CA2106249C - Solutions for the perfusion, preservation and reperfusion of organs - Google Patents

Abstract

Organ perfusion, preservation and reperfusion solution. Single perfusion, storage and reperfusion solution, having substantially the following composition: Constituent Concentration g/l mmol/liter K+ 15 ~ 5% Na+ 100 ~ 10% Mg++ 13 ~ 5% Ca++ 0.25 ~ 5% Chlorides 41.5 ~ 5% - Chloride calcium, 2H2O 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 per adjusted liter pH 7.30 ~ 0.10 ~ 20.degree.C) Theoretical osmolality 360 m Osm/kg Glutathione (reduced GSH) or equivalent 0.5 10 mmol/l, (preferably 3) the reduced glutathione, or equivalent, being in solution under reduced or zero partial oxygen pressure and remaining substantially this value until use.

Description

Solution for infusion, preservation and organ reperfusion.

The present invention relates to solutions of infusion, storage (or storage), and reperfusion of organs, including for heart transplants. She also relates to a method of implementing these solutions applied to the different phases of a transplantation.
One of the main causes of failure of cardiac transplantation comes from the risks of degradation or necrosis of the graft manifesting itself reoxygenation of the transplanted orqan, and are related to ischemia usually prolonged between the beginning explantation in the donor and the end of implantation in the recipient.
An ischemia of four to five hours constitutes for example, in the case of the heart, the upper limit tolerable and does not exclude many accidents.

2 To limit this risk, many authors have proposed and used protective solutions, both for the infusion of the organ to be explanted, only for its conservation at low temperature ,. and his reperfusion during the transplantation.
Examples of these solutions are the solutions x. HTK from Bretschneider . COLLINS *
~
, ST THOMAS
. OW *
~ -. Stanford.
These solutions however only present benefits and provide only partially against the risks that arise during the reperfusion and that is attributed in part to the production metabolism of oxygen-free radicals produced in abundant quantities, especially during the reoxygenation of the ischemic organ.
The risk of cellular oxidative degradation and membranes from the production of these radicals has been the subject of several studies in the field of myocardial protection by cardioplegia. These different work suggested introducing in solutions protective substances used, antioxidant substances.
Different compounds have been proposed, some such as deferoxamine, allopurinol, catalase, peroxidase, as likely to oppose the production of radicals others, such as superoxide dismutase, destroy these radicals, still others, such as vitamin E or equivalents (Trolox) being capable of "neutralize" free radicals.

* Trade-mark ~ 1i ~ 62 49

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

GW Standeven et al., J. Thorac. Cardiovasc.
Surg. 1979, 78, 893-907 Cold-Blood potassium cardioplegia;
- M. Bernier et al., Reperfusion-induced Arrhythmias arid Oxygen-derived Free Radicals, Circulation Research, Vol. 58, No. 3, March 1986, 331-340;
JC 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., Between Myocardial Relationship Glutathione Content and Extent of Ischaemia - Reperfusion Injury, Circulatiori, Vol. 80, No. 6, December 1989, 1795-1803;
- WN 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. Kantamneni 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 MB Forman, Glutathione Redox Pathway and Reperfusion

4 Injury, Circulation, Vol. 78, No. 1, July 1988, 202-213.
He suggests that treatment with N-acetylcysteine (NAC) before reperfusion, can improve recovery postischémiqueõ

So it may seem interesting to use substances acting against the production or the effect of free radicals in the myocardium as part of the cai-dioplegic protection, the choice of compound and modalities of implementation were not obvious and the addition of these compounds, including glutathione, in infusion and myocardial reperfusion solutions in daily hospital practice had not allowed to achieve decisive results.
Ph. Menasché et al., In the Trappers of Free Radicals in Myocardial Protection in Surgery Cardiac, Ann. Cardiol. Angéiol; 1986, 35 (n 7bis), 747-452, however, conclude that preservation of the function left ventricular post-ischemic, due to a solution given cardioplegia could be improved significantly by the addition of antioxidants capable of prevent the formation of free radicals or destroy them or neutralize. On the other hand, the choice of the antioxidant more effective among the many candidates whose superoxide dismutase SOD, peroxidase and glutathione was not obvious, not to mention the side effects or possible toxic. A fortiori when one passes from field of cardioloplegia, in which the durations ischemia is relatively short, in the field of transplantation, the literature did not provide any indication actually usable on the choice and the methods of implementing protective solutions really effective.

Ph. Menasché et al., Then described, in the Belgian patent application BE-A-009101067 perfusion and preservation and / or reperfusion of organs, including the cardiac organ, characterized by the inclusion at least one antioxidant compound, which may be in particular piéqeur free radicals of the oxygen, in particular glutathione in the reduced state, or an analogue, such as N-acetylcysteine, and by a partial pressure of oxygen 1C1 zero or very low and staying substantially this value lowered until use.
However, the different known solutions are do not lend themselves to use, both, for infusion and storage, and for reperfusion, so that in practice, surgical teams are obliged to use at least two different solutions, one for the infusion then the storage of the explanted organ, the other for reperfusion of the organ being implanted.

The present invention proposes to solve these 20 problems and provide protective solutions remarkably effective for the preservation of organs, for technical interventions and in particular for transplantation. Target organs include the heart, as well as the other organs and, in particular, the liver, 25 lung and kidney.
The present invention therefore relates to a single solution of perfusion, storage and reperfusion, having substantially the next composition Constituent (eg Concentration in bags of 1 or 2 1) q / l mmol / liter K + 15 $ 5 Na + 100 10%
Mg ++ 13 t 5%
Ca ++ 0.25 t 5%
Chlorides 41.5 t 5%
- Calcium chloride, 2H20 0.037 - Potassium chloride 1,118 t 5%
- Magnesium chloride 2,642 Histidine (low (a) 4,650 30 t 10%
Mannitol 10.930 60 t 5%
Lactobionate (acid) 28.664 80 t 5%
Glutamate (aci) 2,942 20 f 10%
Water (quantity per liter readjusted at pH 7.30 t 0.10 to + 20 C) Osmolality tea <Drique 360 m Osm / kq Glutathione (re (lysed GSH) or equivalent 0.5 to 10 mmol / l, (from preference 3) reduced glutathione, or equivalent, being in solution under oxygen partial pressure reduced or zero and now substantially at this value up use.
This solution can be used during all the phases of a transplantation, for example as a cardioplegic solution to stop the heart of the donor, as a storage solution for transport 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 overloading calcium in the cells is ensured by the presence of glutamate component that has the ability to activate ATP
under anaerobic conditions, the low concentration of calcium which reduces passive diffusion transfer, high concentration of sodium which limits sodium-exchange calcium, calcium transfer through the channels depending on the electric potential being limited by the weak potassium concentration and the magnesium. This ionic formulation of the extra-cell is made possible by the simultaneous presence effective waterproofs. Mannitol has the capacity to behave like an osmotic agent and as a scavenger of free radicals. Lactobionate provides a more effective prevention of cell growth than the mannitol alone and the total concentration of impurities is equivalent to that of intracellular proteins and non-transferable anions that exert pressure from outside to the inside of the cells.
The pH is preferably weakly acidic (7, 20-7, 40) because it has been discovered that this value improves in yes: re the prevention of calcium overload and, moreover, that unlike more alkaline values, it improves cell protection during shutdown ischemical hypothermic. The solution is advantageously buffered with llistidine because it was discovered that among the buffers usable in humans, histidine is the only which remains truly effective at low temperatures.
Of course the different components of the solution can be replaced by equivalents, under reserve that these provide virtually the same ~ 106 ~ 49 functions.

Among the equivalents of glutathione in the state reduced (GSH) are precursors or substances related substances, its analogues, in particular glutathione monoester and N-acetylcysteine. However others compounds with thiol function that would manifest the same properties could be used.

The solutions according to the invention can be planned for storage, in the form of a preparation ready to use and simultaneously containing the all the components of the solution.
In this case, and in accordance with the invention, the solutions are prepared and stored away from oxygen air, for example by being prepared in solutions degassed, preferably under a nitrogen atmosphere. Storage and preserving solutions according to the invention in airtight containers such as vials or, preferably, plastic bags impervious to air, for example laminated composites of type known in itself.
However, in another embodiment of the invention, the solution according to the invention can be stored in the form of two separate preparations, namely a solution containing reduced glutathione, prepared and kept away from the oxygen of the air, for example in a syringe, and a solution for the other components, which does not necessarily have to be kept safe from oxygen from the air and which can therefore be contained in usual pockets.
Preferably the solution containing glutathione reduced GSH has the following composition (for a liter) (-. ~ T (r ~~

Glutathione (GSH reduced) 185.4 g / 1600 t 10% mmol / 1 Histidine 4,650 ql 30 t 10% mmol / l In this case the solution is advantageously presented in the form of a kit comprising:
- a solution containing, in a pocket or other container, for example a two-liter bag, the different anions and cations, mannitol, lactobionate, glutamate and, preferably, histidine, - a solution contained in a sealed container _LO with oxygen of the air, preferably a syringe, and containing reduced glutathione or the like, and preferably histidine at the same concentration, - and instructions to ensure, when the use, the niëlanqe or the injection of the solution separated containing glutathione in the solution containing the other components.
In the latter case, if the solution containing the other components is not itself at a pressure reduced or no oxygen, the instruction manual specifies advantageously that the final solution, now ready to employment, must be used within a short period of time a few hours, for example three or four hours.
Advantageously, the solutions according to the invention may also comprise, in addition to glutathione reduced, a cornposé opposing the formation of radicals such as metal chelators and especially the deferoxamine (DCI).
By reduced value of oxygen concentration in accordance with. the invention preferably means a -30 maximum dissolved oxygen concentration less than 0.1 ppm.

To achieve, as an example, a solution according to the invention is prepared in an aqueous medium pyrogen-free sterilized, preferably degassed, composition having the following content `i Constituent Concentration g / l mmol / liter K + 15 Na + 100 Mg ++ 13 Ca ++ 0.25 Chloride 41.5 - Calcium chloride, 2H20 0.037 - Potassium chloride 1,118 - Magnesium chloride 2,642 Histidine (basca) 4,650 30 Mannitol 10.930 60 Lactobionate (acid) 28,664 80 Glutamate (aci (ie) 2,942 Water (quantity for one liter readjusted at pH 7,30 Theoretical Osmolality 360 mOsm / kq In a pyrogen-free, sterile, degassed aqueous medium, and under protected conditions of atmospheric oxygen, a solution is produced having the following content, per liter Concentration 215 q / 1 mmol / liter Glutathione (replenishes GSH) 185.4 600 Histidine 4,650 30 ez 106249 This solution is packaged in the form of syringes protected from oxygen from the air and having a capacity of 10 ml of solution for a pocket of two liters or 5 ml for a pocket of one liter.
Preferably the whole is presented in form a kit containing, on the one hand, a solution in a pocket two liters, and secondly a syringe with a ml.
For the use we inject the contents of the 10 syringe in the pocket so as to dilute the contents of the syringe in the poc, he and it leads to a concentration glutathione i-educed of the order of 3 mmol / l.

The solution thus prepared is quickly used to ensure the infusion of the organ to be explanted for get the cardiac arrest. A sufficient volume of solution identical prepared in the same way is used for the preservation of the organ.
During implantation, we still use a identical solution for reperfusion operations, the solution being advantageously mixed with the blood of the patient.

Claims (13)

1. Unique solution for infusion, storage and reperfusion, having substantially the following composition:
Constituent Concentration g / l mmol / liter K + 15 ~ 5%
Na + 100 ~ 10%
Mg ++ 13 ~ 5%
Ca ++ 0.25 ~ 5%
Chlorides 41.5 ~ 5%
- Calcium chloride, 2H2O 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 a liter readjusted at pH 7.30 ~ 0.10 to + 20 ° C

Theoretical Osmolality 360 m Osm / kg Glutathione reduced GSH or equivalent 0.5 to 10 mmol / l, reduced glutathione, or equivalent, being in solution under oxygen partial pressure reduced or zero and now substantially at this value up use. 2. Solution according to claim 1 containing 3 mmol / l GSH reduced glutathione, or equivalent.

3. Solution according to claim 1 or 2 in the form a single preparation ready for use and containing all components, prepared and stored away from oxygen air in a plastic container impermeable to the air. 4. Solution according to claim 1 or 2 in the form a single preparation ready for use and containing all components, prepared and stored away from oxygen air in a waterproof plastic bottle.
the air. 5. Solution according to claim 1 or 2 in the form a single preparation ready for use and containing all components, prepared and stored away from oxygen air in a waterproof plastic bag the air. 6. Solution according to claim 1 prepared and stored as two separate preparations, namely a first preparation containing reduced glutathione, prepared and stored away from the oxygen of the air and a second preparation containing the other ingredients in solution. 7. Solution according to claim 1 prepared and stored as two separate preparations, namely a first preparation containing glutathione reduces GSH being prepared and kept safe from oxygen from the air and having the following composition for one liter:
Glutathione reduced GSH 185.4 g / l 600 ~ 10% mmol / l Histidine 4,650 g / l 30 ~ 10% mmol / l. 8. Solution according to claim 6 or 7, characterized in that the first preparation containing the reduced glutathione is packaged in a sealed syringe in the air. 9. Solution according to claim 6 or 7, characterized in that the first preparation containing the reduced glutathione is packaged in a sealed syringe to the air of 10ml. 10. Solution according to claim 1, characterized in that it is in the form of a kit containing:

- a first preparation contained in a airtight oxygen syringe, containing the reduced glutathione or the like, and histidine, - a second preparation containing, in a pocket or other container, different anions and cations, mannitol, lactobionate, glutamate and histidine, - and instructions to ensure, when the use, mixing or injection of the solution separated containing glutathione in the solution containing the other components. 11. Solution according to one of claims 1 to 10, characterized in that it contains, in addition to glutathione reduced, a compound opposing the formation of radicals. 12. Solution according to one of claims 1 to 10, characterized in that it contains, in addition to glutathione reduced, metal chelators. 13. Solution according to one of claims 1 to 10, characterized in that it contains, in addition to glutathione reduced, deferoxamine.