WO1984001717A1

WO1984001717A1 - Gelatin based synthetic whole blood and a method of making the same

Info

Publication number: WO1984001717A1
Application number: PCT/US1983/000739
Authority: WO
Inventors: Charles S Ecanow; Bernard Ecanow
Original assignee: Neomed Inc
Priority date: 1982-10-29
Filing date: 1983-05-16
Publication date: 1984-05-10
Also published as: IL68744A0; IL68744A; US4539204A; AU564308B2; CA1206093A; EP0124527A1; IT8348451A0; DE3372915D1; IT1228589B; AU1554583A; EP0124527B1

Abstract

A composition of matter which comprises a synthetic whole blood useful as a replacement for whole mammalian blood and a method of making the same. The method of manufacture makes use of two gelatins, two modified fluid gelatins, or one gelatin and one modified fluid gelatin of differing isoelectric points, and selected additives. The invention also contemplates the use of only one gelatin or only one modified fluid gelatin, plus lecithin. The invention also comtemplates using stroma free hemoglobin, microencapsulated stroma free hemoglobin, or synthetic liposomes containing stroma free hemoglobin incorporated in either the coacervate phase or the coacervate system of this invention. The method yields a two phase liquid aqueous system which successfully duplicates the two phase heterogeneous physico-chemical system of naturally occurring whole blood. The disclosed composition possesses many of the physiological capabilities of whole human blood.

Description

GELATIN BASED SYNTHETIC WHOLE BLOOD AND A METHOD OF MAKING THE SAME The present invention relates to a synthetic whole blood useful as a replacement for whole mammalian 5.blood and a method of making the same. It has now been : unexpectedly discovered that a synthetic blood may be made by incorporating either microencapsulated hemoglobin or synthetic liposomes containing stroma free hemoglobin, into the coacervate phase of an appropriate coacervate system or into an appropriate coacervate system. It has also now been unexpectedly discovered that as long as lecithin is present, a synthetic whole blood may be made using only one gelatin or only one modified fluid gelatin, instead of two. It is now recognized that the physical chemical structure of whole human blood has been successfully dup¬ licated in a composition of matter known as Synthetic Whole Blood, as disclosed in Applicants' United States Patent 4,343,797. It is now also recognized that Syn- thetic Whole Blood is a distinct entity, fundamentally different from the preparations referred to in the scien¬ tific literature as "blood substitutes".

An appropriate two-phase aqueous liquid system (i.e. coacervate system) is fundamental to preparation of synthetic whole blood and its companion product, synthetic he atocrit. U.S. Patent 4,343,797 contains the comment, "In the practice of this invention the under¬ lying principle is that any molecule or combination of molecules capable of forming a non-toxic, two-phase, aqueous liquid system can be...used to prepare the

OMPI J requisite coacervate system. " Further study of the dis¬ covery of this principle by the inventors makes it possible to specify this principle in greater detail. The present disclosure further exemplifies this principle. One category of coacervate systems useful to prepare synthetic whole blood contains among its prin¬ cipal components, (1) a suitable protein, i.e. albumin, gelatin, modified fluid gelatin, etc.; (2) a coacervating surface active molecule such as lecithin; each of these components possessing opposing surface charges; and (3) hemoglobin in the form of synthetic liposomes con¬ taining stroma free hemoglobin or stroma free hemoglobin per se, or microencapsulated hemoglobin.

The fundamental components of another category of coacervate systems useful to prepare synthetic whole blood contains (1) two similar or two different protein molecules, i.e. gelatin, modified fluid gelatin, etc., each with a surface charge that opposes the surface charge of the other; and (2) stroma free hemoglobin, microen- capsulated hemoglobin or synthetic liposomes containing stroma free hemoglobin.

Appropriate physiologically useful additives can be readily introduced into the compositions derived from either class of the coacervate systems described above.

A number of considerations warrant the develop¬ ment of an alternative version of the Synthetic Whole Blood preparation, as disclosed in U.S. Patent 4,343,797. Principal among these is the probability that a small but - medically significant number of persons may be sensitive to one or more of the ingredients of the composition re¬ ferred to above.

Aside from the United States Patent 4,343,797, the prior art that has been diligently searched fails to reveal any reference to a preparation which can serve as a whole blood replacement. The literature, however, does contain more than 1500 citations to entities de¬ scribed as "blood substitutes". - These citations refer to studies of such substances as perfluorocarbons, albumin, hydroethyl starch, modified gelatin, etc. (References; Chemical Abstracts; 1970-1982; Index Medicus; 1970- 1982). With the single exception of the references to the gelatin preparations used as "blood substitutes", none of the prior art in the clinical literature appears to have any relevance to the presently claimed invention. No citation was identified which hints at, suggests or implies that a synthetic whole blood can be based on gelatin.

To summarize findings from the clinical prior art, from a physiological point of^*" view, regarding available gelatin "blood substitutes", the molecular structure of gelatin is such that in clinical use, it can only serve as a plasma extender, (expand blood volume). It cannot transport any of the physiological gases. (Reference: Merck Index 1979). Unexpectedly, however, through their research applicants have discovered that gelatin and/or modified gelatin based coacervate systems can trans¬ port essential amino acids, transport physiolog¬ ically important gases and restore or maintain the necessary osmotic pressure. There are however addi¬ tional striking differences. Table I infra of this application lists 13 clinically important variables which distinguish the claimed compositions from the known gelatin "blood substitutes", and which show the similarities between the claimed compositions and whole human blood. In the prior art, is a reference to gelatin based coacervates, Veis, A. and Aranyi, C. , Phase Sep¬ aration in Polyelectrolyte Systems, I; Complex Co¬ acervates of Gelatin; Journal of Physical Chemistry, Volume 64, pages 1203-1205. Examination of this prior art indicates it to be a theoretical study of gelatin based coacervate systems_* It addresses only the condi¬ tions under which gelatins of differing isoelectric points will form coacervates. There is no suggestion nor inference in this prior art that the described coacervate systems have any possible biological or non-biological use. Therefore, the person ordinarily skilled in the art cannot conclude from a study of this prior art that a synthetic whole blood can be based upon it. Given that gelatin solutions are known to be among the available "blood substitutes", it is more probable that this cited prior art would suggest another method of preparing the already known gelatin based plasma extender.

The presently disclosed invention rests upon the applicants' recognition of the biological utility of a coacervate system.

However, this recognition is not of itself suf- ficient to prepare an optimal synthetic whole blood, ready to be administered to mammals, particularly humans. Specific chemical entities, some of which do not of themselves suggest that they are useful in the prepara¬ tion of a synthetic whole blood, must be added to the coacervate system employed in this invention. It is the applicants' position that these additives alter the chemical character and the physiological utility of the coacervate system, resulting in not another version of a gelatin based "blood substitute" but rather in a synthetic whole blood, which can be used as a replace¬ ment for whole mammalian blood.

As it now appears frequently in the literature, the term synthetic liposomes generically covers both stroma free hemoglobin and other stroma free hemo- globin preparations as well as synthetic erythrocytes or lipid encapsulated hemoglobin. Reference: Miller, I. and Djordjevich, L. ; U.S Patent 4,133,874 (1979). With regard to the Miller and Djordjevich reference, the possibility is mentioned that the syn- thetic erythrocytes they have invented can be suspended in isotoniσ saline or Krebs-Ringer solution or in syn¬ thetic plasma materials and used for blood transfusion purposes. Since the vehicles given above contain large quantities of bulk water, there is a strong likelihood that oxygen uptake in such compositions is limited. This stands in direct contrast with the oxygen uptake capability of the presently disclosed invention in which microencapsulated hemoglobin or liposomes containing stroma free hemoglobin is incorporated in the claimed coacervate system or the coacarvate phase of such a system. Both the coacervate system and the coacervate phase of the system have significant oxygen pick up. The addition of stroma free hemoglobin in the form given immediately above serves significantly to enhance the oxygen uptake of these claimed compositions.

This invention to provides an acceptable substi¬ tute for whole mammalian blood, and for preparing an acceptable substitute for whole mammalian blood. Therefore, the present invention provides for a synthetic whole blood comprising two gelatins, two modified fluid gelatins, or one gelatin and one modified fluid gelatin, having different isoelectric points, water, and sufficient alkaline substance to achieve a basic pH, said synthetic whole blood being a substantially non-polar coacervate phase.

Table I

U.S. Patent 4,343,797 Synthetic Synthetic

Whole Synthetic Whole Blood Whole Blood

Properties* Human Blood Whole Blood Gelatin-Gelatin Lecithin-Gelatin

1 Yes Yes Yes -. . Yes

2 Yes Yes Yes Yes

3 Yes Yes Yes Yes

4 Yes Yes Yes Yes

5 Yes Yes Yes Yes

6 Yes, but not equal to Yes Yes Yes that of synthetic whole blood

7 No Yes Yes Yes

8 Yes Yes Yes Yes

9 Increase Can be prep; Can be prepared to Can be prepared to to decrease decrease or in¬ decrease or increas increase % crease % %

10 Yes Yes Yes Yes

11 Yes No No No

12 No Yes Yes Yes

13 No Yes Yes Yes

Table I (Continued)

Gelatin

Lactate including Hydro-

Ringer' s modified Albumin ethyl Perfluoro

Properties* Solution Dextran gelatin 5% Starch chemic ls

1 No No No No No Yes

2 Yes Yes Yes Yes Yes Yes

3 No No No No No No

4 No No No No No No

5 No No No No No Yes

6 No No No No No No

7 No No No No No No

8 No No No No No No

9 Reduction Reduction Reduction Reduction Reduction Reduction

10 Yes Yes Yes Yes Yes Yes

11 Does Not Does Not Does Not Does Not Does Not apply apply apply apply apply Yes

12 No No No No No Yes

13 Yes Yes Yes Yes Yes Yes

i o Mi

Table I (Continued) Properties*

1. Oxygen Transport

2. Carbon Dioxide Transfer

5 3. Oxygen can be held in reserve and released in accordance with physiological tension

4. Hemoglobin can be added or dispersed within the prepara-

10 tion without loss of stability

5. Transfers gasses other than 0„ and C0₂

6. Possesses both polar and non-polar properties

15 7. Dissolves and transports non- polar drug entities without loss of dosage-form stability

8. Transports enzyme systems without loss of stability

20 9. Effect on hematocrit percent after transfusion

10. Essential amino acids can be transported in stable form and desired quantity

25 11. Oxygen uptake ability reduced at low 0₂ partial pressures

12. Transports physiologically useful lipid soluble entities as a stable solution

30 13. Universal donor characteristics

It also provides for a synthetic whole blood comprising two gelatins, two modified fluid gelatins, or one gelatin and one modified fluid gelatin, having different isoelectric points, water, and sufficient alkaline substance to achieve a basic pH, said synthetic whole blood being a two-phase system, said phases being a substantially non-polar coacervate phase and a substantially polar equilibrium water phase.

It also provides for a method to make a synthetic whole blood said method comprising (a) combining water and two gelatins two modified fluid gelatins or one gelatin and one modified fluid gelatin with ifferent isoelectric. points, (b) storing the combination at 15-40°C. , for 12-72 hours, whereby said combination separates into two layers, said lower layer being a substantially non-polar coacervate phase and said upper layer being a substantially polar equilibrium water phase, (c) separating said lower phase from said upper phase, and (d) adjusting the pH of said lower phase to the range of 7.2-7.6..

It also provides for a method to make a synthetic whole blood, said method comprising (a) combining water and two gelatins, two modified fluid gelatins, or one gelatin and one modified fluid gelatin with different isoelectric points, (b) storing the combination at 15-40°C. , for 12-_^72 hours, whereby said combination, separates into two layers, said lower layer being a substantially non-polar coacervate phase and said upper layer being a substantially polar equilibrium water phase, (c) separating said lower phase from said upper phase, (d) adjusting the pH of said lower phase to the range of 7.2-7.6 and (e) combining said lower coacervate phase of step (c) with said previously sep¬ arated upper equilibrium water phase. It also provides for a synthetic whole blood comprising lecithin and either one gelatin or one modified fluid gelatin, water, sufficient alkaline sub¬ stance to achieve a basic pH, and sufficient electrolyte to achieve an isotonicity equal to that of physiological saline solution, said synthetic whole blood being a substantially non-polar coacervate phase.

• It also provides for a synthetic whole blood comprising lecithin and either one gelatin or one modified fluid gelatin, water, sufficient alkaline substance to achieve a basic pH, and sufficient elec¬ trolyte to achieve an isotonicity equal to that of physiological saline solution, said synthetic whole blood being a two-phase system, said phases being a substantially non-polar coacervate phase and a sub¬ stantially polar equilibrium water phase.

It also provides for a method to make a synthetic whole blood, said method comprising (a) combining water and lecithin and either one gelatin or one modified fluid gelatin, (b) mixing in sufficient electrolyte to achieve an isotonicity equal to that of physiological saline solution, and (c) storing the combination at 15-50°C. , for 12-72 hours whereby said combination separates into two layers, said lower layer being a substantially non-polar coacervate phase, and said upper layer being an equilibrium water phase, (d) separating said lower phase from said upper phase and (e) adjusting the pH of said lower phase to the range of 7.2-7.6.

It also provides for a method to make a synthetic whole blood, said method comprising (a) combining water and lecithin and either one gelatin or modified fluid gelatin, (b) mixing in sufficient electrolyte to achieve an isotonicity equal to that of physiological saline solution, and (c) storing the combination at 15-50°C., for 12-72 hours, whereby said combination separates into two layers, said lower layer being a substantially non-polar coacervate phase, and said upper layer being a substantially polar equilibrium water phase, (d) separating said lower phase from said upper phase, (e) adjusting the^'pH of said lower phase to the range of 7.2-7.6 and (f) combining said lower coacervate phase of step (d) with said previously sep¬ arated upper equilibrium water phase.

Some embodiments of the synthetic whole blood may also include 2-15% weight to volume of a hemoglobin selected from stroma free hemoglobin, microencapsulated stroma free hemoglobin, or synthetic liposomes contain¬ ing stroma free hemoglobin.

This invention comprises a composition of matter useful as a substitute for whole natural blood. The claimed invention is comprised of a two-phase aqueous liquid system substantially identical to the physicochemical system of whole natural blood. A substantially non-polar coacervate phase insoluble in and in equilibrium with an associated substantially polar equilibrium water phase are characteristic of both naturally occurring whole blood and the claimed in¬ vention. This invention also comprises a method of making a whole blood substitute, which yields the two- phase system referred to above. The system is composed of an internal suspension phase, herein referred to as the coacervate phase, and an external suspension phase which is the associated equilibrium water phase. When the claimed composition is introduced intravenously, it will disperse in the blood plasma of the recipient, and thereby contribute to the two-phase physicochemical system of the naturally occurring whole blood. The physiochemical characteristics of this invention render it sensitive to and reactive to the oxygen tension of the recipient's blood. Further, it can readily enter and pass through the major blood vessels, capillaries and the microcirculation.

The claimed synthetic whole blood can trans¬ port and transfer oxygen and carbon dioxide much as naturally occurring erythrocytes do, without adversely affecting the percent of the recipient's heraatocrit. In addition, it can carry nutrients, physiological en¬ tities, therapeutic drugs and enzyme systems. .

Upon transfusion this invention can establish, re-establish and/or maintain normal osmotic pressures. The transport characteristics of this composition of matter enable it to serve as a safe and reliable ve- hide. When it is desirable to introduce enzyme systems into the body, such systems as noted above can be added to this invention and infused through conven¬ tional intravenous methods. Enzyme systems introduced through these compositions of matter will perform their normal physiological functions.

The guidelines which determine the quan¬ tities of the claimed synthetic whole blood which may be safely infused are substantially identical to those which govern the use of whole blood. By reason of its mode of manufacture and its physicochemical structure, the claimed whole blood substitute posseses a number of advantages over whole blood. Thus, prior to infusion this invention can be modified to meet many of the specific requirements of given treatment procedures, such as hyperalimentation, intravenous drug therapy, open heart surgery,.,etc. By way of example, additional quantities of stroma free hemoglobin or synthetic liposomes containing stroma free hemoglobin or microencapsulated hemoglobin can be incorporated in a given embodiment of this invention so as to enable more oxygen to be carried for longer periods of time as would be desirable in treatment of certain blood diseases or in instances of prolonged surgery. Electrolytes can be added to the claimed- substance for use in the treatment of cases of severe burns or shock resulting from the loss of blood. In embodiments containing added electrolytes, adjustments to isotonicity are made following such ad¬ ditions. When nutrients must be quickly introduced and/or when the circulatory system is the preferred route for nutrition, essential amino acids and other nutritional agents can be added prior to transfusion. A significant advantage of this invention is that because this invention possess universal donor characteristics, no blood typing is necessary prior to administration of this composition. Other important advantages of this invention may be enumerated as follows: the components of the claimed^' composition are abundant, readily available and relatively inexpensive. Additives can be quickly introduced to previously prepared, stored embodiments. The invention can be used without the need for highly specialized equipment or technology. The constituents of the claimed composition of matter and the method of preparing it eliminates the problems associated with the storage of whole blood In order to explain the invention more fully the following is a general description of the preferred method used to practice this invention. Specific examples of the practice of this invention are also pro¬ vided in the following section of this disclosure. The formulation that follows specifies sub¬ stantially equal proportions of two gelatins, two modified fluid gelatins, or one gelatin and one modified fluid gelatin, with different isoelectric points. How¬ ever, in the practice of this invention unequal pro- portions of the two gelatins, two modified fluid gela¬ tins, or one gelatin and one modified fluid gelatin with different isoelectric points may also be used to pre¬ pare the claimed composition of matter. In the process of manufacture, the component ingredients should be prepared and combined under aseptic conditions.

Mix equal proportions of a 1 to 10% weight to volume solution of gelatin with an isoelectric point of 2 to 6 with a 1 to 10% weight to volume solu¬ tion of gelatin, an isoelectric point of 8.0 to 10.0. In this step, modified fluid gelatins may be used in place of gelatin provided the requirement of differing isoelectric points is observed. The resultant mixture of the two gelatin solutions will be approximately 0.5

r i i

OMPI to 5% weight to volume of each of said gelatins. The mixture is then left undisturbed at 37° C for 24 hours. At the end of this period, the mixture will have separated into two layers, the lower one of which comprises the coacervate phase. The upper layer comprises the equilibrium water phase and may be dis¬ carded. The pH of the coacervate phase is adjusted to 7.4 by the addition, preferably dropwise, of any nontoxic alkaline substance, preferably sodium hy- droxide or sodium bicarbonate. The resulting compo¬ sition can be used as a synthetic whole blood. In the preferred procedure, 2 to 15% weight to volume of stroma free hemoglobin, or that amount of synthetic lip¬ osomes containing stroma free hemoglobin or micro- encapsulated hemoglobin as will result in a 2 to 15% weight to volume of stroma free hemoglobin in the finished product, is added to augment the oxygen transport capability of the composition. If desired, 1 to 10% weight to volume of a nontoxic ionic, or non-ionic surfactant and/or a nontoxic or¬ ganic solvent may be added to the preparation, to en¬ hance the oxygen.transport capability of the composition. A suitable protein such as albumin may also be added. In such instance it is added in the amount of 1 to 5% weight volume.

The non-ionic surfactants that may be used, include any of the nontoxic pluronics or any of the substances known as spans.

The ionic surfactants that- may be used in- elude any of the phospholipids such as lecithin, ceph- alin, isolecithin, sphingomyelin, phosphatidyl serine, phosphatidic acid, phosphatidyl inositol and phos¬ phatidyl choline. Other compounds known to those skilled in the art may also be used. Lecithin is the preferred phospholipid in this invention and is added in the amount of 1 to 10% weight to volume..

Following the addition of any of the above, or any combination of the above, the preparation is

OMPI subjected to vigorous shaking for 3 minutes to achieve uniform dissolution and dispersion of the additive(s). It is highly desired that for the best method that when the ingredients referred to above are added, the amounts of each should be sufficient to reach the saturation point and beyond within the coacervate phase.

If the intended use of the composition in¬ volves an open circuit, prior to infusion, oxygen should be bubbled through the preparation until the desired oxygen concentration is reached. If the synthetic whole blood composition is to be used in a closed system, the desired level of oxygen tension is main¬ tained by bubbling oxygen through the system by the usual means. Another embodiment of this invention also useful as a whole blood substitute is also claimed, which makes use of both layers. The preferred manufacturing; procedure is as follows? The claimed two phase liquid aqueous system is prepared in the man- ner described previously. After the 24 hour period of storage at 37° C the two layers are separated by means of a separatory funnel or other suitable means but, the equilibrium water layer is retained in sterile condition for use in a subsequent manufacturing step. Following the separation procedure, the pH of the co¬ acervate layer is adjusted to 7.25 to 7.4 by the drop- wise addition of sodium hydroxide or sodium bicar¬ bonate. When this step is completed, 2 to 15% weight to volume of stroma free hemoglobin, or that amount of synthetic liposomes containing stroma free hemo¬ globin or microencapsulated hemoglobin is added so that the stroma free hemoglobin in the finished product ranges from 2 to 15% weight to volume, is added and the preparation vigourously mixed. The preparation is then emulsified by adding the previously separated equilibrium water layer and using colloid mill or other suitable means to produce the required emulsion. The particles of the emulsion -16- can range in size from 0.5 to 9 microns in size. In the preferred procedure, the addition of the equilib¬ rium water layer and the emulsifying step follow the addition and mixing of 2 to 15% weight to volume of stroma free hemoglobin, or synthetic liposomes con¬ taining stroma free hemoglobin or microencapsulated hemoglobin. Also, 1 to 10% of a suitable ionic surfactant, preferably lecithin, and 1 to 5% weight to volume of a suitable protein, preferably albumin, may be added.

When preparation of the composition is com¬ pleted, it may be infused to transport physiological gases, restore or maintain osmotic pressure, trans¬ port polar and non-polar drugs, carry enzyme systems, nutriments, etc. Alternatively, it can be stored at from 4 to 10° C until needed. If the composition is to be infused into a human following refrigerated storage it should be warmed to body temperature (37° C). It may be stored at conventional room tem¬ peratures, if the preparation can be maintained in completely sterile condition.

While the above description contains many specifics these should not be construed as limitaions on the scope of the invention but rather as exemplifica¬ tions of preferred embodiments. Accordingly, the scope of this invention should not be determined by the de¬ scribed embodiments but by the appended claims and their legal equivalents. Specific Examples

Examples of how the claimed compositions of matter may be prepared follow.

Sterile conditions are observed during all phases of manufacture. Example 1

Take 4 grams of gelatin, isoelectric point of 9 and add distilled water until a solution of 100 mis is reached. Next, take 4 grams of gelatin, iso- electric point of 4, and add distilled water until a solution of 100 mis is reached. Mix the two solutions thoroughly and incubate, undisturbed at 37 C for 24 hours. Separate the resulting two layers and discard the upper equilibrium water layer. Adjust the pH of the lower (coacervate) layer to 7.4 through the dropwise addition of sodium hydroxide, and add 10% weight to volume of stroma frefe hemoglobin. Disperse the additive by vigorous shaking for 4 minutes. If the preparation is to be infused shortly after manufacture, bubble oxygen through the composition until desired oxygen level is reached.

Example 2 The procedure follows that of Example 1 ex- cept that 5% weight to volume of lecithin is also ad¬ ded to the coacervate layer, and dispersed by shaking the mixture.

Example 3 The procedures follows that of Example 1 ex- cept that 2% weight to volume of albumin is also added to the coacervate layer and dispersed by vigorously shaking the mixture.

Example 4 The procedure follows that of Example 1 ex- cept that 5% weight to volume of stroma free hemoglobin, 5% weight to volume of lecithin, and 1% weight to vol¬ ume of albumin are added to the coacervate layer and dispersed by means of vigorous shaking the.com¬ position. Example 5

Mix equal proportions of 8% weight to volume of gelatin with an isoelectric point of 5, and a gela¬ tin with an isoelectric point, of 9.5 Let the mix¬ ture stand undisturbed for 24 hours at 37 C. At the end of this period, separate the two layers that will have formed and discard the upper layer. Adjust the pH of the lower layer to 7.4 by the dropwise ad¬ dition of sodium hydroxide. To this, add 5% weight to -18- volume of stroma free hemoglobin. Disperse the stroma free hemoglobin by vigorous shaking of the composition.

Example 6 The procedure follows that of Example 5 'ex- cept that 5% weight to volume of lecithin is added to the coacervate layer and dispersed by vigorous shaking of the composition.

Example 7 The procedures follows that of Example 6 ex- cept that 1% weight to volume of albumin is added to the coacervate layer and dispersed by vigorous shaking of the composition.

Example 8 Thoroughly mix equal proportions of 8% weight to volume of modified liquid gelatin with an iso¬ electric point of 5, and a modified liquid gelatin with an isoelectric point of 9. Permit the mixture to stand undisturbed at 37° C for 24 hours. At the end of this period, separate the two layers that will have formed and discard the upper layer. Adjust the pH of the lower layer to 7.4 by the dropwise addition of sod¬ ium hydroxide. Add 10% weight to volume of stroma free hemoglobin and disperse same by vigorous shaking for 4 minutes. Example 9

The procedure follows that of Example 8 ex¬ cept that 5% weight to volume of lecithin is added to the coacervate layer and dispersed through vigorous shaking of the composition. Example 10

The procedure follows that of Example 8 ex¬ cept that 1% weight to volume of albumin is added to the coacervate layer and dispersed through vigorous shaking of the mixture. Example 11

The procedure follows that of Example 8 ex¬ cept that 5% weight to volume of lecithin, and 1% weight to volume of albumin are added to the coacervate layer and dispersed through vigorous shaking of the composi¬ tion.

Example 12 This is the procedure employing encapsulated stroma free hemoglobin. It occurs after the coacervate system has been formed, the phases are separated, and 2 to 5% stroma free hemoglobin has been added to the lower coacervate layer. This procedure thus may be applied to the resultant product of any of Examples 4, 5, 6, 7, 8, 9, 10 and 11. The lower coacervate layer containing the stroma free hemoglobin is combined with the equilibrium liquid water layer and emulsified so that the final emulsion contains- particles (droplets) which can range from 0.5 to 9 microns in size. Next, 1 to 5% formaldehyde solution is added dropwise to the emulsified preparation until the desired degree of shell structuring of the droplets is achieved. The degree of structuring can range from semi-solid or gel-like to rigid, and is achieved either through the amount of formaldehyde added or through the length of the period of storage. After the desired degree of structuring is achieved, the preparation is stored anywhere between 5 to 40 hours at 20° to 40°C. On removal from storage, the preparation will have separated into two layers, the bottom one of which contains microencapsulated globules substantially spherical in shape, contain¬ ing stroma free hemoglobin. The upper layer consists of. equilibrium liquid water. The two layers are separated by means of a separatory funnel or other acceptable means. The microencapsulated spheres are washed with the equilibrium liquid water, until sub¬ stantially all traces of formaldehyde are completely removed. The microencapsulated spheres containing stroma free hemoglobin can then be dispersed in physiological saline solution, in the coacervate phase of any of the herein described coacervate systems, or added to the coacervate phase of the two phase coacervate system. After this step, the composition is then emulsified. The resultant emulsion is pre¬ pared so that the droplets can range in size from 0.5 to 9 microns. When the microencapsulated spheres containing stroma free hemoglobin are incorporated into the two phase coacervate system as described above, the result of the procedure is microen¬ capsulated globules containing stroma free hemoglobin incorporated in droplets of the coacervate phase which in turn is suspended in the equilibrium liquid water phase. In practice, where optimal sustained oxygen uptake and release is desired, minimal structuring of the microencapsulated spheres is preferred. De¬ pending upon the physiological effect to be achieved, differing proportions of microencapsulated spheres of differing degrees of shell hardness can be com¬ bined. This will result in special release effects which can be used when introducing drugs, nutrients, enzyme systems. In other words, the composition can be so prepared as to give the desired specific rate of release of any of the components contained within the microencapsulated spheres. The procedure to in¬ corporate drugs, nutrients, enzyme systems, et cetera, into synthetic blood containing microencapsulated stroma free hemoglobin is the same as the procedure herein described to incorporate drugs, nutrients, enzyme systems, et cetera, into synthetic blood con¬ taining microencapsulated hemoglobin.

Example 13 Take 4 -grams of gelatin, isoelectric point of 9 and add distilled water until a solution of 100 mis is reached. Next, take 4 grains of gelatin, isoelectric point of 4, and add distilled water until a solution of 100 mis is reached. Mix the two solutions thoroughly and incubate, undisturbed at 37° C for 24 hours.

Separate the resulting two layers. Adjust the pH of the lower (coacervate) layer to 7.4 through the drop- wise addition of sodium hydroxide, add 10%. weight to volume of stroma free hemoglobin. Disperse the ad¬ ditive by vigorous shaking.

The preparation is then emulsified by adding the previously separated equilibrium water layer and using a colloid mill to produce the emulsion. The desired emulsion particle size can range from 0.54 to 9 microns 9 microns in size.

Example 14 The procedure follows that of Example 12 ex- cept that 5% weight to volume of lecithin is also added to the coacervate layer.

Example 15 The procedure follows that of Example 12 ex¬ cept that 2% weight to volume of albumin is also added to the coacervate layer.

Example 16 The procedure follows that of Example 12 ex¬ cept that 5% weight ^'to volume of stroma free hemoglobin, 5% weight .to volume lecithin and 1% weight to volume albumin are added to the coacervate layer.

Example 17 Mix equal proportions of 8% weight of volume of modified gelatin, isoelectric point of 5 and with a second modified gelatin, isoelectric point of 9.5, in- cubate 24 hours at 37° C. At the end of this period, separate the two layers that will have formed. Adjust the pH of the lower coacervate layer to 7.4 by the dropwise addition of sodium hydroxide. Add 5% weight to the volume of stroma free hemoglobin. Disperse the stroma free hemoglobin by vigorous shaking.

The previously separated equilibrium water layer is then added to the preparation and a colloid mill is used to produce the emulsion. The desired emul¬ sion particle size can range from 0.5 to 9 microns. Example 18

The procedure follows that of Example 16 ex¬ cept that 5% weight to volume of lecithin is added to the coacervate layer. Example 19 The procedure follows that of Example 16 ex¬ cept that 1% weight to volume of albumin is also added to the coacervate layer. Example 20

The procedure follows that of Example 16 ex¬ cept that 5% weight to volume of stroma free hemoglobin, 5% weight to volume of stroma free hemoglobin, 5% weight, to volume lecithin and 1% weight to volume al- bumin are added to the coacervate layer.

Example 21 The procedure follows that of Example 16 ex¬ cept that (a) 8% weight/volume of modified fluid gela¬ tin, isoelectric point of 5 is mixed with an equal amount of gelatin isoelectric point of 9 and (b) instead of stroma free hemoglobin being dispersed, synthetic lip¬ osomes containing stroma free hemoglobin are dispersed by vigorous mixing into the formed coacervate system.

Example 22 Mix equal proportions of 8% weight to volume of gelatin with an isoelectric point of 5, and a gelatin with an isoelectric point, of 9.5. Let the mixture stand undisturbed for 24 hours at 37° C. At the end of this period, separate the two layers that will have formed and discard the upper layer. Adjust the pH of the lower layer to 7.4 by the dropwise addition of sodium hy¬ droxide. To this, add 5% weight to volume of synthetic liposomes containing stroma free hemoglobin. Disperse the synthetic liposomes containing stroma free hemo- globin by vigorously mixing the coirqposition.

Example 23 Thoroughly mix equal proportions of 8% weight to volume of modified liquid gelatin with an isoelectric point of 5, and a modified liquid gelatin with an isoelectric.point of 9. Permit the mixture to stand undisturbed at 37° C for 24 hours. At the end of this period, separate the two layers that will have formed and discard the upper layer. Adjust the - - pH of the lower layer to 7.4 by the dropwise addition of sodium hydroxide. Add 10% weight to volume of synthetic liposomes containing stroma free hemoglobin and disperse same by vigorous shaking for 4 minutes. Example 24

Mix 5 to 10% weight/volume of gelatin iso¬ electric point 7 to 10 with l/2 to 10% weight to volume of lecithin. Adjust the electrolyte concentration to give an isotonicity equal to that of physiological saline solution. Incubate at 37° C for 24 hours, at the end of which 2 layers will have separated, one of which is the equilibrium water phase and the other is the coacervate phase. Separate the resulting two layers and discard the upper equilibrium water layer. Adjust the pH of the lower (coacervate) layer to 7.4 through the dropwise addition of sodium hydroxide. Add 10% weight to volume of stroma free hemoglobin. Disperse the additive by vigorous shaking for 4 minutes. If the preparation is to be infused shortly after manufacture, bubble oxygen through the composition until desired oxygen level is reached.

Example 25 Mix 1/2 to 10% weight to volume of gelatin or modified fluid gelatin isoelectric point of 5 to 10 with 1/2 to 10% weight to volume of lecithin. Add by mixing in such amounts of a salt of sodium, potassium, calcium and magnesium as will result in the electrolyte balance and isotonicity of physiological saline solution. In¬ cubate at 37 to 50° C for 24 to 36 hours. At the end of the period of incubation, the mixture will have separated into two layers, the bottom one of which is known as the coacervate phase. The upper layer is known as the equilibrium water phase. At this point the two phases may be separated by means of a separatory funnel. Stroma free hemoglobin or liposomes containing, stroma free hemoglobin or microencapsulated hemoglobin is added to the coacervate phase in an amount that will result in a finished product that contains 2 to 15% weight to volume of stroma free hemoglobin. The preparation described immediately above can be used for transfusion or stored at from 4 to 10°C. Alternatively, the coacervate phase which contains the electrolytes and stroma free 5 hemoglobin or liposomes containing stroma free hemoglobin or microencapsulated stroma free hemoglobin in the quantities given above can be combined with the equilibrium water phase and emulsified. The emulsion can be used for transfusion or stored at 4 to 10°C. 10 Example 26

The amounts used were as follows: 5% weight to volume gelatin isoelectric point of 7 and 7% weight to volume of lecithin. All other ingredients were in the amounts given above, for Example 24, and the pro- 15 cedure followed the description given above. The two phases were emulsified as described above. Stroma free hemoglobin was used_> in the amount of 5% weight to volume.

Example 27 This procedure is the same as Example 24 ex- 20.cept that the phases were separated and no emulsifica— tion was used, i.e. the coacervate phase plus the de¬ scribed additives constituted the composition.

Example 28 This procedure is the same as Example 24 ex- 25 cept that modified fluid gelatin was used.

Example 29 The procedure is the same as Example 24 ex¬ cept that modified fluid gelatin was used.

Experiment

The following in vitro experiment was conducted to test the oxygen carrying capacity of the claimed composition. Control substances were comprised of (A) saline solution, and (B) saline solution and 4% weight to volume stroma free hemoglobin. The three compo¬ sitions according to this invention were comprised of

(C) gelatin based coacervate composition of this in¬ vention, (D) gelatin based coacervate composition of this invention plus 4% weight to volume of stroma free hemoglobin, and (E) emulsified gelatin based co¬ acervate composition of this invention plus 4% weight to volume of stroma free hemoglobin.

Oxygen was bubbled through each for 20 minutes at 37°C. The results obtained were as follows:

Oxygen uptake Substance (Volume to volume)

(A) Saline solution 0%

(B) Saline solution and 4% hemoglobin 1% (C) Claimed composition

(Gelatin P.I..3; Gelatin P.1.9)* 10%

(D) Claimed composition and 4% stroma free hemoglobin (Gelatin P.I.3; Gelatin P.I.9)* 22%

(E) Emulsified claimed composition containing 4% hemoglobin 15%

(F). Claimed composition: Coacervate Phase

(Lecithin and Gelatin, P.I.9) 12%

(G) .Claimed Composition: Coacervate Phase

(Lecithin, Gelatin P.1.9; 4% stroma free hemoglobin) 19%

(H) Emulsified claimed composition containing

(Lecithin, Gelatin P.I. and 4% stroma free hemoglobin) 14%

*P.I. is an abbreviation for isoelectric point.

Claims

1. A synthetic whole blood comprising two gel¬ atins, two modified fluid gelatins, or one gelatin and one modified fluid gelatin, having different isoelectric points, water, and sufficient alkaline substance to achieve a basic pH, said synthetic whole blood being a substantially non-polar coacervate phase.

2. The synthetic whole blood of claim 1, wherein one gelatin or modified fluid gelatin has an isoelectric point of 2 to 6 and the other gelatin or modified gelatin has an isoelectric point of 8 to 10; said pH is 7.2 to 7.6; said alkaline substance is sodium hydroxide or sodium bi¬ carbonate; and each of said two gelatins or modified fluid gelatins is present as starting material in an amount of 1-10% weight to volume of water. 3. A synthetic whole blood comprising two gelatins, two modified fluid gelatins or one gelatin and one modified fluid gelatin, having different isoelectric points, water, and sufficient alkaline substance to achieve a basic pH, said synthetic whole blood being a two-phase system, said phases being a substantially ήon-polar coacervate phase and a substantially polar equilibrium water phase.

4. The synthetic -whole blood of claim 3, wherein one gelatin or modified fluid gelatin has an isoelectric point of 2 to 6 and the other gelatin or modified fluid gelatin has an isoelectric point of 8 to 10; said pH is 7.2 to 7.6; .said alkaline substance is sodium hydroxide or sodium bicarbonate; and each of said two gelatins or modified fluid gelatins is present as a starting material in an amount of 1-10% weight to volume of water; and said preparation is an emulsion wherein the particle size is in the range of 0.5-9 microns.

5. The synthetic whole blood of any of claims 1-4, wherein said synthetic whole blood also includes 2-15% weight to volume of a hemoglobin selected from stroma free hemoglobin, microencapsulated stroma free hemoglobin, or synthetic liposomes containing stroma free hemoglobin. 6. The synthetic whole blood of any of claims 1-5, further including an additive selected from 1-10% weight to volume of an ionic surfactant or a non-ionic surfactant or mixtures of the surfactants, 1-10% weight to volume of an organic solvent, or 1-5% weight to volume of a suitable protein.

7. The synthetic whole blood of any of claims

1-6, further including an additive selected from nutrients, physiological entities, therapeutic entities, drugs, enzyme systems, electrolytes, or 0-.

8. A method to make a synthetic whole blood, said method comprising (a) combining water and two gelatins, two modified fluid gelatins, or one gelatin and one modi¬ fied fluid gelatin, with different isoelectric points, (b) storing the combination at 15-40°C, for 12-72 hours where¬ by said combination separates, into-2 layers, said lower layer being a substantially non-polar coacervate phase, and said upper layer being an equilibrium water phase, (c) separating said lower phase from said upper phase_.and (d) adjusting the pH of said lower phase to the range of 7.2-7.6.

9. The method of claim 8, wherein in step (a) one of said gelatins is combined with water to form a 1-10% weight to volume solution, the other said gelatin is com- bined with water to form a 1-10% weight to volume solution, and then said 2 solutions are combined; and one of said gelatins or modified gelatins has an isoelectric point of 2 to 6, and the other of said gelatins or modified gelatins has an isoelectric point of 8 to 10. 10. The method of claim 9, wherein after said pH adjustment, there is also included step (d') comprising the addition of 2-15% weight to volume of stroma free hemo¬ globin, synthetic liposomes containing stroma free hemo¬ globin, or microencapsulated stroma free hemoglobin; 1-10% weight to volume of an ionic surfactant or a non- ionic surfactant or mixtures of the surfactants; 1-10% weight to volume of an organic solvent; or 1-5% weight of a suitable protein. 11. The method of claim 8 or 9, said method also including step (e) comprising the combination of said lower coacervate phase with said previously separated upper equilibrium water phase, thereby forming a two-phase system.

12. The method of claim 11, wherein in step (e), said combination is achieved by emulsification, thereby forming an emulsion, and preferably the particles of said emulsion range in size from 0.5-9 microns. 13. The method of claim 12, wherein prior to step (e), there is also included step (d') comprising the addition of 2-15% weight to volume stroma free hemoglobin, or synthetic liposomes containing stroma free hemoglobin.

14. The method of claim 13, including the steps of (f) adding, preferably dropwise, a 1-5% solution of formal¬ dehyde, until the desired degree of shell structuring of the emulsion particles is achieved, (g) storing said emul¬ sion for 5-40 hours at 20-40°C, whereby the preparation separates into two layers, an upper equilibrium liquid water layer, and a lower layer containing microencapsul¬ ated globules containing stroma free hemoglobin, and (h) washing said microencapsulated globules containing stroma free hemoglobin with said upper equilibrium liquid water, until substantially all traces of the formaldehyde are re- moved.

15. The method of claim 14, further including the steps of (i) dispersing said microencapsulated stroma free hemoglobin in physiological saline solution, in the lower coacervate phase of step (c), or in the two-phase system of step (e), and then (j) emulsifying the resultant preparation, and preferably the emulsion particles are 0.5-9 microns in size. _Λ 16. The method of any of claims 13-15, wherein step (d') also includes the addition of 1-10% weight to volume of an ionic surfactant or a non-ionic surfactant or mixtures of the surfactants; 1-10% weight to volume of an organic solvent; or 1-5% weight to volume of a suitable protein; to said lower coacervate layer after the pH adjustment of step (d).

17. The method of and of claims 8, 9, 10 or 16, wherein step (d') also includes the addition of an addi¬ tive selected from nutrients, physiological entities, therapeutic entities, drugs, enzyme systems, electrolytes, or 0₂.

18. A synthetic whole blood comprising lecithin and either one gelatin or one modified fluid gelatin, water, sufficient alkaline substance to achieve a basic pH, and sufficient electrolyte to achieve an isotonicity equal to that of physiological saline solution, said synthetic whole blood being a substantially non-polar coacervate phase.

19. The synthetic whole blood of claim 18, wherein said gelatin or modified fluid gelatin has an isoelectric point of 2 to 10; said pH is 7.2 to 7.6; said alkaline substance is sodium hydroxide or sodium bicarbonate; said lecithin is present as a starting material as 0.5-10% weight to volume of water; said gelatin or modified fluid gelatin is present as starting material in an amount of 1-10% weight to volume of water; and said electrolyte is a salt of Na, K, Ca, or Mg. 20. A synthetic whole blood comprising lecithin and either one gelatin or one modified fluid gelatin, water, sufficient alkaline substance to^-"achieve a basic pH, and sufficient electrolyte to achieve an-isotonicity equal to that of physiological saline solution, said syn- thetic whole blood being a two-phase system, said phases being a substantially non-polar coacervate phase and a substantially polar equilibrium water phase.

21. The synthetic whole blood of claim 20, wherein said gelatin or modified fluid gelatin has an isoelectric point of 2 to 10; said pH is 7.2 to 7.6; said alkaline substance is sodium hydroxide or sodium bicarbonate; said lecithin is present as a starting material in an amount of 0.5-10% weight to volume of water; said gelatin or modified fluid gelatin is present as a starting material in an amount of 1-10% weight to volume of water; and said electrolyte is a salt of Na, K, Ca, or Mg; and said com- 5 bination is an emulsion wherein the particle size is in the range of 0.5-9 microns.

22. The synthetic whole blood of any of claims 18-21, wherein said synthetic whole blood also includes 2-15% weight to volume stroma free hemoglobin, 1-10%

10 weight to volume of an ionic surfactant or a non-ionic surfactant or mixtures of the surfactants, 1-10% weight to volume of an organic solvent, or 1-5% weight to volume of a suitable protein.

23i The synthetic whole blood of any of claims

15 18-22, further including an additive selected from nu- trients, physiological entities, therapeutic entities, drugs, enzyme systems, electrolytes or 0,.

24. A method to make a synthetic whole blood, said method comprising (a) combining water and lecithin and

20 either one gelatin or modified fluid gelatin, (b) mixing in sufficient electrolyte to achieve an isotonicity equal to that of physiological saline solution, and (c) storing the combination at 15-50°C, for 12-72 hours whereby said combination separates into layers, said lower layer

25 being a substantially non-polar coacervate phase, and said upper layer being an equilibrium water phase, (d) separating said lower phase from said upper-phase, and

(e) adjusting the pH of said lower phase to the range of 7.2-7.6.

30 25. The method of claim 24, further including step

(f) comprising the combination of said lower coacervate phase with said previously separated upper equilibrium water phase.

26. The method of claim 25, wherein in step (f), 35 said combination is achieved by emulsification, thereby forming an emulsion, and preferably the particles of said emulsion range in size rora 0.5-9 microns. 27. The method of any of claims 24, 25, or 26, wherein in step (a) said gelatin or modified fluid gelatin is combined with water to form a 1-10% weight to volume solution, said lecithin is combined with water to form a 0.5-10% weight to volume solution, and then said two solutions are combined; and said gelatin or modified fluid gelatin has an isoelectric point of 2 to 10, and in step (b) said electrolyte is a salt of Na, K, Ca, or Mg.

28. The method of any of claims 7-17, or 24-27, wherein said pH is adjusted to 7.4 by the dropwise addi¬ tion of an alkaline substance, selected from sodium bicarbonate or sodium hydroxide.

29. The method of any of claims 25-28, wherein after the pH adjustment of step (e) there is also included step (e' ) comprising the addition of 2-15% weight to volume stroma free hemoglobin, 1-10% weight to volume of an ionic surfactant or a non-ionic surfactant or mix¬ tures of the surfactants, 1-10% weight to volume of an organic solvent, o 1-5% weight to volume of a suitable protein, to said lower coacervate layer.

30. The method of claim 29, wherein step (e') also includes the addition of an additive selected from nutri¬ ents, physiological entities, therapeutic entities, drugs, enzyme systems, electrolytes, or 0_.