BR112020000781B1 - PRESERVATION SOLUTIONS - Google Patents

Abstract

A preservation solution for the preservation of cells, tissues and/or organs is described, wherein said solution comprises: (i) water for injection; (ii) at least one saccharide; (iii) at least one component with pH buffering properties; (iv) optionally, at least one component with calcium transport blocking properties or an anti-calcium activity; (v) salicylic acid, in free form or in salt form, or aspirin; and (vi) glutamic acid, in free form or in salt form, or glutamine; provided that acetamide is absent and/or if aspirin is present, glutamine is absent and if glutamine is present, aspirin is absent.

Description

FIELD OF INVENTION

[0001] The present invention relates to a preservation solution or a cold storage preservation solution for keeping cells viable without a blood supply, its use to prevent damage to cells, tissues and/or organs in transplants, surgeries , experiments and in vitro, a method for preservation, preservation by washing or washing, a method for treatment, a method for preparation thereof and a kit of parts comprising the solution.

[0002] More particularly, the present invention relates to a preservation solution or a cold storage preservation solution, as described herein, which comprises salicylic acid and/or glutamic acid; and its salts.

BACKGROUND OF THE INVENTION

[0003] Organ transplantation is now available for kidney, liver, heart, lung, pancreas and intestine. In recovery, a transplant organ is flushed through its vasculature with a preservation solution. This solution is designed to facilitate the reduction of organ temperature, prevent cell swelling, remove oxygen free radicals, control pH, reduce ischemic damage, prolong the safe time for which organs can be kept outside the body, and facilitate organ recovery through reperfusion.

[0004] Important washing solutions were introduced by Belzer in 1967 and Collins in 1969, later modified to Euro-Collins (EC), Marshall (1976), Bretschneider (see Isemer et al 1988) and others. The University of Wisconsin (UW) solution, the most successful of all the solutions, was introduced in 1988 by Belzer and his colleagues. There remains a need for enhanced washing preservation and cold storage preservation. Impressive evidence indicates that a high-quality graft provides better immediate function and longer graft life. A simple washing solution containing only sodium phosphate and sucrose was demonstrated by Andrews and Coffey in 1982 and by Coffey and Andrews in 1983 to protect renal tubule morphology against ischemic damage.

[0005] International patent application No. WO 02/41696 (Potts, Lodge) describes a washing preservation solution for preserving cells in the absence of a blood supply, wherein said solution comprises:

[0006] water for injection;

[0007] at least one saccharide, such as a monosaccharide, disaccharide, trisaccharide or polysaccharide;

[0008] at least one component with pH buffer properties (i.e., a pH buffer);

[0009] at least one component with calcium transport blocking properties or an anti-calcium action activity (i.e., a calcium transport blocker);

[0010] optionally, an amino acid, such as glutamine; It is

[0011] optionally, a thromboxane inhibitor to prevent blood clotting, such as aspirin.

[0012] Consequently, there remains a need for an improved commercially viable and effective preservation solution that allows for the prolonged preservation of cells, tissues and organs, including engineered cells, tissues and organs, which provides greater versatility, efficacy and reperfusion in transplants. , in surgeries, including any situation of hot or cold ischemia, such as liver, kidney, small intestine and/or pancreas transplantation; entire limb, entire body or in experimentation.

SUMMARY OF THE INVENTION

[0013] It was found that aspirin (aspirin is the common name for acetylsalicylic acid) in the prior art solution of WO 02/41696 degrades to produce salicylic acid, while glutamine degrades to glutamic acid.

[0014] Aspirin degradation will produce salicylic acid and acetic acid, while glutamine degradation will produce glutamic acid and ammonia. Furthermore, it was found that both acetic acid and ammonia are undesirable in a preservation solution, as they are potentially harmful to cells, tissues and organs; and more likely to generate harmful odors, which may increase with preservation solution storage. Furthermore, it has been found that over time, acetic acid and ammonia can react together in the prior art solution of WO 02/41696 to form acetamide. Acetamide is recognized as a skin irritant and is recognized as a dangerous substance that can damage the liver.

[0015] Thus, by replacing aspirin with salicylic acid and/or replacing glutamine with glutamic acid, or salts thereof, the amount of acetic acid and/or ammonia generated, respectively, can be minimized or removed completely. In examples, it is demonstrated that the use of the solution of the invention for preservation reduces damage to cells and tissues during preservation when compared to comparative solutions.

[0016] In the prior art solution, aspirin is added as an anti-inflammatory thromboxane inhibitor. It has been found that if aspirin is omitted and salicylic acid is added, the salicylic acid will have anti-inflammatory properties (i.e. an anti-inflammatory). Furthermore, in the prior art solution, glutamine is added as a support molecule that can provide energy. It has been found that if glutamine is omitted, it can be replaced with glutamic acid, which will act as an alternative energy substrate for cells, tissues and/or organs.

[0017] Furthermore, we found that, since the new solution of the present invention no longer poses any concern with the degradation of aspirin and/or glutamine, the amounts of replacement components, for example, salicylic acid and/or glutamine glutamic acid can consequently be reduced.

[0018] Therefore, according to a first aspect of the invention, a preservation solution is provided for the preservation of cells, tissues and/or organs, in the absence of a blood supply, wherein said solution comprises: (ii) water for injection; (iii) at least one saccharide, such as a monosaccharide, disaccharide, trisaccharide or polysaccharide; (iv) at least one component with pH buffering properties; (v) optionally, at least one component with calcium transport blocking properties or an anti-calcium activity; (vi) salicylic acid, in free form or in salt form, or aspirin; and (vii) glutamic acid, in free form or in salt form, or glutamine;

[0001] as long as acetamide is absent and/or if aspirin is present, glutamine is absent and if glutamine is present, aspirin is absent.

[0002] The term “absent” or “substantially absent” should be understood by those skilled in the art as below a pharmacologically active concentration; or none detectable by conventional methods known in the art. Such methods should include, but not be limited to, colorimetric testing and GCMS.

[0003] As an additional condition, if only one of the aspirin and glutamine is present, the degradation product of the other will not be present, that is, if there is aspirin, the ammonia will not be present; and if glutamine is present, acetic acid will not be present.

[0004] Salicylic acid, in free form or in the form of salt or aspirin, (v) serves adequately to prevent blood clotting. Suitably, the preservation solution comprises an amount of salicylic acid, in free form or in the form of salt or aspirin (v) to prevent blood clotting.

[0005] Glutamic acid, in free form or in the form of salt or glutamine, (vi) suitably serves as an energy substrate. Suitably, the preservation solution comprises an amount of glutamic acid, in free form or in the form of salt or glutamine (vi) to serve as an energy substrate.

[0006] In a particular aspect of the present invention, the preservation solution as described herein comprises:

[0007] (v) salicylic acid, in free form or in salt form, or aspirin; It is

[0008] (vi) glutamic acid, in free form or in salt form, or glutamine;

[0009] provided that acetamide is absent and/or at least one of acetic acid and ammonia is absent.

[0010] According to a further aspect of the present invention, a preservation solution is provided for the preservation of cells, tissues and/or organs, wherein said solution comprises: (ii) water for injection; (iii) at least one saccharide, such as a monosaccharide, disaccharide, trisaccharide or polysaccharide; (iv) at least one component with pH buffering properties; (v) optionally at least one component with calcium transport blocking properties or an anti-calcium activity; (vi) salicylic acid, in free form or in salt form, or aspirin; and (vii) glutamic acid, in free form or in salt form, or glutamine;

[0001] wherein if (v) is aspirin, (vi) is glutamic acid, in free or salt form, and if (vi) is glutamine, (v) is salicylic acid, in free or salt form. Preferably, the preservation solution comprises a mixture of (i) to (vi) as described herein, thus providing said preservation solution from which acetamide is absent. For example, one of aspirin and glutamine is present and acetamide is absent, and/or one of acetic acid and ammonia is present and acetamide is absent, or both of aspirin and glutamine are absent and both of acetic acid and ammonia are present. absent, and acetamide is absent.

[0002] In one aspect of the invention, a preservation solution as described herein is provided for preserving cells, tissues and/or organs in the absence of a blood supply.

[0003] In embodiments herein, there is provided a prepared preservation solution comprising a preservation solution as described herein, in which acetamide is absent, e.g., one of aspirin and glutamine is present and acetamide is absent and/or one of acetic acid and ammonia is present and acetamide is absent, or both aspirin and glutamine, and both acetic acid and ammonia are absent, and acetamide is absent.

[0004] The prepared preservation solution can be packaged for storage, for example, under anoxic conditions, in the absence of UV light. The prepared preservation solution described here is sterile and packaged for storage under anoxic conditions in the absence of UV light.

[0005] According to one aspect of the invention, the preservation solution is a wash preservation solution.

[0006] According to another aspect of the invention, the preservation solution is a cold storage preservation solution. In one embodiment, (v) is salicylic acid, in free form or in salt form; It is

[0007] (vi) is glutamic acid, in free form or in salt form, or glutamine;

[0008] as long as aspirin is absent, preferably aspirin and acetamide are absent and/or acetic acid and acetamide are absent. In another embodiment, (v) is salicylic acid, in free form or in the form of salt or aspirin; It is

[0009] (vi) is glutamic acid, in free form or in salt form;

[0010] as long as glutamine is absent, preferably glutamine and acetamide are absent and/or ammonia and acetamide are absent.

[0011] In another embodiment, (v) is salicylic acid, in free form or in salt form; It is

[0012] (vi) is glutamic acid, in free form or in salt form;

[0013] as long as glutamine and aspirin are absent, preferably, acetamide is additionally absent and/or acetic acid, ammonia and acetamide are absent.

[0014] In a preferred preservation solution of the invention, aspirin is replaced by salicylic acid, in free form or in salt form, and glutamine is replaced by glutamic acid, in free form or in salt form, so that both the aspirin and glutamine are substantially absent from the solution, preferably, acetamide is additionally absent and/or acetic acid, ammonia and acetamide are absent.

[0015] In another embodiment of the invention, a preservation solution is provided as described herein comprising:

[0016] (v) salicylic acid, in free form or in the form of salt and/or aspirin; It is

[0017] (vi) glutamic acid, in free form or in the form of salt and/or glutamine;

[0018] provided that if aspirin is present, glutamine is absent and if glutamine is present, aspirin is absent and/or that if aspirin and/or acetamide and/or acetic acid are present, the amount of salicylic acid present exceeds that which may be attributed to degradation from aspirin, for example, as determined by the sum of the amounts of any acetic acid and any acetamide present; and/or

[0019] wherein if glutamine and/or acetamide and/or ammonia are present, the amount of glutamic acid present exceeds that which can be attributed to degradation from glutamine, for example, as determined by the sum of the amounts of any ammonia and of any acetamide present.

[0020] In particular, a preservation solution as described herein is provided which comprises:

[0021] (v) salicylic acid, in free form or in the form of salt and/or aspirin; and (vi) glutamic acid, in free form or in the form of salt and/or glutamine; provided that (v) it is salicylic acid, in free form or in the form of salt and aspirin and/or

[0022] (vi) is glutamic acid, in free form or in the form of salt and glutamine;

[0023] provided that if aspirin is present, glutamine is absent and if glutamine is present, aspirin is absent; and/or

[0024] wherein if aspirin and/or acetamide and/or acetic acid are present, the amount of salicylic acid present exceeds that which can be attributed to degradation from aspirin, for example, as determined by summing the amounts of either acid acetic and any acetamide present; and/or

[0025] wherein if glutamine and/or acetamide and/or ammonia are present, the amount of glutamic acid present exceeds that which can be attributed to degradation from glutamine, for example, as determined by the sum of the amounts of any ammonia and of any acetamide present.

[0026] A preservation solution as described herein is also provided which comprises:

[0027] (v) salicylic acid, in free form or in salt form, and, optionally, additionally aspirin; It is

[0028] (vi) glutamic acid, in free form or in the form of salt and/or glutamine;

[0029] provided that if acetamide is absent and/or if aspirin is present, glutamine is absent and if glutamine is present, aspirin is absent; or

[0030] wherein if aspirin and/or acetamide and/or acetic acid are present, the amount of salicylic acid present exceeds that which can be attributed to degradation from aspirin, for example, as determined by summing the amounts of either acid acetic and any acetamide present; and/or

[0031] wherein if glutamine and/or acetamide and/or ammonia are present, the amount of glutamic acid present exceeds that which can be attributed to degradation from glutamine, for example, as determined by the sum of the amounts of any ammonia and of any acetamide present.

[0032] A preservation solution as described here is also provided, comprising:

[0033] (v) salicylic acid, in free form or in salt form and, optionally, additionally aspirin; It is

[0034] (vi) glutamic acid, in free form or in the form of salt and/or glutamine; provided that: when (v) it is salicylic acid, in free form or in the form of salt and aspirin is absent, (vi) it is glutamic acid, in free form or in the form of salt and glutamine.

[0035] According to the invention, a preservation solution as described herein is provided which comprises:

[0036] (v) salicylic acid, in free form or in salt form and, optionally, additionally aspirin; It is

[0037] (vi) glutamic acid, in free form or in the form of salt and glutamine.

[0038] According to the invention, a preservation solution as described herein is provided which comprises:

[0039] (v) salicylic acid, in free form or in the form of salt and aspirin; It is

[0040] (vi) glutamic acid, in free form or in the form of salt and/or glutamine.

[0041] According to the invention, a preservation solution as described herein is provided which comprises:

[0042] (v) salicylic acid, in free form or in the form of salt and aspirin; It is

[0043] (vi) glutamic acid, in free form or in the form of salt and glutamine.

[0044] According to the invention, a preservation solution as described herein is provided which comprises:

[0045] (v) salicylic acid, in free form or in the form of salt and aspirin; It is

[0046] (vi) glutamic acid, in free form or in salt form, or glutamine.

[0047] According to the invention, a preservation solution as described herein is provided which comprises:

[0048] (v) salicylic acid, in free form or in salt form; It is

[0049] (vi) glutamic acid, in free form or in the form of salt and glutamine.

[0050] According to the invention, a preservation solution as described herein is provided which comprises:

[0051] (v) salicylic acid, in free form or in the form of salt and/or aspirin; It is

[0052] (vi) glutamic acid, in free form or in salt form, and, optionally, additionally glutamine; provided that if acetamide is absent and/or if aspirin is present, glutamine is absent and if glutamine is present, aspirin is absent;

[0053] or wherein if glutamine and/or acetamide and/or ammonia are present, the amount of glutamic acid present exceeds that which can be attributed to degradation from glutamine, for example, as determined by the sum of the amounts of any ammonia and any acetamide present and/or

[0054] wherein if aspirin and/or acetamide and/or acetic acid are present, the amount of salicylic acid present exceeds that which can be attributed to degradation from aspirin, for example, as determined by summing the amounts of either acid acetic acid and any acetamide present.

[0055] In accordance with this aspect of the invention, a preservation solution as described herein is provided which comprises:

[0056] (v) salicylic acid, in free form or in the form of salt and/or aspirin; It is

[0057] (vi) glutamic acid, in free form or in salt form, and, optionally, additionally glutamine;

[0058] provided that: when (vi) it is glutamic acid, in free form or in salt form and glutamine is absent, (v) it is salicylic acid, in free form or in salt form, and aspirin.

[0059] According to the invention, a preservation solution as described herein is provided which comprises:

[0060] (v) salicylic acid, in free form or in the form of salt and aspirin; It is

[0061] (vi) glutamic acid, in free form or in salt form, and, optionally, additionally glutamine.

[0062] According to the invention, a preservation solution as described herein is provided which comprises:

[0063] (v) salicylic acid, in free form or in the form of salt and/or aspirin; It is

[0064] (vi) glutamic acid, in free form or in the form of salt and glutamine.

[0065] According to the invention, a preservation solution as described herein is provided which comprises:

[0066] (v) salicylic acid, in free form or in the form of salt and aspirin; It is

[0067] (vi) glutamic acid, in free form or in the form of salt and glutamine. According to the invention, there is provided a preservation solution as described herein comprising:

[0068] (v) salicylic acid, in free form or in salt form, or aspirin; It is

[0069] (vi) glutamic acid, in free form or in the form of salt and glutamine.

[0070] According to the invention, a preservation solution as described herein is provided which comprises:

[0071] (v) salicylic acid, in free form or in the form of salt and aspirin; It is

[0072] (vi) glutamic acid, in free form or in salt form.

[0073] For the avoidance of doubt, it will be understood by those skilled in the art that reference in this document to cells, tissues and organs should include manipulated cells, tissues and organs.

[0074] In one aspect of the invention, at least one component with calcium transport blocking properties or an anti-calcium action activity is present in the preservation solution of the invention.

[0075] In another aspect of the invention, the component with calcium transport blocking properties or an anti-calcium action activity is absent from the preservation solution of the invention.

[0076] In one aspect in the preservation solution of the invention, the amount of salicylic acid, in free form or in salt form, sufficient to prevent blood clotting, is less than 0.5 mmol/l, for example, of about 0.025 to less than 0.3 mmol/l. Alternatively, the amount of salicylic acid sufficient to prevent blood clotting is about 0.025 to about 0.275 mmol/l, preferably about 0.25 mmol/l of salicylic acid, in free form or in the form of salt.

[0077] Alternatively, aspirin, if present, is suitably present in an amount of about 0.3 mmol/l to about 1.0 mmol/l, for example, 0.5 mmol/l. In another aspect in the preservation solution of the invention, the amount of glutamic acid, in free form or in salt form, present is less than about 20 mmol/l. Alternatively, the amount of glutamic acid present is from about 2 to less than 15, or from about 15 to less than 20, such as from about 2 to about 12 mmol/l, preferably about 3 mmol/l. of glutamic acid, in free form or in salt form.

[0078] Alternatively, glutamine, if present, is suitably present in an amount of about 15 mmol/l to about 30 mmol/l, for example, approximately 20 mmol/l. Alternatively, the amount of glutamine present is about 2 to less than 15 mmol/l.

[0079] The salts of salicylic acid and/or glutamic acid, which may be the same or different, will be pharmacologically acceptable salts, that is, salts that have the pharmacologically desired activity of the parent compound. In particular, these salts must be non-toxic to cells, tissues and/or organs. Such salts will generally be formed when an acidic proton present in salicylic acid and/or glutamic acid is replaced by a metal ion, for example, an alkali metal ion or an alkaline earth ion. By way of example only, alkali metal salts include sodium or potassium and the like. By way of example only, alkaline earth metal salts include calcium or magnesium, etc. The wash solution may consist of only these components, in which case it is suitable for preserving universal cell types and function, in particular for preserving simple cellular systems; alternatively, it may be provided together with one or more additional substituents specifically suitable for the preservation of a desired cell type or function, in particular in the preservation of complex cellular systems such as living organs or tissues, more particularly for small or large animals, more particularly human organs such as liver, kidney, small intestine and/or pancreas; and living tissue.

[0080] In accordance with this aspect of the invention, a preservation solution for preserving the liver, kidney, small intestine and pancreas is provided, which includes a combination of (i) water for injection; (ii) a disaccharide; (iii) at least one component with pH buffering properties; (iv) at least one calcium channel or transport blocker; (v) salicylic acid, in free form or in salt form, or aspirin; and (vi) glutamic acid, in free form or in salt form, or glutamine, as previously defined in this document; together with a waterproofing sequestering anion, inorganic solutes, components effective against oxygen free radicals and a colloidal osmotic, and any optional additional components, as defined previously herein.

[0081] A preservation solution for preserving the liver, kidney, small intestine and pancreas is also provided, which includes a combination of (i) water for injection; (ii) sucrose; (iii) Na2HPO4 and NaH2PO4; (iv) diltiazem; (v) salicylic acid, in free form or in salt form, or aspirin; and (vi) glutamic acid, in free form or in salt form, or glutamine, as previously defined in this document; together with lactobionic acid, KOH and NaOH, glutathione and allopurinol and PEG, and any optional additional components as defined previously herein.

[0082] The preservation solution of the present invention has several advantages in terms of improving existing solutions, with reduced damage during preservation, due to the absence of ammonia and toxic acetic acid, and the possibility of extending preservation periods, in addition to the providing a kit to create a specific desired solution, with the associated conveniences and cost implications that will make that solution commercially viable. The present invention has found the preservation solution defined herein to be universally acceptable, based on experience and without attempting to rationalize the underlying preservation mechanism.

[0083] All components of the preservation solution of the present invention meet the national or international purity standards of the Pharmacopoeia, when applicable. Water for injection is typically purified and deionized before sterilization. A saccharide is selected from sucrose, raffinose and mannitol; and combinations thereof. A preferred saccharide is sucrose. A saccharide may be present in an amount of about 50 mmol/l to about 150 mmol/l, for example about 100 mmol/l. A pH buffer is selected from a sodium phosphate buffer, a potassium phosphate buffer and the like, preferably Na2HPO4, NaH2PO4, K2HPO4, KH2PO4 and the like; and combinations thereof. A pH buffer may be present in an amount of from about 15 mmol/l to about 75 mmol/l, for example from about 15 mmol/l to about 20 mmol/l or from about 40 to about 70 mmol /l.

[0084] The preservation solution of the invention is preferably formulated to meet a desired range of pharmacopetically acceptable physical properties. Preferably, the solution has a pH in the range of 6.5 to 7.8, more preferably 6.5 to 7.0, most preferably 6.8 to 7.0. pH is generally measured at room temperature, for example around 20°C.

[0085] An optionally added calcium transport blocker or anti-calcium activity agent is selected from any known calcium transport or channel blocker, such as nicardipine, diltiazem, verapamil, nisoldipine, chlorpromazine or trifluoroperazine; and their combinations and their metabolites. Preferably, a calcium transport or channel blocker is nicardipine and/or diltiazem; or its metabolites. A calcium transport or channel blocker may be present in an amount of about 0.0005 mmol/l to about 1.0 mmol/l, for example, about 0.005 mmol/l. When the calcium transport or channel blocker is nicardipine, nicardipine may be present in an amount of approximately 0.0005 mmol/l to 1.0 mmol/l, for example about 0.005 mmol/l. When the calcium transport or channel blocker is diltiazem, diltiazem may be present in an amount of 0.0005 mmol/l to about 1.0 mmol/l, for example, about 0.022 mmol/l.

[0086] Without being limited to this theory, reference is made here to components by function, based on commonly accepted pharmacological and physiological activity; however, for the avoidance of doubt, the components listed may contribute additional or different function to that assigned, and this should not be seen as a limitation thereof. Furthermore, functional equivalents to those listed may be considered within the scope of this invention. Other components may be present in the preservation solution. Other components, unless otherwise indicated, will typically be present in small amounts, for example in the range of up to 1 mmol/l.

[0087] Preferably, the preservation solution of the invention comprises one or more additional components selected from:

[0088] at least one anion that is largely impermeable to cells, preferably an impermeable sequestering anion; It is

[0089] at least one component with colloidal osmotic properties (it is a colloidal osmotic).

[0090] A waterproofing sequestering anion preferably comprises lactobionate or lactobionic acid. When a waterproofing sequestering anion is present, it may be in an amount of about 15 mmol/l to about 75 mmol/l, for example, from about 15 mmol/l to about 20 mmol/l or about 20 mmol/l. l or from about 40 mmol/l to about 70 mmol/l.

[0091] A colloidal osmotic is preferably selected from polyethylene glycol (PEG), succinylated gelatin (as in Gelofusin), Ficoll (a polysaccharide) and a starch product; and combinations thereof. When a component with colloid osmotic properties is present, it may be in an amount of about 0.5 mmol/l, about 3.0 mmol/l, for example, about 0.75 mmol/l, about 1 .33 mmol/l, like, about 1.0 mmol/l, and 20,000 PM. Alternatively or additionally, the preservation solution of the invention may comprise one or more components selected from:

[0092] inorganic or organic solutes;

[0093] a component or components with calcium chelating properties (i.e., a calcium chelator); It is

[0094] a component or components with iron chelating properties (i.e., an iron chelator).

[0095] Preferably, an inorganic or organic solute comprises an inorganic solute and is an electrolyte that includes cations and/or anions, for example, selected from Na+, K+, CI-, OH- and the like; and combinations thereof. In a preferred aspect of the invention, the solute is an inorganic solute, such as NaCl. When an inorganic or organic solute is present, it may be in an amount of from about 15 mmol/l to about 75 mmol/l, for example from about 15 mmol/l to about 20 mmol/l or from about 40 mmol /l to about 70 mmol/l.

[0096] Preferably, a calcium chelator comprises citrate or EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and an iron chelator comprises EDTA (ethylene tetraacetic diamine).

[0097] Alternatively or additionally, the preservation solution of the invention may comprise one or more components selected from:

[0098] one or more (additional) amino acids, in addition to glutamic acid and/or glutamine;

[0099] at least one component effective against oxygen free radicals or the production of oxygen free radicals;

[0100] at least one component of the energy supply system or that influences the energy supply system or a ketone body; It is

[0101] at least one component that has a cryoprotective action.

[0102] Preferably, an additional amino acid is selected from glycine and n-acetylcysteine, and a combination thereof. When an additional amino acid component is present, it may be in an amount of from about 1 mmol/l to about 30 mmol/l, preferably from about 3 mmol/l to about 12 mmol/l, for example about 10 mmol/l. l.

[0103] Preferred oxygen free radical inhibitors are selected from allopurinol and reduced glutathione, more preferably a combination thereof. When a component effective against oxygen free radicals is present, it may be in an amount of from about 0.2 mmol/l to about 5 mmol/l, for example, from about 3 mmol/l to about 5 mmol/l. Generally, an amount of oxygen free radical inhibitors defined herein refers to the total amount of oxygen free radical inhibitors. For example, when the oxygen free radical inhibitors comprise a combination of allopurinol and reduced glutathione, an amount of the combined oxygen free radical inhibitors may be in an amount of about 0.2 mmol/l to about 5 mmol/l , etc., an energy supply system composed of adenosine. When an energy supply component is present, it may be in an amount of up to about 20 mmol/l, for example, from about 5 to about 20 mmol/l, such as about 5 mmol/l.

[0104] Preferably, a ketone body comprises beta-hydroxy butyrate.

[0105] Cryoprotectants are compounds that, when present in solution, can reduce or inhibit the formation of ice crystals in solutions exposed to temperatures below 0 °C. Thus, the use of a cryoprotectant allows cells, tissues or organs to be stored at a temperature of about -20°C to about 4°C. Preferably, a cryoprotectant is selected from a glycol such as propylene glycol, DMSO, a saccharide, a carbohydrate, a lipid, a glycolipid such as xylomannan, a glycoprotein, protein or polypeptide or a polyol; or a combination thereof. When a cryoprotectant is present, it may be in an amount of about 5 to about 100 mg/ml.

[0106] Alternatively or additionally, the preservation solution of the invention may comprise additional components for a specific function selected from:

[0107] at least one component of the intracellular signal transduction system or that modifies this system, preferably a protein kinase inhibitor or a calmodulin inhibitor; It is

[0108] at least one component that has a membrane stabilizing action, preferably ranolazine, and the like. Preferably, a saccharide component (ii) is present in an amount in the range of 50 to 150 mmol/l, for example approximately 100 mmol/l;

[0109] the pH buffer component (iii) is present in a total amount in the range of 15 to 75 mmol/l, for example, approximately 15 to 20 or 40 to 70 mmol/l;

[0110] the waterproofing sequestering anion is present in a total amount in the range of 15 to 75 mmol/l, for example, approximately 15 to 20 or 40 to 70 mmol/l;

[0111] the inorganic or organic solute component is present in a total amount in the range of 15 to 75 mmol/l, for example, approximately 15 to 20 or 40 to 70 mmol/l;

[0112] the additional amino acid component is present in an amount in the range of 5 to 30 mmol/l, preferably 5 to 12 mmol/l, for example approximately 10 mmol/l; the component effective against oxygen free radicals is present in a total amount in the range of up to 5 mmol/l, for example approximately 3 to 5 mmol/l;

[0113] the energy supply component is present in an amount in the range of up to 20 mmol/l, for example, 5 mmol/l;

[0114] the component with colloid osmotic properties is present in an amount in the range of 0.5 to 3.0 mmol/l, for example, approximately 0.75 to 1.33 mmol/l, such as 1.0 mmol/l and 20,000 PM;

[0115] when the component with calcium transport blocking properties (iv) is present, the calcium transport blocking agent can be, for example, nicardipine or diltiazem. Nicardipine may be present in an amount in the range of 0.0005 to 1.0 mmol/l, for example 0.005 mmol/l; and diltiazem may be present in an amount in the range of 0.0005 to 1.0 mmol/l, for example 0.022 mmol/l.

[0116] In the case of certain components present as 2 or more types, the relative quantities may be critical or non-critical. A preferred component effective against oxygen free radicals is approximately 3 mmol/l reduced glutathione and 0.35 to 0.4, more preferably 0.4 mmol/l allopurinol.

[0117] A preferred inorganic or organic solute component comprises electrolytes as follows:

[0118] Na+ 15 to 150 mmol/l, for example, 30 mmol/l

[0119] K+ 0 to 25 mmol/l, for example, 15 mmol/l

[0120] CI- 0 to 100 mmol/l

[0121] OH- 0 to 75 mmol/l

[0122] Preferably, a solution according to the invention is prepared and stored under anoxic conditions in the absence of UV light.

[0123] It is a particular advantage that the solution as defined comprising components (i) - (vi) can be stored for prolonged periods, and additional components necessary for clinical use (for example, heparin) added immediately before its use. It is especially an advantage of the solution of the present invention that the solution can be stored for prolonged periods without generating undesirable degradation products. More particularly, the solution can be stored without the generation of either or both acetic acid and ammonia, and without the generation of acetamide. Alternatively, the solution can be stored with the generation of one or both of acetic acid and ammonia and/or with the generation of acetamide, in an amount less than the molar equivalent of salicylic acid and glutamic acid, respectively.

[0124] The preservation solution of the invention, as defined herein, preferably comprises the basic components (i) - (vi) combined with additional components for specific functions. The solution for use in organ preservation is particularly of greater complexity than in the preservation of simple cellular systems, however, it has been found that the solution can nevertheless be relatively straightforward.

[0125] Preferably, the preservation solution for intra-abdominal organs such as kidney, liver and pancreas; and also intestine and small intestine; comprises components (i) - (vi) as defined herein together with at least one component selected from an anionic waterproofing sequestering component; and a component with colloid osmotic properties; and more preferably, additionally, one or more components selected from:

[0126] inorganic or organic solute component;

[0127] an additional amino acid component;

[0128] an effective component against oxygen free radicals; It is

[0129] a power supply component.

[0130] Preferably, a preservation solution for preserving the liver, kidney, small intestine and pancreas comprises a combination: (i) water for injection; (ii) a disaccharide; (iii) at least one component with pH buffering properties; (iv) at least one calcium channel or transport blocker; (v) salicylic acid, in free form or in salt form, or aspirin; and (vi) glutamic acid, in free form or in salt form, or glutamine, as previously defined in this document; together with a waterproofing scavenger anion, inorganic solutes, components effective against oxygen free radicals and a colloidal osmotic, and any optional additional components as defined herein;

[0131] more preferably, a combination of

[0132] (ii) water for injection;

[0133] (ii) sucrose;

[0134] (iii) Na2HPO4 and NaH2PO4;

[0135] (iv) diltiazem or nicardipine; (vii) salicylic acid, in free form or in salt form, or aspirin; and (viii) glutamic acid, in free form or in salt form, or glutamine, as previously defined in this document; together with lactobionic acid, KOH and NaOH, glutathione and allopurinol and PEG, and any optional additional components as defined herein; more preferably, it comprises a combination of classes of components given below, more preferably, of the specific type listed, and substantially in the amount listed below, together with any optional additional components as defined herein, when constituted by volume in water:

[0136] provided that if (v) it is aspirin, (vi) it is not glutamine;

[0137] and, more preferably, substantially in the amount listed below, together with any additional optional components as defined hereinbefore, when constituted by volume in water:

[0138] provided that if (v) it is aspirin, (vi) it is not glutamine.

[0139] Furthermore, it has surprisingly been found that the effectiveness of the constituents of a preservation solution according to the invention is improved by replacing certain constituents with their main degradation products. Without being limited to this theory, it appears that the byproducts of this degradation may exert unwanted toxic effects during the use of the preservation solution, both individually and in combination. For example, acetic acid and ammonia form acetamide, which is potentially harmful to the liver. By removing this toxic effect, the effectiveness of the preservation solution is improved.

[0140] Therefore, the finding in accordance with the present invention is that certain substituents are essential for the preservation of the main cellular functions essential for all cell types and have been identified as components (i) to (vi) as defined in this document. While this solution may be highly effective or satisfactory in preserving simple tissues or cellular systems, if one wishes to preserve organs or cellular systems that require or provide unusual or more complex cellular functions, it is necessary to incorporate substituents specifically targeted to preserve the requirement or function provided. be it muscular, electrical, specific membrane activity, energy supply and the like.

[0141] Thus, the invention particularly provides a preservation solution as defined herein for the preservation of the liver, kidney, small intestine and pancreas.

[0142] The solution is suitably prepared by methods as known in the art by simple mixing under pharmacopetically acceptable conditions. Preferably, the components are determined and incorporated at the desired molar concentration.

[0143] It will be appreciated that variation may be specific or nonspecific to the effectiveness of the solution and that an amount of variation that has no effect on fluid performance is considered within the scope of this invention. Selection of the type of component, which requires an amount of verification by routine experimentation, is considered within the scope of this invention.

[0144] In a further aspect of the invention, there is provided a method for preparing a preservation solution comprising adding components (i) - (vi) in sequence to water, accompanying any additional components, with the exception of colloidal osmotic properties unstable components, if any, for example aspirin or glutamine, and dissolution of the mixture; adding the osmotic properties of the colloid component, if any, and making the solution almost in volume; and finally compensating the volume to regulate the pH, sterilizing and cooling to 0 to 4 °C. The solution may be stored, if desired, with the subsequent addition of any unstable components (e.g., aspirin and/or glutamine) immediately before use. The method according to this aspect of the invention may include packaging the prepared solution for storage, preferably under reduced temperature, more preferably in the range of 0 to 12°C, more preferably 0 to 4°C, for example, packaging the prepared solution in an anoxic condition and in the absence of UV light.

[0145] In another aspect, a preservation solution obtained by the method as described herein is provided.

[0146] In embodiments of this document, a preservation solution obtained by a method comprising adding components (i) - (vi) to water is provided.

[0147] In a further aspect of the invention, there is provided for the use of a preservation solution as defined herein as a wash preservation solution or a cold storage preservation solution; for the preservation of cells in the absence of blood supply, in particular to prevent damage to organs, living tissues and cells. The solution is suitable for use on small or large animals or mammals, in particular human cells, tissues and organs.

[0148] The use of the solution may be in transplantation, including organs from donors with or without a heartbeat, in surgery including any situation of hot or cold ischemia, preservation of limbs or the entire body, in experimentation on living tissues, in culture and preservation of engineering cells, tissues and organs and the like. Preferably, the solution is used as a level solution placed in contact with cells, tissues or organs, limbs or the entire body through the vascular system, and optionally also serves as a preservation solution for storage of level cells, tissues and organs. The solution is suitable for long-term preservation of cells, tissues and organs in both hypothermic static storage and hypothermic machine perfusion systems.

[0149] Thus, the invention particularly provides a preservation solution as defined herein for the preservation of the liver, kidney, small intestine and pancreas.

[0150] In a further aspect of the invention, there is provided a method for washing, preserving or washing cells, in particular living cells, tissues or organs whereby the cells, tissues or organs are brought into contact with a solution as defined in this document. In accordance with this aspect of the invention, there is provided a method for washing, preserving or washing cells for simple hypothermic storage, in particular living cells, tissues or organs whereby the cells, tissues or organs are brought into contact with a solution of preservation comprising: (i) water for injection; (ii) at least one saccharide, such as a monosaccharide, disaccharide, trisaccharide or polysaccharide; (iii) at least one component with pH buffering properties; (iv) optionally, at least one component with calcium transport blocking properties or an anti-calcium activity; (v) salicylic acid, in free form or in salt form, or aspirin; and (vi) glutamic acid, in free form or in salt form, or glutamine;

[0151] provided that if acetamide is absent and/or if aspirin is present, glutamine is absent and if glutamine is present, aspirin is absent; more particularly, wherein if (v) is aspirin, (vi) is glutamic acid, in free form or in salt form, and if (vi) is glutamine, (v) is salicylic acid, in free form or in salt form salt;

[0152] in which the cells, tissues or organs are washed with solution, removed from the normal locus, cooled to temperatures typically in the range of zero to 12 ° C and stored.

[0153] According to this aspect of the invention, if acetamide is absent, at least one of the acetic acid and ammonia is absent. According to one aspect of the invention, the method comprises the preservation by washing of living cells, tissues or organs.

[0154] According to another aspect of the invention, the method comprises the preservation in cold storage of living cells, tissues or organs.

[0155] In a preferred method according to this aspect of the invention, aspirin is replaced by salicylic acid, in free form or in salt form, and glutamine is replaced by glutamic acid, in free form or in salt form, so that both aspirin and glutamine are substantially absent from the solution, preferably acetamide is additionally absent and/or acetic acid, ammonia and acetamide are also absent, more particularly wherein (v) is salicylic acid, in free form or in the form of salt, and (vi) is glutamic acid, in free form or in salt form.

[0156] According to a further aspect of the invention, there is provided a method for preserving cells for simple hypothermic storage, in particular living cells, tissues or organs whereby the cells, tissues or organs are brought into contact with a solution of preservation comprising: (i) water for injection; (ii) at least one saccharide, such as a monosaccharide, disaccharide, trisaccharide or polysaccharide; (iii) at least one component with pH buffering properties; (iv) optionally at least one component with calcium transport blocking properties or an anti-calcium activity; (v) salicylic acid, in free form or in salt form, or aspirin; and (vi) glutamic acid, in free form or in salt form, or glutamine; and at least one component with cryoprotective properties (i.e., a cryoprotectant); provided that acetamide is absent and/or at least one of acetic acid and ammonia is additionally absent; more particularly if (v) is aspirin, (vi) is glutamic acid, in free form or in salt form, and if (vi) is glutamine, (v) is salicylic acid, in free form or in salt form;

[0157] in which cells, tissues or organs are washed with solution, removed from their normal location, cooled to temperatures normally in the range between -20 °C and 12 °C and stored.

[0158] According to this aspect of the invention, if acetamide is absent, then at least one of acetic acid and ammonia is absent.

[0159] The method can be for simple hypothermic storage, in which cells, tissues or organs are washed with solution, removed from their normal location, cooled, preferably at temperatures normally in the range between zero and 12 °C and stored. Furthermore, if a cryoprotectant is present, the tissue or organ can be stored at a temperature of about -20°C to about 4°C. Furthermore, the solution can be actively released through the organ. We discovered that cells, tissues or organs can be stored for longer periods than currently practiced, for example, kidney and liver for periods of the order of 48 hours or more. Furthermore, alternatively, the method is for preserving cells, particularly tissues or organs, whereby the cells, tissues or organs have been washed and brought to a hypothermic state and are contacted with the preservation solution by immersion or perfusion.

[0160] Preferably, the method of the invention comprises administering to cells, tissues, organs or a donor a biologically effective amount of the solution of the invention, at an effective rate or at an effective concentration to maintain or improve the function thereof. Preferably, the method is a method for preserving certain cellular, tissue or organ functions, e.g., cellular metabolism, and/or for temporarily interrupting certain functions, e.g., muscle activity, breakdown or excretion of essential cellular components, etc. , excretory products, for example in the form of bile or urine and the like.

[0161] Ischemia is the situation that results from the interruption of blood flow through an organ. The effects are due to a lack of oxygen and nutrients; and accumulation of carbon dioxide and other waste. It is more harmful to body temperature than cold, which is why transplant organs are washed and cooled. Organ donors have often suffered trauma and therefore the donor organ may have been subjected to a period of warm ischemia as a result of the trauma. The experimental addition of a period of warm ischemia before lavage mimics this situation. It is an advantage that our solution provides protection against this warm ischemia. The lavage perfusion is preferably carried out at a pressure of up to 300 mmHg, more preferably in the atmospheric range at 200 mmHg, more preferably in the range of 160 mmHg, more preferably up to 100 mmHg, more preferably up to 50 mmHg.

[0162] The method according to this aspect of the invention comprises the use of the preservation solution as defined herein.

[0163] In a further aspect of the invention, there is provided a kit of parts comprising a preservation solution having, in one part, components (i) - (vi) as defined herein, optionally associated with, in one or more parts additional, individual components selected from one or more of the additional components, as defined herein, for use in preparing one or more solutions for specific purposes; and serving as a universal preservation solution. In embodiments, a kit of parts herein includes a preservation solution having, in a part, components (i) - (vi) as defined herein, of which one or more unstable components are absent and are provided separately, in one or more plus additional parts, for addition immediately before use. According to one aspect of the invention, the kit of parts comprises a level preservation solution with components (i) - (vi) as defined herein.

[0164] According to another aspect of the invention, the parts kit comprises a cold preservation solution with components (i) - (vi) as defined herein.

[0165] The invention is now illustrated in a non-limiting manner with reference to the examples and with reference to the attached figures, in which:

[0166] Figure 1 illustrates the stability of aspirin in a preservation solution: HPLC methods were developed to measure the concentration of aspirin and salicylic acid in the Prior Art solution. Aspirin is rapidly hydrolyzed to salicylic acid and acetic acid during storage. After 8 weeks, none of the parent compounds remain.

[0167] Figure 2 illustrates serum creatinine after renal preservation in an improved preservation solution (solution of the invention) versus a previous formulation (prior art). Lower serum creatinine illustrates reduced organ damage.

[0168] Figure 3 illustrates the ex vivo preservation of porcine kidney in preservation solution of the invention for up to 72 hours outside the body. The parenchyma is well preserved with normal glomeruli and tubules. H&E staining, 40x magnification.

EXAMPLE 1 1.1 Washing Preservation Solutions Used in the Invention

[0169] The preservation solutions involved in this study are shown in Tables 1, 2, 3 and 4, values in mmol/l. The solutions (SOLS) were composed from a flask half filled with water, to which all lactobionates, KOH, sodium phosphate, glutamine or glutamic acid, sucrose, aspirin or salicylic acid, allopurinol and diltiazem were added in sequence and dissolved. . The colloid (PEG) was then added and the solution prepared almost to volume. NaOH was added to set the pH. The solution was then prepared to volume. All solutions were sterilized by filtration and stored in glass bottles at 4 °C. Reduced glutathione was added during preparation or immediately before use. Sources are as indicated above or as disclosed in WO 02/41696.

COMPARATIVE

[0170] The above formulation is as disclosed and described in document WO 02/41696 and as illustrated in Table 1 of this document. UW is an original commercial standard solution (viaSpan/BELZER UW, DU PONT PHARMA).

Composition of Comparative Preservation Solutions

[0171] The composition of comparative preservation solutions is illustrated in Table 1: TABLE 1

Composition of the Cold Preservation Solutions of the Invention

[0172] The composition of the cold preservation solutions of the invention is illustrated in Table 2: TABLE 2

Composition of the Cold Preservation Solutions of the invention with (v) and/or (vi) in reduced quantity

[0173] The composition of the cold preservation solutions of the invention with (v) and/or (vi) in reduced quantity is illustrated in Table 3: TABLE 3

Composition of the Cold Preservation Solutions of the invention with (v) and/or (vi) in reduced quantity

[0174] The composition of the cold preservation solutions of the invention with (v) and/or (vi) in reduced quantity is illustrated in Table 4: TABLE 4

EXAMPLE 2

[0175] The comparative solutions and washing preservation solution of the invention were studied in the following systems.

2.1.1 Generation and impact of toxic metabolites produced by washing preservation solutions described in the prior art

[0176] It was found that the washing preservation solution described in international patent application No. WO 02/41696 generates, upon storage, metabolic products with the potential to be toxic to the cells/tissues/organs under storage. As an example, Figure 1 illustrates that storing the solution for 8 weeks results in 100% of the aspirin converting to salicylic acid and the production of equimolar amounts of harmful acetic acid. In a solution that originally contains 0.5 mM aspirin, this results in the formation of 0.5 mM acetic acid, 30 mg/l. Acetic acid is a known irritant and damages human tissues.

[0177] As an additional example, the presence of ammonia in the prior art solution was demonstrated by colorimetric testing. The Ammonia Assay Kit (AB83360) provides a rapid, simple, sensitive and reliable assay suitable for high-throughput ammonia and ammonium research and testing. In the assay, ammonia and ammonium are converted into a product that reacts with the OxiRed probe to generate color (Àmax = 570 nm) that can be easily quantified by the plate reader. The kit can detect 1 nmol (~20 μM) of total ammonia and ammonium, which is much more sensitive than measuring ammonia with an NADPH-based assay. Using this protocol, it is demonstrated that the Prior Art Solution contains approximately 2.6 mM ammonia. This is many times greater than the known toxicity of ammonia, 300 μM is known to be toxic to human cells1. On the other hand, in the solution of the invention, ammonia is substantially absent, below the lowest level of quantification of the test, less than 2 nmol.

[0178] Ammonia toxicity may occur, causing a disturbance in the absorption of potassium by cells. This can cause deleterious effects on all organs and tissues; change in pH, membrane potential and metabolism2. In vivo, ammonia would normally be detoxified by the hepatic urea cycle. However, this cannot occur during storage or use of the preservation solution, as the metabolic activity of cells is intentionally suppressed during cold preservation. This means that the ammonia in the prior art solution remains during the preservation period and there is a significant potential for cellular damage through reperfusion. In a solution of the present invention, ammonia is absent and this damage cannot occur.

[0179] In a solution that originally contains 20 mM glutamine or approximately 2.6 mM ammonia and 0.5 mM aspirin, or up to 0.5 mM acetic acid, this results in the formation of up to 0.5 mM of acetamide, 30 mg/l. Acetamide is known to be harmful to human tissues. 1Heeneman et al, Journal of Immunological Methods (1993) 166, 1, pages 85 to 91 2Dasarathy, S., Mookerjee, R.P., Rackayova, V. et al. Metab Brain Dis (2017) 32: 529. https://doi.org/10.1007/sl 1011 -016-9938-3

2.1.2 Absence of toxic metabolites in the washing preservation solutions of the invention

[0180] In the solutions of the invention in which aspirin is absent, SOL 1.1, SOL 1.2, SOL 2.1, SOL 2.2, SOL 3.1 and SOL 3.2, acetic acid is substantially absent even after storage, below the lowest level of quantification of the test.

[0181] In other solutions of the invention in which aspirin is added immediately before use, acetic acid is substantially absent or at reduced levels during the period of use.

[0182] In a solution of the invention from which glutamine is absent, SOL 1.1, SOL 1.3, SOL 2.1, SOL 2.3, SOL 3.1 and SOL 3.3, ammonia is substantially absent even after storage, below the lowest level of test quantification, less than 2 nmol.

[0183] In other solutions of the invention in which glutamine is added immediately before use, ammonia is substantially absent or at reduced levels during the period of use.

[0184] Furthermore, in all solutions of the invention aspirin or glutamine or both are absent, SOL 1.1, SOL 1.2, SOL 1.3, SOL 2.1, SOL 2.2, SOL 2.3, SOL 3.1, SOL 3.1, SOL 3.2 and SOL 3.3, and acetamide is substantially absent even after storage.

2.2 Pig Transplant Model

[0185] For a true assessment of the quality of preservation solutions, it is necessary to employ a whole animal model to evaluate the clinical results of transplantation using the test solutions. The porcine model is more appropriate for this. In summary, the protocol involves recovery of the organ (liver, kidney, small intestine, or pancreas), preservation by washing in test (invention) or control solutions (comparison), and then implantation (allo or autotransplantation, depending on the organ considered). Organ function and the recipient's overall health are monitored after surgery for a clinically appropriate period of time.

[0186] The following table provides an indication of the measurements used to evaluate the functionality of the transplanted organ in the recipient animal. In addition, the animal's general health and well-being (body weight, food consumption, etc.) are monitored. At the end of the study, the animal is sacrificed and the main organs are collected for histological analysis.

[0187] Creatinine is an important marker in the blood, directly related to kidney function. A functional kidney will maintain a low creatinine concentration; a high level indicates a poorly functioning kidney. After transplantation, serum creatinine should increase and only return to normal levels if/when kidney function is restored. The magnitude of the increase reflects the magnitude of the preservation injury. The speed at which creatinine levels return to normal reflects how quickly the kidney returns to normal function; this in itself is a key measure of how well the organ has been preserved. Figure 2 illustrates a recent study in which the invention's preservation solution significantly outperforms the prior art solution; peak creatinine is significantly lower and kidney function returns to normal much more quickly. This is evidence of much reduced tissue damage in the solution of the invention.

HISTOLOGY

[0188] Histology was performed on tissue samples collected from abdominal organs (liver, kidney, pancreas, and small intestine) of animals perfused at the University of Wisconsin (UW) (commercially available as Belzer UW® cold storage solution) or in solution of use of the invention for up to 72 hours. Standard comparison solutions, such as UW, are typically capable of preserving tissue for approximately 12 to 24 hours. The tissue samples stained with hematoxylin and eosin (Figure 3) illustrate the normal morphology and good preservation of the samples by the preservation solution of the invention for up to 72 hours. Better retention of cytoplasmic content is observed and the nuclear ghost was also present in the UW group, absent in the preservation solution of the invention. Other nuclear ghosts are apparent in UW samples at 48 and 72 hours, absent in the sample preservation solution. This is evidence that the invention's preservation solution causes greatly reduced cellular damage over preservation times.

Claims (20)

1. Preservation solution for the in vitro preservation of cells, tissues and/or organs, in the absence of a blood supply, characterized by the fact that it comprises: (i) water for injection; (ii) at least one saccharide; (iii) at least one component with pH buffering properties; (iv) optionally, at least one component with calcium transport blocking properties or an anti-calcium activity; (v) salicylic acid, in free form or in salt form, or aspirin; and (vi) glutamic acid, in free form or in salt form, or glutamine; provided that acetamide is absent and/or if aspirin is present, glutamine is absent and if glutamine is present, aspirin is absent. 2. Preservation solution, according to claim 1, characterized by the fact that if (v) it is aspirin, (vi) it is glutamic acid, in free form or in salt form. 3. Preservation solution, according to claim 1, characterized by the fact that if (vi) it is glutamine, (v) it is salicylic acid, in free form or in salt form. 4. Preservation solution according to claim 1, characterized by the fact that the solution is a discharge preservation solution. 5. Preservation solution according to claim 1, characterized by the fact that the solution is a cold storage preservation solution. 6. Preservation solution according to claim 1, characterized by the fact that aspirin is absent. 7. Preservation solution according to claim 6, characterized by the fact that (v) it is salicylic acid, in free form or in salt form; and (vi) is glutamic acid, in free form or in salt form, or glutamine; and aspirin is absent, and/or in which acetic acid and/or acetamide are absent. 8. Preservation solution according to claim 1, characterized by the fact that glutamine is absent. 9. Preservation solution according to claim 8, characterized by the fact that (v) it is salicylic acid, in free form or in salt form, or aspirin; and (vi) is glutamic acid, in free form or in salt form; and glutamine is absent, and/or in which ammonia and/or acetamide are absent. 10. Preservation solution according to claim 1, characterized by the fact that aspirin and glutamine are absent. 11. Preservation solution, according to claim 10, characterized by the fact that (v) is salicylic acid, in free form or in salt form; and (vi) is glutamic acid, in free form or in salt form, provided that acetamide is absent and/or acetic acid and ammonia are absent. 12. Preservation solution according to claim 10, characterized by the fact that acetamide is absent and/or aspirin and glutamine are absent. 13. Preservation solution according to claim 1, characterized by the fact that at least one component with calcium transport blocking properties or an anti-calcium action activity is present. 14. Preservation solution according to claim 1, characterized in that the at least one component with calcium transport blocking properties or an anti-calcium action activity is absent. 15. Preservation solution, according to claim 1, characterized by the fact that the amount of salicylic acid, in free form or in salt form, is less than 0.5 mmol/l. 16. Preservation solution according to claim 1, characterized by the fact that the saccharide is selected from one or more of sucrose, raffinose and mannitol. 17. Preservation solution according to claim 1, characterized in that the pH buffer is selected from a sodium phosphate buffer, a potassium phosphate buffer. 18. Preservation solution according to claim 1, characterized by the fact that the calcium transport blocker or anti-calcium activity agent is selected from calcium transport channel blocker, nicardipine, diltiazem, verapamil, nisoldipine, chlorpromazine or trifluoroperazine. 19. The preservation solution of claim 1, wherein the preservation solution includes at least one anion that is largely impermeable to cells. 20. Preservation solution according to claim 1, characterized by the fact that the preservation solution includes at least one component with calcium chelating properties.