WO2011148024A2 - Composición para la preservación fría de órganos - Google Patents
Composición para la preservación fría de órganos Download PDFInfo
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- WO2011148024A2 WO2011148024A2 PCT/ES2011/070375 ES2011070375W WO2011148024A2 WO 2011148024 A2 WO2011148024 A2 WO 2011148024A2 ES 2011070375 W ES2011070375 W ES 2011070375W WO 2011148024 A2 WO2011148024 A2 WO 2011148024A2
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- preservation
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Definitions
- the present invention relates to a composition for the cold preservation of organs for transplantation and to the uses, methods and kits for the preparation thereof.
- Organ transplantation is widely used for organs such as the heart, lung, pancreas, intestine (colon) and in particular kidney and liver.
- organs such as the heart, lung, pancreas, intestine (colon) and in particular kidney and liver.
- the growing demand for organs and a shortage of donors have led to a growing waiting list and a greater need to use sub-optimal donor organs.
- the organs obtained for transplantation must be stored and transported between hospitals. Time is needed for histo-compatibility tests between donor and recipient, and for the preparation of the recipient patient.
- the length of time that organs and tissues can be maintained outside the body varies, depending on the organ, the age and health of the donor, the method of conservation and temperature.
- cold preservation or preservation using special preservation solutions. These solutions are used in a first phase, prior to extraction, for an infusion of the organ (flushing) in order to remove and replace the donor's blood, while cooling it to 4 ° C.
- cold preservation solutions are used to store and preserve the extracted organ, also in hypothermia (around 4 ° C), until the moment of transplantation.
- Cold preservation seeks to conserve the organ by suppressing the metabolism and secondly avoiding and minimizing the alterations that cold ischemia itself could cause. Maathuis and cois.
- US4798824 discloses a cold preservation solution comprising gluconate and hydroxy ethyl starch.
- US4879283 discloses the University of Wisconsin solution (UW or Belzer solution), which comprises as differential components: lactobionate and raffinose, as waterproofing agents to reduce cell swelling; and hydroxy-ethyl starch, to prevent edema.
- UW or Belzer solution the University of Wisconsin solution
- lactobionate and raffinose as waterproofing agents to reduce cell swelling
- hydroxy-ethyl starch to prevent edema.
- cold preservation with current preservation solutions has important limitations. Tissue damage and the inflammatory response caused by cold preservation can increase immunogenicity and initiate rejection of the organ.
- cold ischemia contributed to an increase in the mortality rate due to renal failure and accelerated acute rejection (Salahudeen AK. Am J Physiol Renal Physiol.
- Cold ischemia is related to changes in osmoregulation, energy and aerobic metabolism. A reduction in ATPase-Na-K activity and ATP levels allows intracellular accumulation of sodium and water, causing cell swelling. Likewise, the lactic acid caused by glucose metabolism causes lysosomal instability and impaired mitochondrial function, causing swelling, activation of the mitochondrial apoptotic pathway and free radical production. On the other hand, once the transplant has been carried out, a new damage caused by reperfusion of the organ is added, possibly more sensitive due to the cold ischemia to which it has been subjected. This process of damage involves an increase in reactive oxygen free radicals, the inflammatory response and cytosolic calcium, as well as an activation of proteolytic enzymes, for example, caspases.
- the invention relates to a composition for cold preservation of organs comprising, together or separately,
- CT-1 cardiotrophin 1
- the invention relates to a method for the preparation of a cold organ preservation composition comprising adding cardiotrophin 1 or a functionally equivalent variant thereof to a cold organ preservation solution.
- the invention relates to the use of cardiotrophin 1 or a functionally equivalent variant thereof for the preparation of a composition for the protection and / or cold preservation of organs for transplantation.
- the invention relates to the use of a composition according to the invention for the protection and / or cold preservation of organs for transplantation.
- the invention relates to a composition for cold preservation of organs according to the invention for use in the protection and / or cold preservation of organs for transplantation.
- the invention also relates to a method for cold preservation of an organ comprising contacting said organ with a composition according to the invention.
- the invention relates to an isolated cold preserved organ obtained by a method of the invention.
- the invention relates to a kit for the preparation of a composition for cold preservation of organs comprising:
- B Renal levels of tumor necrosis factor alpha (TNFa), expressed in pg / mL.
- C Renal levels of inducible nitric oxide synthase (iNOS), expressed in arbitrary units.
- SOA free radical super-oxide anion
- TNFa tumor necrosis factor alpha
- iNOS inducible
- B Renal levels of tumor necrosis factor alpha (TNFa), expressed in pg / mL.
- C Renal levels of inducible nitric oxide synthase (iNOS), expressed in arbitrary units.
- SOA superoxide anion free radical
- TNFa tumor necrosis factor alpha
- iNOS inducible nitric oxide synth
- SOA superoxide anion
- TNFa tumor necrosis factor alpha
- FIG. 4 Study of the effect on the kidney of cold preservation with a UW solution containing 0.1 mg / L of CT-1 (UW [CT1]) and with a solution from the University of Wisconsin (UW) as a control, in an experimental model of homologous and orthotopic renal transplantation in rats.
- the pre-established reperfusion times for the different groups were respectively, on days t (d): 1, 3, 7 and 14.
- a group of simulated rats (Sim) was included. n> 15 transplants per group.
- SOA superoxide anion free radical
- FIG. 6 Comparative study of the effect of cold preservation (4 ° C) in the heart of Wistar rats of the University of Wisconsin solution (S / CT-1) and the University of Wisconsin solution with 0.2 mg / L of CT-1 (C / CT-1) at different times expressed in hours.
- n 5 animals per group.
- iNOS oxide synthase inducible nitric
- B Activation of the NFKB transcriptional factor measured by tissue levels of p65 NFKB, expressed in arbitrary units.
- C Tissue levels of the soluble form of the adhesion molecule ICAM-1 (sICAM-1), corrected by tubulin, expressed in arbitrary units.
- sICAM-1 Tissue levels of the soluble form of the adhesion molecule ICAM-1
- SOA superoxide anion free radical
- C) and I) Renal levels of inducible nitric oxide synthase (iNOS) at 24 and 48 hours, respectively, corrected by tubulin, expressed in arbitrary units.
- D) and J) Renal levels of the soluble form of the adhesion molecule ICAM-1 (ICAM-1) at 24 and 48 hours, respectively, corrected by tubulin, expressed in arbitrary units.
- NFKB pro-inflammatory signaling pathways
- NFKB pro-inflammatory cytokines
- IL-6 inflammatory cytokines
- VCAM-1, ICAM-1 proinflammatory adhesion molecules
- compositions of the invention are suitable for cold preservation of kidneys for transplantation, since they prevent tubular necrosis caused by ischemia reperfusion, thereby improving renal functionality (reduction of levels of serum creatinine and increased creatinine clearance) and survival (Figure 4).
- the invention relates to a composition for cold preservation of organs comprising, together or separately,
- CT-1 cardiotrophin 1
- cold organ preservation composition refers to a liquid composition, preferably sterile, containing cardiotrophin 1 (or a functionally equivalent variant of the same) and a cold organ preservation solution.
- the CT-1 or its functionally equivalent variant
- the composition of the invention comprises the two components (i) and (ii) formulated separately, which in this case must be previously combined before administration.
- compositions according to the invention are compositions suitable for cold preservation of organs.
- cold preservation of organs means the ability to conserve, protect, preserve and / or store organs in hypothermic conditions, minimizing cell damage in said organs, through the use of agents that produce a suppression of metabolism and prevent or counteract the alterations that cold ischemia itself can cause in the organ.
- cardiotrophin 1 and cold organ preservation solutions are suitable agents for cold organ preservation.
- CT-1 refers to a cytokine belonging to the interleukin 6 family, capable of binding and activating signaling mediated at least by the receptor complex LIFR consisting of the gpl30 / LIFRp heterodimer.
- CT-1 means the protein defined by the sequence of the NCBI database with access number NP 001321.1 corresponding to the sequence SEQ ID NO: 1, corresponding to the isoform 1 of human cardiotrophin; or the protein defined by the sequence of the NCBI database with access number NP 001 136016.1, corresponding to isoform 2 of human cardiotrophin.
- the CT-1 is a CT-1 of human origin, preferably of sequence SEQ ID NO: 1.
- the invention contemplates the use of functionally equivalent variants of CT-1.
- functionally equivalent variant of CT-1 is meant any molecule that shares with CT-1 one or more of the functions described in the present invention associated with CT-1, both in vitro and in vivo , and that has a minimum identity in the amino acid sequence.
- the CT-1 variants can be both natural and artificial.
- natural variant refers to all those variants of the human CT-1 mentioned above that appear naturally in other species, that is, the orthologs of CT-1.
- Such natural variants include, without limitation, the mouse CT-1, which corresponds to the sequence with access number Q541U3 / Q60753 in the NCBI database or to the isoform of 196 amino acids corresponding to the sequence with access number P83714 in the NCBI database; rat CT-1, which corresponds to the sequence with access number Q63086; the macaque CT-1, which corresponds to the predicted sequence with access number XP 001103315; the dog CT-1, which corresponds to the predicted sequence with access number XP 849072; the horse CT-1, which corresponds to the predicted sequence with access number XP 001915457; CT-1 of bovine origin, which corresponds to the predicted sequence with access number XP 592709; and chimpanzee CT-1, which corresponds to the predicted sequence with accession number XP 592709.
- Natural variants of CT-1 suitable for use in the present invention can also be derived from said sequences by insertion, replacement or deletion of one. or more amino acids and include natural alleles (such as the A92T variant of human CT-1), variants resulting from alternative processing and secreted and truncated forms that appear naturally.
- the CT-1 useful in the present invention can, therefore, be of natural sequence, when it comprises a polypeptide having the same amino acid sequence as nature-derived CT-1.
- Such natural sequence polypeptides can be isolated from nature or can be produced by recombinant and / or synthetic means.
- the CT-1 of the invention can be a recombinant protein obtained by the expression of a polynucleotide encoding CT-1 or a functionally equivalent variant thereof in a heterologous organism, such as a bacterium, yeast or insect cell or mammal.
- Said recombinant protein can be obtained as a fusion protein with an amino-terminal histidine tail that facilitates its subsequent purification.
- the expression and purification of said proteins can be carried out according to methods known to those skilled in the art and described in the state of the art.
- the CT-1 is of human origin and corresponds to the sequence SEQ ID NO: 1.
- the CT-1 is rat, preferably it is a fusion protein with an amino-terminal histidine tail, more preferably SEQ ID NO: 2.
- CT-1 may be a functionally equivalent artificial variant of CT-1 that can be obtained by recombinant and / or synthetic means.
- the variants of CT-1 contemplated in the present invention show at least some of the functions of CT-1 such as, without limitation:
- NFkB proinflammatory signaling pathways
- TNFa inflammatory cytokines
- iNOS inflammatory cytokines
- VCAM-1 proinflammatory adhesion molecules
- ICAM-1 proinflammatory adhesion molecules
- functionally equivalent variants of CT-1 contemplated in the context of the present invention include polypeptides that show at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, 95%, 97%, 99% similarity or identity with the different natural variants of CT-1 mentioned above.
- the degree of identity between two polypeptides is determined using computer-implemented algorithms and methods that are widely known to those skilled in the art.
- the identity between two amino acid sequences is preferably determined using the BLASTP algorithm (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., Et al., J., 1990, Mol Biol. 215: 403-410).
- Cardiotrophin 1 of the composition according to the invention is in a pharmacologically effective concentration.
- pharmaceutically effective concentration is meant that concentration capable of producing one or more of the effects listed above and attributable to CT-1, and can be determined by standard techniques based on the methods detailed in the present description.
- cardiotrophin 1 is in a concentration between 0.001 g / L and 5,000 g / L; preferably between 0.005 g / L and 1 000 g / L; more preferably between 0.010 g / L and 0.500 g / L; even more preferably between 0.08 g / L and 0.20 g / L; and even more preferably between 0.1 g / L and 0.2 g / L.
- cardiotrophin 1 is in a concentration between 0.001 mg / L and 5,000 mg / L; preferably between 0.005 mg / L and 1,000 mg / L; more preferably between 0.010 mg / L and 0.500 mg / L; even more preferably between 0.08 mg / L and 0.20 mg / L; and even more preferably between 0.1 mg / L and 0.2 mg / L.
- cold organ preservation solution refers to a solution suitable for preserving, preserving and storing organs for transplantation under hypothermia conditions, which contains substances that minimize the damages that occur in the organs both in the process of cold ischemia, while waiting to be transplanted to the recipient, as in the reperfusion process during the transplant.
- cold organ preservation solutions exhibit their protective properties under conditions of hypothermia.
- “Hypothermia conditions” means temperatures below the physiological temperature between 0 ° C and 25 ° C.
- the solution and cold preservation composition of organs of the invention are at a temperature between 0 ° C and 25 ° C, preferably between 0 ° C and 15 ° C, more preferably between 2 ° C and 7 ° C, even more preferably at about 4 ° C.
- Cold ischemia means, in the present invention, tissue damage and inflammatory response caused in the organ due to its cold preservation, that is, due to the hypothermia conditions to which it is subjected.
- the term “reperfusion”, in the context of the present invention refers to the restoration of blood flow in the organ after a period of cold ischemia.
- suppression of metabolism is meant, in the present invention, the action of stopping or minimizing the function and metabolic activity of the organ.
- the cold organ preservation solution and by extension the compositions of the invention, allow to preserve solid organs of any species, such as, without limitation, mammal (primate, cows, horses, pigs, sheep, goats, dogs, cats, rodents , rabbits, etc.), birds, but preferably of human origin, such as heart, lung, pancreas, small intestine, colon, kidney. Such organs may come from a living donor or from a corpse.
- the cold organ preservation solution and, by extension the composition of the invention is especially suitable for the cold preservation of kidneys for transplantation.
- the cold organ preservation solution comprises:
- the cold organ preservation solution has a pH between 6.5 and 8.5; and an osmolarity between 300 and 410 mOsm / L, both parameters being measured at 20 ° C.
- buffering agent is meant, in the context of the present invention, an agent capable of controlling the pH of the solution, thus avoiding severe acidosis that can contribute to the ischemic damage of the organ.
- Buffer agents suitable for The present invention are phosphate (monoacidic and diacidic), bicarbonate, sulfate, histidine, hisidine-HCl, HEPES, citrate and a combination thereof.
- the cold organ preservation solution comprises a buffering agent selected from the group of phosphate, bicarbonate, sulfate, histidine, histidine-HCl, HEPES, citrate and a combination thereof.
- the preservation solution comprises as a buffer agent a monoacidic phosphate and / or a diacidic phosphate and / or histidine-HCl.
- impervious agent and “waterproofing agent” are used interchangeably in the present invention and refer to those agents with osmotic capacity and relatively impervious to cell membranes capable of maintaining extracellular tonicity preventing the entry of water into the cells , so that they prevent and / or reduce cell swelling.
- Suitable waterproofing or waterproofing agents for the present invention are histidine, glucose, sucrose, mannitol, trehalose, gluconate, citrate, lactobionate, raffin s a and a combination thereof, such as a combination of lactobionate and raffinose; of lactobionate and mannitol; of trehalose and gluconate; gluconate and glucose; of gluconate and raffinose; of mannitol and citrate; of mannitol and histidine; of lactobionate, mannitol and histidine.
- the cold organ preservation solution comprises an impervious or waterproofing agent selected from the group of histidine, glucose, sucrose, mannitol, trehalose, gluconate, citrate, lactobionate, raffinose and a combination thereof; preferably selected from lactobionate, raffinose, mannitol and a combination thereof; more preferably it comprises lactobionate and mannitol; even more preferably it comprises lactobionate and raffinose; most preferably it comprises lactobionate.
- the cold organ preservation solution comprises an impervious agent that is selected from the group of lactobionate, raffinose and combinations of both.
- Electrolytes Suitable for use in the present invention are sodium, potassium, magnesium, calcium, chloride and a combination thereof.
- the cold organ preservation solution comprises an electrolyte selected from the group of sodium, potassium, magnesium, calcium, chloride and a combination thereof.
- the cold organ preservation solution comprises glucose as a waterproofing or waterproofing agent, and is preferably the Euro-Collins (EC) solution.
- Euro-Collins solution refers to a cold preservation solution of intracellular organs comprising high concentrations of glucose and potassium and a low concentration of sodium.
- the Euro-Collins solution was originally described by GM Collins in 1969 (Collins GM et al, 1969, Lancet, 2: 1219) and modified by the Eurotransplant Foundation in 1976 by removing magnesium (Annual Report Eurotransplant International Foundation.
- the typical composition of the Euro-Collins solution can be the following: 195 mM glucose, 15 mM K 2 HP0 4 , 43 mM KH 2 P0 4 , 10 mM NaHC0 3 , 15 mM chloride, 115 mM potassium and 10 mM sodium. .
- said composition may vary, so that in the context of the present invention any solution cataloged as such in the literature or commercially available under that designation will be considered as Euro-collins solution.
- the cold organ preservation solution is a solution where component (a) is selected from the group of phosphate, bicarbonate, sulfate, histidine, histidine-HCl, HEPES, citrate and a combination thereof. ; component (b) is selected from the group of histidine, glucose, sucrose, mannitol, threhalosa, gluconate, citrate, lactobionate, raffinose and a combination thereof; and / or component (c) is selected from the group of sodium, potassium, magnesium, calcium, chloride and a combination thereof.
- the parameters that characterize the cold preservation solution of organs of the invention are its pH and its osmolarity.
- pH refers to the measure of the acidity or alkalinity of a solution.
- the pH typically ranges from 0 to 14 in aqueous solution, the solutions being acidic with pH below 7 and alkaline those having pH greater than 7.
- the pH 7 indicates the neutrality of the solution, where the solvent is water.
- the determination of the pH of a solution can be done precisely by means of a potentiometer (or pH meter) and also approximately by means of indicators, by methods widely known in the state of the art. Since the pH value can vary with temperature, in the context of this invention the pH measurement is performed at 20 ° C.
- the cold preservation solutions of the invention have a pH measured at 20 ° C between 6.5 and 8.5; preferably between 7.0 and 7.5.
- osmolarity in the context of the present invention, is a measure of the total concentration of substances in solution, defined as the number of osmoles of solute per liter of solution, which indicates the possible variation of the osmotic pressure that is will produce in the cells when introducing the solution in the organism.
- the osmolarity can be calculated from the value of the osmolality, the latter measured by an osmometer by methods known to those skilled in the art. Since osmolarity is temperature dependent, in the context of the present invention the calculation of osmolarity is performed at 20 ° C.
- the osmolarity of the cold organ preservation solution of the invention measured at 20 ° C, is between 300 and 410 mOsm / L; preferably between 310 and 390 mOsm / L; more preferably between 320 and 360 mOsm / L.
- the cold organ preservation solution may contain other components.
- the cold organ preservation solution additionally comprises at least one colloid agent, at least one metabolic agent, at least one amino acid, at least one antioxidant agent, at least one vitamin and / or at least one antibiotic.
- colloid agent are understood those substances constituted by a multitude of small particles that are suspended in a liquid, but that do not dissolve in it, and that are unable to pass through semipermeable membranes, so they are useful to prevent the expansion of the interstitial space during reperfusion.
- colloid agents among others, hydroxyethyl starch (also known as pentafraction or HES), polyethylene glycols, dextran and albumin can be incorporated.
- the colloid agent is HES.
- the colloid agent is polyethylene glycol.
- the colloid agent is selected from the group of hydroxyethyl starch (HES), polyethylene glycol or a combination of both.
- metabolic agent or “metabolic substrate” is meant, in the context of the present invention, an energy precursor agent.
- amino acid is meant an organic molecule with an amino group and a carboxylic group.
- metabolic agents and amino acids suitable for incorporation into the solutions of the invention there are listed, among others, adenosine, glutamate or glutamic acid, glucose, ketoglutarate, insulin, pyruvate, aspartate and tryptophan.
- antioxidant agent an oxygen reactive species sequestrant capable of retarding or preventing the oxidation of other molecules.
- Antioxidants can include, among others, allopurinol, glutathione, mannitol, trolox, vitamin E, tryptophan, alpha-tocopherol and / or ascorbic acid.
- vitamin refers to a substance not synthesized by the organism and precursor of coenzymes that intervenes as a catalyst for biochemical reactions.
- vitamins that can be used in the present invention are, without limitation, ascorbic acid, biotin, calcium pantothenate, choline chloride, inositol, ergocalciferol, folic acid, menadione, nicotinamide, nicotinic acid, pyridoxal, riboflavin, thiamine, vitamin A , vitamin B12 and vitamin E.
- antibiotic is meant a chemical produced by a living being or synthetic derivative of it that, at low concentrations, kills or prevents the growth of certain kinds of sensitive microorganisms.
- antibiotics useful in the present invention are, without limitation, trimethoprim-sulfamethoxazole, penicillin, etc.
- the cold preservation composition of organs of the invention may also contain other pharmaceutically acceptable components, that is, approved by a federal or state government regulatory agency or included in the US Pharmacopoeia. or other pharmacopoeia generally recognized for use in animals and, more particularly, in humans.
- pharmaceutically acceptable components are, without limitation, diluents, adjuvants, excipients or vehicles for administration of the composition.
- Anticoagulants such as heparin, may also be included.
- the composition additionally contains sodium heparin.
- Cold organ preservation solutions can be obtained extemporaneously by the addition of the desired components, but they can also be cold organ preservation solutions known in the state of the art, and some of them commercially available.
- cold preservation solutions of organs known in the state of the art that can be used are, without limitation, the University of Wisconsin (UW) solution (Belzer FO and Southard JH.
- the cold organ preservation solution comprises lactobionate and HES.
- the cold preservation solution of the invention comprises lactobionate and a polyethylene glycol.
- the cold preservation solution comprises lactobionate, raffinose and HES and in particular said solution is the solution of the University of Wisconsin (UW).
- the term "University of Wisconsin (UW) solution”, as used in the present invention refers to a cold organ preservation solution comprising HES as a colloid agent and lactobionate and raffinose as waterproofing agents.
- the University of Wisconsin solution also typically contains at least one buffering agent such as KH 2 P0 4 , and at least one electrolyte such as chloride, magnesium sulfate, potassium and sodium.
- the typical composition of said solution includes antioxidant agents such as allopurinol and glutathione, and additives such as adenosine.
- the University of Wisconsin solution developed by Belzer and Southard comprises HES (50 g / L) as a colloid agent, lactobionate (100 mM) and raffinose (30 mM) as an impervious agent, KH 2 P0 4 (25 mM) as buffer, chloride (20 mM), magnesium sulfate (5 mM), potassium (120 mM) and sodium (25 mM) as electrolytes, allopurinol (1 mM) and glutathione (3 mM ) as anti-oxidant agents and adenosine (5 mM) as additive.
- the composition of said solution may vary, being both in the documents of the state of the art (US4879283) and in commercial solutions, UW solutions that lack some non-essential component, such as chloride ion, or whose components have concentrations slightly variable. Therefore, the University of Wisconsin solution useful in the present invention includes all those solutions that are marketed as a Wisconsin solution or those listed in the literature as such, regardless of whether their composition conforms to that described by Belzer and Southard
- the cold preservation solution of the invention comprises lactobionate, rafmosa and a polyethylene glycol, and in particular said solution is the solution of the Instit ⁇ Georges López (IGL-1).
- the cold preservation solution comprises lactobionate, rafmosa and mannitol, and in particular said solution is the Celsior solution.
- the cold preservation solution comprises gluconate, trehalose and HES, and in particular said solution is the Kyoto solution (ET-Kyoto).
- the cold preservation solution comprises gluconate, trehalose and a polyethylene glycol, and in particular said solution is the Polisol solution.
- the cold preservation solution comprises histidine and mannitol, and in particular said solution is the Bretschneider solution (HTK, histidine-tryptophan-ketoglutarate).
- the cold preservation solution comprises citrate and mannitol, and in particular it is the Marshall solution (HOC or hyperosmolar citrate).
- HOC Marshall solution
- the compositions of the invention may be formed by a single composition in which the cardiotrophin-1 (CT-1) or the functionally equivalent variant thereof is dispersed in the cold organ preservation solution.
- CT-1 cardiotrophin-1
- the invention contemplates that both components are separated in the form of "parts kit" so that the final composition can be prepared at the right time, for example at the time prior to use, by combining both components .
- kit refers to a combination of a set of components suitable for obtaining the compositions according to the invention, together with their containers and containers suitable for commercial sale, etc.
- suitable component for obtaining the compositions according to the invention to any compound that can be used for obtaining (i) and (ii), and includes, without limitation, means, additives , CT-1, electrolytes, waterproofing, buffers, cold organ preservation solutions, etc.
- the kit may comprise:
- a container with the cold preservation solution of constituted or partially constituted organs an additional package comprising cardiotrofma 1 (or a functionally equivalent variant); and, optionally, other packages with constituents of the cold preservation solution of organs or with additional additives, wherein the mixing of the components for the preparation of the cold preservation composition of organs of the fully constituted invention is carried out at the time prior to its use;
- the means, constituents and additives for the preparation of the cold preservation composition of organs according to the invention must be pharmaceutically acceptable.
- pharmaceutically acceptable means approved by a Federal or state government regulatory agency or included in the US Pharmacopoeia. or other generally recognized pharmacopoeia, for use in animals and, more particularly, in humans.
- means and additives is meant diluents, adjuvants, excipients or vehicles with which the composition of the invention is administered.
- Such media and additives may be sterile liquids.
- the invention relates to a kit for the preparation of a composition for cold preservation of organs comprising:
- kit of the invention as regards the compositions for the cold preservation of organs obtained by said kit have been described previously in the context of the compositions of the invention.
- the invention in another aspect of the invention, relates to a method for the preparation of a cold organ preservation composition comprising adding cardiotrophin 1 or a functionally equivalent variant thereof to a solution. of cold preservation of organs.
- cold organ preservation composition comprising adding cardiotrophin 1 or a functionally equivalent variant thereof to a solution. of cold preservation of organs.
- cardiotrophin 1 or a functionally equivalent variant thereof
- cold organ preservation solution as well as its components and parameters that characterize it, have been defined and described in detail previously in the context of the compositions of the invention.
- the cold organ preservation solution comprises
- the cold organ preservation solution has a pH between 6.5 and 8.5; and an osmolarity between 300 and 410 mOsm / L, both parameters being measured at 20 ° C.
- the preparation of said cold organ preservation composition can be done extemporaneously, at the time prior to its use, by adding a therapeutically effective amount of cardiotrophin 1 or a functionally equivalent variant thereof to a cold organ preservation solution.
- Said cold organ preservation solution may be a commercial solution, a solution known in the state of the art, or a solution obtained by mixing at least one buffering agent, at least one waterproofing or waterproofing agent, and at least one electrolyte. , to obtain a solution with a pH between 6.5 and 8.5; and an osmolarity between 300 and 410 mOsm / L, both parameters measured at 20 ° C.
- the composition according to the invention can also be prepared beforehand and kept cold until used.
- the cold organ preservation composition prepared by this method may be any composition of those described in the context of the first aspect of the invention.
- the method of preparing the composition of the invention is carried out using cardiotrophin 1 of human origin, more particularly, cardiotrophin 1 corresponding to SEQ ID NO: 1.
- the method is carried out so that the concentration of final cardiotrophin 1 in the composition is in a concentration between 0.001 g / L and 5,000 g / L; preferably between 0.005 g / L and 1 000 g / L; more preferably between 0.010 g / L and 0.500 g / L; even more preferably between 0.08 g / L and 0.20 g / L; and even more preferably between 0.1 g / L and 0.2 g / L.
- the method is carried out so that the concentration of final cardiotrophin 1 in the composition is in a concentration between 0.001 mg / L and 5,000 mg / L; preferably between 0.005 mg / L and 1,000 mg / L; more preferably between 0.010 mg / L and 0.500 mg / L; even more preferably between 0.08 mg / L and 0.20 mg / L; and even more preferably between 0.1 mg / L and 0.2 mg / L.
- the invention relates to a method for cold preservation of an organ comprising contacting said organ with a cold organ preservation composition comprising
- the cold organ preservation solution has been explained in detail in relation to the compositions of the invention.
- the cold organ preservation solution comprises
- the cold organ preservation solution has a pH between 6.5 and 8.5; and / or an osmolarity between 300 and 410 mOsm / L, both parameters being measured at 20 ° C.
- Cold preservation of an organ means the ability to conserve, protect, preserve and / or store an organ under hypothermia, minimizing cell damage by suppressing metabolism and avoiding or counteracting alterations caused in the organ both for cold ischemia and for the reperfusion of said organ.
- Said cold preservation of an organ can be carried out on the human or animal body from which it derives, outside said body or both on the body and outside it.
- the organ is isolated.
- isolated means an organ that has been extracted from the donor and is outside the human or animal body from which it is derived.
- contacting an organ with a cold preservation composition of organs according to the invention, is meant by making said composition come into contact with said organ, either by washing, immersion or perfusion.
- Cold organ preservation methods and especially those directed to organs for transplantation, generally comprise a first stage of perfusion of said organ with a cold organ preservation liquid.
- perfusion is meant, in the context of the present invention, intravascular lavage of the organ performed through a cannula inserted therein in order to remove donor blood debris that could hinder subsequent stages of preservation, and also in order to replace said blood with the cold preservation liquid, so that it cools the organ to the desired temperature.
- Said infusion can be performed on the organism of a living or dead donor or on the isolated organ.
- the inventors have shown that perfusion of the organ with a composition according to the invention is sufficient to preserve and / or preserve it properly without the need for subsequent treatments with the same composition. Therefore, in a particular embodiment of the invention the composition is contacted with the organ by perfusion in vivo in the donor. In one embodiment yet more particularly, the organ perfused in vivo with said composition is not maintained after immersion extraction in a composition according to the invention.
- in vivo perfusion is understood as the perfusion that is carried out on the body of a human or living animal donor.
- the composition is contacted with the organ by infusion when said organ is isolated from the donor.
- the isolated organ perfused with said composition is not maintained after immersion extraction in a composition according to the invention.
- the isolated organ perfused with said composition is immersed in a composition according to the invention.
- Perfusion in the isolated organ is understood once it has been removed from the donor.
- the organ may have been perfused "in vivo" by a "cold organ preservation solution”, then be perfused again, once isolated, with the composition according to the invention. Subsequently, it is not immersed in the composition according to the invention.
- the composition is contacted with the organ by perfusion in the donor and, after extraction, by immersion.
- the present invention also contemplates physical interventions on a human or animal organism in which the maintenance of the life and health of said organism is not of capital importance.
- the organ comes from a dead donor.
- the composition is contacted with the organ by perfusion in the dead donor.
- the organ perfused with said composition is not maintained after immersion extraction in a composition according to the invention.
- the composition is contacted with the isolated organ extracted from the dead donor by perfusion and, after such perfusion, by immersion.
- the composition is contacted with the isolated organ extracted from the dead donor by infusion and, after said perfusion, is not immersed in a composition according to the invention.
- a human or animal body in which the vital activity of the entire brain including the brain stem has irreversibly ceased, proven by well-defined neurological clinical protocols and supported by known specialized tests. by the person skilled in the art.
- the term “dead” in this document includes, in addition to corpses, organisms in a state of brain death or brain death whose cardiac and ventilatory activity is artificially maintained.
- the term “donor”, in the context of this invention, refers to human or animal organisms from which the organ for transplantation comes.
- washing in the context of the present invention, is meant the treatment of the external and / or internal surface of the organ with a cold organ preservation composition or solution for a short period of time.
- immersion in the context of the present invention, it is meant to immerse the organ in the preservation solution.
- the organ may be immersed, for example, in a container containing the composition of the invention in an amount sufficient to cover said organ.
- the organ can be preserved stored in hypothermic perfusion machines.
- hypothermic perfusion machines are devices that maintain a controlled flow of continuous or pulsatile form with a cold organ preservation composition according to the invention.
- composition of the invention can also be perfused during the reperfusion period, that is, when the organ that has suffered cold ischemia recovers blood flow.
- the organ may remain in contact with the composition of the invention for a period of time ranging from seconds to hours or days. Typically, the organ and the composition are kept in contact outside the body for at least 150 seconds, at least 30 minutes, at least 3 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, etc., preferably between 24 and 48 hours, more preferably 24 hours.
- the composition according to the invention remains in contact and the organ must be kept at a temperature between 0 and 25 ° C, preferably between 0 ° C and 15 ° C, more preferably between 2 ° C and 7 ° C, and even more preferably at about 4 ° C.
- perfusion of a human kidney can be performed for 10-15 minutes using a volume of cold preservative liquid of up to 10 times the weight of the kidney. Subsequently, this kidney can be stored in a cold preservation solution for a period that can range from a few seconds to 72 hours, depending on the needs.
- the organ may alternatively be subjected to perfusion, immersion or washing in the composition according to the invention; or a combination thereof.
- a first perfusion of the organ is performed with the cold preservation composition of the invention and subsequently the organ is stored and preserved in said solution until the moment of transplantation.
- the first stage of infusion can be carried out on the organ of a living or dead donor. Therefore, in another particular embodiment the The composition is contacted with the organ by perfusion in vivo in the donor and, after extraction, by immersion. In another particular embodiment the composition is contacted with the organ by perfusion in the dead donor and, after extraction, by immersion.
- the invention also contemplates the possibility that the perfusion is carried out with a cold preservation solution devoid of cardiotrophin-1 and subsequently the compositions according to the invention are used for the maintenance of the organ by immersion or washing.
- the cold organ preservation composition is contacted with the organ by washing, immersion, perfusion or a combination thereof.
- the cold organ preservation composition used in this method is a cold preservation composition as detailed above in the context of the compositions of the invention.
- the organ that contacts the method of the invention can be any solid organ of any species, such as, without limitation, mammal (primate, cows, horses, pigs, sheep, goats, dogs, cats, rodents, rabbits, etc), birds, but preferably it is of human origin. Said organ may come from a living donor or from a corpse of the same or another species different from the recipient.
- Suitable organs for the cold preservation method of the invention can be, without limitation, heart, lung, pancreas, small intestine, colon, kidney.
- the organ that contacts the composition of the invention is the kidney.
- the organ is the lung.
- the organ is the heart.
- the invention relates to the use of cardiotrophin 1 or a functionally equivalent variant thereof for the preparation of a composition for the protection and / or cold preservation of organs for transplantation.
- the cold organ preservation composition is a composition as described above in the context of the compositions of the invention.
- the invention relates to the use of a composition according to the invention for the protection and / or cold preservation of organs for transplantation.
- the invention relates to a composition for cold preservation of organs according to the invention for use in the protection and / or cold preservation of organs for transplantation.
- Protection and / or cold preservation of organs for transplantation means the ability to conserve, protect, preserve and / or store an organ under hypothermia, minimizing cell damage by suppressing metabolism and avoiding or counteracting alterations caused in the organ both by cold ischemia and by the reperfusion of said organ produced at the time of transplantation.
- the cold protection and / or preservation of organs for transplantation is performed in vivo in the donor, preferably by perfusion in vivo.
- Said protection and / or preservation in vivo takes place on the donor's human or animal body, usually by perfusion of the composition according to the invention.
- the protection and / or cold preservation of organs for transplantation is carried out in two stages: first, an in vivo perfusion is carried out in the donor with the "cold organ preservation solution", which is not It contains cardiotrophin, and subsequently the organ is removed. Once isolated, in a second stage, the organ is re-perfused with the composition of the invention.
- the cold protection and / or preservation is performed by perfusion in vivo in the donor or in the organ isolated from the composition of the invention and, additionally, after the donor is removed, the organ for transplantation it is immersed in said composition.
- the cold protection and / or preservation is carried out by perfusion of the composition according to the invention in the isolated organ of the donor and, additionally, by immersion of said organ in said composition.
- the present invention also contemplates physical interventions on a human or animal organism in which the maintenance of the life and health of said organism is not of capital importance.
- the cold protection and / or preservation of organs for transplantation is performed by perfusion on the human or animal body of a dead donor.
- a human or animal body in which the vital activity of the entire brain including the brain stem has irreversibly ceased, proven by well-defined neurological clinical protocols and supported by known specialized tests. by the person skilled in the art.
- the term "dead” in this document includes, in addition to corpses, organisms in a state of brain death or brain death whose cardiac and ventilatory activity is artificially maintained.
- the organ for transplantation is isolated.
- isolated is meant an organ that has been extracted from the human or animal organism from which it is derived and that is outside the organism.
- the organs for transplantation that are used in the method of the invention can be any solid organ of any species, such as, without limitation, mammal (primate, cows, horses, pigs, sheep, goats, dogs, cats, rodents, rabbits, etc), birds, but preferably it is of human origin.
- Said organ may come from a living donor or from a corpse of the same or another species different from the recipient.
- Suitable organs for the cold preservation method of the invention may be, without limitation, heart, lung, pancreas, small intestine, colon, kidney.
- the organ that is protected and / or preserved for transplantation with the composition of the invention is the kidney.
- the organ that is protected and / or preserved for transplantation with the composition of the invention is the lung.
- the organ is the heart.
- compositions of the invention reduce the damages associated with cold ischemia and post-transplant reperfusion in said organs.
- the authors of the present invention have shown that the compositions of the invention are suitable for cold preservation of kidneys for transplantation, since they prevent tubular necrosis caused by ischemia-reperfusion, thus improving renal functionality (level reduction of serum creatinine and increased creatinine clearance) and survival ( Figure 4) ⁇ Therefore, in another aspect, the invention relates to a cold-preserved isolated organ obtained by the methods according to the present invention.
- organ refers to a differentiated part of the organism with a specific function. Examples include, but are not limited to, parts that have specific functions such as breathing, secretion or digestion. These organs are "isolated", that is, outside the animal or human organism from which they derive. Preserved organs isolated in accordance with the present invention include, without limitation, heart, kidney, lung, heart, heart-lung, intestine, spleen and thymus.
- the isolated preserved organ is a kidney.
- the isolated preserved organ is a lung.
- the isolated preserved organ is a heart.
- Preserved organs are particularly useful for use in transplant-based therapies.
- a portion of the kidney was separated and washed with homogenizing buffer [0.05M monobasic potassium phosphate and lmM EDTA, with 0.25% sodium colalate; pH 7.8], at a temperature between 0 and 4 ° C to minimize oxidative processes. Subsequently, it was weighed and homogenized with an amount of the same homogenization buffer in a 1: 10 w / v ratio. The homogenate was centrifuged at 100,000 g for 60 minutes, at a temperature of 4 ° C. The soluble fraction obtained was divided into aliquots and frozen at -80 ° C for preservation until analysis. These samples were used to determine renal levels of superoxide anion (SO A), TNFa and IL-6.
- SO A superoxide anion
- kidney Another portion of the kidney was separated for protein determination by Western-blot.
- the sample was first homogenized in lysis buffer (25 mM HEPES, pH 7.5; 150mM NaCl; lmM EDTA; IGEPAL CA-630 1%; 10% glycerol; 2 lOmM MgCl; 0.25% sodium deoxycholate; aprotinin 10 ⁇ g / ml, 10 ⁇ g / ml leupeptin; 10MM phenylmethyl sulfonyl fluoride (PMSF); 100mM Na 2 V0 4 sodium orthovanadate; and 25mM sodium fluoride (NaF), at a rate of 1 mL / 100 mg of tissue and for 30 -60 minutes on ice. It was subsequently centrifuged for 25 minutes at 12,000 g and at 4 ° C; The supernatant was collected and frozen in aliquots at -80 ° C until the time of use.
- lysis buffer 25 mM
- a cuvette was prepared for spectrometry (1 ml cuvettes) by adding 100 ⁇ of cytochrome C (75 ⁇ ), 20 ⁇ of superoxide dismutase (SOD; approximately 264 U), 25 ⁇ of sample, and buffer solution (potassium phosphate 0, 1 M + 0.1 mM EDTA; pH 7.8) to complete a volume of 1,000 ⁇ ;
- cytochrome C 75 ⁇
- SOD 20 ⁇
- buffer solution a volume of 1,000 ⁇ was completed
- TNFa in renal tissue, serum and preservation fluid were determined by ELISA using a DuoSet ELISA Development System kit TNFa / TNFSFlA (R&D Systems; Minneapolis, USA), according to the manufacturer's instructions.
- IL-6 levels were determined by ELISA with a commercial kit DuoSet ELISA Development System rat IL-6 (R&D Systems).
- tissue samples were diluted (1: 20) in lysis buffer and a Bio-Rad kit was used, based on the Lowry method.
- the reaction was carried out in 96-well plates (Microtest TM 96, Becton Dickinson Labware, NJ, USA) using 5 for both the samples and the straight pattern. Each of the measures was performed in triplicate.
- a calibration curve was previously developed from standard solutions of known concentration of BSA. Then 25 of reagent A (supplemented with 20 of reagent S per each mL of reagent A was added, since the samples contained detergent: IGEPAL CA-630). Subsequently 200 ⁇ ⁇ reagent B.
- the mixture was incubated at least 15 minutes and the absorbance at 750 nm was measured were added.
- the separation of proteins by electrophoresis is based on the Laemmli method.
- the proteins were loaded on a 1.5 mm thick Tris-glycine-polyacrylamide gel containing a 17% loading gel of acrylamide mix (29.2% acrylamide, 0.8% bis-acrylamide>) and a Acrylamide mix gel of varying thickness, depending on the size of the protein and the volume of sample to be loaded.
- a molecular weight marker (Low / Broad range, Bio-Rad Laboratories, CA, USA or BenchMark TM Pre-Stained Protein Ladder, Invitrogen Corporation, CA, USA) was also loaded.
- the amount of tissue extract used was 200 ⁇ g of protein / well.
- Each sample was mixed with an equal volume of loading buffer (1% 2-mercaptoethanol, 2% sodium dodecyl sulfate [SDS], 10% glycerol, 125 mM Tris pH 6.8, bromophenol blue 0.0005% w / v ), denaturing the proteins at 100 ° C for 5 minutes and then depositing a small amount of loading buffer in the wells without showing, and filling the remaining space of all the wells with electrophoresis buffer (192 mM glycine, 0.1 SDS %, 25 mM Tris base, pH 8.3).
- electrophoresis buffer 192 mM glycine, 0.1 SDS %, 25 mM Tris base, pH 8.3
- the electrophoresis was carried out in an electrophoresis buffer applying a constant voltage of 100 V.
- the proteins separated and included in the acrylamide gel were transferred to a membrane.
- the transfer was carried out in a cuvette with transfer solution (190 mM glycine, 20 mM Tris pH 8.3) maintaining a constant amperage of 400 mA, having previously embedded the material (blotting papers and sponges) and balancing the gel and membrane of nitrocellulose (Hybond-ECL Amersham Biosciences) in the transfer buffer for 15 minutes.
- TTBS wash buffer (0.1% TWEEN-20 v / v; 150 mM NaCl; 20 mM Tris pH 7.5
- TTBS wash buffer 0.1% TWEEN-20 v / v; 150 mM NaCl; 20 mM Tris pH 7.5
- Incubation with the primary antibody at the appropriate dilution and temperature in the corresponding buffer under stirring constant, it was performed during different times depending on each protein.
- 4 washes of 5 minutes were made with wash buffer, and then the membrane was incubated with the corresponding secondary antibody, at the appropriate dilution, for a specified time with each protein. At the end, another 4 washes of 5 minutes were made with wash buffer.
- the detection of the specific bands of the different proteins was carried out by means of a chemiluminescence assay, for which a mixture of commercial development solutions (Amersham Biosciences, United Kingdom) was used in a 1: 1 ratio, kept under constant agitation. for 1 minute at room temperature and then the developing solution was discarded. The membrane was then placed in a camera to capture the image (ImageQuant RT ECL imager, GE Healthcare). Once the image was captured, the optical density of the bands obtained with the program was quantified (ImageQuant TL software).
- FU is the urinary flow (ml / min)
- CrO the concentration of creatinine in urine
- CrP the concentration of creatinine in plasma
- CT-1 cardiotrophin 1
- EC EuroCollins solution
- UW University of Wisconsin
- rat CT-1 provided by DRO Biosystems, was used to supplement the media. It was produced in E. Coli BL21 (Invitrogen, Barcelona, Spain) as a fusion protein with an amino-terminal histidine tail. Expression of the recombinant protein was induced with IPTG (Sigma) for a final concentration of 0.5 mM in 2 hours at 25 ° C. After homogenizing the bacteria pellet, the His-CT-1 fusion protein was purified by nickel affinity column chromatography (Qiagen, Barcelona, Spain).
- the sequence of the CT-1 used is SEQ ID NO: 2 MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDRWGSMSQR EGSLEDHQTD SSFSFLPHLE 60 AKIRQTHNLA RLLTKYADQL LEEYVQQQGE PFGLPGFSPP RLPLAGLSGP APSHAGLPVS 120 ERLRQDAAAL SALPALLDAV RRRQAELNPR APRLLRSLED AARQVRALGA AVETVLAALG 180 AAARGPVPEP VATSALFTSN SAAGVFSAKV LGLHVCGLYG EWVSRTEGDL GQLVPGGVA 239
- the EC solution was purchased from Laboratorios Esteve (Barcelona, Spain) and the UW solution was purchased from Bristol-Myers-Squibb (ViaSpan; Bristol-Myers-Squibb S.L .; Madrid; Spain).
- Tables 1 and 2 present the composition of the different cold preservation compositions of organs that were prepared. Table 1. Composition of the cold preservation solution of EC organs and of the EC composition [CT1] according to the invention.
- composition was used in cold preservation tests of kidney with EC [CT1] (Example 2, Figure 1).
- Table 2 Composition of the cold preservation solution of UW organs and of the UW composition [CT1] according to the invention.
- the preservation composition Cold organ contained 0.1 mg / L of CT-1.
- the cold organ preservation composition contained 0.2 mg / L of CT-1.
- Example 2 Cold preservation studies with a cardiotrophin 1 concentration of 0.1 mg / L. Wistar rats (225-300 g / rat males were used for the first study, and 225-250 g / rat males for the second study).
- Rats were anesthetized by intraperitoneal injection with ketamine chloride (75 mg / kg) + diazepam (50 mg / kg) and atropine (20 mg / kg).
- a middle laparotomy was performed and the retroperitoneum was dissected to expose the abdominal aorta and renal arteries to the aortic bifurcation (iliac).
- the renal vessels were dissected by ligation of the lower adrenal and gonadal branches; and the ureter was dissected in its proximal half, respecting the periurethral fat.
- the aorta was clamped below the renal artery outlet, thus maintaining its vascularization.
- Aortic catheterization was performed by transverse aortotomy on its anterior face, prior to distal ligation at the height of its bifurcation.
- the aorta was occluded by ligation above the left renal artery, while the clamp was removed to initiate cold perfusion (4 ° C) with the cold preservation composition of organs to be tested and with 1 mg / Kg of heparin Sodium
- the drainage was carried out through the renal vein, which was sectioned at its junction with the vena cava.
- the ureter in the dissected path
- the renal artery were sectioned.
- the kidney was removed and immersed in a container containing the cold preservation composition to be tested, in an amount sufficient to cover the organ, maintained at a temperature of 4 ° C.
- SOA superoxide anion free radical
- TNFa tumor necrosis factor
- iNOS nitric oxide synthase
- mice Male Wistar rats of 225-250 g / rat were used. Prior to extraction, a cold kidney and liver perfusion was performed with the preservation composition at 4 ° C. Subsequently, in the same surgical act, the two organs were removed separately. First the liver ligaments were sectioned. Subsequently, the bile duct was sectioned to simulate transplant donor surgery, and the vein and right renal artery were ligated. A double ligation of the two ends of the pyloric vein was then performed and dissected. Then 1 ml of heparinized saline was administered by intravenous injection.
- the aorta was channeled with a 20G catheter and perfused at a rate of 4 ml / min with 12 mL of The cold preservation composition.
- the inferior vena cava was sectioned to allow the perfusion fluid to escape, and then the suprahepatic vena cava and the portal vein.
- the left renal artery and vein were ligated, the left kidney was removed and immersed in the preservation composition at 4 ° C for a preset time.
- the liver was further perfused with 6 ml more, extracted from its cavity and finally submerged in a container with the cold preservation composition at 4 ° C for the preset time. After the preset cold preservation time, samples were taken for analysis.
- a composition according to the invention containing 0.2 mg / L of CT-1 was tested in a solution from the University of Wisconsin (UW [CT1]) and a solution from the University of Wisconsin as a control (UW).
- Example 4 Study of cold preservation and renal transplantation with a composition with a concentration of cardiotrophin 1 of 0.1 mg / L.
- the donor animal was anesthetized with a combination of ketamine hydrochloride 50 mg / kg (Ketolar, Parke-Davis), diazepam 50 mg / kg (Valium, Roche), and atropine 25 mg / kg (Atropine, Braun).
- the animals were placed on a thermo-statized surface and after identification and direction of the kidney, a small portion of perirenal and periureteral fat was preserved to facilitate handling.
- the ureter was sectioned and cannulated with a plastic tube (0.68 mm OD, 0.28 mm ID, ref. 427400, Intramedic, Clay Adams).
- the kidney was perfused with the cold preservation composition to be tested at 4 ° C at a rate of 0.74 mL / min for 150 seconds. Then the renal artery and vein was cut and a tube was inserted polyethylene (1.57 mm OD, 1.14 mm ID, ref. 427430, Intramedic® Clay Adams) in the renal vein.
- kidney was removed, it was kept in the cold preservation composition (4 ° C) for 24 hours.
- the recipient rat was anesthetized as described above. After laparotomy, the left kidney was removed, the preserved kidney was washed and placed orthotopically in the recipient rat, and an anastomosis of the renal arteries was performed using a single strand of polyamide filament. Veins and ureters donors and recipients were anastomosed using plastic tubes previously placed in the donor kidney. The peritoneum and abdominal muscle were closed in a single plane with continuous vicryl 5/0 suture. The skin was closed with staples. The anastomosis reperfusion did not last more than 10 min, and the total in the receiving rat no more than 30 minutes. The animal was kept warm with an incandescent lamp until its movement and reactions were normal.
- the blood reperfusion was maintained for a pre-established time, at the end of which blood, urine and kidney samples were taken.
- Example 5 Study of cold and pulmonary cardiac preservation with a concentration of cardiotrophin 1 of 0.2 mg / L
- the cardiotrofma used was rat CT-1. After the cold preservation during the pre-established time, the samples were prepared and analyzed for analysis of the different objective parameters. The frozen lungs and heart were homogenized in 5 volumes of tris-HCl buffer (50 mM, pH 7.4) at 4 o C (also containing 0.50 ml / 1 Triton X-100) for 2 minutes at 13,000 rpm with a Ultra-Turrax T25 homogenizer (IKA, Staufen). After centrifuging, the supernatant was obtained where the protein concentration was measured by the Lowry method. In this case, the induction of iNOS, the levels of p65 NFKB, the levels of PNFKB and the production of SOA were evaluated for the lung. For the heart, the induction of iNOS, p65 NFKB levels, ICAM levels, SOA production and TNFa production were evaluated.
- the antibodies used in this case come from commercial houses other than those used in the previous experiments and are:
- Figure 5A shows that preservation with Wisconsin without CT-1 for 24, and even more, for 48 hours induces an increase in synthase expression inducible nitric oxide (i OS) in rat lungs (corrected by tubulin). This increase is not observed when preserved with Wisconsin with CT-1.
- i OS synthase expression inducible nitric oxide
- Figure 5B shows that preservation with Wisconsin without CT-1 for 24 and 48 hours induces a small increase in p65 NFKB expression in rat lungs. This increase is not observed when preserved with Wisconsin with CT-1.
- Figure 5C shows that preservation with Wisconsin without CT-1 for 24 and 48 hours induces a marked increase in tissue levels of fospo-p65 NFKB (p NFKB). This increase is not observed when lungs are preserved with Wisconsin with CT-1.
- Figure 5D shows that the preservation of the rat lungs with Wisconsin without CT-1 for 24 and 48 hours induces a marked increase in the concentration of superoxide anion. This increase is much smaller when preserved with Wisconsin containing CT-1.
- Figure 6 A shows that preservation with Wisconsin without CT-1 for 24, and even more, for 48 hours induces an increase in the expression of inducible nitric oxide synthase (corrected by tubulin) in the rat heart. This increase was no different when the preservation was made with the University of Wisconsin solution containing CT-1.
- Figure 6B shows that preservation with Wisconsin without CT-1 for 48 hours induces an increase in the expression of p65 NFKB in the rat heart. This increase was not observed when preserved with Wisconsin containing CT-1.
- Figure 6C shows that preservation with Wisconsin without CT-1 for 24 and 48 hours induces a marked increase in tissue levels of ICAM-1 in the rat heart. This increase is similar when preserved with Wisconsin containing CT-1.
- Figure 6D shows that preservation with Wisconsin without CT-1 for 24 and 48 hours induces a marked increase in the concentration of superoxide anion (SOA) in the rat heart This increase is significantly lower when preserved with Wisconsin containing CT-1.
- SOA superoxide anion
- Figure 7 shows that when CT-1 is added to the preservation liquid (University of Wisconsin solution), the amount of TNF- ⁇ that appears in the preservation liquid is less than when CT-1 is not added. This is observed both at 24 and 48 hours of preservation (at time 0 no TNF- ⁇ is detected in the preservation liquid).
- Example 6 Comparison of the protective effect of cardiotrophin 1 in the washing liquid, the preservation liquid or both in the renal damage induced by cold preservation with a concentration of cardiotrophin 1 of 0.2 mg / L
- Wistar rats (225-250 g / rat male) were used. Rats were anesthetized by intraperitoneal injection with ketamine chloride (75 mg / kg) + diazepam (50 mg / kg) and atropine (20 mg / kg). A middle laparotomy was performed and the retroperitoneum was dissected to expose the abdominal aorta and renal arteries to the aortic bifurcation (iliac). Subsequently, the renal vessels were dissected by ligation of the lower adrenal and gonadal branches; and the ureter was dissected in its proximal half, respecting the periurethral fat.
- the aorta was clamped below the exit of the renal artery, thus maintaining its vascularization.
- Aortic catheterization was performed by transverse aortotomy on its anterior face, prior to distal ligation at the height of its bifurcation.
- the aorta was occluded by ligation above the left renal artery, while the clamp was removed to initiate cold perfusion (4 ° C) with the cold organ preservation composition (University of Wisconsin solution (UW) with or without CT-1, and with 1 mg / kg of sodium heparin).
- UW Universality of Wisconsin solution
- kidney was perfused, it was separated by aortic and ureter section and immersed in 20 ml of the preservation solution (University of Wisconsin solution (UW) with or without CT-1 0.2 mg / L, and with 0 , 25 mg / L of sodium heparin). The kidney was kept in the preservation fluid for 24 or 48 hours.
- UW Universal of Wisconsin solution
- Each group was composed of 5 animals.
- the kidney was removed from the preservation medium, washed with cold saline and immediately frozen in liquid nitrogen.
- SOA superoxide anion free radical
- TNFa tumor necrosis factor
- iNOS tissue levels of nitric oxide inducible synthase
- ICAM-1 adhesion molecule the NFKB transcriptional factor
- CT-1 the addition of CT-1 to the cold organ preservation solution for transplantation, be it EuroCollins or the University of Wisconsin solution, decreases the production of oxygen free radicals by the preserved kidney, especially when it has been subjected to very long preservation times (24-48 hours). Since EuroCollins and UW preservation solutions are very different in their composition, it is reasonable to think that CT-1 will be equally effective with other cold preservation solutions.
- CT-1 when the preserved kidney is transplanted to a syngeneic rat, the addition of CT-1 to the cold preservation solution improves the survival of the animal, renal function, histological damage, oxidative stress, proinflammatory cytokine production, iNOS induction, neutrophil infiltration and activation of the NFKB transcriptional factor, compared to the kidneys that have been preserved in a cold preservation solution without CT-1.
- CT-1 to the cold preservation solution prevents and reduces damage due to cold ischemia and posttransplant ischemia-reperfusion damage that causes acute kidney necrosis in the kidney, improving kidney function and survival.
- the experiments allow us to conclude that adding CT-1 to the cold preservation solution of the lung-heart block causes a lower activation of NFKB and a lower production of anion superoxide (SOA) in both heart and lung, as well as a lower release of TNF- ⁇ towards the preservation fluid, which shows that there is less induction of the oxidative and inflammatory processes stimulated by cold ischemia and preservation.
- SOA anion superoxide
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Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2800756A CA2800756A1 (en) | 2010-05-24 | 2011-05-24 | Composition for the cold storage of organs |
BR112012030015A BR112012030015A2 (pt) | 2010-05-24 | 2011-05-24 | composição para a preservação de órgãos a frio, método para a preparação de uma composição de preservação de órgãos a frio, uso da cardiotrofina-1 ou uma variante equivalente funcionalmente da mesma, método para a preservação de órgãos a frio, órgão isolado preservado a frio e kit para a preparação de uma composição para a preservação de órgãos a frio |
JP2013511708A JP2013526600A (ja) | 2010-05-24 | 2011-05-24 | 冷臓器保存組成物 |
MX2012013665A MX2012013665A (es) | 2010-05-24 | 2011-05-24 | Composicion para la preservacion fria de organos. |
RU2012156019/13A RU2012156019A (ru) | 2010-05-24 | 2011-05-24 | Композиция для холодовой консервации органов, способ получения композиции для холодовой консервации органов, способ холодовой консервации органов для трансплантации, холодоконсервированный изолированный орган, набор для получения композиции для холодовой консервации органов |
EP11745806.7A EP2578082A2 (en) | 2010-05-24 | 2011-05-24 | Composition for the cold storage of organs |
CN201180036103.8A CN103025155B (zh) | 2010-05-24 | 2011-05-24 | 器官冷藏组合物 |
AU2011257148A AU2011257148A1 (en) | 2010-05-24 | 2011-05-24 | Composition for the cold storage of organs |
US13/699,792 US9049856B2 (en) | 2010-05-24 | 2011-05-24 | Cold organ preservation composition and method of use |
Applications Claiming Priority (2)
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ESP201030782 | 2010-05-24 | ||
ES201030782 | 2010-05-24 |
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WO2011148024A2 true WO2011148024A2 (es) | 2011-12-01 |
WO2011148024A3 WO2011148024A3 (es) | 2012-03-01 |
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PCT/ES2011/070375 WO2011148024A2 (es) | 2010-05-24 | 2011-05-24 | Composición para la preservación fría de órganos |
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US (1) | US9049856B2 (es) |
EP (1) | EP2578082A2 (es) |
JP (1) | JP2013526600A (es) |
CN (1) | CN103025155B (es) |
AU (1) | AU2011257148A1 (es) |
BR (1) | BR112012030015A2 (es) |
CA (1) | CA2800756A1 (es) |
MX (1) | MX2012013665A (es) |
RU (1) | RU2012156019A (es) |
WO (1) | WO2011148024A2 (es) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2013021093A1 (es) * | 2011-08-10 | 2013-02-14 | Digna Biotech, S.L. | Uso de la cardiotofina-1 para el tratamiento de enfermedades renales |
Families Citing this family (5)
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CN107148215A (zh) * | 2014-05-30 | 2017-09-08 | 思佰益药业股份有限公司 | 器官保存液 |
CN104719285B (zh) * | 2015-04-16 | 2016-08-31 | 中国农业科学院特产研究所 | 卵巢常温保存液及保存方法 |
CN104938479B (zh) * | 2015-05-29 | 2018-02-09 | 广州赛莱拉干细胞科技股份有限公司 | 组合物及其用途、脐带保存制剂及其制备方法 |
TR201615137A2 (tr) * | 2016-10-25 | 2017-02-21 | Abdurrahman Coskun | Naki̇l öncesi̇ organ koruma solüsyonu |
JP7454493B2 (ja) * | 2017-08-28 | 2024-03-22 | フォーム 62 エルエルシー | 組織の保存のための方法および組成物 |
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US4798824A (en) | 1985-10-03 | 1989-01-17 | Wisconsin Alumni Research Foundation | Perfusate for the preservation of organs |
US4879283A (en) | 1985-10-03 | 1989-11-07 | Wisconsin Alumni Research Foundation | Solution for the preservation of organs |
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US5208145A (en) * | 1991-05-06 | 1993-05-04 | The Salk Institute For Biological Studies | Assay for the presence, in cultured cells, of functional ligand-gated channels, and substances which enhance intracellular lithium accumulation by cultured cells via such channels |
JP3773148B2 (ja) * | 1997-10-29 | 2006-05-10 | キッコーマン株式会社 | 虚血性疾患予防、治療剤及び臓器保存剤 |
ES2200646B1 (es) * | 2001-09-21 | 2005-05-01 | Fundacion Para La Investigacion Medica Aplicada | Uso de la cardiotrofina en enfermedades hepaticas. |
WO2005040391A1 (en) * | 2003-10-27 | 2005-05-06 | Murdoch Childrens Research Institute | Compositions and methods for differentiating stem cells |
WO2006052133A2 (en) * | 2004-11-12 | 2006-05-18 | Doorzand Airdrive B.V. | Composition for cold preservation and perfusion of organs |
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2011
- 2011-05-24 MX MX2012013665A patent/MX2012013665A/es not_active Application Discontinuation
- 2011-05-24 CA CA2800756A patent/CA2800756A1/en not_active Abandoned
- 2011-05-24 US US13/699,792 patent/US9049856B2/en not_active Expired - Fee Related
- 2011-05-24 JP JP2013511708A patent/JP2013526600A/ja active Pending
- 2011-05-24 RU RU2012156019/13A patent/RU2012156019A/ru not_active Application Discontinuation
- 2011-05-24 AU AU2011257148A patent/AU2011257148A1/en not_active Abandoned
- 2011-05-24 BR BR112012030015A patent/BR112012030015A2/pt not_active IP Right Cessation
- 2011-05-24 CN CN201180036103.8A patent/CN103025155B/zh not_active Expired - Fee Related
- 2011-05-24 WO PCT/ES2011/070375 patent/WO2011148024A2/es active Application Filing
- 2011-05-24 EP EP11745806.7A patent/EP2578082A2/en not_active Withdrawn
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2013021093A1 (es) * | 2011-08-10 | 2013-02-14 | Digna Biotech, S.L. | Uso de la cardiotofina-1 para el tratamiento de enfermedades renales |
Also Published As
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RU2012156019A (ru) | 2014-06-27 |
US20130130225A1 (en) | 2013-05-23 |
AU2011257148A1 (en) | 2013-01-10 |
US9049856B2 (en) | 2015-06-09 |
EP2578082A2 (en) | 2013-04-10 |
MX2012013665A (es) | 2013-04-22 |
CN103025155A (zh) | 2013-04-03 |
WO2011148024A3 (es) | 2012-03-01 |
BR112012030015A2 (pt) | 2016-08-16 |
JP2013526600A (ja) | 2013-06-24 |
CN103025155B (zh) | 2015-03-18 |
CA2800756A1 (en) | 2011-12-01 |
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