CH686870A5 - infusion solution, preservation and organ perfusion. - Google Patents

Description

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CH 686 870 A5

Description

The present invention relates to perfusion, preservation (or storage) and organ perfusion solutions, including for heart transplants. It also relates to a process for implementing these solutions applied to the different phases of a transplant.

One of the main causes of failure of heart transplants comes from the risks of damage, even necrosis, of the graft which appear during the reoxygenation of the transplanted organ, and which are linked to ischemia generally prolonged between the beginning explantation in the donor and the end of implantation in the recipient.

A four to five hour ischemia constitutes, for example in the case of the heart, the tolerable upper limit and does not exclude many accidents.

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

Examples of these solutions are the solutions

. HTK from Bretschneider. COLLINS. ST THOMAS. UW

. Stanford.

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

The risk of oxidative cellular and membrane degradations resulting from the production of these radicals has been the subject of several studies in the field of myocardial protection by cardioplegia. These various studies have suggested the introduction of antioxidant substances into the protective solutions used. Different compounds have been proposed, some, such as deferoxamine, allopu-rinol, catalase, Peroxidase, as being capable of opposing the production of free radicals, others, such as superoxide dismutase, which can destroy these radicals , still others, such as vitamin E or equivalents (Trolox) being capable of "neutralizing" free radicals.

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

- G.W. Standeven et al., J. Thorac. Cardiovasc. Surg. 1979, 78, 893-907 Cold-Blood potassium cardioplegia;

- M. Bernier et al., Reperfusion-induced Arrhythmias and Oxygen-derived Free Radicals, Circulation Research, Vol. 58, No. 3, March 1986, 331-340;

- J. C. Chatham et al., Depletion of Myocardial Glutathione: Its effects on heart function and metabo-lism 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., Rôle 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 M. B. Forman, Glutathione Redox Pathway and Reperfusion Injury, Circulation, Vol. 78, no.1, July 1988, 202-213. He suggests that treatment with N-acetylcysteine (NAC) before reperfusion can improve postischemic recovery.

If it may therefore appear interesting 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 the compound and the methods of implementation did not seem obvious and the addition of these compounds, including glutathione, in myocardial infusion and reperfusion solutions in daily hospital practice had not produced decisive results.

Ph. Menasché et al., In Free Radical Trappers in Myocardial Protection in Cardiac Surgery, Ann. Cardiol. Angéiol; 1986, 35 (no. 7bis), 747-452, however conclude that the preservation of the post-ischemic left ventricular function, due to a given cardioplegic solution, could be significantly improved by the addition of antioxidants capable of preventing formation free radicals or destroy or neutralize 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, not to mention possible side or toxic effects. A fortiori when one

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CH 686 870 A5

passing from the field of cardioloplegia, in which the durations of ischemia are relatively short, to the field of transplantation, the literature did not provide any really usable indication on the choice and the methods of implementation of really effective protective solutions.

Ph. Menasché et al., Then described, in the Belgian patent application BE-A 009 101 067 solutions for perfusion and for preservation and / or reperfusion of organs, including the cardiac organ, characterized by inclusion of at least one antioxidant compound, which can in particular be a scavenger of free oxygen radicals, in particular glutathione in the reduced state, or an analog, such as N-acetylcysteine, and by a partial pressure of oxygen zero or very lowered and is kept substantially at this lowered value until use.

However, the various known solutions do not lend themselves well to use, both for perfusion and storage, and for reperfusion, so that, in practice, surgical teams are forced to use at least two different solutions. , one for the perfusion then the storage of the explanted organ, the other for the reperfusion of the organ during implantation.

The present invention proposes to solve these problems and to provide remarkably effective protective solutions for the preservation of organs, with a view to surgical operations and in particular transplantation. Target organs include the heart, as well as other organs, including the liver, lung, and kidney.

The present invention therefore relates to a single perfusion, storage and reperfusion solution, having substantially the following composition:

Constituent (for example in bags of 1 or 2 I) 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 per liter readjusted to pH 7.30 ± 0.10 at + 20 ° C)

Theoretical osmolality 360 m Osm / kg

Glutathione (reduced GSH) or equivalent 0.5 to 10 mmol / l, (preferably 3)

the reduced glutathione, or equivalent, being in solution under reduced or zero partial pressure of oxygen and remaining substantially at this value until use.

This solution can be used during all phases of a transplant, for example as a cardioplegic solution to stop the donor heart, as a storage solution for transport and hypothermic storage of the organ during reimplantation, either in the form of a colloid solution, that is to say preferably after dilution with blood.

In this solution, the prevention of calcium overload in the cells is ensured by the presence of the glutamate component which has the capacity to activate ATP under anaerobic conditions, the low concentration of calcium which reduces transfer by passive diffusion, the high concentration of sodium which limits the sodium-calcium exchange, the transfer of calcium through the channels depending on the electrical potential being limited by the low potassium concentration as well as by the magnesium content. This ionic formulation of extracellular type is made possible by the simul-tanned presence of effective waterproofing agents. Mannitol has the dual ability to act as an osmotic agent and as a free radical scavenger. Lactobionate provides more effective prevention of cell swelling than mannitol alone, and the total concentration of waterproofing agents is equivalent to that of intracellular proteins and non-transferable anions that exert pressure from outside to inside cells.

The pH is preferably weakly acidic (7.20-7.40) because it has been discovered that this value also improves the prevention of calcium overload and, moreover, that, unlike values

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CH 686 870 A5

more alkaline, it improves cell protection during hypothermic ischemic arrest. I_a solution is advantageously buffered with histidine because it has been discovered that among the buffers which can be used in humans, histidine is the only one which remains truly effective at low temperatures.

Of course, the various components of the solution can be replaced by equivalents, provided that these perform practically the same functions.

Among the equivalents of glutathione in a reduced state (GSH) are its precursors or related substances, its analogues, in particular glutathione monoester and N-acetylcysteine. However, other thiol-functional compounds which exhibit the same properties could be used.

The solutions according to the invention can be provided for storage, in the form of a single preparation ready for use and simultaneously containing all of the components of the solution.

In this case, and in accordance with the invention, the solutions are prepared and stored away from oxygen in the air, for example being prepared in degassed solutions, preferably under a nitrogen atmosphere. The solutions according to the invention are stored and preserved in airtight containers such as bottles or, preferably, airtight plastic bags, for example in laminated composites of a type known per se.

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 stored away from oxygen. air, for example in a syringe, and a solution for the other components, which does not necessarily have to be stored away from the oxygen of the air and which can therefore be contained in usual bags.

Preferably the solution containing the reduced glutathione GSH has the following composition (for one liter):

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 pocket, the various anions and cations, mannitol, lactobionate, glutamate and, preferably, histidine,

- a solution contained in a container sealed against oxygen from the air, preferably a syringe, and containing reduced glutathione or the like, and preferably histidine at the same concentration,

- and instructions for ensuring, during use, mixing or injecting the separate solution containing glutathione into the solution containing the other components.

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

Advantageously, the solutions according to the invention can also comprise, in addition to reduced glutathione, a compound opposing the formation of radicals such as metal chelators and in particular deferoxamine (INN).

By reduced value of the oxygen concentration in accordance with the invention is preferably meant a maximum dissolved oxygen concentration of less than 0.1 ppm.

To produce, by way of example, a solution according to the invention, a composition having the following content is prepared in a sterile, sterile, preferably degassed aqueous aqueous medium:

Glutathione (reduced GSH) Histidine

185.4 g / l 4.650 g / l

600 ± 10% mmol / l 30 ± 10% mmol / l

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CH 686 870 A5

Component

Concentration

g / i mmol / liter

K +

15

Na +

100

Mg ++

13

Ca ++

0.25

Chlorides

41.5

- Calcium chloride, 2H2O

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 a liter readjusted to pH 7.30) Theoretical osmolality 360 m Osm / kg

In a degassed sterile pyrogen-free aqueous medium, and under conditions protected from atmospheric oxygen, a solution having the following content is produced, per liter.

Concentration

g / i mmol / liter

Glutathione (reduced GSH)

185.4

600

Histidine

4.650

30

This solution is packaged in the form of syringes protected from the oxygen of the air and having a capacity of 10 ml of solution for a bag of two liters or 5 ml for a bag of one liter.

Preferably the whole is presented in the form of a kit containing, on the one hand, a solution in a two-liter 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 pocket so as to dilute the contents of the syringe in the pocket and a reduced glutathione concentration of the order of 3 mmol / l is obtained.

The solution thus prepared is quickly used to ensure the perfusion of the organ to be explanted to obtain cardiac arrest. A sufficient volume of identical solution prepared in the same way is used for the preservation of the organ.

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

Claims (8)

Claims 1. Single infusion, storage and reperfusion solution, having substantially the following composition: 5 5 10 15 20 25 30 35 40 45 50 55 60 65 CH 686 870 A5 Constituent mmol / liter K + Na + Mg ++ Ca ++ Chlorides - Calcium chloride, 2H2O - Potassium chloride - Magnesium chloride Histidine (base) Mannitol Lactobionate (acid) Glutamate (acid) 15 ± 5% 100 ± 10% 13 ± 5% 0.25 ± 5% 41.5 ± 5% 0.037 1,118 2,642 4.650 10,930 28,664 2.942 ± 5% 30 + 10% 60 ± 5% 80 ± 5% 20 ± 10% Water (quantity per liter readjusted to pH 7.30 ± 0.10 at + 20 ° C) Theoretical osmolality 360 m Osm / kg Glutathione in a reduced state or its precursors or glutathione monoester or N-acetyl-cysteine: 0.5 to 10 mmol / l, reduced glutathione, or its precursors or glutathione monoester or N-acetylcysteine, being in solution under reduced or zero partial oxygen pressure and being maintained at approximately this value until use. 2. Solution according to claim 1, characterized in that the reduced glutathione concentration is 3 mmol / l. 3. Solution according to one of claims 1 or 2 in the form of a single preparation ready for use and containing all the components, in airtight containers such as bottles or bags in airtight plastic material. . 4. Solution according to one of claims 1 or 2, in the form of two separate preparations, namely a preparation containing reduced glutathione, and a preparation containing the other components in solution. 5. Solution according to claim 4 characterized in that the preparation containing the reduced glutathione GSH has the following composition (for one liter): 6. Solution according to one of claims 4 and 5, characterized in that the preparation containing reduced glutathione is packaged in an airtight syringe, in particular of 10 ml. 7. Solution according to claim 6, characterized in that it is in the form of a kit containing: - a preparation containing, in a pocket or other container, the various anions and cations, mannitol, lactobionate, glutamate and histidine, - a preparation contained in a syringe tight to oxygen of air, containing reduced glutathione or its precursors or glutathione monoester or N-acetylcysteine and histidine. 8. Solution according to one of claims 1 to 7 characterized in that it contains, in addition to reduced glutathione, a compound opposing the formation of radicals such as metal chelators and in particular deferoxamine (INN). Glutathione (reduced GSH) Histidine 185.4 g / l 4.650 g / l 600 ± 10% mmol / l 30 ± 10% mmol / l 6