Preserving agent for organs or tissue and preservation method for organs or tissue

Abstract

This invention discloses a preserving agent for organs or tissue comprising (A) quercetin, and (B) at least one sugar selected from the group consisting of fructose and sucrose; a preserving solution for organs or tissue comprising the preserving agent; and a preservation method for organs or tissue comprising the step of immersing an organ or tissue in a liquid mixture comprising (A) quercetin, and (B) at least one sugar selected from the group consisting of fructose and sucrose.

Claims

1. A preserving solution for organ or tissue, comprising (A) quercetin; and (B) at least one sugar selected from the group consisting of fructose and sucrose, wherein the quercetin concentration is 1 to 100 μg/mL, and the sugar concentration is 0.025 to 0.4M.

2. The preserving solution according to claim 1, wherein the organ or tissue is heart, liver, kidney, pancreas, or pancreatic islet.

3. The preserving solution according to claim 1, further comprising at least one solvent selected from the group consisting of a physiological saline solution, an infusion solution, a buffer solution, a cell culture solution, an organ or tissue preserving solution, and a modena solution.

4. The preserving solution according to claim 3, further comprising at least one component selected from the group consisting of antibiotics, antibacterial agents, antioxidants, serums, sugars, lipids, vitamins, proteins, peptides, amino acids, pH indicators, chelating agents, and osmotic pressure regulators.

5. The preserving solution according to claim 1, wherein the quercetin concentration is 1 to 10 μg/mL.

6. A preservation method for organ or tissue, comprising immersing an organ or tissue in a liquid mixture comprising (A) quercetin and (B) at least one sugar selected from the group consisting of fructose and sucrose, wherein the quercetin concentration is 1 to 100 μg/mL, and the sugar concentration is 0.025 to 0.4M.

7. The method according to claim 6, wherein the organ or tissue is heart, liver, kidney, pancreas, or pancreatic islet.

8. The method according to claim 6, wherein the quercetin concentration is 1 to 10 μg/mL.

Description

DESCRIPTION OF EMBODIMENTS

(1) The present invention is specifically explained below.

(2) The preserving agent for organs or tissue of the present invention is characterized by comprising (A) quercetin; and (B) at least one sugar selected from the group consisting of fructose and sucrose.

(3) Further, the preservation method for organs or tissue of the present invention is characterized by comprising the step of immersing an organ or tissue in a liquid mixture comprising (A) quercetin; and (B) at least one sugar selected from the group consisting of fructose and sucrose.

(4) The quercetin and sugars used in the present invention may be chemically synthesized by a known method. Further, since quercetin and sugars are contained in organisms such as plants, they may be obtained by being extracted from such an organism using a known method. Further, quercetin and sugars may be obtained from commercial suppliers.

(5) The “low-temperature damage” in the present specification means cell damage caused by a low temperature, and “a low-temperature damage protection effect” means an effect to protect cells from such a low-temperature damage. Therefore, in light of these meanings, the preserving agent of the present invention may be referred to as a “low-temperature damage protection agent.”

(6) In the present invention, the organ or tissue may be derived from any animal. Among various animals, organs or tissue derived from a mammal (humans, apes, bovine, swine, sheep, goats, horses, dogs, cats, rabbits, mice, rats, or the like) are preferable.

(7) The organ or tissue is preferably applied to organ transplantation or tissue transplantation. Examples of organs include heart, lung, liver, kidney, pancreas, and small intestine; preferable examples include heart, liver, kidney and pancreas; and particularly preferable examples include heart and liver. Examples of tissue include cornea, skin, bones, blood vessels, heart valves, amniotic membranes, pancreatic islets, and the like; preferable examples include pancreatic islets.

(8) In addition to quercetin and at least one sugar selected from the group consisting of fructose and sucrose, known additives may be suitably incorporated in the preserving agent of the present invention.

(9) The preserving solution for organs or tissue of the present invention is characterized by comprising the preserving agent described above. In the preservation of organs or tissue in the present invention, quercetin and at least one sugar selected from the group consisting of fructose and sucrose are used in the form of a liquid mixture (i.e., fluid mixture, preferably a solution) (corresponding to the preserving solution in this specification). The liquid mixture generally contains a solvent in addition to quercetin and at least one sugar selected from the group consisting of fructose and sucrose. The solvent is not particularly limited, and examples thereof include a physiological saline solution, infusions (electrolyte infusion, nutrient infusion, carbohydrate infusion, amino acid infusion, glucose solution, Ringer's solution, Ringer's acetate solution, Ringer's lactate solution, etc.), a buffer solution (PBS, Tris-buffer solution, Hepes buffer solution, MOPS buffer solution, PIPES buffer solution, etc.), a cell culture solution (RPMI 1640, DMEM etc.), organ or tissue preserving solution (EC solution, UW solution, ET-Kyoto solution, etc.), a modena solution, etc.

(10) The quercetin concentration in the preserving solution of the present invention is generally 0.001 to 1000 μg/mL, preferably 0.01 to 100 μg/mL. The sugar concentration in the preserving solution of the present invention is generally 0.01 to 0.8M, and preferably 0.025 to 0.4M (when the preserving solution contains both fructose and sucrose, the sugar concentration means the total concentration thereof). The preserving solution of the present invention may contain other previously known components, such as antibiotics, antibacterial agents, antioxidants, serums, sugars, lipids, vitamins, proteins, peptides, amino acids, pH indicators, chelating agents, osmotic pressure regulators, and the like.

(11) The present invention is preferably carried out at a temperature (low temperature) at which organs or tissue does not freeze. The temperature at which organs or tissue does not freeze cannot be unconditionally defined as it varies depending on the components contained in the preserving agent or preserving solution, the composition, the preservation period, the organs or tissue to be preserved, and the like. From the viewpoint of exhibiting the low-temperature damage protection effect of the present invention, for example, the temperature is −15° C. to 20° C., preferably 0° C. to 20° C., and more preferably 0° C. to 10° C.

(12) When cooling the organs or tissue, the liquid mixture may be cooled in advance before the organs or tissue is immersed therein, or after the organs or tissue is immersed therein, insofar as the organs or tissue does not freeze. Further, once the liquid mixture containing an organ or tissue is cooled, the liquid mixture is maintained at the same temperature insofar as the organ or tissue does not freeze. However, it is not necessary to keep it at a constant temperature at all times; it is acceptable that the temperature may fall out of the above range only for a short period of time.

(13) By using a liquid mixture containing quercetin and at least one sugar selected from the group consisting of fructose and sucrose of the present invention, a low-temperature damage protection effect can be ensured. Since the present invention allows organs or tissue to be preserved under conditions suitable for preservation while inhibiting low-temperature damage derived from such conditions, the present invention enables preservation of organs or tissue under appropriate conditions.

EXAMPLES

(14) The present invention is more specifically explained below in reference to Examples. However, the present invention is not limited to these examples etc.

Test Example 1: Effects of Combined Use of Quercetin and Sugar in an Organ Preservation Test

(15) The protective action of a preserving solution for organs or tissue used in organ transplantation or tissue transplantation was assumed, and formulations of preservative solutions containing UW solution, and a combination of quercetin and sugar were evaluated using liver damage degree as an index.

(16) Williams solution (GIBCO, No. 12551-032) only, Williams solution containing quercetin (Sigma-Aldrich, 337951-25G) (10 μg/mL), Williams solution containing quercetin (10 μg/mL) and fructose (0.05M to 0.4M) (Kanto Chemical Co., Inc., Code No. 16065-00) (0.05M to 0.4M), or Williams solution containing quercetin (10 μg/mL) and sucrose (0.025M to 0.2M) (Nacalai Tesque, Inc., Code No. 30404-45) (0.025M to 0.2M) was used as an organ perfusion solution. Subsequently, UW solution (VSP1000, VIASPAN (cold storage solution) 1000 mL: Astellas Pharma Inc.) only, UW solution containing quercetin (10 μg/mL), UW solution containing quercetin (10 μg/mL) and fructose (0.05M to 0.4M), or UW solution containing quercetin (10 μg/mL) and sucrose (0.025M to 0.2M) was used as an organ preserving solution.

(17) SD rats (male, body weight=255.4 g to 339.0 g) were injected with heparin from the portal vein under anesthesia. Thereafter, they were perfused with a physiological saline solution (8 cmH.sub.2O, about 100 mL) at room temperature, and then perfused with each organ perfusion solution (8 cmH.sub.2O, about 100 mL) at room temperature. Thereafter, the liver was excised, immersed in 20 mL of each organ preserving solution, and preserved at 4° C. A part of each organ preserving solution was collected at 1 day after, 3 days after, 5 days after, and 7 days after the preservation was started, and ALT (alanine aminotransferase) (IU/L) was measured as an index of the degree of liver damage due to low-temperature preservation. The measured ALT are shown in Tables 1 and 2 (average value of n=3±standard deviation).

(18) TABLE-US-00001 TABLE 1 Effects of Combined Use of Quercetin and Sugar (Liver Preservation Effects) ALT Value in Organ Immersion Solution Concentration in (IU/L) Addition 1 day 3 days 5 days 7 days Quercetin Sugar after after after after (1) — — 292 ± 100 404 ± 172 416 ± 166 440 ± 172 (2) 10 μg/mL — 65 ± 71 90 ± 82 98 ± 77 117 ± 71  (3) 10 μg/mL 0.05M 18 ± 8  37 ± 13 55 ± 3  83 ± 13 Fructose (4) 10 μg/mL 0.1M 30 ± 21 57 ± 23 84 ± 52 128 ± 93  Fructose (5) 10 μg/mL 0.2M 24 ± 8  49 ± 13 59 ± 12 92 ± 15 Fructose (6) 10 μg/mL 0.4M 21 ± 8  43 ± 16 53 ± 14 72 ± 10 Fructose

(19) TABLE-US-00002 TABLE 2 Effects of Combined Use of Quercetin and Sugar (Liver Preservation Effects) ALT Value in Organ Immersion Solution Concentration in (IU/L) Addition 1 day 3 days 5 days 7 days Quercetin Sugar after after after after (1) — — 399 ± 125 577 ± 207 583 ± 211 616 ± 215 (2) 10 μg/mL — 62 ± 68 108 ± 121 126 ± 135 148 ± 144 (3) 10 μg/mL 0.025M 18 ± 16 35 ± 33 58 ± 64 79 ± 72 Sucrose (4) 10 μg/mL 0.05M 28 ± 5  53 ± 12 62 ± 15 84 ± 21 Sucrose (5) 10 μg/mL 0.1M 21 ± 15 40 ± 35 52 ± 44 82 ± 74 Sucrose (6) 10 μg/mL 0.2M 34 ± 21 55 ± 22 77 ± 20 134 ± 56  Sucrose

(20) The ALT in the preservation in the UW solution containing quercetin and trehalose was greater than the ALT in the preservation in the UW solution containing quercetin and fructose or in the UW solution containing quercetin and sucrose.

Test Example 2: Effects of Combined Use of Quercetin and Sugar in an Organ Preservation Test

(21) The same test as in Test Example 1 was performed using heart instead of liver, and the formulations of preservative solutions containing UW solution, and a combination of quercetin and sugar were evaluated using heart damage degree as an index.

(22) Williams solution only, Williams solution containing quercetin (10 μg/mL) and fructose (0.2M), or Williams solution containing quercetin (10 μg/mL) and sucrose (0.1M) was used as an organ perfusion solution. Subsequently, UW solution only, UW solution containing quercetin (10 μg/mL) and fructose (0.2M), or UW solution containing quercetin (10 μg/mL) and sucrose (0.1M) was used as an organ preserving solution.

(23) SD rats (male, body weight=257.4 g to 280.3 g) were injected with heparin from the large vein under the suprahepatic portion under anesthesia. Thereafter, they were perfused with a physiological saline solution (8 cmH.sub.2O, about 100 mL) at room temperature, and then perfused with each organ perfusion solution (8 cmH.sub.2O, about 100 mL) at room temperature. Thereafter, the heart was excised, immersed in 20 mL of each organ preserving solution, and preserved at 4° C. A part of each organ preserving solution was collected at 1 day after, 2 days after, 3 days after, and 4 days after the preservation was started, and LDH (lactase dehydrogenase) (IU/L) was measured as an index of the degree of heart damage due to low-temperature preservation. The measured LDH are shown in Table 3 (average value of n=3±standard deviation).

(24) TABLE-US-00003 TABLE 3 Effects of Combined Use of Quercetin and Sugar (Heart Preservation Effects) LDH Value in Organ Immersion Solution Concentration in (lU/L) Addition 1 day 2 days 3 days 4 days Quercetin Sugar after after after after (1) — — 213 ± 54 312 ± 71 403 ± 83  528 ± 113 (2) 10 0.2M 134 ± 56 190 ± 76 238 ± 78 287 ± 81 μg/mL Fructose (3) 10 0.1M 116 ± 15 172 ± 12 248 ± 40 319 ± 40 μg/mL Sucrose

Test Example 3: Effects of Combined Use of Quercetin and Sugar in an Organ or Tissue Preservation Test, Histopathological Change

(25) The formulations of preserving solutions containing UW solution, and a combination of quercetin and sugar were evaluated using a hepatic histopathological change as an index.

(26) Williams solution only, Williams solution containing only quercetin (10 μg/mL), Williams solution containing only fructose (0.2M), Williams solution containing only sucrose (0.1M), Williams solution containing quercetin (10 μg/mL) and fructose (0.2M), or Williams solution containing quercetin (10 μg/mL) and sucrose (0.1M) was used as an organ perfusion solution. Subsequently, as an organ preserving solution, UW solution only, UW solution containing only quercetin (10 μg/mL), UW solution containing only fructose (0.2M), UW solution containing only sucrose (0.1M), UW solution containing quercetin (10 μg/mL) and fructose (0.2M) or UW solution containing quercetin (10 μg/mL) and sucrose (0.1M) was used.

(27) SD rats (male, body weight=264.1 g to 317.2 g) were injected with heparin from the portal vein under anesthesia. Thereafter, they were perfused with a physiological saline solution (8 cmH.sub.2O, about 100 mL) at room temperature, and then perfused with each organ perfusion solution (8 cmH.sub.2O, about 100 mL) at room temperature. Thereafter, the liver was excised, immersed in 20 mL of each organ preserving solution, and preserved at 4° C. 1 day after and 2 days after the preservation was started, all liver samples were immobilized using a 10% phosphate buffer formalin aqueous solution, and embedded in paraffin according to the standard method. Each sample was sliced into about 6 μm and a hematoxylin-eosin staining sample was prepared, followed by microscopic examination. The degrees of the observed histopathological change were converted to scores as shown below, and summed as cumulative values. Tables 4 and 5 show the results.

(28) Score 0: no change was observed, Score 0.5: slight change was observed, Score 1.0: small change was observed, Score 2.0: intermediate change was observed, Score 3.0: great change was observed.

(29) TABLE-US-00004 TABLE 4 Effects of Combined Use of Quercetin and Sugar (1 Day After Preservation at 4° C. Was Started) Histopathological Change (Cumulative Score of n = 3) Sporadic Diffuse Single Cell Sporadic Concentration in Addition Vacuolar Cloudy Single Cell Quercetin Sugar Degeneration Swelling Necrosis (1) — — 3.0 7.0 4.0 (2) 10 μg/mL — 3.0 1.5 0.0 (3) — 0.2M Fructose 2.0 2.5 1.0 (4) — 0.1M Sucrose 3.0 2.0 0.5 (5) 10 μg/mL 0.2M Fructose 0.0 0.0 0.0 (6) 10 μg/mL 0.1M Sucrose 0.0 0.0 0.0

(30) TABLE-US-00005 TABLE 5 Effects of Combined Use of Quercetin and Sugar (2 Days After Preservation at 4° C. Was Started) Histopathological Change (Cumulative Score of n = 3) Sporadic Diffuse Single Cell Sporadic Concentration in Addition Vacuolar Cloudy Single Cell Quercetin Sugar Degeneration Swelling Necrosis (1) — — 3.0 8.0 5.0 (2) 10 μg/mL — 3.0 2.0 0.5 (3) — 0.2M Fructose 1.5 1.0 0.5 (4) 0.1M Sucrose 3.0 2.5 1.0 (5) 10 μg/mL 0.2M Fructose 0.0 0.0 0.0 (6) 10 μg/mL 0.1M Sucrose 0.0 0.0 0.0

Test Example 4: (Effects of Combined Use of Quercetin and Sugar in an Organ or Tissue Preservation Test, Rat Orthotopic Liver Transplantation)

(31) The formulations of preserving solutions containing UW solution, and a combination of quercetin and sugar were evaluated using a rat orthotopic liver transplantation.

(32) Williams solution only, and Williams solution containing quercetin (10 μg/mL) and sucrose (0.1M) were used as organ perfusion solutions. Subsequently, UW solution only, and UW solution containing quercetin (10 μg/mL) and sucrose (0.1M) were used as organ preserving solutions.

(33) Donor SD rats (male, body weight=273.8 g to 335.7 g) were injected with heparin from the portal vein under anesthesia. Thereafter, they were perfused with a physiological saline solution (8 cmH.sub.2O, about 100 mL) at room temperature, and then perfused with each organ perfusion solution (8 cmH.sub.2O, about 100 mL) at room temperature. Thereafter, the liver was excised, immersed in 20 mL of each organ preserving solution, and preserved at 4° C. 1 day after the preservation was started, the livers were orthotopically transplanted to recipient rats. Two hours after the transplantation, the blood ALT was measured as an index of the degree of liver damage due to low-temperature preservation. The blood ALT is shown in Table 6 (average value of n=3±standard deviation). Further, all livers were simultaneously immobilized using a 10% phosphate buffer formalin aqueous solution, and embedded in paraffin according to the standard method. Each sample was sliced into about 6 μm, and a hematoxylin-eosin staining sample was prepared, followed by microscopic examination. The degrees of the observed histopathological change were converted to scores as shown below, and summed as cumulative values. Table 7 shows the results.

(34) Score 0: no change was observed, Score 0.5: slight change was observed, Score 1.0: small change was observed, Score 2.0: intermediate change was observed, Score 3.0: great change was observed.

(35) TABLE-US-00006 TABLE 6 Effects of Combined Use of Quercetin and Sugar (ALT in Rat Orthotopic Liver Transplantation Blood (IU/L)) Preserving Agent Blood ALT (IU/L) (1)UW 119 ± 13 (2)UW + Quercetin 10 μg/mL + 0.1M Sucrose 78 ± 8

(36) TABLE-US-00007 TABLE 7 Hepatic Histopathological change (Cumulative Score of n = 3) Partial Sinusoidal Sporadic Endothelial Cell Single Cell Preserving Agent Partial Bleeding Degeneration Necrosis (1)UW 4.0 3.0 3.0 (2)UW + 10 μg/mL 0.0 0.0 0.0 Quercetin + 0.1M Sucrose