SK6282000A3 - Physiologically acceptable aqueous solutions and methods for their use - Google Patents

Abstract

Physiologically acceptable aqueous solutions and methods for their use are provided. The subject solutions comprise: electrolytes; a dymamic buffering system and an oncotic agent; and do not comprise a conventional biological buffer. The subject solutions find 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

This application is linked to application number .........

filed on June 2, 1997 (Attorney Reference Number 06537/012001), which follows Application No. 08 / 780,974 filed 9.

No. 08 / 364,699, which is incorporated herein by reference

The subject of January 1997, which, which follows the application herein, is proposed to the application followed by the application number application number 08 / 253,384, number 08 / 071,533 and conclusions as citations.

The present solutions contain an electrolyte, a system and do not contain a portion of the patient's solution.

oncotic active ingredient, conventional buffers. Described in a number of applications, especially blood to be physiological and their use, dynamic buffering solutions will find solutions when replaced by substitution

BACKGROUND OF THE INVENTION

Physiologically acceptable solutions find application in a variety of applications in medicine, biomedical research and related fields. An example would be the use of physiologically acceptable solutions as a plasma substitute for surgical procedures where a large volume of blood plasma needs to be replaced. Another 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 by-pass device, where the addition of an additional circulating volume results in an empty volume which must be filled with a compatible blood substituent, i. a solution that increases the patient's blood volume.

Physiologically plasma substitutes with unacceptable changes in precipitates which inadequate amounts by introducing substances acceptable must be mixed in their usable solutions as freely mixed with the constituents such as clogged small blood vessels, without the destruction of blood components (blood cells creating water, ionic or imbalances). damage the cells of the body and platelets) of molecular tissue or cause severe physiological damage, such as a disproportionate increase or decrease in heart rate, nerve function, muscle activity, and the like.

The first blood plasma substitutes developed were prepared from mammalian blood. Although their use has been successful because they originate from a natural source, these derivatives may contain a number of pathogenic substances such as HIV-type pathogens, hepatitis B and other pathogens, e.g. prions whose presence is associated with Cruetzfeldt Jacob disease and the like.

Today there are a variety of synthetic blood and plasma substitutes. Although synthetic blood plasma substitutes are useful in many 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 surgery such as heart surgery and the like. It is also preferred that these solutions be heat sterilized.

A number of physiologically acceptable solutions, in particular blood substitutes and their use, are described in U.S. Pat. Patent Nos: RE 34 077; 3,937,821; 4,001,101; 4,061,736; 4,216,205; 4,663,166; 4,812,310; 4,908,350; 4,923,442; 4,927,806;

082 831; 5,084,377; 5,130,230; 5,171,526; 5,210,083; 5,274,001; 5,374,624 and 5,407,428.

Other citations 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, Proc. 12 ^th Congr. Int: Soc. Blood Transf. (1969); Spahn, Anesth. Analg. (1994) 78: 1000-1021; Biomedical Advances in Aging (1994) (Plenum Press) Chapter 19; Wagner et al., Clin. Pharm. (1993) 12: 335; ATCC Catalog of Bacteria & Bacteriophages (1992) p. 486; and 06-3874-R7-Rev. May (1987) Abbott Laboratories of North Chicago, IL 60064, USA.

Other citations 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 Proceedings (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; Proc. 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 relates to physiological solutions and their use. The disclosed solutions comprise an electrolyte, a dynamic buffer 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 application in a variety of applications, particularly when a portion of the patient's blood is to be replaced with a substitution solution. The aqueous solutions themselves are first described in detail, followed by a description of representative applications in which they are used.

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, although not described, is within the scope of the appended claims. It is also obvious that the terminology for describing particular variants cannot be considered as limiting. 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 be plural unless otherwise expressly stated. Also, unless expressly stated, technical and scientific terms have their usual meaning fully understood by one of ordinary skill in the art.

The aqueous solutions of the present invention are physiologically acceptable, meaning that they can be introduced into the bloodstream of a patient without causing a toxic reaction.

These solutions will have

A pH in the range of from 4 to 10, more preferably from

4.5 to 9 and most preferably from 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 concentration of sodium ions will range from about 70 to 160, more preferably from about 110 to 150, and in some particular variations, from 130 to 150 mM. The concentration of chloride ions in solution will range from about 70 to 170, but usually from 80 to 160, more preferably from about 100 to 135, and in some particular variations, from 110 to 125 mM. The concentration of potassium ions in solution will be at physiological concentrations and lower, wherein the physiological concentration is from 3.5 to 5, more preferably from 4 to 5 mM, and the lower concentration is from 0 to about 3.5, more preferably from about 2 to 3 mM. wherein in many particular variations the amount of potassium ions will range from 1 to 5, more preferably from 2 to 3 mM. In some particular variations, the amount of potassium ions will be greater than 5 mM and even greater than 5 mM

5.5 mM, but usually does not exceed 5.5 mM. The proposed solution also contains calcium ions in concentrations ranging from 0.5 to 6.0 mM and in most cases ranging from 0.5 to 4.0 mM, usually from 2.0 to 2.5 mM. In some particular variations, it will range from 4.0 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, 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 of from 50 mM to 3.0 M, more preferably from 200 mM to 2.5 M, and most preferably from 1.0 to 3.0 M.

2.5 M and magnesium ions in the range from 40 mM to 1.0 M, more preferably from 0.1 to 0.9 M, and most preferably from 0.3 to 0.7 M.

The present invention also describes solutions that contain elevated levels of potassium and magnesium electrolytes (known as supersaturate solutions). An increased concentration level is an imaginary concentration of potassium ions in the range of 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 1.0 M, more preferably from 0.1 to 0.9 M and most preferably from 0.3 to 0.7 M. These solutions may then, in some particular variations, contain bicarbonate, wherein the bicarbonate is present in concentrations ranging from 0.1 to 40 mN, 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, wherein the term dynamic buffer system is used to refer to one or more substances 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 that perform this function also in in vivo systems. The concept of dynamic buffer systems is based on the discovery of the inventors that substances without an apparent buffering capacity in the biological range, such as lactic, acetic or glucanic acid, 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 basically bloodless conditions. The dynamic buffer system of the present invention depends in part on the oxidation 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, ie, the dynamic buffer system of the present invention is capable of maintaining 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, their salts and / or esters. Carboxylic acids, salts and / or esters thereof are those of the formula RCOOX wherein R is alkyl, alkenyl or aryl, branched or unbranched, containing from 1 to 30 carbon atoms which may be substituted and one of the carbon atoms contains a lactic acid, gluconic acid chain , lemon, pyruvate or other biological metabolites, and wherein X is hydrogen or sodium or another, biocompatible ionic substituent that may be associated with 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 about 0.1 mM to about 0.1 mM

Ί mM, preferably from 0.5 mM to 30 mM, and most preferably from 1.0 mM to 10.0 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 the appropriate value in solution in vitro. By conventional biological buffer is meant a component that maintains a pH within the biological range of 7-8 in a cell-free system. Examples of conventional biological buffers are N-2-hydroxyethylpiperazine-N'-2-hydroxypropanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid. (MOPS), 2 - ((2-hydroxy-1,1bis (hydroxymethyl) ethyl) amino) ethanesulfonic acid (TES), 3- (N-tris (hydroxymethyl) ethylamino) -2-hydroxyethyl) -1-piperazinepropanesulfonic acid acid (EPPS), tris (hydroxymethyl) aminomethane (THAM) and tris (hydroxymethyl) methylaminomethane (TRIS). Conventional biological buffers have a pH 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 substantial advantages. For example, a lower concentration of buffers composed of natural biological components is necessary to maintain in vivo pH, as opposed to conventional biological buffers. Conventional biological buffers can cause some problems due to their toxicity. The absence of biological buffers also allows the final thermal sterilization of the solutions. Generally, solutions used in medicine may be heat-sterilized, heat-sterilized or ultimately sterilized prior to administration to patients as described herein by heating the solution at elevated pressure to about 120 ° C for 15 minutes, i. at such a pressure and temperature and over a period of time that guarantees the disposal of virtually all

The terms heat process the bacteria and inactivate virtually all the viruses in solution. This process is normally carried out 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 at 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 oncotic active ingredient. The oncotically active component is a molecule of sufficient size to prevent its loss from the bloodstream by entering the interstitial space of the body's tissues. Oncotic active ingredients as a group may be exemplary blood plasma substitutes. The substances useful as the oncotic active ingredients in the solution of the present invention may be natural or synthetic and are polymeric substances having an average molecular weight of at least 40,000, preferably at least 100,000 and more, most preferably about 200,000, and oncotic active agents may also be used. having a molecular weight of 300,000 or more. Examples of oncotically active ingredients suitable for use in the solution of the present invention are protein components 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, wherein non-antigenic polysaccharides are more suitable.

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

Hydroxyethyl starches are disclosed in several specific solution variants of the present invention. The average molecular weight of the hydroxyethyl starches usable in the solution of the present invention should be in the range of 10,000 d to 1,000,000 d or more, their molecular weight typically being in the range of 40,000 d to 1,000,000 d, preferably 100. 000 d to 900 000 d, most preferably from about 200 000 d to 800 000 d.

Most preferred are formulations having a molecular weight of the oncotically active ingredient in the form of hydroxyethyl starch in the range of from 50,000 d to 1,000,000 d, preferably from 100,000 d to 900,000 d, and most preferably from about 200,000 d to 800,000 d. The degree of substitution should be in the range of 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 hydroxyethyl starches with 6 to 7 hydroxyethyl groups for each 10 glucose units. Thus, another class of preferred solutions will contain hydroxyethyl starches with 4 to 5 hydroxyethyl groups for every 10 glucose units, and yet another class of preferred solutions will contain hydroxyethyl starches with 7 to 8 hydroxyethyl groups for every 10 glucose units.

The most preferred oncotically active ingredient is Hetastarch (McGaw, Inc.), a waxy starch-based artificial colloidal solution consisting essentially of amylopectin with hydroxyethyl ether groups attached to glucose units linked by an alpha (1-4) bond and molar substitution of about 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 about 0.45 hydroxyethyl groups per glucose unit and an average molecular weight (measured by HPSEC technique as described in PDR 1996) ranging from about 150,000 to 350,000 d, with 80% between 10 000 and 2 000 000 d.

Another suitable oncotic active ingredient is Hexastarch with a molar substitution of about 0.64 hydroxyethyl groups per glucose unit and an average molecular weight of about 220,000 d.

In some particular variations, the hydroxyethyl starches may be selected from the original hydroxyethyl starch fraction as a particular size fraction, generally the fraction being at least one of less than 1,000,000 daltons or an average molecular weight greater than 50,000 daltons. Conventional fractionation processes can be used to prepare these fractions.

The concentration of the oncotic active agent in the solution is sufficient to maintain (when considered concurrently with sodium, calcium and magnesium chloride, organic ions from organic sodium salts and hexose sugars discussed above) colloidal osmotic pressure close to normal human serum, approximately 28 mm Hg. Generally, the amount of the oncotic active ingredient in the solution will range from 0.5 to 30, usually from about 1 to 25, and preferably from 2 to 8%. When the oncotically active ingredient is hydroxyethyl starch, the amount present in the solution will range from 1 to 30, more typically from 2 to 15, and preferably from 4 to 8%.

In one variation of the present invention, the solution comprises two or more oncotic active ingredients with different rates of clearance from the bloodstream. The solution of the present invention, which contains two or more oncotic active ingredients with different rates of clearance, 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 Hetastarch (average molecular weight 40,000 to 200,000) and Dextran 40 with an intravascular persistence of approximately 2 to 3 hours (Mesmer (1989)).

The solution of the present invention may also contain other optional ingredients that may be added to the solution to be suitable for the particular application. One of these usually present ingredients is sugar. Typically, the sugar is hexose, such as glucose, fructose and galactose, with glucose being most preferred. In some particular variations of the present invention, nutritional hexoses are used in the form of a mixture of sugars. The 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 the most preferred glucose concentration being 5 mM. Sometimes it is necessary to increase the concentration of hexoses to reduce fluid retention in the subject's tissues. The hexose concentration can then be increased to 50 mM or more, but usually not more than 60 mM, preferably not more than 55 mM, if it is necessary to prevent edema in the subject being treated, except when used as a cryoprotective agent.

The solution according to the invention may also contain precipitants which are capable of accelerating or initiating the formation of a blood clot. The most preferred precipitants for use in solution according to the present invention are vitamin

K, factors I, II,

V, VII, VIII, VIIIC, IX,

X, XI, XII

XIII, protein C, von Willebrand factor,

Fitzgerald factor,

Fletcher factor and proteinase inhibitors.

The concentration of the precipitants is determined by the person skilled in the art according to 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-carrying agent at a concentration so low that it is not toxic to the patient. The agent capable of binding and carrying 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. A sufficient amount of an oxygen-binding agent is an amount sufficient to allow a subject to remain free of subsequent circulatory and physiological disorders to survive and recover from trauma, disease, or injury. In normal humans, and at normal body temperature it is at least 5-6 ml O _2/100 ml of intravascular fluid. The oxygen-binding agent may be hemoglobin extracted from a substrate of human or animal origin, recombinant hemoglobin, hemocyanine, chlorocruonin and hemerythrin and other naturally occurring respiratory pigments, extracted from natural substrates or prepared by recombinant DNA or in vitro techniques. These substances can be modified by a variety of methods known in the art, including chemical crosslinking or covalent attachment to polyethylene glycol. If the oxygen binding agent is hemoglobin, it is desirable to have 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, wherein the term cryoprotective agent means any substance that protects the structural integrity of the tissue under hypothermic conditions, e.g. at a temperature below zero, where in some particular variations, the cryoprotective agent is a substance that at least partially disrupts the formation of water molecules into crystals such that the freezing point of such a composition containing the agent is lower than the freezing point of the composition not containing it. The cryoprotective agent may be alcohols, especially low molecular weight aliphatic alcohols, usually C 1 -C ₆ alcohols, preferably C 1 -C 4 alcohols such as methanol, ethanol and the like, polyols, including linear, branched and cyclic polyols, aliphatic polyols, polyols such as sugars with the most preferred ethylenediol, propanediol, glycerol and the like, sugars, arabinose, xylose, preferably low molecular weight including diols, trioles and others (described in more detail by polyols are diols, butanediol, trioles including erythrose, threose, ribose, lyxose, alose, atrose , glucose, mannose, supra), in particular e.g.

gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, disaccharides, most preferably dimethylamine, silicone-containing formamide such as silanes and the like fluorocarbon and fructose, sucrose, glucose, e.g.

is sorbose trimethylamine oxide, lactose, others (TMAO), dimethylformamide and the like, such as urea, maltose tagatose, DMSO, clathrates, derivatives thereof. The cryoprotective agent can be converted into a solution by elevated pressure and / or by a suitable surfactant, the choice of which is within the skill of the art. These agents are usually present in an amount sufficient to achieve the desired cryoprotective effect, the particular amount of the appropriate agent depending on the agent used.

As regards the polyol, e.g. diol, generally speaking, it should be present in an amount of from 0.2 M to 1.0 M, or from 0 to 30%. Propanediol ranges from about 0.2 M to 0.6 M, the most preferred concentration of propanediol being about 0.4 M. 1.2 Propanediol is most preferred when low temperatures are needed to protect the donor and patient, however, also use 1,3 propanediol. The final concentration of TMAO in solution will be in the range of 0.2 M to 7.0 M. When glycerol is used, it will be in concentrations from 0 to 40%, preferably from 5 to 30%, and most preferably from 5 to 20%. When DMSO is used, it will be at a concentration of from 0 to 40%, preferably from 5 to 30%, and most preferably at a concentration from 5 to 20%. When using sugar (mostly glucose), the concentration will be in the range of 0.6 M to 1.4 M, but the most preferred concentration for some variants is 1.0 M. In one group of preferred variants, the solution of the present invention will contain at least two components of the following, magnesium ions, sugar, e.g. glucose and medium to high molecular weight hydroxyethyl starch.

Solution variants useful in the present invention:

Solution A

Hetastarch with high molecular weight

weight (average about 350,000 to 900,000) 1 until 10% Ca ⁺⁺ 1 until 6 mM K ⁺⁺ 1 until 5 mM Mg ⁺⁺ 0 until 10 mM lactic acid 1 until 40 mM glucose 0 until 50 mM

Solution B

Hetastarch with high molecular weight

weight (average about 350,000 to 900,000) 1 until 10% Ca ⁺⁺ 1 until 6 mM K ⁺⁺ 1 until 5 mM Mg ⁺⁺ 0 until 10 mM lactic acid 1 until 40 mM

glucose 0 to 50 mM bicarbonate 5 to 10 mM Cryoprotective solutions

Solution I Hetastarch with high molecular weight weight (average about 350 000 to 900 000) 1 until 10% Ca ⁺⁺ 1 until 6 mM A ⁺⁺ 1 until 5 mM Mg ⁺ * 0 until 10 mM lactic acid 1 until 40 mM glucose 0 until 50 mM bicarbonate 5 until 10 mM glycerol 10 i to 20% Solution II Hetastarch with high molecular weight weight (average about 350 000 to 900 000) 1 until 10% Ca ⁺⁺ 1 until 6 mM K ⁺⁺ 1 until 5 mM Mg ⁺⁺ 0 until 10 mM lactic acid 1 until 40 mM bicarbonate 5 until 10 mM glycerol 1C i to 20% Solution III Hetastarch with high molecular weight weight (average about 350 000 to 900 000) 1 until 10% Ca ⁺⁺ 1 until 6 mM A ⁺⁺ 1 until 5 mM Mg ⁺⁺ 0 until 10 mM lactic acid 1 until 40 mM glucose 0 until 50 mM bicarbonate 5 until 10 mM glycerol 10 until 20% DMSO 5 ' until 15%

Various substitutes may be used in the preparation of the solutions of the present invention, mixed at once or added sequentially, as it seems most preferred. The resulting resulting solution may finally be heat sterilized as described above. As also mentioned above, the solution may also contain non-heat-sterilizable substances, for example a source of bicarbonate, the bicarbonate being part of a dynamic buffer system. In this case, sodium bicarbonate will be added as a sterile solution to the autoclaved solution. Also, if it is necessary to add a blood clot or oxygen transfer agent, these are added to the autoclaved masterbatch in the form of a sterile solution.

For further description of the invention, the solution according to the invention is always considered in the form of an aqueous solution. For the following description of the invention, it is believed that one of ordinary skill in the art will be able to use this mixture in the dry state and determine the amount of sodium chloride and other organic sodium salts to be sufficient to dissolve the dry form in saline that be a usable solvent.

The solution of the present invention is useful in many different applications. The disclosed solution is useful in situations where it is necessary to replace at least a portion of the patient's body fluid (or tissue) with a solution.

The application may be a surgical procedure, including processes requiring a lowering of the patient's body temperature to less than normal, replacement for blood, maintenance of body integrity after death, such as protection of 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 patient's body temperature is reduced well below normal temperature. For example, during surgery and transplantation of organs from donor carcasses at low temperatures, the body fluid may be temporarily replaced with the supercooled solution of the present invention, which solution will only be used during the procedure to protect the recipient's organs from damage.

The solution of the present invention may be administered intravenously or intraarterially into a eutermic subject that has an oxygen overpressure of up to 100%, or to a subject undergoing processes during which its body temperature is significantly reduced below its normal body temperature, or when hyperbaric is not used. oxygen. Once the solution is applied to the bloodstream of the recipient, additional agents, such as cardioplegics, may be added directly to the subject's circulatory system, directly to 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 cardiac contractility, arresting cardiac fibrillation, or complete inhibition of myocardial or cardiac muscle contractile activity.

Cardioplegics are agents that cause myocardial contraction to stop, and inhibition of contractions can be maintained by anesthetics such as lidocaine, procaine and nonocaine, and monovalent ions such as potassium at appropriate 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 to treat the subject), the subject may be infused with a mixture of the solution of the present invention together with previously obtained blood from the treated subject or blood donor.

During the heating of the subject, the whole blood is introduced until a normal hematocrit, usually exceeding about 20%, is reached. Upon reaching an acceptable hematocrit, perfusion is discontinued, and the subject is revived by standard procedures upon completion of surgery.

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

The solution can be used in many different ways and for various surgical procedures. It can be used in fine neurosurgery, where a clearly visible surgical site is a prerequisite and a reduction in central nervous system activity is necessary and maintained during surgery on a patient whose body core and / or cerebral temperature has been substantially reduced.

The solution can be used to maintain a subject (having a significantly reduced blood volume, e.g., 20% to 98% of its blood) at normal body temperatures in a pressurized environment, at elevated oxygen contents above normal atmospheric levels of up to 10% oxygen. The object is maintained in an oxygen-rich atmosphere either continuously or periodically until sufficient blood components have been formed to allow the subject to survive at atmospheric pressure and normal oxygen concentration. The solution of the present invention can also be used to maintain a subject at a reduced body temperature and reduced metabolic rate following a serious lethal injury until appropriate medical or surgical procedures have been performed. Furthermore, this solution can 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 discovered that it is possible to replace substantially all of the blood from the 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 of the blood of the mammalian subject was replaced when the subject's hematocap fell below 10%.

The hematocap may be less than 10% when oxygen is delivered to the subject artificially, or substantially less than 10% in the hyperbaric oxygen chamber. The solution of the present invention can of course also be used to maintain a patient with a hematocrylate in excess of 10%.

The process of replacing substantially all of the patient's blood can take place at his or her standard body temperature. Furthermore, this process can be performed by gradually cooling the patient and reducing his body temperature to below normal. The subcooling may be carried out, for example, by immersing the subject in an ice water bath, in salt ice, or with a cooling blanket. The subject may be further super-cooled by cooling the solution of the present invention prior to its introduction into the bloodstream of the patient.

In the process of replacing substantially whole blood from the bloodstream of a mammalian subject according to the present invention, it is desirable that the subject be super-cooled while introducing the solution into the bloodstream using an arterial catheter that introduces the present invention into the bloodstream of the patient and venous catheter leaving. blood and patient perfusate. Essentially, whole blood can be drawn in this way, which can be determined by hematocryption of the fluid flowing from the venous catheter. Once all of the patient's blood has been removed, perfusion can be stopped. The process of replacing substantially all of the blood from the bloodstream of a mammalian subject may take place in hyperbaric O ₂ . The subject is placed in an oxygen pressurized hyperbaric chamber at concentrations greater than 20%, more preferably 100%. The pressure in the hyperbaric chamber is maintained most of the time from 0.5 pounds per square inch above normal atmospheric pressure to about twice the standard atmospheric pressure. In one particular variation, the procedure is carried out on the subject in a hyperbaric chamber at an elevated pressure of about 0.07 to 2.0 atmospheres above normal atmospheric pressure (0.5 to 30 pounds per square inch (psi)) in a 100% oxygen atmosphere. If necessary, the pressure in the hyperbaric chamber may be reduced to normal during the wound closure. The subject is then further held in the hyperbaric chamber at high oxygen concentrations. The pressure is gradually reduced to lower values, but is still hyperbaric. It is preferred to maintain the pressure from 10 psi to 5 psi for several hours to days. Thereafter, the pressure is again gradually reduced to below 5 psi, more preferably below 0.5 psi, and is maintained at this value for a longer period of time, about several days.

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

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, 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 coordinated contractility. In addition, using the solutions of the present invention with these additives, it is possible to reduce the body temperature of the complete mammalian subject to below freezing (0 ° C) and to recover from the frozen state so that it is able to coordinate cardiac contractions, respiration and even conscious reactions. Other organs are expected to maintain a high degree of biological integrity, i. they remain in a viable 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 accuracy of the values used (eg quantity, temperature, etc.), but a certain amount of experimental error and deviation is obvious. Unless otherwise indicated, parts are meant by weight, molecular weight is average molecular weight, temperature is in ° C, and pressure is at least about standard atmospheric.

Example 1

Solution according to the invention

A. Solution L solution

The composition of L solution consisting of 143.0 mM Na ^+, Ca ⁺⁺

2.5 mM, Mg ⁺⁺ 0.45 mM, K ⁺⁺ 3.0 mM, lactic acid 28.0 mM, and glucose 5.0 mM. This solution is filtered to remove insoluble material, transferred to autoclavable containers and heat sterilized at 120 ° C for 15 minutes.

B. HL (BioTime Hextend ™ - Lactate)

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

C. HLB (BioTime Hextend ™ -lactate-bicarbonate)

This is a HL solution with 5 ml / 1M NaHCO 3 solution.

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

It is a solution with HL solution, where instead of 6%

Hetastarch is used Dextran 40.

E. AL (BioTime Albextend ™)

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

F. HL-Heme

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

G. L-Heme composition

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

Example 2

Blood replacement using HL-DL solution

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

Example 3

Reviving the dog after blood substitution in an ice bath

A male dog weighing 26.8 kg was anesthetized by injection of nembutal and then intubated. It was transferred to the operating room, ventilated and catheterized by venous, Foley, arterial, and Swan-Ganz catheters and after i.v. injection of heparin, his right femur artery and vein cannulated. He was connected to the esophageal tube and given an antacid. Temperature sensors were placed in the esophagus and anus. Using i.v. methylprednisole was injected. After wrapping the animal in a cooling blanket, hypothermia was started. The cannula of the animal was connected to a bypass circuit, which consisted of a vortex blood pump and an oxygenator with a built-in heat exchanger, a secondary insertion for insertion. ml) and protected from being replaced by HLB solution.

blood (225 overheating. Blood was fast

Bypass circuit containing 1.05 towards the animal and cooling of the solution started

They were gradually replaced in the animal

HLB was opened to the thermal core. 33 1 HLB solution.

Once substantially all of the blood had been removed, sufficient 2 M KCl blood was introduced into the animal to stop cardiac contractions. When the freezing temperature was reached, the hematocrit fell below 1%. The temperature of the animal's esophagus 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 warmed. As soon as the body temperature exceeded 10 ° C, the animal's blood was drawn back to the bloodstream, along with the blood of the donor. The hematocrit increased with increasing temperature. The animal was given lidocaine and bicarbonate, the heart defibrillated and breathing started. Once the blood pressure and body temperature were at normal levels, the animal was disconnected from the by-pass and given the protamine and Lasix. A few hours after warming 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 24 kg male Papio annubis baboon was anesthetized first with ketamine and acepromazine i.m. and then pentotalom. It was then immobilized with pancuronium bromide. The animal was intubated, ventilated, and catheterized by a venous, Foley, and arterial catheter. The baboon was wrapped in a cooling blanket and supercooling began. After i.v. injection of heparin, his right femur artery and bilateral femur vein were cannulated. Temperature sensors were placed in his esophagus, anal opening, and brain. The animal was connected to ECG, somatosensory induced potentials (SSEPs) and EEG. He was given intravenous dexometasone.

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 2.0 L of Plasmalyte (commercially available electrolyte solution) was opened towards the animal and cooling of the heat core began.

As soon as the temperature of the deep esophagus dropped below 13 ° C, another 2 liters of Plasmalyt containing

12.5 g of mannitol, which replaced the previously running Plasmalyt. This diluted blood was stored for later heating. Immediately thereafter, 10 L of HLB was added to completely replace Plasmalyt. Once the zero temperature was reached, the hematocap was less than 1%. As soon as 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 kept in circulatory arrest conditions for 45 minutes. After this time, circulation was resumed.

After a hypothermic period, 4.2 L of HLB solution was flushed through the animal's circulatory system and the venous effluent was maintained and the animal was gradually warmed. Once the 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 bloodstream to Plasmalyt. The previously rinsed solution as well as blood from the donor and freshly thawed plasma began to be returned to the bloodstream. The animal's hematocrit increased with increasing temperature. Immediately after reaching normal rectal and esophagus temperature, the heart fibrils during warming and begins to normalize. The animal also began to breathe normally. Immediately after reaching normal blood pressure and body temperature, the animal received an injection of protamine i.v., was disconnected from the bypass, the cannulae and catheters removed and the wound sutured.

The temperature of the deep esophagus of the test animal was 3 hours below 15 ° C, below 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, lift and eat pieces of banana and drink apple juice. It was alive and healthy for one week after the experiment, when it was killed and examined histologically.

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

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

One advantage of using ABL solution for bypass and blood collection is that the patient returns their own diluted blood during heating and that albumin functions as a natural component that maintains oncotic balance without the need to stimulate the patient's own mechanisms for albumin synthesis.

Example 6

Emergency blood replacement in a hemorrhagic subject using HL-Heme solution

A patient suffering from blood loss is infused with an HL solution containing 5 mg / l of a vitamin K clotting factor and 30 g / l of 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 degrades Hetastarch as it is replaced by its own synthesized albumin and thus the oncotic potential will remain stabilized during treatment. The use of a solution containing precipitants and oxygen transfer components can completely replace the use of donated blood.

Example 7

Use of blood clotting in a mammal with diluted blood

Six young female rats (227-262) were anesthetized, their right femur arteries and veins were cannulated and 40-60% of their blood was replaced with HL solution. As soon as the hematocrit was reduced to 16-22%, the animals were slowly injected i.v. 6 ml Trasylol® (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 but one animals survived at least one day. Eight control animals were similarly perfused with HL solution but without Trasylol® injection. The mean blood loss was 4.8 ± 0.54 ml and only 3 of the 8 animals survived. Mortality (P <0.02) and blood loss (P <0.002) in HL perfused animals not receiving Trasylol ® were significantly higher compared to untreated controls.

Example 8

Replacement of dog blood with HLB solution at zero temperature

A dog weighing 25-30 kg was attached to a partial cardiopulmonary bypass. The animal's surface and core were cooled to a temperature in the region of 0 ° C (1-3 ° C). The blood of the dog was replaced under hypothermic conditions with HLB solution as described in Example 1. The collected blood was stored. The body temperature of the animal was reduced to a very low value (about 4 ° C) and then the animal was heated. As he warmed up, his blood was gradually returned and the animal was revived.

Preparation: The dog was catheterized into the right radial vein, i.v. he was injected with pentotal, inserted an endotracheal tube, and was ventilated with isofluorane (or Fleter) in 100% oxygen. Lactate infusion was initiated to a value titrated for canine arterial blood pressure (about 40 mL / hr i.v.). The dog was then wrapped in an undercoat and cooled with recirculating ice water. His true carotid artery was catheterized to monitor his blood pressure (CPA). 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 for urine collection and urine levels were measured during the procedure. Then, a 2 lumen, 4F, Swan Ganz catheter was implanted into the right jugular vein or right femur vein, which 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 (CVP). If necessary, the central venous pressure can also be measured by a catheter inserted into the barachial vein. The left femoral artery and vein were prepared and prepared for cannulation. The animal was given heparin (about 5,000 µl). The venous Biomedicus venous return cannula (15-19 F) was inserted into the femural vein and the Biomedicus arterial 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 the 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. Solu-Delta-Cortef (Upjohn, veterinary prednisolone succinate sodium), 80 mg was then administered via i.v. injections. The eyes were covered with Terrimycin (or Lacrylub) and DiGel (or Maalox, 20 ml) was added via the esophagus tube.

Measurements: Blood gas levels, pH and hematocrush were measured in each blood sample, and in some cases also levels of ions, enzymes, and some other substances. The esophagus and rectal temperatures as well as the inlet temperature of the arteries and the outlet temperature of the veins were monitored. In addition, blood pressure, pulmonary pressure, central venous pressure, ECG and air pressure were 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 on a computer.

Components of the bypass circuit: The 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 tank with filter and a secondary heat exchanger with integrated bubble trap (Electromedics), animal. The drain segment is located adjacent the inlet to the venous reservoir and terminates in a check valve. This allows rapid and efficient blood / blood replacement. There is an A-V sliding segment that allows circulation until the by-pass is connected.

The venous reservoir can be filled with either a one liter separatory funnel through the primary inlet or from dual infusion bags through one of the cardiotomy inlets. The arterial line from the oxygenator to the arterial cannula and the A-V switch is composed of 1/4 inch tubes, the venous return line, drain and main pump inlet are 3/8 inch tubes. In segments where a certain stress may occur, thick-walled or spiral-reinforced tubes are used.

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

Another bypass circuit device: ice water pumped from one of the two 10 gallon insulated reservoirs is essential for cooling the oxidizer and the secondary heat exchanger. Furthermore, a cooling blanket is supplied from the reservoirs. Water is circulated through the cooling blanket prior to surgery. At the start of the by-pass, room temperature water is circulated through the heat exchangers.

The temperature is gradually reduced by adding ice to the cartridges to maintain a 7-10 ° C difference between the temperature of the esophagus and the circulating blood temperature. After complete replacement of the blood (i.e., the hematocrit drops below 4%), maximum circulation of ice water is initiated.

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

The oxidizer is filled with sterile 100% oxygen.

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

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

The blood stream is gradually cooled until the esophagus temperature reaches 20 ° C. At this 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 as soon as its volume in the reservoir drops below 250 ml, about 6 liters of HLB solution are 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 placed in bottles and stored for reinfusion. The very dilute fraction (about 5.5 L) is discarded.

After changing 4 liters (i.e. after adding 2 L of L solution and 2 L of HLB solution), 20 meq KCl is added through the stopcock on the secondary heat exchanger to stop the heart. During replacement, the inflow is adjusted such 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 rate will be maintained to maintain the isovolemic flow rate (constant reservoir level and above pressures, i.e. pulmonary pressure <5 mm Hg and central venous pressure <5 mm Hg).

Once almost all the blood has been 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 an increased hematocrit is observed at this stage, this mixture may be replaced by replacement with 2 to 4 liters of HLB solution as described above.

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

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

The animal is infused with blood from autotransfusion or donor blood, carefully heated with a difference not exceeding 10 ° C, never 40 ° C. The heart starts spontaneously beating or defibrillated. Once the body temperature (esophagus and rectal) reaches about 35 ° C, physiological conditions stabilize and the animal can be disconnected.

Example 9

Concentrated aqueous solutions of magnesium and potassium to protect the heart

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

These solutions can be used in hypothermic total body flushing temperatures to ensure its arrest. This solution with HLB solution at low better heart protection throughout can also be used as a cardioplegic agent along with HLB solution in standard surgeries that do not affect the whole body or to protect the heart for transplantation after total body flushing with ice solution HLB.

For the preparation of solutions with a high concentration of Mg and K their salts, MgSO ₄ and KCl are used. These substances 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 the range of 7.2 to 8.4). Other compounds useful for the production of solutions with a high concentration of Mg and K are KHCO 3, K ₂ PO _4, lactobionate, potassium citrate, potassium acetate, potassium gluconate, may also be added to the solution containing high concentrations of K ⁺ and Mg ⁺⁺ NaHCO3 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. About 1 to 2 ml of this solution is injected iv into a vein or artery to induce cardiac arrest in small, chilled, blood-substitute hamsters. This volume corresponds to about 30 to 50% of the total hamster blood volume. It has been experimentally confirmed that when this solution is administered intravascularly just after washing the organism, it induces cardiac arrest. It was found that about 9.5 ml of a 50% MgSO ₄ .H ₂ O solution (ie about 2M) and 27 ml of KCl administered intra-arterially chilled dog (weight 25 kg, blood volume measured about 2.8 L), whose blood was replaced HLB greatly improves the protection of his heart compared to when K and Mg are administered additionally or when a dilute solution is used (ie after first diluting a concentrated K / Mg solution of about 500 ml of the HLB solution in the oxidizer and further diluting the solution in the circuit containing an additional 850 mL of HLB to form a solution containing 6.2 mM Mg ⁺⁺ and 17.4 mM K ⁺ ).

A solution containing about 37 mL of a solution of 0.55 M MgSO ₄ .H ₂ O and 1.48 M KCl was administered to a portion of the bypass circuit (heat exchanger) that was directly attached to the subject's femural artery. In view of this dog experiment, it is expected 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 a hamster is administered 0.1 ml of a high K ⁺ and Mg ⁺⁺ concentration intraarterially, it effectively provides protection for his heart only when used immediately after hypothermal replacement of his blood with HLB.

The invention describes the use of solutions 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 the HLB solution. The applied concentrations of K ⁺ and Mg ⁺⁺ are more than

Vyššieχ above (for both ions), protective solutions.

as previously used

1. Hamster recovery using HLB and concentrated Mg / K solution

A 55 g female hamster, fed overnight, was anesthetized with 0.03 ml (100 mg / ml) of 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 vein effluent was collected. The blood was replaced with 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% oxygen began as soon as the temperature dropped below 10 ° C. About 7 ml or two volumes of blood are required to replace whole blood in the circulatory system. After perfusion was stopped, 0.2 ml (i.v.) of 0.5 M KCl with 0.1 M MgSO 4 was applied. The heart stopped immediately. The animal was kept at a heartbeat for 5 hours and 30 minutes. Then 3 ml of HLB solution and donor blood were perfused. During warming, normal heart and lung activity resumed. Upon awakening, the animal normally responded to stimuli such as light compression of the abdomen or paws.

2. A 70 g hamster, digested overnight, was anesthetized with 0.03 ml (100 mg / ml) of ketamine 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 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% oxygen began as soon as the temperature dropped below 10 ° C. About 7 ml or two volumes of blood are required to replace whole blood in the circulatory system.

A 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.

Before the appearance of a high Mg / K solution, it was not possible to re-invigorate the hamster after 5 hours of cardiac arrest.

3. A 70 g female hamster, fed overnight, was anesthetized with 0.03 ml (100 mg / ml) of 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 vein effluent was collected. The blood was replaced with 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% oxygen began as soon as the temperature dropped below 10 ° C. About 5 ml is needed to replace whole blood in the circulatory system. Then, intraarterially administered 0.2 ml of 0.5 M KC1 in 0.09 M MgSO _fourth 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 of 4 ml of HLB and donor blood was initiated and the animal gradually warmed. During heart warming, it began to work spontaneously, showing a normal ECG signal, followed by normal lung function, animal responses, 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

Use of HLB solution for occasional perfusion with additional bicarbonate

During supercooling to a very low temperature and blood replacement during cardiac arrest, recovery of some neurological functions takes longer than when the HLB solution slowly circulates continuously. Hamster experiments showed an improvement when alternating perfusion with HLB containing bicarbonate and pure HLB solution that reduced acidosis. This also prevents measurability and improves the restoration of brain functions, as shown in hamsters after a long cardiac arrest.

It is also described that slow perfusion with ice solution

HLB allows to prolong the period of cardiac arrest in a dog compared to complete arrest.

Hamsters were found to be 5.5 mM additional bicarbonate suitable for long-term circulatory arrest to reduce acidosis.

1. A 70 g hamster, digested overnight, was anesthetized with 0.03 ml (100 mg / ml) of ketamine and covered with crushed ice so that the rectal temperature dropped to about 13 ° C. Then, the animal was 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 NaHCCl) was introduced into the artery and the vein effluent was collected. The blood was replaced with 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% oxygen began as soon as the temperature dropped below 10 ° C. About 7 ml or two volumes of blood are required to replace whole 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 mM MgSO ₄ . After 4 hours and 40 minutes, the animal was reperfused with 2 ml of HBL followed by donor blood. The animal failed to revive.

2. The same experiment as in Example 1 was performed except that intermittent bicarbonate perfusion was practiced 2 hours and 30 minutes after the circulation stopped.

A 70 g hamster, digested overnight, was anesthetized with 0.03 ml (100 mg / ml) of ketamine 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 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% oxygen began as soon as the temperature dropped below 10 ° C. About 7 ml or two volumes of blood are required to replace whole blood in the circulatory system. Perfusion was then performed with a solution of HLB (containing an additional 50 mM glucose) and 5 mM NaHCO 3 to adjust the resulting pH to 8.5. After perfusion, the heart was stopped with 0.5 ml of 50 mM KCl and 10 mM MgSO ₄ . After 2 hours and 30 minutes perfusion with HLB solution (ie 2 ml HLB) was applied for 15 minutes. After 4 hours and 40 minutes, 2 ml of HBL was perfused, followed by donor blood and the animal was heated. The animal has completely restored its physiological functions. Cardiac arrest lasted longer than 5 hours.

Example 11

Cryoprotective solution

1. Preparation of the solution

(a) Method 1:

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

(b) Method 2:

For 1 liter of a 15% glycerol solution in HLB mix: HES 60 g / l, NaCl 6.72 * g / l, dissolve in half the final volume, ie in 500 ml deionized water. Then, while stirring gradually dissolve 0.09 g / 1 _MgCl2, 0.37 g / 1 _CaCl2, 0.22 g / 1 KC1, 0.9 g / 1 glucose, 4.03 ml / 1 of sodium lactate (60 % syrup), 0.42 g / l or 0.84 g / l NaHCO3 (additional bicarbonate may contribute to reduced acidity). Add 850 ml deionized water. Add 150 mL of 100% glycerol. Filter through a 0.2 micron filter.

the amount of NaCl added is adjusted 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

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

A 60 g female hamster, fed overnight, was anesthetized with 0.03 ml (100 mg / ml) of 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 vein effluent was collected. 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. Ventilation of the animal with 100% oxygen began as soon as the temperature dropped below 10 ° C. After perfusion of about 5 ml 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 -15 ° C cooling bath. After 30 minutes the rectal temperature dropped to

-2.2 ’C.

The animal was then heated until it melted, perfused with HBL and then with donor blood. After heating, normal ECG appeared, followed by breathing and normal animal reactions. A number of extensive lesions (microhemorrhagic) were observed in the brain after necropsy, which demonstrated that the brain was essentially frozen under these conditions. Nevertheless, the animal managed to revive and it responded.

3. Cryoprotective solution containing 10% glycerol and 10% DMSO

A 60 g female hamster, fed overnight, was anesthetized with 0.03 ml (100 mg / ml) of ketamine 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. The HLB solution was introduced into the artery and vein effluent was collected. The blood was replaced with 1.5 ml of ice-cold HLB solution and then cryoprotective HLB solution with 10% glyceroloma 10% DMSO. Once about 65% of the blood was removed, the animal's temperature was lowered to 1 ° C. Ventilation of the animal with 100% oxygen began as soon as the temperature dropped below 10 ° C. After perfusion of about 6.5 to 7.0 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 -15 ° C cooling bath. After 30 minutes the rectal temperature dropped to -1.5 ° C.

The animal was then heated until it melted, perfused with HBL and then with donor blood. After heating, normal ECG appeared, followed by breathing and normal animal reactions. A number of extensive lesions (microhemorrhagic) were observed in the brain after necropsy, which demonstrated that the brain was essentially frozen under these conditions. Nevertheless, the animal managed to revive and it responded.

All publications and patent applications cited herein are hereby incorporated by reference, each of which is individually cited by reference. References to publications describing the prior art prior to the date of administration of the present invention demonstrate 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 (12)

PATENT CLAIMS A physiologically acceptable aqueous solution comprising: electrolytes, a dynamic buffering system, and at least one oncotically active ingredient, wherein the solution comprises at least two components of the following, magnesium, sugar in an amount of no greater than about 50 mM and a medium or high molecular, oncotically active ingredient. 2. A physiologically acceptable aqueous solution comprising: electrolytes, a dynamic buffer system and at least one oncotic active ingredient, characterized in that the oncotic active ingredient is a medium or high molecular weight hydroxyethyl starch. Solution according to claims 1 and 2, characterized in that the dynamic buffer system comprises carboxylic acids, their salts and esters. A 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, further comprising a cryoprotective component. Solution according to all of the preceding claims, characterized in that the oncotically active ingredient is a polysaccharide. A solution according to any preceding claim, wherein the polysaccharide is hydroxyethyl starch. 8th solution by of claim 7 No. u it T ý m that this hydroxyethyl starch is a hydroxyethyl medium molecular weight starch. 9th solution by of claim 7 No. u it T ý m that this hydroxyethyl starch is a hydroxyethyl high molecular weight starch. The solution of claim 7, wherein the hydroxyethyl starch contains 6 to 7 hydroxyethyl groups for every 10 monomer units and is present at a concentration of about 6%, wherein the solution contains no more than 50 mM glucose. The solution of claim 7, wherein the hydroxyethyl starch contains 4 to 5 hydroxyethyl groups for every 10 monomer units and is present at a concentration of about 6%, wherein the solution contains no more than 50 mM glucose. The solution of claim 7, wherein the hydroxyethyl starch contains 7 to 8 hydroxyethyl groups for every 10 monomer units and is present at a concentration of about 6%, wherein the solution contains no more than 50 mM glucose. A solution according to any preceding claim, wherein the electrolytes comprise sodium, potassium, calcium, chloride ions and magnesium. 14. solution by any prevent precedence claim, v y wit u j ú c i sa t further It contains precipitant. 15th process by any previous claim, v y wit u j ú c i sa t that in the introduction into the of the circulatory system of a patient or part thereof, the use of a physiologically acceptable aqueous solution according to any preceding claim as a blood plasma substitute brings about an improvement. 16. A physiologically acceptable solution according to claim 1, wherein said oncotic active ingredient is a low or medium molecular, oncotic active ingredient.