CZ294795B6 - Physiologically acceptable aqueous solution and method for its use - Google Patents

Abstract

In the present invention, there is disclosed a physiologically acceptable aqueous solution comprising: electrolytes; a dynamic buffering system; and at least one oncotic agent from a primary source of oncotic active component, wherein the weight fraction molecular weight ranges within 50 000 do 1 000á000, wherein said aqueous solution further comprises at least two of magnesium, a sugar in an amount of not more than about 50 mM and a low or medium molecular weight oncotic agent ranging within 50 000 to 200 000 or high molecular weight oncotic agent ranging within 300 000 to 1 000á000, and further wherein said solution does not comprise a conventional biological buffer. The subject solution finds use in a variety of applications, particularly in those applications where at least a portion of a host's blood volume is replaced with a blood substitute.

Description

Physiologically acceptable aqueous solution and its use

Technical field

The subject of the present invention are physiological solutions for plasma substitution and their use. The solutions described herein comprise: an electrolyte, a dynamic buffering system, and an oncotic active ingredient, which solutions do not contain conventional biological buffers. The disclosed solutions will find utility in a variety of applications, particularly when a portion of the patient's blood is to be replaced with a substitution solution.

BACKGROUND OF THE INVENTION

Physiologically acceptable solutions find use in a variety of applications in medicine, biomedical research and related fields. An example would be the use of physiologically acceptable plasma replacement solutions in surgical procedures where a large volume of blood plasma needs to be replaced in a patient. A further use of these solutions is to compensate for blood loss caused by surgery or injury, or to maintain a tissue, organ, group of organs or the entire subject in a hypothermic or frozen state. These solutions are also necessary if the patient's blood passes through an external device, such as a cardiopulmonary bypass device, where an additional volume is created as a result of the addition of an additional circulating volume, which must be filled with a compatible blood substituent, i.e. a blood volume. a solution that increases the patient's blood volume.

Physiologically acceptable solutions, which can be used as plasma substitutes, must be freely mixed with blood, without unacceptable changes in its components, such as the formation of precipitates that would clog small vessels, by destroying a disproportionate amount of blood components (blood cells, platelets). an ionic or molecular imbalance that would damage the cells of the body and tissues, or causing severe physiological damage, such as disproportionately increasing or decreasing heart rate, nerve function, muscle activity, and the like.

The first blood plasma replacements developed were prepared from mammalian blood. Although their use has been successful since they originate from a natural source, such derivatives may contain a number of pathogenic substances, such as viral pathogens of the HIV type, hepatitis B and other pathogens, eg prions, whose presence is associated with Creutzfeldt-Jakob disease and the like.

Today there are a number of synthetic blood and plasma substitutes. Although synthetic blood plasma substitutes are useful in a variety of applications, none of them can be used in all cases.

Therefore, there is still a need to develop new physiologically acceptable aqueous solutions useful as blood plasma substitutes. Specifically, these are solutions useful in hypothermic surgeries such as heart surgery and the like. It is also desirable that such a solution be heat-sterilizable.

Relevant literature

A number of physiologically acceptable solutions, in particular blood substitutes, and methods of using them are described in US Patent Nos. RE 34 077; 3,937,821; 4,001,101; 4,061,736; 4,216,205;

663 166; 4,812,310; 4,908,350; 4,923,442; 4,927,806; 5,082,831; 5,084,377; 5,130,230;

171 526; 5,210,083; 5,274,001, 5,374,624 and 5,407,428.

Other references describing physiologically acceptable solutions including blood substitutes are: Bishop et al., Transplantation (1978) 25: 235-239; Messmer et al., Characteristics, Effects and Side Effects of Plasma Substitutes 51-74; Rosenberg, Why 12 ^th Congr. Int: Soc. Blood Transf. (1969); Spahn, Snesth. Analg. (1994) 78: 1000-1021; Biomedical Advances In Aging (1994)

-1 CZ 294795 B6

Chapter 19; Wagner et al., Clin. Pharam. (1993) 12: 335; ATCC Catalog of Bacteria & Bacteriophages (1992) p 486; and 06-3874-R7-Rev. May (1987) Abbott Laboratories, North Chicago, IL 60064, U.S.A.

Other references describing physiologically acceptable solutions including hypothermal applications are: Bailes et al., Cryobiology (1990) 27: 615-696 (pp. 622-623); Belzer et al., Transplantation (1985) 39: 118-121; Collins, Transplantation Proceeding (1977) 9: 1529; Fischer et al., Transplantation (1985) 39: 122; Kallerhoff et al., Transplantation (1985) 39: 485; Leavit et al., FASB J (1990) 4. A963, Ross et al., Transplantation (1976) 21: 498; Segall et al., FASB J. (1991) Smith; Why Royal Soc. (1956) 145: 396; Waitz et al., Fasb J. (1991) 5.

lehninger, Biochemistry (2 ^nd Ed. 1975}, p. 829ff) describes an overview of blood components and their substitutes.

SUMMARY OF THE INVENTION

The present invention provides a physiologically acceptable aqueous solution for use in medicine and related fields, characterized in that it comprises electrolytes selected from the group consisting of sodium, chloride, potassium and calcium ions, a dynamic buffer system comprising a carboxylic acid, a salt or ester thereof, and at least one mass fraction of a polymeric oncotic active ingredient from a primary source of oncotic active ingredient, said weight fraction having a molecular weight range of 50,000 to 1,000,000, said solution comprising at least two magnesium, sugar in an amount of no more than 50 mM and oncotic an active ingredient with a low and medium molecular weight in the range of 50,000 to 200,000 or a high molecular weight in the range of 300,000 and 1,000,000 and beyond, wherein the range does not comprise a biological buffer.

The disclosed solutions comprise: an electrolyte, a dynamic buffering system, and an oncotic active ingredient, which solutions may further comprise either sugar or bicarbonate and will contain either magnesium or sugar. The disclosed solutions will find utility in a variety of applications, particularly when a portion of the patient's blood is to be replaced with a substitution solution. Next, aqueous solutions per se are described in detail first, followed by representative applications in which these solutions will find use.

It should be noted that the present invention is not limited to the specific variations of the invention described below, since a variation of the present invention may be used which is not described but is within the scope of the appended claims. It goes without saying that the terminology used to describe specific variants cannot be taken as a limit. The object and scope of the present invention is defined by the appended claims.

It is to be understood that all terms used in the singular shall take the form of a plural unless expressly stated otherwise. Also, unless expressly stated, technical and scientific terms have their usual meaning, as will be readily apparent to one of ordinary skill in the art.

The aqueous solutions of the present invention are physiologically acceptable, which means that they can be introduced into the patient's bloodstream without causing a toxic reaction. Thyroid solutions will have a pH in the range of 4 to 10, but more preferably 4.5 to 9, and most likely 5 to 8.5.

The proposed solution may contain a number of electrolytes, including sodium ions, chloride ions, potassium ions and, in certain circumstances, magnesium ions. The sodium ion concentration will range from 70 to 160, more preferably from 110 to 150, and in some particular variations, from 130 to 150 mM. The concentration of chloride ions in the solution will range from 70 to 170, but usually from 80 to 160, more preferably from 100 to 135, and in some particular variations, from 110 to 125 mM. The concentration of potassium ions in solution will be in physiologic concentrations

The term "physiological" concentration means from 3.5 to 5, but more preferably from 4 to 5 mM, and "lower" concentration is from 0 to 3.5, but more preferably from 2 to 3 mM. wherein in a number of specific variations the amount of potassium ions will range from 1 to 5, but more preferably from 2 to 3 mM. In some particular variations, the amount of potassium ions may be greater than 5 mM, even greater than 5.5 mM, but usually does not exceed 5.5 mM. The proposed solution also contains calcium ions in the concentration range of 0.5 to 6.0 mM and in most cases in the range of 0.5 to 4.0 mM, usually 2.0 to 2.5 mM. In some particular variations, it will range from 4.0 to 6.0 mM, more often from 4.5 to 6.0 mM. The proposed solutions may also contain magnesium ions. When present, the magnesium ions are comprised in the range of from 0 to 10 mM, but more preferably from 0.3 to 3.0, and most preferably from 0.3 to 0.45 mM.

In some particular variations, the solutions of the present invention will contain elevated levels of both potassium and magnesium ions. By elevated level is meant a concentration of potassium ions in the range from 50 mM to 3.0 M, but more preferably from 200 mM to 2.5 M, and most preferably from 1.0 to 2.5 M, and magnesium ions in the range from 40 mM to 1 M, 0 M, but more preferably from 0.1 to 0.9 M, most preferably from 0.3 to 0.7 M.

The present invention also provides solutions containing elevated levels of potassium and magnesium electrolytes (known as "super-weak" solutions). By an increased concentration level is meant a concentration of potassium ions in the range from 50 mM to 3.0 M, but more preferably from 200 mM to 2.5 M, and most preferably from 1.0 to 2.5 M, and magnesium ions from 40 mM to 1 M, These solutions may, in some particular variations, contain bicarbonate, wherein the bicarbonate will be present in a concentration range of from 0.1 to 40 mM. more preferably from 0.5 to 30 mM and most preferably from 1 to 10 mM.

The disclosed solution also comprises a dynamic buffer system, the term dynamic buffer system being used to refer to one or more agents operating in combination to maintain the pH of the solution within the range common to in vivo systems. It is preferable that these components are normal biological substances fulfilling this function even in in vivo biological systems. The concept of dynamic buffering systems rests on the volume of the inventors that substances without a clear buffering capacity in the biological range, such as lactic, acetic or gluconic acids, that are metabolized in the in vivo system, cooperate with other components of solutions to maintain a biologically acceptable pH in animals. even at hypothermic temperatures and essentially bloodless conditions. The dynamic buffer system of the present invention depends in part on oxygenation and removal of carbon dioxide (CO ₂ ). The dynamic buffer system of the present invention does not or substantially does not have the ability to function as a buffer outside the biological systems, i. the dynamic buffer system of the present invention is capable of maintaining the biological pH in vivo, but not in a cell-free environment.

A critical component of the dynamic buffer systems of the present invention are the carboxylic acids, salts or esters thereof. By carboxylic acids, their salts and or esters are meant compounds of the formula RCOOX, wherein R is alkyl, alkenyl or aryl, branched or unbranched, containing from 1 to 30 carbons which may be substituted and one of the carbon atoms contains a lactic acid chain, glucone, lemon, pyruvate and / or other biological metabolites, and wherein X is hydrogen or sodium or other biocompatible ionic substituent that may be associated at the oxygen position.

The dynamic buffer system may further comprise a source of bicarbonate, typically sodium bicarbonate (NaHCO ₃ ). If present, its concentration ranges from 0.1 mM to 40 mM, but more preferably from 0.5 mM to 30 mM, and most preferably from 1 mM to 10 mM.

The solution of the present invention does not contain a conventional biological buffer. By "conventional buffer" is meant a substance that maintains the pH at an appropriate value in solution in vitro. By " conventional biological buffer " is meant a component that maintains a pH in a biological solution of 7 to 8 in a cell-free system. Examples of conventional biological buffers are N-2-hydroxy

Ethylpiperazine-N-2-hydroxypropanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid (MOPS), 2 - ((2-hydroxy-1,1-bis (hydroxymethyl) ethyl) amino) ethanesulfonic acid (TES), 3- (N-tris (hydroxymethyl) ethylamino) -2-hydroxyethyl) -1-piperazinepropanesulfonic acid (EPPS), Tris (hydroxymethyl) aminomethane (THAM) and Tris (hydroxymethyl) methyl aminomethane ( TRIS). Conventional biological buffers have a pK in the physiological range and function most efficiently in this field. These buffers operate independently of normal biological processes and are most effective in cell-free systems.

The absence of conventional biological buffers in the solution of the present invention brings several significant advantages. For example, lower concentrations of buffers composed of natural biological components are necessary to maintain in vivo pH, as opposed to conventional biological buffers. Conventional biological buffers can also cause some problems due to their toxicity. The absence of biological buffers also allows the final thermal sterilization of the solutions. In general, medical solutions should be heat sterilized prior to administration to patients. The terms "thermally sterilized" or "finally thermally sterilized" as used herein describe the process of heating the solution to 120 ° C for 15 minutes at elevated pressure, i. at such a pressure and temperature and for a period of time that guarantees the destruction of substantially all bacteria and the inactivation of substantially all viruses in solution. This process normally takes place in an autoclave and is referred to as "autoclaving". The reason for heat sterilization is the effort to dispose of all possible infectious agents present in the solution.

Some infectious microorganisms are known to be able to survive temperatures up to 100 ° C. It is known in the art that heating the solution at elevated pressure to 120 ° C for 15 minutes is sufficient to achieve sterility.

The solution will also contain an oncotically active ingredient. This oncotic active ingredient is a molecule of sufficient size to prevent its loss from the bloodstream by entering the interstitial space of the body's tissues. As a group, oncotic active ingredients may be exemplary blood plasma substitutes. The substances useful as oncotic active ingredients in the solution of the present invention may be natural or synthetic and will be polymeric substances having an average molecular weight of at least 40,000, but usually at least 100,000 or more, most likely 200,000, wherein the oncotic active a molecular weight of 300,000 or more can also be used. Examples of oncotically active ingredients suitable for use in the solution of the present invention are protein ingredients such as albumin, e.g., human serum albumin, and linked or high molecular weight hemoglobin, polysaccharides such as glucan-based polymers and the like, organic polymers, e.g. PVP, PEG, etc., and the like, with non-antigenic polysaccharides being more suitable.

Polysaccharides useful as the oncotic active ingredients in the solution of the present invention are hydroxyethylated starches, hydroxymethyl alpha (1-4) or (1-6) polymers, D-glucose polymers such as alpha (1-6) bonded dextrans, cyclodextrins, hydroxypropyl starches, hydroxyacetyl starches and the like.

Hydroxyethylated starches are disclosed in several particular solution variants of the present invention. The average molecular weight of the hydroxyethylated starches usable in the solution of the present invention should be in the range of 10,000 to 1,000,000 or more, typically having a molecular weight of from 40,000 to 1,000,000, more preferably from 100,000. up to 900,000, and most likely from 200,000 to 800,000.

Most preferred are formulations in which the molecular weight of the oncotically active ingredient in the form of hydroxyethylated starch is in the range of from 50,000 to 1,000,000, but more preferably from 100,000 to 900,000, and most likely from 200,000 to 800,000. from 4 to 10, but in some particular variations it may be 7 to 10, and sometimes 4 to 5, and sometimes 6 to 7. One class of preferred solutions thus contains hydroxyethylated starches with 6 to 7 hydroxyethylated groups for every 10 glucose units. Another class of preferred solutions

Thus, it will then contain hydroxyethylated starches having 4 to 5 hydroxyethylated groups for every 10 glucose units, and yet another class of preferred solutions will contain hydroxyethylated starches having 7 to 8 hydroxyethylated groups for every 10 glucose units.

Essentially the most suitable oncotic active ingredient is Hetastarch (McGaw, Inc.), A waxy starch-derived artificial colloid composed essentially entirely of amylopectin with hydroxyethylated ether groups, attached to kglucose units, linked by an alpha (1-4) bond and having a molar substitution around 0.7 hydroxyethyl groups per glucose unit. The colloidal properties of the 6% (w / w) solution of Hetastarch roughly correspond to human serum albumin.

Another suitable oncotic active ingredient is Pentastarch with a molar substitution of 0.45 hydroxyethyl groups per glucose unit and an average molecular weight (measured by HPSEC technique as described by PDR 1996) ranging from 150,000 to 350,000, with 80% between 10,000 and 2,000. 000 000.

Another suitable oncotic active ingredient is Hexastarch with a molar substitution of the 0.64 hydroxyethyl group glucose unit and an average molecular weight of about 220000.

In some particular variations, the hydroxyethylated starches may be selected from the original hydroxyethylated starch fraction as a particular size fraction, generally that fraction will be at least one of molecular weight less than 1,000,000 or a fraction with an average molecular weight greater than 50,000. For these fractions, conventional fractionation processes can be used.

The concentration of the oncotic active agent in the solution is sufficient to maintain (when taken together with sodium, calcium and magnesium chloride, the organic ions from organic sodium salts and hexose sugars discussed above) of the colloidal osmotic pressure close to normal human serum, approximately 28 mm Hg. In general, the amount of oncotic active ingredient in the solution will be in the range of 0.5 to 30, usually from 1 to 25, and most preferably from 2 to 8% by weight. If the oncotic active ingredient is hydroxyethyl starch, the amount present in the solution will be in the range of 1 % to 30, more preferably from 2 to 15, and most preferably from 4 to 8 wt.

In one variation of the present invention, the solution comprises two or more oncotic active ingredients with different rates of clearance. Such a solution of the present invention, comprising two or more oncotic active ingredients with different rates of clearance from the bloodstream, has additional advantages in restoring blood oncotic pressure in hypovolaemic patients over an extended period of time while starting production of blood plasma proteins in the injured subject. Synthetic oncotic active ingredients with a long half-life are, for example, Hetastarch (molecular weight 300,000 to 1,000,000) and dextran 70, which have an intravascular persistence of approximately 6 hours (Mesmer (1989) Bodensee Symposium on Microcirculation (Hammersen & Messmer, eds.) Karger , NY page 59). Synthetic oncotic active ingredients with a relatively short half-life are low- and medium-molecular fractions of Heastarch (average molecular weight 40,000 to 200,000) and dextran 40 with an intravascular persistence of approximately 2 to 3 hours (Mermes (1989)).

The solution of the present invention may also contain other optional ingredients that may be added to the solution to make the solution suitable for a particular application, one of which is usually present is sugar. The sugar is usually hexose, such as glucose, fructose and galactose, with glucose being most suitable. In some particular variations of the present invention, nutritional hexoses in the form of a mixture of sugars are used. Sugars are usually, but not necessarily, present in solution in physiological amounts. By "physiological amount" or "physiological level" is meant a sugar concentration in the range of 2 mM to 50 mM, with a glucose concentration of 5 mM being most suitable. Sometimes you need to increase

Concentration of hexoses to reduce fluid retention in the tissues of the subject. The hexose concentration can then be increased to 50 mM and above, but usually not more than 60 mM, and even more preferably not more than 55 mM, if it is necessary to prevent edema in the treated subject, except when used as a cryoprotective agent.

The solution of the present invention may also contain clotting factors capable of accelerating or initiating the formation of a blood clot. The most suitable clotting factors for use in the solution of the present invention are vitamin K, Factors I, II, V, VII, VIII, VIIIC, IX, X, XI, XII, XIII, protein C, von Willebrand factor, Fitzgerald reactor, Fletcher factor and proteinase inhibitors. The concentration of clotting factors is determined by one skilled in the art depending on the specific treatment requirements. For example, when using vitamin K, the patient should be given 5 to 10 mg.

The solution of the present invention may also contain an oxygen-transporting component at a concentration low enough not to be toxic to the patient. The agent capable of binding and transporting oxygen should be present in an amount sufficient to transport oxygen to the tissues of the subject to be treated so as not to result in a toxic reaction. The term "sufficient amount" of oxygen-binding agent is an amount that allows a subject to remain free of circulatory and physiological disturbances to survive and recover from trauma, disease, or injury. In normal humans at normal temperature that is at least 5-6 ml 0 _2/100 ml of intravascular fluid. The oxygen-binding component may be hemoglobin extracted from a substrate of human or animal origin, recombinant hemoglobin, hemocyanin, chlorocruonin and hemerythrin and other naturally occurring respiratory pigments extracted from natural substrates or prepared by recombinant DNA or in vitro techniques. These materials can be modified by a variety of techniques known in the art, including chemical crosslinking, or covalent attachment to polyethylene glycol. If the oxygen-binding component is hemoglobin, it is desirably present in a concentration in the range of 20 to 200 g / l.

The solution of the present invention may also contain one or more cryoprotective agents, and the term "cryoprotective agent" means any substance that protects the structural integrity of a tissue under hypothermal conditions, eg, a temperature below zero, where in some particular variations the cryoprotective agent is a substance which disrupts at least partially the formation of water molecules into crystals such that the freezing point of such a composition containing the respective agent is lower than the freezing point of the non-containing agent. The cryoprotective agents may be: alcohols, especially low molecular weight aliphatic alcohols, usually C1 to C6 alcohols, but more preferably C1 to C4 alcohols such as methanol, ethanol and the like, polyols including linear, branched and cyclic polyols, especially low molecular weight aliphatic polyols including diols , trioles and other polyols such as sugars (described in more detail above), the most preferred polyols being diols, particularly ethanediol, propanediol, butanediol, trioles such as glycerol and the like, sugars including erythrose, threose, ribose, arabinose, xylose , lyxose, allose, atrose, glucose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose and disaccharides such as sucrose, lactose, maltose, but glucose, other substances are most suitable such as trimethylamine, trimethylamine oxide (TMAO), DMSO, urea, formamide, dimethylformamide and the like, clathrates, silicon-containing substances such as silanes and the like, fluorocarbon compounds and derivatives thereof, etc., wherein the cryoprotective agent may be brought into solution by elevated pressure and / or by a suitable surfactant, the choice of which depends on the person skilled in the art. These agents are usually present in an amount sufficient to achieve the desired cryoprotective effect, with the particular amount of the appropriate agent being dependent on the agent used. When it is a polyol, e.g. a diol, it can generally be said to be present in an amount ranging from 0.2 M to 1 M, or from 0 to 30% by weight. 2 M to 0.6 M and a concentration of about 0.4 M is most suitable for the use of propanediol. 1,2 propanediol is most suitable when low temperatures are needed to protect the donor and patient organs of the present invention, but 1,3 propanediol can also be used. The TMAO will be present in the solution at a final concentration ranging from 0.2 M to 7 M. If glycerol is used, it will be present in concentrations from 0 to 40% by weight, rather

However, from 5 to 30 wt. When DMSO is used, it will be at a concentration of from 0 to 40% by weight, but more preferably from 5 to 30% by weight. When using sugar (mostly glucose), the concentration will be in the range of 0.6 M to 1.4 M, but the most suitable concentration for some specific variants is 1.0 M. In one group of the preferred variants, the solution of the present invention will comprise at least two of the following components, magnesium ions, a sugar such as glucose and medium to high molecular weight hydroxyethylated starch.

The following solution design worthy of consideration:

Solution A:

High molecular weight Hetastarch (average 350,000 to 900,000) Ca ⁺⁺ K ⁺ Mg ⁺⁺ _ Lactic acid glucose 1 to 10% 1 to 6 mM 1 to 5 mM 0 to 10 mM 1 to 40 mM 0 to 50 mM

Solution B:

High molecular weight Hetastarch (average 350,000 to 900,000 Ca ⁺⁺ K ⁺ Mg ⁺⁺ lactic acid glucose bicarbonate 1 to 10% 1 to 6 mM 1 to 5 mM 0 to 10 mM 1 to 40 mM 0 to 50 mM 5 to 10 mM

Cryoprotective solutions

Solution I:

High molecular weight Hetastarch (average 350,000 to 900,000) Ca ⁺⁺ K ⁺ Mg ⁺⁺ Lactic acid Glucose Bicarbonate Glycerol 1 to 10% 1 to 6 mM 1 to 5 mM 0 to 10 mM 1 to 40 mM 0 to 50 mM 5 to 10 mM 10 to 20%

Solution II:

High molecular weight Hetastarch (average 350,000 to 900,000) Ca ⁺⁺ K ⁺ Mg ⁺⁺ Lactic acid bicarbonate glycerol 1 to 10% 1 to 6 mM 1 to 5 mM 0 to 10 mM 1 to 40 mM 5 to 10 mM 10 to 20

Solution III:

High molecular weight Hetastarch (average 350,000 to 900,000)

Ca ⁺⁺

K ⁺

Mg ⁺⁺ lactic acid glucose bicarbonate glycerol

DMSO up to 10% to 6 mM to 5 mM to 10 mM to 40 mM to 50 mM to 10 mM to 20% to 15%

Various substitutes may be used in the preparation of the solutions of the present invention, they may be agitated, or added sequentially as it seems most appropriate. The resulting resulting solution may ultimately be heat sterilized as described above. As also mentioned above, the solution may also contain non-thermally sterilizable substances, such as a source of bicarbonate, the bicarbonate being part of a dynamic buffering system. In these cases, sodium bicarbonate will be added as a sterile solution to the autoclave solution. Likewise, if a blood clotting factor or oxygen transfer agent is to be added, they are added as a sterile solution to the autoclave masterbatch.

For the purpose of describing the invention, the solution of the present invention is always considered in the form of an aqueous solution. For the following description of the invention, it is believed that one skilled in the art will be able to use this mixture in dried form and determine the amount of sodium chloride and organic sodium salts as needed to achieve the amount of sodium chloride contained in normal saline solution that can be used. according to the invention as a solvent for dissolving the dry form.

The solution of the present invention is useful in a variety of applications. The disclosed solution may find use in situations where at least a portion of the patient's (or its tissue) body fluid needs to be replaced with a replacement solution, and these applications may be: surgery, including processes requiring lowering the patient's body temperature to less than normal, a substitute for maintaining physical integrity after death, such as protecting tissues and organs by cold, partial chemoperfusion, and the like.

The solution can be used as a circulating solution in conjunction with oxygen or hyperbaric oxygen at normal body temperature or during processes where the body temperature of the patient's body is reduced considerably below normal body temperature. For example, during surgery and transplantation of organs from dead donors at low temperatures, the body fluid may be temporarily replaced by the supercooled solution of the present invention, which solution will be used only for the duration of the procedure to protect the recipient's organs from damage.

The solution of the present invention can be administered intravenously or intraarterially into a eutermic subject found at elevated oxygen concentrations of up to 100% by weight, or to a subject undergoing processes during which its body temperature is significantly lowered below its normal body temperature, whether hyperbaric oxygen is used or not. Once the solution is applied to the recipient ' s bloodstream, additional agents, such as cardiplegics, may be added directly to the subject's circulatory system, directly into the subject's myocardium, or sequentially added with the solution of the present invention. These agents are added to achieve the desired physiological effects, such as maintaining proper contractile heart activity, arresting cardiac fibrillation, or complete inhibition of contractile myocardial or cardiac muscle activity.

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Cardioplegics are agents that stop myocardial concentration with anesthetics such as lidocaine, procaine, and novocaine and monovalent ions such as potassium cation at concentrations suitable to inhibit myocardial concentrations. The concentration of potassium ions sufficient to maintain this effect usually does not exceed 15 mM and magnesium may be present at concentrations not exceeding 0.5 mM.

During the recovery of the subject (after a period of subnormal temperature or cryogenic intervention, with a solution of the present invention for treating the subject, the subject may be infused with a mixture of the solution of the present invention simultaneously with blood obtained previously from the treated subject or from blood donors.

As the subject is warmed up, the whole blood is delivered to the subject until normal hematocrit, usually exceeding about 20%, is reached. However, once an acceptable hematocrit is achieved, perfusion is discontinued and the subject is revived after completion of surgical procedures using standard techniques.

Generally, the solutions of the present invention are administered by intravenous infusion (if the subject is maintained at normal temperature), or a hypothermic subject, by a circulation device such as a centrifugal pump, a roller pump, a peristaltic pump or other known and available circulation device.

The solution can be used in a variety of ways and in various surgical procedures. It may find use in fine neurosurgery, where a clearly visible surgical site is a precondition and a reduction in central nervous system activity is necessary and maintained when the procedure is performed on a patient whose body core and / or cerebral temperature has been substantially reduced.

The solution may be used to maintain a subject (having a greatly reduced blood volume, e.g., 20% to 98% of its blood) at normal body temperatures in a pressurized environment at elevated oxygen levels above normal atmospheric levels of up to about 100% oxygen. The object is maintained in an oxygen-rich atmosphere either continuously or periodically until sufficient blood components are formed to allow the subject to survive at atmospheric pressure and normal oxygen concentration. The solution of the present invention may also be used to maintain a subject at reduced body temperature and reduced metabolic rate under severe fatal injury until appropriate medical or surgical procedures can be performed. Furthermore, this solution may be used in patients with a rare blood group or tissue feature until a suitable donor has been found and the blood or organ can be replaced.

Surprisingly, it has been found that it is possible to replace substantially all of the blood from mammalian bloodstream with a solution of the present invention and to maintain such a subject alive without reintroducing blood into its circulatory system. Essentially, all blood of a mammalian subject is considered to be replaced when the subject's hematocrit falls below 10%. The hematocrit may be less than 10% when O ₂ is artificially delivered to the subject, or substantially less than 10% in the hyperbaric oxygen chamber. Of course, the solution of the present invention can also be used to maintain a patient with a hematorkite exceeding 10%.

The process of replacing substantially all of the patient ' s blood can take place at their standard body temperature. It is also possible to carry out this process by gradually cooling the patient and reducing his body temperature to below normal. The subcooling may take place, for example, by immersing the subject in an ice water bath, salted ice crumb, or using a cooling sheet. The subject may be further subcooled by cooling the solution of the present invention before introducing it into the patient's bloodstream.

In the process of the present invention to replace substantially all blood from the bloodstream of a mammalian subject, it is desirable that the subject be super-cooled when introducing the solution into the

Blood circulation using an arterial catheter that introduces the solution of the present invention into the bloodstream of a patient, and a venous catheter through which blood and perfusate flows from the patient.

Essentially, all blood can be collected in this manner, as determined by the hematocrit of the fluid flowing from the venous catheter. Once all the patient's blood has been removed, perfusion can be stopped. The procedure for replacing substantially all of the blood from the bloodstream of a mammalian subject may be performed with hyperbaric O ₂ . The subject is placed in a hyperbaric chamber pressurized with oxygen at concentrations greater than 20%, preferably 100%. The pressure in the hyperbaric chamber is maintained for most of the time in the range of 3.5 kPa above normal atmospheric pressure up to about twice the standard atmospheric pressure. In one particular variation, the procedure is carried out on a subject in a hyperbaric chamber at an elevated pressure of 3.5 to 200 kPa above normal atmospheric pressure in a 100% oxygen atmosphere. If necessary, the pressure in the hyperbaric chamber may be reduced to normal during wound closure. The subject is then further maintained in a hyperbaric chamber and at high oxygen concentrations. The pressure is gradually reduced to lower values, but is still hyperbaric. Preferably, the pressure is maintained from 70 to 35 kPa for several hours to days. Thereafter, the pressure is again gradually lowered below 7 kPa, and more preferably below 3.5 kPa, and is maintained at this value for a further period of about several days.

The solution can also be used to maintain the physiological integrity of a donor organ immediately after brain death. The subject is supercooled, its blood is removed and replaced with a circulating mixture of less than 37 ° C, or with a supercooled solution of the present invention. The use of this solution thus minimizes the ischemia of living organs. By maintaining the circulating supercooled solution of the present invention in a subject's circulatory system at a reduced temperature and with or without placing the subject in a hyperbaric oxygen chamber, living organs can be maintained for a relatively long time thereby maximizing the number of organs that can be effectively used for transplantation to potential recipients. .

In another variation of the present invention, it has been found that with the use of certain additives such as propanediol and glucose at high concentrations, the temperature of the donor organs and even the donor heart can be brought below the freezing point of water (0 ° C) and kept in a usable state. ie. able to work and maintain a coordinated contractile function. In addition, using the solutions of the present invention with these additives, it is possible to reduce the body temperature of a complete mammalian subject below the freezing point of water (0 ° C) and recover from a frozen state capable of coordinating cardiac contractions, respiration and even conscious reactions. Other organs are believed to maintain a high degree of biological integrity; they remain in a life-capable state.

The following examples are given by way of illustration only and should not be construed as limiting the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following examples are given to those skilled in the art with complete conclusions and descriptions of how to ensure the synthesis of the solution of the present invention and should not be construed as limiting the present invention. Attention should be paid to the value of the values used (eg quantity, temperature, etc.), but a certain amount of experimental error and deviation is self-evident. Unless otherwise indicated, parts are meant by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at least about standard atmospheric.

Example 1. A solution according to the invention

1. Solution L solution

The formulation with solution L is composed of: Na ⁺ 143 mM, Ca ⁺⁺ 2.5 mM, Mg ⁺⁺ 0.45 mM, K ⁺ 3.0 mM, lactic acid 28 mM, glucose 5 mM, this solution is filtered, to remove undissolved material and transfer to autoclavable containers and heat sterilized at 120 ° C for 15 minutes.

2. HL (BioTime Hextend ™ - Lactate)

It is a formulation with solution L with the addition of 60 g / l of high-molecular-weight Heterstarch.

3. HLB (BioTime Hextend ™ - Lactate-bicarbonate)

It is a solution with HL solution with addition of 5 ml / 11 M NaHCO ₃ solution.

4. HL-DL (BioTime Hetadex ™ - Lactate)

It is a solution with HL solution where 6% Dextran 40 is used instead of 6% Hetastarch.

5. Al (BioTime Albextend ™)

It is a formulation with solution L with the addition of 50 g / l albumin. The ABL formulation is the same as the AL formulation with the addition of 5 mL / 1 M NaHCO ₃ solution.

6. HL-Heme

It is an HL solution formulation with the addition of 20 to 200 g / l hemoglobin.

7. L-Heme

It is a formulation with solution L with the addition of 20 to 200 g / l hemoglobin.

Example 2. Blood replacement with HL-DL solution

A female rat weighing 240 g was anesthetized i.m. injections containing a mixture of catemin, xylazine and acepromazine. The animal was placed on a surgical table and its right femur artery and vein were cannulated. The animal was perfused isovolemically with 10 ml of HL-D1 solution until its hematocrit reached 17.2%. The cannula was removed, the vessels were sutured and the wound closed. The animal tolerated perfusion very well and was completely active and ate normally. 3 days after the procedure the animal has recovered completely and is currently alive and healthy.

Example 3. Recovery of a dog after blood replacement in an ice bath

A male dog weighing 26.8 kg was anesthetized by nembutal injection and then intubated. It was transferred to the operating room, ventilated and catheterized by venous, Foley, arterial and SwanGanz catheters and after i.v. injection of hepatrin, the right femur artery and vein were cannulated. He was attached an esophageal tube and given an antacid. Temperature sensors were placed in the esophagus and the anal opening. Using i.v. methyl prednisol was injected. The animal was wrapped in a cooling blanket and surface supercooling was started. The cannulas of the animal were connected to a bypass circuit, which consisted of a vortex blood pump and an oxygenator with a built-in

A heat exchanger, a secondary, in-line heat exchanger and a connection for introducing blood and blood substituents. All blood (225 ml) was collected and protected from overheating. Blood was rapidly replaced by HLB solution. The bypass circuit, containing 1.05 liters of HLB solution, was opened towards the animal and cooling of the thermal core began.

Thirty-three liters of HLB solution were gradually replaced in the animal. Once substantially all of the blood had been removed, the animal was given enough blood of 2 m KCl to stop cardiac contractions. Once the ice temperature was reached, the hematocrit fell below 1%. The oesophageal temperature of the animal was less than 10 ° C for 4 hours and 5 minutes with a minimum temperature variation of 0.7 ° C.

After the hypothermic period, the animal was heated. As soon as the body temperature exceeded 10 ° C, the animal's blood previously collected, along with the blood of the donor, began to return to the bloodstream. Hematocrit increased with increasing temperature. Lidocaine and bicarbonate were administered to the animal, the heart defibrillated and breathing started. Once the blood pressure and body temperature were at normal levels, the animal was disconnected from the bypass and given protamine and Lasix. A few hours after heating, the animal was healthy and communicative. At present, the animal is already completely healthy.

Example 4. Reviving baboon after blood replacement in an ice bath

A male baboon of the species Papio annubis, 24 kg in weight, was first anesthetized with ketamine and acepromazine i.m. and then i.v. pentotalem. It was then immobilized with pancuronium bromide. It was intubated, ventilated, and catheterized by a venous, Folay, and arterial catheter. The animal was wrapped in a cooling blanket and surface supercooling started. After i.v. injection of heparin, his right femur artery and bilateral femur vein were cannulated. Temperature sensors were placed in the esophagus, the anal opening and the brain. The animal was connected to ECG, somatosensory-induced potentials (SSEPs) and EEG. Dexometasone was administered intravenously to the animal.

The cannulae were connected to a bypass circuit, which consisted of a vortex blood pump and an oxygenator with a built-in heat exchanger and a connection for rapid introduction of blood and blood substituents. All blood (300 ml) was collected and protected from overheating. The blood was rapidly replaced with 300 ml of saline. The bypass circuit, containing 2 liters of Plasmalyt (commercially available electrolyte solution), was opened towards the animal and cooling of the thermal core began.

When the temperature of the deep esophagus dropped below 13 ° C, an additional 2 liters of Plasmalyt containing 12.5 g of mannitol were added to replace the Plasmalyt, which had previously been run through the system. This diluted blood was stored for later heating. Immediately thereafter, 10 L of HLB was added, completely replacing the Plasmalyt. Once zero temperature was reached, hematocrit was less than 1%. When the animal's brain temperature dropped to 3.4 ° C and the deep esophagus temperature dropped to 2.8 ° C, the blood pump was stopped and the animal was maintained under circulatory arrest conditions for 45 minutes. After this time, circulation was restored.

After a hypothermic period, 4.2 liters of HLB solution were flushed through the animal's circulatory system until the vein eluent was maintained and the animal was gradually heated. When body temperature reached 15 ° C, 2 liters of Plasmalyt were added to the bloodstream to replace the HLB solution. Mannitol (6.25 g / l) was added to the plasma for circulation. Thereafter, the previously rinsed solution began to be returned to the bloodstream, as well as blood from the donor and freshly thawed plasma, and the animal's hematocrit increased with body temperature. Once the rectal and oesophageal temperature reached normal, the heart fibrillated during warming and began to beat normally. The animal also began to breathe normally. When blood pressure and body temperature reached normal, the animal was injected with protamine i.v., disconnected from the bypass, the cannula and catheters removed and the wound sutured.

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The deep oesophageal temperature of the test animal was less than 15 ° C for three hours and less than 10 ° C for 2 hours and 17 minutes with the lowest temperature measured at 2.8 ° C (Table 3). The following morning, the animal was able to sit upright in its cage, pick up and eat pieces of banana and drink apple juice. It was alive and healthy for one week after the experiment, when it was sacrificed and examined histologically.

Example 5. Blood substitution by a two-solution system in a patient undergoing surgery - cardiopulmonary bypass

The patient was anesthetized, cannulated and prepared for cardiopulmonary bypass. The patient was then wrapped in a cooling blanket and the body surface was cooled to 30 ° C. The patient was then connected to a bypass with a circuit filled with ABL solution. The patient's body was subcooled until his deep esophagus temperature reached 20 ° C. Blood is collected with ABL solution and immediately replaced with HBL solution. The body is then cooled and maintained under stable conditions as long as surgery is performed on the heart or brain. Thereafter, the patient is heated and the HBL solution is gradually replaced with the ABL solution and then the ABL solution and its blood as previously collected. Finally, 5 to 10 mg of vitamin K is administered.

One advantage of using ABL solution for bypass connection and blood collection is that the patient is replaced with his / her own diluted blood during heating and albumin functions as a natural component maintaining oncotic balance without the need to stimulate the patient's own albumin synthesis mechanisms.

Example 6. Emergency replacement of blood and hemorrhagic subject with HL-Heme solution

The patient suffering from blood loss is infused with an HL solution containing 5 mg / l clotting factor - vitamin K and 30 g / l hemoglobin as an oxygen-carrying agent. The patient's blood pressure is stabilized and a normal oxygen supply to the patient's tissues is ensured. The patient's body gradually breaks down Hetestarch, as it will be replaced by its own synthesized albumin, and thus the oncotic potential will remain stable during the treatment period. The use of a solution containing clotting factors and oxygen transfer components can completely replace the use of donated blood.

Example 7. Use of clotting factors in blood-diluted mammals

Six young female rats (227-262) were anesthetized, their right femoral arteries and veins were cannulated, and 40 to 60% of their blood was replaced with HL solution. Once the hematocrit was reduced to 16-22%, the animals were slowly injected in 6 ml of Trasylol ^R (10,000 KIU / ml). Their tails were separated 30 mm from the tip. The mean blood loss was 0.39 ± 0.06 (mean ± SEM) ml and all animals survived for at least one day except for one. Eight control animals were similarly perfused with the HL solution but without the injection of Trasylol ^(R) . The mean blood loss was 4.8 ± 0.54 ml and only 3 out of 8 animals survived. In comparison to untreated controls, the nortality (P <0.02) and blood loss (P <0.002) in HL perfused animals not receiving Trasylol ^(R) were significantly higher.

Example 8. Replacement of blood in a dog with a solution of HLB at zero temperature.

A dog weighing 25 to 30 kg was prepared for partial cardiopulmonary bypass. The animal's surface and core were cooled to a temperature in the region of 0 ° C (1-3 ° C). Dog blood was replaced with HLB solution under hypothermic conditions as described in Example 1. The collected blood was stored. The body temperature of the animal was regulated to a very low value (about

The blood was preserved. The body temperature of the animal was regulated to a very low value (about 4 ° C) and then the animal was heated. During warming, his blood was again gradually returned and the animal was revived.

Preparation: The dog was catheterized into the right radial vein, i.v. he was injected with pentothal, an endotracheal tube was inserted and ventilated with isofluorane (or Flether) in 100% oxygen. Lactate infusion was triggered to a value titrated for canine arterial blood pressure (about 40 mL / hr i.v.). The dog was then confiscated in a subcooling blanket and cooled with recirculated ice water. His right carotid was catheterized to monitor his blood pressure (CAP) and a three-way cock was added to allow blood samples to be taken every 10 to 60 minutes throughout the procedure. A Foley catheter was inserted to collect urine and urine levels were measured during surgery. Then, a 2 lumen, 4F, Swan Ganz catheter was implanted in the right jugular vein or right femur vein that leads from the right ventricle to the pulmonary artery. The distal end was used to measure pulmonary pressure (PAW), the proximal end was used to measure central venous pressure (PVP). If necessary, the central venous pressure can also be measured by a catheter inserted into the brachial vein. The left femural artery and vein were prepared and prepared for cannulation. The animal was given heparin (about 5000 µl). The venous Biomedicus venous return cannula (15-19 F) was inserted into the femur vein and the arterial Biomedicus cannula (12-15 F) into the femural artery. The activated blood clotting rate (ACT) was measured every 45 minutes (until the blood was replaced) and heparin was added gradually so that this value did not fall below 400 seconds. A thermocouple was attached approximately to the center of the esophagus tube and the unit was inserted so that the tube enters the stomach. The second thermocouple was placed rectally. ECG wires were connected. Then, Sol-Delta-Cortef (Upjohn, veterinary prednisolone succinate sodium) 80 mg was administered via i.v. injection. The eyes were covered with Terrimycin (or Lacrylub) and DiGel (or Maalox, 20 ml) was added via the esophagus tube.

Measurements: Blood gases, Ph, and hematocrits were measured in each blood sample, and in some cases, levels of ions, enzymes, and some other substances. The esophagus and rectal temperatures as well as the inlet temperature of the arteries and vein exit temperature were monitored. Blood pressure, pulmonary pressure, central venous pressure, ECG, and air pressure were also measured.

The temperature was displayed digitally and stored as a function of time in the computer data system. Individual pressures and ECGs can be displayed as wavy lines in real time or as numeric data and stored in a computer.

Components of the bypass circuit. This circuit consists of a Biomedicas centrifugal blood pump and flow meter, Terumo hollow fiber membrane oxidizer with built-in heat exchanger, a solid Shiley venous filter cartridge and a secondary heat exchanger with an integrated bubble trap (Elekctromedics), located as close to the animal as possible. The drain segment is located next to the inlet to the venous reservoir and terminated by a check valve. This allows rapid and effective blood / blood substitute replacement. There is an A-V sliding segment that allows circulation when a bypass is not connected.

The venous reservoir can be filled either by a single separating funnel through the "primary" inlet or from dual infusion bags through one of the cardiotomy inlets. The arterial conduit from the oxygenator to the arterial cannula and A-V switch is composed of 1/4 inch tubing, the venous return line, drain, and the main pump inlet is 3/8 inch tubing. Thick-walled tubes or “spiral reinforcement” tubes are used in these segments, where some stress may occur.

The loop leading into the patient is wrapped twice and the entire circuit (except the factory-sterilized tank, secondary heat exchanger and oxidizer) is sterilized in ethylene oxide vapors in six basic pieces (main pump component, flow meter, central bypass loop, inlet funnel, infusion line and air filter line).

- 14GB 294795 B6

Other bypass circuit equipment: Ice water pumped from one of two 401-liter insulated reservoirs is necessary for cooling the oxidizer and the secondary heat exchanger. Furthermore, a cooling blanket is supplied from the reservoirs. Before the surgery begins, water is circulated through the cooling blanket. At the start of the bypass, room temperature water is circulated through the heat exchangers.

The temperature is gradually lowered by adding ice to the reservoir so as to maintain a temperature of 7-10 ° C between the oesophageal temperature and the circulating blood temperature. After the blood has completely blocked (the hematocrit drops below 4%), maximum circulation of ice water is started.

Before preheating, the ice is removed from the reservoirs and the heater is activated. The temperature of the heating current is limited to a maximum of 10 ° C higher than the temperature of the returning blood; it is influenced by the temperature thermostat setting.

The oxidizer is filled with sterile 100% O ₂ .

Blood replacement: The circuit is filled with 2 liters of solution L (Example 1) and recirculated through the A-V segment so that temperature and gas balance would stabilize. The cannulae are connected to the arterial and venous conduit of the bypass circuit and the conduit remains closed. The quilt is wrapped around a patient who is supercooled until the deep esophagus temperature reaches 35 ° C.

The terminals are removed and the bypass is opened filled with solution L and diluted with blood at room temperature (about 25 ° C). At the beginning of hypothermia, gas anesthesia is stopped and the dog is anesthetized with 2.5% pentotal.

The blood stream is gradually cooled until the oesophageal temperature reaches 20 ° C, at which point the blood supply from the reservoir is stopped and pure solution L is infused. During this exchange two more liters of solution L are added to the reservoir and once its volume in the reservoir drops below 250 ml, about 6 liters of HLB solution is added in several steps until the blood is completely removed (HTC less than 2%, visual observation). About 4 liters of the blood / blood substitute mixture are collected in bottles and stored for reinfusion. The heavily diluted fraction (about 5.5 liters) is discarded.

Once 4 liters have been replaced (i.e. after adding 2 liters of solution L and 2 liters of HLB solution), 20 meq KCl is added to the circulation through the stopcock on the secondary heat exchanger, thereby stopping the heart. During the exchange, it is adjusted so that the pulmonary pressure is kept below 5 mm Hg and the outflow rate is the same as the inflow, i. approaching isovolemic values. At the end of the exchange, the reservoir volume will be about 500 ml, the pulmonary pressure will be less than 5 mm Hg and the central venous pressure will be less than 5 mm Hg. The flow will be maintained to maintain the isovolemic flow rate (constant reservoir level and pressures above, i.e. pulmonary pressure <5 mm Hg and central venous pressure <5 mm Hg).

Once almost all blood is removed (HCT less than 4%, visual observation), the cooling current can be reduced to 0 ° C (by filling the reservoir with ice) and the dog is rapidly cooled to a minimum temperature. If increased hematocrit is observed at this stage, this mixture may be replaced in exchange for 2 to 4 liters of HLB solution as above.

Blood samples are taken throughout the procedure and the pH, HCT, sometimes the concentration of electrolytes and other blood components are monitored.

After 1 to 2 hours of blood replacement and cooling, the temperature of the dog's body will be 1 to 4 ° C and will begin to heat. The dog will be heated by first removing the ice from the reservoir and heating the contents while heating the blanket. When the oesophageal temperature reaches 15 ° C, 4 liters of solution L with 25 mg mannitol will replace the HLB solution and are immediately replaced with 4 liters of the collected blood / substitute mixture. The washed-out solution is removed.

- 15GB 294795 B6

The animal is infused with autotransfusion or donor blood, cautiously heated with a difference of no more than 10 ° C and never 40 ° C. The heart begins to beat spontaneously or is defibrillated. Once the body temperature (esophagus and rectal) reaches about 35 ° C, physiological parameters stabilize, and the animal can be disconnected.

Example 9. Concentrated aqueous solutions of magnesium and potassium for heart protection

The invention described herein is a novel product, especially aqueous solutions containing high concentrations of K ⁺ and Mg ²⁺ , which are used in combination with an ice-cold HLB solution to replace blood in subjects undergoing surgery at reduced temperature, subjects undergoing cardioplegia or in organ donors to better protect organs such as the heart from transplantation.

These solutions can be used in hypothermic total flushing of the body with HLB at low temperatures to provide better protection for the heart during cardiac arrest. This solution can also be used as a cardioplegic agent in conjunction with HLB solution in standard surgical procedures that do not affect the whole body, or to protect the heart for transplantation after total body flushing with ice cold HLB solution.

In the preparation of solutions with high concentrations of Mg and K, their salts, MgSO ₄ and KCl are used. These agents are currently available in a form approved for use in human patients by the US FDA. Of course, MgCl ₂ can replace MgSO ₄ and KOH is useful to provide a basic pH of about 7.8 (in a solution of 7.2 to 8.4). Other substances useful for preparing a solution with a high concentration of K and Mg, are KHCO 3, K ₂ PO _4, lactobionate, potassium citrate, potassium acetate and potassium gluconate, as well as may be a solution containing a high concentration of K ⁺ and Mg ⁺⁺ , NaHCO ₃ added. The result is in any case a solution containing 1.5 M potassium and 0.5 M magnesium. First, a solution containing 50 mM KCl and 10 mM MgSO ₄ in HLB solution is prepared. 1-2 ml of this solution is injected iv into a vein or artery to induce cardiac arrest in small hypothermia blood substitute hamsters. This volume corresponds to 30 to 50% of the total volume of hamster blood. It has been experimentally confirmed that this solution, when administered intravascularly just after washing the body, induces cardiac arrest. It was found that about 9.5 ml of a 50% MgSO ₄ solution. H ₂ O (i.e. about 2M) and 27 ml of KCl, administered intraarterially to a supercooled dog (weighing about 25 kg, found a blood volume of 2.8 liters) whose blood has been replaced by HLB, greatly improves the protection of his heart K and MG are administered additionally, or when a dilute solution is used (i.e., after a first dilution of a concentrated K / Mg solution of about 500 ml of HLB solution in the oxidizer with a further dilution of the solution containing a further 850 ml of HLB) , 2 mM Mg ⁺ and 17.4 mM K ⁺ ).

A solution containing 37 ml of 0.55 M MgSO _fourth H ₂ O and 1.48 KKCl were administered to a portion of the bypass circuit (heat exchanger) that was directly attached to the subject's femural artery. Due to this dog experiment, it is anticipated that the use of this solution in patients will improve their heart protection while their blood is replaced by HLB at low temperatures. Likewise, when 0.1 ml of a high concentration of K ⁺ and Mg ^{++ is administered} intraarterially to a hamster, it effectively provides protection to its heart only when used immediately after hypothermic replacement of its blood with HLB.

The invention describes solutions used containing extremely high concentrations of K ⁺ and Mg ⁺⁺ which can be administered safely and efficiently to protect the patient's heart while hypothermia and blood substitute for HLB solution. The concentrations of K ⁺ and Mg ⁺⁺ that are applied are more than 1 times higher (for both ions) than in previously used protective solutions.

1. Recovery of the hamster using HLB and concentrated Mg / K solution

The female hamster, weighed 55 g, was digested overnight with ketamine 0.03 ml (100 mg / ml) and covered with crushed ice so that the rectal temperature dropped to about 13 ° C. The animal was then transferred

-16GB 294795 B6 on a surgical pad under a stereomicroscope. Cannulas were inserted into the carotid and jugular vein. The HLB solution was introduced into the artery and the vein drain was collected. The blood was replaced with an ice cold HLB solution. Once about 65% of the blood was removed, the animal's temperature was lowered to 1 ° C. Animal ventilation with 100% O ₂ began as soon as the temperature dropped below 10 ° C. About 7 ml or two volumes of blood are required to replace complete blood in the circulatory system. After perfusion was stopped, 0.2 ml (iv) of 0.5 M KCl with 0.1 M MgSO ₄ was applied. The heart stopped immediately. The animal was maintained at cardiac arrest for 5 hours and 30 minutes. 3 ml of HLB solution and blood from the donor were then perfused. During warming, normal heart and lung activity resumed. Upon awakening, the animal normally responded to stimuli such as slight abdominal grip or paws.

2. A 70 g hamster, digested overnight, was anesthetized with 0.03 ml (100 mg / ml) ketamine and covered with crushed ice to bring the rectal temperature to about 13 ° C. The animal was then transferred to a surgical pad under a stereomicroscope. Cannulas were inserted into the carotid and jugular vein. The HLB solution (with the addition of 50 mM glucose and 5 mM NaHCO ₃ ) was introduced into the artery and the vein drain was collected. The blood was replaced with an ice-cold HLB solution. Once about 65% of the blood was removed, the animal temperature was lowered to 1 ° C. Animal ventilation with 100% O ₂ began as soon as the temperature dropped below 10 ° C. About 7 ml or two volumes of blood are required to replace complete blood in the circulatory system.

The solution containing 0.02 ml 1 M MgSO ₄ and 0.1 M KCl is diluted with 0.1 ml HLB. The resulting solution, 0.22 ml, is administered iv. This corresponds to the administration of a mixture of 0.5 M KCl with 0.09 M MgSO ₄ . After 5 hours of cardiac arrest, the animal is perfused with 3 ml of HLB and donor blood. During warming, normal heart and lung activity resumed.

Previously, before the discovery of a high Mg / K concentration, it was not possible to re-recover the hamster after 5 hours of cardiac arrest.

3. A 70g female hamster was digested overnight with 0.03 ml (100 mg / ml) ketamine and covered with crushed ice until the rectal temperature dropped to about 13 ° C. The animal was then transferred to a surgical pad under a stereomicroscope. Cannulas were inserted into the carotid and jugular vein. The HLB solution was introduced into the artery and the vein drain was collected. The blood was replaced with an ice cold HLB solution. Once about 65% of the blood was removed, the animal's temperature was lowered to 1 ° C. Ventilation of the animal with 100% O ₂ began as soon as the temperature dropped below 10 ° C. About 5 ml are needed to replace complete blood in the circulatory system. Then 0.2 ml of 0.5 M KCl with 0.09 M MgSO ₄ was applied intraarterially. Then, 1 ml of HLB was injected to follow the "cardioprotectant" into the circulatory system and tissues. The animal was kept at cardiac arrest for 1 hour. Thereafter, perfusion was started with 4 ml of HLB and blood from the donor and the animal was gradually heated. The heart began to work spontaneously during warming, giving a normal ECG signal, followed by normal lung function, animal responsiveness, normal mobility and long-term survival. Previously, long-term survival was rarely observed in this type of experiment without the use of a solution containing high concentrations of Mg / K.

Example 10. Method of using HLB solution in occasional perfusion with additional bicarbonate

During a period of hypothermia to a very low temperature and when blood is replaced during circulatory arrest, recovery of some neurological functions takes longer compared to the case where the HLB solution is constantly circulating slowly. Improvements were found in hamster experiments when perfusing alternated with HLB, containing bicarbonate with a pure solution of HLB, which reduced acidosis. It also prevents stiffness and improves the restoration of brain functions as demonstrated in hamsters after a long cardiac arrest.

It is also described that slow perfusion of an ice cold HLB solution in dogs allows the period of cardiac arrest to be prolonged compared to complete arrest.

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Hamsters were found to have additional bicarbonate, 5 mM, suitable for reducing acidosis after prolonged circulatory arrest.

1. The hamster, weighed 70 g overnight, was anesthetized with ketamine 0.03 ml (100 mg / ml) and covered with crushed ice so that the rectal temperature dropped to about 13 ° C. The animal was then transferred to a surgical pad under a stereomicroscope. Cannulas were inserted into the carotid and jugular vein. HLB solution (with the addition of 50 mM glucose and 5 mM NaHCO ₃ ) was introduced into the artery and the vein effluent was collected. The blood was replaced with an ice cold HLB solution. Once about 65% of the blood was removed, the animal's temperature was lowered to 1 ° C. Ventilation of the animal with 100% O ₂ began as soon as the temperature dropped below 10 ° C. About 7 ml or two volumes of blood are required to replace complete blood in the circulatory system. Addition of 5 mM NaHCO ₃ adjusted the resulting pH to 8.5. After perfusion, the heart is stopped with 0.5 ml of 50 mM KCl and 10 mM MgSO ₄ . After 4 hours and 40 minutes, the animal was reperfused with 2 ml of HB1 followed by donor blood. The animal failed to resuscitate.

2. The same experiment as in Case 1 was performed except that intermittent bicarbonate perfusion was practiced for 15 minutes, 2 hours and 30 minutes under circulatory arrest. A hamster weighing 70 g, digested overnight, was anesthetized with ketamine 0.03 ml (100 mg / ml) and covered with crushed ice so that the rectal temperature dropped to about 13 ° C. The animal was then transferred to a surgical pad under a stereomicroscope. Cannulas were inserted into the carotid and jugular vein. HLB solution (with the addition of 50 mM glucose and 5 mM NaHCO ₃ ) was introduced into the artery and the vein effluent was collected. The blood was replaced with an ice cold HLB solution. Once about 65% of the blood was removed, the animal's temperature was lowered to 1 ° C. Animal ventilation of 100% O ₂ began as soon as the temperature dropped below 10 ° C. About 7 ml or two volumes of blood are required to replace complete blood in the circulatory system. Perfusion was then performed with an HLB solution (containing additionally 50 mM glucose) and 5 mM NaHCO ₃ to adjust the resulting pH to 8.5. After perfusion, the heart is stopped with 0.5 ml of 50 mM KCl and 10 mM MgSO ₄ . After 2 hours and 30 minutes, perfusion was applied with the HLB solution for 15 minutes (i.e., 2 ml HLB). After 4 hours and 40 minutes, perfusion was started with 2 ml HLB and then blood from the donor, and the animal was heated. The animal has completely restored its physiological functions. Cardiac arrest time was greater than 5 hours.

Example 11. Cryoprotective solution

1. Preparation of the solution

Method 1:

To prepare 50 mL of 10% glycerol in HLB, 45 mL of HLB is transferred to the reaction vessel. 5 ml of 100% glycerol is then added. The solution is stirred and finally filtered through a 0.2 or less micron filter.

b. Method 2:

Per 1 liter 15% glycerol solution in HLB mix: HES 60 g / l, NaCl 6.72 * g / l, dissolved in half the final volume, ie. in 500 ml deionized water. After stirring to dissolve gradually _MgCl2, 0.09 g / 1 _CaCl2, 0.37 g / 1 KC1 0.22 g / 1, glucose 0.9 g / 1 sodium lactate (60% syrup) 4 , 03 ml / l, NaHCO ₃ , 0.42 g / l or 0.84 g / l (additional bicarbonate may help reduce acidity. Add 850 ml deionized water. Add 150 ml 100% glycerol. Filter through a 0.2 micron filter. .

* the amount of NaCl added is adjusted according to the NaCl value given for starch, note: in the case of 50 mM extra glucose, 9 g / l glucose should be added with stirring.

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2. Cryoprotective solution of 10% glycerol in HLB, allowing recovery of partially frozen hamsters

A 60 g female hamster, digested overnight, was anesthetized with ketamine 0.03 ml (100 mg / ml) and covered with crushed ice until the rectal temperature dropped to about 13 ° C. The animal was then transferred to a surgical pad under a stereomicroscope. Cannulas were inserted into the carotid and jugular vein. The HLB solution was introduced into the artery and the vein drain was collected. The blood was replaced with ice-cold HLB solution with 10% glycerol. Once about 65% of the blood was removed, the animal's temperature was lowered to 1 ° C. Animal ventilation of 100% O ₂ began as soon as the temperature dropped below 10 ° C. After about 5 ml of cryoprotective solution was perfused, the heart was stopped with 1.5 ml of 50 mM KCl with 10 mM MgSO ₄ , administered intravenously. The animal was then transferred in a plastic bag and immersed in a cooling bath set at -15 ° C. After 30 minutes the rectal temperature dropped to -2.2 ° C.

The animal was then heated to thaw, perfused with HLB and then blood from the donor. After heating, a normal ECG signal appeared, followed by breathing with normal animal reactivity. A number of extensive lesions (microhemorrhagic) were observed in the brain following necropsy, demonstrating that the brain was essentially frozen under these conditions. Despite this, the animal managed to revive and it responded.

3. A cryoprotective solution containing 10% glycerol and 10% DMSO

A 60 g female hamster, digested overnight, was anesthetized with ketamine 0.03 ml (100 mg / ml) and covered with crushed ice until the rectal temperature dropped to about 13 ° C. The animal was then transferred to a surgical pad under a stereomicroscope. Cannulas were inserted into the carotid and jugular vein. The HLB solution was introduced into the artery and the vein drain was collected. The blood was replaced with 1.5 ml of ice-cold HLB solution followed by cryoprotective HLB solution with 10% glycerol and 10% DMSO. Once about 65% of the blood was removed, the animal's temperature was lowered to 1 ° C. Animal ventilation of 100% O ₂ began as soon as the temperature dropped below 10 ° C. After perfusion of about 6.5 to 7.5 ml of cryoprotective solution, the heart was stopped with 1.5 ml of 50 mM KCl with 10 mM MgSO ₄ , administered intravenously. The animal was then transferred in a plastic bag and immersed in a cooling bath set at -15 ° C. After 30 minutes the rectal temperature dropped to -1.5 ° C.

The animal was then heated to thaw, perfused with HLB in the blood from the donor. After heating, a normal ECG signal appeared, followed by breathing with normal animal reactivity. A number of extensive lesions (microhemorrhagic) were observed in the brain following necropsy, demonstrating that the brain was essentially frozen under these conditions. Despite this, the animal managed to revive and it responded.

All publications and patent applications cited herein are incorporated by reference, each of which is individually cited. Citations relating to publications describing the prior art prior to the date of administration of the present invention are evidence that the present invention is not disclosed in the prior art.

Although the present invention has been described in some cases in great detail, it will be apparent to those skilled in the art that these are only guidelines and illustrations and that many changes and modifications can be made without departing from the scope of the present invention.

Claims (16)

PATENT CLAIMS A physiologically acceptable aqueous solution for use in medicine and related fields, characterized in that it comprises electrolytes selected from the group of sodium, chloride, potassium, magnesium and calcium ions, a dynamic buffer system comprising a carboxylic acid, a salt or an ester thereof, at least one weight fraction of the polymeric oncotic active ingredient from a primary source of oncotic active ingredient, wherein said weight fraction has a molar mass range of 50,000 to 1,000,000, said solution comprising at least two magnesium, sugar not more than 50 mM, and oncotic active a low and medium molecular weight component in the range of 50,000 to 200,000 or a high molecular weight in the range of 300,000 to 1,000,000; and further wherein the solution does not contain a biological buffer. A physiologically acceptable aqueous solution for use in medicine and related fields, characterized in that it comprises electrolytes, a dynamic buffering system and at least one fraction of the oncotic active ingredient from the primary source of the oncotic active ingredient, said weight fraction having a molar mass range of 50 000 to 1,000,000, and wherein the oncotic active ingredient is a medium or high molecular weight hydroxyethylated starch. Solution according to claims 1 and 2, characterized in that the dynamic buffering system comprises carboxylic acids, their salts and esters. The solution according to claims 1, 2 and 3, characterized in that the dynamic buffer system further comprises bicarbonate. A solution according to any preceding claim, wherein the solution further comprises a cryoprotective component. A solution according to any preceding claim, wherein the oncotic active ingredient is a polysaccharide. A solution according to any preceding claim, wherein the polysaccharide is hydroxyethylated starch. A solution according to claim 7, wherein the hydroxyethylated starch is a medium molecular weight hydroxyethylated starch. A solution according to claim 7, wherein the hydroxyethylated starch is a high molecular weight hydroxyethylated starch. The solution of claim 7, wherein the hydroxyethylated starch contains 6 to 7 hydroxyethylated groups for each 10 monomer units and is present at a concentration of about 6% by weight, wherein the solution contains no more glucose than 50 mM. The solution of claim 7, wherein the hydroxyethylated starch contains 4 to 5 hydroxyethylated groups for every 10 monomer units and is present at a concentration of about 6% by weight, wherein the solution contains no more glucose than 50 mM. The solution of claim 7, wherein the hydroxyethylated starch contains 7 to 8 hydroxyethylated groups for each 10 monomer units and is present at a concentration of about 6% by weight, wherein the solution contains no more glucose than 50 mM. -20EN 294795 B6 A solution according to any preceding claim, wherein the electrolytes comprise sodium, potassium, calcium, chloride ions and magnesium. 5 A solution according to any preceding claim, further comprising a clotting factor. Use of a physiologically acceptable aqueous solution according to any preceding claim for the manufacture of a blood plasma substitute for introduction into a patient's circulatory system, or a portion thereof. O 16. A physiologically acceptable solution according to claim 1, wherein said oncotic active ingredient is a low or medium molecular oncotic active ingredient.