Solution for preserving and/or rinsing an organ to be transplanted

Abstract

This invention relates to an aqueous solution for preserving and rinsing organs to be transplanted comprising: sodium (Na+) ions at a concentration between 30 and 150 mmol.Math.L.sup.−1; potassium ions (K+) at a concentration between 10 and 40 mmol.Math.L.sup.−1; polyethylene glycol with a molecular weight of 35,000 g.Math.mol-1 (PEG 35000) at a concentration between 2 and 5 g.Math.L.sup.−1.

Claims

1. An aqueous solution comprising: sodium (Na.sup.+) ions at a concentration between 30 and 150 mmol.Math.L.sup.−1; potassium ions (K.sup.+) at a concentration between 10 and 40 mmol.Math.L.sup.−1; polyethylene glycol with a molecular weight of 35,000 g.Math.mol.sup.−1 (PEG 35000) at a concentration between 2 and 5 g.Math.L.sup.−1; raffinose at a concentration between 25 and 35 mmol.Math.L.sup.−1 and allopurinol at a concentration between 0.5 and 1.5 mmol.Math.L.sup.−1, or mannitol at a concentration between 40 and 80 mmol.Math.L.sup.−1, or mannitol at a concentration between 40 and 80 mmol.Math.L.sup.−1 and histidine at a concentration between 25 and 35 mmol.Math.L.sup.−1, and wherein the aqueous solution is an organ preservation and rinsing solution.

2. The solution of claim 1, further comprising glutathione, as an antioxidant agent, at a concentration between 1 and 11 mmol.Math.L.sup.−1.

3. The solution of claim 1, further comprising zinc ions (Zn.sup.2+) at a concentration between 0.170 and 0.210 mmol.Math.L.sup.−1.

4. The solution of claim 1 further comprising nitrite ions (NO.sup.2−) at a concentration between 5 and 100 nmol.Math.L.sup.−1.

5. The solution of claim 1, wherein the solution has a pH between 7.2 and 7.6.

6. The solution of claim 1 further comprising an impermeant anion, a sugar, a cell membrane stabilizer, a buffer solution, and/or an energy source.

7. The solution of claim 1 further comprising: lactobionic acid at a concentration between 80 and 120 mmol.Math.L″.sup.−1; sulphate ions (SO.sub.4.sup.2−) at a concentration between 4 and 6 mmol.Math.L.sup.−1; phosphate ions (PO.sub.4.sup.3−) at a concentration between 20 and 30 mmol.Math.L.sup.−1; and adenosine at a concentration between 4 and 6 mmol.Math.L.sup.−1.

8. The solution of claim 1 further comprising: PEG 35000 at a concentration of 5 g.Math.L.sup.−1; glutathione at a concentration of 9 mmol.Math.L.sup.−1; Zn.sup.2+ ions at a concentration of 0.191 mmol.Math.L.sup.−1; NO.sup.2− ions at a concentration of 50 nmol.Math.L.sup.−1; raffinose at a concentration of 30 mmol.Math.L.sup.−1; lactobionic acid at a concentration of 100 mmol.Math.L.sup.−1; SO.sub.4.sup.2− ions at a concentration of 5 mmol.Math.L.sup.−1; PO.sub.4.sup.3− ions at a concentration of 25 mmol.Math.L.sup.−1; adenosine at a concentration of 5 mmol.Math.L.sup.−1; and allopurinol at a concentration of 1 mmol.Math.L.sup.−1.

9. The solution of claim 1 further comprising: PEG 35000 at a concentration of 5 g.Math.L.sup.−1; glutathione at a concentration of 9 mmol.Math.L.sup.−1; Zn.sup.2+ ions at a concentration of 0.191 mmol.Math.L.sup.−1; NO.sup.2− ions at a concentration of 50 nmol.Math.L.sup.−1; mannitol at a concentration of 60 mmol.Math.L.sup.−1; lactobionic acid at a concentration of 100 mmol.Math.L.sup.−1; SO.sub.4.sup.2− ions at a concentration of 5 mmol.Math.L.sup.−1; PO.sub.4.sup.3− ions at a concentration of 25 mmol.Math.L.sup.−1; and adenosine at a concentration of 5 mmol.Math.L.sup.−1.

10. The solution of claim 1 further comprising: PEG 35000 at a concentration of 5 g.Math.L.sup.−1; glutathione at a concentration of 9 mmol.Math.L.sup.−1; Zn.sup.2+ ions at a concentration of 0.191 mmol.Math.L.sup.−1; NO.sup.2− ions at a concentration of 50 nmol.Math.L.sup.−1; mannitol at a concentration of 60 mmol.Math.L.sup.−1; histidine at a concentration of 30 mmol.Math.L.sup.−1; lactobionic acid at a concentration of 100 mmol.Math.L.sup.−1; SO.sub.4.sup.2− ions at a concentration of 5 mmol.Math.L.sup.−1; PO.sub.4.sup.3− ions at a concentration of 25 mmol.Math.L.sup.−1; and adenosine at a concentration of 5 mmol.Math.L.sup.−1.

11. The solution of claim 7, wherein the SO.sub.4.sup.2− ions provided by MgSO.sub.4 and the PO.sub.4.sup.3− ions provided by KH.sub.2PO.sub.4.

12. The solution of claim 8, wherein the SO.sub.4.sup.2− ions provided by MgSO.sub.4 and the PO.sub.4.sup.3− ions provided by KH.sub.2PO.sub.4.

13. The solution of claim 9, wherein the SO.sub.4.sup.2− ions provided by MgSO.sub.4 and the PO.sub.4.sup.3− ions provided by KH.sub.2PO.sub.4.

14. The solution of claim 10, wherein the SO.sub.4.sup.2− ions provided by MgSO.sub.4 and the PO.sub.4.sup.3− ions provided by KH.sub.2PO.sub.4.

Description

(1) The invention and the advantages deriving therefrom will be better understood from the following figures and examples provided as a non-limiting illustration of the invention.

(2) FIG. 1 represents the quantification of aldehyde dehydrogenase-2 (ALDH 2) activity after 24 hours of static hypothermic preservation of a liver in a UW®, IGL-0, IGL-1 solution or solution according to this invention (INV).

(3) FIG. 2 represents the quantification of transaminase content in liver tissue after 24 hours of static hypothermic preservation of a liver in UW®, IGL-1 solution or solution according to this invention (INV).

(4) FIG. 3 represents the quantification of the glutamate dehydrogenase (GLDH) content in liver tissue after 24 hours of static hypothermic preservation of a liver in UW®, IGL-1 solution or solution according to this invention (INV).

(5) FIG. 4 represents the determination of the amount of cells present in the rinse effluent of a healthy liver or a steatotic liver, rinsed with IGL-1® solution or solution according to this invention (INV).

(6) FIG. 5 represents the quantification of the number of red blood cells remaining in the liver tissue after rinsing a healthy liver or a steatotic liver with UW®, IGL-1® solution or solution according to this invention (INV).

(7) FIG. 6 represents the evaluation of aldehyde dehydrogenase-2 (ALDH 2) activity in the liver after dynamic hypothermic preservation with PERF-GEN® solution or solution according to this invention (INV).

(8) FIG. 7 represents the evaluation of hepatic parenchyma degradation by measuring the aspartate aminotransferase (ASAT) content in the liver after dynamic hypothermic preservation with PERF-GEN® solution or solution according to this invention (INV).

(9) FIG. 8 represents the evaluation of hepatic parenchyma degradation by measuring the glutamate dehydrogenase (GLDH) content in the liver after dynamic hypothermic preservation with PERF-GEN® solution or solution according to this invention (INV).

EXAMPLE EMBODIMENT OF THE INVENTION

(10) 1/Preparation of the Preservation and Rinsing Solution According to this Invention

(11) A solution according to this invention is prepared by mixing the ingredients according to the formulation (per 1 liter) in Table 1:

(12) TABLE-US-00001 TABLE 1 Ingredient Concentration PEG 35000 (molecular weight 35,000 0.14 mM (5 g .Math. L.sup.−1) g .Math. L.sup.−1) Na.sup.+ (provided by NaOH) 125 mM K.sup.+ (provided by KH.sub.2PO.sub.4) 25 mM Zn.sup.2+ (provided by ZnCl.sub.2) 0.191 mM Glutathion 9 mM NO.sup.2− (provided by NaNO.sub.2) 50 nM Raffinose 30 mM Lactobionic acid 100 mM SO.sub.4.sup.2− (provided by MgSO.sub.4) 5 mM PO.sub.4.sup.3− (provided by KH.sub.2PO.sub.4) 25 mM Adenosine 5 mM Allopurinol 1 mM

(13) The preparation of the solution consists in dissolving all the ingredients, under magnetic agitation, in an aqueous solution, and the pH of the obtained solution is adjusted to 7.4.

(14) 2/Comparison of the Viscosity of the Solutions of the Prior Art Compared to the Solution According to this Invention

(15) The viscosity was determined by the European Pharmacopoeia method in Chapter 2.2.9 “Viscosity—Capillary tube method”.

(16) TABLE-US-00002 TABLE 2 Solution 1 IGL-1 ® INV Perf-Gen ® Use Static preservation Rinsing + Dynamic preservation Static preservation + dynamic preservation Oncotic agent PEG 35 (1 g .Math. L.sup.−1) PEG 35 (5 g .Math. L.sup.−1) HES (50 g .Math. L.sup.−1) Viscosity (cP) 1.2 1.4 2.4

(17) These results show that, unexpectedly, the significant increase in the PEG concentration in the INV solution compared to the IGL-1® solution does not induce an increase in the viscosity of the solution.

(18) The viscosity of the solution according to this invention is therefore adapted to its use for static (such as IGL-1®) and dynamic (such as Perf-Gen®) preservation.

(19) 3/Static Preservation of a Liver under Hypothermia Condition

(20) To assess the effectiveness of the preservation solution according to this invention, evaluations were carried out on healthy livers or livers suffering from steatosis.

(21) 3.1/Experimental Conditions

(22) The liver of normal (healthy) and obese rats (Zucker rats; steatotic liver and referred to as “Ob” or “fatty” in the figures) aged 10 to 12 weeks was collected and stored according to techniques known to the skilled person.

(23) The purpose of these experiments is to compare the performance of the solution according to this invention (INV Solution) on the preservation of the liver in hypothermia ex vivo compared to an IGL-0 solution (IGL-1® solution formulated without PEG) or to prior art solutions, namely: Belzer UW® solution (hereinafter referred to as UW); IGL-1® solution.

(24) The organ is then stored statically in the rinsing solution (100 mL; HTK®, IGL-1 ® or INV) for 24 hours at 4° C.

(25) Different parameters were measured to evaluate the effectiveness of the solution according to this invention on liver preservation in hypothermia conditions.

(26) 3.2/Quantification of Aldehyde Dehydrogenase-2 Content in Liver Tissue

(27) Mitochondrial aldehyde dehydrogenase 2 (ALDH 2) is a major enzyme in aldehyde metabolism that protects against toxic accumulation of aldehyde at the cellular level, for example, by converting acetaldehyde to acetic acid. The activation of aldehyde dehydrogenase-2 (ALDH2) is associated with protection of the cells of the organ to be transplanted.

(28) After 24 hours of preservation in UW®, IGL-0 (IGL-1 ® solution formulated without PEG), IGL-1® or INV solutions, an analysis of ALDH2 activation by enzyme kit was performed on healthy livers.

(29) The results are shown in FIG. 1.

(30) The data show that preserving the liver in an INV solution induces an increase in ALDH2 activity in tissue compared to IGL-0, IGL-1 ® and UW® solutions. The result is protection of the organ against damage caused by ischemia.

(31) The formulation of IGL-0 and UW solutions is PEG-free. These results therefore show that the PEG 35 used at a concentration according to this invention in the INV solution provides better organ protection (value of p<0.05).

(32) 3.3/Quantification of Transaminase Content in Liver Tissue

(33) Transaminases (alanine aminotransferase, aspartate aminotransferase . . . ) are enzymes synthesized by hepatocytes and released in case of hepatocellular lesion or necrosis. The transaminase concentration is therefore a marker of the effectiveness of liver preservation with a preservation solution.

(34) After 24 hours of preservation in UW®, IGL-1® or INV solutions, a quantitative analysis of transaminase levels by enzyme kit was performed on steatotic livers.

(35) The results are shown in FIG. 2.

(36) The data show that the transaminase concentration is lower after 24 hours of preservation with the INV solution according to this invention than with the UW® solution. In other words, the INV solution provides better preservation than the UW® solution.

(37) 3.4/Quantification of Glutamate Dehydrogenase Content in Liver Tissue

(38) Glutamate dehydrogenase (GLDH) is a liver-specific mitochondrial enzyme that plays an important role in amino acid catabolism. It participates in the deamination of glutamic acid (or glutamate) to α-ketoglutarate acid. An increase in serum GLDH concentration indicates a degradation of the hepatic parenchyma and more specifically a degradation of the mitochondria.

(39) After 24 hours of preservation in UW®, IGL-1® or INV solutions, a quantitative analysis of GLDH levels by enzyme kit was performed on steatotic livers.

(40) The results are shown in FIG. 3.

(41) The data show that the GLDH concentration is 2.5 times lower after 24 hours of preservation with the INV solution compared to the IGL-1® solution and 5 times lower than with the UW® solution. These results indicate that the INV solution provides better preservation of the liver to be transplanted than solutions of the prior art.

(42) 4/Rinsing a Liver to be Transplanted

(43) 4.1/Experimental Conditions

(44) The liver of normal (healthy) and obese rats (Zucker rats; steatotic liver and referred to as “Ob” or “fatty” in the figures) aged 10 to 12 weeks was collected then washed using techniques known to the skilled person.

(45) The purpose of these experiments is to compare the rinsing performance of the solution according to this invention (INV Solution) against the solutions of the prior art, namely: Belzer UW® solution (hereinafter referred to as UW); HTK® Preservative Solution (for histidine-tryptophan-ketoglutarate or Custodiol® HTK solution); IGL-1 ® solution.

(46) The liver is rinsed by influx of the rinsing solution through the aorta and efflux through the portal vein.

(47) Different parameters were measured to evaluate the effectiveness of the solution according to this invention on hepatic rinsing.

(48) 4.2/Determination of the Amount of Cells Present in the Liver Tissue Rinse Effluent

(49) The rinsing performance of the solution according to this invention (INV) is evaluated at: T0 (aortic dissection after the total flushing volume has passed through the aorta in all experimental groups except HTK); T1 (after the total rinse volume has passed through the aorta in the HTK group); T2 (after the total rinse volume has passed through the portal vein); and T24 (24 hours post-ischemia), on healthy and steatotic livers, compared to IGL-1® solution.

(50) The results are shown in FIG. 4.

(51) The data show that, for the healthy liver, the effluent obtained after rinsing the liver with INV solution is more concentrated in cells, in this case in red blood cells, than IGL-1® solution at T0, T1 and T2. The INV effluent is therefore “dirtier”, more concentrated in red blood cells, than IGL-1 effluent throughout the liver harvesting procedure. The INV solution according to this invention therefore ensures a better liver flushing than the IGL-1® solution.

(52) As known to the skilled person, the rinsing of a steatotic liver is less effective than that of a healthy liver due to cellular and tissue damage. The results show that the INV solution induces a decrease in cell concentration in the effluent over time. These data therefore reflect that the solution according to this invention ensures an effective rinsing of the steatotic liver.

(53) 4.3/Quantification of the Number of Red Blood Cells Present in the Liver Tissue after Rinsing

(54) A histological analysis was conducted to quantify the number of red blood cells remaining in: healthy liver tissue, after harvesting and rinsing with HTK®, IGL-1 and INV solutions; and steatotic (“fatty”) liver tissue, after harvesting and rinsing with UW and INV solutions.

(55) The results are shown in FIG. 5.

(56) For a healthy liver, the data show that the liver rinsed with INV solution contains 7 times less red blood cells than the liver rinsed with the HTK® solution and 2 times less than the liver rinsed with the IGL-1® solution.

(57) For a steatotic liver, the data show that the liver rinsed with INV solution contains more than 1.5 times less red blood cells than the liver rinsed with the UW® solution.

(58) These results confirm that the solution according to this invention provides a better hepatic flushing than the solutions of the prior art.

(59) 5/Dynamic Infusion of a Liver

(60) 5.1/Dynamic Infusion in Hypothermic Liver Condition

(61) A hypothermic infusion machine is used to preserve a liver to be transplanted, this device allows to implement the HOPE (Hypothermic Oxygenated Perfusion) protocol which ensures passive oxygenation of the hypothermic infusion, i.e. without oxygen transporter, which protects mitochondrial integrity, and allows to reduce ischemia-reperfusion lesions in the liver.

(62) The liver to be transplanted is stored in a preservation solution for 7 hours, then placed in a hypothermic infusion machine for 1 hour to be subjected to the HOPE protocol. The liver is then reperfused with a Krebs solution in normothermia.

(63) The Perf-Gen® solution is one of the prior art solutions used in a hypothermic infusion machine. This solution includes hydroxyethyl starch as oncotic agent and glucose as osmotic agent.

(64) The purpose of these experiments is to compare the performance of the solution according to the invention (INV Solution) against the Perf-Gen® solution.

(65) Different parameters were measured to evaluate the efficacy of the solution according to this invention on liver preservation under dynamic infusion conditions in hypothermia:

(66) 5.2/Assessment of ALDH2 Content in Liver Tissue

(67) The ALDH2 concentration was measured in healthy liver tissue to assess the protective properties to the cells of the organ to be transplanted of the INV solution used in an infusion device according to the HOPE protocol (see point 3.4), compared to the Perf-Gen® solution.

(68) The results are shown in FIG. 6.

(69) The data show that the INV solution according to this invention induces a significantly higher ALDH2 activity compared to the Perf-Gen® solution after a dynamic infusion in hypothermia.

(70) 5.3/Assessment of Hepatic Parenchyma Degradation

(71) 5.3.1/Aspartate Aminotransferase

(72) The content in aspartate aminotransferase (ASAT), a particular type of transaminase, was measured by enzyme kit 1 hour after being placed under HOPE protocol (see point 3.4).

(73) The results are shown in FIG. 7.

(74) The data show a lower ASAT content in hepatic tissue infused with the solution according to this invention compared to the use of the prior art solution.

(75) 5.3.2/GLDH The GLDH content was measured by enzyme kit, at the launch of the HOPE protocol and then every 15 minutes, at 0, 15, 30, 45 and 60 minutes.

(76) The results are shown in FIG. 8.

(77) The data show that infusion with the INV solution ensures a decrease in GLDH content compared to the Perf-Gen® solution.

(78) In conclusion, infusion with the solution according to this invention of a liver in a hypothermic infusion machine leads to lower levels of ASAT and GLDH, reflecting a decrease in the degradation of liver tissue compared to the Perf-Gen® solution.

(79) 6/Conclusion

(80) The solution according to this invention is suitable for use for static preservation in hypothermia of an organ to be transplanted and/or for dynamic rinsing of the organ in a hypothermic infusion device. In addition, in these 2 contexts of use, the INV solution according to this invention is more effective than the solutions of the prior art. INV allows protecting the organ to be transplanted and reducing damage to liver tissue caused by ischemia and reperfusion