rhe basic ingredients of continuous perfusion are ~1~ pulsatile flow, (2~
hypothermia, (3) membrane oxygenation9 and (4) a perfusate containing both albumin and lipids. With minor modifications, all presently used clinical preservation units share these basic principles~ There are se~eral advantages to continuous perusion in clinical transplantation. First, perfusion provides enough time to make cadaveric transplantation a partly elective procedure~ Second, it allows viability testing prior ~o implantation. A significan~ improvemen~ in the results of cadaveric renal transplantation could be expected if the preservation tîme could be extended to the 5 to 7 days required for present methods of mixed lymphocyte culture testing~
The ability to successfully prese~ve human kidneys for two to three days by either simple cold storage after inital flushing with an intracellular electrol~Yte solution or by o pulsatile perfusion with an electrolyte-protein solution has allowed sufficient time for histo-compatibility testing of the donor and recipient ~ kidney sharing among transplant centers t careful preoperative preparation of the recipient, time for preliminary donor culture results to become available, and vascular repairs of the kidney grant prior to implantation. Kidneys preserved ~or 72 hours using hypothermic pulsatile per~uslon with cryoprecipitated plasma proved to be a significant advance for human kidney preservation and is currently the preferred method of .
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preservation. Kidney organ preservation with ice-cold intracellular electrolyte flush solution followed by simple cGld storage has been satisfactorily employed for human kidney preservation ~or up to 61 hours.
Serum albumin, in various forms s is used exclusively for clinical organ preser~ration to produce the necessary oncotic pressure~ These forms include cryoprecipitated plasma, plasma protein fraction, human serum albumin, and silica gel-treated plasma. However, because these perfusates are prepaFed from naturally derived matel~a]s7 variation is unavo;dable. It would be particularly advantageous if a perfusate containing a synthetic colloid was available.
In the past, a large number of synthetic colloidal materials have been experimentally tested for effectiveness in kidney preservation. These colloids include dextrans, polyvinyl pyrrolidine s pluronics 9 hydroxyethyl starch ~HES) Ficoll, gum arabic, and polyethylene glycol. None of these were as effective as serum albumin . However ~ HES was effective ~or 24 hours OI preservatioll and in some cases for ~0 72 hours of preservation~ These colloidal materials were all tested in saline~based perfusates. Recently, excellent 72-hour preservation of canine kidney was observed with a perfusate containing gluconate anions in place of chloride with human serum albumin (HSA~ for colloid osmotic support.
In accordance with the present invention a method of preserving kidneys using a perfusate containing HES in place of human serum albumin is disclosed.

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As indicated hereinabove, serum albumin (HSA) based perfusates have been the standard for preservation of kidneys both experimentally and clinically for the past 17 years. Unfor~unately preservation periods of only three days 5 could be obtained with these types of perfusates. Although both of these methods preserve kidney viability for up to three days, longer preservation times are difficult to obtain consistently. Moreover, even though these methods preserve viability or up to three days, the }ddneys are damaged as 10 indicated by the elevated post-transplantatioll serum creatinine levels and time required to return those elevated le-rels to normal. Early perfusates were chosen from electrolyte solutions readily available for intravenous in~usion and were basically OI extracellular composition.
Heretofore, acceptable methods for renal preservation have not been available. Those that have been proven clinically effective are limited to short-term storage (three days~ and significantly reduced viability. The present invention describes the biochemical composition of the 2 0 perfusate best suited for the hypothermically perfused Icidneys and a novel synthetic colloid osmotic agent that yields significantly improved long-term preservation.
_rief Description of the Drawings FIG 1 is a comparison of human serum albumin and 25 hydroxyethyl starch perfusates on renal function after three days of perfusion preservation in accordance with the prior art;

FIG 2 shows the effects oiE dialysis of hydroxyethyl starch on post-transplant renal function after five days of perfusion preservation in accordance with the present invenffon, wi~h the creatinine level indicated;
FIG 3 shows the effects of dialysis of hydroxyethyl starch on post-transplant renal function after seven days of perfusion preservation in accordance with the present invention~
Detailed Description of the Preferred Em~odiment In accordance with the present invention the preferred colloid is hydroxyethyl starch having a weight average molecular weight of ~rom about 150, 000 to ahout 350, 000 daltons and degree of subsfftution of from about 0 . 4 to about Q. 7. A more preferred colloid is hydroxyethyl starch having a weight average molecular weight of from about 2û0, OOD to about 300, 000 daltons ~ In accordance with one embodiment of the present invention ~ the hydroxyethyl starch is dialyzed against distilled-deionized water or otherwise treated to remove several contaminants previously unknown to have an adverse affect on the efIectiveness of hydroxyethyl starch preparations. The materials removed by the dialysis process are the very smallest hydroxyethyl starch components, including the ethylene glycol and ethylene chlorohydrin side products ~f the hydroxyethylation as well as the resldual acetone and sodium chloride. Ethylene glycol and ethylene chlorohydrin are known to be toxic. Hence, their removal, e-ren if present in small amount, is desirable.

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In accordance with the present invention, the kidney perfusate composition includes, but is not limited to, the ~ollowing:
Table 1 5% hydroxyeth~rl starch having a molecular weight of from about 20û, 000 to about 300, 000 and a degree of ~u~stitu~iorl of ~Pom n . ~ to 0 . 7 0 25mM KH2PO4 3mM glutathlone 5mM adenosine lOmM glucose lOmM HEPES butter 5mM magnesium gluconate 1. SmM CaCl2 105mM sodium gluconate 200,000 units penicillin 40 units insulin 16mg l:)examethasone 12mg Phenol Red pH 7.4-7.5 The post-transplant serum creatinine levels in dogs receiving a kidney preserved ~or three days with the hydroxyethyl starch (HES) perfusate is compared with the albumin (HSA) perfused kidney in FIG 1 With HE5 as part of the perfusate, there is a slight elevatiorl of the serum creatine during the first 2-4 days post-transplant followed by a rapid return to normal.
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Extendirlg the preservation time with this basic perfusate was unsuccessi;ll until the starch was dialized pric>r to preparation of the perfusate. As shown in FIG 2, the dialyzed starch gave successful preser~ation although the kidneys exhibited somewha~ elevated seruM creatinine values.
Seven day preservatioll of kidneys was accomplished with the HES-based perfusate. As shown in Figure 3, five of 17 dogs (30%~ survived seven day preservation. In all 17 dogs, none showeà signs OI endothelial damage. Thus 9 the HES-based perfusate extend~ the preservation time beyond that whîch was possible with the HSA perfusate.
In additîon to the HES, the perIusate is radic~lly different from other commonly used perfusates. Chloride has been replaced with the larger molecular weight ~and less permeable) gluconate to suppress hypo~hermic induced cell swelling~ Adenosine and RO~ are included to stimulate ATP
syntheses. Glutathion is added to suppr0ss the loss of ~lutathione from perfused organs and to act as an antioxidant~ The K~ concentration is elevated to 25mM to suppress the loss of intracellular K at hypothermia and PO4 and HEPES are used as buffers. The role of some of these agents have been shown to be bene~lcial to kidneys during preservation.
Example 1 One hundred grams of hydroxyethyl starch was dissolved in one liter of distilled-deionized water to make a 10~ w /w solution . The HES solution was placed in dialysis bags (34 mm x 18 inches) having a molecular weight cut-off of 50, 000 daltons, placed in a 10-15 liter container of , : , : ' ' ,, ~. - . ., ' `
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distilled-deionized water, and stirred for 72 hours. The water was changed da~ly, and the HES was collected and frozen at -20~C unffl used.
Example 2 Adult mongrel do~s (15 to 25 kg~ were used in all cases. Surgical procedures and hypothermic pulsatile perfusion preservation were performed ~y conventional procedures. The composition of ~he perfus~2te is shown in Table l~ Kidneys were transplanted after 72 hours of preser~raffon followed by îmmediate contralateral nephrectomy.
Serum creatinine level was determined daily after the transplant~
~ccordingly, the present invention provides extended clinical organ preservation time and 9 as a synthetic colloid, minimizes the variation which results from perfusates prepared rom naturally derived mateIials.
The present invention has been described in detail and w~th particular reference to the preferre~ embodiments;
however, it will be understood by one having ordinary skill in the art that changes can be made thereto without departing from the spirit and scope thereof.

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SUPPLEMENTARY DISCLOSURE
Further to the invention hereinbefore described, it would bedesirable and advantageous to provide a preservation solution useful for all donor organs~ both for _ situ organ cooling in the donor and for co]d-storage after the organ is harvested.
According to a further feature of the present invention we provide a method of preserving organs using a perfusate or storage so]ution containing a specific synthetic hydroxyethyl starch (HES) in place of human serum a]bumin. A suitable l~ eomposition is provided. There is disclosed a cold-storage so]ution and perfusate that has provided 72 hour preservation for the pancreas, 48 hour storage for the kidney and at least 24 hour preservation for the liver.
Freezing and continuous aerobic perfusion are theoretically the on]y means of obtaining tru]y long-term preservation (from a month to years). Simple co]d-storage has a specific time ]imit beyond which the organ is no longer viable. Hypothermia decreases the rate at which intracellular enzymes degrade essential cellular components necessary for organ viability.
~ypothermia does not stop metabolism; rather, it simply slows reaction rates and ce]l death.
Calme, et al., Brit. Med. J. 1963; 2:651-655~ showed that the simple cooling of ischemic kidneys with cold blood preserved function for 12 hours. Collins (Lancet 1969; 2:1219-1222) showed that using an appropriate f]ush-out solution further increased storage time for kidneys by a factor of 3 (to 30 hours). Th`e failure of this solution to preserve other organs, such as the pancreas, liver, and heart, is be]ieved to be due to organ-specific metabolic differences.
To be appropriate and effective, the flush-out so]ution must have a composition that 1) minimizes hypothermic-induced cel]
swe]]ing, 2) prevents intracellu]ar acidosis, 3) prevents the _ 9 _ ; , '':
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~ ~323~2 expansion of extrace]lular space during the flush-out period, 4) prevents injury from oxygen-free radicals, especial]y during reperfusion, and 5) provides substrates for regenerating high-energy phosphate compounds during reperfusion.
Hypothermic-induced cell swelling is due to the accumulation of water. This tendency to swell can be co~mteracted by adding 110 to 140 mmol (110 to 140 mOsm/kg osmotic force) of substances that are impermeable to the cell (impermeants). This concentration of impermeants approximately equals the 1~ c~ncentration of glucose in Collins co]d-storage solution (120 mM) and of impermeants in other cold-storage solutions. Thus a key component of successful cold-storage so]utions is the appropriate concentration of an effective impermeant.
A second important consideration for successful cold-storage is the prevention of intracellular acidosis. Ischemia, even in the cold, stimulates glycolysis and glycogeno]ysis (Pasteur effect); it also increases the production of lactic acid and the concentration of hydrogen ions. Tissue acidosis is fatal to cells and can induce lysosomal instability, activate lysosomal enzymes, and alter mitochondrial properties. The prevention of intracellular acidosis is, therefore, an important prerequisite Eor good preservation. Some studies have shown that the effective buffering of cold-storage solutions or the use of f]ush-out solutions with an alkaline pH improves the storage of livers (Lie, TS et al., Transplant Proc. 1984, 16:134-137) and pancreases tAbstract, Am. Soc. of Transplant Surgeons 13th Annual Meeting, May 28-29, 1987).
An effective co]d flush-out solution must prevent the expansion of the extracel]ular space, expansion that can occur during the _ situ flushing of donor organs and after the organs have been harvested. Such expansion can compress the capillary system and cause the flush-out solution to be poorly distributed , 34~
in the tissue. Most cold-storage solutions do not contain substances that exert oncotic support (albumin or other colloids). The components of the f]ush-out solution, therefore, rapidly diffuse into extrace]lu]ar spaces and cause tissue edema.
Thus, the ideal _ situ f]ush-out solution should contain substances that create colloidal osmotic pressure, and allows the free exchange of essential constituents of the f]ush-out solution without expanding the extracellular space.
A fourth important consideration for effective co]d-storage 1~ is injury from oxygen-free radicals during reperfusion; but the exact role of these agents is sti]l unclear. It is believed that oxygen-free radicals may be of little significance in human livers and kidneys because endogenous xanthine oxidase has a relatively low activity compared with the high endogenous activity of superoxide dismutase, which scavenges superoxide anions. In contrast, injury induced by oxygen-free radicals may be extremely important in lungs and intestines, which are sensitive to such damage.
A final important consideration is energy metabolism.
~ Adenosine triphosphate (ATP) rapidly degrades during hypothermic storage, and this degradation results in the formation of end products (adenosine, inosine, and hypoxanthine) to which the plasma membrane is freely permeable. Organ reperfusion necessitates the rapid regeneration of Na-pump activity, which requires ATP. The availability of ATP precursors, therefore, may be important for successfu] organ preservation.
There are important differences in the metabolism of the kidney, liver, and pancreas, and these differences may influence how well these organs are preserved. The suppression of cell swe].ling necessitates the use of an effective impermeant.
Glucose, the main impermeant in Collins so]ution is not effective for the liver or pancreas and readily enters cells, Southard, et ~323~L2 al., Cryobiology 1986; 23:477-~82. Mannitol, another commonly used impermeant, is about as permeab]e as glucose in the liver.
Thus, one reason why co]d-storage solutions that depend on glucose or mannitol are not effective for the ]iver and pancreas is that these solutions do not contain effective impermeants.
In accordance with the further feature of the present invention, the solution for preservation of organs contains the anion lactobionate and raffinose as impermeants to the cell, has a so]ution osmolality of about 320 mOsm/L, K+ of 120 mM and Na+
l~ o 30 mM. The preferred col]oid is a modified hydroxyethyl starch having a weight average molecular weight of from about ~50,000 to about 350,000 daltons and degree of substitution of from about 0.4 to about 0.7. A more preferred colloid is hydroxyethyl starch having a weight average molecular weight of from about 200,000 to about 300,000 daltons. The preferred colloid is substantially free of hydroxyethy] starch having a molecular weight of less than about 50,000 daltons.
In accordance with one embodiment of the present invention, the hydroxyethyl starch is dialysed against distilled-deionized ~0 water or otherwise treated to remove several contaminants previously unknown to have an adverse affect on the effectiveness oE hydroxyethyl starch preparations. The materia]s removed by the dialysis process are the very smallest hydroxyethyl starch components, including the ethylene glycol and ethylene chlorohydrin side products of the hydroxyethylation as wel] as the residual acetone and sodium chloride. Ethy]ene g]ycol and ethylene chlorohydrin are known to be toxic. Hence, their removal, even if present in small amount, is desirab]e.
In a preferred embodiment, the preservation solution and perfusate composition includesj but is not limited to, the following:

~ ~ ~2 3 SubstanceAmount in 1 Liter K+-lactobionate 100 mmol ~H2 PO4 25 mmol MgSO4 5 mmo]
Raffinose 30 mmol Adenosine 5 mmol Glutathione 3 mmol 10 Insulin 100 U
Bactrim 0.5 mL
Dexamethasone 8 mg Allopurinol 1 mM
Hydroxyethyl starch having a molecular 50 g weight of about 200,000 to about 300,000 daltons and a degree of substitution of from about 0.4 to 0.7 The solution is brought to pH 7.~ at room temperature with NaOH. The final concentrations are Na+ = 30 + 5 mM, K+ - 120 + 5 mM, mOsm/liter = 320 _ 5. Bactrim = trimethoprim (16 mg/mL) and sulfamethoxazole (80 mg/mL). The hydroxyethyl starch can be present in the range of about 3 to 8%.
Accordingly, the present invention provides extended clinical organ preservation time and, as a synthetic col]oid, minimizes the variation which results from perfusates prepared from naturally derived materials.
The present invention has been described in detail and with particular reference to the preferred embodiments; however, it will be understood by one having ordinary skill in the art that changes can be made thereto without departing from the spirit and scope thereof.

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PREPARATION OF HYDROXYETHYL STARCH (HES) . . . ~
One hundred grams of hydroxyethyl starch were dissolved in one liter of distilled-deionized water to make a 10% w/w solution. The HES so]ution was placed in dialysis bags (34 mm x 18 inches) having a molecular weight cut-off of 50,000 daltons, placed in a 10-15 liter container of distil]ed-deionized water, and stirred for 72 hours. The water was changed daily, and the ~ES was col]ected and frozen at -20C until used.
l~ EXAMPLE 4 Female mongrel dogs weighing 15-25 kg were used for the experiment.
Operative procedure. Anaesthesia was induced with pentathol and maintained with halothane. Through a midline incision, the left segment (tail) of the pancreas was harvested as previously described. The graft with the spleen attached was transp]anted to the iliac vessels either immediately after flush-out (control) or after 48 and 72 hours of cold-storage. The pancreatic duct ~0 was left open allowing pancreatic juices to drain freely into the abdominal cavity. No anticoagulants were used. The right segment of the pancreas was removed at the time of transp].antation.
Experimental protocol. All dogs received 0.5 g Mandol I.V.
before harvest and during the first three days posttrans-plantation. Dogs were fed a standard dog food diet containing Viokase. The animals were divided into three groups. Group I
(control): After harvesting and washout, the grafts were immediately transpLanted; group 2 (48-hour co]d-storage); group 3 (72-hour cold-storage). Blood glucose concentration was determined daily during the firs-t posttransplant week, and biweekly thereafter. Intravenous glucose tolerance test (IVGTT) ~ 2 ~Z 3~
was performed 24 hours, 2 weeks, and 4 weeks after transp]anta-tion. The grafts were removed after 4 weeks, and 2-3 days ]ater and IVGTT was performed. For the IVGTT, glucose (0.5 g/kg body weight) was injected and b]ood g]ucose determined after 1, 5, 10, 20, 30, 60~ 90, and 120 minutes. The K value was calculated from the blood g]ucose concentration obtained from the 5-60 minute measurements (9). Glucose va]ues greater that 150 mg% for more than 2 days and a l~ va]ue less than 1.0 were considered signs of diabetes.
Preservation. The composition of the preservation solution is shown in Tab]e 2. After remova], the pancreas was f]ushed with approximately 250-300 mL of flush-out solution from a height of 60 cm. The graft was placed in a double plastic bag, covered with preservation solution, and placed in an ice-water bath.
Statistics. Statistical evaluation was made using the student's T test. The values given are means + SEM.
RESULTS
All grafts were well perfused immediately following transplantation. In the preserved grafts, varying degrees of intralobu]ar edema deve]oped after 5-10 minutes of reperfusion.
The sp].een was we]l perfused in al] cases. As shown in Table 3, five dogs died; three in the control group and two in group 3 (72-hour preserved). The causes of death were unrelated to the transplant and all dogs died with functioning grafts.
At the time of pancreatectomy, al] grafts (even controls) showed various degrees of fibrosis, as did the sp]een. Arterial and venous thrombosis was not evident in any of the grafts.
Posttransplant blood g]ucose values and results of IVGTT are shown in Table 3 for each animal. The mean values (+SEM) for each group studied are also shown in Table 3. The mean blood glucose value during the first posttransplant week was highest in group 3 i~ .
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(124+6 mg~) and this value was significantly different (P<0.05) when compared with values for group 1 (94+7 mg~) and group 2 (10+7 mg%). The mean K value on day 1 was also significantly lower (P<0.05) in group 3 (185+0.15~) compared with group 1 (2.44+0.14%) and group 2 (2.53~0.22%). In group 3, the K values tested on days 14 (1.7+0.1~) and 28 (1.61+0.19~) remained similar to the K value on day 1 (P=NS).
In groups l and 2, the K values declined and at 2 weeks posttransplant; there were no significant differences between the three groups. By the fourth week, the K value for group 3 was better than for group 2, but somewhat lower when compared with the control group (statistical significance could not be shown because of the small numbers in group 1).
All dogs became hyperglycemic (blood glucose greater than 200 mg~ following removal of the pancreas indicating that the transplanted organ was solely responsible for glucose homeostasis. Four animals from group 3 where observed for long-term survival. One dog died 7 weeks posttransplant from pneumonia, but remained normoglycemic.
Two animals were sacrificed at 3 and 4 months, and one dog was kept for 6 months. All showed no signs of diabetes and were normoglycemic.

24 HOUR LIVER PRESERVATI~:)N
Clinical liver preservation is limited to about 6-10 hours and increasing this to 24 hours or more could have significant impact on liver transplantation. The isolated perfused rabbit liver was used to assess the quality of preservation following cold storage in collins solution, Cambridge plasma protein fraction (PPF), Marshalls solution, and the solution of the present invention (preservation solution). Bile produ~tion during normothermic perfusion of cold stored livers was the most useful parameter of viability and the rate of bile production ~mL/100 gm/hr ~ S~) in control vs 24 hour cold stored rabbits livers is shown in the table.

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Control Cambridge PPF Eurocollins Marshalls Preservation Solution 5.4+1.7 1.8_+ 0.9 1.9 + 1.3 3.1~0.5 4.4 + 0.5 The described preservation solution was superior to other cold storage solutions on the basis of bile production after 24 hours cold storage (2-4C) and normothermic reperfusion.
The ultimate test of successful liver preservation is the transplantation model. The preservation solution was, therefore, tested in the canine orthotopic liver transplant model. Following simple flush perfusion with the solution, three consecutive canine livers were stored for 24 to 26 hours. Transplantation was performed using a combined "cuff"
and suture technique. All livers immediately took on a satisfactory appearance and all three dogs woke promptly and were on their feet within 4 hours of the end of the procedure.

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Platelet counts were normal 6 hours postoperatively.
Bilirubin and enzyme values 6 hours and the subsequent 7 days are recorded in the table (mean + SD) and show a rapid return of normal liver function. One dog died on postoperative day 5 due to intussusception.

6 Hrs Day 1 Day 3 Day 5 Day 7 Bilirubin my % 0.6+0.3 0.7+0.6 0.9+0.7 0.5+0.4 0.4+0.2 SGOT 2148+983 1835+1145 61+16 55~40 45+21 Alk. Phos. 186+14 217+47 273+126 311+64 315+48 KIDNÆY PRESERVATION
Based on this experience we have investigated the potential usefulness of the described cold storage (CS) solution for kidney preservation and its effect on renal function after reperfusion was investigated: 1) in the isolated perfused dog kidney model (IPK); and 2) in the canine autotransplant model.
1) Dog kidney~ were either cold stored for 48 hours in Eurocollins (EC) or the described cold storage solution (CS).
Kidney function was determined during reperfusion with the IPR model using an oxygenated modified albumin containing Krebs-Henseleit solution at 37C over a period of 90 minutes.
Urine samples were collected every 10 minutes and analyzed.
GFR tcreatinine clearance), urine/plasma protein (U/P) and fractional sodium reabsorption (% Na) were calculated.
\ Results are shown in Table 4 as mean~ with standard deviations in parenthesis.
~oth cold stored kidney groups had decreased renal function at time of reperfusion (compared to control kidneys), In contrast to EC-stored kidneys, CS-stored kidneys improved GFR and sodium reabsorption significantly during IPK. This improvement in function suggests that kidneys preserved in CS are able to repair cold ischemic damage more rapidly than kidney~ stored in EC.

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2) Eight consecutive dog kidneys preserved for 48 hours in CS have been autotransplanted. Three animals were sacrificed due to technical complications (arterial thrombosis, intussusception. Posttransplant serum S creatinines (mean + SD) of the five survivors are shown in Table 5.
The study indicates good preservation of renal function for 48 hours of cold storage with the CS solution. This solution, therefore, is capable of preserving the kidney, pancreas and liver and can be used for either simple cold storage or continuous perfusion.

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Claims (24)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A perfusate for the preservation of organs intended for implantation in an animal comprising:
5% hydroxyethyl starch 25mM KH2PO4 3mM glutathione 5mM adenosine 10mM glucose 10mM HEPES butter 5mM magnesium gluconate 1.5mM CaCl2 105mM sodium gluconate 200,000 units penicillin 40 units insulin 16mg Dexamethasone 12mg Phenol Red plt 7.4-7.5 wherein the hydroxyethyl starch is substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone; and the perfusate has an osmolality of about 320 mOSm/l.

2. The perfusate of Claim 1 wherein the hydroxyethyl starch has a weight average molecular weight of about 200,000 daltons and a degree of substitution of from about 0.4 to about 0.7.

3. A perfusate for the preservation of organs intended for implantation in an animal comprising:
a pharmacologically acceptable perfusate solution having an osmolality of about 320 mOSm/l; and 5 weight percent hydroxyethyl starch, wherein said starch is substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone and has a molecular weight of about 200,000 daltons.

4. A composition of matter comprising hydroxyethyl starch having a molecular weight of from about 150,000 to about 350,000 daltons, degree of substitution of from about 0.4 to about 0.7, and being substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone.

5. The composition of matter of Claim 4 wherein the hydroxyethyl starch has a molecular weight of from about 200,000 to about 300,000 daltons.

6. A perfusate for the preservation of organs intended for implantation in a patient requiring such implantation, including:
5% hydroxyethyl starch having a molecular weight of from about 200,000 to about 300,000 25mM KH2PO4 3mM glutathione 5mM adenosine 10mM glucose 10mM HEPES buffer 5mM magnesium gluconate 1.5mM CaCl2 105mM sodium gluconate 200,000 units penicillin 40 units insulin 16mg Dexamethasone 12mg Phenol Red pH 7.4-7.5 wherein the hydroxyethyl starch is substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone; and the perfusate has an osmolality of about 320 mOSm/l.

7. The perfusate of claim 6 wherein the hydroxyethyl starch has a weight average molecular weight of about 200,000 daltons and a degree of substitution of from about 0.4 to about 0.7.

8. A perfusate for the preservation of organs intended for implantation in a patent requiring such implantation, including:
a pharmacologically acceptable perfusate solution having an osmolality of about 320 mOsm/l; and 5 weight percent hydroxyethyl starch, wherein said starch is substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone and has a molecular weight of about 200,000 daltons.

9. The perfusate of claim 8 wherein the solution includes a gluconate salt.

10. The perfusate of claim 9 wherein the solution includes an electrolyte for maintenance of cell viability and a drug to minimize infection.

11. The perfusate of claim 10 including glucose and insulin.

12. A method for extending the preservation of organs intended for implantation, said method comprising infusing said organ with a perfusate including:
a pharmacologically acceptable perfusate solution having an osmolality of about 320 mOsm/l; and 5 weight percent hydroxyethyl starch, wherein said starch is substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone and has a molecular weight of about 200,000 daltons.

13. A composition of matter comprising hydroxyethyl starch having a molecular weight of from about 150,000 to about 350,000 daltons, degree of substitution of from about 0.4 to about 0.7, and being substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone, and being substantially free of hydroxyethyl starch having a molecular weight of less than about 50,000 daltons.

CLAIMS SUPPORTED BY SUPPLEMENTARY DISCLOSURE

14. A solution for the preservation and storage of organs intended for implantation in a patient requiring such implantation, comprising:
a pharmacologically acceptable storage solution having a solution osmolality of about 320 mOsm/liter; and about 5 weight percent hydroxyethyl starch, wherein said starch is substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone and has a molecular weight of from about 150,000 to about 350,000 daltons.

15. The solution of claim 14 wherein the starch is substantially free of hydroxyethyl starch having a molecular weight of less than about 50,000 daltons.

16. The solution of claim 15 which includes an electrolyte for maintenance of cell viability.

17. The solution of claim 16 which includes a lactobionate salt.

18. The solution of claim 17 including raffinose.

19. A method for the preservation and storage of organs intended for implantation in a patient requiring such implantation, said method comprising:
flushing said organ with a solution having an osmolality of about 320 mOsm/liter and about 5 weight percent hydroxyethyl starch, said starch being substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone and having a molecular weight of from about 150,000 to about 350,000 daltons; and storing said organ in said solution until implantation in said patient.

20. The method of claim 19 in which the starch in said solution is substantially free of hydroxyethyl starch having a molecular weight less than about 50,000 daltons.

21. The method of claim 20 in which the solution includes an electrolyte for maintenance of cell viability.

22. The method of claim 21 in which the solution includes a lactobionate salt.

23. The method of claim 22 in which the solution includes raffinose.

24. The method of claim 23 in which the solution includes glutathione.