A SUBSTITUTE FOR HUMAN BLOOD AND A METHOD OF MAKING THE SAME

Abstract: The method of this invention yields a two phase liquid aquous system which replicates the two phase heterogeneous physico-chemical system of naturally securing whole human blood. The method produces: (1) a composition of matter which comprises an oxygen transport solution; (2) when pyridoxalated-polymerized hemoglobin and other specific additives are incorporated, said composition comprises a synthetic blood useful as a physiological substitute for human blood; and (3) when said blood substitute is processed further, yields a microencapsulated composition with time released characteristics that can transport oxygen. The invention overcomes the obstacles that have prevented the use of pyridoxalated-polymerized hemoglobin and other forms of modified hemoglobin in the preparation of oxygen transport solutions.

Description: A SUBSTITUTE FOR HUMAN BLOOD AND A METHOD OF MAKING THE SAME The invention concerns an improved composition useful as a substitute for human blood and as an oxygen transport system, and methods of preparation. In response to an evident need, a number of oxygen transporting solutions have recently been developed. Each reflects a different approach. At this point of development, most of these preparations exhibit either manufacturing or clinical difficulties. In some in¬ stances, both problems are present. Perfluorocarbon based compositions were among the earliest of these oxygen transport solutions. While such compositions ' possess oxygen carrying capability, difficulties in dwell time, in administering the prepara¬ tion and the suspicion of a toxic potential has raised serious questions regarding the safety and utility of this product. The effort to replicate erythrocytes through the development of liposomes containing stroma free hemoglobin represents a second approach. (Ref. Djorejevich, L; Miller, L, Lipid Encapsulated Hemoglobin as a Synthetic Erythrocyte. Fed. Proc. 1977, 36:567). The evidence to date suggests that in this approach, un- desireable and erratic effects are known to follow when the hemoglobin of the composition attaches to the exterior of the liposome during the process of manu¬ facture or leaks from the encapsulating liposome after the product is introduced into the circulation of the recipient. In either event, free hemoglobin is liberat¬ ed into the circulation of the recipient. The possible consequences of this event are well known to clinicians andothers skilled in the art. A third approach to the development of an oxygen transporting fluid is based upon efforts to modify the hemoglobin molecule through the process of pyridoxalation and polymerization. See: DeVenuto, F. and Zegna, A. Preparation and Evaluation of the Pyridoxalated-Polymerized Hemoglobin Molecule. Surg. Res. 35-205, 212 (1983). At least two major difficulties, appear to be associated with solutions containing modified hemoglobin. The first involves the problem of oxygen release; the second is the loss of the composition from the vascular space. The problem of the toxicity of this composition has apparently not been explored. These difficulties raise doubts about the utility of presently known oxygen transporting solutions based on modified hemo- globin. The fourth approach to the development of an oxygen transporting solution is based upon the applicants* inventions disclosed in U.S. Patent Nos. 4,343,797 and 4,439,424. The process involved in this approach makes use of a two phase liquid aqueous heterogeneous system, and yields a substitute for human blood. The herein disclosed invention represents a significant scientific advance in that through its use of a coacervate system the problems associated with liposome preparations and pyridoxalated-polymerized hemoglobin solutions are avoided. The manufacturing sequence of this invention is designed: (1) to yield a coacervate system which can serve as an oxygen transport solution; (2) to produce, when specific additives, including many of the forms of hemoglobin, i.e. stroma free hemoglobin, lipsome en¬ capsulated hemoglobin, pyridoxilated-polymerized hemo¬ globin etc. are incorporated in the coacervate system, a composition suitable as a substitute for human blood is produced; and (3) to provide, if desired, a form of microencapsulated hemoglobin with an approximate equival¬ ence to the cytoplasm of erthrocytes or packed red cells. The oxygen carrying solution herein described can also restore and maintain normal oncotic pressure when in- fused into the circulatory system. For purposes of this application, the ap¬ plicants draw a distinction between synthetic blood products disclosed in U.S. Patent Nos. 4,343,797 and 4,439,424, and oxygen transport solutions. For the most part the oxygen release curve of the latter differs significantly from that of synthetic whole blood. It is an object of this invention to provide a composition of matter which can serve as an oxygen transport solution, and further, when specific ad- ditives are incorporated in said composition, said com¬ position can serve as a substitute for human blood. It is another object to provide a convenient method for the manufacture of these compositions based on the process of σoacervation. It is a further object to provide a composition that has physiological characteristics equivalent to those of packed red cells, and further a convenient method of preparing this composition. More¬ over this invention provides a composition with char¬ acteristics similar to the cytoplasm of erythrocytes and a method of producing the same. In particular, the invention provides a com¬ position of matter which is useful as an oxygen trans¬ port system or, when appropriate additives are incor¬ porated, as a blood substitute; said preparation char- acterized by a non-toxic two phase liquid system, both said phases being aqueous; a) one of said phases being a relatively non- polar coacervate phase having physio¬ logical and physicochemical properties substantially equivalent to cytoplasm, as present in red blood cells; b) the other of said phases being a relatively polar liquid aqueous phase having physio¬ logical and physicochemical properties substantially equivalent to blood plasma; c) said relatively non-polar coacervate phase being insoluble in and in equilibrium with said relatively polar liquid aqueous phase, d) said two phase liquid system having physiological and physicochemical prop¬ erties which enable it to serve as an ef¬ fective oxygen transport system, e) . said two phase system, when prepared to contain hemoglobin and other additives in the coacervate phase of the coacervate system, has physiological and physico¬ chemical properties substantially similar to human blood. Moreover the invention provides a method of preparing a composition of matter which is useful as a blood substitute, said method characterized by the steps of (a) combining albumin and.aphospholipid in water; (b) thoroughly mixing the components; (c) storing said mixture undisturbed until the composition of step (a) separates into two layers, one above the other, the lower layer being a substantially non-polar coacervate phase, and the upper layer being an equilibrium water phase; (d) continuing the separation process until no increases in the volume of the coacervate phase can be observed; (e) centrifuging the composition until in¬ spection reveals a clear demarcation of the two phases; and (f) separating the two phases. This invention comprises compositions of mat- ter and methods by means of which they can be prepared. The claimed inventions comprise a combination of en¬ dogenous components and water which yields a two phase heterogeneous physico-chemical coacervate system similar to that of human blood. This coacervate system is the basis of the herein disclosed discovery. In practice, any appropriate non toxic co¬ acervate system can be used to manufacture the products of this invention, and further, any endogenous bio¬ logical surface active agent or derivative thereof, such as albumin, lecithin, gelatin etc. , can be used to prepare a coacervate system appropriate for the method and product of this invention. Appropriate non- toxic exogenous components, i.e. acacia gel, can also be used.to prepare suitable coacervate systems. . he method of making this invention begins with the preparation of a two phase aqueous liquid system, also referred to as " a coacervate system. When the prep¬ aration of the coacervate system is completed, it will consist of two phases: (1) an internal suspension, relatively non polar phase, commonly referred to as the coacervate phase; and (2) an associated, relatively polar external suspension or equilibrium phase. Both phases are in equilibrium with and insoluble in each other. The coacervate phase of this two phase system can comprise from about 0.5% to 99.5% by volume of the system; correspondingly, the associated equilibrium phase can comprise from about 0.5% to 99.5% by volume of the system. The preferred proportions are 50% volume to volume of each of these components. The coacervate phase of the claimed coacervate system possesses physiological properties equivalent to the cytoplasm of erythrocytes or packed red cells. As such, this phase of the coacervate system has sig¬ nificant oxygen transport capability. The equilibrium phase possesses physiological and physiochemical prop¬ erties equivalent to the plasma of human blood. The claimed two phase aqueous liquid system, (i.e. coacervate system) functions as an oxygen transport, solution and may be drawn off during the process of manufacture. When components such as ap¬ propriate proteins, electrolytes, sterols, any of sev¬ eral available forms of hemoglobin and an oxygen re¬ leasing entity are added to the coacervate system, the system achieves a functional physiological equivalence approaching that of human blood. If a composition with properties similar to the cytoplasm of erytrocytes or of packed red cells is desired, the disclosed coacer¬ vate systemand additives are subjected to warming and/or •other procedures. In such process the end product is microencapsulated hemoglobin. Enzymes, nutrients and drugs are among the ad¬ ditives that may be incorporated in the preparation of the blood substitute. Virtually any of the known forms of hemoglobin which are free of stromal toxicities may be used: i.e., stroma free hemoglobin, liposomes con¬ taining stroma free hemoglobin, polymerized hemoglobin, pyridoxalated-polymerized hemoglobin, microencapsulated ■ stroma free hemoglobin, etc. Human and other forms of mammalian blood, i.e. bovine blood, etc. comprise the sources of the hemoglobin component. The human source is preferred. When modified hemoglobin, i.e., the pyridoxala- ted-polymerized form, is incorporated in the claimed preparation as an additive, the problems presently as- sociated with such forms of hemoglobin, i.e. oxygen re- lease, at normal oxygen tensions and loss of hemoglobin solution from the vascular system, are eliminated. This is accomplished through the addition of an oxygen releasing molecule, such as di-phospho-glycerate to the coacervate system in the course of the manufacturing process. As used in this invention, di-phospho-gly¬ cerate acts to release oxygen from hemoglobin precisely as it does in the body. Small quantities of urea may also be added, if desired, during the preparation of this composition to further the release of .oxygen from the said composition. Loss of the oxygen transport solution from the vascular space is prevented in this invention by two factors: (1) by emulsifying the preparation wherein the resulting emulsified droplets in which the hemo¬ globin is contained are manufactured to a size of ap¬ proximately 7 microns, i.e., the size of normally oc¬ curring erythrocytes. Emulsified droplets of this size permit oxygenation of tissues, prevent escape of the solution from the circulation and allow entry of the hemoglobin bearing droplets into the microcirculation. The method of preparation, however, provides for the droplet size, if desired, to vary from 100 millimicrons to 15 microns. Loss of the solution from the vascular space is also prevented in this invention through the ef¬ fects of electrical charges present on the surface of droplets of the finished product and on the surfaces of arterial and venous branches of the circulatory system. Thus, the surfaces of blood vessels are negatively charged; the electrical charge on the surface of the emulsified droplets of the claimed composition is also negative. The resultant repulsant effect serves to pre¬ vent the loss of the solution from the circulatory system. In the finished product, the composition may by comprised of emulsified droplets of the same size or of any combination of sizes, depending upon the in¬ tended use. Thus, in a given version of this invention a preponderance of emulsified droplets of a size smaller than 0.6 microns may be indicated; example: when it is desired that the claimed composition penetrate in- farcted area(s) in the vascular system. While the disclosed invention indicates that equal proportions of albumin and lecithin are preferred in the preparation of the claimed composition, it is possible to produce coacervate systems using unequal proportions of albumin and lecithin. In the case of such usage, however, the resulting coacervate system may not have the optimal yield of the coacervate phase. However, the coacervate phase of such systems may pos¬ sess other desireable characteristics known to those skilled in the art, e.g., oxygen transport. The claimed invention also contains a pro¬ cess in the manufacturing procedure which yields de- rivative compositions. One of these is the physio¬ logical equivalent of the cytoplasm of erythrocytes. When hemoglobin is added to this preparation, the equivalent of packed red .cells is produced. The deriva¬ tive preparations can be subjected to microencapsula- tion procedures and to a heating step. The heating step will act to harden the surface of the coacervate phase droplets of the composition to any desired degree. This results in compositions with sustained release characteristics. If desired, achemical process using non-toxic members of the aldehyde group may be used. The heating procedure is preferred. With the exception of the inventors' contri¬ bution to the prior art, the scientific literature con¬ tains no reference to a two phase heterogeneous physico- chemical system which permits the effective incorpora- tion of modified forms of hemoglobin and further, which can serve as a useful substitute for human blood. In addition, there is no literature reference to a composi¬ tion which possesses physico-chemical properties that approximate those of the cytoplasm of erythrocytes or of packed red cells. In order to explain the claimed invention, the following is a general example of a preferred method of preparation. Specific examples appear in the fol- lowing section of this disclosure. In the preparation of the disclosed composition, the component ingredients are prepared and combined un¬ der sterile conditions. All water used in the manu¬ facturing process must be sterile and pyrogen free. The preparation of the appropriate coacervate system constitutes the first s.tep of the method neces¬ sary to produce the product of this invention. The preferred ingredients of this step are albumin and a suitable phospholipid. In this method lecithin is pre- ferred. However, other phospholipids known to those skilled in the art such as cephalin, isolecithin, sphingomyolin, phosphatidyl serine, phosphatidic acid, phosphatidyl inosital, phosphatidyl choline may also be used. In the preferred method, equal weight to volume proportions of albumin and lecithin are added to an amount of sterile water that will yield 100 mis. of aqueous solution. The mixture is then thoroughly mixed by vortex mixer. The preferred proportions for each component, i.e. albumin and lecithin, are 3% weight to volume. Unequal proportions of albumin and lecithin can yield a coacervate system. However, this method is not preferred. In the preferred method of preparation any quantity of albumin and lecithin can be used, provided the requirement of the proportions of the ingredients is observed and quantity of water used is adjusted ac¬ cordingly. Following thorough mixing the solution is stored in suitable containers. In the preferred method, the solution is stored undisturbed until the maximum yield of the coacervate phase of the coacervate system has been achieved. Maximum yield is the point at which no significant increase in the volume of the coacer- vate phase can be observed. This determination can be made by direct visual.inspection or other suitable means. As is known to those skilled in the art, longer periods of storage produce greater yields of the coacer¬ vate phase. The storage step may take place at tempera¬ tures ranging from freezing point to about 4 degrees C. and up to room temperature or higher. In the preferred method, storage takes place at a temperature of from about 4 degrees to 10 degrees C. . When it is .observed that the maximum yield of the coacervate phase has been achieved, the coacervate system is centrifuged until observation indicates that a clear division exists at the interface of the two phases of the coacervate system. If an oxygen trans- port solution is desired, the system is emulsified, the particle size of which may range from 100 milli¬ microns to 10 microns. The composition is placed in re¬ frigerated storage until needed. If the manufacturing objective is to produce a synthetic blood, the fol- lowing steps are initiated. The two phases are then separated-by means of a separatory funnel. The equilibrium phase is set aside for subsequent recombination with the coacervate phase. Any of the previously preferred to forms of hemo- globin is then mixed into the coacervate phase in an amount that will produce life sustaining oxygen ten¬ sions in the finished product. Thus any suitable form of hemoglobin known to those skilled in the art may be used: i.e. stroma free hemoglobin, liposomes contain- ing stroma free hemoglobin, polymerized hemoglobin, pyridoxalated-polymerized hemoglobin, stroma free micro¬ encapsulated hemoglobin or mixtures thereof. In this disclosure pyridoxalated-polymerized hemoglobin is pre¬ ferred. It is noted that the source of the hemoglobin component may be human or bovine. After this step, any oxygen liberating en¬ tity, such as di-phospho-glycerate is added, and mixed into the coacervate phase. The amount added may range from 1% or less to 6% or more weight to volume. In this disclosure 4% weight to volume of di-phospho- glycerate is preferred. The next step consists of recombining the equilibrium phase with the coacervate phase which now includes the additives described above. This is fol- lowed by a step in which the preparation is emulsified and an electrolyte is added. Any of the electrolytes known to those skilled in the art, i.e. sodium chloride, potassium chloride, magnesium chloride, or calcium chloride may be used. The purpose of this addition is to render the composition isotonic with human blood. Sodium chloride is the preferred electrolyte and is added in that quantity that will produce the desired isotonicity. At this point, if desired, 1 mg per cent of urea my be added. This component can act to facili- tate the release of oxygen from the hemoglobin present in the claimed composition. If desired, a sterol from the following group is added: chlosterol, ergosterol, 7-dehydrocholesterpl, 0< sitosterol, p sitosterol, γ sitosterol, campesterol or mixtures thereof. Choles- terol is preferred. 0.1 to 10 mg. per cent of choles¬ terol may be added to the preparation to improve the stability of the composition. The preferred amount of cholesterol added to the composition s 1 mg. per cent. Following this step the pH of the preparation is adjusted to 7.4 to 7.5 by the drop by drop addition of either HCl or sodium bicarbonate, depending upon the pH of the preparation. Any other suitable non toxic acidifying or alkalizing agent may be used in place of hydrochloric acid or sodium bicarbonate, however, the agents named are preferred. Upon completion of this step, the compo- sition is again emulsified using either a colloid mill, sonification or other emulsifying technique known to those skilled in the art. This step produces emul¬ sified droplets which contain the hemoglobin component. The droplets can range in size from less than 100 milli- microns to 15 microns and above; the preferred size is that of normal erythrocytes. However, the invention pro¬ vides for the possibility that specific medical treat¬ ments may require that the size of the droplets be of smaller dimensions. If desired, enzymes, nutrients and drugs may be added to the coacervate phase of the com¬ position or to the composition at this stage of manu¬ facture. If the manufacturing objective is to produce a composition that has the physiological properties of erythrocytes or packed red cells, the first step of that process consists of warming the preparation described immediately above. This step is accomplished by warming the preparation in a water bath or controlled oven to a temperature ranging from 15 to 50 C for from 20 seconds to 3 hours in order to produce a cross linking of the albumin and lecithin of the composition. The effect of this process is a hardening of the surface of the emul¬ sified droplets. The degree of hardness obtained is a function of the duration and temperature of the warming step. Thus, subjecting the composition for relatively shorter periods of time to higher temperatures will yield approximately the same degree of hardening of the emulsified droplet surfaces as subjecting the compo¬ sition to' lower temperatures for relatively longer periods of time. In point of fact, a spectrum of de- grees of surface hardness is possible at this point of manufacture by varying the variables of time and tem¬ perature. In this invention the degree of structuring or hardening of the surface of the emulsified droplets can range from fluid-like to semi-solid, i.e. gel¬ like to rigid. When the desired degree of surface hardness of the emulsified droplets has been achieved, the droplets are filtered from the emulsion. The fil¬ trate is discarded. The droplets are removed from the filter bed, washed thoroughly with normal saline or other suitable solution and then dried by any of the con¬ ventional methods. If desired, differing proportions of the dried preparation with differing degrees of shell hardness may be combined, during the process of re¬ constituting the preparation with normal saline or other suitable solutions. Alternatively, droplets of the same degree of surface hardness may be used in the process of reconstitution. In either formulation, the composition will possess special oxygen release proper¬ ties, and will be capable of prompt, sustained and/or prolonged effects. Cross linking may also be achieved through chemical means known to those skilled in the art, i.e., through the use of gluteraldehyde, etc. The method of heating is preferred in this invention. If it is de¬ sired to produce a product that has physiological prop¬ erties similar to the cytoplasm of erythrocytes, the pro¬ cedure described above is followed except that the hemo- globin component is omitted. When the manufacturing steps are completed, the products, i.e., the oxygen transport solution, the blood substitute, or either of the derivative compo¬ sitions, can be transfused into the circulatory system, where the individually described functions will be carried ' out: transport of physiological gases, restora¬ tion of blood pressure, transport of drugs and enzyme systems etc. Alternatively, each composition can be stored, preferably at from 4 to 10 degrees C until need¬ ed. * If the composition is to be infused into a human or animal following refrigerated storage, it should be warmed to body temperature (37 degrees C) before in¬ fusion. SPECIFIC EXAMPLES Examples of how the claimed composition(s) of matter may be prepared follow: EXAMPLE 1 5% weight to volume proportions of albumin and lecithin are added to an amount of sterile water that will yield 100 mis of aqueous solution. The mixture is then thoroughly mixed by vortex mixer. Following thorough mixing, the solution is stored undisturbed until the maximum yield of the co¬ acervate phase of the coacervate system has been achieved. The storage.step takes place at 4 degrees C. When it is observed that the maximum yield of the coacervate phase has been achieved, the coacervate system is centrifuged until observation indicates that a clear division exists at the interface of the two phases of the coacervate system. The two phases are then separated by means of a separatory funnel. The equilibrium phase is set aside for subsequent recombina¬ tion with the coacervate phase. 15 grams of pyridoxa¬ lated-polymerized hemoglobin are then dispersed into the coacervate phase. After this step, 4% weight to volume di-phospho-glycerate is added and mixed into the co- acervate phase. The next step consists of reco bining the equilibrium phase and the coacervate which contains the above named components and emulsifying the preparation, and adding that quantity of sodium chloride as will render the composition isotonic with human blood. At this point, 1 mg. per cent of urea is added. 1 mg. of cholesterol is added as the following step. The compo¬ sition is then mixed vigorously until all additives are dispersed. Following this step, the pH of the preparation is adjusted to 7.4 to 7.5.by the drop by drop addition of either HCl or sodium bicarbonate, depending upon the pH of the preparation at this stage of manufacture. Upon completion of this step, the composition is again emulsified using a colloid mill. The resulting emulsified droplets which contain the hemoglobin compon¬ ent are prepared to be 7 microns in size. EXAMPLE 2 .200 mis of 5% solution' of albumin is added to 200 mis. of a 3% solution of lecithin and mixed thoroughly. The remaining steps of the procedure follow those of Example 1. EXAMPLE 3 The procedure of Example 1 is followed ex¬ cept that the urea adding step is omitted. EXAMPLE 4 The procedure of Example 1 is followed ex¬ cept that the cholesterol adding step isomitted. EXAMPLE 5 The procedure of Example 1 is • followed ex¬ cept that 15 grams of stroma free hemoglobin are added in¬ stead of the pyridoxalated-polymerized hemoglobin. EXAMPLE 6 The procedure of Example 1 is followed ex¬ cept that liposomes containing a total of 15 grams of stroma free hemoglobin are used in place of pyridoxa- lated-polymerized hemoglobin. EXAMPLE 7 200 mis. of a 5% solution of albumin is added to 200 mis. of a 7% solution of lecithin and mixed thoroughly. The remaining steps of the procedure fol- low Example 1. EXAMPLE 8 200 mis. of a 3% solution of albumin is thoroughly mixed with 200 mis of a 3% solution of iso- lecithin. The solution is then stored undisturbed at . 4 degrees C for 24 hours. The remaining steps of the procedure follow Example 1. EXAMPLE 9 The procedure of Example 1 is followed ex¬ cept that the steps involving the addition of cholesterol and urea are omitted. EXAMPLE 10 The procedure of Example 1 is followed to completion. The resulting composition is then sub¬ jected to a warming step. This consists of placing the solution in a water bath at 25 degrees C for five min¬ utes. At the end of this period the droplets of the composition are filtered from the emulsion, and washed thoroughly with normal saline solution and dried by conventional means. 100 mis of normal saline solution are added to the product resulting from this process thereby reconstituting a composition, the physio¬ logical properties of which are equivalent to the cyto¬ plasm of packed red -cells. EXAMPLE 11 The procedure of Example 10 is followed! ex¬ cept that the warming stage is carried out at 30 degrees C -π- for 1 minute. EXAMPLE 12 The procedure of Example 1 is followed ex¬ cept that 2% weight to volume of di-phospho-glycerate is used. EXAMPLE 13 The procedure of Example 1 is followed ex¬ cept that 1 mg. per cent of ergosterol is used in place of cholesterol. EXAMPLE 14 The procedure of Example 1 is followed ex¬ cept that the emulsified droplets in the finished pro¬ duct are prepared to be 100 millimicrons in size. EXAMPLE 15 The procedure follows Example 1 except that after the emulsification step, essential amino acids such as L-lysine, L-tryptophan, L-histidine, L-phenyl- alanine, L-leucine, L-isoleucine, L-threonine, L-valine, L-orgine, and L-methionine can be added in the amounts and mixtures as indicated by the needs of the individual situation. EXAMPLE 16 The procedure follows Example 1 except that hemoglobin and di-phospho-glycerate components are omitted from the manufacturing process. This example produces a composition which approximates the physio¬ logical properties of the cytoplasm of erythrocytes.

Claims: CLAIMS 1. A composition of matter which is useful as an oxygen transport system or, when appropriate additives are incorporated, as a blood substitute; said prepara¬ tion characterized by a non-toxic two phase liquid system, both said phases being aqueous; a) one of said phases being a relatively non- polar coacervate phase having physiologi¬ cal and physiocochemical properties sub¬ stantially equivalent to cytoplasm, as present in red blood cells; b) the other of said phases being a relatively polar liquid aqueous phase having physio¬ logical and physicochemical properties substantially equivalent to blood plasma; c) said relatively non-polar coacervate phase being insoluble in and in equilibrium with said relatively polar liquid aqueous phase, d) said two phase liquid system having physio¬ logical and physicochemical properties which enable it to serve as an effective oxygen transport system, e) said two phase system, when prepared to contain hemoglobin and other additives in the coacervate phase of the coacervate system, has physiclogical and physico- chemical properties substantially similar to human blood. 2. A composition according to claim 1, where¬ in the relatively non-polar coacervate phase comprises from 0.5% to 99.5% by volume, and the relatively polar liquid aqueous phase comprises from 0.5% to 99.5% by volume, of the two phase liquid system. 3. A composition according to claim 1, wherein when the phases are emulsified, said relatively non- polar coacervate phase is in the form of coacervate droplets suspended in said relatively polar liquid aqueous phase. 4. A composition according to claim 3, where¬ in said droplets are essentially of a size within the range from 100 millimicrons to 10 microns. 5. A composition according to claim 1, wherein the pH of the two phase liquid system is in the range of from 7.35 to 7.45. 6. A composition according to claim 1, where¬ in the two aqueous phases comprise a protein or protein derivatives with surface active properties, an electrolyte, a surface active agent and water. 7. A composition according to claim 6, wherein the protein or protein derivative is selected from albumin, gelatin or modified fluid gelatin. 8. A composition according to claim 6, wherein the electrolyte is selected from sodium chloride, magnesium chloride, calcium chloride, potassium chloride and mixtures thereof. 9. A composition according to claim 6, wherein the surface active agent is a phospholipid or a derivative thereof. 10. A composition according to claim 9, wherein the phospholipid is selected from lecithin, cephalin, isolecithin, sphingomyelin, phosphatidyl serine, phosphatidic acid, phosphatidyl inositol, phos¬ phatidyl choline or mixtures thereof. 11. A composition according to claim 9, wherein the. phospholipid is lecithin, the protein is albumin, and wherein the concentration of albumin in said two phase aqueous system is equal to, less than, or greater than the concentration of lecithin. 12. The use of a composition as defined in any of claims 1 to 11, as an oxygen transport solution based on the two phase heterogeneous aqueous system. 13. A composition as defined in any of claims 1 to 11, wherein one or both of the aqueous phases include urea, electrolytes, hemoglobin, di-phospho- glycerate, sterols, or mixtures or combinations thereof. 14. A composition according to claim 13, wherein the hemoglobin component is selected from stroma free hemoglobin, microencapsulated stroma free hemoglobin, liposomes containing stroma free hemoglobin, polymerized hemoglobin, pyridoxalated-polymerized hemo¬ globin, or mixtures thereof, or hemoglobin derived from bovine or other appropriate mammalian sources. 15. A composition according to claim 13, wherein the hemoglobin is pyridoxalated-polymerized hemoglobin. 16. A composition according to claim 15, including di-phospo-glycerate. 17. A composition according to claim 13, wherein the sterol is selected from cholesterol, ergo¬ sterol, 7-dehydrocholesterol, <f** sitosterol,-^0 sitosterol, ^sitosterol or campesterol, or mixtures thereof. 18. A composition according to claim 13, wherein the electrolytes are selected from NaCl, KCl, MgCl, CaCl- or mixtures thereof. 19. The use of a composition as defined in any of claims 1 to 11 and 13 to 18 as a substitute for human blood. 20. The use of the coacervate phase of the composition as defined in any of claims 1 to 11 and 13 to 18,- that is physiologically equivalent to packed red cells. 21. The use of the coacervate phase of the composition as defined in any of claims 1 to 11, that is physiologically equivalent to the cytoplasm of erythrocytes. . T e use of a compos t on as ef ned in claim 1 for introducing and transporting enzymes, drugs and nutrients into the circulatory system of a recipient. 23. A method of preparing a composition of matter which is useful as a blood substitute, said method characterized by the steps of (a) combining albumin and phospholipid in water; (b) thoroughly mixing the components; (c) storing said mixture un¬ disturbed until the composition of step (a) separates into two layers, one above the other the lower layer being a substantially non-polar coacervate phase, and the upper layer being an equilibirum water phase; (d) continuing the separation process until no increase in the volume of the coacervate phase can be observed; (e) centrifuging the composition until inspection reveals a clear demarcation of the two phases; and (f) separating the two phases. 24. The method of claim 23, wherein the phospholipid is selected from lecithin, cephalin iso- lecithin, sphingomyelin; phosphatidyl serine, phosphatidic acid, phosphatidyl inositol, phosphatidyl choline, or mixtures thereof. 25. The method of claim 23, wherein the phospholipid is lecithin, and the concentration of albumin is equal to, greater than, or less than lecithin. 26. The method of claim 23, 24 or 25, in¬ cluding the steps of adding hemoglobin free of stromal toxicities to the coacervate phase in a quantity that will result in a concentration of hemoglobin in the finished product that will range from 1% to 20% weight to volume. 27. The method of claim 26, wherein the hemo¬ globin is selected from stroma free hemoglobin, lipo- somes containing stroma free hemoglobin, polymerized hemoglobin, pyridoxalated-polymerized hemoglobin, micro encapsulated hemoglobin or mixtures thereof, or hemo¬ globin derived from bovine or other appropriate mam¬ malian sources. 28. The method of claim 27, wherein the hemoglobin is pyridoxalated-polymerized hemoglobin. 29. The method of claim 28, including the step of adding from 0.5% to 10% weight to volume of di-phospho-glycerate to the coacervate phase, after the addition of pyridoxalated-polymerized hemoglobin. 30. The method of claim 29, including the further step of combining the equilibirum phase of the coacervate system and the associated coacervate phase now containing said additives. 31. The method of claim 30, including the furhter step of emulsifying the composition. 32. The method of claim 31, including the further step of adding an electrolyte in an amount that will render the isotonicity of the preparation equal to that of human blood. 33. The method of claim 32, wherein the elec¬ trolyte is selected from sodium chloride, potassium chloride, calcium chloride,, magnesium chloride , or mixtures thereof. 34. The method of claim 33, including the further step of adding o.1 to 1 mg. urea. 35. The method of claims 33 or 34, including the step of adding a sterol. 36. The method of claim 35, wherein the sterol is selected from cholesterol, ergosterol, 7- dehydrocholesterol cfe sitosterol, > sitosterol, ^sito¬ sterol, campesterol, and mixtures thereof. 37. The method of claim 35, including the step of adding from 0.1 to 10 mg. per cent cholesterol. 38. The method of any of claims 23 to 37, in¬ cluding the step of adjusting the pH of the* preparation to 7.35 to 7.4 by the dropwise addition of either hydro- chloric acid or sodium chloride. 39. The method of claim 38, including the further step of emulsifying the composition after said pH adjustment. 40. The method of claim 39, wherein the par¬ ticles of said emulsion range from 100 millimicrons to 10 microns. 41. The method of any of claims 31, 39 or 40, wherein the emulsified composition is subjected to apro- cess to harden the surfaces of the emulsified droplets contained within said emulsion. 42. The method of claim 41, wherein the hard¬ ening process is based upon either a physical or a chemical procedure. 43. The method of claim 42, wherein.the physi¬ cal process comprised of a warming step. 44. The method of claim 43, wherein the composition is subjected to a warming procedure in which the composition is placed in a water bath, the tempera¬ ture of which is from 15°C to 50 C. 45. The method of claim 44, wherein the warm¬ ing period is from 20 seconds to 3 hours. 46. The method of any of claims 41 to 45, wherein the surface hardened droplets are filtered from the composition. 47. The method of claim 46, wherein the emul¬ sified droplets containing the hemoglobin component are washed thoroughly. 48. The method of claim 47, wherein the washed particles of the composition are dried. 49. The method of claim 48, wherein the composition is reconstituted by the addition of any physiologically suitable solution. 50. The method of claim 49, wherein the solu¬ tion is normal saline solution.

Inventors: ECANOW BERNARD, ECANOW CHARLES S, ECANOW, BERNARD, ECANOW, CHARLES, S.
Applicants: NEOMED INC, NEOMED, INC.
Application year: 1985
Main classification: A61K37
IPC codes: A61K9, A61K38