GB2270614A

GB2270614A - Solution for the perfusion, preservation and reperfusion of organs

Info

Publication number: GB2270614A
Application number: GB9319264A
Authority: GB
Inventors: Philippe Menasche
Original assignee: Pasteur Merieux Serum et Vaccines SA
Current assignee: Sanofi Pasteur SA
Priority date: 1992-09-18
Filing date: 1993-09-17
Publication date: 1994-03-23
Anticipated expiration: 2013-09-17
Also published as: GB2270614B; ATA189793A; IT1272638B; ES2070089A1; CA2106249A1; GB9319264D0; FR2695827A1; CA2106249C; CH686870A5; FR2695827B1; ES2070089B1; DE4331711C2; AT407216B; DE4331711A1; ITMI931994A0; BE1007500A3; ITMI931994A1

Abstract

Single solution for perfusion, storage and reperfusion, essentially having the following composition: <IMAGE> Water (quantity for one litre readjusted to pH 7.30 +/- 0.10 at +20 DEG C> Theoretical osmolality 360 mOsm/kg Glutathione (reduced GSH) or equivalent 0.5 to 10 mmol/l, (preferably 3) Reduced glutathione, or equivalent, being in solution under reduced or zero partial pressure of oxygen and being essentially kept at this value up to the time of use.

Description

2270614 Solution for the perfusion, preservation and reperfusion of

organs.

The present invention relates to solutions f or the perfusion, preservation (or storage) and reperfusion of organs, including for heart transplantations. It also relates to a process f or using these solutions applied to the different phases of a transplantation.

One of the principal causes of failure of heart transplantations results f rom the risks of degradation, or even necrosis, of the transplant which manifest themselves during reoxygenation of the transplanted organ and which are linked to the generally prolonged ischaemia between the beginning of the explantation in the donor and the end of the implantation in the recipient.

An ischaemia of four to five hours constitutes, f or example in the case of the heart. the tolerable upper limit and does not exclude numerous complications.

To limit this risk, many authors have proposed and used protective solutions, both for the perfusion of the organ to be explanted, and for its preservation at low temperature, and its reperfusion during the transplantation.

Examples of these solutions are the solutions:

Bretschneider ETK COLLINS ST THOMAS UW Stanford.

However. these solutions have only limited advantages and at best offer only a partial protection against the risks which appear during reperfusion and which are partly attributed to the metabolic production of oxygen free radicals which are produced in large quantities, especially during reoxygenation of the ischaemic organ.

The risk of cellular and membrane oxidative degradations resulting from the production of these radicals has been the subject of several studies in the field of myocardial protection by cardioplegia. These

2 different studies have suggested introducing antioxidants into the protecting solutions used. Various compounds have -been proposed, some, such as deferoxamine, allopurinol, catalase and peroxidase, as being capable of preventing the production of free radicals, others, such as superoxide dismutase, being capable of destroying these radicals, still others, such as vitamin Eor equivalents (Trolox) being capable of "neutralizing" the free radicals.

Among these latter compounds are also molecules carrying thiol groups such as N-acetylcysteine or reduced glutathione (GSH), the latter having been considered as scavenger for free radicals. However. the literature is divided on the usefulness of glutathione. See:

is - G.W. Standeven et al., J. Thorac. Cardiovasc.

Surg. 1979, 78, 893-907 Cold-Blood potassium cardio plegia; _ M. Bernier et al., Reperf us ion- induced Arrhyth mias and Oxygen-derived Free Radicals, Circulation Research, Vol. 58, No. 3, March 1986, 331-340; i.C. Chatham et al., Depletion of Myocardial Glutathione; Its effects on heart function and metabolism during ischaemia and reperfusion, Cardiovascular Research, 1988. 22, 833-839; - A. Blaustein et al.,, Myocardial Glutathione Depletion Impairs Recovery After Short Periods of Ischaemia, Circulation, Vol. 80. No. 5. November 1989; - A. Singh et al.. Relation Between Myocardial Glutathione Content and Extent of Ischaemia Reperfusion Injury, Circulation, Vol. 80, No. 6, December 1989, 1795-1803; - W.N. Wicomb et al., Role of Glutathione in 24-hour Heart Storage by Microperfusion Using a New Polyethylene Glycol Solution. J. Mol. Cell Cardiol. 22 (supplement V) 1990, p. 82; - V. Kantamnen! et al., Extended Preservation of Canine Myocardium Using UW Solution, J. Mol. Cell Cardiol. 1990 (Suppl. V); 22: 22 (Abstr.).

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 the reperfusion can improve postischaemic 5 recovery.

While it may therefore appear advantageous to use substances which act against the production or the effect of free radicals in myocardium within the framework of cardioplegic protection, the choice of compound and the modes of application did not appear to be obvious and the addition of these compounds, including glutathione, into the solutions for myocardial perfusion and reperfusion in daily hospital procedure had not made it possible to arrive at conclusive results.

Ph. Menasch6 et al., in Pi6geurs de Radicaux Libres dans la Protection Myocardique en Chirurgie Cardiaque [Scavengers for Free Radicals in Myocardial Protection in Heart Surgery], Ann. Cardiol. Ang6iol; 1986r 35 (No. 7A), 747-752, conclude however that the preservation of the postischaemic left ventricular function, due to a given cardioplegic solution, could be -significantly improved by the addition of antioxidants capable of preventing the formation of free radicals or of destroying or neutralizing them. On the other hand, the choice of the most effective antioxidant among the many candidates, including superoxide dismutase SOD, peroxidase and glutathione, was not obvious, without mentioning the possible side or toxic effects. A fortiori when instead of the field of cardioplegia, in which the durations of ischaemia are relatively short, the field of transplantation is considered, the literature provided no real applicable indication on the choice and ways of using really effective protective solutions. Ph. Menasch6 et al. then described, in Belgian 35 Patent Application BE-A-009101067, solutions for the perfusion and preservation And/or reperfusion of organs, including the heart organ, characterized by the inclusion of at least one antioxidant compound, which compound may be especially a scavenger for oxygen free radicals, 4 especially glutathione in the reduced state, or an ana logue, such as N-acetylcysteine, or by a zero or highly reduced partial pressure of oxygen and being essentially kept at this reduced value up to the time of use.

However, the various solutions known are unsuit able f or use both f or perfusion and storage, and f or reperfusion, such that. in practice, surgical teams are obliged to use at least two different solutions. one for reperfusion and then storage of the explanted organ, the other for reperfusion of the organ during implantation.

The present invention aims to resolve these problems and to provide protecting solutions which are remarkably ef f ective f or the preservation of organs. with a view to surgical operations and in particular transis plantation. The organs of interest comprise the heart, as well as-the other organs and, especially the liver, the lungs and the kidneys.

The subject of the present invention is therefore a single solution for perfusion, storage and reperfusion, 20 essentially having the following compo.sition:

- Constituent (for example Concentration in 1 or 21 bags) g/1 mmol/litre K+ is 5% Ne 100 10% Mg' 13 5% Ca' 0.25 5% Chlorides 41.5 5% - calcium chloride, 2H20 0.037 - potassium chloride 1.118 5% - magnesium chloride 2.642 Histidine (base) 4.650 30 10% Mannitol 10.930 60 5% Lactobionate (acid) 28.664 80 5% Glutamate (acid) 2.942 20 10% Water (quantity for one litre readjusted to pE 7.30 0.10 at +20C) Theoretical osmolality 360 mOsm/kg Glutathione (reduced GSH) or equivalent 0.5 to 10 mmol/1, (preferably 3) Reduced glutathione, or equivalent, being in solution under reduced or zero partial pressure of oxygen and being essentially kept at this value up to the time of use.

This solution may be used for all the phases of a transplantation, for example as cardioplegic solution for stopping the heart of the donor, as storage solution for the transportation and hypothermic storage of the organ during reimplantation, either in the form of a colloidal solution or, preferably, after dilution with blood.

In this solution, the prevention of calcium excess in the cells is ensured by the presence of the glutamate component which has the capacity to activate ATP under anaerobic conditions, t he low concentration of 35 calcium which reduces transfer by passive diffusion, the high concentration of sodium which limits sodium-calcium exchange, the electric potential -dependent transfer of calcium by the channels being limited by the low concentration of potassium as well as by the magnesium content. This. extracellular type ionic formulation is made possible by the simultaneous presence of effective imper- meabilizers. Mannitol has the dual capacity of behaving as an osmotic agent and as a scavenger for free radicals. Lactobionate ensures a more effective prevention of cellular swelling than mannitol alone and the total concentration of impermeabilizers is equivalent to that of intracellular proteins and non-transferable anions which exert a pressure from the outside towards the inside of the cells.

The pH is preferably weakly acidic (7.20-7.40) because of the fact that it has been discovered that this value further improves the prevention of calcium excess and, furthermore, that. contrary o more alkaline values, it improves the protection of cells during the hypothermic ischaemic arrest. The solution is advantageously buffered with histidine because it has been discovered that among the buffers which can be used in man, histidine is the only one which remains really effective at low temperatures.

Of course, the various components of the solution can be replaced with equivalents, provided that the latter ensure practically the same functions.

Among the glutathione equivalents in the reduced state (GSH) are its precursors or related substances, its analogues, especially glutathione monoester and N-acetylcysteine. However, other compounds with a thiol func- tional group which might manifest the same properties could be used.

The solutions conforming to the invention may be designed for storage in the form of a single ready-foruse preparation and simultaneously containing all the components of the solution.

In this case, and in conformity with the invention, the solutions are prepared and kept protected from atmospheric oxygen, by being for example prepared in degassed solutions, preferably under a nitrogen atmosphere. The storage and the preservation of the solutions according to the invention are performed in air-tight containers such as air-tight bottles or pref erably bags made f rom plastic material, f or example f rom 5 laminated composites of a type known per se.

However, in another embodiment of the invention, the solution according to the invention may be stored in the form of two separate preparations, namely a solution containing reduced glutathione, prepared and kept protected from atmospheric oxygen, for example in a syringe, and a solution for the other components, which does not necessarily have to be kept protected from atmospheric oxygen and which can therefore be contained in normal bags.

is Preferably, the solution containing reduced glutathione GSH has the following composition (per litre):

Glutathione (reduced GSH) 185.4 g/1 600 10% mmol/1 Histidine 4.650 g/1 30 10% mmol/1 In this case,, the solution is advantageously provided in the form of a kit comprising:

- a solution containing, in a bag or other container, for example a two-litre bag, the different anions and cations, mannitol, lactobionate, glutamate and, preferably, histidine, - a solution contained in a container sealed from atmospheric oxygen, preferably a syringe. and containing reduced glutathione or an analogue, and preferably histidine at the same concentration.

- and instructions to ensure, during use, the mixing or injection of the separate solution containing the glutathione into the solution containing the other components.

In this latter case, if the solution containing the other components is not itself at a reduced or zero oxygen pressure, the instruction leaflet advantageously specifies that the final solution, now ready for use, 8 should be used within a short period of a few hours, for example three or four hours.

Advantageously, the solutions according to the invention may also contain, in addition to reduced glutathione, a compound which prevents the formation of radicals such as metal chelators and in particular deferoxamine (DCI).

Reduced value of oxygen concentration in conformity with the invention is understood to mean, preferably, a maximum dissolved oxygen concentration of less than 0.1 ppm.

In order to produce, by way of example, a solution according to the invention, a composition having the following content is prepared in a sterilized pyrogen- free aqueous medium:

Constituent Concentration g/1 mmol/litre V' 15 Ne 100 Mg' 13 Ca' 0.25 Chlorides 41.5 - calcium chloride, 2H20 0.037 - potassium chloride 1.118 magnesium chloride 2.642 Histidine (base) 4.650 30 Mannitol 10.930 60 Lactobionate (acid) 28.664 80 Glutamate (acid) 2.942 20 Water (quantity for one litre readjusted to pE 7.30) Theoretical osmolality 360 mOsmlkg In a degassed sterile pyrogen-free aqueous medium, and under conditions protected from atmospheric oxygen, a solution having the following content is prepared, per litre 9 - Concentration g/1 mmol/litre Glutathione (reduced GSH) 185.4 600 Histidine 4.650 30 This solution is packaged in the f orm of syringes protected from atmospheric oxygen and capable of holding ml of solution for a two-litre bag or 5 ml for a one-litre bag.

-Preferably, the whole is provided in the form of a kit containing, on the one hand, a solution in a two-litre bag and, on the other hand, a syringe with a content of 10 ml.

For use, the contents of the syringe are injected into the bag so as to dilute the contents of the syringe is in the bag and this results in a reduced glutathione concentration of the order of 3 mmol/1.

The solution thus prepared is rapidly used in order to ensure the perfusion of the organ to be explanted in order to obtain the cardiac arrest. A sufficient volume of identical solution prepared in the same manner is used for preserving the organ.

During the implantation, an identical solution is again used for the reperfusion procedures, the solution being advantageously mixed with the patient's blood.

Claims

1 Single solution for perf usion, storage and reperfusion, essentially having the following composition:

Constituent Concentration g/1 mmol/litre K+ is 5% Ne 100 10% Mg++ 13 5% C&+ 0.25 5% Chlorides 41.5 5% - calcium chloride, 2H20 0.037 - potassium chloride 1.118 5% - magnesium chloride 2.642 Histidine (base) 4.650 30 10% Mannitol 10.930 60 5% Lactobionate (acid) 28.664 80 5% Glutamate (acid) 2.942 20 10% Water (quantity for one litre readjusted to pE 7.30 0.10 at +20OC) 20 Theoretical osmolality 360 mOsmlkg Glutathione (reduced GSH) or equivalent 0.5 to 10 mmol/1, (preferably 3) Reduced glutathione, or equivalent, being in solution under. reduced or zero partial pressure of oxygen and being essentially kept at this value up to the time of use.

2. Solution according to Claim 1 in the f orm, of a single preparation ready for use and containing all the components, prepared and kept protected from atmospheric oxygen in air-tight containers such as air-tight bottles or bags made from plastic material.

3. Solution according to Claim 1, prepared and stored in the form of two separate preparations, namely a preparation containing reduced glutathione, prepared and kept protected f rom atmospheric oxygen, and a preparation containing the other components in solution.

4. Solution according to Claim 3, characterized in that the preparation containing reduced glutathione GSH has the following composition (per-litre):

Glutathione (reduced GSH) 185.4 g/1 600 10% mmol/1 Histidine 4.650 g/1 30 10% mmol/1.

5. Solution according to one of Claims 3 and 4 characterized in that the preparation containing the reduced glutathione is packaged in an airtight syringe, especially with a content of 10 ml.

6. Solution according to Claim 5, characterized.in that it is provided in the form of a kit containing:

- a preparation containing, in a bag or other container, the different anions and cations, mannitol, lactobionate, glutamate and histidine, - a preparation contained in a syringe sealed from atmospheric oxygen. containing reduced glutathione or an analogue, and histidine, - and instructions to ensure, during use,, the mixing or injection of the separate solution containing the glutathione into the solution containing the other components.

7. Solution according to one of Claims 1 to 6, characterized in that it contains, in addition to reduced glutathione, a compound which prevents the formation of radicals such as metal chelators and especially in particular deferoxamine (DCI).