Stable plasmin compositions for organ preservation and conditioning

Abstract

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

Description

Technical Field

[0001] The present invention details novel compositions for preserving and / or reconditioning organs prior to transplantation. Background Art

[0002] For many patients with terminal diseases, organ transplantation is the only viable treatment option. Naturally, an adequate supply of high-quality donor organs is crucial for any successful transplantation program. Static cold storage (SCS) is one of the most commonly used methods for effectively preserving and transporting viable organs for transplantation. SCS involves flushing the obtained organ with a preservation solution at 0 to 4°C, and then soaking it in a preservation solution at the same temperature until transplantation. The low temperature environment is responsible for reducing cell metabolism, and the preservation solution reduces cell metabolism and provides cell protection. SCS preservation solutions are typically some variants of buffers (e.g., histidine or phosphate) and antioxidants (e.g., tryptophan-ketoglutarate, glutathione-mannitol), characterized in that the concentrations of potassium ions and sodium ions are different.

[0003] Although the SCS approach can suffer from many limitations, including tissue damage induced by long-term cryopreservation, difficulty in assessing donor organ function and viability, and reduced chance of organ repair, its use remains highly prevalent in the transplantation field.

[0004] The scarcity of brain-dead organ donors makes it challenging to have a continuous and consistent supply of viable organs for transplantation. A compromise solution to this problem is to condition organs donated after cardiac death (DCD) and has led to the use of extended criteria donor organs. Therefore, the use of machine perfusion / ex vivo organ perfusion (EVOP) for donor organ functional assessment prior to transplantation and ex vivo organ repair and conditioning has become increasingly popular.

[0005] EVOP involves perfusing an organ with a controlled flow of perfusion solution. This method helps maintain organ microvasculature tone, provides oxygen and nutrients to support tissue metabolism, and removes toxic metabolic waste products. Given that the rate of cellular respiration is proportional to the ambient temperature, the temperature at which EVOP is performed can have an effect on the organ's preservation time and the organ's responsiveness to conditioning. There are three main types of EVOP: normothermic machine perfusion at 35 to 38°C, subnormothermic machine perfusion at 20 to 34°C, and hypothermic machine perfusion at 0 to 8°C.

[0006] The importance of perfusate composition in ex vivo conditioning and maintaining stable organ function cannot be overstated. Over time, blood-based perfusates have been largely replaced by safer and more reproducible alternatives such as chemical solutions. TM Is one of the most widely used solutions for machine perfusion of the lungs and supplements Steen Solution with pharmacologically active substances TM / Other organ perfusates are a new area of ​​research that holds great promise in the field of organ transplantation. One such example is US20190059362 to CSL Behring LLC, which discloses a lung perfusate solution containing alpha-1 antitrypsin.

[0007] The increasing use of marginal and DCD organs has shown a steady increase in the popularity of EVOP as a means of assessing donor organ function. In addition, EVOP has shown the potential to enable graft repair and conditioning.

[0008] Although SCS and EVOP have been successful in providing patients with viable organ transplants, both practices can be rendered ineffective by the presence of thrombi in the organs used for transplantation. Thrombosis of transplanted organs presents a high risk because thrombi reduce the perfusion of the graft, thereby reducing the effectiveness of the storage and EVOP processes. In addition, thrombosis in donor organs is always 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 becoming ineffective due to thrombi and microthrombi. After the death of a DCD organ donor, the organ is harvested and perfused with a cold preservation solution. Such a step prevents the formation of new thrombi, but does not destroy existing thrombi formed during the dying period or immediately after death. Microthrombi can also be observed in organs from brain-dead donors as a result of metabolic events before and after brain death.

[0009] Therefore, the challenge of providing a stable perfusate and / or storage solution capable of removing thrombi and other vascular occlusions formed in a donor organ prior to transplantation remains an unmet need. Summary of the invention

[0010] In a first aspect, the present invention relates to a stable composition for organ conditioning, the stable composition comprising:

[0011] Plasmin,

[0012] Lysine mimetics, and

[0013] A pharmaceutically acceptable organ preservation solution having a pH of about 6 to about 8,

[0014] The molar ratio of plasmin:lysine mimetic is about 1:1 to about 1:10,000.

[0015] In a second aspect, the invention relates to a method for preserving or conditioning an organ prior to transplantation, the method comprising contacting the organ with a composition of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a schematic diagram of a number of plasmin variants and mutants within the scope of the present invention;

[0017] Figure 2A ) is a graph of plasmin solubility in the presence of varying concentrations of tranexamic acid; B) is a graph of plasmin solubility when formulated in tranexamic acid or L-arginine and diluted into organ preservation solution.

[0018] Figure 3 is a graph of plasmin activity in the presence of different concentrations of tranexamic acid;

[0019] Figure 4. A) is a graph of plasmin activity over a 24 hour period at 0°C; B) is a graph of plasmin activity when plasmin is formulated in tranexamic acid and stored at various temperatures for 24 hours after dilution into organ preservation solution; C) is a graph of activity when plasmin is formulated in tranexamic acid or ε-aminocaproic acid and stored at 5°C for 1 month after dilution into organ preservation solution.

[0020] Figure 5 is a graph of the percentage of livers surviving after 24 hours (POD1) in the control group and the plasmin-treated group; and

[0021] Figure 6 Graphs of liver function as assessed by alkaline phosphatase levels and blood glucose levels after 24 hours (POD1) in the control group and the plasmin-treated group are provided. DETAILED DESCRIPTION

[0022] The word "comprising" and its variations and the word "having / including" used in this document when referring to the present invention are used to specify the presence of stated features, integers, steps or components, but do not exclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0023] Those skilled in the art will appreciate that some specific embodiments disclosed herein should not be understood in isolation, and this specification is intended to be used to interpret the disclosed embodiments in combination with each other rather than individually. Therefore, each embodiment can be used as a basis for modifying or limiting other embodiments disclosed herein.

[0024] Concentration, amount and other numerical data can be represented or shown in range form in this article.It should be understood that such range form is used only for convenience and simplicity, and should therefore be flexibly interpreted as not only including the numerical value clearly recorded as range boundary, but also including all single numerical values ​​or sub-ranges included in the range, as each numerical value and sub-range are clearly recorded.As an example, the numerical range of "10 to 100" should be interpreted as not only including the value of 10 to 100 clearly recorded, but also including single values ​​and sub-ranges in the shown range.Therefore, included in the numerical range is a single value (such as 10,11,12,13 ... 97,98,99,100) and a sub-range (such as 10 to 40,25 to 40 and 50 to 60, etc.).This same principle is applicable to the scope of only recording a numerical value, such as "at least 10".In addition, no matter how the breadth of the described range or feature is, such an explanation should be applicable.

[0025] Compositions of the present invention

[0026] In a first aspect, the present invention provides a stable composition for organ conditioning, the stable composition comprising:

[0027] Plasmin,

[0028] Lysine mimetics, and

[0029] A pharmaceutically acceptable organ preservation solution having a pH of about 6 to about 8,

[0030] The molar ratio of plasmin:lysine mimetic is about 1:1 to about 1:10,000.

[0031] " Stable composition " used herein relates to such composition: make plasmin component neither immediately nor precipitate out from solution over time, thereby allow to preserve organ longer than prior art composition.Any method known in the art can be used to determine whether composition is stable, for example, by any method for determining plasmin precipitation, for example turbidity, the turbidity is the process of measuring the intensity loss of light through sample caused by the scattering effect of insoluble particles.Turbidity can be determined, for example, by measuring the absorbance at 405nm wavelength.Other methods suitable for determining precipitation are, for example, refraction method, visual inspection (on black & white background light box), microflow imaging (Micro Flow Imaging, MFI), dynamic light scattering (Dynamic Light Scattering, DLS) and high-speed centrifugation for finding visible precipitate.

[0032] "Organ conditioning" in this specification means preserving or improving the condition of an organ intended for transplantation.

[0033] As shown in SEQ ID NO: 1, the native circulating human plasminogen is a single-chain protein containing 791 amino acid residues with 24 intrachain disulfide bridges, 5 kringle domains, a serine protease domain and a preactivation peptide (PAP). The positions of these domains relative to SEQ ID NO: 1 are summarized in Table 1 below.

[0034] Table 1

[0035] Residue (according to SEQ ID No: 1) describe length No. 1 to 79 PAP domain 79 No. 84 to 162 Three rings 1 79 No. 165 to 243 Three Rings 2 79 No. 275 to 333 Three Rings 3 78 No. 358 to 435 Three Rings 4 78 No. 462 to 541 Three Rings 5 80 No. 562 to 789 S1 Peptidase 228

[0036] In vivo, plasminogen exists as Glu-plasminogen and Lys-plasminogen, depending on whether the N-terminal amino acid is glutamic acid or lysine. Glu-plasminogen is composed of the entire amino acid sequence specified by the gene sequence (excluding the precursor peptide). Lys-plasminogen is the result of cleavage of Glu-plasminogen mainly between Lys-77 and Lys-78. Glu-plasminogen is the main form of plasminogen present in human plasma.

[0037] Once plasminogen is secreted into the plasma, it can be converted into 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 the plasminogen zymogen. The resulting plasmin molecule is a two-chain, disulfide-linked serine protease with trypsin-like specificity (cleavage after Lys and Arg).

[0038] The amino-terminal heavy chain of plasmin is composed of five kringle domains, each of which contains about 80 amino acid residues. The kringle domains are responsible for the interaction of plasmin with other proteins, such as polymerized fibrin and the plasmin inhibitor α2-antiplasmin.

[0039] The C-terminal light chain of plasmin is a typical serine protease, homologous to trypsin and containing the classic serine protease catalytic triad: His603, Asp646 and Ser741.

[0040] As used herein, "plasmin" refers to a serine protease also known as plasmin or lysofibrin, which is activated by the zymogen plasminogen. Activation is the result of proteolytic cleavage between amino acids 561 and 562 (numbering relative to human Glu-plasminogen). Plasmin carries a heavy chain containing 5 kringle domains and a light chain containing a catalytic domain.

[0041] The term "plasmin" as used herein should be interpreted as meaning a therapeutically effective amount of wild-type (human) plasmin protein, a functional mutant thereof, or a functional fragment thereof, or a combination of these.

[0042] "Functional mutants" used herein relate to the sequence of such plasmin: there is addition, substitution, deletion or a combination thereof in the amino acid sequence of plasmin and / or has been chemically modified relative to the plasmin sequence and retains substantially equivalent catalytic capabilities. Preferably, the functional mutants of plasmin show the following catalytic capabilities: at least 60%, preferably 70%, advantageously 80%, more preferably 90%, more preferably 95%, even more preferably 97% and even more preferably 98%, advantageously 99%. In a specific embodiment, the functional mutants of plasmin have 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.

[0043] "Functional fragment" used herein relates to a part of the plasmin sequence containing a functional catalytic domain. As long as the catalytic domain remains functional, the truncation of the plasmin molecule (outside and / or inside the plasmin catalytic domain) is possible, so such truncation leads to the formation of a "proteolytically active derivative" of plasmin. Therefore, one or more of the 5 three ring domains may be completely or partially missing. Therefore, the present invention contemplates the truncated plasmin or plasmin lacking one or more three ring domains and / or lacking one or more three ring domains as some examples of functional fragments of plasmin or proteolytically active derivatives of plasmin. Some examples of truncated variants of plasmin include, but are not limited to, "midi plasmin (midiplasmin)", "mini plasmin (miniplasmin)", "micro plasmin" and "δ-plasmin".

[0044] Figure 1 Schematic representations of many plasmin variants and mutants within the scope of the invention are disclosed. Mutants with various arrangements of kringle domains 1 to 5 tethered to the serine protease component are within the scope of the invention. Minor changes in the amino acid sequence are inconsequential, provided that the Figure 1 SEQ ID NO: 1, Table 1 and Figure 1 The information summarized in provides the skilled person with sufficient guidance and clarity as to which plasmin mutants fall within the scope of the present invention.

[0045] According to the present invention, "plasmin" includes but is not limited to:

[0046] - (human) Glu-plasmin,

[0047] - (human) Lys-plasmin,

[0048] -midi-plasmin,

[0049] -mini-plasmin,

[0050] -Microplasmin

[0051] -δ-plasmin and combinations thereof.

[0052] "Midi-plasmin" as used herein relates to plasmin lacking the kringle domains 1 to 3 (eg Christensen et al., 1995, Biochem J 305, 97-102).

[0053] "Mini-plasmin" as used herein relates to plasmin lacking the kringle domains 1 to 4 (Christensen et al., 1979, Biochim Biophys Acta 567, 472-481; Powell & Castellino, 1980, J Biol Chem 255, 5329)).

[0054] "Microplasmin" as used herein relates to a low molecular weight form of plasmin produced by specific autolytic cleavage of the plasmin molecule in alkaline solution and substantially lacking all kringle domains. Suitable microplasmins are those disclosed in U.S. Pat. No. 4,774,087, or the commercial plasmin mutant Ocriplasmin with CAS No. 1048016-09-6.

[0055] As used herein, "delta-plasmin" refers to plasmin having an N-terminal domain homologous to the kringle domain of native human plasminogen, which exhibits similar structural and functional characteristics to the native kringle domain of plasminogen (US Pat. No. 8,420,079). Delta plasmin lacks kringle domains 2 to 5.

[0056] In one embodiment, plasmin is (human) Glu-plasmin. In some embodiments, plasmin is (human) Lys-plasmin. In other embodiments, plasmin is a mixture of Glu-plasmin and Lys-plasmin. The term "plasmin for pharmaceutically acceptable organ preservation solution" as used herein refers to a solution used in the perfusion and / or preservation of an organ for transplantation, and is characterized by the presence of a colloidal component (e.g., human serum albumin, dextran 40, mannitol or other carbohydrate-based substances) that maintains a turgor pressure close to that of human blood / serum / plasma, the physiological ion concentration of which regulates osmotic concentration, a buffer for maintaining normal physiological pH, and in some cases an energy source (e.g., carbohydrates such as glucose). The description "pharmaceutically acceptable organ preservation solution" is included in static refrigerated solutions and perfusates used in normothermic machine perfusion, subnormothermic machine perfusion, and hypothermic machine perfusion.

[0057] Advantageously, the stable compositions for organ preservation of the present invention are effective in reducing thrombus and / or vascular obstruction in the organ for transplantation because they comprise plasmin mixed with a pharmaceutically acceptable organ preservation solution. The composition can be used for the preservation and / or ex vivo perfusion of an organ, for example, for the preservation of an organ transplanted in a recipient or re-implanted in a subject during operation, processing, storage and / or transportation. The compositions of the present invention are particularly advantageous because they are stable, so that the plasmin components neither immediately nor precipitate out of the solution over time, thereby allowing the preservation of the organ longer than the prior art compositions. Naturally, compositions that contribute to a longer preservation time before transplantation will be used to increase the number of viable organs that can be used for transplantation.

[0058] In some embodiments, the pH of the pharmaceutically acceptable organ perfusate at room temperature may be about 7.0 to 8.0, such as about 7.2 to 7.9, such as about 7.4 to 7.85. All pH measurements expressed herein should be interpreted as being measured in water at 25°C.

[0059] In some embodiments, the osmolarity of the pharmaceutically acceptable organ perfusate can be about 200 to 400 mOsm / Kg, such as about 250 to about 350 mOsm / Kg, such as about 270 to about 330 mOsm / Kg. In certain embodiments, the osmolarity of the pharmaceutically acceptable organ perfusate can range from about 275 to about 300 mOsm / kg. The osmolarity of the solution can be measured using a pressure meter called an osmometer (e.g., a Precision Systems Osmette TM or Advanced Instruments ) can be easily determined by analytical instruments.

[0060] The term "osmolarity" is a measure of the total concentration of a substance in a solution, defined as the number of osmoles of solute per liter of solution, which represents the potential change in osmotic pressure that will occur in cells after the solution is introduced into the body. Osmolarity can be calculated from osmolarity values, the latter being measured by an osmometer using methods known to those skilled in the art. Since in the context of the present invention osmolarity is dependent on temperature, osmolarity is calculated at 20°C.

[0061] "Lysine mimetics" as used herein refers to non-natural, natural derivatives and / or analogs of amino acids comprising 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). Mimetics as used herein means a compound that binds to the lysine binding site of plasmin.

[0062] In some embodiments, the lysine mimetic can be selected from tranexamic acid, ε-aminocaproic acid, L-lysine, L-arginine, L-ornithine, γ-aminobutyric acid, 5-aminopentanoic acid, 7-aminoheptanoic acid, glycylglycine, triglycine, N-α-acetyl-L-arginine, betaine, sulfanilic acid, pharmaceutically acceptable salts thereof, and combinations of these. 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-aminopentanoic 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.

[0063] 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-amino 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-arginine and glycylglycine; L-lysine and triglycine; L-arginine and L-ornithine; L-arginine and gamma-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 gamma-aminobutyric acid; L-ornithine and 5-aminovaleric acid; L-ornithine and 7-aminoheptanoic acid; L-ornithine and glycylglycine glycine; L-ornithine and triglycine; gamma-aminobutyric acid and 5-aminovaleric acid; gamma-aminobutyric acid and 7-aminoheptanoic acid; gamma-aminobutyric acid and glycylglycine; gamma-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.

[0064] In a more preferred embodiment, the composition comprises tranexamic acid and ε-aminocaproic acid.

[0065] In another preferred embodiment, the composition comprises three lysine mimetics.In a more preferred embodiment, the composition of the invention comprises ε-aminocaproic acid, tranexamic acid and L-arginine.

[0066] For example, the lysine mimics can be selected from tranexamic acid, ε-aminocaproic acid, L-arginine, its pharmaceutically acceptable salts, and these combinations. In another preferred embodiment, the lysine mimics is tranexamic acid. In another preferred embodiment, the lysine mimics is ε-aminocaproic acid. In another preferred embodiment, the lysine mimics is L-arginine.

[0067] In certain embodiments, the lysine mimetic is a compound of formula (i) or a pharmaceutically acceptable salt thereof:

[0068]

[0069] in:

[0070] Each of R1 to R5 and R 1’ To R 5’ Each of them is the same or different and is independently selected from 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 alkylsulfonyl, C1-C5 haloalkylsulfonyl, C1-C6 alkylsulfonamido, C1-C6 haloalkylsulfonamido, C1-C6 bis(alkyl)sulfonamido, C1-C5 bis(haloalkyl)sulfonamido, 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 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.

[0071] In other embodiments, the lysine mimetic may be a compound of formula (ii) or a pharmaceutically acceptable salt thereof:

[0072]

[0073] in:

[0074] R 11 To R 15 Each of the R 11’ To R 15’Each of them is the same or different and is independently selected from 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 alkylsulfonyl, C1-C5 haloalkylsulfonyl, C1-C6 alkylsulfonamido, C1-C6 haloalkylsulfonamido, C1-C6 bis(alkyl)sulfonamido, C1-C5 bis(haloalkyl)sulfonamido, 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 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.

[0075] 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 another embodiment, 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 some specific 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 other 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.

[0076] The composition of any of the preceding claims, wherein the lysine mimetic is present at a concentration of about 0.1 μM to about 0.6 M. In some embodiments, the lysine mimetic is present at a concentration of about 10 μM to about 0.4 M. In other embodiments, the lysine mimetic is present at a concentration of about 100 μM to about 0.2 M. In other embodiments, the lysine mimetic is present at a concentration of about 1 mM to about 0.1 M. For example, the lysine mimetic may be present at a concentration of about 10 μM to about 1 mM. In some specific embodiments, the lysine mimetic may be present at a concentration of 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.

[0077] In some embodiments, the lysine mimetic can be selected from tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof and the lysine mimetic can be present at a concentration of about 10 μM to about 100 mM. In other embodiments, the lysine mimetic can be selected from tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof and the lysine mimetic can be present at a concentration of about 50 μM to about 1 mM.

[0078] In another preferred embodiment, the lysine mimic is tranexamic acid and exists at a concentration of 10 μM. In another preferred embodiment, the lysine mimic is tranexamic acid and exists at a concentration of 25 μM. In another preferred embodiment, the lysine mimic is tranexamic acid and exists at a concentration of 50 μM. In another preferred embodiment, the lysine mimic is tranexamic acid and exists at a concentration of 0.1 mM. In another preferred embodiment, the lysine mimic is L-arginine and exists at a concentration of 0.75 mM. In another preferred embodiment, the lysine mimic is L-arginine and exists at a concentration of 1.5 mM. In another preferred embodiment, the lysine mimic is L-arginine and exists at a concentration of 3 mM. In another preferred embodiment, the lysine mimic is L-arginine and exists at a concentration of 6 mM. In another preferred embodiment, the lysine mimic is ε-aminocaproic acid and exists at a concentration of 0.1 mM.

[0079] 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.

[0080] In some embodiments of the compositions of the present invention, the molar ratio of plasmin:lysine mimics can be from about 1:1 to about 1:1,000. For example, the molar ratio of plasmin:lysine mimics can be from about 1:1 to about 1:500. In other embodiments, the molar ratio of plasmin:lysine mimics can be from about 1:1 to about 1:100. In certain embodiments, the molar ratio of plasmin:lysine mimics can be from about 1:1 to about 1:50. In some specific embodiments, the molar ratio of plasmin:lysine mimics can be from about 1:10 to about 1:10,000. In other embodiments, the molar ratio of plasmin:lysine mimics can be from about 1:10 to about 1:1,000. For example, the molar ratio of plasmin:lysine mimics can be from about 1:10 to about 1:100. In other embodiments, the molar ratio of plasmin:lysine mimics can be from about 1:10 to about 1:50. In some specific embodiments, the molar ratio of plasmin: lysine mimics can be from about 1:30 to about 1:10,000. In other embodiments, the molar ratio of plasmin: lysine mimics can be from about 1:30 to about 1:1,000. For example, the molar ratio of plasmin: lysine mimics can be from about 1:30 to about 1:100. In other embodiments, the molar ratio of plasmin: lysine mimics can be from about 1:30 to about 1:50. In other embodiments, the molar ratio of plasmin: lysine mimics can be from about 1:50 to about 1:10,000. In other embodiments, the molar ratio of plasmin: lysine mimics can be from about 1:50 to about 1:1,000. For example, the molar ratio of plasmin: lysine mimics can be from about 1:50 to about 1:100. In a preferred embodiment, the molar ratio of plasmin: lysine mimics is 1:50. In another preferred embodiment, the molar ratio of plasmin: lysine mimics is 1:10.

[0081] In some embodiments, the lysine mimetic can be selected from tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof and the molar ratio of plasmin:lysine mimetic can be from about 1:1 to about 1:1,000. For example, the molar ratio of plasmin:lysine mimetic can be from about 1:1 to about 1:500. In other embodiments, the molar ratio of plasmin:lysine mimetic can be from about 1:1 to about 1:100. In certain embodiments, the molar ratio of plasmin:lysine mimetic can be from about 1:1 to about 1:50.

[0082] In other embodiments, the lysine mimetic can be selected from tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof and the molar ratio of plasmin:lysine mimetic can be from about 1:10 to about 1:10,000. In other embodiments, the molar ratio of plasmin:lysine mimetic can be from about 1:10 to about 1:1,000. For example, the molar ratio of plasmin:lysine mimetic can be from about 1:10 to about 1:100. In other embodiments, the molar ratio of plasmin:lysine mimetic can be from about 1:10 to about 1:50.

[0083] In certain embodiments, the lysine mimetic can be selected from tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof and the molar ratio of plasmin:lysine mimetic can be from about 1:30 to about 1:10,000. In other embodiments, the molar ratio of plasmin:lysine mimetic can be from about 1:30 to about 1:1,000. For example, the molar ratio of plasmin:lysine mimetic can be from about 1:30 to about 1:100. In other embodiments, the molar ratio of plasmin:lysine mimetic can be from about 1:30 to about 1:50.

[0084] In other embodiments, the lysine mimetic can be selected from tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations thereof and the molar ratio of plasmin:lysine mimetic can be from about 1:30 to about 1:1,000. In other embodiments, the molar ratio of plasmin:lysine mimetic can be from about 1:30 to about 1:100. For example, the molar ratio of plasmin:lysine mimetic can be from about 1:30 to about 1:70. In other embodiments, the molar ratio of plasmin:lysine mimetic can be from about 1:50. In other embodiments, the molar ratio of plasmin:lysine mimetic can be from about 1:10. In some embodiments, the osmotic concentration of the pharmaceutically acceptable organ perfusate can be from about 200 to 400mOsm / kg, for example, from about 250 to about 350mOsm / kg, for example, from about 270 to about 330mOsm / kg. In certain embodiments, the osmolarity of a pharmaceutically acceptable organ perfusate may range from about 275 to about 300 mOsm / kg. The osmolarity of a solution may be measured using an analytical instrument called an osmometer (e.g., a Precision Systems Osmette TM or Advanced Instruments ) can be easily determined.

[0085] In some embodiments of the compositions of the present invention, the pharmaceutically acceptable organ preservation solution may be STEEN TM Solution or its derivatives. STEEN TMThe composition of the solution is described, for example, in US Pat. No. 7,255,983 B2.

[0086] In other embodiments of the composition of the present invention, the pharmaceutically acceptable organ preservation solution can be a cold storage solution selected from the group consisting of: Euro Collins (EC) solution (Annual Report Eurotransplant International Foundation. Leiden, The Netherlands: Eurotransplant International Foundation, 1976); University of Wisconsin (UW) solution Also known as Beizer (Belzer FO and Southard JH. Transplantation, 1988; 45: 673, US 4879283); Solution, also known as HTK (histidine-tryptophan-ketoglutarate) solution or Bretschneider solution (Bretschneider HJ. Thorac Cardiovasc Surg, 1980; 28: 295); Solution (CEL), Menasche P et al. Eur J Cardiothorac Surg, 1994; 8: 207); Institut Georges López solution (IGL-1) (Ben Abdennebi H et al. Transpl Int 2002; 15: 348); Marshall solution (HOC or hypertonic citrate) (Southard JH, Belzer FO. Annu Rev Med 1995; 46: 235); Solution (Müller C et al. Transplantation, 1999; 68: 1139-43); 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 Let al. World J Gastroenterol, 2007; 13: 3684-3691); sucrose phosphate buffer (Lam FT et al. Transplantation, 1989; 47: 767); St. Thomas Hospital solution 1 and 2 (STH-1, STH-2) (Michel P et al. J Heart Lung Transplant, 2002; 21: 1030-9); Lyon preservation solution (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: 1030-9); KPS-1 solution and combinations thereof. In one embodiment, the pharmaceutically acceptable organ preservation solution may be a cold storage solution selected from the following: University of Wisconsin (UW) solution, Solution, Solutions and combinations thereof. For example, a pharmaceutically acceptable organ preservation solution may be In another preferred embodiment, the organ preservation solution is HTK solution. In another preferred embodiment, the organ preservation solution is KPS-1 solution. In another preferred embodiment, the organ preservation solution is Beizer (UW) solution.

[0087] The composition of the solutions disclosed herein is widely known in the art. Okada et al Surg Today (2012) 42: 152–156 discloses the composition of EP-TU and EC solutions. Guibert et al Transfus Med Hemother 2011; 38: 125–142 discloses the composition of EC, UW (Viaspan), Celsior, Custodial and IGL-1. Muhlbacher et al Transplantation Proceedings, 31, 2069–2070 (1999) discloses the composition of EC, UW, HTK and Celsior. Ferng et al Journal of Cardiothoracic Surgery (2017) 12: 7 discloses the composition of Celsior, Perfadex and Somah solutions. The document Chen et al Yonsei Med J Vol 45, No 6 (2004) p1107 discloses ET-Kyoto, EC and UW solutions. The document Jing et al Acta Pharmacologica Sinica (2018) 39: 845–857 discloses the composition of EC, UW, HTK / Custodial, Celsior, Perfadex, EP-TU and ET-Kyoto solutions.

[0088] In some embodiments of the compositions of the present invention, the pharmaceutically acceptable organ preservation solution may include at least one additive selected from the group consisting of physiologically acceptable salts, buffers, colloidal carbohydrates, antioxidants, and combinations thereof. The solution may additionally include a source of energy and nutrients for the organ. Suitable substances include, but are not limited to, glucose, amino acids, vitamins, lipids, and combinations thereof.

[0089] In some embodiments, the physiologically acceptable salt may include ions selected from the group consisting of sodium ions, potassium ions, calcium ions, magnesium ions, hydrogen carbonate ions, bicarbonate ions, chloride ions, and combinations thereof. For example, the physiologically acceptable salt may be selected from sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium dihydrogen phosphate, sodium bicarbonate, sodium hydroxide, and combinations thereof.

[0090] In certain embodiments, the buffer can 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 can be a combination of histidine and tryptophan. In some specific embodiments, the buffer can comprise a phosphate-based buffer in combination with one of the amino acids listed above. In another preferred embodiment, the buffer comprises a phosphate-based buffer.

[0091] In other embodiments, the colloidal carbohydrate can be selected from a dextran compound, mannitol, and a combination thereof. For example, the dextran can be dextran 40.

[0092] In other embodiments, the antioxidant may be selected from alpha-ketoglutarate, glutathione, and combinations thereof.

[0093] In certain embodiments of the compositions of the present invention, the pharmaceutically acceptable organ preservation solution may comprise an additive selected from the group consisting of albumin, a dextran compound, a physiologically acceptable salt, and combinations thereof. In a preferred embodiment, the additive is heparin. In another preferred embodiment, the additive is zosyn. 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.

[0094] In some embodiments, the concentration of albumin can 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 can be from about 5 mg / mL to about 30 mg / mL. In other embodiments, the concentration of albumin can 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, about 90 mg / mL, or about 100 mg / mL.

[0095] In certain embodiments, 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. For example, the dextran compound may comprise a dextran having a molecular weight of about 40 kDa.

[0096] In certain embodiments, the concentration of the dextran compound can 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 can be about 5 mg / mL.

[0097] In some embodiments, the physiologically acceptable salt may include ions selected from the group consisting of sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, bicarbonate ions, chloride ions, and combinations thereof. For example, the physiologically acceptable salt may be selected from sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium dihydrogen phosphate, sodium bicarbonate, sodium hydroxide, and combinations thereof.

[0098] In some particularly preferred embodiments, the physiologically acceptable salt is present in a concentration range that is considered normal in human blood, human serum or human plasma. For example, the physiologically acceptable salt comprises an ion selected from the following: a sodium ion at a concentration of about 135 mM to about 150 mM, a potassium ion at a concentration of about 3 mM to about 5 mM, a chloride ion at a concentration of about 95 mM to 110 mM, a hydrogen carbonate ion at a concentration of 20 mM to 30 mM, a bicarbonate ion at a concentration of 20 mM to 30 mM, a calcium ion at a concentration of 2 mM to 3 mM, a phosphate ion at a concentration of 1 mM to 1.5 mM, and combinations thereof.

[0099] In certain embodiments, the physiologically acceptable salt is selected from the following: sodium chloride at a concentration of 75mM to 150mM, potassium chloride at a concentration of 0.4mM to 5mM, calcium chloride at a concentration of 1mM to 2mM, magnesium sulfate at a concentration of 1mM to 2mM, sodium bicarbonate at a concentration of 10mM to 20mM, and combinations thereof.

[0100] Those skilled in the art will appreciate that the specific embodiments disclosed in paragraphs

[0013] to

[0045] should not be read in isolation, and that the present description is intended to disclose these embodiments in combination with other embodiments rather than individually.

[0101] Method of the present invention

[0102] In a second aspect, the invention provides a method for preserving or conditioning an organ prior to transplantation, the method comprising contacting the organ with a composition of the invention.

[0103] As used herein, "organ" refers to any part of an organism that is usually self-contained and has a specific vital 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 liver.

[0104] In one embodiment, an organ is perfused with a composition of the invention (as part of an ex vivo organ preservation or conditioning procedure).

[0105] In some embodiments, the organ is isolated. In some embodiments, the composition is contacted with the organ by washing, soaking, perfusion, or a combination thereof. In another embodiment, the composition is contacted with the organ by in vivo perfusion in a donor. In another embodiment, the composition is contacted with the organ by perfusion when the organ is isolated from the donor. In another embodiment, the organ is from a deceased donor.

[0106] In another embodiment, the organ is contacted with the composition of the present invention at a lower temperature (as part of a static cold storage process or a sub-normothermic perfusion process). For example, the organ may be contacted with the composition of the present invention or perfused with the composition of the present invention at a temperature selected from about 20 to 37°C and about 0 to 10°C. In one embodiment, the organ may be contacted with the composition of the present invention or perfused with the composition of the present invention at a temperature of about 0 to 10°C. In certain embodiments, the organ may be contacted with the composition of the present invention at a temperature of about 0°C. Advantageously, in the stable formulations of the present invention, plasmin exhibits proteolytic activity at a temperature of about 0 to 10°C and even at 0°C.

[0107] Those skilled in the art will appreciate that some of the previously disclosed specific embodiments should not be interpreted in isolation, and that this specification is intended to disclose these embodiments in combination with other embodiments rather than individually. For example, each of the embodiments disclosed in this specification should be interpreted as being explicitly combined with each of the embodiments disclosed in other parts of this specification or any arrangement of 2 or more embodiments disclosed herein.

[0108] All terms and embodiments previously disclosed with respect to the compositions of the present invention are equally applicable to the methods of the present invention.

[0109] Example

[0110] It should be apparent to one of ordinary skill in the art that the embodiments disclosed below represent only generalized embodiments and that other arrangements and methods of reproducing the present invention are possible and encompassed by the present invention.

[0111] Example 1: In vitro solubility and activity studies

[0112] Plasmin is stable in acidic solutions, but once it is added to neutral solutions, the stabilizing effect of the acidic pH diminishes and plasmin begins to precipitate out of solution. Full-length plasmin solution (11 mg / ml, pH 3.4) was added to human plasma at room temperature with varying concentrations of tranexamic acid (TXA) at a ratio of 1:10, and the resulting turbidity was measured in a microtiter plate by absorbance at 405 nm.

[0113] according to Figure 2A It is evident that turbidity / plasmin precipitation decreases with increasing TXA concentrations. In particular, once plasmin is added to the test plasma at a fully diluted concentration of 1 mg / mL, TXA concentrations of 0.5 mM and above significantly reduce the level of plasmin precipitation.

[0114] according to Figure 2B, it can be concluded that plasmin formulated with tranexamic acid or L-arginine is soluble in a variety of commonly used organ preservation and conditioning solutions. Plasmin formulated as 10 mg / mL was directly diluted with tranexamic acid (1mM, 2.5mM, 5mM or 10mM TXA) or L-arginine (75mM, 150mM, 300mM or 600mM Arg) at 1:100 into HTK, KPS-1 or Belzer University of Wisconsin (UW) organ preservation solutions without other additives. For each figure, the turbidity measurement results at 405nm are shown for the following: single organ preservation solutions without additives, soluble plasmin of 10 mg / mL without excipients, and plasmin diluted to the organ preservation solution at 1:100 plus specified excipients. After the specified incubation time, the turbidity of each preparation diluted in the preservation and conditioning solution was tested. Diluted plasmin formulated with the indicated excipients remained soluble in all cases and at all time points and did not increase in turbidity when stored at 4°C for at least 24 hours.

[0115] The full-length plasmin is prepared according to the procedures / methods known to those skilled in the art and described in detail in Section 18.3, Novokhatny, V. et al., Acid Stabilised Plasmin as a Novel Direct-Acting Thrombolytic, Ch 18, pp. 259 to 271, Production of Plasma Proteins for Therapeutic Use, Eds. J. Bertolini, et al., Wiley, 2013 [Print ISBN: 9780470924310 | Online ISBN: 9781118356807], the contents of which are incorporated herein by reference. The "full-length plasmin" of this specification refers to a mixture of Lys-plasmin and Glu-plasmin, and does not include Figure 1 The mutants outlined in . Figure 1 The truncated plasmin mutants outlined in can be recombinantly prepared using preparation methods within the general knowledge of the skilled person and purified according to the procedures disclosed in Novokhatny et al. (supra).

[0116] S-2403 is a commercially available plasmin-specific chromogenic substrate. This substrate was used to measure plasmin activity in the presence of TXA, as Figure 3As shown in . In the presence of different TXA concentrations (0.001mM to 100mM), the proteolytic activity of plasmin at a concentration of 1mg / mL showed unchanged kinetics. Therefore, it was concluded that TXA can be formulated with plasmin without any deleterious effect on the proteolytic activity of plasmin.

[0117] To evaluate the compatibility of plasmin as an additive in refrigerated solutions, the activity of plasmin at a concentration of 1 mg / mL was evaluated at multiple time points when stored at 0°C. Figure 4A , from which it is clearly seen that plasmin at a concentration of 1 mg / mL exhibits unchanged kinetics when stored at 0° C. over a period of 24 hours.

[0118] The results in Figure 4b show that plasmin formulated in tranexamic acid and directly diluted into a variety of commonly used organ preservation solutions maintains plasmin activity for at least 24 hours at temperatures commonly used in organ transplant preservation and conditioning methods. 10 mg / mL plasmin formulated with 5 mM tranexamic acid was directly diluted to HTK, KPS-1 or Belzer University of Wisconsin (UW) organ preservation solutions without other additives at 1:100 (final concentration: 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 (striped bars) for a period of 24 hours. The activity at each specified time was assessed using a commercial S-2403 plasmin-specific chromogenic substrate.

[0119] The results in Figure 4c clearly show that plasmin formulated in tranexamic acid or ε-aminocaproic acid and directly diluted into a variety of commonly used organ preservation solutions maintains plasmin activity for at least 1 month when stored at 5°C. Plasmin formulated at 10 mg / mL was diluted directly into HTK, KPS-1 or Belzer University of Wisconsin (UW) organ preservation solutions without other additives at 1:100 with 10 mM ε-aminocaproic acid (eACA), 1 mM tranexamic acid (TXA), 5 mM tranexamic acid (TXA) or 10 mM tranexamic acid (TXA) and maintained at 5°C for 1 month. When formulated with these excipients and diluted into organ preservation and conditioning solutions, significant plasmin activity was maintained. Activity at each indicated time was assessed using a commercial S-403 plasmin-specific chromogenic substrate.

[0120] Example 2: Rat liver transplantation study

[0121] Rat liver recoveryMale Lewis rats (200 to 300 grams) were used as donors. General anesthesia was induced using 5% isoflurane during the operation and maintained at 1% to 2% by a nose cone. The abdomen was opened using a cruciate incision. The left phrenic vein was dissected, ligated and divided. The posterior caudate lobe was dissected from the stomach and spleen. The common bile duct was dissected and cannulated using a 24G angiocath tied with a 5-0 silk tie. The hepatic artery was dissected near the porta hepatis and divided between the knots. The portal vein was dissected and the two nearest tributaries were ligated and divided between the knots. Heparin (250 units) was injected systemically through the inferior vena cava and allowed to circulate for 3 minutes. The portal vein was clamped for 30 minutes. After the in situ ischemic period, the portal vein was cannulated and fixed with 5-0 silk. Then 50 ml of ice-cold HTK solution ( The liver was flushed with scissors (no additives). During flushing, the lower IVC was quickly opened with scissors to drain the blood effluent from the liver. Once flushing was completed, the liver was explanted by dissecting in a clockwise manner, dividing the vessels and ligaments starting from the upper IVC, the left and right coronary ligaments, and the posterior attachment of the spine (only in non-grafts).

[0122] Refrigerated group . After the liver is removed, the liver is kept at room temperature (original group; in the second group, this is done on ice), and a vascular cuff is inserted into the portal vein and the infrahepatic inferior vena cava. The invagination process takes 10 to 15 minutes. After this, a 200 ml back table flush is performed using HTK containing albumin (control) or plasmin: tranexamic acid (1:50 molar ratio, 0.1 mg / mL plasmin). The liver is then stored on ice for 1 hour. After the cold storage period, a final pre-transplant flush consisting of 100 ml HTK (no additive) is performed before transplantation.

[0123] Machine perfusion group . After the liver is removed, sub-normothermic machine perfusion is started at room temperature. DEVOL solution perfusate is used, and its matrix is ​​composed of Duosol 4555 and 4% human albumin. Additives include heparin, zosyn, dexamethasone, bicarbonate and calcium gluconate. In the experimental group, plasmin: tranexamic acid (1:50 molar ratio) is added to 200ml perfusate to produce a final concentration of 0.1mg / mL plasmin. After machine perfusion for 1 hour, vascular cuffs are inserted into the portal vein and infrahepatic inferior vena cava (original group; in the second group, this is done on ice) at room temperature. Before transplantation, 100ml HTK (without additives) is used for final pre-transplantation flushing.

[0124] Liver transplant process The liver was transplanted in an orthotopic manner as previously described by Abraham et al. (Abraham N et al.. Front Med (Lausanne). 2022; 9:804834. doi: 10.3389 / fmed.2022.804834), the contents of which are incorporated herein by reference.

[0125] Study End Points Subject survival was recorded on postoperative day 1. Surviving subjects were sacrificed under general anesthesia. Midline laparotomy was performed and ascites volume and ascites hematocrit were recorded. Exsanguination was performed by collecting blood from the vena cava, which was centrifuged and used to measure liver function tests. Liver grafts and bile ducts were biopsied and preserved in formalin for histological examination.

[0126] result After 1 hour of cold storage in the presence of plasmin / tranexamic acid, a significant survival benefit was observed at 24 hours after transplantation (80% vs 14.2% survival, P=0.0293), see Figure 5 The low number of survivors in the control group precluded meaningful comparisons of the levels of bile duct injury.

[0127] Liver function panel assessments in the machine perfused group showed signs of improved function after postoperative day 1 (POD1). After 1 hour of machine perfusion, there was a significant decrease in 24-hour serum alkaline phosphatase with the administration of plasmin / tranexamic acid (mean 216.6 vs 56 U / L, P = 0.0006). In addition, at 24 hours, glucose homeostasis was improved in recipients of plasmin-treated grafts (mean 163 vs 82.3 mg / dl, P = 0.0067). These results were Figure 6 Draw in.

[0128] No significant bleeding complications were observed after transplantation in either organ preservation mode.

[0129] sequence

[0130] The sequences mentioned in the foregoing text are summarized below in fasta format. In the case where there are differences between the sequences listed herein and the corresponding sequences in the attached sequence listing, for the purpose of error correction, the sequences listed herein shall be the prevailing sequences.

[0131] SEQ ID NO: 1 Human plasminogen protein sequence

[0132]

Claims

1. A stable composition for organ conditioning, comprising: Plasmin, Lysine mimetics, and A pharmaceutically acceptable organ preservation solution having a pH of about 6 to about 8, The molar ratio of plasmin:lysine mimetic is about 1:1 to about 1:10,000.

2. The composition according to claim 1, wherein the plasmin is selected from plasmin, a functional mutant thereof, a functional fragment thereof, or a combination thereof.

3. The composition according to claim 2, wherein the functional fragment of plasmin is selected from the group consisting of midi-plasmin, mini-plasmin, microplasmin and delta-plasmin.

4. The composition according to claim 2, wherein the plasmin is selected from the group consisting of Glu-plasmin and Lys-plasmin.

5. The composition of any one of claims 1 to 4, wherein the lysine mimetic is selected from tranexamic acid, ε-aminocaproic acid, L-lysine, L-arginine, L-ornithine, γ-aminobutyric acid, 5-aminopentanoic acid, 7-aminoheptanoic acid, glycylglycine, triglycine, N-α-acetyl-L-arginine, betaine, sulfanilic acid, pharmaceutically acceptable salts thereof, and combinations of these.

6. The composition according to any one of claims 1 to 5, wherein the lysine mimetic is selected from tranexamic acid, ε-aminocaproic acid, L-arginine, pharmaceutically acceptable salts thereof, and combinations of these.

7. The composition according to any one of claims 1 to 6, wherein the lysine mimetic is a compound of formula (i) or a pharmaceutically acceptable salt thereof: in: Each of R1 to R5 and R 1’ To R 5’ Each of them is the same or different and is independently selected from 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 alkylsulfonyl, C1-C5 haloalkylsulfonyl, C1-C6 alkylsulfonamido, C1-C6 haloalkylsulfonamido, C1-C6 bis(alkyl)sulfonamido, C1-C5 bis(haloalkyl)sulfonamido, 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 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.

8. The composition according to any one of claims 1 to 6, wherein the lysine mimetic is a compound of formula (ii) or a pharmaceutically acceptable salt thereof: in: R 11 To R 15 Each of the R 11’ To R 15’ Each of them is the same or different and is independently selected from 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 alkylsulfonyl, C1-C5 haloalkylsulfonyl, C1-C6 alkylsulfonamido, C1-C6 haloalkylsulfonamido, C1-C6 bis(alkyl)sulfonamido, C1-C5 bis(haloalkyl)sulfonamido, 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 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.

9. The composition of any of the preceding claims, wherein plasmin is present at a concentration of about 0.01 mg / mL to about 50 mg / mL.

10. The composition of any one of the preceding claims, wherein the lysine mimetic is present at a concentration of about 0.1 μM to about 0.6M.

11. The composition of any one of the preceding claims, wherein the pharmaceutically acceptable organ perfusate has an osmolarity of about 200 to about 400 mOsm / Kg.

12. The composition of any of the preceding claims, wherein the pharmaceutically acceptable organ perfusate has an osmolarity of about 250 to about 350 mOsm / Kg.

13. The composition of any one of the preceding claims, wherein the osmolarity of the pharmaceutically acceptable organ perfusate is about 270 to 330 mOsm / Kg.

14. The composition according to any one of the preceding claims, wherein the pharmaceutically acceptable organ preservation solution comprises an additive selected from the group consisting of albumin, a dextran compound, a physiologically acceptable salt, and combinations thereof.

15. The composition of claim 14, wherein the albumin is at a concentration of about 1 mg / mL to about 100 mg / mL, about 5 mg / mL to about 85 mg / mL, about 10 mg / mL to about 70 mg / mL, about 20 mg / mL to about 60 mg / mL, about 5 mg / mL to about 30 mg / mL, about 60 mg / mL to about 90 mg / mL, about 50 mg / mL to about 100 mg / mL, about 55 mg / mL to about 85 mg / mL, about 60 mg / mL to about 80 mg / mL, about 65 mg / mL to about 85 mg / mL, 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, about 90 mg / mL, or about 100 mg / mL.

16. The composition of any one of claims 14 to 15, 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.

17. The composition of any one of claims 14 to 16, wherein the dextran compound comprises dextran having a molecular weight of about 40 kDa.

18. The composition of any one of claims 14 to 17, 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.

19. The composition of any one of claims 14 to 18, wherein the concentration of the dextran compound is about 5 mg / mL.

20. The composition of any one of claims 14 to 19, wherein the physiologically acceptable salt comprises ions selected from the group consisting of sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, bicarbonate ions, chloride ions, and combinations thereof.

21. The composition of any one of claims 14 to 20, wherein the physiologically acceptable salt is selected from sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium dihydrogen phosphate, sodium bicarbonate, sodium hydroxide, and combinations thereof.

22. The composition of any one of claims 14 to 21, wherein the physiologically acceptable salt is present in a concentration range considered normal in human blood, human serum or human plasma.

23. according to the composition described in any one of claims 14 to 22, wherein the physiologically acceptable salt comprises ions selected from the group consisting of sodium ions at a concentration of about 135mM to about 150mM, potassium ions at a concentration of about 3mM to about 5mM, chloride ions at a concentration of about 95mM to about 110mM, bicarbonate ions at a concentration of about 20mM to about 30mM, calcium ions at a concentration of about 2mM to about 3mM, phosphate ions at a concentration of about 1mM to about 1.5mM, and combinations thereof.

24. The composition of any one of claims 14 to 23, wherein the physiologically acceptable salt is selected from the group consisting of sodium chloride at a concentration of about 75 mM to about 150 mM, potassium chloride at a concentration of about 0.4 mM to about 5 mM, calcium chloride at a concentration of about 1 mM to about 2 mM, magnesium sulfate at a concentration of about 1 mM to about 2 mM, sodium bicarbonate at a concentration of about 10 mM to about 20 mM, and combinations thereof.

25. The composition according to any one of claims 1 to 24, 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.

26. The composition of claim 25, wherein the physiologically acceptable salt comprises ions selected from the group consisting of sodium ions, potassium ions, calcium ions, magnesium ions, bicarbonate ions, bicarbonate ions, chloride ions, and combinations thereof.

27. The composition of any one of claims 25 to 26, 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.

28. A composition according to any one of claims 25 to 27, wherein the buffer comprises a phosphate-based buffer.

29. The composition according to any one of claims 25 to 28, wherein the colloidal carbohydrate may be selected from the group consisting of dextran compounds, mannitol, and combinations thereof.

30. The composition of any one of claims 25 to 29, wherein the antioxidant may be selected from alpha-ketoglutarate, glutathione, and combinations thereof.

31. The composition of any one of claims 1 to 30, wherein the pharmaceutically acceptable organ preservation solution is a STEEN solution.

32. The composition of any one of claims 1 to 30, wherein the pharmaceutically acceptable organ preservation solution is a cold storage 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), EP-TU solution, ET-Kyoto solution, and combinations thereof.

33. The composition of claim 32, wherein the pharmaceutically acceptable organ preservation solution is a HTK / Custodiol solution.

34. The composition of claim 32, wherein the pharmaceutically acceptable organ preservation solution is KPS-1.

35. The composition of claim 32, wherein the pharmaceutically acceptable organ preservation solution is Belzer University of Wisconsin (UW).

36. A method of preserving or conditioning an organ prior to transplantation, the method comprising contacting the organ with a composition as claimed in any one of claims 1 to 35.

37. The method of claim 36, wherein the organ is perfused with the composition of any one of claims 1 to 35.

38. The method of claim 37, wherein the organ is isolated.

39. The method of any one of claims 37 or 39, wherein the composition is contacted with the organ by washing, soaking, perfusion, or a combination thereof.

40. The method of claim 36, wherein the composition is contacted with the organ by in vivo perfusion in the donor.

41. The method of claim 36, wherein the composition is contacted with the organ by perfusion while the organ is separated from the donor.

42. The method of any one of claims 36 to 41, wherein the organ is from a deceased donor.

43. The method of any one of claims 36 to 42, wherein the organ is contacted with or perfused with the composition of the invention at a temperature selected from 20°C to 37°C, 0°C to 10°C, and about 0°C.

44. The method of any one of claims 36 to 43, wherein the organ is liver, kidney, lung or heart.

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