CA2055827C

CA2055827C - Solutions for the perfusion, preservation and reperfusion of organs

Info

Publication number: CA2055827C
Application number: CA002055827A
Authority: CA
Inventors: Philippe Menasche
Original assignee: Pasteur Merieux Serum et Vaccines SA
Current assignee: Imtix Sangstat
Priority date: 1990-11-20
Filing date: 1991-11-19
Publication date: 1999-06-15
Anticipated expiration: 2011-11-19
Also published as: CH683485A5; GB2249937A; ITMI912991A0; IT1251995B; DE4138040A1; ITMI912991A1; GB9124488D0; AT399440B; ATA228791A; ES2050580B1; CA2055827A1; ES2050580A1; GB2249937B; BE1006834A3

Abstract

Perfusion and preservation and/or reperfusion solutions for surgery and organ transplantation, including that of the heart, a feature of which solutions is the inclusion of at least one antioxidant compound, such as a trapping agent for free oxygen radicals, in particular glutathione in the reduced state or N-acetylcysteine, said solutions possessing a zero or greatly reduced partial pressure of oxygen which is maintained substantially at this value up to the time of use. The solution is preferably presented in flexible bags impermeable to oxygen.

Description

2~~~~~~
Solutions for the perfusion, preservation and reperfusion of organs.
The present invention relates to solutions for the perfusion, preservation {or storage) and/or reperfusion of organs, including those employed in heart transplantation. It also relates to a method for using these solutions applied in the different phases of a transplantation.
One of the- main causes of failure of heart transplants originates from the risks of degradation, or even of necrosis, of the graft, which manifest themselves during reoxygenation of the transplanted organ and which are linked to the ischemia, generally prolonged, occur ring between initiation of the explantation from the donor and completion of the implantation in the recipient.
An ischemia of four to five hours constitutes, for example in the case of the heart, the upper tolerable limit, and does not rule out a large number of accidents.
To limit this risk, many authors have proposed and used protective solutions, both for perfusion of the organ to be explanted and for its preservation at low temperature and its reperfusion during transplantation.
Examples of these solutions are the following solutions:
Bretschneider's HTK
~ Collins ~ St. Thomas ~ UW
~ Stanford These solutions, however, possess only limited advantages, and afford at most only a partial protection against the risks which appear during reperfusion, and which are attributed in part to the metabolic production of free oxygen radicals produced in copious amounts, in particular during reoxygenation of the ischemic organ.
The risk of oxidative cell and membrane degra-dations originating from the production of these radicals has been the subject of several studies in the field of myocardial protection by cardioplegia. These various investigations have suggested the introduction into the protective solutions used of substances capable of counteracting the production or the effect of free radicals, and in particular antioxidant substances. Various compounds have been proposed, some, such as deferoxamine, allopurinol, catalase and peroxidase, as being capable of counteracting free radical production, others such as superoxide dismutase being capable of destroying these radicals, yet others such as vitamin E or equivalent (Trolox*) being capable of "neutralizing" the free radicals.
These latter compounds also include molecules bearing thiol groups, such as N-acetylcysteine or reduced glutathione (GSH), which has been considered to be a free-radical "trapping" agent (scavenger). However, the literature is divided on the value of glutathione.
Thus, G.W. Standeven et al., in J. Thorac.
Cardiovasc. Surg. 1979, 78, 893-907 Cold-Blood potassium cardioplegia, found little difference in the level of protection afforded by the addition of glutathione.
In contrast, M. Bernier et al., in Reperfusion-induced Arrhythmias and Oxygen-derived Free Radicals, Circulation Research, Vol. 58, no. 3, March 1986, 331-340, find that the addition of L-methionine, super-oxide dismutase, catalase, mannitol, glutathione or deferoxamine to the perfused isolated rat heart reduces the risk of fibrillation or of ventricular tachycardia during reperfusion.

-2a-J.C. Chatham et al., in Depletion of Myocardial Glutathione: Its effects on heart function and metabolism during ischaemia and reperfusion, Cardiovascular Research, 1988, 22, 833-839, concludes that a depletion of glutathione during ischemia of rat hearts does not appear to result in a worsening of the metabolic impairment.
A Blaustein et al., in Myocardial Glutathione Depletion Impairs Recovery After Short Periods of ' :_~'~ 20497-654 ~0~~~~'~

Ischaemia, Circulation, Vol. 80, no. 5, November 1989, conclude that a depletion of glutathione in the isolated rat heart, obtained by injection of diethyl maleate, leads to a poor recovery of systolic function, and that an improvement may be obtained in the case of reperfusion with a solution supplemented with glutathione.
A. Singh et al., in Relation Between Myocardial Glutathione Content and Extent of Ischaemia - Reperfusion Injury, Circulation, Vol. 80, no. 6, December 1989, 1795-1803, show an increase in the GSH content in pigs perfused intravenously with glutathione five minutes before and during cardiac reperfusion, and find an improvement in the local recovery.
W.N. Wicomb et al., in Role of Glutathione in 24 hour Heart Storage by Microperfusion Using a New Poly ethylene Glycol Solution, J. Mol. Cell. Cardiol. 22 (Supplement V) 1990, p. 82, report an improvement in the recovery of the isolated rabbit heart preserved in a protective solution comprising GSH glutathione, the simple addition of glutathione during reperfusion not being effective.
V. Kantamneni et al. , in Extended Preservation of Canine Myocardium Using UW Solution, J. Mol. Cell.
Cardiol. 1990 (Suppl. V); 22:22 (Abstr), conclude that solutions (UW solutions and modified UW solutions) containing glutathione, which show some degree of efficacy in the preservation of isolated organs such as liver, kidney and pancreas, do not bring about signifi-cant improvements compared to modified Collins solution not containing this compound, and that these solutions were unable to permit a significant increase in the period of preservation of the heart in dogs.
The addition of N-acetylcysteine is studied by M.B. Forman, Glutathione Redox Pathway and Reperfusion Injury, Circulation, Vol. 78, no. 1, July 1988, 202-213.
He suggests that a treatment with N-acetylcysteine (NAC) before reperfusion can improve postischemic recovery.
While it may hence appear advantageous to use substances acting against the production or the effect of 2(~~j'~~'~

free radicals in the myocardium in the context of cardioplegic protection, the choice of compound and the procedure for its use do not appear to be obvious, and the addition of these compounds, including glutathione, to myocardial perfusion and reperfusion solutions in daily hospital practice has failed to yield decisive results.
Ph. Menasch~ et al., in les Pi~geurs de Radicaux Libres dans la Protection Myocardique en Chirurgie Cardiaque [Free-Radical Trapping Agents in Myocardial Protection in Cardiac Surgery], Ann. Cardiol. Ang~iol;
1986, 35 (no. Ibis), 447-452, conclude, however, that the preservation of postischemic left ventricular function, due to a given cardioplegic solution, could be signifi-cantly improved by adding antioxidants capable of pre-venting the formation of free radicals or of destroying or neutralizing them. In contrast, the choice of the most effective antioxidant from among the many candidates, including superoxide dismutase SOD, peroxidase and glutathione, is not obvious, not to mention the possible side effects or toxic effects. A fortiori, when we turn from the field of cardioplegia, in which the periods of ischemia are relatively short, to the field of transplan-tation, the literature provides no genuinely useable information about the choice and procedure for use of genuinely effective protective solutions.
An objective of the present invention is to solve these problems and to provide exceptionally effective protective solutions for the preservation of organs for the purpose of surgical operations and especially of transplantation. The organs in question comprise the heart, as well as the other organs, and in particular the liver, lung and kidney.
The present invention provides, to this end, for a perfusion and storage solution for the explanted organ and a reperfusion solution for the organ undergoing implantation, a feature of both solutions being the inclusion of at least one antioxidant compound which can be a trapping agent for free oxygen radicals, said 2Q~~

solutions possessing a zero or greatly reduced partial pressure of oxygen which is maintained substantially at this value up to the time of use.
As a special preference, the free-radical sca~en-ger- compound is a compound comprising thiol functions.
Preferred thiols include glutathione in the reduced state (GSH) or its precursors or related substances, and in particular N-acetylcysteine (NAC), glutathione analogs and in particular glutathione monoester.
Other compounds containing a thiol function may be used, in particular diethyldithiocarbamate, its analogs and derivatives, as well as converting enzyme inhibitors.
Another compound which is useful in the invention is vitamin E or its analogs or derivatives.
According to the invention, the solutions are prepared and stored protected from aerial oxygen, being, for example, prepared in the form of outgassed solutions, preferably under a nitrogen atmosphere. The storage and preservation of the solutions according to the invention are carried out in airtight containers such as bottles or, preferably, airtight bags made of plastic, for example made of laminated composites of a type known per se.
Advantageously, the solutions according to the invention can also contain, apart from free-radical inhibitory thiols, a compound counteracting radical formation, such as metal chelators and especially deferoxamine (INN).
Reduced value of the oxygen concentration accord-ing to the invention is preferably understood to mean a maximum concentration of dissolved oxygen of less than 0.1 ppm.
In the case where glutathione is used, the reduced glutathione content of the solution, is preferably of the order of 0.5 to 10, and advantageously of the order of 3, mmo1/l. In the case where the thiol is NAC, the content is preferably of the order of 10 to 80, and advantageously of the order of 40, mmol/1.

2~~~~~'~

The invention is preferably implemented in different forms, depending on whether it is applied to the perfusion and preservation of the explanted organ, or to the reperfusion of the implanted organ.
In the case of a perfusion and preservation preparation according to the invention, the solution is made up so as to prevent the formation of cell edema and the appearance of oxidative lesions while limiting the calcium overload. Furthermore, for some organs, and in particular the heart, the solution is capable of playing the part of a metabolic inhibitor.
Advantageously, the calcium content is low, preferably below 0.5 mM, and it is preferable for the solution to contain magnesium, preferably at a content above 10 mM. In addition, a lower pH, in particular 7.40 ~ 0.40, is preferred. In the case where the per-fusion and preservation solution is intended for the heart, a potassium concentration preferably equal to at least 10 mM is provided, it being possible for the potassium, where appropriate, to be absent for the other organs.
In an especially effective and advantageous embodiment of the invention, a solution for the perfusion and preservation (storage) of the heart according to the invention contains the following compounds:
A Perfusion and storage solution (1) Constituent Concentration (mmol/liter) Na+ 90-120 Mgr 10-20 Cap 0.005-1.2 C1' 100-160 Mannitol 50-200 Glutamate 4-26 or GSH 0.2 to 0.5-10 Osmolarity 270-450 (for example 370) mOsm/1 pH 7.40 ~ 0.40 (at 20°C) 2~~~~~~

In the case of a reperfusion preparation accord-ing to the invention, the solution is made up so as to continue to limit cell edema and oxidative lesions. It is also contrived so as to reestablish calcium homeostasis.
The preferred pH is 7.70 ~ 0.30. In the case of the heart, it is made up so as to prolong metabolic inhi-bition, and will retain potassium at a concentration preferably above 10 mM.
Thus, in an advantageous embodiment, the reper-fusion solution contains the following compounds:
B Reperfusion solution (2) Constituent Concentration ~mmol/liter~
R+ 10-30 Na+ 90-120 Mgr 0-20 Cap 0.005-1.2 Mannitol 50-200 Glutamate 4-26 or GSH 0.2 to 0.5-10 Osmolarity 270 to 450 (for example 370) mOsm/1 pH 7.70 ~ 0.30 at 28°C
The preferred embodiment of the perfusion and preservation solution is:
C Perfusion and storage solution (1) Constituent Concentration (mmol/liter) R+ 12 Na+ 10 0 3 0 Mgr'" 13 Cap' 0.25 C1- 110 . 4 Mannitol 109.8 Glutamate 20 GSH 0.5 to 3 Osmolarity 370 mOsm/1 _ 2~~~°~
_8_ pH 7.40 (at 20°C) and the preferred embodiment of the reperfusion solution is D Reperfusion solution (2) Constituent Concentration ~(mmol/literl K+ 14.9 Na+ . , 10 0 Mg** _ _ _ Cap"' 1.2 C1- 97 . 5 Mannitol 136 Glutamate 20 GSH 0.5 to 3 Osmolarity 370 mOsm/1 pH 7.70 at 28°C
Naturally, the compounds thus defined may be replaced by compounds having equivalent functions, the molar contents preferably remaining substantially unchanged.
Thus, glutamate, whose function is to stimulate anaerobic energy production by myocardial cells, may be replaced, in particular, by aspartates, succinates, fumarates and malates.
Mannitol, an impermeant compound whose function is to limit edema, may be replaced by other substances playing the part of an impermeant compound in the inter stitial medium, increasing osmotic pressure, such as lactobionate (by reducing proportionately the chloride concentration), raffinose or sucrose. Since the chosen impermeant substance must not be metabolized or taken up by the organ, mannitol is suitable for the heart whereas it will be ruled out for the liver.
The relatively acid pH of the perfusion and preservation solution, preferably of the order of 7.40 at 28°C, is preferably produced without a buffer.
The pH of the reperfusion solution, preferably adjusted to 7.70 at 28°C, may be optionally produced using a buffer (in particular bicarbonate, histidine).

2~~~~~~
_ g _ Preferably, the two solutions according to the invention are presented, in a single package, in the form of one or more containers per solution, preferably deformable bags having airtight walls, of total volume 1500 to 2000 ml.
The subject of the invention is also a method for using the solutions according to the invention for heart transplantation, wherein cardiac arrest of the organ to be explanted is induced by perfusion of the perfusion and preservation solution for a few minutes, wherein the organ removed is placed in a container, bag or bottle filled with said solution protected from the air for preservation at low temperature, wherein the organ is perfused again using said solution during the grafting of the transplanted heart, and wherein the graft is then reperfused after the graft has been installed, this time using the reperfusion solution according to the inven-tion, for a period preferably of the order of 5 minutes, after which the systemic circulation is reestablished.
The subject of the invention is also a method of use for the transplantation of organs other than the heart, and in particular liver, kidney and lung, wherein perfusion of the organ to be explanted is performed for a few minutes with the perfusion and preservation solution, the subsequent operations of preservation and reperfusion being similar to those in the abovementioned case of the heart.
Other advantages and features of the invention will become apparent on reading the description which follows, given as an example without implied limitation.
1 Preparation of the perfusion and preservation solution An outgassed, sterile, pyrogen-free aqueous medium is prepared under conditions of protection from atmospheric oxygen, and having the composition C.
This composition possesses on average a maximum concentration of dissolved oxygen < 0.1 ppm and a pH of 7.40 at 20°C. This solution is packaged in plastic bags impermeable to atmospheric oxygen, or in plastic bags - to -which are permeable to oxygen but are themselves contained in a bag of a plastic/aluminum complex which is impermeable to atmospheric oxygen.
2. Preparation of the reperfusion solution Taking the same precautions as in Example 1, a reperfusion solution having the composition D is prepared.
This composition can contain a bicarbonate or histidine buffer maintaining the pH at 7.70 at 28°C.
This solution is placed in similar bags of volume 100 ml.
3. Test of solutions containincr NAC on isolated rat heart preparations Fifty isovolumic preparations of isolated hearts from Sprague-Dawley rats weighing 300 grams were used, the hearts being connected rapidly to a non-recirculating Langendorff perfusion column to establish a retrograde perfusion using an oxygenated (95~ Oz, 5~ COZ) Krebs Henseleit buffer, to establish a retrograde perfusion at a pressure of 100 cm of water. The left ventricular pressure, its derivative and the end-diastolic pressure were recorded continuously. The coronary flow rate was measured by noting the venous coronary flow rate. Left ventricular stimulation was maintained at a frequency of 320/min.
After a twenty-minute monotoring period, thirty hearts were arrested by perfusion using the preservation and perfusion solution at 4°C, and then rapidly placed in glass containers filled with the same solution and surrounded by ice. The hearts were maintained therein for five hours, with a mean myocardial temperature of 2°C at the end of storage. The hearts were then reconnected to the perfusion circuit and subjected to an additional ischemia for one hour at between 15 and 18°C. The hearts were divided into three groups, including a control group which received a first perfusion of a solution which was identical but devoid of NAC on establishment of the post-storage ischemia and an additional perfusion of 25 ml of this solution immediately before unclamping the aorta, 2~~~~2'~

the perfusions being performed at 4°C and 8°C, respec-tively. The same protocol was observed for the second group of hearts, except for the fact that the solution contained 0.4 M NAC. In the third group, a solution containing NAC at 28°C was distributed in a single dose at the end of the ischemia. In this latter group, the NAC
concentration was adjusted to 0.072 M so that all the hearts treated received the same amount of this sub-stance, that is to say approximately 1.80 millimoles. The bottles containing the solutions as well as the connect-ing tubes were protected from light in order to avoid oxidation. In all three groups, the perfusions after storage were delivered at a pressure of 30 cm of water.
After a period of ischemia of six hours, the hearts were reperfused for one hour at 37°C.
For all three groups, the stimulation was stopped and the left intraventricular balloon deflated during the period of ischemia so as to simulate clinical conditions .
After reperfusion, stimulation was reestablished.
Isovolumetric measurements of the coronary flow rate, the left ventricular pressure, its first derivative and the telediastolic pressure were performed three times during the monotoring period and then at 5, 30, 45 and 60 minutes during the reperfusion.
The results were as follows:
- Coronary flow rate: after sixty minutes of reperfusion, the flow rate of all the hearts was reduced significantly (p < 0.001) relative to the pre-ischaemic reference values. However, the best recovery of coronary flow rate was noted in the groups treated with NAC
(Table I).
- Left ventricular function: the left ventricular pressure decreased significantly after the ischemia and reperfusion in the hearts of all three groups (Table I).
However, the hearts of the control group (Group I) and those which had received only the reperfusion supple-mented with NAC at the end of the ischemic episode after storage (Group III) manifested significantly greater pressure losses (p < 0.001) than those protected with the 2~~~Q2~

multidose solution enriched with NAC (Group II) during the last hour of overall ischemia ( p < 0 . O1 ) . In addi-tion, throughout the reperfusion period, a significantly greater pressure was developed in Group II. The effects of the treatment on the postischemic derivative dP/dt were similar to those for the pressure developed, and the largest values were obtained for Group II.
- Left ventricular diastolic pressure: at the end of the arrest-storage-ischemia protocol, the hearts of the control Group I as well as those of Group III under went a significant loss of compliance. In contrast, postischemic contracture was significantly decreased in Group TI.
The results of this experiment demonstrate the substantial advantage of using the protective solutions according to the invention in a realistic model capable of extrapolation to the sphere of human transplantation.
4. Tests of solutions containing GSH on isolated rat heart preparations The tests are conducted on rat heart prepara-tions, with isovolumetric contraction, divided into three groups 1, 2 and 3. Cardiac arrest is obtained by per-fusion of the perfusion and storage solution at +4°C. The hearts are then stored by immersion for 5 h in the solution at +2°C. A one-hour period of ischemia at l5-18°C is then established, with initial perfusion with administration of the perfusion and storage solution at the beginning of this period of ischemia, and admini-stration of the reperfusion solution at the end of the hour of ischemia, that is to say immediately before unclamping the aorta. The temperatures of the solutions are 4 and 28°C, respectively. They are administered under a pressure of 30 cm H20. After the period of ischemia, the hearts are reperfused for one hour at 37°C.
The solutions used are as follows:
Group 1 (control): solution identical to the solution C
but devoid of GSH, then solution D devoid of GSH, Group 2: solution C, then solution D, Group 3: solution D supplemented with deferoxamine, then 2~~~~~~

solution D supplemented with deferoxamine.
The results are recorded in Table II. They show the spectacular improvement produced by the solutions C, distinguished by the presence of GSH and the low oxygen concentration.
In the table:
CF - coronary flow rate (ml/min), Pdiast - diastolic pressure (mm Hg), Pdev - pressure developed (mm Hg), dP/dt - first derivative of the pressure (mm Hg. sec-1) .
5. Use of the solutions in human heart transplantation The solutions are prepared according to Examples 1 and 2. After the establishment of transthoracic access to the explanted heart, 1000 to 2000 ml of the solution C at 4°C are perfused into the heart via the aorta for three to four minutes. The heart is removed, then installed in a jar provided for this purpose so as to be immersed in the solution C, and the jar is cooled to the customary preservation temperature of +4°C.
When the graft has been transported to the prepared recipient maintained by means of an extra-corporeal circulation, grafting of the heart is carried out and, during this operation, an antero- or retrograde perfusion of the solution C is performed at +4°C in order to reinforce cardiac arrest. The perfusion volume is generally of the order of 1000 to 2000 ml.
When installation of the graft is complete, the heart is reperfused via the aorta using the solution D at 28-27°C for a period of 5 min on average, the volume used being o f the order o f 10 0 0 ml . At the end o f this per-fusion, the aorta is unclamped, circulation is re-established and severing of the extracorporeal circula-tion is performed in the customary manner.

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TABLE II
A. Analysis of variance ~( I ~ CF ( ml /min L,( n = 7 ) 1) Group 1 = 12.6 ml/min ~ 0.5 ml/min.
2) Group 2 = 14.2 ~ 0.4 ml/min.
3) Group 3 = 10.6 ~ 0.4 ml/min.
Row (line) effect F > 19,1173 => p < 0.001 Column (time) effect F > 6.3592=> p < 0.001 (1) versus (3) p < 0.01 (2) versus (3) p < 0.001 (II1 Pdiast (mmHQ) (n = 71 1) Group 1 = 33.1 ~ 2.9 mmHg 2) Group 2 = 16.9 ~ 1.4 mmHg 3) Group 3 = 21.7 ~ 2.2 mmHg Line effect - F > 13.1426 p < 0.001 Time effect - N.S.
(1) versus (2) p < 0.001 (1) versus (3) p < 0.01 (2) versus (3) N.S.
2 0 ~( I I I ) Pdev ( mmHct ) ( n = 7 ) Group l = 84.0 t 3.0 mmHg Group 2 = 104.5 t 4.1 mmHg Group 3 = 116.8 ~ 3.6 mmHg Line effect F > 20.6411 p < 0.001 Time effect N.S.
(1) versus (2) p < 0.001 (1) versus (3) p < 0.001 (2) versus (3) N.S.

~IV)~ dP/dtmax ~+) ~n = 7 ) Group 1 = 2932 ~ 96 mmHg sec-1 Group 2 = 3418 ~ 122 mmHg sec-1 Group 3 = 3479 ~ 173 mmHg sec-1 Line effect F > 4.7966 p < 0.01 Time effect N.S.
(1) versus (2) p < 0.05 (1) versus (3) p < 0.05 (2) versus (3) N.S.
B. Scheff~ test at 60 min of reperfusion ~(I) CF (ml/min) (n = 7~
Group 1 = 11.3 ~ 0.8 ml/min.
Group 2 = 12.6 ~ 0.8 ml/min.
Group 3 = 8.9 ~ 0.7 ml/min.
(1) versus (3) N.S.
(2) versus (3) p < 0.02 lII) Pdiast (mmHq) (n = 7) Group 1 = 30.4 ~ 5.0 mmHg Group 2 = 14.9 t 1.9 mmHg Group 3 = 19.6 ~ 4.0 mmHg (1) versus (2) p < 0.05 (1) versus (3) N.S.
(2) versus (3) N.S.
(III) Pdev (1) mmHcL(n = 7) Group 1 = 80.1 ~ 5.3 mmHg Group 2 = 107.6 ~ 8.5 mmHg Group 3 = 120.7 ~ 8.9 mmHg (1) versus (2) p < 0.05 (1) versus (3) p < 0.01 (2) versus (3) N.S.

,~ IVY dP/dt max (mmHQ sec 1' ( n = 7 ~
Group 1 = 2714 ~ 110 mmHg sec-1 Group 2 = 3429 t 260 mmHg sec-1 Group 3 = 3500 ~ 300 mmHg sec-1 (1) versus (2) p < 0.05 (1) versus (3) p < 0.05 (2) versus (3) N.S.
C. Comparison of the reference values with thevalues measured after 60 min of perfusion (Student's testl re ~(I) CF ml/min ~
( Group 1 = 14.4 0.7 v 11.3 0.8 ml/min p 0.05 Group 2 = 15.0 0.4 v 12.6 0.8 ml/min p 0.05 Group 3 = 15.1 0.3 versus 8.9 0.7 ml/min p 0.001 LII ~~ Pdiast (mmHct) (1) 11.3 ~ 0.7 versus 30.4 ~ 5.0 mmHg p < 0.01 (2) 10.6 t 0.7 versu-s 14.9 mmHg t 1.9 N.S.
(3) 9.6 ~ 0.5 versus 19.6 t 4.0 mmHg p < 0.05 ( I I I ) Pdev ( mmHq.~, Group 1 = 132.1 ~ 3.1 versus 80.1 ~ 5.3 mmHg p < 0.001 Group 2 = 134.4 ~ 2.7 versus 107.6 ~ 8.5 mmHg p < 0.05 Group 3 = 149.3 ~ 2.9 versus 120.7 ~ 8.9 mmHg p < 0.05 (IV) dP/dt max (mmHq sec-1) (1) 4281 ~ 147 versus 2714 t 110 mmHg sec-1 p < 0.001 (2) 4714 ~ 200 versus 3429 ~ 260 mmHg sec-1 p < 0.01 (3) 4191 ~ 289 versus 3500 t 300 mmHg sec-1 N.S.

Claims

1. A solution for the perfusion, preservation or reperfusion of an organ wherein the solution comprises at least one antioxidant compound and a zero or greatly reduced partial pressure of oxygen which is maintained substantially at this reduced value up to the time of use.

2. A solution according to claim 1 wherein the organ is a heart.

3. A solution according to claim 1 wherein the antioxidant compound is a trapping agent for free oxygen radicals.

4. A solution according to claim 1, 2 or 3 in which the antioxidant compound is a compound comprising a thiol function.

5. A solution according to claim 4, in which the thiol is glutathione in the reduced state (GSH) or a glutathione analog.

6. A solution according to claim 4, in which said free-radical trapping compound is N-acetylcysteine (NAC) or an analog thereof.

7. A solution according to any one of claim 1 to 6, which is packaged in an airtight bottle or flexible bag.

8. A solution according to any one of claims 2 to 7, which further comprises a compound counteracting radical formation.

9. A solution according to any one of claims 2 to 7, which further comprises deferoxamine (INN).

10. A solution according to any one of claims 2 to 7, which further comprises a metal chelator.

11. A solution according to any one of claims 1 to 10, in which the maximum concentration of dissolved oxygen is 0.1 ppm or less.

12. A solution according to claim 5, in which the reduced glutathione concentration is between 0.5 and 10 mmol/1.

13. A solution according to claim 5, in which the reduced glutathione concentration is about 3 mmol/1.

14. A solution according to claim 6, in which the NAC
content is between 0.01 mol/1 and 0.08 mol/1.

15. A solution according to claim 6, in which the NAC
content is about 0.04 mol/1.

16. A solution according to any one of claims 1 to 13, which contains at least one constituent preventing the formation of cell edema, and means limiting the calcium overload, said solution having a pH of the order of 7.40 +
0.40.

17. A solution according to claim 16 wherein the constituent preventing the formation of cell edema is an impermeant compound.

18. A solution as claimed in claim 16 or 17, for the heart, which contains at least one constituent which is a metabolic inhibitor.

19. A solution as claimed in claim 16 or 17 for the heart, which contains potassium.

20. A solution according to any one of claims 16 to 19 comprising either A:
Constituent Concentration (mmol/liter) K+ 10-30 Na+ 90-120 Mg++ 10-20 Ca++ 0.005-1.2 Cl- 100-160 Mannitol 50-200 Glutamate 4-26 or GSH 0.2 to 0.5-10 or C:
Constituent Concentration (mmol/liter) K+ 12 Na+ 100 Mg++ 13 Ca++ 0.25 Cl- 110.4 Mannitol 109.8 Glutamate 20 GSH 0.5 to 3

21. A solution according to claim 20, for the liver, wherein said mannitol is replaced by another impermeant compound.

22. A solution according to any one of claims 1 to 13, which contains a constituent capable of preventing the formation of cell edema, and means for reestablishing calcium homeostasis, the solution having a pH of the order of 7.70 +
0.30.

23. A solution according to claim 22 wherein constituent capable of preventing the formation of cell edema is an impermeant compound.

24. A solution for the heart according to any one of claims 1 to 13, which contains a constituent capable of preventing the formation of cell edema, as well as a metabolic inhibitor.

25. A solution according to claim 24 wherein the constituent capable of preventing the formation of cell edema is an impermeant compound and the metabolic inhibitor is potassium.

26. A solution according to any one of claims 22 to 25 comprising either Constituent Concentration (mmol/liter) K+ 10-30 Na+ 90-120 Mg++ 0-20 Ca++ 0.005-1.2 Cl- 100-160 Mannitol 50-200 Glutamate 4-26 or GSH 0.2 to 0.5-10 or:
Constituent Concentration (mmol/liter) K+ 14.9 Na+ 100 Mg++ ---Ca++ 1.2 Cl- 97.5 Mannitol 136 Glutamate 20 GSH 0.5 to 3

27. A solution according to claim 26, for the liver, wherein said mannitol is replaced by another impermeant compound.

28. A kit comprising a perfusion and preservation solution and a reperfusion solution and means to contain said perfusion and preservation solution separate from said reperfusion solution, wherein each of said perfusion and preservation solution and said reperfusion solution is a solution according to any one of claims 1 to 27.