GB2249937A - Solutions for the perfusion, preservation and reperfusion of organs - Google Patents

Solutions for the perfusion, preservation and reperfusion of organs Download PDF

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GB2249937A
GB2249937A GB9124488A GB9124488A GB2249937A GB 2249937 A GB2249937 A GB 2249937A GB 9124488 A GB9124488 A GB 9124488A GB 9124488 A GB9124488 A GB 9124488A GB 2249937 A GB2249937 A GB 2249937A
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solution
reperfusion
perfusion
preservation
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Philippe Menasche
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Sanofi Pasteur SA
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Pasteur Merieux Serum et Vaccines SA
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A61K31/175—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine having the group, >N—C(O)—N=N— or, e.g. carbonohydrazides, carbazones, semicarbazides, semicarbazones; Thioanalogues thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/14—Alkali metal chlorides; Alkaline earth metal chlorides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A61K38/063—Glutathione

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Abstract

Perfusion and presentation 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

224 99 37 Solutions for the perfusion, preservation and reperfusion of
organs.
The present invention relates to solutions f or 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, occurring 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 degradations originating from the production of these radicals 2 - 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 e:Cfect--of free radicals, and in particular antioxidant substances. Various compounds have been proposed, some, such as def eroxamine, 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 (acavenger).- However, the literature isdivided 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 Reperfusioninduced Arrhythmias and Oxygen-derived Free Radicals, Circulation Research, Vol. 58, no. 3, March 1986, 331-340, find that the addition of L-methionine, superoxide' dismutase, catalase, mannitol, glutathione or deferoxamine to the perfused isolated rat heart reduces the risk of fibrillation or of ventricular tachycardia during reperfusion.
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 i 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 24hour Heart Storage by Microperfusion Using a New Polyethylene 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 significant 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 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 repetfus.Jon solutions in daily hospital practice has failed to yield decisive results.
Ph. Menaschd et al., in les Pi6geurs de Radicaux Libres dans la Protection Myocardique en Chirurgie Cardiaque [Free-Radical Trapping Agents in Myocardial Protection in Cardiac Surgery], Ann. Cardiol. Angdiol; 1986, 35 (no. 7bis), 447-452, conclude. however, thatthe preservation -of postischemic left ventricular function, due to a given cardioplegic solution, could be significantly improved by adding antioxidants capable of preventing 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 f ield of transplantation, the literature provides no genuinely useable information about the choice and procedure for use of genuinely effective protecti.,ve 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 - 5 be used 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 scavenger:-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 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 according to the invention is preferably understood to mean a maximum conc entration 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, mmol/1. 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.
6 - The invention is preferably implemented in dif f erent f orms, depending on whether it is applied to the perfusion and pres,;arvation 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 mX. In addition, a lower pH, in particular 7.40 0.40, is preferred. In the case where the perfusion 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/li Na xg+ Ca+ ClMannitol Glutamate NAC or GSH 10-30 90-120 10-20 0.005-1.2 100-160 50-200 4-26 10-80 0.2 to 0.5-10 Osmolarity pH 270-450 (for example 370) mOsm/1 7.40 0.40 (at 20"e) In the case of a reperfusion preparation according 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 c-ase of the heart, it is made up so as to prolong metabolic inhibition, and will retain potassium at a concentration preferably above 10 mM.
Thus, in an advantageous embodiment, the reperfusion solution contains the following compounds:
B Reperfusion solution (2) 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 NAC 10-80 or GSH 0.2 to 0.5-10 Osmolarity 270 to 450 (for example 370) mOsm/1 pH 7.70 0.30 at 280C The preferred embodiment of the perfusion and preservation solution is:
c Perfusion and storage solution (1) 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 Osmolarity 370 mOsm/1 - 8 pH 7.40 (at 20OC) and the preferred embodiment of the reperfusion solution is Reperfusion solution (2) Concentration limal/litglrl 14.9 100 D Constituent K+ Na"' Mg Ca++ ClMannitol Glutamate GSH 1.2 97.5 136 20 0.5 to 3 Osmolarity pH 370 mOsm/1 7.70 at 28'C Naturally, the compounds thus def ined may be replaced by compounds having equivalent functions, the molar contents preferably remaining substantially unchanged.
Thus, glutamate, whose function is to stimulate aTiaerrobic 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 28C, is preferably produced without a buffer.
The pH of the reperfusion solution, preferably adjusted to 7.70 at 280C, may be optionally produced using a buffer (in particular bicarbonate, histidine).
I! i - 9 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 invention, 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.
Preparation of the Derfusion 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 200C. This solution is packaged in plastic bags impermeable to atmospheric oxygen, or in plastic bags 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 solut ion 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 28C.
This solution is placed in similar bags of volume 100 M1.
3. Test of solutions containing 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 nonrecirculating Langendorff perfusion column to establish a retrograde perfusion using an oxygenated (95% 02, 5% C02) KrebsHenseleit 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 3 2 0 /min.
Af ter 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 f or five hours, with a mean myocardial temperature of VC 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 18C. 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, the perfusions being performed at 4C and 8'C, respectively. The same protocol was observed for the second group of hearts, except for the fact that the solution contained 0.04 M NAC. In the third group, a solution containing NAC at 2VC was distributed ina 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 substance, that is to say approximately 1.80 millimales. The bottles containing the solutions as well as the connecting 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 370C.
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 1). However, the hearts of the control group (Group I) and those which had received only the reperfusion supplemented 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 multidase solution enriched with NAC (Group I1) during the last hour of overall ischemia (p < 0. 01). In addition, 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 arres t- storageis chemia protocol, the hearts of the control Group I as well as those of Group III underwent a significant loss of compliance. In contrast, postischemic contracture was significantly decreased in Group II.
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 containincr GSH on isolated rat heart preparations The tests are conducted on rat heart preparations, with isovolumetric contraction, divided into three groups 1, 2 and 3. Cardiac arrest is obtained by perfusion of the perfusion and storage solution at +4C. The hearts are then stored by immersion for 5 h in the solution at +20C. A one-hour period of ischemia at 1518C is then established, with initial perfusion with administration of the perfusion and storage solution at the beginning of this period of ischemia, and administration 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 28C, respectively. They are administered under a pressure of 30 cm H20. After the period of ischemia, the hearts are reperfused for one hour at 37C.
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 - 13 solution D supplemented with deferoxamine.
The results are recorded in Table II. They show the spectacular improvement produced by the solutions C, disting-uished 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 (nun Hg), dP/dt first derivative of the pressure (mm Hg. sec-').
5. Use of the solutions in human heart transDlantation 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 40C 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 +4C.
When the graft has been transported to the prepared recipient maintained by means of an extracorporeal circulation, grafting of the heart is carried out and, during this operation, an antero- or retrograde perfusion of the solution C is performed at +4C 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 28-27C for a period of 5 min on average, the volume used being of the order of 1000 ml. At the end of this perfusion, the aorta is unclamped, circulation is reestablished and severing of the extracorporeal circulation is performed in the customary manner.
Table I
Effect of NAC on coronary flow rate and left ventricular pressure developed Group N = 10 Left ventricular pressure developed (mm H9) Coronary flow rate (ml/min) Normal Reperfusion Normal Reperfusion Control 14.6 0.8 10.5 0.4 130.7 2.1 63.6 j.3 Solution without NAC Second group 14.8 0.6 12.1 0.5 126.4 2.8 101.5 3.4 (0. 04 M) NAC Third group 16.0 0.5 13.4 .0.4 129.7 2. 0 69.8 3.6 1 (0. 07 M) NAC p < 0.05 with respect to the reference cardioplegia. p < 0.01 with respect to the reference cardioplegia. p < 0.001 with respect to the reference cardioplegia and the reperfusion enriched with NAC; 11 1 1 TABLE II
A. Analvsis of variance (I) CF (ml/min) (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 Column (time) effect (1) versus (3) p < 0.01 (2) versus (3) p < 0.001 -(II) Pdiast (mmHcr) (n = 7)1) Group 1 = 33.1 2.9 mmHg 2) Group 2 = 16.9 1.4 mmHg 3) Group 3 = 21.7 2.2 mmHg F > 19,1173 => p < 0.001 F > 6.3592-> p < 0.001 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.
(III) Pdev (mmHcr) (n = 7) Group-1 = 84.0 3.0 mmHg Group 2 = 104.5 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.
16 - (IV) dP/dtmax (n =-21 Group 1 = 2932 96 mmHg sec Group 2 = 3418 122 mmHg sec-1 Group 3 = 3479 173 mmHg sec-1 Line effect Time effect (1) versus (2) p < 0.05 (1) versus (3) p < 0.05 (2) versus (3) N.S.
F > 4.7966 p < 0.01 N.S.
B. Scheff6 test at 60 min of relDerfusion (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 (II) Pdiast (mmHcT) (n = 11 Group 1 = 30.4 5.0 mmHg Group 2 = 14.9 1.9 mmHg Group 3 = 19.6 4.0 mmHg 25 (1) versus (2) p < 0.05 (1) versus (3) N.S. (2) versus (3) N.S.
(III) Pdey (1) mmHcr (n f:_-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.
(IV) dP/dt max (mmHcr sec-1) (n = 7) Group 1 = 2714 110 mmHg sec-1 Group 2 = 3429 260 nmiHg 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 the values measured after 60 min of reloerfusion (Student's test) (1) 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 (II) Pdiast (mmHcr) (1) 11.3 0.7 versus 30.4 5.0 mmHg p < 0.01 (2) 10.6 0. 7 versus 14.9 mmHg 1.9 N.S. (3) 9.6 0.5 versus 19.6 4.0 mmHg p < 0.05 (III) Pdey (imnHgl 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 nmHg p < 0.05 Group 3 149.3 2.9 versus 120.7 8.9 mmHg p < 0.05 1 (IV) dP/dt max (inmHcr sec-n (1) 4281 147 versus 2714 110 mmHg sec-' p < 0.001 (2) 4714 200 versus 3429 260 mraHg sec-' p < 0.01 (3) 4191 289 versus 3500 300 nmiHg see-' N.S.

Claims (27)

1. A solution for use in the perfusion, preservation and/or reperfusion of a vital human or animal organ, which solution comprises a physiologically acceptable aqueous medium containing at least one antioxidant compound, characterised in that said solution is maintained substantially free of dissolved oxygen.
2. A solution for the perfusion and preservation and/or reperfusion of organs including the heart, features of the solution being the inclusion of at least one antioxidant compound which can be, in particular, a trapping agent for free oxygen radicals, and a zero or greatly reduced partial pressure of oxygen which is maintained substantially at this reduced value up to the time of use.
3. A solution as claimed in claim 1 or claim 2 in which the antioxidant compound is a free-radical trapping agent.
4. A solution as claimed in claim 3 wherein the antioxidant compound comprises one or more thiol functions.
5. A solution as claimed in any preceding claim, in which the antioxidant compound is glutathione in the reduced state (GSH) or a glutathione analog.
6. A solution as claimed in claim 5, in which the reduced glutathione concentration is between 0.5 and 10 mmol/1, and in particular of the order of 3 mmol/1.
7. A solution as claimed in claim 3, in which said free-radical trapping compound is N-acetylcysteine (NAG) or an analog thereof.
8. A solution as claimed in claim 7. in which the NAG content is between 0.01 mol/1 and 0.08 mol/1, and in particular of the order of 0.04 mol/1.
19 -
9. A solution as claimed in any preceding claim, which contains a free- radical inhibitory thiol and a compound counteracting radical formation.
10. A solution according to claim 9 in which said compound counteracting radical formation is a metal chelato
11. A solution according to claim 10 in which said metal chelator is deferoxamine.
12. A solution as claimed in any preceding claim, in which the maximum concentration of dissolved oxygen is 0.1 ppm or less.
13. A solution as claimed in any preceding claim for perfusion or preservation, which contains at least one constituent such as an impermeant compound preventing the formation of cell edema, and means limiting the calcium overload, said solution having a pH of the order of 7.4o + o.4o.
14. A perfusion and preservation solution as claimed in claim 13, for the heart, which contains at least one constituent which is a metabolic inhibitor.
15. A solution as claimed in claim 14 in which said metabolic inhibitor is potassium.
16. A perfusion and preservation solution as claimed in claim 1, which is of the formulation A or C.
17. A perfusion and preservation solution as claimed in any of claims 13 to 16 modified for perfusion or preservation of the liver, which solution does not contain mannitol but instead contains another impermeant compound.
18. A solution as claimed in any one of claims 1 to 12 for reperfusion which contains a constituent such as an impermeant compound 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.
19. A solution for the heart as claimed in any one of claims 1 to 12 for reperfusion which contains a constituent such as an impermeant compound capable of preventing the formation of cell edema, as well as constituent which is a metabolic inhibitor, in particular postassium.
20. A reperfusion solution as claimed in claim 18 and claim 19, which possesses the formulation B or D.
21. A reperfusion solution as claimed in claim 20 for reperfusion of the liver, which solution does not contain mannitol but instead contains another impermeant compound.
22. A solution as claimed in any preceding claim packaged in a gas-tight container which container is substantially impervious to oxygen.
23. A solution according to claim 22 wherein said container is an airtight bottle or flexible bag.
24. A kit comprising a perfusion solution and a reperfusion solution, which kit comprises said solutions in separate containers characterised in that said solutions are as defined in any of claims 1 to 23.
25. A method of maintaining a human or animal organ or tissue in a vital state, which comprises contacting said organ or tissue with a solution as defined in any of claims 1 to 23.
26. A method of producing a solution as claimed in any of claims to 1 to 23 which comprises removing dissolved oxygen from a dissolved-oxygen containing solution comprising a physiologically acceptable aqueous medium and at least one antioxidant.
27. The use of a solution comprising a physiologically acceptable aqueous medium containing at least one antioxidant compound in the manufacture of a solution for use in the perfusion, preservation and/or reperfusion of a vital human or animal organ, characterised in that said solution is substantially free of dissolved oxygen.
GB9124488A 1990-11-20 1991-11-18 Solutions for the perfusion, preservation and reperfusion of organs Expired - Lifetime GB2249937B (en)

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FR9014424A FR2669189B1 (en) 1990-11-20 1990-11-20 PERFUSION AND STORAGE SOLUTIONS FOR CARDIAC TRANSPLANTATIONS.
FR9109027A FR2679107B1 (en) 1991-07-17 1991-07-17 ORGANIC PERFUSION, PRESERVATION AND REPERFUSION SOLUTIONS.

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FR2695827A1 (en) * 1992-09-18 1994-03-25 Pasteur Merieux Serums Vacc Solution for perfusion, preservation and reperfusion of organs.
EP0664953A1 (en) * 1994-01-28 1995-08-02 Senju Pharmaceutical Co., Ltd. Lung graft preservative composition and a method for viable preservation of lung grafts
EP0687411A1 (en) * 1994-06-14 1995-12-20 Senju Pharmaceutical Co., Ltd. Hepatic graft preservative composition and a method for viable preservation of the hepatic graft
EP0697169A1 (en) * 1993-05-07 1996-02-21 Chugai Seiyaku Kabushiki Kaisha Organ preservative
US7510823B2 (en) 2000-11-22 2009-03-31 The Leeds Teaching Hospitals Nhs Trust Flush preservation solution

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DE19706111C2 (en) * 1997-02-17 1999-02-18 Fresenius Medical Care De Gmbh Solution for storage of organs
US5990153A (en) * 1997-05-05 1999-11-23 Wood; John G. Ultrasonicated α-lipoic acid solutions for attenuating microvascular injury
US6045990A (en) * 1998-07-09 2000-04-04 Baust; John M. Inclusion of apoptotic regulators in solutions for cell storage at low temperature
DE19834087C1 (en) * 1998-07-29 2000-03-30 Mirzaie Sedaposhteh Massoud Aqueous preservative solution for storage of animal tissue, especially porcine heart valves, contains sodium, potassium, magnesium and calcium salts, glucose and chelate former
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Cited By (11)

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FR2695827A1 (en) * 1992-09-18 1994-03-25 Pasteur Merieux Serums Vacc Solution for perfusion, preservation and reperfusion of organs.
BE1007500A3 (en) * 1992-09-18 1995-07-18 Pasteur Merieux Serums Vacc Solution infusion, conservation and organ reperfusion.
EP0697169A1 (en) * 1993-05-07 1996-02-21 Chugai Seiyaku Kabushiki Kaisha Organ preservative
EP0697169A4 (en) * 1993-05-07 1996-04-17 Chugai Pharmaceutical Co Ltd Organ preservative
US6030766A (en) * 1993-05-07 2000-02-29 Chugai Seiyaku Kabushiki Kaisha Organ preservation composition comprising nicaraven and methods of use
EP0664953A1 (en) * 1994-01-28 1995-08-02 Senju Pharmaceutical Co., Ltd. Lung graft preservative composition and a method for viable preservation of lung grafts
US5556842A (en) * 1994-01-28 1996-09-17 Senju Pharmaceutical Co., Ltd. Method for viable preservation of lung grafts
EP0687411A1 (en) * 1994-06-14 1995-12-20 Senju Pharmaceutical Co., Ltd. Hepatic graft preservative composition and a method for viable preservation of the hepatic graft
US5660976A (en) * 1994-06-14 1997-08-26 Senju Pharmaceutical Co., Ltd. Method for preserving a liver by perfusion with or storage in a solution containing ascorbyl tocopheryl phosphodiester
US7510823B2 (en) 2000-11-22 2009-03-31 The Leeds Teaching Hospitals Nhs Trust Flush preservation solution
US8236486B2 (en) 2000-11-22 2012-08-07 The Leeds Teaching Hospital NHS Trust Flush preservation solution

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GB2249937B (en) 1994-02-16
ITMI912991A1 (en) 1993-05-11
DE4138040A1 (en) 1992-05-21
ES2050580A1 (en) 1994-05-16
IT1251995B (en) 1995-05-27
BE1006834A3 (en) 1995-01-03
GB9124488D0 (en) 1992-01-08
ES2050580B1 (en) 1994-12-16
CA2055827A1 (en) 1992-05-21
CH683485A5 (en) 1994-03-31
ATA228791A (en) 1994-10-15
ITMI912991A0 (en) 1991-11-11
AT399440B (en) 1995-05-26
CA2055827C (en) 1999-06-15

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