Method for monitoring the viability of a graft

The present invention relates to a method of monitoring (monitoring) the oxygenation of a graft, pending its transplantation.

The delivery of grafts requires particularly strict hygienic and temperature conditions in order to maintain the graft in a state to be implanted. The conventional graft delivery procedure includes a first explantation step during which the graft is taken from a donor under aseptic conditions, generally in the operating room. The graft is then placed in a sealed pot which is placed in a first plastic bag hermetically sealed by a clip closure. This set is then placed in a second plastic bag of the same type and closed in the same way. Everything is placed in an insulating transport cooler filled with a cooling substance (for example ice and / or eutectic gels) which makes it possible to maintain the graft at a temperature slightly above 0 ° C. The sachets covering the sealed jar protect the graft from any contact with the cooling substance and with the ambient air potentially carrying germs. Arrived at destination, the assembly consisting of the two sachets and the pot containing the graft is removed from the insulating transport cooler and introduced into the implantation room, which is also aseptic.

This procedure is therefore delicate: the packaging must be sterile, adapted to the organ, and the transport must be carried out quickly.

Despite this, the transplant has a limited lifespan, which varies according to the organ (for example 4 hours for a heart, 10 hours for a liver and lungs; 36 hours for a kidney).

There is therefore a need for a method making it possible to increase the viability of the graft, including during its transport. This method must be simple to implement, and must be compatible with any transport route (road but also air). This method should make it possible to physiologically assess the graft in a global and rapid manner.

The Applicant has now found a process that responds to this problem. This process is simple to carry out, and makes it possible to prolong the life of the graft. In particular, the method according to the invention makes it possible to evaluate the oxygenation of the graft.

The subject of the invention is therefore a method for monitoring (or monitoring) the oxygenation of a graft, comprising:

a) providing an organ preservation solution in a sealed container.

Preferably, the organ preservation solution is mixed with at least one oxygen carrier, in order to obtain a composition. Preferably, the organ preservation solution is mixed with at least one oxygen transporter chosen from extracellular hemoglobin from Annelids, its globins and its globin protomers, in order to obtain a composition, in the sealed container;

b) immersing the graft in the solution or the composition obtained in a), to obtain a second composition;

c) introducing an oxygen probe into the solution or the composition obtained in a), or into the second composition of step b); and

d) the closure of the leaktight container,

steps c) and d) being carried out simultaneously or in any order.

The method according to the invention is thus concerned with a physiological parameter, ie the quantity of dissolved dioxygen present in the medium surrounding the graft. It thus accurately reflects the viability of the graft.

The method according to the invention may comprise a step e) of transporting the sealed container, in particular to the place of transplantation of the graft to a recipient.

The recipient is preferably a mammal. Preferably, the recipient is a human, in particular awaiting a transplant, or else a non-human mammal, for example a pig.

The method according to the invention comprises a step a) of supplying an organ preservation solution in a sealed container. Such an organ preservation solution is in particular as described below.

Preferably, the organ preservation solution is mixed with at least one oxygen carrier. Preferably, the organ preservation solution comprises at least one oxygen carrier. Such an oxygen carrier is advantageously chosen from molecules which bind oxygen in a reversible manner. Preferably such a transporter is chosen from the extracellular hemoglobin of Annelids, its globins and its globin protomers.

Preferably, the method according to the invention thus comprises a step a) of mixing an organ preservation solution with at least one oxygen transporter, preferably at least one molecule chosen from extracellular hemoglobin from Annelids. , its globins and its globin protomers, in order to obtain a composition, in a sealed container.

The composition of step a) thus comprises:

- at least one oxygen transporter, preferably at least one globin, a protomer of globin or an extracellular hemoglobin of Annelids, and

- an organ preservation solution.

The oxygen transporter according to the invention is preferably at least one molecule chosen from extracellular hemoglobin of Annelids, its globins and its globin protomers. The extracellular hemoglobin of Annelids is present in the three classes of Annelids: the Polychaetes, the Oligochaetes and the Achetes. We speak of extracellular hemoglobin because it is naturally not contained in a cell, and can therefore circulate freely in the blood system without chemical modification to stabilize it or make it functional.

The extracellular hemoglobin of Annelides is a giant biopolymer with a molecular weight between 2000 and 4000 kDa, made up of about 200 polypeptide chains between 4 and 12 different types that are generally grouped into two categories.

The first category, comprising 144 to 192 elements, groups together the so-called “functional” polypeptide chains which carry an active site of the heme type, and are capable of reversibly binding oxygen; they are globin-type chains whose masses are between 15 and 18 kDa and which are very similar to the a and b type chains of vertebrates.

The second category, comprising 36 to 42 elements, groups together the so-called “structural” or “linker” polypeptide chains having little or no active site but allowing the assembly of subunits called twelfths or protomers.

Each hemoglobin molecule consists of two superimposed hexagons which have been called hexagonal bilayer (hexagonal-bilayer) and each hexagon is itself formed by the assembly of six subunits (or "twelfths" or "protomers" ) in the shape of a drop of water. The native molecule is made up of twelve of these subunits (dodecamer or protomer). Each subunit has a molecular mass between 200 and 250 kDa, and constitutes the functional unit of the native molecule.

Preferably, the extracellular hemoglobin of Annelids is chosen from extracellular hemoglobins of Polychete Annelids, preferably from extracellular hemoglobins of the Arenicolidae family and extracellular hemoglobins of the Nereididae family. Even more preferentially, the extracellular hemoglobin of Annelides is chosen from the extracellular hemoglobin of Arenicola marina and the extracellular hemoglobin of Nereis, more preferably the extracellular hemoglobin of Arenicola marina.

According to the invention, the composition can also comprise at least one globin protomer of the extracellular hemoglobin of Annelids. Said protomer constitutes the functional unit of native hemoglobin, as indicated above.

Finally, the composition can also comprise at least one globin chain of the extracellular hemoglobin of Annelids. Such a globin chain can in particular be chosen from globin chains of the Ax and / or Bx type of extracellular hemoglobin from Annelids.

The extracellular hemoglobin of Annelids and its globin protomers have intrinsic superoxide dismutase (SOD) activity, and therefore do not require any antioxidants to function, unlike the use of mammalian hemoglobin, for which the molecules Antioxidants are contained inside the red blood cell and are not related to hemoglobin. On the other hand, the extracellular hemoglobin of Annelids, its globin protomers and / or its globins do not require a cofactor in order to function, unlike the hemoglobin of mammals, in particular human. Finally, the extracellular hemoglobin of Annelides, its globin protomers and / or its globins not having a blood typing, they make it possible to avoid any problem of immunological reaction.

As demonstrated in the examples, the extracellular hemoglobin of Annelides, in particular the extracellular hemoglobin of Arenicola marina, allows oxygen to be transferred to the graft for several hours, for example at least 10 hours, preferably at least 15 hours. hours, preferably at least 20 hours, preferably at least 21, 22, 23, 25 or 28 hours, especially compared to the preservative solution alone.

In addition, the extracellular hemoglobin of Annelides, in particular the extracellular hemoglobin of Arenicola marina, makes it possible to maintain the p02 of the solution or composition in which the graft bathes at a constant level, for several hours, for example at least. 10 hours, preferably at least 15 hours, preferably at least 20 hours, preferably at least 21, 22, 23, 25, 28 or 30 hours.

The organ preservation solution helps to maintain the basic metabolism of the constituent cells of the transplant. It meets a threefold objective: to wash the arterial blood of the graft, bring the graft homogeneously to the desired storage temperature, and protect and prevent lesions caused by ischemia and reperfusion and optimize recovery of function. The organ preservation solution is therefore clinically acceptable.

The organ preservation solution is an aqueous solution having a pH between 6.5 and 7.5, comprising salts, preferably chloride, sulfate, sodium, calcium, magnesium and potassium ions; sugars, preferably mannitol, raffinose, sucrose, glucose, fructose, lactobionate (which is a waterproofing agent), or gluconate; antioxidants, preferably glutathione; active agents, preferably xanthine oxidase inhibitors such as allopurinol, lactates, amino acids such as histidine, glutamic acid (or glutamate), tryptophan; and optionally colloids such as hydroxyethyl starch, polyethylene glycol or dextran.

According to a preferred embodiment of the invention, the organ preservation solution is chosen from:

- the solution from the University of Wisconsin (UW or Viaspan ® ), which has an osmolality of 320 mOsmol / kg and a pH of 7.4, of the following formulation for one liter, in water: Potassium lactobionate: 100 mM

KOH: 100 mM

NaOH: 27 mM

KH2PO4: 25 mM

MgS0 4 : 5 mM

Raffinose: 30 mM

Adenosine: 5 mM

Glutathione: 3 mM

Allopurinol: 1 mM

Hydroxyethyl starch: 50 g / L,

IGL-1 ® , having an osmolality of 320 mOsm / kg and a pH of 7.4, with the following formulation, for one liter in water:

NaCl: 125 mM

KH2PO4: 25 mM

MgS0 4 : 5 mM

Raffinose: 30 mM

Potassium lactobionate: 100 mM

Glutathione: 3 mM

Allopurinol: 1 mM

Adenosine: 5 mM

Polyethylene glycol (molecular weight: 35 kDa): 1 g / L,

- Celsior ® , with an osmolality of 320 mOsm / kg and a pH of 7.3, with the following formulation for one liter in water:

Glutathione: 3 mM

Mannitol: 60 mM

Lactobionic acid: 80 mM

Glutamic acid: 20 mM

NaOH: 100 mM

Calcium chloride dihydrate: 0.25 mM

MgS04: 1.2 mM

KOI: 15 mM

Magnesium chloride hexahydrate: 13 mM

Histidine: 30 mM,

- SCOT 15 Multi Organs Abdominals ® and SCOT 30 Vascular Grafts ® from Macopharma, both comprising in particular high molecular weight polyethylene glycol (20 kDa),

BMPS Belzer ® , or Belzer machine infusion solution, or KPS1, comprising in particular 100 mEq / L of sodium, 25 mEq / L of potassium, a pH of 7.4 at room temperature, and having an osmolarity of 300 mOsm / L,

- Custodiol ® HTK Solution, with the following formulation for one liter in water, the pH being 7.20 at room temperature, and the osmolality being 310 mOsm / kg:

NaCl: 18.0 mM

KCI: 15.0 mM

KH2PO4: 9 mM

Hydrogenated potassium 2-ketoglutarate: 1.0 mM

Magnesium chloride hexahydrate: 4.0 mM

Histidine, HCl, H2O: 18.0 mM

Histidine: 198.0 mM

Tryptophan: 2.0 mM

Mannitol: 30.0 mM

Calcium chloride dihydrate: 0.015 mM,

- Euro-Collins ® , having an osmolality of 355 mOsm / kg and a pH of 7.0, and with the following formulation for one liter in water:

Sodium: 10 mM

Potassium: 1 15 mM

Chloride: 15 mM

H2PO4 -: 15 mM

HPO4 2 -: 42.5 mM

HCO3 -: 10 mM

Glucose: 194 mM,

- Soltran ® , having an osmolality of 486 mOsm / kg and a pH of 7.1, and of the following formulation for one liter in water:

Sodium: 84 mM

Potassium: 80 mM

Magnesium: 41 mM

Sulphate: 41 mM

Mannitol: 33.8 g / l

Citrate: 54 mM

Glucose: 194 mM,

- Perfadex ® , with an osmolarity of 295 mOsmol / L and of the following formulation in water:

50g / L of dextran 40 (molecular weight: 40,000),

Na +: 138 mM,

K +: 6 mM,

Mg2 +: 0.8 mM,

Cl-: 142 mM,

S0 4 2 : 0.8 mM,

(H2PO4- + HPO4 2 ): 0.8 mM and

Glucose: 5 mM,

- Ringer lactate ® , of the following formulation, in water, the pH being between 6.0 and 7.5 at room temperature, and having an osmolarity of 276.8 mOsmol / L:

Na +: 130 mM,

K +: 5.4 mM,

Ca2 +: 1.8 mM,

Cl-: 1 1 1 mM,

Lactates: 27.7 mM,

- Plegisol ® , of the following formulation, in water:

KOI: 1.193 g / L,

MgCl 2 , 6 H 2 0: 3.253 g / L,

NaCl: 6.43 g / L,

CaCl 2 : 0.176 g / L,

- Solution from Hôpital Edouard Henriot, with the following formulation in water, the pH being equal to 7.4 at room temperature, and having an osmolarity of 320 mOsmol / L:

KOH: 25mM,

NaOH: 125mM,

KH 2 P0 4 : 25mM,

MgCl 2 : 5mM ,

MgS0 4 : 5mM ,

Raffinose: 30mM,

Lactobionate: 100mM,

Glutathione: 3mM,

Allopurinol: 1 mM,

Adenosine: 5mM,

Hydroxyethyl starch 50g / L,

- and the Steen® solution, comprising human serum albumin, dextran and extracellular electrolytes with a low concentration of potassium.

All of these organ preservation solutions are commercial products.

Preferably, the composition of step a) has a pH of between 6.5 and 7.6, and comprises:

- at least one globin, a globin protomer or an extracellular hemoglobin from Annelids, preferably from Arenicolidae,

- calcium ions, preferably in an amount between 0 and 0.5 mM;

- KOH, preferably in an amount between 20 and 100 mM;

- NaOH, preferably in an amount between 20 and 125 mM;

- KH2PO4, preferably in an amount between 20 and 25 mM;

- MgCl2, preferably in an amount between 3 and 5 mM;

- at least one sugar chosen from raffinose and glucose, preferably in an amount between 5 and 200 mM;

- adenosine, preferably in an amount of between 3 and 5 mM;

- glutathione, preferably in an amount between 2 and 4 mM;

- allopurinol, preferably in an amount between 0 and 1 mM; and

- at least one compound chosen from hydroxyethyl starch, polyethylene glycols of different molecular weights and human serum albumin, preferably in a quantity of between 1 and 50 g / L.

Typically, the extracellular hemoglobin of Annelides, its globin protomers and / or its globins, is present at a concentration, relative to the final volume of composition, of between 0.001 mg / ml and 100 mg / ml, preferably between 0.005 mg / ml and 20 mg / ml, more preferably between 0.5 mg / ml and 5 mg / ml, in particular 1 mg / ml.

Typically, the composition of step a) has an osmolarity of between 250 and 350 mOsm / L, preferably between 275 and 310 mOsm / L, preferably about 302 mOsm / L.

The sealed container used in the process according to the invention, in particular in step a), is any container suitable for transporting grafts. Such containers are known from the prior art. In particular, the container is as described in application FR2994163. Preferably, the sealed container corresponds to the Biotainer 2.8L kit. It can be included in a carrying bag, such as that marketed under the name Vitalpack® EVO ™ by E3 Cortex.

Preferably, the sealed container is a pot (or rigid primary packaging) - of sufficient size to contain the graft and the composition of step a) - closed by a lid provided with a handle. Preferably, the cover comprises an opening, preferably circular, allowing the passage of the oxygen sensor. This opening is tight: the edges of the opening are preferably coated with a tight seal and allow the oxygen sensor to be attached.

Preferably, the sealed container is placed in a flexible plastic container as defined below, defining a first hermetic internal volume called useful volume and a second hermetic volume called reserve volume adjacent to the first volume, a sealing element extending between the two volumes to define a hermetic border between the two volumes. In particular, the sealed container is placed in the useful volume. Finally, the sealed container, placed in the useful volume of the container, can be placed in a transport bag. A refrigerant substance, especially used during transport, can be placed in the container.

Preferably, the flexible plastic container defines a first sealed interior volume (useful volume) and a second sealed volume (reserve volume) adjacent to the first volume, a sealing element extending between the two volumes to define a boundary. airtight between the two volumes. The first volume comprises at its end opposite the second volume a device for opening and hermetically closing a first access to the first volume, the second volume being shaped so that a cutout through the second volume releases two grippable portions of the container, the spacing of which removes the hermetic border between the two volumes to form a second access to the first volume distinct from the first access. Thus, cutting the reserve volume protects the sealing element for any liquid retention, in particular from the refrigerant substance used during transport. In particular, any traces of liquid remain on the outer wall of the container, the interior of the gripping portions (and therefore the sealing element) being preserved from any pollution. The spacing of the gripping portions ensures that no pollution can migrate towards the sealing element.

The introduction of the sealed container through a first access and its extraction through a second access preserves the sealed container, by preventing the latter from being exposed to possible contamination of the first access which would have taken place during packaging operations.

Advantageously, such a container is produced by superposing two sheets of flexible plastic material having free edges secured to each other. In this way, the container can easily be made to the dimensions of the content (sealed container). The joined edges of the plastic sheets can be joined together using peelable bonds. This then allows, by simple traction, a corollary opening of the bag over its entire length and releases the content without it being necessary to roll up the packaging around the sealed container. Advantageously, the sealing element comprises a peelable connection between two plastic surfaces.

At the end of step a), an organ preservation solution is thus obtained contained in a sealed container.

At the end of step a), a composition is preferably obtained, based on hemoglobin, globin, or annelid globin protomer and an organ preservation solution, contained in a sealed container. .

Step b) then comprises the immersion of the graft in this composition. The graft can be any organ that can be transplanted. Preferably, the graft is a kidney, a heart, a pancreas, a lung, a liver or else a heart-lung unit.

At the end of step b), a graft is then obtained which is immersed in the solution obtained in step a) or in the composition obtained in step a). Preferably, the graft is completely immersed in the solution or the composition.

Thus, the amount of solution or composition used varies according to the volume of the graft. For example, the composition (milliliter): graft (gram) weight ratio is between 2: 1 and 4: 1.

Then, the method according to the invention comprises the introduction of an oxygen probe into the solution or composition obtained in a), or into the composition of step b): this is step c).

It is important to note that the oxygen probe is introduced directly into the composition, and not on the graft. Indeed, the classic monitoring of graft oxygenation typically includes the evaluation of the rate of oxygen consumption of the total organ (WOOCR for whole organ oxygen consumption rate), and uses an oxygen probe which is placed directly on the graft irrigation systems, for example on the artery and vein (therefore upstream and downstream) of the graft. Such a manipulation is not necessarily easy to implement, takes a certain time (at least a few minutes), and can be damaging for the graft.

On the contrary, according to the process of the present invention, the oxygen probe is directly introduced into the composition or the solution in which the graft is bathed. This avoids any invasive step in the graft.

Thus, step c) of the method according to the invention preferably comprises the introduction of a single oxygen probe into the solution or composition obtained in a), or into the

composition of step b). The oxygen sensor is preferably single. In particular, the method according to the invention does not use two oxygen probes.

The oxygen probe introduced is in particular not brought into contact with the graft, in particular is not placed directly on a graft irrigation system (ie artery or vein). Preferably, the oxygen probe is introduced into the organ preservation solution comprising at least one oxygen carrier, in which the graft is bathed.

The oxygen probe, or oximeter, used makes it possible to measure the concentration of molecular oxygen in the liquid mixture obtained in a) or b), therefore to measure the quantity of dissolved oxygen present in the solution or composition of step a) or in the composition of step b). This measure avoids any invasive step in the graft.

Preferably, the oxygen probe is a Clark electrode. It comprises a probe head coated with a membrane, the probe head consisting of an electrode composed of a platinum cathode and a silver anode immersed in an electrolyte (in particular an alkaline solution of sodium phosphate Na3P04, for example at 50 g / L). The electrode - electrolyte assembly is separated from the liquid medium by the membrane, which is permeable to dioxygen but impermeable to water and ions.

The operating principle is as follows: a potential difference (for example 800 mV) is established between anode and cathode, the oxygen present between the electrodes is reduced. The intensity of the resulting current is proportional to the oxygen concentration in the electrolyte.

Preferably, according to another embodiment, the oxygen probe is a sensor for measuring dissolved oxygen by optical measurement, in particular by luminescence. In this case, it does not include a membrane or an electrolyte. Such a probe is commercially available, in particular under the reference Optod (Digisens range) by Ponsel.

Preferably, the oxygen sensor is a portable model, preferably a pocket model. Preferably this is the ProfiLine Oxi 3205 model from WTW.

Preferably, the probe is waterproof. Preferably, it is attached to the lid of the sealed container.

According to the present invention, in step c), the oxygen probe is placed directly in the composition, therefore in the medium in which the graft is immersed. It is much simpler and more convenient, and faster. In addition, this step is not harmful to the graft, because it is strictly non-invasive.

According to one embodiment, the oxygen probe can be directly introduced into the solution or composition obtained in step a), then the graft is added to said composition.

According to another embodiment, the graft is first added to the solution or composition of step a), then the oxygen probe is introduced into the resulting mixture. Indeed, since the probe is in particular fixed to the lid of the sealed container, it can be introduced into the mixture at the same time as the step of fixing the cover on the sealed container, therefore at the same time as step d).

The method according to the invention comprises a step d) of closing the sealed container. According to the invention, steps c) and d) can be carried out simultaneously or in any order.

As indicated above, in the case where the probe is fixed on the lid of the sealed container, it can be introduced into the mixture at the same time as the step of fixing the lid on the sealed container; in this case, steps c) and d) are simultaneous.

If the probe is not yet attached to the cover, it can be inserted:

either before the container is closed: in this case, step c) takes place before step d); or after the container has been closed: in this case, step d) takes place before step c).

Once the container is closed, the graft can thus be transported under good conditions to its destination.

In particular, thanks to the presence of the oxygen probe in the composition, the monitoring of the oxygenation of the graft is carried out in real time.

Furthermore, by the presence of a globin, a protomer of globin or an extracellular hemoglobin of Annelids in the composition, the transport can be effected by any means (ground or air transport), and not requires no special condition. This is therefore advantageous compared to the use of gaseous oxygen, which is present in specific containers (bottles in general) maintained at a given pressure, and therefore less easy to transport (in particular by air).

Transport step e) can be carried out by placing the sealed container in a suitable container. Such a container is known from the prior art, and is suitable for transporting grafts. Preferably, this container is a transport bag, for example as described in application EP1688124. It is more particularly a bag for transporting a graft for transplantation comprising at least one internal wall delimiting at least two compartments each having an openable part, a first compartment being intended to receive one or more vials and / or jars of biological samples from the donor (for example blood) protected by a block of flexible and elastic material, while a second compartment contains an isothermal tank intended to receive the sealed container according to the invention.

In particular, transport is carried out by placing the sealed container in a bag marketed under the name Vitalpack® EVO ™ by E3 Cortex.

According to the present invention, the graft can be stored in dynamic perfusion.

The method according to the invention can also comprise, after step d) and / or e), a step e ′) of establishing a calibration curve representing the pO2 of the composition obtained in a) in which the graft is submerged, optionally normalized with respect to the weight of the graft, as a function of time.

The p02 is in particular expressed in mmHg or in bar or in%.

Obtaining this calibration curve makes it possible to deduce, for a given graft, the optimal duration of oxygenation. For example, for a kidney, obtaining a calibration curve makes it possible to deduce the maximum duration of oxygenation, if a pO2 of at least 50% is desired.

Thus, the present invention also relates to a method for determining the viability of a graft, comprising the use of the calibration curve described above. This curve is in particular obtained according to the method described above.

Such a method for determining the viability of a graft comprises in particular the following steps:

(i) providing an organ preservation solution in a sealed container. Preferably step (i) comprises mixing an organ preservation solution with at least one oxygen transporter chosen from extracellular hemoglobin from Annelids, its globins and its globin protomers, in order to obtain a composition, in a sealed container;

(ii) immersing the graft in the solution or the composition obtained in (i);

(iii) introducing an oxygen probe into the solution or the composition obtained in (i), or into the composition of step (ii);

(iv) closing the sealed container, steps (iii) and (iv) being carried out simultaneously or in any order; then

(v) transport of the sealed container, in particular to the place of transplantation of the graft to a recipient,

and wherein the maximum time elapsing between step (ii) and the end of step (v) is determined according to the calibration curve described above, keeping said pO2 at a physiologically acceptable value.

By "physiologically acceptable pO2 value" is meant a value which allows the viability of the graft.

It should be noted that all the operating conditions and embodiments of steps (i) to (v) are as described for steps a) to e) above.

The invention is now illustrated with the aid of the examples which follow.

Example 1: Study of conservation of a pig kidney in a preservation solution with or without annelid hemoglobin

The aim of this study is to establish a link between the effects of extracellular hemoglobin from Arenicola marina (M101) on the reduction of ischemia / reperfusion lesions in static cold preservation and the mechanism of action of molecule. In order to establish this link, sequential measurements are performed both at the functional level and at the cellular level.

METHODS

1. HEMQ2life®

Arenicola marina extracellular hemoglobin was used to formulate a commercial product, HEM02life® (Hemarina SA), an additive to preservative solutions. HEM02life® is manufactured in accordance with EU Good Manufacturing Practice for Medicines.

2. Preservation of the kidney

Both kidneys were explanted from the same animal (pig) 18 minutes after the circulatory arrest.

The kidneys were washed with 200 ml of UW (Bridge to Life) organ preservation solution or 200 ml of UW + 1 g / L HEM02life®. The kidneys were weighed after tightening. The kidneys were immediately immersed in an organ reservoir

hermetically sealed and filled with 800 ml of their respective solutions (standard solution: UW and UW + HEM02life® 1 g / L) at 6 ° C.

Then the reservoirs were transported to the laboratory under hypothermic conditions at 4 ° C and successive measurements for PO2 and biomarkers start at 1 hour.

Two other reservoirs (controls) are used to measure the same parameters with no kidney inside, and serve as controls for both UW and UW + HEM02life® 1g / L The reservoirs were placed on a shaking table with shaking slow.

3. Analyzes

Functional analyzes of M101

The sequential measurement was carried out at 1 h, 4 h, 6 h, 24 h, 30 h, 48 h, 55 h: HEM02life® functional analyzes.

Binding to oxygen: the functionality of M101 is followed by spectrophotometry allowing the characterization of oxyhemoglobin (Hb02) and deoxyhemoglobin (deoxy-Hb). The absorption spectra are recorded over the 370-640 nm range (UVmc2, SAFAS, Monaco) according to the method described by Thuiller et al. 201 1, Supplementation With a New Therapeutic Oxygen Carrier Reduces Chronic Fibrosis and Organ Dysfunction in Kidney Static Preservation: A New 02 Therapeutic Molecule Improves Static Kidney Preservation. Am J Transplant. 201 Sep 1; 1 1 (9): 1845-60.

P02 and pH monitoring

Sequential measurements were taken every hour from 1 h to 12 h; 24 hours to 36 hours and 48 to 55 hours for the dissolved pH and G02 of the preservation solution.

Dissolved O2 (d02) and pH are measured using an O2 sensor (WTW Oxi 3205) and a pH sensor (WTW pH31 10) directly in the closed (airtight) tank.

RESULTS

The results are in Figures 1 and 2.

Functional analyzes of M101

The functional analyzes show that the spectral signature of M101 of tO ​​at 52h reveals the presence of hemoglobin in the oxyHb form. The molecule remains in the oxyHb form from the start until 52h, which means that there is oxygen available in the preservation solution.

The spectral signature of M101 from 52h to 55h is characteristic of deoxyHb and shows that the molecule has transferred all of its oxygen to the solution.

P02 and pH monitoring

For the controls, the pO2 was measured at 100% dissolved 02 in the two reservoirs at tO and does not decrease for 55 h at 6 ° C.

This means that there is no O2 consumption under these kidney-free conditions.

For the kidneys, their respective weight is 273.4g (UW + HEM02life® 1 g / L) and 268.0g (UW). The temperature of the room during the experiment is maintained at 6 ° C.

The p02 is indexed to 100% dissolved 02 at 6 ° C at the start of the experiment. During the first hour, the pO2 rapidly decreases to 50% in both solutions.

The results on p02 are in FIG. 1. The p02 continues to decrease sharply in the solution which does not contain HEM02life® to reach 0% after 24 h. The evolution of p02 in the preservation solution containing HEM02life® is slowed down and then stabilized for 1 to 30 hours at approximately 50% dissolved oxygen (p50). This plateau therefore corresponds to the situation in which p02 = p50. It is only after 30h that the dissolved oxygen slowly drops back to 0% at 52h.

These results, coupled with the functional results, show that HEM02life® is a good carrier of oxygen and is able to distribute it as it is stored, from tO up to 52 hours. At 52h, the parallel measurements of p02 and the functional analysis show that at this time, dissolved G02 is at 0% in the preservation solution, which means that HEM02life® has delivered all of its transported oxygen. HEM02life® is a very good oxygen donor to a fluid. The molecule distributes oxygen to maintain 50% of dissolved 02 from 1 h to 30 h, then until the exhaustion of the oxygen transported from 30 h to 52 h. A decline is observable at 30h and dissolved G02 slowly decreases to reach 0% at 52h. Without HEM02life®, 50% of the p02 is reached after 1 h, and the p02 already reaches 0% after 24 h.

The results on pH are in Figure 2. In the reservoir not containing kidney, the pH was measured. It is very stable in the two reservoirs containing UW (pH 7.4), and UW + HEM02life® 1 g / L (pH 7.5).

In tanks with kidneys, the pH is very stable in the solution to which HEM02life® 1 g / L has been added, around 7.4, from the start up to 55 hours. The pH in UW preservative solution without HEM02life® 1 g / L decreases from 7.4 to 7.1 in 55 hours. The difference is probably explained by the acidosis of the reservoir containing the kidney without HEM02life® 1 g / L.

These results clearly demonstrate the beneficial use of HEM02life® at 1 g / L in addition to the low temperature preservation solution. The evolution of p02 shows that HEM02life® transfers oxygen 28 hours more than the preservation solution alone. In addition, HEM02life® maintains the oxygen dissolved in the 50% solution for 30 hours, ie at a constant level allowing much better preservation of the organ. Biochemical analyzes confirm these results.

CLAIMS

1. A method of monitoring the oxygenation of a graft, comprising:

a) providing an organ preservation solution and mixing this solution with at least one oxygen carrier, in order to obtain a composition, in a sealed container;

b) immersing the graft in the composition obtained in a) to obtain a second composition;

c) the introduction of an oxygen sensor into the composition obtained in a), or into the second composition of step b); and

d) the closure of the leaktight container,

steps c) and d) being carried out simultaneously or in any order.

2. The method of claim 1, wherein:

step a) comprises mixing the organ preservation solution with at least one oxygen transporter chosen from extracellular hemoglobin from Annelids, its globins and its globin protomers, in order to obtain a composition, in the sealed container; and

step b) comprises immersing the graft in the composition obtained in a).

3. Method according to claim 1 or 2, comprising a step e) of transporting the sealed container, in particular to the place of transplantation of the graft to a recipient.

4. Method according to one of claims 1 to 3, wherein step c) comprises the introduction of a single oxygen sensor in the composition obtained in a), or in the second composition of step b). .

5. Method according to one of claims 1 to 4, wherein the oxygen probe of step c) is a Clark electrode or sensor for measuring dissolved oxygen by optical measurement, in particular by luminescence.

6. Method according to one of claims 1 to 5, wherein the oxygen probe of step c) comprises a probe head coated with a membrane, the probe head consisting of an electrode composed of a cathode. in platinum and a silver anode immersed in an electrolyte, said membrane being permeable to oxygen but impermeable to water and to ions.

7. Method according to one of claims 1 to 6, comprising a step e ′) of establishing a calibration curve representing the p02 of the composition obtained in a) in which the graft is immersed, optionally normalized with respect to the weight of the graft, as a function of time.

8. A method of determining the viability of a graft, comprising the use of the calibration curve obtained according to the method of claim 7.

9. The method of claim 8, comprising:

(i) providing an organ preservation solution, preferably in admixture with at least one oxygen transporter, preferably selected from extracellular hemoglobin of Annelids, its globins and its globin protomers, in order to 'obtain a composition, in a sealed container;

(ii) immersing the graft in the composition obtained in (i);

(iii) introducing an oxygen sensor into the composition obtained in (i), or into the composition of step (ii);

(iv) closing the sealed container, steps (iii) and (iv) being carried out simultaneously or in any order; then

(v) transport of the sealed container, in particular to the place of transplantation of the graft to a recipient,

and wherein the maximum time elapsing between step (ii) and the end of step (v) is determined according to the calibration curve obtained according to the method of claim 3, keeping said p02 at a value physiologically acceptable.

10. Method according to one of claims 2 to 9, wherein the extracellular hemoglobin of Annelids is chosen from extracellular hemoglobins of Polychete Annelids, preferably from extracellular hemoglobins of the Arenicolidae family and extracellular hemoglobins of the family of Nereididae, more preferably among the extracellular hemoglobin of Arenicola marina and the extracellular hemoglobin of Nereis, more preferably the extracellular hemoglobin of Arenicola marina.

1 1. Method according to one of claims 1 to 10, wherein the organ preservation solution is an aqueous solution having a pH between 6.5 and 7.5, comprising salts, preferably chloride, sulfate, sodium ions, calcium, magnesium and

potassium; sugars, preferably mannitol, raffinose, sucrose, glucose, fructose, lactobionate (which is a waterproofing agent), or gluconate; antioxidants, preferably glutathione; active agents, preferably xanthine oxidase inhibitors such as allopurinol, lactates, amino acids such as histidine, glutamic acid (or glutamate), tryptophan; and optionally colloids such as hydroxyethyl starch, polyethylene glycol or dextran.