METHOD FOR MONITORING THE VIABILITY OF A GRAFT

Abstract

The present invention relates to a method for monitoring the oxygenation of a graft, comprising: a) mixing an organ storage solution preferably with 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; b) immersion of the graft in the composition obtained in a); c) the introduction of an oxygen probe in the composition obtained in a), or in the composition of step b); and d) the closure of the hermetic container, steps c) and d) being carried out simultaneously or in any order.

It also relates to a method for determining the viability of a graft.

Claims

1. Method of monitoring the oxygenation of a graft, comprising: a) providing an organ storage solution and mixing the organ storage solution with at least one oxygen carrier, to obtain a composition, in a sealed container, b) immersing the graft in the composition obtained in step a) to obtain a second composition; c) introducing an oxygen probe in the composition obtained in a), and/or in the second composition of step b); and d) hermetically closing the sealed container, steps c) and d) being carried out simultaneously or in any order.

2. Method according to claim 1, wherein: the at least one oxygen carrier is an extracellular hemoglobin a globin from annelids or a globin protomer from annelids.

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

4. Method according to claim 1, wherein step c) comprises introducing a single oxygen probe in the composition obtained in a), or in the second composition obtained in step b).

5. Method according to claim 1, wherein the oxygen probe of step c) is a Clark electrode or sensor for measuring dissolved oxygen by optical measurement.

6. Method according to claim 1, 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 of platinum and with a silver anode immersed in an electrolyte, said membrane being permeable to oxygen but impermeable to water and to ions.

7. Method according to claim 1, comprising a step e′) of establishing a calibration curve representing the pO2 of the composition obtained in a) in which the graft is immersed, optionally normalized relative to the weight of the graft, as a function of time.

8. (canceled)

9. Method of determining the viability of a graft comprising: (i) providing an organ-storage solution with at least one oxygen carrier, in order to obtain a composition, in a sealed container; (ii) immersing the graft in the composition obtained in (i) to obtain a second composition; (iii) introducing an oxygen probe in the composition obtained in (i), and/or in the second composition of step (ii); (iv) closing the sealed container, steps (iii) and (iv) being carried out simultaneously or in any order; then (v) transporting the sealed container, to the place of transplantation of the graft to a recipient, vi) during steps ii) to v), monitoring the dissolved oxygen in the second composition, wherein the amount of dissolved oxygen indicates the viability of the graft.

10. Method according to claim 2, wherein the extracellular hemoglobin of annelids is an extracellular hemoglobin of a Polychete Annelid.

11. Method according to claim 1, wherein the organ-storage solution is an aqueous solution having a pH between 6.5 and 7.5, comprising salts; sugars; antioxidants; active agents; and, optionally, colloids.

12. The method of claim 5, wherein the optical measurement is luminescence.

13. The method of claim 9, wherein the at least one oxygen carrier is an extracellular hemoglobin of annelids, a globin of annelids or a globin protomer of annelids.

14. The method of claim 10, wherein the Polychete Annelid is of the Arenicolidae family or the Nereididae family.

15. The method of claim 14, wherein the Arenicolidae family is Arenicola marina.

16. The method of claim 9, further comprising a step of establishing a calibration curve representing the pO2 of a composition obtained in which the graft is immersed, optionally normalized relative to the weight of the graft, as a function of time.

17. The method of claim 11, wherein: the salts include chloride, sulfate, sodium, calcium, magnesium and jarassium; the sugars include mannitol, raffinose, sucrose, glucose, fructose, lactobionate and gluconate; the antioxidants include glutathione; the active agents include xanthine oxidase inhibitors and amino acids; and, the colloids include hydroxyethyl starch, polyethylene glycol or dextran.

18. The method of claim 17, wherein the xanthine oxidase inhibitors include allopurinol and lactates; and the amino acids include histidine, glutamic acid and tryptophan.

Description

EXAMPLE 1: STORAGE STUDY OF A PIA KIDNEY IN A PRESERVATIVE SOLUTION WITH OR WITHOUT ANNELID HEMOGLOBIN

[0191] 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 storage and the mechanism of action of the molecule. In order to establish this link, sequential measurements are performed at both the functional level and the cellular level.

[0192] Methods

[0193] 1. HEMO2life®

[0194] Arenicola marina extracellular hemoglobin was used to formulate a commercial product, HEMO2life® (Hemarina SA), an additive to storage solutions. HEMO2life® is manufactured in accordance with EU Good Manufacturing Practice for Medicines.

[0195] 2. Storage of the Kidney

[0196] Both kidneys were explanted from the same animal (pig) 18 minutes after the circulatory arrest.

[0197] The kidneys were washed with 200 ml of UW (Bridge to Life) organ-storage solution or 200 ml of UW+1 g/l HEMO2life®. The kidneys were weighed after tightening. The kidneys were immediately immersed in a tightly closed organ reservoir and filled with 800 ml of their respective solutions (standard solution: UW and UW+HEMO2life® 1 g/l) at 6° C.

[0198] Then the reservoirs were transported to the laboratory under hypothermic conditions at 4° C. while successive measurements for pO2 and biomarkers start at 1 hour.

[0199] Two other reservoirs (controls) are used to measure the same parameters with no kidney inside, and serve as controls for both UW and UW+HEMO2life® g/l.

The reservoirs were placed on a shaking table with slow shaking.

[0200] 3. Analyzes

[0201] Functional Analyzes of M101

[0202] The sequential measurement was carried out at 1 h, 4 h, 6 h, 24 h, 30 h, 48 h, 55 h: HEMO2life® functional analyzes.

[0203] Binding to oxygen: the functionality of M101 is followed by spectrophotometry allowing the characterization of oxyhemoglobin (HbO.sub.2) 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. 2011, Supplementation With a New Therapeutic Oxygen Carrier Reduces Chronic Fibrosis and Organ Dysfunction in Kidney Static Storage: A New O2 Therapeutic Molecule Improves Static Kidney Storage. Am J Transplant. 2011 September; 11 (9): 1845-80.

[0204] pO2 and pH Monitoring

[0205] Sequential measurements were taken every hour from 1 h to 12 h; 24 h to 36 h and 48 to 55 h for the pH and dissolved O2 of the storage solution.

[0206] Dissolved O2 (dO2) and pH are measured using an O2 sensor (WTW Oxi 3205) and a pH sensor (WTW pH3110) directly in the closed (hermetic) tank.

[0207] Results

[0208] The results are in FIGS. 1 and 2.

[0209] Functional Analyzes of M101

[0210] The functional analyzes show that the spectral signature of M101 from t0 to 52 h reveals the presence of hemoglobin in the oxyHb form. The molecule remains in the oxyHb form from the start until 52 h, which means that there is oxygen available in the storage solution.

[0211] The spectral signature of M101 from 52 h to 55 h is characteristic of deoxyHb and shows that the molecule has transferred all of its oxygen to the solution.

[0212] pO2 and pH Monitoring

[0213] For the controls, the pO2 was measured at 100% dissolved O2 in the two reservoirs at t0 and does not decrease for 55 hours at 6° C.

[0214] This means that there is no O2 uptake in these kidney-free conditions.

[0215] For the kidneys, their respective weight is 273.4 g (UW+HEMO2life® 1 g/l) and 268.0 g (UW). The room temperature during the experiment is kept at 6° C.

[0216] The pO2 is indexed to 100% dissolved O2 at 6° C. at the start of the experiment. The first hour, the pO2 decreases rapidly to 50% in both solutions.

[0217] The results on pO2 are in FIG. 1. The pO2 continues to decrease sharply in the solution which does not contain HEMO2life® to reach 0% after 24 h. The evolution of pO2 in the storage solution containing HEMO2life® 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 pO2=p50. It is only after 30 h that the dissolved oxygen slowly drops back to 0% at 52 h.

[0218] These results, coupled with the functional results, show that HEMO2life® is a good carrier of oxygen and is able to distribute it as it is stored, from t0 up to 52 hours. At 52 h, parallel pO2 measurements and functional analysis show that at this time, dissolved O2 is at 0% in the storage solution, which means that HEMO2life® has delivered all of its transported oxygen. HEMO2life® is a very good donor of oxygen to a fluid. The molecule distributes oxygen to maintain 50% of dissolved O2 from 1 h to 30 h, then until the oxygen transported is exhausted from 30 to 52 h. A decline is observable at 30 h and the dissolved O2 slowly decreases to reach 0% at 52 h. Without HEMO2life®, 50% of the pO2 is reached after 1 h, and the pO2 already reaches 0% after 24 h.

[0219] The results on pH are in FIG. 2. In the reservoir not containing kidney, the pH was measured. It is very stable in the two reservoirs containing UW (pH of 7.4), and UW+HEMO2life® 1 g/l (pH of 7.5).

[0220] In reservoirs with kidneys, the pH is very stable in the solution to which HEMO2life® 1 g/l has been added, around 7.4, from the start up to 55 h. The pH in UW storage solution without HEMO2life® 1 g/L decreases from 7.4 to 7.1 in 55 h. The difference is probably explained by the acidosis of the reservoir containing the kidney without HEMO2life® 1 g/l.

[0221] These results clearly demonstrate the beneficial use of HEMO2life® at 1 g/L in addition to the low temperature storage solution. The evolution of pO2 shows that HEMO2life® transfers oxygen 28 h more than the storage solution alone. In addition, HEMO2life® maintains dissolved oxygen in the 50% solution for 30 h, i.e. at a constant level allowing much better storage of the organ. Biochemical analyzes confirm these results.