Ex vivo perfusion treatment method and ex vivo perfusion composition using activated protein c for excized organ

Abstract

In mechanical perfusion of an organ to be used for organ transplantation, a method for storing the organ while maintaining the function of the organ in a satisfactory condition has been demanded. Provided is a composition containing activated protein C (APC). The composition is mixed with a perfusion fluid when the organ is perfused ex vivo.

Description

Method for extracorporeal perfusion treatment of isolated organs using activated protein C and composition for extracorporeal perfusion

[0001] RELATED APPLICATIONS This application claims the benefit of priority from Application No. 2023-120981, filed with the Japan Patent Office on July 25, 2023. The priority application is incorporated herein by reference in its entirety.

[0002] The present invention relates to a composition containing activated protein C (APC), which is mixed with a perfusion solution when an organ is perfused extracorporeally. The present invention also relates to an extracorporeal treatment method using activated protein C in organ transplantation using a harvested organ. More specifically, the present invention relates to an extracorporeal (pre-transplant) perfusion method in which activated protein C, which has anticoagulant and cytoprotective effects, is perfused during machine perfusion preservation, which is thought to be more effective in solid organ transplantation using a harvested organ.

[0003] Against the backdrop of a serious donor shortage occurring on a global scale, the use of extended criteria donors (ECD), which were not previously used for transplantation, is rapidly increasing. According to statistics from 2019, transplants using organs harvested from non-heart-beating donors after cardiac death (DCD), a type of ECD, accounted for 23% of solid organ cadaver transplants in 22 major countries around the world.

[0004] On the other hand, the increased use of these high-risk organs, including DCD, has led to a high incidence of primary non-function (PNF) and early allograft dysfunction (EAD).

[0005] The occurrence of PNF / EAD mentioned above has exposed the limitations of static cold storage (SCS), which has been the global standard method for over half a century for transplantation of organs donated from brain-dead donors. Donor organs are usually immersed in a cold storage solution below 10°C and stored for a certain period of time.

[0006] Against this background, the development of machine perfusion (hereinafter referred to as "MP") preservation as an alternative organ preservation method to SCS has become a global interest.

[0007] Organ transplantation is unique in that the donor organ must be removed from the body, making not only ischemic injury but also ischemia / reperfusion injury (IRI) associated with resumption of blood flow inevitable. Organs removed under standard brain death, i.e., while the heart is beating, can withstand the cold storage time in SCS, but ECD, such as DCD, fatty liver, or aged organs, ultimately induces PNF / EAD.

[0008] In contrast, MP reduces IRI and exerts organ-protective effects by reducing ischemic injury through oxygenated perfusion, supplying nutrients through continuous perfusion, and removing harmful metabolic waste products.

[0009] MPs are classified according to the temperature range in which they are used: body temperature (normothermic MP: NMP, usually 35-37.5°C), hypothermic MP (hypothermic MP: HMP, usually 4-12°C), and intermediate temperature range (subnormothermic MP: SNMP, usually 20-25°C). NMP and HMP have been used in clinical trials in the field of liver transplantation; the former can be administered during organ transport or after arrival at the facility, whereas the latter is often administered after arrival at the facility (end-ischemic) after organ transport by SCS as usual (Non-Patent Document 1).

[0010] Adding a substance that has a cell or tissue protecting effect to the preservation solution may also be effective in reducing IRI in donor organs.

[0011] Protein C (hereinafter referred to as "PC"), a precursor of activated protein C (hereinafter referred to as "APC"), is a vitamin K-dependent plasma protein that is activated by thrombin cleavage of 12 amino acid residues at its N-terminus. In vivo, there are a group of anticoagulant factors that control excessive blood coagulation reactions, and APC is one of these anticoagulant factors.

[0012] In addition to its anticoagulant activity due to limited proteolysis of activated blood coagulation factor V and activated blood coagulation factor VIII, APC also has cytoprotective effects (anti-apoptotic effects, anti-inflammatory effects, etc.) mediated by its receptors on cells, PAR-1 and EPCR (Non-Patent Document 2).

[0013] In Japan, pharmaceuticals containing APC as the main ingredient have been on the market since 2001, and are indicated for the treatment of deep vein thrombosis, acute pulmonary thromboembolism, and purpura fulminans caused by congenital PC deficiency.

[0014] The effectiveness of APC in preserving isolated organs has been reported in animal disease models (Non-Patent Documents 3 and 4). Non-Patent Document 4 reports its protective effect against small-for-size syndrome after partial liver transplantation, which mimics adult living donor liver transplantation. Specifically, an excessively small partial liver equivalent to 20% of the total liver was cold-preserved for 6 hours using an organ preservation solution containing histidine-tryptophan-ketoglutaric acid (HTK) solution plus APC, and then transplanted into recipient rats. The results showed that the APC-treated group had a significantly higher survival rate 7 days after transplantation compared to a control group without APC, and the liver function index (AST / ALT) was also better (Non-Patent Document 4).

[0015] Thus, although the possibility of adding APC during simple cold immersion preservation has been suggested, from a scientific perspective, the function of APC under ice cooling (usually <4°C) and simple immersion cannot be expected to be as effective as that of the present invention, which performs machine perfusion at body temperature, which is considered to be the optimal temperature for enzymes (proteins).In addition, there have been no research reports focusing on the extracorporeal treatment of ECD organs (including DCD) that can be achieved through perfusion preservation.

[0016] Hata et al., Organ Biology 2022, 29:21-28. Mosnier LO. et al, Blood 2012, 120:5237-5246. Griffin JH. et al, Blood 2015, 125:2898-2907. Kuriyama N. et al, Liver Transplantation 2010, 16:1-11.

[0017] In recent years, transplants using DCD from non-heart-beating donors have been rapidly increasing overseas, and according to statistics from 2019, DCD transplants accounted for a whopping 23% of solid organ cadaver transplants in 22 major countries around the world. SCS, which has been the global standard for half a century, has a high risk of PNF / EAD for these high-risk organs, making it an urgent issue.

[0018] Another issue that needs to be resolved is the inferior post-transplant outcomes of DCDs and other organs that are eligible for expanded ECD transplantation, which were not previously used for transplantation, compared to transplants from brain-dead donors (before cardiac arrest).

[0019] The development of MP for organs for transplantation may provide a new therapeutic window for (isolated) extracorporeal treatment of organs. The present disclosure provides a composition comprising activated protein C (APC), which is mixed with a perfusion fluid when an organ is perfused extracorporeally. [Item 1] A composition comprising activated protein C (APC), which is mixed with a perfusion solution when perfusing an organ extracorporeally; [Item 2] A perfusion solution comprising activated protein C (APC), which is used to perfuse an organ extracorporeally; [Item 3] A method for perfusing an organ extracorporeally, which comprises circulating a perfusion solution through an extracorporeal organ connected to inflow and outflow tubes, wherein the perfusion solution comprises activated protein C; [Item 4] The composition according to Item 1 or the perfusion solution according to Item 2, wherein the perfusion solution is performed at body temperature; [Item 5] The method according to Item 3, wherein the perfusion is performed at body temperature; [Item 6] The composition according to Item 1 or 4, or the perfusion solution according to Item 2 or 4, wherein the extracorporeal organ is the liver; [Item 7] The method according to Item 3 or 5, wherein the extracorporeal organ is the liver; [Item 8] A composition comprising activated protein C (APC), [Item 9] An organ preservation solution containing activated protein C (APC), wherein an organ in a living body is treated with the organ preservation solution and the treated organ is perfused ex vivo; [Item 10] A machine perfusion device using the composition according to Item 1, 4 or 6, or the perfusion solution according to Item 2, 4 or 6.

[0020] This invention enables extracorporeal treatment during organ preservation. Donor organs that were previously discarded due to being deemed untransplantable can now be used for transplantation, which is expected to alleviate the chronic global donor shortage. It also makes it possible to improve post-transplant outcomes in ECD, including DCD.

[0021] Figure 1 shows the experimental design. After 50 minutes of donor warm ischemia time (dWIT), rat livers were procured and subjected to 2 hours of static cold storage (SCS, 4°C) in UW, followed by 2 hours of normothermic machine perfusion (NMP) with oxygenated semidiluted blood (37°C), followed by 1 hour of SCS. In the APC group, activated protein C (APC) was added to the perfusion / preservation solution at 100 nmol / L, whereas in the control group, it was not added. The liver graft was then orthotopically transplanted into the recipient, and arterial reconstruction was performed. Figure 2 shows a photograph of the perfusion apparatus. Figure 3 shows a schematic diagram of the rat liver NMP system. The liver was placed in a pre-warmed chamber and perfused through the portal vein (PV). The portal vein flow rate was adjusted by controlling the speed of a roller pump according to the liver weight. Carbogen (95% O ) was added while the perfusate flowed through the gas-permeable silicone tubing of the perfusion system. ２ + 5% CO ２) and oxygenated. A bubble trap was installed just before the PV, and a manometer was installed to monitor PV pressure (PVP). Figure 4 shows the composition of the perfusion preservation solution. Figure 5 shows a graph of the time course of lactate concentration in NMP. Figure 6 shows a graph of the time course of oxygen consumption in NMP. Figure 7 shows a graph of the time course of portal vein perfusion pressure in NMP. Figure 8 shows a graph of the time course of pH in NMP. Figure 9 shows a graph of the time course of glucose consumption in NMP. Figure 10 shows a graph of the release of transaminases into the perfusate in NMP. Figure 11 shows the pathological findings (HE staining and Suzuki injury score) after NMP. D-E and F-G: Hematoxylin-eosin (HE) staining in the control group and APC group, respectively. H: Suzuki injury score (mean ± standard error [SEM], n = 5 for each group). The original magnification is ×40 for D and F, and ×200 for E and G. The scale bars represent 300 μm for D and F, and 40 μm for E and G. Figure 12 shows platelets and fibrin thrombi (CD41 immunohistochemical staining and phosphotungstic acid-hematoxylin (PTAH) staining) in the hepatic parenchyma of DCD livers after NMP. A-C: Graphs showing the detection of platelet thrombi by CD41 (cluster of differentiation 41) immunohistochemical staining and quantification of the positive area. D-F: Graphs showing the detection of fibrin thrombi by PTAH staining and quantification of the positive area. A and B: Representative immunohistochemical staining for CD41, showing platelet thrombi in the control group and the APC group, respectively. D and E: PTAH staining of fibrin thrombi in the control group and the APC group, respectively. The original magnification is ×400, and the scale bars represent 40 μm. C and F: Quantification graphs of each staining-positive area (mean ± standard error [SEM], n = 5 for each). Figure 13 shows transmission electron microscopy images of DCD liver tissue sections after NMP (A: control group, B: APC group). Original magnification: ×8000. Scale bar: 500 nm. Figure 14 shows the damage to the micro- and ultrastructure of intrahepatic bile ducts (bile canaliculi and their microvilli) in DCD livers after NMP.A-B: Immunohistochemical staining of biliary glycoprotein carcinoembryonic antigen-associated cell adhesion molecule 1 (CEACAM1, CD66a) (original magnification: ×400, scale bar: 40 nm). C: Quantification graph of stained area (mean ± standard error [SEM], n = 5 for each). D-E: Scanning electron micrographs of bile canaliculi (original magnification: ×13,000, scale bar: 1 μm). Figure 15 shows the measured values ​​and time course of each marker suggesting ultrastructural damage of intrahepatic bile ducts in DCD livers after NMP. F: Bile production in NMP. G: Lactate dehydrogenase (LDH, a marker of intrahepatic bile duct injury) release in NMP. H: Direct bilirubin excretion in NMP (a comprehensive marker of glucuronidation, biliary transport, and biliary excretion). All data are shown as mean ± standard error [SEM] (n = 5 for each). Figure 16 shows sinusoidal endothelium in DCD livers after NMP. AB: Immunohistochemical staining of CD31 (original magnification: ×400, scale bar: 40 μm). C: Quantification graph of stained area (mean ± standard error of mean [SEM], n=5 for each). DE: Scanning electron micrographs of hepatic sinusoids (original magnification: ×5,000, scale bar: 1 μm). Figure 17 is a graph showing the progression of survival rate after transplantation.

[0022] DETAILED DESCRIPTION OF THE INVENTION Hereinafter, embodiments for carrying out the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0023] In one aspect of the present invention, a composition comprising activated protein C (APC) is provided, the composition being mixed with a perfusion solution when perfusing an organ extracorporeally. In another aspect, a perfusion solution comprising activated protein C (APC) is provided, the perfusion solution being used to perfuse an organ extracorporeally. In yet another aspect, a method of perfusing an organ extracorporeally is provided, the method comprising circulating a perfusion solution through an extracorporeal organ connected to inflow and outflow tubing, the perfusion solution comprising activated protein C. In yet another aspect, the use of activated protein C in organ perfusion is provided. In yet another aspect, a method of treating an organ with activated protein C during perfusion is provided.

[0024] In the present invention, organ "transplantation" refers to the transfer and transplantation of a donor's organ into a recipient, and the type of transplant is not particularly limited. Transplant types are classified according to the relationship between the donor and the recipient and the condition of the donor. Examples of classification based on the relationship between the donor and the recipient include autotransplantation and allotransplantation, and allotransplantation includes allotransplantation and xenotransplantation. Examples of classification based on the condition of the donor include living donor transplantation and cadaveric transplantation, and cadaveric transplantation includes brain-dead donor transplantation and cardiac arrest transplantation.

[0025] In the present invention, the "organ" is not particularly limited, and examples thereof include the liver, heart, kidney, lung, pancreas, small intestine, and eyeball.

[0026] In the present invention, "perfusion" of an organ refers to the inflow and outflow of a perfusion solution into and from the organ. In one embodiment of the present invention, perfusion is performed by connecting tubes such as cannulas for the inflow and outflow of perfusion solution to the blood vessels of the organ to be transplanted, and inflowing and outflowing the perfusion solution into and from the organ. Those skilled in the art can select appropriate tubes for the inflow and outflow of perfusion solution and use them for perfusion. When perfusing the liver, the tube for the perfusion solution is selected from, for example, the portal vein, inferior vena cava, bile duct, and hepatic artery.

[0027] In the present invention, the "perfusion solution" and "organ preservation solution" can be appropriately selected by those skilled in the art from compositions known or equivalent thereto, depending on the type of target organ, etc. For example, a cell-free solution or a solution containing an oxygen carrier such as red blood cells can be used. The "perfusion solution" and "organ preservation solution" may be Bretschneider's solution, Bretschneider's HTK solution, Euro-Collins solution, St. Thomas' Hospital solution, ET-Kyoto solution, Steen solution, or UW-Gluconate solution, respectively, and may optionally contain blood, plasma, or serum.

[0028] In the present invention, perfusion conditions can be appropriately set by those skilled in the art according to the type of target organ, etc., using conditions known to those skilled in the art or equivalent conditions. For example, the temperature, perfusion rate, perfusion volume, pH of the perfusate, oxygen content of the perfusate, and the presence or absence of removal of waste products or the degree of removal can be appropriately set.

[0029] The perfusion temperature may be selected from, but is not particularly limited to, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or any temperature therebetween. In the present invention, perfusion is performed at, for example, 4-12°C, 20-25°C, 20-30°C, 10-35°C, or 35-38°C.

[0030] In the present invention, the concentration of activated protein C contained in the organ preservation solution or perfusion solution is not particularly limited as long as it is a concentration that improves or exerts the organ preservation or therapeutic effect. For example, 0, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000 nM or more.

[0031] As used herein, a "composition" or "solution" containing activated protein C (APC) is a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective. The composition may include, but is not limited to, a buffer, an excipient, a stabilizer, or a preservative.

[0032] In the present invention, the origin of the organ to be transplanted is not particularly limited, and it may be derived from mammals such as humans, pigs, cows, monkeys, baboons, dogs, cats, rats, and mice, and is preferably derived from humans.

[0033] Activated protein C circulates in blood vessels as its precursor, protein C (hereinafter sometimes referred to as PC). Once the coagulation system is activated and thrombin is formed, thrombin binds to thrombomodulin (hereinafter sometimes referred to as TM), a membrane protein on vascular endothelial cells, activating PC and converting it into activated protein C, which has serine protease activity. Activated protein C selectively deactivates activated factor V and activated factor VIII of the blood coagulation system on cell membrane phospholipids, exerting a potent anticoagulant effect. The anticoagulant effect of activated protein C is enhanced in the presence of the cofactor protein S (hereinafter sometimes referred to as PS). The coagulation control mechanism involving PC, TM, PS, etc. is referred to as the protein C anticoagulant system. Activated protein C can be produced by any method, including, but not limited to, activating PC isolated from human blood or obtained by genetic engineering, isolating activated protein C from human blood, or directly preparing activated protein C using genetic engineering.

[0034] There are no particular limitations on the method for activating PC to activated protein C, and it can be carried out, for example, by activation with thrombin isolated from human or bovine blood, or by activation with recombinant thrombin.

[0035] Methods for producing activated protein C derived from blood include, but are not limited to, the following: a method in which PC purified from human plasma by affinity chromatography using an anti-PC antibody is activated with human thrombin and then purified using cation chromatography; a method in which PC purified from human plasma using Kisiel, through the steps of barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex column chromatography, dextran sulfate agarose chromatography, and polyacrylamide gel electrophoresis, is activated to produce activated protein C; and a method in which PC purified from a commercially available factor IX complex preparation containing PC by affinity chromatography using an anti-PC antibody is activated to produce activated protein C.

[0036] Activated protein C may be used as long as it is activated during organ preservation, such as perfusion. Alternatively, PC may be appropriately activated and used. Because PC is activated to activated protein C by minute amounts of thrombin contained in organs, using PC is also an option. The amount of activated protein C used for organ perfusion, the timing of mixing with the perfusion solution, and other factors can be appropriately selected by those skilled in the art depending on the type of organ, perfusion time, and other conditions. Examples of perfusion times include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 360, 384, 408, 432, 456, and 480 hours or more, but are not particularly limited as long as the perfusion time is sufficient to achieve organ preservation effects.

[0037] In one embodiment of the present invention, the use of activated protein C in the perfusion solution may reduce IRI or improve organ function and recipient survival rate after transplantation. In one embodiment of the present invention, organ function or the degree of injury can be measured by collecting blood or tissue from the perfusion solution, bile, urine, tissue, etc. during perfusion preservation, or from the recipient after transplantation, thereby confirming the state of organ preservation. According to the present invention, the state of the organ is maintained or improved. In one embodiment of the present invention, the use of activated protein C in the perfusion solution may improve organ microcirculation. Maintaining or improving the preservation state may enable longer-term perfusion compared to when APC is not used. In one embodiment of the present invention, it is predicted that microcirculation is better after perfusion preservation than at the time of extraction, and that new therapies using ex vivo drug perfusion will be effective.

[0038] For example, according to the present invention, in high-risk DCD transplants, adding activated protein C, a therapeutic agent, to NMP can improve the donor organ's ex vivo transplantability, thereby expanding the donor pool (increasing the number of transplantable organs) and improving recipient post-transplant outcomes. The activated protein C activity exhibited at body temperature (around 37°C) is higher than that of SCS, which is typically stored on ice, and NMP, which is continuously perfused, is expected to have a greater protective effect than SCS that is simply immersed. In other words, ex vivo organ treatment is expected to be possible precisely because it is performed at body temperature, where various proteins can exert their functions, and because of the continuous perfusion.

[0039] In this specification, the term "about" means within a range of ±10%, preferably ±5%.

[0040] The present invention will be specifically and in detail explained below by showing examples. However, the examples are used to illustrate the present invention and are not intended to limit the present invention.

[0041] Example 1 The present inventors combined a rat DCD model with a machine perfusion device and set up two groups: one with APC added to the organ preservation solution and one without, to evaluate the effect of ex vivo treatment of DCD livers with APC.

[0042] Experimental Procedure (Figure 1) The experiment was conducted according to the experimental protocol shown in Figure 1. Specifically, donor rats were induced to cardiac arrest by diaphragmatic incision, and then the aorta was clamped to subject the abdominal organs to 50 minutes of warm ischemic time. The donors were divided into two groups: a control group (no APC added) and an APC group (100 nM APC added) in UW solution (University of Wisconsin), an organ preservation solution used for blood flushing within the donor's body and for preservation after extraction. Liver grafts were extracted from rats in each group and cold-preserved in SCS for 2 hours.

[0043] Perfusion apparatus (Figure 2), schematic diagram of NMP system (Figure 3), and composition of perfusion preservation solution (Figure 4). Figure 2 shows a photograph of the perfusion apparatus, Figure 3 shows a schematic diagram of an example of an NMP system for rat liver, and Figure 4 shows the composition of the perfusion preservation solution. Experiments were conducted according to Figures 3 and 4. Specifically, in the APC group, the perfusion solution was prepared so that APC was 100 nM. Perfusion preservation was performed at body temperature (NMP). The apparatus consisted of an organ chamber that held the liver graft and automatically adjusted the temperature, a roller pump that maintained a constant perfusion rate, an oxygenator that supplied oxygen to the perfusion preservation solution, and a bubble trap.

[0044] After cold storage in the SCS, the liver graft was transferred to an organ chamber, and the portal vein, inferior vena cava, and bile duct were cannulated and connected to a perfusion device, followed by sub-thermal machine perfusion (NMP) at 37°C for 2 hours.

[0045] After 2 hours of NMP, an additional hour of preservation in SCS was performed in order to mimic the clinical requirements of actual human liver transplantation in an animal model.

[0046] Evaluation items during NMP of transplanted liver (Figs. 5-10) Portal vein perfusion pressure was continuously monitored using a manometer during the 2-hour NMP. Perfusion fluid was also sampled periodically, and lactate levels (an index of hepatic microcirculation and aerobic metabolism), oxygen consumption (an index of metabolic function of the transplanted liver), PVP, pH, glucose consumption rate, and transaminase levels (an index of liver injury) were measured over time. (Fig. 5) Lactate release into the perfusate. (Fig. 6) Inflow O2 as a function of flow rate and liver weight. ２ and outflow O ２Liver O calculated from the difference ２ Consumption rate. (Fig. 7) PVP as an index of total vascular resistance. (Fig. 8) Time-dependent change in perfusate pH. (Fig. 9) Glucose consumption rate calculated from the difference in pre- and post-hepatic glucose concentrations normalized by flow rate and liver weight as a marker of liver metabolic function. For Figs. 5-9, differences between groups were analyzed using a two-way repeated measures ANOVA (PVP, P<0.01; O ２ The data were evaluated by a two-way repeated measures ANOVA (AST, P < 0.01; ALT, P < 0.01; LDH, P < 0.05) followed by a Bonferroni post hoc test (*P < 0.05; †P < 0.01; ‡P < 0.001 vs. control group, n = 6 for each group, mean ± standard error of the mean [SEM]). The data for Figure 10 were evaluated by a two-way repeated measures ANOVA (AST, P < 0.01; ALT, P < 0.01; LDH, P < 0.05) followed by a Bonferroni post hoc test (*P < 0.05; †P < 0.01; ‡P < 0.001 vs. control group, n = 6 for each group, mean ± SEM).

[0047] After orthotopic transplantation of the liver grafts into recipient rats, the survival rates of both groups were compared over 14 days. Blood and liver tissue samples were collected from the recipients after transplantation to evaluate liver function and injury.

[0048] Changes in Lactate Levels Over Time Figure 5 shows the changes in lactate levels in NMP over time. 120 minutes after the start of NMP administration, lactate levels in the APC group were significantly lower than those in the control group. Lactate levels in the perfusate under liver isolation conditions are a good indicator of anaerobic liver metabolism. In other words, the worse the microcirculation in the donor liver is, the less oxygen is distributed, and the higher the lactate levels. Furthermore, it is known that the liver normally metabolizes approximately 70% of the lactate produced in the body, and a decline in liver function worsens lactic acidosis. While lactate levels in the control group showed a temporary increase over time, they decreased over time with the addition of APC, indicating that the addition of APC improved the donor liver's liver function, which would have otherwise suffered from liver dysfunction after transplantation.

[0049] Changes in oxygen consumption over time Figure 6 shows the changes in oxygen consumption over time in NMP. For example, the oxygen consumption of the APC group at 60 minutes in NMP was significantly higher than that of the control group, suggesting that the addition of APC improved metabolic activity and microcirculation.

[0050] Changes in portal vein perfusion pressure over time Figure 7 shows the changes in portal vein perfusion pressure over time during NMP. NMP: The portal vein pressure in the APC group at 120 minutes was significantly lower than that in the control group, suggesting that the addition of APC improved hepatic microcirculation and reduced tissue edema.

[0051] Changes in pH in NMP over time Figure 8 shows the changes in pH in NMP over time. Compared to the control, which became significantly acidic after the start of NMP, the pH of the perfusate remained significantly neutral, suggesting a decrease in anaerobic metabolism, i.e., an improvement in microcirculation due to the addition of APC.

[0052] Changes in glucose consumption in NMP over time Figure 9 shows changes in glucose consumption in NMP over time. Compared to the control group, in which glucose consumption did not increase even after the start of NMP, glucose consumption increased in the APC group after 30 minutes, suggesting a significant improvement in metabolic activity at 90 minutes after NMP.

[0053] Figure 10 shows the time course of liver injury. The concentrations (leakage) of AST, ALT, and LDH in the perfusion solution during NMP administration were plotted against time. (A) Aspartate aminotransferase (AST), (B) Alanine aminotransferase (ALT), and (C) Lactate dehydrogenase (LDH) released into the perfusion solution were significantly lower in the APC group than in the control group after 2 hours of NMP administration. Hepatic parenchymal cell (AST, ALT) and non-parenchymal cell (LDH, AST) injury in the APC group was significantly lower than in the control group.

[0054] Pathological findings after NMP (HE staining and Suzuki injury score). Figure 11 shows HE staining (D-G) and Suzuki injury score (H) of liver tissue after NMP. In the control group (D, E), pathological changes such as sinusoidal endothelial cell detachment, sinusoidal thrombus obstruction and congestion, and panlobular hepatocyte vacuolization and necrosis were observed. However, no such pathological findings were observed in the APC group (F, G), demonstrating the protective effect of APC perfusion histomorphologically. Furthermore, a significant difference in injury score (H) was confirmed between the control and APC groups (mean ± standard error of mean [SEM], n = 5 for each group). *P < 0.05 by unpaired Student's t-test.

[0055] Figure 12 shows platelets and fibrin clots in the liver parenchyma of DCD livers after NMP (CD41 immunohistochemical staining and phosphotungstic acid-hematoxylin (PTAH) staining). Significant differences were confirmed in the staining-positive areas (C and F) between the control and APC groups (quantification of the positive areas using ImageJ2 version 2.14.0 with Fiji, mean ± standard error [SEM], n = 5 each). *P < 0.05 by unpaired Student's t-test).

[0056] Figure 13 shows tissue sections (A: control group, B: APC group) observed under a transmission electron microscope. In the control group, hepatocyte mitochondria showed severe swelling with low electron density, and the endoplasmic reticulum was also disrupted. In contrast, these adverse changes were significantly alleviated in the APC group.

[0057] Figure 14 shows the microscopic and ultrastructural damage to intrahepatic bile ducts in DCD livers after NMP. In contrast to the severely attenuated CEACAM-1 staining in control DCD livers (A), APC-treated livers showed significantly denser and thicker staining (B), indicating significantly better preserved bile canaliculi (*P<0.05, unpaired Student's t-test, mean ± standard error of the mean [SEM], n=5 each, C). In control livers, microvilli formation within bile canaliculi became significantly sparser, and their height was significantly reduced (D). In contrast, microvilli formation was significantly better preserved in APC-treated livers, with both density and height maintained (E).

[0058] Figure 15 shows the time course of markers suggesting microstructural and ultrastructural damage to the intrahepatic bile ducts (bile canaliculi and their microvilli) in DCD livers after NMP. Bile production during 2 hours of NMP was significantly higher in the APC group than in the control group (F. Difference between groups by two-way repeated measures ANOVA: P<0.01; time point assessment: ‡P<0.001 and ¶P<0.0001 by Bonferroni's post-hoc test). Lactate dehydrogenase (LDH) release into bile was confirmed as a marker of intrahepatic bile duct injury (G. Difference between groups by two-way ANOVA: P<0.05 by post-hoc test; †P<0.01). Direct (conjugated) bilirubin excretion during 2 h NMP was confirmed as a comprehensive marker of hepatic glucuronidation, biliary transport, and biliary excretion in DCD (H. † P < 0.01, group control by unpaired Student's t test).

[0059] Figure 16 shows the sinusoidal endothelium of DCD livers after NMP. CD31 is highly expressed at endothelial cell-cell junctions and is essential for maintaining the continuity of the endothelial lining. In control livers, CD31 expression was severely damaged, irregular, and absent (A), whereas it was significantly better preserved along the sinusoidal lining in APC-treated livers (B and C, quantified using ImageJ2 version 2.14.0 with Fiji, mean ± standard error [SEM], n = 5 each, *P < 0.05 vs. control). In control livers, sinusoidal endothelial cells were significantly destroyed and sloughed off (D). In contrast, in APC-treated livers, the sinusoidal lining and its pores were better preserved (E).

[0060] Figure 17 shows the change in animal survival rate (50 min dWIT) after orthotopic liver transplantation of DCD livers. The survival rate after orthotopic liver transplantation of DCD livers (50 min dWIT) was significantly better in the APC group than in the control group (P < 0.001 by log-rank test, n = 7 for each group). In the control group, 60% of cases died the day after transplantation and all cases died by day 4, whereas in the APC-added group, more than 80% survived even on day 14 after transplantation.

[0061] If we can achieve a higher rate of organ transplants from non-heart-beating donors, it will lead to a dramatic improvement in the critical shortage of donors for solid organ transplants such as hearts, lungs, livers, and kidneys, and will bring about a dramatic revolution in transplant medicine.

Claims

1. A composition comprising activated protein C (APC), which is mixed with a perfusion fluid when perfusing an organ ex vivo.

2. A perfusion solution comprising activated protein C (APC), which perfusion solution is used to perfuse an organ extracorporeally.

3. A method of perfusing an organ extracorporeally, comprising the step of circulating a perfusion fluid through an extracorporeal organ connected to inflow and outflow tubing, the perfusion fluid comprising activated protein C.

4. The composition of claim 1 or the perfusion solution of claim 2, wherein the perfusion is performed at body temperature.

5. The method of claim 3, wherein the perfusion is performed at body temperature.

6. A composition according to claim 1 or 4, or a perfusion solution according to claim 2 or 4, wherein the extracorporeal organ is the liver.

7. The method according to claim 3 or 5, wherein the extracorporeal organ is the liver.

8. A composition comprising activated protein C (APC), wherein the composition is mixed with an organ preservation solution, the resulting mixture is used to treat an organ in vivo, and the treated organ is perfused ex vivo.

9. An organ preservation solution comprising activated protein C (APC), wherein an in vivo organ is treated with said organ preservation solution and the treated organ is perfused ex vivo.

10. A machine perfusion device using a composition according to claim 1, 4 or 6, or a perfusion solution according to claim 2, 4 or 6.

Images