Stable plasmin composition for organ preservation and reconditioning

Abstract

Disclosed herein are methods and compositions for preserving or reconditioning organs prior to transplantation. The compositions of the invention contain plasmin and / or functionally active variants thereof formulated in a pharmaceutically acceptable organ preservation solution. The compositions of the invention are stable and non-toxic to organs intended for transplantation.

Description

Technical Field

[0001] The present invention details innovative compositions for the preservation and / or reconditioning of organs before transplantation.

Background Art

[0002] For many end-stage disease patients, organ transplantation is the only viable treatment option. Naturally, a sufficient supply of high-quality donor organs is essential for the success of transplantation programs. Static cold storage (SCS) is one of the most frequently used methods for the effective preservation and transportation of viable organs for transplantation. SCS involves flushing the obtained organ with a preservation solution at 0-4°C and immersing the organ in the preservation solution at the same temperature until transplantation. The low-temperature environment is a factor that reduces cell metabolism, and the preservation solution reduces cell metabolism and provides cryoprotection. SCS preservation solutions are usually variants of buffers (e.g., histidine or phosphate) and antioxidants (e.g., tryptophan-ketoglutaric acid, glutathione-mannitol), characterized by different potassium and sodium ions.

[0003] Despite having several limitations, including tissue damage induced by long-term hypothermic preservation, difficulty in evaluating the function and viability of donor organs, and a reduced opportunity for organ repair, the SCS method is still widely used in the field of transplantation.

[0004] The shortage of brain-dead organ donors makes it difficult to continuously and consistently supply viable organs for transplantation. A compromise solution to this problem is the reconditioning of organs provided after cardiac death (DCD), which has led to the use of extended criteria donor organs. As such, pre-transplant functional evaluation, ex vivo repair, and organ reconditioning of donor organs are becoming increasingly common using machine perfusion / extracorporeal organ perfusion (EVOP).

[0005] EVOP controls the flow of the perfusion solution to perform organ perfusion. This method promotes the maintenance of organ microvascular system tension, the supply of oxygen and nutrients to support tissue metabolism, and the removal of toxic metabolic waste. Considering that the respiratory rate of cells is proportional to the ambient temperature, the temperature at which EVOP is performed may affect the organ preservation period and the responsiveness of the organ to reconditioning. EVOP has three main categories, namely, normothermic machine perfusion at 35 - 38 °C, subnormothermic machine perfusion at 20 - 34 °C, and hypothermic machine perfusion at 0 - 8 °C.

[0006] It cannot be overemphasized how important the perfusion fluid composition is in the ex vivo reconditioning of organ function and the maintenance of stable organ function. Over time, blood-based perfusion fluids have been almost completely replaced by safer and more reproducible alternatives such as chemical solutions. Steen Solution (trademark) is one of the most widely used solutions for lung machine perfusion, and supplementing Steen Solution (trademark) / other organ perfusion fluids with pharmacologically active substances is a new research area with great expectations in the field of organ transplantation. An example is U.S. Patent Application Publication No. 20190059362 (CSL Behring LLC), which discloses a lung perfusion fluid containing alpha-1 antitrypsin.

[0007] As the use of marginal organs and DCD organs increases, the popularity of EVOP as a means of evaluating donor organ function is steadily growing. In addition to this, EVOP also shows potential for the repair and reconditioning of transplanted tissues.

[0008] Although SCS and EVOP have been successful in providing viable organ grafts to patients, the presence of thrombi in the organs for transplantation can invalidate both procedures. Thrombosis of the transplanted organ is associated with a high risk in that the thrombi reduce perfusion to the graft, thereby reducing the effectiveness of the preservation and EVOP processes. Moreover, donor organ thrombosis is associated with graft dysfunction and poses a significant risk of embolic complications to the organ recipient. DCD organs are at a particularly high risk of being invalidated by thrombi and microthrombi. After the death of the DCD organ donor, the organ is removed and perfused with a cold preservation solution. In such a process, the formation of new thrombi is prevented, but existing thrombi formed during the terminal period or immediately after death are not destroyed. Microthrombi may be seen in organs from brain-dead donors as a result of metabolic events around the time of brain death.

[0009] Accordingly, the problem of providing a stable perfusion fluid and / or preservation solution that can remove thrombi and other vascular occlusions formed in the donor organ before transplantation remains an unmet need. SUMMARY OF THE INVENTION

[0010] In a first aspect, the present invention relates to plasmin, a lysine mimetic, and a pharmaceutically acceptable organ preservation solution having a pH of from about 6 to about 8 comprising, wherein the molar ratio of plasmin:lysine mimetic is from about 1:1 to about 1:10,000, and relates to a stable composition for organ reconditioning.

[0011] In a second aspect, the present invention relates to a method of preserving or reconditioning an organ before transplantation, the method comprising contacting the organ with the composition of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

Figure 1

Figure 2A

Figure 2B

Figure 3

Figure 4A

Figure 4B

Figure 4C

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0013] The words "comprises / comprising" and the words "having / including", as used herein with respect to the present invention, are used to identify the presence of the described features, integers, steps or components, but do not preclude the presence or addition of one or more other features, integers, steps or components or groups thereof.

[0014] It is understood by those skilled in the art that the specific embodiments disclosed herein should not be read in isolation and that this specification is intended that the disclosed embodiments be read in combination with each other rather than individually. As such, each embodiment may serve as a basis for modifying or limiting other embodiments disclosed herein.

[0015] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. Such a range format is used merely for convenience and brevity and is thus to be flexibly interpreted as including not only the numerical values explicitly recited as the limits of the range but also all the individual numerical values or sub-ranges subsumed within that range as if each numerical value and sub-range were explicitly recited. By way of illustration, the numerical range “10 to 100” is to be interpreted as including not only the explicitly recited values from 10 to 100 but also the individual values and sub-ranges within the indicated range. Thus, this numerical range includes the individual values, e.g., 10, 11, 12, 13, ···, 97, 98, 99, 100, as well as sub-ranges, e.g., 10 to 40, 25 to 40, and 50 to 60, etc. This same principle applies to ranges reciting only a single numerical value, such as “at least 10”. Further, such an interpretation applies regardless of the width or the property being described of the range.

[0016] The compositions of the present invention In a first aspect, the present invention plasmin, a lysine mimetic, and a pharmaceutically acceptable organ preservation solution having a pH of about 6 to about 8 comprising, wherein the molar ratio of plasmin:lysine mimetic is from about 1:1 to about 1:10,000, provides a stable composition for organ reconditioning.

[0017] As used herein, the "stable composition" refers to a composition in which the plasmin component does not precipitate from the solution and does not precipitate either immediately or over time, enabling longer preservation of organs compared to prior art compositions. Any method known in the art can be used to determine whether a composition is stable. For example, it can be done by any method for determining plasmin precipitation, such as turbidity, which is a process of measuring the decrease in the intensity of light transmitted through a sample caused by the scattering effect of insoluble particles. Turbidity can be determined, for example, by measuring the absorbance at a wavelength of 405 nm. Other methods suitable for determining precipitation include, for example, refractive index measurement, visual inspection (visual inspection on a black and white background light box), microflow imaging (MFI), dynamic light scattering (DLS), and high-speed centrifugation to search for visible pellets.

[0018] As used herein, "organ reconditioning" means preserving an organ for transplantation purposes or improving the state of that organ.

[0019] As defined in SEQ ID NO: 1, naturally circulating human plasminogen is a single-chain protein containing 791 amino acid residues and has 24 intra-chain disulfide bridges, 5 kringle domains, a serine protease domain, and a pre-activation peptide (PAP). The positions of these domains with respect to SEQ ID NO: 1 are outlined in Table 1 below.

[0020]

Table 1

[0021] In the body, plasminogen exists as Glu-plasminogen and Lys-plasminogen depending on whether the N-terminal amino acid is either glutamic acid or lysine. Glu-plasminogen is composed of the entire amino acid sequence (excluding the precursor peptide) specified by the gene sequence. Lys-plasminogen results from the cleavage of Glu-plasminogen mainly between Lys-77 and Lys-78. Glu-plasminogen is the major form of plasminogen present in human plasma.

[0022] When secreted into the plasma, plasminogen is converted to plasmin by the action of tissue-type plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA). t-Pa / u-PA cleaves the Arg561-Val562 peptide bond in plasminogen. The resulting plasmin molecule is a two-chain disulfide-bonded serine protease with trypsin-like specificity (cleaving after Lys and Arg).

[0023] The amino-terminal heavy chain of plasmin is composed of five kringle domains, each containing approximately 80 amino acid residues. The kringle domains are responsible for the interaction of plasmin with other proteins such as multimeric fibrin and the plasmin inhibitor α2-antiplasmin.

[0024] The C-terminal light chain of plasmin is a typical serine protease, homologous to trypsin, and contains the classical serine protease catalytic triad (His603, Asp646, and Ser741).

[0025] "Plasmin", as used herein, is also known as fibrinolysin or lysfibrin, and is a serine protease resulting from the activation of chymogen plasminogen. The activation is the result of proteolytic cleavage between amino acid 561 and amino acid 562 (numbering for human Glu-plasminogen). Plasmin has a heavy chain containing five kringle domains and a light chain containing a catalytic domain.

[0026] The term "plasmin" as used herein is to be interpreted as meaning a therapeutically effective amount of wild-type (human) plasmin protein, a functional variant thereof or a functional fragment thereof, or a combination thereof.

[0027] "Functional variant", as used herein, refers to a sequence of plasmin having an addition, substitution, deletion or combination thereof in the amino acid sequence and / or a sequence of plasmin that is chemically modified with respect to the sequence of plasmin but retains substantially equivalent catalytic ability. Preferably, the functional variant of plasmin exhibits at least 60%, preferably 70%, advantageously 80%, more preferably 90%, more preferably 95%, even more preferably 97%, even more preferably 98%, advantageously 99% of the catalytic ability. In certain embodiments, the functional variant of plasmin has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 1.

[0028] "Functional fragment", as used herein, refers to a portion of the sequence of plasmin that contains a functional catalytic domain. Cleavage (truncation) of the plasmin molecule (truncation outside and / or inside the catalytic domain of plasmin) is possible as long as the catalytic domain is functional, and such cleavage (truncation) forms a "proteolytic activity derivative" of plasmin. Thus, one or more of the five kringle domains can be deleted in whole or in part. Therefore, truncated (truncated) plasmin or a functional fragment of plasmin lacking one or more kringle domains and / or lacking a part of one or more kringle domains is contemplated by the present invention as an example of a functional fragment of plasmin or a proteolytic activity derivative of plasmin. Examples of truncated (truncated) variants of plasmin include, but are not limited to, "midiplasmin", "miniplasmin", "microplasmin" and "delta-plasmin".

[0029] Figure 1 discloses a schematic diagram of some plasmin variants and plasmin mutants within the scope of the present invention. Variants having various permutations of kringle domains 1-5 linked to a serine protease component are within the scope of the present invention. Minor changes in the amino acid sequence are irrelevant as long as the motifs outlined in Figure 1 are maintained. The information outlined in SEQ ID NO:1, Table 1 and Figure 1 provides sufficient direction and clarity to those skilled in the art as to which plasmin mutants are included within the scope of the present invention.

[0030] According to the present invention, "plasmin" includes, but is not limited to, -(human) Glu-plasmin, -(human) Lys-plasmin, -midiplasmin, -miniplasmin, - Microplasmin, - delta - Plasmin, and combinations thereof are included.

[0031] "Midip lasmin", as used herein, refers to plasmin lacking kringle domains 1 - 3 (e.g., Christensen et al., 1995, Biochem J, 305, 97 - 102).

[0032] "Miniplasmin", as used herein, relates to plasmin lacking kringle domains 1 - 4 (Christensen et al., 1979, Biochim Biophys Acta, 567, 472 - 481; Powell & Castellino, 1980, J Biol Chem, 255, 5329).

[0033] "Microplasmin", as used herein, results from specific autolytic cleavage of plasmin molecules in an alkaline solution and refers to a low - molecular - weight form of plasmin that basically lacks all kringle domains. Suitable microplasmin is that disclosed in U.S. Patent No. 4,774,087 or the commercially available plasmin mutant Ocriplasmin having CAS number 1048016 - 09 - 6.

[0034] "Delta - Plasmin", as used herein, refers to plasmin having an N - terminal domain homologous to the kringle domain of native human plasminogen, which exhibits structural and functional properties similar to those of the native kringle domain of plasminogen (U.S. Patent No. 8,420,079). Delta - Plasmin lacks kringle domains 2 - 5.

[0035] In one embodiment, the plasmin is (human) Glu-plasmin. In some embodiments, the plasmin is (human) Lys-plasmin. In other embodiments, the plasmin is a mixture of Glu-plasmin and Lys-plasmin. As used herein, the term "pharmaceutically acceptable organ preservation solution" refers to a solution utilized for the perfusion and / or preservation of organs for transplantation, and typically is characterized by the presence of a colloid component (e.g., human serum albumin, dextran 40, mannitol, or other carbohydrate-based substances) for maintaining a colloidal osmotic pressure close to that of human blood / serum / plasma, physiological ion concentrations for adjusting the osmolality, a buffer for maintaining a normal physiological pH, and optionally an energy source (e.g., a carbohydrate such as glucose). The expression "pharmaceutically acceptable organ preservation solution" includes static cold storage solutions, as well as static cold storage solutions and perfusates utilized in normothermic machine perfusion, subnormothermic machine perfusion, and hypothermic machine perfusion.

[0036] Advantageously, the stable composition for organ preservation of the present invention contains plasmin mixed with a pharmaceutically acceptable organ preservation solution, and thus is effective in reducing thrombi and / or vascular occlusion in organs for transplantation. The composition can be used for the preservation and / or ex vivo perfusion of organs, for example, for organ preservation during the period of organ manipulation, processing, preservation, and / or transportation for transplantation to a recipient or re-transplantation to a subject. The composition of the present invention is particularly advantageous in that it is stable such that it allows for longer preservation of organs compared to prior art compositions, since the plasmin component does not precipitate either immediately or over time from the solution. Naturally, a composition that facilitates a longer preservation time prior to transplantation serves to increase the number of viable organs available for transplantation.

[0037] In some embodiments, the pharmaceutically acceptable organ perfusion fluid may have a pH of about 7.0 to 8.0 at room temperature, for example, a pH of about 7.2 to 7.9, for example, a pH of about 7.4 to 7.85. All pH measurements shown herein are to be interpreted as being measured in water at 25°C.

[0038] In some embodiments, the pharmaceutically acceptable organ perfusion fluid may have an osmolarity of about 200 to 400 mOsm / Kg, for example, an osmolarity of about 250 to about 350 mOsm / Kg, for example, an osmolarity such as about 270 to about 330 mOsm / Kg. In certain embodiments, the pharmaceutically acceptable organ perfusion fluid may have a range of osmolarity of about 275 to about 300 mOsm / Kg. The osmolarity of a solution can be readily measured using an analytical instrument called an osmometer, for example, the Osmette™ from Precision Systems, or the OsmoTECH® from Advanced Instruments.

[0039] The term "osmolarity" is a measure of the total concentration of substances in a solution and is defined as the number of osmoles of solute per liter of solution, indicating the potential change in osmotic pressure that occurs intracellularly when the solution is introduced into the body. Osmolarity can be calculated from the osmolarity values measured by an osmometer using various methods known to those skilled in the art. Since osmolarity is temperature-dependent, in the context of the present invention, osmolarity is calculated at 20°C.

[0040] "Lysine mimetic" as used herein refers to non-natural, natural derivatives and / or analogs of amino acids containing a C5-6 aliphatic or aromatic ring and at least two basic amine functional groups (i.e., at least one basic amine functional group in addition to the N-terminal amine). Mimetic, as used herein, means that these compounds bind to the lysine binding site of plasmin.

[0041] In some embodiments, the lysine mimetic may be selected from the group consisting of tranexamic acid, ε-aminocaproic acid, L-lysine, L-arginine, L-ornithine, γ-aminobutyric acid, 5-aminovaleric acid, 7-aminoheptanoic acid, glycylglycine, triglycine, N-α-acetyl-L-arginine, betaine, sulfanilic acid, pharmaceutically acceptable salts thereof, and combinations thereof. In another preferred embodiment, the lysine mimetic is tranexamic acid. In another preferred embodiment, the lysine mimetic is ε-aminocaproic acid. In another preferred embodiment, the lysine mimetic is L-arginine. In another preferred embodiment, the lysine mimetic is L-lysine. In another preferred embodiment, the lysine mimetic is L-ornithine. In another preferred embodiment, the lysine mimetic is γ-aminobutyric acid. In another preferred embodiment, the lysine mimetic is 5-aminovaleric acid. In another preferred embodiment, the lysine mimetic is 7-aminoheptanoic acid. In another preferred embodiment, the lysine mimetic is glycylglycine. In another preferred embodiment, the lysine mimetic is triglycine. In another preferred embodiment, the lysine mimetic is N-α-acetyl-L-arginine. In another preferred embodiment, the lysine mimetic is betaine. In another preferred embodiment, the lysine mimetic is sulfanilic acid.

[0042] Preferably, in some embodiments, the composition comprises two lysine mimetics, more preferably tranexamic acid and ε-aminocaproic acid; tranexamic acid and L-lysine; tranexamic acid and L-arginine; tranexamic acid and L-ornithine; tranexamic acid and γ-aminobutyric acid; tranexamic acid and 5-aminovaleric acid; tranexamic acid and 7-aminoheptanoic acid; tranexamic acid and glycylglycine; tranexamic acid and triglycine; ε-aminocaproic acid and L-lysine; ε-aminocaproic acid and L-arginine; ε-aminocaproic acid and L-ornithine; ε-aminocaproic acid and γ-aminobutyric acid; ε-aminocaproic acid and 5-aminovaleric acid; ε-aminocaproic acid and 7-aminoheptanoic acid; ε-aminocaproic acid and glycylglycine; ε-aminocaproic acid and triglycine; L-lysine and L-arginine; L-lysine and L-ornithine; L-lysine and γ-aminobutyric acid; L-lysine and 5-aminovaleric acid; L-lysine and 7-aminoheptanoic acid; L-lysine and glycylglycine; L-lysine and triglycine; L-arginine and L-ornithine; L-arginine and γ-aminobutyric acid; L-arginine and 5-aminovaleric acid; L-arginine and 7-aminoheptanoic acid; L-arginine and glycylglycine; L-arginine and triglycine; L-ornithine and γ-aminobutyric acid; L-ornithine and 5-aminovaleric acid; L-ornithine and 7-aminoheptanoic acid; L-ornithine and glycylglycine; L-ornithine and triglycine; γ-aminobutyric acid and 5-aminovaleric acid; γ-aminobutyric acid and 7-aminoheptanoic acid; γ-aminobutyric acid and glycylglycine; γ-aminobutyric acid and triglycine; 5-aminovaleric acid and 7-aminoheptanoic acid; 5-aminovaleric acid and glycylglycine; 5-aminovaleric acid and triglycine; 7-aminoheptanoic acid and glycylglycine; 7-aminoheptanoic acid and triglycine; or glycylglycine and triglycine.

[0043] In a more preferred embodiment, the composition comprises tranexamic acid and ε-aminocaproic acid.

[0044] In another preferred embodiment, the composition comprises three lysine mimetics. In a more preferred embodiment, the composition of the present invention comprises ε-aminocaproic acid, tranexamic acid and L-arginine.

[0045] For example, the lysine mimetic may be selected from the group consisting of tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof. In another preferred embodiment, the lysine mimetic is tranexamic acid. In another preferred embodiment, the lysine mimetic is ε-aminocaproic acid. In another preferred embodiment, the lysine mimetic is L-arginine.

[0046] In certain embodiments, the lysine mimetic has the following general formula (i):

Chemical formula

[0047] In other embodiments, the lysine mimetic is of the following general formula (ii): [Chemical formula] wherein R 11 -R 15 each of, and each R 11’ -R 15’are the same or different and independently selected from the group consisting of hydrogen, halogen, C1-C5 alkyl, C1-C5 haloalkyl, OH, C1-C5 alkoxy, C1-C5 haloalkoxy, SH, C1-C5 alkylthio, C1-C5 haloalkylthio, C1-C6 alkylsulfinyl, C1-C6 haloalkylsulfinyl, C1-C5 alkylsulfonyl, C1-C5 haloalkylsulfonyl, C1-C6 alkylsulfonamide, C1-C6 haloalkylsulfonamide, C1-C6 bis(alkyl)sulfonamide, C1-C5 bis(haloalkyl)sulfonamide, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, NH(C1-C5 haloalkyl), N(C1-C5 haloalkyl)2, N(C1-C5 alkyl)(C1-C5 haloalkyl), C(O)NH(C1-C6 alkyl), C(O)N(C1-C6 alkyl)2, C(O)NH(C1-C6 haloalkyl), C(O)N(C1-C6 haloalkyl)2, C(O)N(C1-C6 alkyl)(C1-C6 haloalkyl), C(O)H, C(O)C1-C6 alkyl, C(O)C1-C6 haloalkyl, C(O)O(C1-C6 alkyl), C(O)O(C1-C6 haloalkyl), OC(O)C1-C6 haloalkyl, OC(O)C1-C6 alkyl, SF5, SCN, cyano, and nitro) The compound, or a pharmaceutically acceptable salt thereof, may be used.

[0048] In some embodiments of the present invention, plasmin may be present in the composition of the present invention at a concentration of about 0.01 mg / mL to about 50 mg / mL. For example, plasmin may be present in the composition of the present invention at a concentration of about 0.01 mg / mL to about 30 mg / mL. In some embodiments, plasmin may be present in the composition of the present invention at a concentration of about 0.01 mg / mL to about 20 mg / mL. In other embodiments, plasmin may be present in the composition of the present invention at a concentration of about 0.05 mg / mL to about 20 mg / mL. In yet further embodiments, plasmin may be present in the composition of the present invention at a concentration of about 0.05 mg / mL to about 10 mg / mL. In other embodiments, plasmin may be present in the composition of the present invention at a concentration of about 0.05 mg / mL to about 5 mg / mL. In certain embodiments, plasmin may be present in the composition of the present invention at a concentration of about 0.05 mg / mL to about 2.5 mg / mL. In further embodiments, plasmin may be present in the composition of the present invention at a concentration of about 0.05 mg / mL to about 1 mg / mL. In a preferred embodiment, plasmin is present in the composition of the present invention at a concentration of 0.1 mg / mL.

[0049] In the composition according to any of the preceding claims, the lysine mimetic is present at a concentration of from about 0.1 μM to about 0.6 M. In some embodiments, the lysine mimetic is present at a concentration of from about 10 μM to about 0.4 M. In other embodiments, the lysine mimetic is present at a concentration of from about 100 μM to about 0.2 M. In further embodiments, the lysine mimetic is present at a concentration of from about 1 mM to about 0.1 M. For example, the lysine mimetic may be present at a concentration of from about 10 μM to about 1 mM. In certain embodiments, the lysine mimetic may be present at a concentration of from about 50 μM to about 100 μM. In another preferred embodiment, the lysine mimetic is present at a concentration of 10 μM. In another preferred embodiment, the lysine mimetic is present at a concentration of 25 μM. In another preferred embodiment, the lysine mimetic is present at a concentration of 50 μM. In another preferred embodiment, the lysine mimetic is present at a concentration of 0.1 mM. In another preferred embodiment, the lysine mimetic is present at a concentration of 0.75 mM. In another preferred embodiment, the lysine mimetic is present at a concentration of 1.5 mM. In another preferred embodiment, the lysine mimetic is present at a concentration of 3 mM. In another preferred embodiment, the lysine mimetic is present at a concentration of 6 mM.

[0050] In some embodiments, the lysine mimetic may be selected from the group consisting of tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof, and the lysine mimetic may be present at a concentration of about 10 μM to about 100 mM. In other embodiments, the lysine mimetic may be selected from the group consisting of tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof, and the lysine mimetic may be present at a concentration of about 50 μM to about 1 mM. In another preferred embodiment, the lysine mimetic is tranexamic acid and is present at a concentration of 10 μM. In another preferred embodiment, the lysine mimetic is tranexamic acid and is present at a concentration of 25 μM. In another preferred embodiment, the lysine mimetic is tranexamic acid and is present at a concentration of 50 μM. In another preferred embodiment, the lysine mimetic is tranexamic acid and is present at a concentration of 0.1 mM. In another preferred embodiment, the lysine mimetic is L-arginine and is present at a concentration of 0.75 mM. In another preferred embodiment, the lysine mimetic is L-arginine and is present at a concentration of 1.5 mM. In another preferred embodiment, the lysine mimetic is L-arginine and is present at a concentration of 3 mM. In another preferred embodiment, the lysine mimetic is L-arginine and is present at a concentration of 6 mM. In another preferred embodiment, the lysine mimetic is ε-aminocaproic acid and is present at a concentration of 0.1 mM.

[0051] In a preferred embodiment, the concentration of tranexamic acid is between 0.001 mM and 100 mM. In another preferred embodiment, the composition comprises tranexamic acid at a concentration greater than 5 μM.

[0052] In some embodiments of the composition of the present invention, the molar ratio of plasmin:lysine mimetic may be from about 1:1 to about 1:1,000. For example, the molar ratio of plasmin:lysine mimetic may be from about 1:1 to about 1:500. In other embodiments, the molar ratio of plasmin:lysine mimetic is from about 1:1 to about 1:100. In certain embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:1 to about 1:50. In certain embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:10 to about 1:10,000. In further embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:10 to about 1:1,000. For example, the molar ratio of plasmin:lysine mimetic may be from about 1:10 to about 1:100. In even further embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:10 to about 1:50. In certain embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:30 to about 1:10,000. In further embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:30 to about 1:1,000. For example, the molar ratio of plasmin:lysine mimetic may be from about 1:30 to about 1:100. In even further embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:30 to about 1:50. In other embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:50 to about 1:10,000. In further embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:50 to about 1:1,000. For example, the molar ratio of plasmin:lysine mimetic may be from about 1:50 to about 1:100. In a preferred embodiment, the molar ratio of plasmin:lysine mimetic is 1:50. In another preferred embodiment, the molar ratio of plasmin:lysine mimetic is 1:10.

[0053] In some embodiments, the lysine mimetic may be selected from the group consisting of tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof, and the molar ratio of plasmin:lysine mimetic may be from about 1:1 to about 1:1,000. For example, the molar ratio of plasmin:lysine mimetic may be from about 1:1 to about 1:500. In other embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:1 to about 1:100. In certain embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:1 to about 1:50.

[0054] In other embodiments, the lysine mimetic may be selected from the group consisting of tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof, and the molar ratio of plasmin:lysine mimetic may be from about 1:10 to about 1:10,000. In further embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:1 to about 1:1,000. For example, the molar ratio of plasmin:lysine mimetic may be from about 1:10 to about 1:100. In even further embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:10 to about 1:50.

[0055] In certain embodiments, the lysine mimetic may be selected from the group consisting of tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof, and the molar ratio of plasmin:lysine mimetic may be from about 1:30 to about 1:10,000. In further embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:30 to about 1:1,000. For example, the molar ratio of plasmin:lysine mimetic may be from about 1:30 to about 1:100. In even further embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:30 to about 1:50.

[0056] In further embodiments, the lysine mimetic may be selected from the group consisting of tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof, and the molar ratio of plasmin:lysine mimetic may be from about 1:30 to about 1:1,000. In further embodiments, the molar ratio of plasmin:lysine mimetic may be from about 1:30 to about 1:100. For example, the molar ratio of plasmin:lysine mimetic may be from about 1:30 to about 1:70. In other embodiments, the molar ratio of plasmin:lysine mimetic may be about 1:50. In other embodiments, the molar ratio of plasmin:lysine mimetic may be about 1:10. In some embodiments, the pharmaceutically acceptable organ perfusion fluid may have a weight molar osmotic concentration of about 200 to 400 mOsm / Kg, for example, a weight molar osmotic concentration of about 250 to about 350 mOsm / Kg, for example, a weight molar osmotic concentration such as about 270 to about 330 mOsm / Kg. In certain embodiments, the pharmaceutically acceptable organ perfusion fluid may be in the range of about 275 to about 300 mOsm / Kg weight molar osmotic concentration. The weight molar osmotic concentration of the solution can be readily determined using an analytical instrument called an osmometer, such as the Osmette™ from Precision Systems or the OsmoTECH® from Advanced Instruments.

[0057] In some embodiments of the compositions of the present invention, the pharmaceutically acceptable organ preservation solution may be the STEEN™ solution or a derivative thereof. The composition of the STEEN™ solution is described, for example, in U.S. Patent No. 7,255,983 (B2).

[0058] In other embodiments of the composition of the present invention, pharmaceutically acceptable organ preservation solutions include Euro Collins (EC) solution (Annual Report Eurotransplant International Foundation, Leiden, the Netherlands: Eurotransplant International Foundation, 1976), University of Wisconsin (UW) solution [ViaSpan (registered trademark)] (which is also known as Belzer) (Belzer FO and Southard JH, Transplantation, 1988; 45: 673, U.S. Patent No. 4,879,283), HTK / Custodiol (registered trademark) solution (which is also known as HTK (histidine-tryptophan-ketoglutaric acid) solution or Bretschneider solution) (Bretschneider HJ, Thorac Cardiovasc Surg, 1980; 28: 295), Celsior (registered trademark) solution (CEL) (Menasche P et al., Eur J Cardiothorac Surg, 1994; 8: 207), Institut Georges Lopez solution (IGL-1) (Ben Abdennebi H et al., Transpl Int, 2002; 15: 348), Marshall solution (HOC or hyperosmotic citrate) (Southard JH, Belzer FO, Annu Rev Med, 1995; 46: 235), Perfadex (registered trademark) solution (Muller C et al., Transplantation, 1999; 68: 1139-43), IGL-1 (registered trademark) solution, EP-TU solution (Okada et al., Surg Today (2012), 42: 152-156), ET-Kyoto solution (Chen F et al., Yonsei Med J, 2004; 45: 1107-1114), Polysol solution (Wei L et al., World J Gastroenterol, 2007; 13: 3684-3691), sucrose phosphate buffer (Lam FT et al., Transplantation, 1989; 47: 767), St.The refrigerated storage solution may be selected from the group consisting of Thomas Hospital Solution 1 and 2 (STH-1, STH-2) (Michel P et al., J Heart Lung Transplant, 2002;21:1030-1039), Lyon Preservation Solution (LYPS) (Michel P et al., J Heart Lung Transplant, 2000;19:1089-1097), Somah Solution (Ferng et al., Journal of Cardiothoracic Surgery (2017), 12:7), Stanford Solution (STF) (Michel P et al., J Heart Lung Transplant, 2002;21:1030-1039), KPS-1 Solution, and combinations thereof. In one embodiment, the pharmaceutically acceptable organ preservation solution may be a refrigerated storage solution selected from the group consisting of University of Wisconsin (UW) Solution, HTK / Custodiol® Solution, Celsior® Solution, and combinations thereof. For example, the pharmaceutically acceptable organ preservation solution may be HTK / Custodiol® Solution. In another preferred embodiment, the organ preservation solution may be HTK Solution. In another preferred embodiment, the organ preservation solution is KPS-1 Solution. In another preferred embodiment, the organ preservation solution is Belzer (UW) Solution.

[0059] The composition of the solution disclosed in this specification is widely known in the art. Okada et al., Surg Today (2012), 42:152-156 disclose the composition of the EP-TU solution and the EC solution. Guibert et al., Transfus Med Hemother, 2011;38:125-142 disclose the composition of EC, UW (Viaspan), Celsior, Custodial, and IGL-1. Muhlbacher et al., Transplantation Proceedings, 31, 2069-2070 (1999) disclose the composition of EC, UW, HTK, and Celsior. Ferng et al., Journal of Cardiothoracic Surgery (2017), 12:7 disclose the composition of the Celsior solution, the Perfadex solution, and the Somah solution. Chen et al., Yonsei Med J, Vol. 45, No. 6 (2004), page 1107 disclose the composition of the ET-Kyoto solution, the EC solution, and the UW solution. Jing et al., Acta Pharmacologica Sinica (2018), 39:845-857 disclose the composition of the EC solution, the UW solution, the HTK / Custodial solution, the Celsior solution, the Perfadex solution, the EP-TU solution, and the ET-Kyoto solution.

[0060] In some embodiments of the composition of the present invention, the pharmaceutically acceptable organ preservation solution may comprise at least one additive selected from the group consisting of physiologically acceptable salts, buffers, colloidal carbohydrates, antioxidants, and combinations thereof. The solution may further comprise an energy source and nutrients for the organ. Suitable materials include, but are not limited to, glucose, amino acids, vitamins, lipids, and combinations thereof.

[0061] In some embodiments, the physiologically acceptable salt may contain ions selected from the group consisting of sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, hydrogen carbonate ions, chloride ions, and combinations thereof. For example, the physiologically acceptable salt may be selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium dihydrogen phosphate, sodium bicarbonate, sodium hydroxide, and combinations thereof.

[0062] In certain embodiments, the buffer may be an amino acid selected from the group consisting of histidine, tryptophan, N-acetylhistidine, glycine, alanine, arginine, aspartic acid, glutamic acid, and combinations thereof. For example, the amino acid may be a combination of histidine and tryptophan. In certain embodiments, the buffer may include a phosphate buffer in combination with one of the amino acids listed above. In another preferred embodiment, the buffer includes a phosphate buffer.

[0063] In other embodiments, the colloidal carbohydrate may be selected from the group consisting of dextran compounds, mannitol, and combinations thereof. For example, the dextran may be dextran 40.

[0064] In yet another embodiment, the antioxidant may be selected from the group consisting of α-ketoglutaric acid, glutathione, and combinations thereof.

[0065] In certain embodiments of the composition of the present invention, the pharmaceutically acceptable organ preservation solution may contain additives selected from the group consisting of albumin, dextran compounds, physiologically acceptable salts, and combinations thereof. In a preferred embodiment, the additive is heparin. In another preferred embodiment, the additive is zocin. In another preferred embodiment, the additive is dexamethasone. In another preferred embodiment, the additive is bicarbonate. In another preferred embodiment, the additive is calcium gluconate.

[0066] In some embodiments, the concentration of albumin may be from about 1 mg / mL to about 100 mg / mL, from about 5 mg / mL to about 85 mg / mL, from about 10 mg / mL to about 70 mg / mL, from about 20 mg / mL to about 60 mg / mL. In other embodiments, the concentration of albumin may be from about 5 mg / mL to about 30 mg / mL. In further embodiments, the concentration of albumin may be from about 60 mg / mL to about 90 mg / mL. In another embodiment, the concentration of albumin is from about 50 mg / mL to about 100 mg / mL, from about 55 mg / mL to about 85 mg / mL, from about 60 mg / mL to about 80 mg / mL, from about 65 mg / mL to about 85 mg / mL, from about 70 mg / mL to about 80 mg / mL, about 50 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, or about 100 mg / mL.

[0067] In certain embodiments, the dextran compound comprises dextran having a molecular weight of about 1 kDa to about 250 kDa, about 20 kDa to about 150 kDa, about 50 kDa to about 100 kDa, about 1 kDa, about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 150 kDa, about 200 kDa, or about 250 kDa. For example, the dextran compound may comprise dextran having a molecular weight of about 40 kDa.

[0068] In certain embodiments, the concentration of the dextran compound may be about 1 mg / mL to about 55 mg / mL, about 2 mg / mL to about 25 mg / mL, about 2 mg / mL to about 20 mg / mL, about 2 mg / mL to about 10 mg / mL, about 2 mg / mL, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL. In some embodiments, the concentration of the dextran compound may be about 5 mg / mL.

[0069] In some embodiments, the physiologically acceptable salt may comprise ions selected from the group consisting of sodium ion, potassium ion, calcium ion, magnesium ion, bicarbonate ion, hydrogen carbonate ion, chloride ion, and combinations thereof. For example, the physiologically acceptable salt may be selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium dihydrogen phosphate, sodium bicarbonate, sodium hydroxide, and combinations thereof.

[0070] In a particularly preferred embodiment, the physiologically acceptable salts are present in a concentration range considered normal in human blood, human serum or human plasma. For example, the physiologically acceptable salts may include ions selected from the group consisting of sodium ions at a concentration of about 135 mM to about 150 mM, potassium ions at a concentration of about 3 mM to about 5 mM, chloride ions at a concentration of about 95 mM to 110 mM, bicarbonate ions at a concentration of 20 mM to 30 mM, bicarbonate ions at a concentration of 20 mM to 30 mM, calcium ions at a concentration of 2 to 3 mM, phosphate ions at a concentration of 1 mM to 1.5 mM, and combinations thereof.

[0071] In certain embodiments, the physiologically acceptable salts are selected from the group consisting of sodium chloride at a concentration of 75 mM to 150 mM, potassium chloride at a concentration of 0.4 mM to 5 mM, calcium chloride at a concentration of 1 mM to 2 mM, magnesium sulfate at a concentration of 1 mM to 2 mM, sodium bicarbonate at a concentration of 10 mM to 20 mM, and combinations thereof.

[0072] It should be understood by those skilled in the art that the specific embodiments disclosed within the scope of paragraphs

[0013] to

[0045] should not be read in isolation, and that this specification is intended to disclose these embodiments in combination with other embodiments, as opposed to being disclosed individually.

[0073] The method of the present invention In a second aspect, the present invention provides a method for preserving or reconditioning an organ prior to transplantation, the method comprising contacting the organ with a composition of the present invention.

[0074] "Organ", as used herein, typically refers to any part of an organism that is self-sufficient and has a specific life function. In a preferred embodiment, the organ is selected from the group consisting of kidney, liver, heart, lung, pancreas and intestine. In a more preferred embodiment, the organ is the liver.

[0075] In one embodiment, the organ is perfused with the compositions of the invention (as part of an ex vivo organ preservation process or an organ conditioning process).

[0076] In some embodiments, the organ is isolated. In some embodiments, the composition contacts the organ by washing, immersion, perfusion, or a combination thereof. In yet another embodiment, the composition contacts the organ by in vivo perfusion in its donor. In another embodiment, the composition contacts the organ by means of perfusion when the organ is isolated from its donor. In yet another embodiment, the organ is from a deceased donor.

[0077] In a further embodiment, the organ is contacted with the compositions of the invention at a lower temperature (as part of a static cold storage process or a subnormothermic perfusion process). For example, the organ may be contacted with the compositions of the invention, or perfused with the compositions of the invention, at a temperature selected from the group consisting of about 20-37°C and about 0-10°C. In one embodiment, the organ may be contacted with the compositions of the invention, or perfused with the compositions of the invention, at a temperature of about 0-10°C. In a particular embodiment, the organ may be contacted with the compositions of the invention at a temperature of about 0°C. Advantageously, plasmin exhibits proteolytic activity at a temperature of about 0-10°C, and even at 0°C, in the stable formulations of the invention.

[0078] Those skilled in the art should understand that the specific embodiments disclosed above should not be read alone, and this specification is intended to disclose these embodiments not individually but in combination with other embodiments. For example, each embodiment disclosed in this specification should be read as being explicitly combined with each embodiment disclosed in other parts of this specification, or as being explicitly combined by arbitrarily rearranging two or more embodiments disclosed in this specification.

[0079] All of the terms and embodiments disclosed above in connection with the composition of the present invention are equally applicable to the method of the present invention.

Example

[0080] Detailed examples of the present invention In the examples disclosed below in this specification, only generalized examples are presented, and other configurations and methods capable of reproducing the present invention are also possible, and it will be readily apparent to those skilled in the art that they are encompassed by the present invention.

[0081] Example 1: In vitro solubility and activity studies Plasmin is stable in acidic solutions, but once added to a neutral solution, the stabilizing effect of the acidic pH decreases and plasmin begins to precipitate from the solution. Solutions of full-length plasmin (11 mg / ml, pH 3.4) and various concentrations of tranexamic acid (TXA) were added to human plasma at a ratio of 1:10 at room temperature, and the resulting turbidity was measured by absorbance at 405 nm in a microtiter plate.

[0082] It is clear from Figure 2A that turbidity / plasmin precipitation decreases as the TXA concentration increases. In particular, when plasmin is added to test plasma at a fully diluted concentration of 1 mg / mL, the level of plasmin precipitation significantly decreases at TXA concentrations of 0.5 mM or higher.

[0083] From Figure 2B, it can be concluded that plasmin formulated with tranexamic acid or L-arginine can be dissolved in various commonly used organ preservation solutions and organ conditioning solutions. Plasmin formulated at 10 mg / mL with tranexamic acid (1 mM, 2.5 mM, 5 mM, or 10 mM TXA) or L-arginine (75 mM, 150 mM, 300 mM, or 600 mM Arg) was directly diluted 1:100 into HTK organ preservation solution, KPS-1 organ preservation solution, or Belzer University of Wisconsin (UW) organ preservation solution without other additives. Each graph shows the turbidity measurements at 405 nm of the individual organ preservation solutions without additives, 10 mg / mL soluble plasmin without excipients, and organ preservation solutions diluted 1:100 with plasmin and the indicated excipients. Each formulation diluted in the preservation / conditioning solution was examined for turbidity after the indicated incubation times. Diluted plasmin formulated with the indicated excipients remained soluble in all cases and at all time points and did not increase in turbidity for at least 24 hours when stored at 4°C.

[0084] Full-length plasmin was prepared according to procedures / methodologies known to those skilled in the art and detailed below: Section 18.3 (Chapter 18, pages 259-271) of Novokhatny, V. et al., Acid Stabilised Plasmin as a Novel Direct-Acting Thrombolytic, Production of Plasma Proteins for Therapeutic Use (edited by J. Bertolini et al., Wiley, 2013) [Print ISBN: 9780470924310 / Online ISBN: 9781118356807] (the contents of which are incorporated herein by reference). As used herein, "full-length plasmin" means a mixture of Lys-plasmin and Glu-plasmin, excluding the variants outlined in Figure 1. The truncated plasmin variants outlined in Figure 1 can be recombinantly prepared using manufacturing processes within the general common knowledge of those skilled in the art and purified according to the procedures disclosed by Novokhatny et al. (supra).

[0085] S-2403 is a commercially available plasmin-specific chromogenic substrate. Using this substrate, plasmin activity was measured in the presence of TXA as illustrated in Figure 3. The proteolytic activity of plasmin at a concentration of 1 mg / mL showed no change in kinetics in the presence of various TXA concentrations (0.001 mM - 100 mM). Therefore, it was concluded that TXA does not have a detrimental effect on the proteolytic activity of plasmin even when formulated together with plasmin.

[0086] To evaluate the compatibility of plasmin as an additive in refrigerated storage solutions, the activity of plasmin at a concentration of 1 mg / mL was evaluated at multiple time points during storage at 0°C. The results are plotted in Figure 4A, and it is clear that plasmin at a concentration of 1 mg / mL showed no change in kinetics while being stored at 0°C for 24 hours.

[0087] The results in Figure 4b show that plasmin formulated with tranexamic acid and directly diluted in various commonly used organ preservation solutions maintains plasmin activity for at least 24 hours at temperatures commonly used in organ graft preservation and reconditioning methods. Plasmin formulated at 10 mg / mL with 5 mM tranexamic acid was directly diluted 1:100 into HTK organ preservation solution, KPS-1 organ preservation solution, or Belzer University of Wisconsin (UW) organ preservation solution without other additives (final concentrations of 0.1 mg / mL plasmin and 0.05 mM tranexamic acid), and maintained at 5°C (white bars), 25°C (black bars), or 37°C for 24 hours (hatched bars). Activity at each indicated time was evaluated using a commercially available S-2403 plasmin-specific chromogenic substrate.

[0088] The results in Figure 4c clearly show that plasmin formulated with tranexamic acid or ε-aminocaproic acid and directly diluted in various commonly used organ preservation solutions maintains plasmin activity for at least one month when stored at 5°C. Plasmin formulated at 10 mg / mL with 10 mM ε-aminocaproic acid (eACA), 1 mM tranexamic acid (TXA), 5 mM tranexamic acid (TXA), or 10 mM tranexamic acid (TXA) was directly diluted 1:100 into HTK organ preservation solution, KPS-1 organ preservation solution, or Belzer University of Wisconsin (UW) organ preservation solution without other additives (final concentrations of 0.1 mg / mL plasmin and 0.1 mM eACA, 0.01 mM TXA, 0.05 mM TXA, or 0.1 mM TXA), and maintained at 5°C for one month. Significant plasmin activity is maintained when formulated with these excipients and diluted in organ preservation fluids and organ reconditioning solutions. Activity at each indicated time was evaluated using a commercially available S-2403 plasmin-specific chromogenic substrate.

[0089] Example 2: Rat liver transplantation study Rat liver harvestMale Lewis rats (200 - 300 grams) were used as donors. General anesthesia was induced using 5% isoflurane and maintained at 1 - 2% via a nose cone during the procedure. The abdomen was opened by a midline laparotomy. The left diaphragmatic vein was incised, ligated, and divided. The posterior caudate lobe was dissected from the stomach and spleen. The common bile duct was incised and cannulated using a 24G angiocath tied with 5 - 0 silk suture. The hepatic artery was incised near the umbilicus and divided during suturing. The portal vein was incised, the two closest tributaries were ligated, and divided during suturing. Heparin (250 units) was injected systemically via the inferior vena cava and circulated for 3 minutes. The portal vein was clamped for 3 minutes. After this in - situ ischemia period, the portal vein was cannulated and fixed with 5 - 0 silk suture. Subsequently, the liver was flushed with 50 ml of ice - cold HTK solution (Custodiol®, without additives). During the flush, the inferior IVC was sharply opened with scissors to drain the blood effluent from the liver. When the flush was complete, the liver was incised clockwise and explanted by dividing the vessels and ligaments starting from the superior IVC, the left and right coronary mesenteries, and the posterior attachments to the spine (only in non - transplantation cases).

[0090] Cold storage cohort After liver explantation, the liver was kept at room temperature (first cohort; in the second cohort this was done on ice) while vascular cuffs were inserted into the portal vein and the large intra - hepatic vein. This dissection process took 10 - 15 minutes. Subsequently, a 200 - ml back - table flush was performed using HTK and albumin (control) or plasmin:tranexamic acid (1:50 molar ratio, 0.1 mg / mL plasmin). Then, the liver was stored on ice for 1 hour. After the cold storage period, a final pre - transplantation flush consisting of 100 ml of HTK solution (without additives) was performed.

[0091] Machine perfusion cohortAfter liver explantation, subnormothermic machine perfusion was initiated at room temperature. DEVOL solution perfusate was used. Its base consisted of Duosol 4555 and 4% human albumin. Additives included heparin, zocin, dexamethasone, bicarbonate, and calcium gluconate. In the test group, plasmin:tranexamic acid (1:50 molar ratio) was added to 200 ml of perfusate to achieve a final concentration of 0.1 mg / mL plasmin. After 1 hour of machine perfusion, vascular cuffs were inserted into the portal vein and the intrahepatic vena cava at room temperature (first cohort; in the second cohort, this was done on ice). A final pre-transplant flush wash was performed with 100 ml of HTK solution (without additives).

[0092] Liver transplantation procedure The liver was transplanted in an orthotopic fashion as previously described by Abraham et al. (Abraham N et al., Front Med (Lausanne), 2022;9:804834, doi:10.3389 / fmed.2022.804834), the content of which is incorporated herein by reference.

[0093] Study endpoint The survival of the subjects was recorded on the first postoperative day. Surviving subjects were sacrificed under general anesthesia. Median laparotomy was performed, and the ascites volume and hematocrit of the ascites were recorded. Total blood sampling was performed by venipuncture from the large vein, which was centrifuged and used for measurement of liver function test values. Liver grafts and bile ducts were biopsied for histological examination and formalin-fixed.

[0094] Results After 1 hour of cold storage in the presence of plasmin / tranexamic acid, a significant survival benefit at 24 hours post-transplantation (80% vs. 14.2% survival, P = 0.0293) was observed. See Figure 5. Due to the small number of survivors in the control group, a meaningful comparison of bile duct injury levels could not be made.

[0095] In the evaluation of the liver function panel in the machine perfusion cohort, signs of improved function were observed after the first postoperative day (POD1). After 1 hour of machine perfusion, there was a significant decrease in serum alkaline phosphatase at 24 hours with plasmin / tranexamic acid administration (mean: 216.6 vs 56 U / L, P = 0.0006). Furthermore, glucose homeostasis was improved at 24 hours in recipients of plasmin-treated grafts (mean: 163 vs 82.3 mg / dl, P = 0.0067). These results are plotted in Figure 6.

[0096] No obvious hemorrhagic complications were observed after transplantation in either mode of organ preservation.

[0097] Sequence The sequences shown in the foregoing text are outlined below in fasta format. If there is a discrepancy between the sequences listed in the text and the sequences corresponding to the attached sequence listing, the sequences listed in the text shall be the prioritized sequences for error correction. [Table 2]

Claims

1. Plasmin, Lysinmimetic, and A pharmaceutically acceptable organ preservation solution with a pH of approximately 6 to 8. It includes, The molar ratio of plasmin to lysine mimetic is approximately 1:1 to approximately 1:10,000. Plasmin is selected from the group consisting of Glu-plasmin and Lys-plasmin. A stable composition for organ reconditioning.

2. Lysine mimetic, A substance selected from the group consisting of tranexamic acid, ε-aminocaproic acid, L-lysine, L-arginine, L-ornithine, γ-aminobutyric acid, 5-aminovaleric acid, 7-aminoheptanoic acid, glycylglycine, triglycine, N-α-acetyl-L-arginine, betaine, sulfanilic acid, pharmaceutically acceptable salts thereof, and combinations thereof, or General formula (i): 【Chemistry 1】 (In the formula, R １ -R ５ each of, and each R １’ -R ５’ is the same or different and independently is hydrogen, halogen, C １ -C ５ alkyl, C １ -C ５ haloalkyl, OH, C １ -C ５ alkoxy, C １ -C ５ haloalkoxy, SH, C １ -C ５ alkylthio, C １ -C ５ haloalkylthio, C １ -C ６ alkylsulfinyl, C １ -C ６ haloalkylsulfinyl, C １ -C ５ alkylsulfonyl, C １ -C ５ haloalkylsulfonyl, C １ -C ６ alkylsulfonamide, C １ -C ６ haloalkylsulfonamide, C １ -C ６ bis(alkyl)sulfonamide, C １ -C ５ bis(haloalkyl)sulfonamide, NH ２ , NH(C １ -C ６ alkyl), N(C １ -C ６ alkyl) ２ , NH(C １ -C ５ haloalkyl), N(C １ -C ５ haloalkyl) ２ , N(C １ -C ５ alkyl)(C １ -C ５ haloalkyl), C(O)NH(C １ -C ６ alkyl), C(O)N(C １ -C ６ Alkyl) ２ , C(O)NH(C １ -C ６ Haloalkyl), C(O)N(C) １ -C ６ (Haloalkyl) ２ , C(O)N(C １ -C ６ (Alkyl) (C １ -C ６ Haloalkyl), C(O)H, C(O)C １ -C ６ Alkyl, C(O)C １ -C ６ Haloalkyl, C(O)O(C) １ -C ６ Alkyl), C(O)O(C １ -C ６ Haloalkyl), OC(O)C １ -C ６ Haloalkyl, OC(O)C １ -C ６ (Selected from the group consisting of alkyl, SF5, SCN, cyano, and nitro) A compound of or a pharmaceutically acceptable salt thereof, General formula (ii): 【Chemistry 2】 (In the formula, Each of R11-R15, and each of R11'-R15', are the same or different and independently of hydrogen, halogen, C1-C5 alkyl, C1-C5 haloalkyl, OH, C1-C5 alkoxy, C1-C5 haloalkoxy, SH, C1-C5 alkylthio, C1-C5 haloalkylthio, C1-C6 alkylsulfinyl, C1-C6 haloalkylsulfinyl, C1-C5 alkylsulfonyl, C1-C5 haloalkylsulfonyl, C1-C6 alkylsulfonamide, C1-C6 haloalkylsulfonamide, C1-C6 bis(alkyl)sulfonamide, C1-C5 bis(haloalkyl)sulfonamide, NH2 NH(C1-C6 alkyl), N(C1-C6 alkyl)2, NH(C1-C5 haloalkyl), N(C1-C5 haloalkyl)2, N(C1-C5 alkyl)(C1-C5 haloalkyl), C(O)NH(C1-C6 alkyl), C(O)N(C1-C6 alkyl)2, C(O)NH(C1-C6 haloalkyl), C(O)N(C1-C6 haloalkyl)2, C(O)N(C1-C6 alkyl)(C1-C6 haloalkyl), C(O)H, C(O)C1-C6 alkyl, C(O)C1-C6 haloalkyl, C(O)O(C (Selected from the group consisting of 1-C6 alkyl, C(O)O (C1-C6 haloalkyl, OC(O)C1-C6 haloalkyl, OC(O)C1-C6 alkyl, SF5, SCN, cyano, and nitro) A compound of or a pharmaceutically acceptable salt thereof. The composition according to claim 1.

3. The composition according to claim 1 or 2, wherein plasmin is present at a concentration of about 0.01 mg / mL to about 50 mg / mL.

4. The composition according to claim 1 or 2, wherein lysine mimetic is present at a concentration of about 0.1 μM to about 0.6 M.

5. The composition according to claim 1 or 2, wherein the pharmaceutically acceptable organ perfusion fluid has a molar osmotic pressure concentration between approximately 200 and approximately 400 mOsm / kg.

6. The composition according to claim 1 or 2, wherein the pharmaceutically acceptable organ preservation solution comprises an additive selected from the group consisting of albumin, dextran compounds, physiologically acceptable salts, and combinations thereof.

7. The composition according to claim 6, wherein the albumin concentration is approximately 1 mg / mL to approximately 100 mg / mL, approximately 5 mg / mL to approximately 85 mg / mL, approximately 10 mg / mL to approximately 70 mg / mL, approximately 20 mg / mL to approximately 60 mg / mL, approximately 5 mg / mL to approximately 30 mg / mL, approximately 60 mg / mL to approximately 90 mg / mL, approximately 50 mg / mL to approximately 100 mg / mL, approximately 55 mg / mL to approximately 85 mg / mL, approximately 60 mg / mL to approximately 80 mg / mL, approximately 65 mg / mL to approximately 85 mg / mL, approximately 70 mg / mL to approximately 80 mg / mL, approximately 50 mg / mL, approximately 60 mg / mL, approximately 65 mg / mL, approximately 70 mg / mL, approximately 75 mg / mL, approximately 80 mg / mL, approximately 85 mg / mL, approximately 90 mg / mL, or approximately 100 mg / mL.

8. The composition according to claim 6, wherein the dextran compound comprises a dextran having a molecular weight of about 1 kDa to about 250 kDa, about 20 kDa to about 150 kDa, about 50 kDa to about 100 kDa, about 1 kDa, about 5 kDa, about 10 kDa, about 20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 150 kDa, about 200 kDa, or about 250 kDa.

9. The composition according to claim 6, wherein the concentration of the dextran compound is about 1 mg / mL to about 55 mg / mL, about 2 mg / mL to about 25 mg / mL, about 2 mg / mL to about 20 mg / mL, about 2 mg / mL to about 10 mg / mL, about 2 mg / mL, about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, or about 25 mg / mL.

10. The composition according to claim 6, wherein the physiologically acceptable salt comprises an ion selected from the group consisting of sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, sodium chloride ions, and combinations thereof.

11. The composition according to claim 6, wherein the physiologically acceptable salt is selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium dihydrogen phosphate, sodium bicarbonate, sodium hydroxide, and combinations thereof.

12. The composition according to claim 1 or 2, wherein the pharmaceutically acceptable organ preservation solution comprises at least one additive selected from the group consisting of physiologically acceptable salts, buffers, colloidal carbohydrates, antioxidants, and combinations thereof.

13. The composition according to claim 12, wherein the physiologically acceptable salt comprises an ion selected from the group consisting of sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, sodium chloride ions, and combinations thereof.

14. The composition according to claim 12, wherein the buffer is an amino acid selected from the group consisting of histidine, tryptophan, N-acetylhistidine, glycine, alanine, arginine, aspartic acid, glutamic acid, and combinations thereof, or the buffer comprises a phosphate buffer.

15. The composition according to claim 12, wherein the colloidal carbohydrate is selected from the group consisting of dextran compounds, mannitol, and combinations thereof.

16. The composition according to claim 12, wherein the antioxidant is selected from the group consisting of α-ketoglutaric acid, glutathione, and combinations thereof.

17. The composition according to claim 1 or 2, wherein the pharmaceutically acceptable organ preservation solution is STEEN solution, or a refrigerated preservation solution selected from the group consisting of Euro Collins (EC) solution, HTK / Custodiol solution, Celsior solution, Perfadex solution, KPS-1, Belzer University of Wisconsin (UW) solution, EP-TU solution, ET-Kyoto solution, and combinations thereof.

18. A method for preserving or reconditioning an organ before transplantation, comprising contacting the organ with the composition described in claim 1 or 2.

19. The method according to claim 18, wherein the organ is perfused extracorporeally with the composition.

20. The method according to claim 18, wherein the composition is brought into contact with an organ by washing, immersion, perfusion, or a combination thereof.

21. The method according to claim 18, wherein the organ is brought into contact with or perfused with the composition at a temperature selected from the group consisting of 20 to 37°C, 0 to 10°C, and about 0°C.

22. The method according to claim 18, wherein the organ is the liver, kidney, lung, or heart.

Images