CA1312009C - Extra pure semi-synthetic blood substitute - Google Patents

Abstract

ABSTRACT

A blood substitute and plasma expander comprising a cross-linked, substantially endotoxin-free homoglobin solution and process for preparing same. The process comprises fractionating whole blood, separating out a stromal-free, sterile hemoglobin solution, chromatographical-ly separating endotoxins from said hemoglobin solution and crosslinking the resulting endotoxin-free hemoglobin solu-tion.

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Description

~ITIE OF THE: I~NTION
~:XTR~ pUR~ SEMI-SYNTHETIC lBI~)OD SUBST~
,~Ac~GRouND QF THE IN~N~E;EQ~

Field o~ the Tnventiono This inYention rela'ces to a process for producing a novel ~;~mi- ynthetic ~blood substitute ~nd the r~ovel ~emi-~;ynthetic blood ~ubstitute resulting there~rom. The novel semi synthetic blood ~ubstitute i~ a B~emoglobin lpreparation charac~erized by ~l~æ purity, it6 exceptionally low 1~VE~1S of endotoxin, the absence ~f ns:~n hemoglobin proteins, and its m~lecular ~rc~ss--linking profile. The E~emi-synthetic blood i;ubstitute has no toxic z~ctivity when used in a substitute ~ashion and posse~ses the property of rever6ibly binding gaseou~ liyands 6uch as oxygen and is useful ~or transporting and l!;upplying oxygen to vital tissues and organ~. Addition-ally, the blood 6ubstitute ~;erves as a blood plasma expander ~or ~anage~ent ~ di~ease and ~or ~aintaining circulatory integrity. A ~urther a~pect o~ the invention i~ the pre-ur~or or intermediate, the ~ubstantially pure, phospholipid-.

~ 3 ~

free, endotoxin-free hemoglobin solution in uncross~linked form.

Description of ~ackqround Materials Complex multicellular organisms are equipped with specialized tissues which are concerned with the processes of nutrition and excretion. It is the primary function of blood to provide a link between various organs and cells of the body. Blood, red cells, plasma and other components maintain a constant cellular environment by circulating through every tissue and continuously delivsring nutrients to the tissues and removing waste products and various tissues which are concerned with the tissue secretions from themO PHYSIOLOGY, Third Edition, Edited by Edward ~. Selkurt, Page 223 ~1971).
Blood is a viscous fluid composed of cells and plasma. More than 99% of the cells are red blood cells. The major function of red blood cells is to transport hemoglobin, which in turn carries oxygen from the lungs to the tissues and C02 from the tissues to the lungs. Normal red blood cells contain approximately 34 grams of hemoglobin per 100 ml of cells. Ea~h gram of hemoglobin is capable of combining with approximately 1.33 ml of oxygen. See Guyton, A. C., BASIC HUMAN PHYSIOL~&Y: NORMAL FUNCTION IN MECHANISMS OF
DISEASE, Paqes 84-85 (1971).
Because of the critical and ongoing need for a thera-peutic agent use~ul as a blood substitute for carrying and supplying oxygen and as a blood plasma expander, intense research e~forts have been directed to the development of an adequate blood substitute. The need for a blood substitute exists for replacing blood lost by acute hemorrhage, blood losses occur.ing during surgical operations, resuscitation procedures after acidental bl~od loss, and the like.
~urther, as a plasma expander, a blood substitute serves as a ~3~J~

therapeutic to treat volume deficiency shock, as an alle~iant in anaphylactic and allergic shock, and ~or replacing plasma lost after burns and as a result of severe diarrhea.
~ emoglobin in solution ha~ the capabiliy to transport oxygen and, theoretically, could be used as a substitute for red blood cells. Because hemoglobin solutions ar~ oncoti~
c~lly active, these solutions al~o expand plasma volume, thereby providing a function as a plasma expander as well.
Thus the ability to be oncotically active and transport oxygen suggests that hemoglobin ~olutions would be desirable for a resuscikation fluid where rapid initial treatment of hypovolemia and tissue hypoxia is reguired. However, in order to function as an adequate resuscitation fluid, hemoglobin solutions must be capable of maintaining tissue oxygenation for specified periods of time.
Hemoglobin present in the blood of mammals has a fundamental property in solution of reversible oxygenation, In its natural form, ma~malian hemoglobin is a conjugat~d, non-crosslinked protein having a molecular weight of approxi-mately 68,000 and structurally co~prised of two pairs of sub~units. Each sub-unit contains a heme group and a polypeptide chain, called globin. In mammals, hemoglobin is present in erythrocytes, along with stroma which consists of proteins, phospholipids and cholesterol. See CLINICAL
HEMATOLOGY, By Wintrobe, 6 Ed. Payes 138-199, (1967).
The reversible binding of oxygen requires the interac-tion between four chains of hemoglobin (tetrameric hemo-globin) which results from the ability of the protein to exist as two different quarternary structures (relaxed and tense) that have different oxygen affinities (Perutz, M. F., Prog. Clin. Biol. Res~ 1: 3 (1975)). The two different oxygen af~inities permit hemoglobin to on-load oxygen when the oxygen tension is high ~approximately 100 ~n Hg pO2) and to off-load oxygen when the oxygen tension is low (approxi-~ 3 ~

mately 40 mm Hg pO2) and give rise to a characteristicsigmoidal shape to the o~ygen-hemoglobin dissociation curve.
It is now known that the tense state of some hemoglobin in red cells is stabilized by the presence of organic phosphates such as 2,3-diphosphoylycerate ~2,3-DPG), with the tense state of hemogl~bin in solutlon not ~tabili~ed du~e to the absence of 2,3-DP~. Accordingly, hemoglobin in solution has a lower P50 than hemoglobin in its natural form (Arnone, A., Nature 237: 146 (1972).
~ queous hemoglobin exists in equilibrium between the tetrameric (MW 68,000) and dimeric (MW 34,000) ~orms (Bunn, H. F. et al., Trans. Assn. Am._Physicians 81: 187 (1968)) D
The dimers are excreted by the kidney and resul~ in rapid intravascular elimination of hemoglobin solutions, with such solutions having a 2-4 hour plasma half~ e. Accordingly, efforts hav~ been directed to overcome the inhexent limita-tions of hemoglobin solutions by m~lecular modification of the hemoglobin. The purpose of the molecular modification is to stabilize hemoglobin to prevent dimer formation and to maintain the tense conformational state. Bunn et al.~ supra, demonstrated that cross-linking hemoglobin reduced renal elimination and increased intravascular retention time. Bunn et al. utiliæed bis (Nmaleimidomethyl~ ether; however, the resulting hemoglobin solution had a high oxygen a~finity, i.e., a P50 o~ 3 ~m Hg. Pyridoxal-5-phosphate has been demonstrated to have an analogous effect to 2,3-DPG in lowering oxygen affinity, resulting in a P50 of 26-30 mm Hg (Benesch, R. E., Biochem. 11: 256~ (1972)). However, unlike 2,3-DPG, pyridoxal phosphate does not act as a cross~linking agent, resulting in intravascular retention times similar to that o~ unmodified hemoglobin (Greenburg, A. ~. et al., Surqery 86: 13 (1979)). Thus it was thought that pyridoxyla-tion and cross-linking would be required to produce a blood suh~titute having low oxygen af~inities (P50 equal to 20-30 2 ~

mm Hg~ and adequate intravascular retention times ~hal~
disappearance times of 20 ox more hours).
In 1~85, the Congress of the United States, Office of Technolo~y Assessment (OT~)I issued a report enti~led ~'Blood Policy and Technology." At chapter 6 o~ this report~
alternative sources of blood products were di cu~sed, with the conclusion that the impetus to develop alternati~e blood sources and substitutes based on economic, safety, and availability considerations was a necessity. According to the report, the ideal red blood cell substitute would have ~ix properties: 1) an oxygen dissociation curve and oxygen-carrying capacity similar to that of intact red blood cells;
2) be non-toxic and non-antigenic; 3) have good flow charac-teristics: 4) remain in the circulation for a long period of time; 5) have a long shelf life; and 6~ be cost effective in comparison to present red blood cell transfusions. The report also concluded that no substitute yet developed fulfills all these criteria.
Four basic approaches have been utilized to develop an adequate blood substitute. In one approach, a class of synthetic compounds called perfluoro chemicals are being developed. In a second approach, synthesized analogues of hemoglobin are being developed. Investigators are also attempting to ass~mble a red cell by encapsulating hemoglobin in lipid vesicles called liposomPs. Finally, purified hemoglobin has been chemically modified to prolong its circulation and enhance its oxygen bindin~-dissociation properties.
According to the OTA report, supra, to date, none o~
these approaches has proven satisfactory. The fluorocarbons are removed by the circulatory ~ystem as foreign substances, and they become ludged in the liver, spleen, and other tissues. Artificial cells made of membrane encapsulated hemoglobin have not been used for many reasons. The use of ~ 3 ~

microcapsules made from synthetic polymers such as poly-styrene, ethylcellulos~, and silicone rubber introduces biologically incompatable materials into a living system.
The cell walls of the capsules tend to leak, it is difficult to control permeability of the wall, and these capsules are too rigid and too large to pass through the capilla~ bed.
The use of blood and blood fractions is fraught with disadvantages. For example~ the use of whole blood often is accompanied by the risk of transmission of hepatitis-produc-ing virus and AIDS-producing virus which complicate the patient's recovery in the first instance and is fatal in the second. Additionally, the use of whole blood requires blood-typing and cross-matching to avoid immunohematological problems and interdonor incompatibility.
The blood fraction plasma (BFP) which is a physiologi-cally balanced colloidal solution that Pulfills many of the requirements of a blood volume expander, cannot be safely used for this purpose. The high incidence and the risk of transmitting homologous serum hepatitis associated with plasma is so great, that its use is no longer warranted.
The blood component hemoglobin possesses osmotic activity and the ability to transport and exchange oxygen, but it has the disadvantage of rapid elim;nation from circulation by the rena~ route and through vasculax walls, resulting in a very short, and therefore, unsatisfactory half-life.
The literature, both patent and non-patent, is replete with efforts to produce a ~atisfactory blood substitute from polymerized, cross-linked, stromal free hemoglobin. Bonsen et al., U.S. 4,001,200, and Bonsen et al., U.S~ 4,001,401 disclose polymerized, cross-linked, "stromal-free" hemoglobin and pharmaceutical compositions (and methods Por using same) comprising the polymerized, cross-linked, "stromal-free"
hemoglobin. The process for producing the polymerized, cross-linked, "stromal-freel' hemoglQbin of Bonsen et_ al.
comprises lysing red blood cells t filtering through diatoma-ceous earth to remove stroma, dialy~ing to remove residual low molecular weight salts and metabolytes, polymerizing to form water soluble, cross-linked, macrsmolecular, stromal-free hemoglobin, with a ~inal sterilization by ~iltering through a filter having a pore ~ize of about 0.20 to 0.45 microns. Included among the cross-linking agents disclosed by Bonsen et al. are dialdehydes such as glyoxal, malonic dialdehyde, succinic dialdehyde, glutaraldehyde, adipal-dehyde, 3-methyl ylutaraldehyde, propyladipaldehyde, phthalic dialdehyde, terephthaldehyde and malonic dialdehydeO
Bonsen et al. (III, U.S. 4,053,590~, extends the disclosure of Bonsen et al~ ('200) and Bonsen et al. ('401) with a discussion of physiologically acceptable polymeric plasma substitutes as carriers for the blood substitute.
Further, applications for use as an artificial oxygen exchange solution in conventional oxygenators such as cardiac by-pass, extracorporeal circulatory assist devices, and hollow-fiber and sheet type membrane devices ~or use in assisting the circulation in ill patients, is su~gested.
Additionally, the polyhemoglobin is sugg~sted as a sourc~ of protein and oxygen in the microbiological assay of foods for aerobic bacillus and staphyllococcus to ensure the food i8 safe for animal and human consumption and as a storing and preserving solution for viable isolated perfused mammalian organs for their eventual transplant into a recipient.
Bonhard et al., U.S. 4,136,093 discloses a hemo~lobin preparation suitable for intravenous injection comprising a substantially pyrogen-free condensation product of hemoglobin and pyridoxal phosphate. The hemoglobin preparation is claimed to have a retention time in the blood system of from 2 to 9 hours. The product is produced by washing red blood cells with a weakly alkaline solution, hemolyzing, and a^l treating the resulting material with a cation exchange resin.
The material is separated fro~ the resin, diluted to a hemoglobin concentration of about 5-9%l adjusted to a pH of about 7 to ~, treated with pyridoxalA5-phosphate and, optionally, treated with a solution of a borohydride and then a dialdehyde to cross-link the hemoglobin molecul~s. The non-pyrogenic nature of the infusion solution is obtain~d by, as a minimum, repeated washings with the weakly alkaline solution.
In Bonhard et al., U.S. 4,336,248, hPmoglobin molecules were coupled to increase their intravascular residenc~ time without significantly diminishing the oxygen transport ability of the molecule. The hemoglobin molecules are coupled to one another and/or to serum proteins and gelatin derivatives using dialdehydes such as aliphatic dialdehydes of 3A8 carbon atoms. Optionally, pyridoxal phosphate may be added subsequently. The coupled hemoglobin molecules are recovered by ammonium sulphate precipitation.
In Simmonds et al., U.S. 4,401,652, there is disclosed a process for preparing a "stromal-free" hemoglobin solutivn.
The Simmonds et al. process is particularly adapted for large scale production of "stromal-freel' hemoglobin, with reduced methemoglobin formationO The process comprises washing blood cells to remove non-cellular components, removing leukocytes, typically by filtration through a suitable adsorbent which preferentially retains the leukocytes, lysing the remaininy red blood cells ultrasonically or mechanically, precipitation of the hemoglobin by mixture with a polyvalent cation, a polysulphate, and a polyvalent anion, and final puri~ication by filtration and dialysis. The resulting hemoglobin solution is "substantially pure" and "~ree of stroma" and other lipoprotein cellular constituents and contains less than 5% methemoglobin.

~ :3 ~ ~ ~ v; I

Tye, U.S. 4,529,719, discloses "stromal-freel' tetrameric hemoglobin which is cross-linked with certain bis-disalicyl esters and modified with pyridoxyl-5'-phosphate followed by reduction to produce bis-diamide covalently cross-linked, pyridoxyl-5'-phosphate covalently modified, tetrameric hemoglobin. The modified cross-linked "stromal-free"
tetrameric hemoglobin is disclosed to be disease-free and capable of transporting oxygen to perfused tissue and remains in the intravascular space. Additionallyl the product is suggested to be free from cell surface antigens, making it suitable for transfusion in place of red blood cells.
The modified cross-linked, "stromal-free" tetramexic hemoglobin of Tye is produced by starting with red blood cells of freshly drawn, outdated, or frozen packed cells or whole blood. The blood is drawn in sterile fashion into ~ontainers with ufficient anticoagulant activity to prevent clot formation. ~emoglobin from a variety of mammalian sources, such as human, bovine, ovine, or porcine are disclosed to be useful. Any non-heme protein is removed, preferably by zinc precipitation. Hemoglobin is released from the red blood cells by hypotonic lysis followed by ultrafiltration. The filtered hemoglobin is passed through a subsequent filtration step to remove virus particles, protein aggregates, and stromal elementsO The typical f ilter has a ~ominal pore size of 0.020 microns and an exclusion for ylobular proteins of 1,000,000 Daltons. Zinc iron is added to precipitate the hemoglobin and the precipitate concen-trated by filtration. The non-heme protein is removed in the filtrate. The resulting hemoglobin is then cross-linked using the bis-disalicyl esters and treated with pyridoxyl-5'-phosphate, followed by reduction of the reversible Schiff base covalent bond.
Rothe et al., U.S. 4,526,715 discloses a method for producing highly purified hemoglobin ~olutions free of plasma ^S ~ r`

--:LO--proteins and residual stromal lipids prepared from human blood or from animal blood in quantitles large enough for clinical applications. The disclosed pxocess comprises contacting red blood cells with a washing solution, hemolys-ing by introduction of the concentrated red blood cells into 2-3 times the volume of water, separating the strom~ from the hemoglobin by ultrafiltration, and concentration in a third filtration ~tage utilizing a ~econd ultrafiltration unit having a permeability of l0,000 to 50,000 Daltons.
However, in spite of the recent advances in the prepara-tion of "stromal-free,11 cross-linked hemoglobin origin blood substitutes, the need has continued to exist for a blood substitute which is substantially free of endotoxins, phospholipids, and non-hemoglobin proteins, which is capable of l) transporting adequate amounts of oxygen to tissue under ambi~nt conditions; 2) having an oncotic activity eguivalent to that of whole blood; 33 having an adequate intravascular retention time; 4~ transfusible to all recipients without cross-matching or sensitivity testing; 5) free from di~ease agents such as bacteria and virus particles (hepatitis, AIDS, etc.); and 6) storable with minimum amounts of refrigeration.

SUMMARY OF THE INVENTION

Recognizing the long-standing need in the field to develop a blood substitute comprising an oncotically active protein solution capable o~ transporting oxygen and readily available when massive transfusions are required, the inventors endeavored to develop a blood substitute based on a hemoglobin solution. Further, recognizing that the massive demands for such a blood substitute would require volumes of startiny material far in excess of that which could poten-tially be made available as discarded human blood, a further goal of the present invention was to generate a process for ~ ~ 3 ~

creating such a blood substitute wherein nonhuman mammalian blood sources would be suitable as starting materials.
With these goals in mind, the following invention has resultPd, a ~emi-synthetic blood substitute comprising monomeric mammalian hemoglobin in cross-linked form, said semi-synthetic blood substitute bein~ substan-tially Pree of endotoxins, phospholipid~ and non-hemoglobin proteins such as enzymes. An additional aspect of the present invention comprises the process by which the afore-mentioned blood substitute is prepared. Essentially, the blood substitute i5 prepared from a mammalian blood fraction by a process comprising 1) separation of red blood cells from the mammalian blood fraction; 2) hemolysis of the red blood cells to produce a composite of monomeric hemoglobin and stroma, including phospholipids; 3) separation by filtration of the hemoglobin, contaminated with at least a portion of the phospholipid; 4) purification of the monomeric hemoglobin by high performance liquid chromatography ~HPLC~ to separate the hemoglobin from all other proteins residual of the red blood cells, as well as the phospholipid, enzyme and endo-to~in contaminants; 5~ cross-linking (polymerizing or aggregating) the monomeric hemoglobin, and 6) partially separating the cross-linked hemoglobin from the non-cross-linked hemoglobin. An essential aspect of the present process comprises conducting the above steps under conditions which result in a product which is substantially free of endotoxins, phospholipids and non-hemoglobin proteins such as enzymesl and has a defined molecular weight distribution of greater than about 90% between 68,000 daltons and 500,000 daltons.
The resulting product (hereinafter "Invention Hemo-globin") is a blood substitute which is substantially free of endotoxins, has vascular persistence of at least two days, has the property of reversibly binding gaseous ligands such ~ 3 ~
~2-as oxygen and is useful for transporting and supplying oxygen to vital tissues and organs. A5 such, the blood substitute of the present invention is use~ul as a blood expander and resuscitating fluid in the management of disease and for maintaining circulatory integrity where needed, i.e., in response to sudden and massive blood loss.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures l~-lH are flow sheets describing the process of Example I. Figure lA refers to the blood collection phase.
Figure lB refers to the blood separation phase. Figure lC
refers to the microporous filtration phase. Figure lD refers to the ultrafiltration phase. Figure lE refers to 'the column chromatography phase. Figures lF and lG refer to the cross-linking phase. Figure lH refers to the storage and process fluid preparation phase.
Figure ~ is a graphic comparison of platelet numbers between three groups of rabbits, with percent change from baseline represented on the ordinate and time represented on the abscissa. Tl represents the baseline platelet level; T2 represants the platelet level at 15 minutes post-transfusion;
T3 represents the platelet level at 1 hour post-transfusion;
T4 represents platelet level at 3 hours post-transfusion; and T5 represents platelet level at 24 hours post transfusion.
The solid circles represent the mean value ~ standard error ~or platelet levels ~or 5iX rabbits having one-third of estimated blood volume replaced with a hemoglobin solution containing between one and two endotoxin units (EU)/ml: the clear circles represent the mean value + standard error for platelet levels of four rabbits that had one-third of blood volume replaced with 5% Plasma Protein Fraction (commercial product found to contain 0~05-0.15 EU/ml); and the square~
represent the mean value ~ standard error for ~ix rabbits ~ 3 1 r~ J ` ~ ~J~
-13~

that had one-third of the estimated blood volume replaced with pure polymerized hemoglobin according to the present invention, said hemoglobin containing 0-0.35 EU/ml.
Figure 3 is a graphic representation comparing white blood cell levels for one-third transfused rabbits from baseline to 24 hours. As in Figure 2, the ordina~e repre-sents percent change, with the abscissa represen ing time.
Tl represents the baseline platelet level; T2 represents the platelet level at 15 minutes post-transfusion; T3 represents the platelet level at 1 hour post-transfusion; T4 represents platelet level at 3 hours post-transfusion; and T5 represents platelet level at 24 hours post-transfusion. The solid circles represent the mean value ~ standard error of 6 rabbits that had one-third of the estimated blood volume replaced with a hemoglobin solution containing between 1 and 2 EU/ml; the clear circles represent the mean value ~
standard error of four rabbits that had one-third of the estimated blood volume replaced with 5% Plasma Protein Fraction (commercial product found to contain 0.05-0.15 EU/ml); and the s~uares represent the mean value + standard error of 6 rabbits that had one-third of the blood volume replaced with pure polymerized hemoglobin according to the present invention, containing 0-0.35 EU/ml.
Figure 4 is a graphic comparison of ~erum fibrinogen levels between three groups of rabbits. The ordinate represents the percent change in fibrinogen levels; the abscissa represents the time period, with Tl representing baseline; T2 the value at 15 minutes post-transfusion; T3 the value at one hour post~transfusion; T~ the value at 3 hours post~transfusion and T5 the value at 24 hours post-krans-fusion. The 601id circles repres0nt the mean value ~
standard error ~or six rabbits with one-third of the esti-mated blood volume replaced with a hemoglobin solution that contained between 1 and 2 EU/ml; the clear circles represent the mean value + standard erxor for four rabbits that had one-third of blood volume xeplaced with 5% Plasma Protein Fraction (commercial produ t found to contain 0.05-0.15 EU/ml~; and the ~quares represent the mean value ~ standard error of six rabbits that had one-third of blood volume replaced with pure polymerized hemoglobin, containihg 0-0.35 EU/~l.
Figure 5 represents a graphic eomparison of prothrombin levels between three groups of rabbits. The ordinate represents the percent change in prothrombin levels, with the abscissa representing time. Tl represents the baseline prothrombin level; T2 represents prothrombin levels at 15 minutes post-transfu~ion; T3 represents prothrombin levels at 1 hour post-transfusion; T4 represents prothrombin levels at 3 hours post-transfu~ion; and T5 represents prothrombin levels at 24 hours post-transfusion. Solid circles represent the mean value + standard error of six rabbits that had one-third of the estimated blood volume replaced with a hemoglobin containing between 1 and 2 EU/ml: the clear circles represent the mean value ~ standard error of ~our rabbits that had one-third of the estimated blood volume replaced with 5% Plasma Protein Fraction (commercial product found to contain 0.05-0.~5 EU/ml); and the squares represent the mean value + standard error of six rabbits that had one-third of the estimated blood volume replaced with pure polymerized hemoglobin, containing 0-0.35 EUJml.
Figure 6 is a graphic representation comparing serum creatinine levels between three groups of rabbits. The ordinate represents the percent change in serum creatinine levels, with the abscissa representing time. Tl represents the baseline serum creatinine level; T2 represents serum creatinine levels at 15 minutes post-transfusion; T3 repre-sents ~erum creatinine levels at 1 hour post-transfusion; T~
repre~ents serum creatinine levels at 3 hours post-trans-fusion; and T5 represents serum creatinine levels at 24 hours post-transfusion. Solid circles represent the ~ean value +
standard error of six rabbits that had one-third of the estimated blood volume replaced with a hemoglobin solutisn containing betwean 1 and 2 EU/ml (Chromogenic LAL test); the clear circles represent the mean value ~ standard~error of four rabbits that had one-third of the estimated blood volume replaced with 5% Plasma Protein Fraction (commercial product found to contain 0.05-0.15 EU/ml); and the squares represent the mean value + standard error of ~ix rabbits that had one-third of the estimated blood volume replaced with pure polymerized hemoglobin, containing 0-0.35 EU/ml.
Figure 7 represents a graphic comparison of the changes in hematocrit (Hct) following a 50% hemorrhage-transfusion in monkeys. The ordinate repre~ents the hematocrit with the abscissa representing the time in days. Hematocrits of hemoglobin-transfused monk~ys are noted with the solid circles; hematocrits o~ Plasma Protein Fraction are denoted with the open circles.
~ igure 8 is a graphic representation of white blood cell percent, red blood cell percent, hemoglobin percent, ~nd hematocrit percent, from time 0 through day 21, o~ a dog which underwent a 40~ exchange trans~usion with a cross-linked hemoglobin solution of the present invention.
Figure 9 is a graphic representation o~ white blood cell percent, red blood cell percent, hemoglobin percent, and hematocrit percent, from time 0 through day 1~, o~ a dog which underwent a 25% exchange transfusion with a cross-linked hemoglobin solution according to the present inven-tion.
Figure 10 is a graphic representation of white blood cell percent, red blood cell percent, hemoglobin percent, and hematocrit percent, ~rom time 0 through day 78, of a dog which underwent a 33% exchange transfusion with a cross-~ ~ ~ 2 '~

linked hemoglobin solution according to the pres~nt inven-tion.
Eigure 11 is a graphic representation of white blood cell percent, red blood cell percent, hemoglobin percent, and hematocrit percent, from time O through day 47, of a dog which underwent a 33% transusion with 5% huma~ albumin solu~ion.
Figure 12 is a graphic representation o~ white blood cell percent, red blood cell percent, hemoglobin percent, and hematocrit percent, from time O through day 23, o~ a dog which underwent a 50% blood volume hemorrhage, followed by immediate replacement with 5% albumin solution as a ~irst step in the exchange transfusion. This was followed by a rapid removal of another 50% of the hlood vol~me followed by replacement with a cross-linked hemoglobin solution of the present invention.
Figure 13 is a graphic representation o~ white blood cell percent, red blood cell percent, hemoglobin percent, and hematocrit percent, from time O through day 30, of a dog which underwent a 50% hemorrhage followed by immediate replacement with an equal volume of a cross-linked h~moglobin solution of the present invention and a second 50% hemorrhage followed by replacement with an equal volume composed of a cross-linked hPmoglobin ~olukion of the present invention and a 5% albumin solution, mixed in equal parts.
Figure 14 is a graphic representation of Table V o~
Example VIII. The ordinate represents the percentage of total Invention ~emoglobin remaining in each one of its compositional ~ubgroup molecular weights. The abscissa represents time in hours.
Figure 15 is a graphic representation of the data collected from khe experimental work of Example IX. The graph demonstrates that as red blood cells are progressively exchanged for Invention Hemoglobin, beginning at or below a hematocrit o~ 20~, there i5 an expected increase in th2 total plasma hemoglobin concentration.
Figure 16 i~ a graphic representation of data collected from the experimental work of Example IX. Seven test animals and six control animals were excha~ged from i~itial hemato-crit levels with non oxygen bearing volume re~ acemen~s (i.e., Ringer's lactate and hydroxyethyl starch solutions) down to approximately 20%. Both groups showed a similar decrease from initial values to approximateiy 20%. Below 20%, khe control group showed a progressive decrease in arterial oxygen content associated with progressive decrease in hematocrit. The six control animals did not survive. In contrast, the test group displayed noticeably higher arterial oxygen content which was maintained despi~e the progressive decrease in hematocrit. The seven test animals survived and appeared clinically normalO In Figure 16, the ordinate represents arterial oxygen content while the abscissa represents hematocrit.
Figure 17 is a graphic representation of experimental data collected from Example IX. The ordinate represents mixed venous oxygen content, while the abscissa represents hematocrit.
Figure 18 is a graphic representation of data collected from Example IX. The ordinate represents contribution to oxygen content; the abscissa represents hematocrit. The graph demonstrates the contribution of Invention Hemoglobin and of sheep red blood cells to arterial oxygen content at various hematocrit levels. In the seven test animals, as Invention Hemoglobin increased and hematocrit decreased, an increase in percentage of arterial oxygen content was contributed by the Invention Hemoglobin. ~t hematocrit levels in the range of 3%, almost 90% of arterial oxygen content is contributed by the I~vention Hemoglobin, with the ~ ~ ~ 2 ~' bf~ FJ`

remainder being contributed by the residual sheep red blood c~lls and diluted plasma.
Figure 19 is a graphic representation of oxygen delivery during exchange transfusion. The ordinate represents the percent of ini~ial value; the abscissa repxesents tim~ in hours. During the first exchange with Ringer's lactate solution, hematocrit as well as oxygen delivery decreased.
~t this point t- >), exchanges were begun with Invention Hemoglobin solution. While sheep hematocrit decreased further, oxygen delivery increased back towards baseline levels, in association with the Invention Hemoglobin solution infusion. This demonstrates that at the end of the exchange, oxygen delivery is due principally to the Invention Hemo-globin and not to the residual hematocrit (approximately 3%~.
Figure 20 is a graphic representation of data collected from Example X. In both Figures 20(A) and 20(B), the ordinate represents hematocrit percent to blood volume while the abscissa repr~sents time in minutes. Both the test and control groups showed a similar decrease in hematocrit during the exchange transfusion.
Figure 21 is a graphic representation of experimental data collected during the course of Example X. Figure 21~A) is data collected from four test dogs, while Figure 21(B) is data collected from three control dogs transfused with hydroxyethyl starch solution (HES). The test group shows a progressive increase in plasma (free) hemoglobin to approxi-mately 6 percent during exchange, in contrast to the control group. The ordinate represents the grams per 100 ml. of blood volume.
Figure 22 is a graphic representation of data collected from Example X. Comparison of four test dogs (upper figure) ~nd three control dogs (lower figure) demonstrates that the test animals receiving Inv0ntion Hemoglobin maintained stable Gardiac outputs in contrast to the control group which showed increasing cardiac outputs associated with declining hemato-crit and arterial oxygen content. In Figure 22, the ordinate represents the liters per minute of cardiac output (flow) while the abscissa represents time in minutes.
Figure 23 represents a graphic repre~-entation of data collectsd during the performance of Example X. A comparison of four test dogs (upper ~igure) and three control dogs (lower figure) shows reduced but ade~uate and well-maintained arterial oxyyen content during the exchange in the test group in contrast to the control group, which had progressive decline in oxygen content associated with the decreasing hematocrit. In Figure 23, the ordinate represents arterial oxygen content while the abscissa represents time in minutes.
Fiyure 24 represents a graphic comparison of four test dogs (upper figure) and three control dogs (lower figure~.
The ordinate in Figure 23 represents venous oxygen content while the abscissa represents time in minutes. The test group shows reduced but adequate and well-maintained venous oxygen contents during the exchange in contrast to the control group which had progressive decline in oxygen content associated with the decreasing hematocrit.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The product of the present invention comprises a mammalian blood substitute which is a cross-linked h~moglobin solution having a molecular weight distribution of greater than about 90% in the range of 68,000 to 500,000 Daltons, osmolarity as measured by freezing point depression in the range of 180-320 milliosmoles per liter of solution, a final hemoglobin content of 5-25, preferably 9-13 grams per deciliter, a methemoglobin content of less than about 20% and preferably le~s than about 10%, physiologic levels o~ sodium chloride and potassium chloride, less than about 1 nanomole 3 ~ ~ f~

o~ phospholipid per milliliter, le~s than about 1 part per million o~ cross-linking agent, a P5~ in the range of about 18-35, preferably about 24-32 mm Hg, and an intravascular half-li~e of at laast 4 days, with at least a portion of the material remaining in the body ~or at least 6 to 8 days.
The term "P50" is recognized in the art to de~cribe the interaction between oxygen and hemoglobin and represents the partial pressure of oxygen (pO2) at a 50% saturation of hemoglobinO This interaction is frequently represented as an oxygen dissociation curve with the percent saturation of hemoglobin plotted on the ordinate axis and the partial pressure of oxygen in millimeters of mercury (mm Hg) or torrs plotted on the abcissa.
By the term "intravascular half-life" is intende~ the period of time in which the initial amount of hemoglobin in an in ViYo environment falls to half its initial value.
The blood substitute is further characterized by a cross-linking profile on gel permeation chromatography of 50-70% cross-linking, with no material having a molecular weight o~ less than 68,000 being detectable.
The profile for gel permeation chromatography of the blood substitute can be characteri~ed by integration from low molecular weight to total excluded volume where the amount o~
cross-linking i5 from 50% to 75 or 80~. A preferred embodi-ment of the invention shows a molecular weight distribution of greater than about 90~ in the range from 6B,OOO MW to 500,000 MW where no more than 10 to 15% of the mat~rial is in the excluded volume which is in the range of 400~000 to 500,000 MW and higher. After careful filtration, the gel permeation chromatogram also shows that almost none of the ~aterial, if any, is below the 68,000 MW level. The initial 6B,OOO molecular weight peak oE pure homoglobin as measured by gel permeation chromatography is broadened after polymeri-zation such that the retention time of 6~,000 MW is somewhat -21~

complexed so that it is larger - up to 90,000 MW. Integra-tion can be performed on this final peak such that it is found that at least 20% will be in the 68,000 MW range. This fraction does not cause a toxic response in the animal, but is merely excrsted by the kidneys and can be shown in th urine upon sampling.
Additionally, the blood substitute is substantially endotoxin free and pyrogen free as well, and does not cause any of the following abnormal and detrimental chemical and physiologic functions _n vivo: (1) does not activate complement; (2) does not cause hemorrhagic disorders;
(3) does not cause abnormal platelet function or aggregation;

(4) does not cause abnormal prothrombin times (PT~; (5) does not cause abnormal partial thromboplastin times; (6) does not interfere with blood typing or cross-matching; (7) is non-toxic to the kidneys in 3.5 grams per kilogram per body weight or 8 grams per deciliter circulating blood volume;
(8) exhibits circulating persistence of at l~ast seven days;
and (9) acts as a stimulus to accelerated erythropoiesis.
By the term "blood substitute" i5 intended to be a material having the ability to transport and supply oxygen to vital organs and tissues and to maintain intravascular oncotic pressure. Accordingly, the term encompasses materi-als known in the art as "plasma expanders" and "resuscitation fluids" as well.
The term "cross-linked" or "pol~merized" is intended to encompass both int~r-molecular and intramolecular polyhemo-globin, with at least 50% of the polyhemoglobin of greater than tetrameric form.
The term "substantially endotoxin free", for the purposes of the present invention, may be described function-ally as a ~lood substitute which contains less than 1.0 endotoxin units per milliliter of solution, at a concentra-tion of 10 grams of hemoglobin per deciliter o~ solution.

1 3 ~

This blood ~ub~ ute, when used as ~ repl~ce~ent ~or approximately one-third of the total blood v~lume of a rabbit, produces a percent ~hange in blood platelet levels, over time, wh~ch i~ ~ubstantially ~imilar to Curve delta-delta of Figure 2, or a percent change in whi~e blood cell level~, over time, which is s~bstantially similar~ to Curve delta-delta of Fiyure 3, or a percent change in ~ibrinogen levels, over time, which is ~ub~tanti~lly ~imilar to Curve delta-delta of Figure 4, or a percent change in pro-thrombin levels, over time, which i6 ~imilar to Curve del~a-delta of Figure 5, or a percent change $n serum creatinine levels~
over time, which i8 substantially ~imilar to Curre delta-delta of ~igure 6.
~ n a preferred ~mbodiment, the "~ubstantially endotoxin free" blo~d ~ubstitute o~ the invention will co~tain le~s than 0.5, ~nd preferably less than 0.25, ~ost pr~ferably le~s than 0.02 end~ oxin units per milliliter 9f solution (EU/ml) as ~easured by the Limulus ~mebocytic Lysate (L~L3 assay. The L~L assay is described by Nachum et al.~ L b~r~t~r~
13:112-117 (1982) and Pearson III et al., ~ioscien.ce, 30:
461-464 (1980)~.
By the term ~endotoxin(s~" is intended the generally cell-bound lipopolysaccharides produced as a part of the outer layer o bacterial c~ll walls, which under ~any conditions are toxic. When in;ected into an animal, endo-toxins cause fever, diarrhea, hemorrhagic ~hock, and other tiBSUe damage.
By the term "endotoxin unit" (EU) is intend~d that ~eaning given by the United States Pharmacopeial Convention of 1983, Page 3014, which defined EU as the activity con tained in 0.2 nan~grams of the U.S. reference ~tandard lot EC-2. One vial oP EC-2 contains 5,000 EU~
The pre~ent invention further involves the process for producing the ~emi-fiynthetic, 6ubstantially ~tromal-free :~3~ 2~

blood substitute. The process comprises the steps o~ (1) obtaining the blood raw product, (2) fractionating the blood raw product to produce a red blood cell ~raction which is substantially free from white blood cells and platelets, (3) mechanically disrupting the red blood cell ~raction to produce a hemoglobin-containing solution, (~) clari~ying the hemoglobin-containing solution to produce a hemoglobin solution which is substantially free of cellular debris, (5) microporously filtering the hemoglobin solution which is substantially ~ree of cellular debris to produce a partially sterilized hemoglobin-containing solution, (6) ultrafiltering the partially sterilized hemoglobin-containing solution to produce a size-separated hemoglobin-containing solution, (7) chromatographically separating the size separated hemoglobin-containing solution to produce a hemoglobin substantially ~ree of phospholipids and non-hemoglobin proteins, said hemoglobin retained on the chromatographic column, (8) eluting the substantially phospholipid~-~ree hemoglobin from the column to produce a substantially endotoxin-free hemo-globin solution, (9) cross-linking 6aid substantially endotoxin-~ree hemoglobin solution to produce the cross-linked blood substitute, and (10) partially separating the cross-linked blood ~ubstitute by ~iltration, all ~teps done in a substantially endotoxin-free environment.
Each of the proce~s ~teps will be described in greater detail below.

I. THE PROCESS

A. Blood Collection Starting point in the present invention is an erythro-cyte (red blood cell) source. As such, the starting material may be freshly drawn human blood, outdated old blood ~rom ~3~ a~, blood banks, placentas, or packed erythrocytes obtained from human donor c~nters. Additionally, erythrocytes obtained from animal blood are ~ntirely suitable as well. Accordingly, blood from a variety o~ ~ources such as bovine, ovine, or porcine may be used. Because of its ready availability, bovine blood obtained ~rom slaughterhouses is the pre~erred erythrocyte source.
The unique approach of the present invention has re~uired special techniques in the collection and handling of blood in large quantities. Large collection trochars are used which extract the blood in a sterile manner. The trochars reyuire careful insertion and handling and are connected to tubing approximately 2 feet in length. In order to insert the trochar the hide must be cut away, peel~d back, and the trochar then inserted in the animal's major vessels close to the heart with care not to puncture the esophagus.
Avoiding the introduction of bacteria and the maintainance of endotoxin-free or low endotoxin level material is important.
This is accomplished using individual containers that are pre-charged with an anticoagulant and that are depyrogenated and re-checked for endotoxins. Typical anticoagulants include sodium citrate. In all cases, endotoxin levels of the containers must be less than .01 endotoxin units as detected by LAL.
This solution is then charged to small vessels that can hold between 2 to 10 gallons of gather~d blood in a sterile ~anner and, therefore, maintain the blood in an endotoxin-~ree state. The collected blood in its container is capped off immediately to avoid exposure to the environ-ment. Upon completion of the collection process, the material is chilled, typically to about flC, to limit bacterial ~rowth. There is no pooling of blood at this time;
the blood is later checked for endotoxins and sterility to ensure that (1) no one cow is sick; or (2) a bad collection J~ a J

technique has not contaminat2d the entire batch or collection for that dayO ~lthough the abova collection method is preferred, there are many collection methods which are suitable and a~ailable to one with ordinary skill in the art.

B. Red Cell Separation The blood is brought to a processing center at which time each vessel is sampled and checked by L~ analysis for endotoxin levels. If the endotoxin level is higher than 6-7 EU per ml the blood is discarded. Only if the individual blood container tests at below that endotoxin level is the material approved for secondary processing.
Typical secondary processing of the prior art was to suspend the blood (ACD anticoagulated blood) in a saline solution of physiologic salt concentration and centrifuge in order to separate efectively the plasma proteins and white cells ~rom the red cells. This suspension process is per~ormed through several "washing" steps, i.e~, 2-4 times, in an attempt to remove all free proteins. In the prucess of the present invention, however, it was found that this approach was untenable ~or practical manufacturing scale up;
in fact, to separate the hemoglobin product free ~rom many contaminants, it is not necessary to do this washing process at all.
In the preferrsd process, the whole blood ~rom the animal, once it has been checked for endotoxins, is passed through a semi-continuous type centrifuge where the red cells, white cells and plasma can be effectively separated on as large a scale as desired~ The process employs a bowl-type semi-continuous centrifuge where the bowl is maintained at 15,000 to 18,000 rpms, i.e., a Sharples AS-15 unit. The bowl and the top configuration are set up with an opening at a particular radius that permits a discrete layer separation i 3 l 2 ~ ? ~' such that red cells, white cells and plasma can be removed through the operation. While the Sharples bowl-type centrifuge is pref~rred, typical separation apparatus also suitable include basket centrifuges such as are manufa~tured by Bec~man Instruments.
To prepare ~or this operation the centrifuge ~s depyro-genated, i.e. using, typically, a 0.5 molar sodium hydroxide for at least 1 hour prior to installation into the machine housing container. The top spouts or collection devices are handled in a similar fashion, thus allowing for complete depyrogenation of all contact surfaces the blood may en-counter. Once the parts have been put through the depyrogen-ating process, the system is assembled. A lobe pump or peristaltic pump with sanitary design is used to fl~sh fluid through the entire system and collection ports; typically, a solution of 0.5 molar sodium hydroxide i~ used, but other depyrogenating solutions known to the art are suitable as well. At the completion of the flush, it is necessary to reduce the pH to a range which is conducive to the handling of the hemoglobin solution. This is accomplished through a water purge which reduces the pH to a range of approximately 7-9. In some cases it has been necessary, because of density differences between the depyrogenating solution and water, to use an acid solution to help neutralize the strong base employed in the depyrogenation step. All these solutions must be depyrogenated and checked prior to use in the washes.
Once the pH level has been brought below 9, samples are obtained from the effluent streams of the centrifuge and endotoxin testing is performed. When an endotoxin level of .Ol EU/ml or less is achieved, the system is ready for the separation o~ the blood. ~owever, in this separation it is critical that the flow rate of the blood into the centrifuge be at a suf~icient rate to limit the amount of sedimentation of red blood cells caused in the Sharples centrifuge. If the U~ J
--27 ~

flow rate is too low, the red cells will settle into the bowl of the centrifuge and not be separated or collectible into a separate container. In a centrifuge with a 4'~ diameter bowl, a ~low rate in excess o~ 2.5 to 3 liters per minute, but not excseding 6 liter~ per minute, is required to limit sedimen-tation. I~ a larger bowl or different g forces are~employed, then different flow rates are required, the particular parameters being within the skill of the art.
Once all parameters have been established, the blood from the various batches or various cows is introduced into the system and the effluent (separated red cells) is col-lected under sterile conditions in a separate container.
Howe~er, at this point the effluent is pooled and is no longer treated on an individual animal basis. To eliminate any variations of pH or potential initial entrapment of bacteria on the centrifuge, positive pressure sterile nitrogen is applied to the chamber where the bowl is spin-ning. For true sterilization of the system, a steam sterili-zation cycle may be applied by introducing steam into the bowl spinning chamber and steaming for up to ~n hour prior to use. After comple~ion of steaming, the system is cooled i.e., through glycol coolant tubes, typically to about 4C.
(After harYesting from the animal, it is important that the blood be brought to and maintained at a temperature just above freezing, typically about 4C.) In the separation of the red cells it is important that the collection chamber, i.e., spout area, where the red cells are collected at the top o~ the high speed spinning bowl, be configured such that high impact o the cells occurs. In striking these ~urfaces, the red cells become broken through a mechanical degradation, as opposed to using a hypotonic solution. (In a hypotonic solution, the red cells swell and cause the membrane to rupture ~rom hydraulic forces.) This ~s a change from the normal operation of swelling cells to ~ 3 ~

lyse them by hydraulic pressure, to one of mechanical degradation. This mechanical degradation is ex remely rapid and does not generate the high degree Df free small cell membrane components found by other methods. The red cells are collected in a vessel and prepared for the second centrifuge operation.

C. Red Cell Clarification Once the blood has been processed and the red cells have been separated from the white cells and plasma, the mechani-cally disrupted red cells are diluted using pure depyrogen-ated water which has been maintained at low temperature, i.e., about 4C. Typically, disrupted the red cells are diluted by at least 50%. The red cells are then introduced into the second separation step; typically a similar type of centrifuge to the first operation may be used. In a prefer-red embodiment, a Sharples centrifuge with a 4" bowl operat-ing in a semi-continuous mode and spinning at 15,000 to 18,000 rpms is used. The flow rate, however, is substan-tially decreased: 0.5 liters per minute or less is recom-mended. Unlike the first processing step, this step employs a different typ~ of top configuration to the centrifugation bowl. No separation of two layers such as the plasma white cell and red cell composition is effected at this step.
This clarification step results in the separation of all cell debris from the liberated hemoglobin solution. The same care taken in the first step for depyrogenation and sterility must be employed in the second step. Once this material has been collected from this second step, it is ready for microporous ~iltration.

~1 3 ~ r~

D. Microporous Filtration The microporous filtration must be operated dif~erently than a pressure ~iltration mode. In a practical sense, pressure filtration is not acceptable to industrial scale processing of hemoglobin ~olutions. To employ ml~roporous filtration suecessfully, either a plate and frame ~iltration or hollow fiber filtration system may be used: however, it must be operated ~uch that the pressure drop across the membrane (the transmembrane pressure) is carefully maintained to within about 5 pounds per square inch (psi). If the pressure drop exoeeds the tolerance level by l to 2 psi, the membrane rapidly becomes plugged with the remaining cell debris and the flux rate across the membrane drops to an unacceptable level for industrial purification in a semi-con-tinuous mode.
While tangential ~low of this material across the membrane is at a flow rate of 2 to 5 liters per minute, the flux through the membrane is on the order of 0.1 to 0.2 liters per minute. This operational rate is maintained to eliminate cellular debris from building up on the membrane.
When the concentration of solution tangential to the membrane decreases to less than 10% of the initial solution, the remaining solution is discarded or it is re-diluted with water to extract additional product and thereby produce a higher yield of hemoglobin from the system.
The filtration system may utilize lobe or peristaltic pumps with sanitary design, thus decreasing and limiting seals and sha~ts that may cause introduction of bacteria and pyrogen contamination. Other pump designs known to the art for sanitary pumping may be used, however. Such pumps include centrifugal, gear, and tubular diaphragm pumps.
The membrane systems are pre-treated to ensure depyro-genation and proper pH. I~ handled improperly, pyrogens are :~ 3 ~

added at this point and it becomes more and more di~ficult to remove them throughout the remaining processiny steps.
Depyroyenation and p~ control is accvmplished by using standard sanitation procedures and depyrogenation procedures, i.e., typically with sodium hydroxide and voluminous washings with pyrogen-free water to bring the pH to within a~ceptable ranges for handling of the hemoglobin solution (<pH 9).
While handling o~ a transmembrane pressure limitation in such a manner i5 not well known and has only been practiced in the last few years on a selected basis with tissue fluid process ing, suitable techniques are within the skill of the art.
In a preferred embodiment, a filtrate side restriction is employed such that flux rate is limited to its steady state (non-plugging) condition. If a fluid stream is applied to a tangential flow membrane system, and the inlet pressure is approximately 20 psig, the outlet pressure is 0 psig, and the filtrate side of the membrane is 0 psig, giving an average transmembrane pressure (ATP) o~ 10 psi., the solution to be filtered has a tendency to ~ill and extrude into the porous membrane surface. I~ will ~ssentially plug the membrane and will not be swept clear by the tangential shear created by the cross-flow of 1uid. By restricting the outlet ~filtrate~ BO that the ATP is only 1 to 2 psi, the tangential ~low sweeps th~ sur~ace clear and flux across the membrane remains constant yet low when compared to initial flux rates with high ATP. The flow undex steady state conditions may be 0.2 liters per minute, with 1 to 2 psi ATP, and 1 to 1.2 liters per minute with 20 psi ATP. However, the 20 psi ATP will stay constant and cause the flux to rapidly fall to zero flux within minutes.
With the completion o~ this first microporous filtration step, the ~olution has been at least partially sterilized and substantially all cell debris above 0.45 micron has been removed. In ~ome cases it may be required to make the 3~-~oluticn sterile ~t t~i~ point. In these cases; ~fter the 0.45 ~icron ~icroporou~ filt~ation ha~ besn c~mpleted a 0.22 ~icr~n ~iltration may be ~ployed ~n the ~ame ~anner ~ the 0.4~ ~icron filtration. TAe re~ulting ~olution is n~w ready ~or the ~olecular 6eparations which f~llow.

E. Ultrafiltration ~ he next ~tep includee the carePul ~tagin~ oP 100,000 molecular weight ~iltration (measured in Daltun~) using membranes which ~f~ectively retain everything greater ~han 100,000 molecular weight and which permit everything less than 100,000 ~olecular weight to pass through. Typical membranes are commercially available ~rom ~illipore Corpora-tion, and are ~old under the trade nam2 Durapore. Everykhing bslow thes~ levels is ~iltered t~rough the ~embrane ~y tem.
Hemoglobin (about 67-68,090 ~olecular weight) passes thr~ugh t~i~ ~embrane eystem ~nd i~ collected ~n tankageO
ThiQ l~rge ~embrane filtration operation reguires careful monit~ring because, over a period o~ hours, the membrane will be~ome plugqed and ~iltration ~lux will decrease rapidly. It i5, therefore, ne~essary to ~lush the ~embrane on a regular basis with a pure water solution. The Ilushing reduces oell debri~ w~ich ~nay otherwis~ c:oat anù
occlude the membrane, thereby reducing hemoglobin ~;olution ~lux rate. The tangential cro~ low time cycling ~ver this ~embrane can be up to 2 hours and does not affect the ~ethemoglobin level or the viability of the hemoglobin for its intended purpose. ~hen the ~luid volume aPter the ~icroporous filtration has been reduced to about 30% of its volume during ultrafiltration, 6terile pyrogen free water may be ~dded to obtain greater yield of hemoglobin 601ution.
~aximum dilution i6 about 50%. This ~aterial ~ay ~l~o be di~carded. If the 30% original material is diluted, it again * Trade-mark . i~
., ~ ~2$~`~

may be reduced t~ about 30~, at which ti~e ik i~ di~carded.
The filtered intermediat~ held in ~terile, pyr~gen~free tankage for ~ubsequ~nt operation~. A typical devi~e for effecting the ultrafiltration ~tep is a Millipore* Pellicon cas~ette with a Durapore ~e~brane; however, other device~
known to the art ~ay be used as well.
The next ultrafiltration Bt2p, requires a removal of material below 68,000 ~olecular weight. ~hi6 i~olates ~mall molecule ~emoglobin ~nd other ~mall prot~ins that may have been carried over from the whole bls~d plasma. In all ca~es the hemoglobin 601utiDn i~ Daintained ~t a concentration o~
~bout 5 to 15 grams per deciliter. Filtration accomplished at thi~ step provides some degree o~ concentration. At high concentration, low ~lux rates are exhibited. In both ultrafiltration op~rations where 100,000 mw and 30,000 mw me~branes are e~ployed, the nece~sary depyrogenatio~ ~teps and ~ubseguent checking a~ter washing with pyrogen ~ree water are usually reguiredO
In the 1~0,000 ~w ~eparation ~tep pyrogens ~ay be removed ~ince ~ome pyrogen~ ~re betwe~n 100,000 and 1 million ~w. With the depyr~genation of the 30,000 ~olecular w2i~ht membrane ~nd the preparation of this membrane packet for its filtration process, the hemoglobin ~olution has pass~d over thi tangential flow 8y6tem to allow the per~usion of ~mall ~lecules through the ~embrane. Recycle may or may not be used in ~his operation, ~l~hough i~ is requir~d in the 100,000 ~w filtr~tion ~tep. The retentate (material re-tained), is held $n a ~torage tank and checked for endo-toxins. In ~11 cases the endotoxin~ ~ust be below 0.5 EU per ml because subsequent operations makes re~oval o~ high levels o~ pyrogens quite di~ficult. This ~aterial is ~tored under a sterile nitrogen or ~rgon atmosphere that ~aintains st~bility in the tankage ~ystem. '~ypically the ~ethemoglobin level ~6 below 1% at this point in the process. The ~iltration steps * Trade-mark .~1~

~ 3 ~

must be performed at low temperatures, typically at about 4C. Following the filtration, the material i~ either ~rozen or directly aliguoted to lot sizes for large scale chromato graphic processing.

F. Chromatography Prior to the chromatographic ~eparation, the material is in a concentrated state of no less than 2 grams per deci-liter and not greater than 11 grams per deciliter. The chromatographic system includes pumps, a gradient generator, columns and detectors.
A typical pumping system comprises a diaphragm pumping system with a range of 1 ko 5 liters per minute pumping capability. Such a system includes a Pulsafeeder 8480 stainless steel diaphragm pump or equivalent. For the feed ~ystem a smaller pump is used where the flow will range from 0.1 liters per minute to 1.5 liters per minute~ This pump is typically a smaller volume pump and would be of a tubular diaphra~m design. ~ typical pump for this oper~tion is a Pulsafeeder 7120. ~o configure the chromatographic system such that it operates properly it is required that two large systems are asse~bled so that one would be used as thP
operating system for chromatographing the material while the other system is used for flushing, cleaning and regeneration of the column.
~ solvent composition generation system has been fabricated and is comprised of flow control valves that deliver to the applicable pumping system a proportional amount of two ~luids generating a fluid composition gradient over time of a specific ionic ~trength. Ionic interaction is used to effect an ion exchange chromatographic separation on the column syst2m. Fabrication o~ this or equivalent systems is within khe ~;kill oî the art.

~ 3 :~ 2 i~
-3~-A typical flow control valve is a Baumann flow control valve, which has been programmed to operate using a standard programmable controller, for example a Texas Instrument 530 programmable controller. All piping and tubing to the system is of sanitary nature and ~ade of 316L tubing approximately 1/2" to 1" in diameter. The feed system through the~gradient yenerator and through the pump are presented to a separation segment or column as is known in the art.
The column is typically made from stainless pipe. ~he stainless pipe can be interconnected with tubing of 1/2"
diameter, such that it will comprise one long column for effecting a separation. The pipe or column is typically lined with teflon to give compliance to the internal surface which is helpful in effecting packing of the media internal to the column system.
The chromatographic system affluent can be monitored by a splitting o~ the stream and passing that small representa~
tive amount khrough a refractive ind~x detector like a model R401 from Waters Associates or an ultraviolet detector, typically a 441 Waters ~ssociates model number. These systems can be used to monitor the effluent stream from the column to detect the point at which the protein o~ interest is being eluted.
Once all parameters have been established and guidelines have been set, there is no need for a detector in the system and fraction collection may be achieved through simple time elution profiles.
These materials may be either fabricated or purchased from various suppliers of industrial grade piping and tubing.
The column is fabricated to achieve uniform distribution of sample being presented to the top of the column and, in conjunction with that, uniform samplQ collection from the effluent of the column. The length to diameter ratio is significant in that cre~ting a column that is too long or too ~ 3 ~ 2 ~ . . ) short will significantly affect the efficiency of the separation and equilibration Eor doing the ion exchangeO
The column i~ comprised of ~eparation media whi~h allows for some irreversible adsorption of phospholipids ~irrever-sible in the simple operation mode3 a~d a discrete ion exchange separation using a specified gradient~ elution pattern of solvent. The separation media comprise particles of silica gel of from about 50 to 150 microns in size; the flow arross this material is in the range of about 2.5 liters per minute.
The silica gel is of an average pore size of 300 angstrom units, as measured by BET nitrogen absorption. This silica gel is available from various manufacturers i.e., W.R.
Grace Dlvison Cbemical Co. This gel is the preferred substrate on which to build the derivatiæed surface which gives the functionalized property for separation of the hemoglobin solutionO
To produce the separation media, it is necessary to derivatize the silica surface irst with a special ~ilane which creates a diol chemical type surface on the silica surface. This diol can be typically achieved by creatin~ a glycidoxypropyltrimethoxysilane coating to the surface, with techniques which are well known in the field of chromato-graphy, typically by suspending the silica and the silane in a vessel which has been partially diluted with water. The reaction is a water base reaction and this polymer will coat onto the surface of the silica. This reaction to coat the silica re~uires a 20 hour reaction time at approx. 70C.
Once this coating has been achieved on the silica, the material may be simply washed by a series of methanol and acetone washes to create a clean permanently bonded, diol coated silica. The material is then dryed and prepared for the 6econd step or ~eries of steps where different monomers are coated onto the surface and the surface will be deriva--36~

tized to have a quat~rnary amine type surface property for doing or preforming the spPcific kind of ion exchange separation. The organic stationa~y phase is a t~in skin of cross-linked polymer. The cross-linked polymer that i6 put onto the surface is built up from two different hydrophilic vinyl monomers. For example, one may use a monome~ such as n-methyl/alacrylamide in 48% water ~olution (Silar Labs), and methylamdiopropyltrimethylamonium/chloride.
The two monomers have various capabilities; one monomer will copolymerize with another functional monomer, i.e., one having the ion exchange or absorption properties desired. It will cross-link with other polymer chains and anchor the cross-linked polymer to the silica sur~ace.
The speciPic ~onomers chosen for this purpose have a vinyl functionality and a reactive groups that react in such a way that they can react with each other, forming the bridyes that are necessary to coat to the surface and the coating of a stationary phase consisting of an amine func-tional group, thereby producing an ion exchange capabili y in the desired range.
Once these two monomers have been suspended in the aqueous solution, as well as with a methanol solution of the silica, the ~uspension solution is evaporated away leaving the monomers coated onto and into the silica gel. At this stage the mixture is resuspended in a new solution which also includes a radical initiation system, such as a Dupont product, Vazo 64. To initiate the reaction, the reaction mixture is heated to the point where it must be maintained at 70~75C, no higher and no lower.
At this temperature the reaction proceeds and the polymer i8 coated onto and bonded to surface, including the functional groups that produce the surface property used in the chromatographic media. When the reaction has been completed, it i~ necessary to remove unreacted monomer with a series of washes with several solYents~ such as acetone and methanol. Following the completion of all these washes the material is dried and ready for use.
A typical column diameter is 6" and a typical column length is 2 feet. However, suitable variations are within the skill of the art. The maximum operating pressu~e is 500 psi. The injection is made by pumping the solution onto the column, typically at a rate of 1 liter per minute for approximat~ly 1 minute, then injection is terminated.
Therefore, the load factor is no greater than 1 liter of material at 7 grams per deciliter. At the completion of loading the hemoglobin solution, an isocratic flow of buffer (e.g. tris buffer at pH 8.9 to 9.0) is applied to the column and continues to flow through the column until such time as the gradient or ~ariable composition flow i5 started. The buffer, as the primary eluant, is then diluted over time.
Typically, the eluant is made up from a Tris buffer base solution which is made in a concentration of 108 grams per liter Tris with a p~ of about 8.6-9.2. The temperature range for elution is about 3-10C. These ranges are significant since changing the temperature range also changes the pH of the elution solution. The secondary solution for eluting the material of interest may be prepared using a solution of Tris buffer, highly purified in the same manner as the previous buffer. In addition, this buffer also contains salt to a 1 molar concentration. This solution is also pH adjusted to be identical to the original pH solution, which is in the range of 8.6 to 9.2. Release of phospholipids takes place prior to the elution of the hemoglobin, with endotoxins eluting after the hemoglobin peak of interest has been collected.
The chromatographic selection techni~le is done by W
absorption, refractive index, typically using equipment as described above, or visible observation of the effluent stream. Typically, the first portion of the eluting hemo-globin is discarded to waste; then the collection of e~fluent begins and continues until the peak or the response has bee~
reduced to 20% to 10% of its peak amplitude. This consti-tutes the fraction to be collected and the fraction of interest for purification. If the coll~ction point runs beyond the appropriate retention time, then other`proteins and/or endotoxins may be collPcted and the product may be render~d unusable. Similarly, if collected be~ore a peak retention time, the material may contain unacceptable levels of endotoxins. The phospholipid count and extraneous sub-components of hemoglobin are discarded, both the pre-retention peaks and the post-retained peaks. ~his collection process allows intermediate product material which has been diluted approximately 40 to 1, in a pH range of 8.9 to 9Ø
In this pH range it is necessary to c~ncentrate the material rapidly. In this dilute state, the appearance and formation of methemoglobin occurs at a rapid rate. To effect this concentration, a membrane of 10,000 mw ~r less can be used. Either plate and frame tangential flow or hollow-fiber flow sytems are acceptable. Typical systems include a Millipore Pellicon cassette. When concentration levels of 7 to 10 grams per deciliter are achieved and a methemoglobin level of less than 1.5% are achieved, the fractions are collected for long-term storage. At this point the ~aterial may also be transported into a reactor system for the subsequent polvmerization reaction.
Following the collection in the chromatographic system, the chromatographic column undergoes a seguence o~ washes to prepare it for a second loading of unpurified material. If this column preparation is not per~ormed, various subcom-ponents and contaminants will elute and render subsequent runs invalid. Typically, the wash is a~complished by using a 100% pyrogen-free 0.5-1.0 molar NaCl ~a~h for a period of at least 5 minutes, or 3 column volumes, and no more than 10 minutes, or 6 column volumes. At the completion of the buffer yradient and salt flush, the fluid phase is returned to initial conditions of 100% tris buffer which is 0.18 grams per liter of ~ris buffer, and pH is adjusted to approximately 8.9 + .1 for the hemoglobin elution process. Although ranges of the hemoglobin pH have been studied, the 8.9-9 pH range of the chromatographic system yields the highest and best isolation of a pure hemoglobin analogue. At lower ranges (8.6-8.4), hemoglobin is eluted in a pure tate but the loadability of material onto the separation m~terial is drastically decreasedO At pH levels of 9.5-11, the formation o~ methemoglobin occurs at a rate which makes it untenable to maintain low methemoglobin levels. Further, there is the potential Por cross-contaminationO Over a period o~ 2 hours the methemcglobin level may increase 5% at this higher pH
range. The matPrial eluting from the column is a hemoglobin solution which is substantially free of other proteins~
endotoxins, and phospholipids. This material has utility in its own right as an intermediate product in the production of a cross-linked, substantially endotoxin free, substantially phospholipid-~ree semi-synthetic ~losd substitute.
Long term storage of hemoglobin solution a~ter concen-tration with sodium chloride and tris buffer has been carried out for periods o~ as short as 1 day and as long as 6 months.
The results have shown no product degradation or increase in the methemoglobin level if the product is maintained at -20C. The solution, however, upon thawing over a 2 to 24 hour period, may exhibit an increase in methemoglobin. If left in an un~rozen state, the methemoglobin level will continue to rise. In other studies where material had low p~
(pH 7 and below) the methemoglobin level increase is drama-tic, i.e., a 10 perc~ntage point increase within 3 hours.
~ he hemoglobin solution typically has the following ¢haracteristics:

HEMOGLOBIN SO~UTION SPECIFICATIONS

~emoglobin g/dl 7 - 15 Oxyhemoylobin ~0 - 100%
Carboxyhemoglobin O - 2%
~ethemoglobin 0 - 10%
pH 6.5 - 9.0 Endotoxin EU/ml < 0.01 Molecular weight daltons 68,000 Phospholipids < 1 nanomole/ml TLC Plat iodine developed -- clear Amino Acid Analysis No foreign protein amino acids N-Terminal sequencing ~8%~ conforms to Bovine hPmoglobin sequence Page Gel Single Band? (no virus contamination) Salt concentrations may vary High performance chromatography 99.9%+ hemoglobin protein G. Polymerization (Cross-linking) Reaction When material has been either specifically allocated for polymerization reaction or has been thawed from the frozen state, it is introduced into a sterile pyrogen-free reactor having impeller blades positioned to effect rapid mixing and high shear. (A typical apparatus is a 3 liter Applicon fermenter with a flat bladed impeller positioned one inch from the bottom of the reactor and with 5 one-hal~ înch baffles positioned about the reactor. This is necessary to ~ 3 ~

preven~ l~rge polymer ~ormation when ~he cross-linking ~gen~
is added.) The hemo~lobin ~lu~ion added to the reaetDr i~
put on ~ rec~rculation ~yste~ ~nd the he~globin 601ution i~
withdrawn ~ro~ the react~r and p~ssed acr~s an ex~lu~ion ~e~brane, typically a 10,000 ~w exclusiQn ~ilter, and returned to the rea~tor in a low 2 ¢nviro~men~ he reactor may be blanketed with an inert ga~ . argonO~ This l~st pr~cedure is ~ce:omplished by drawing a vacuum on the reactor ~nd placing ~n ~rg~n blanket on the liguid in this r~act~r. Extreme caution i~ taken to eliminate introduction of bacteria at this point; the ~aterial i6 pyroge~-free, exhibiting no endotoxi~s by LAL ~nalysi~.
A 6terile pyroyen-fr~e bu~er (pH 8.9-9.1~ i~ then ~dded to the reactor through a depyrogenating ~embrane ~ilter, typically a 10,~00 molecule w~ight ~ilter. Simultaneously a lO,OOo mw concentration loop i~ cycled ts balance the volume o~ intrcduced ~luid and the ~xiting fluid from the rea~tor ~ystem.
The reaction buf~er which is being used to neutralize the high pH is a physiologic ~omposition of sodium, ~hloride, and potassium, with typical Yalues o~ 120 milli~quivalents codium, 120 ~illieguivalent~ ~hloride ~nd 4 millie~uivalents pot~ssium. The pH of the ~olution is adjusted with HCl and *ris base to a ~ o~ about 4.7 to 5.2. If the pH i~ too low during the pH reducing process, large ~mounts of methemo-globin or~ at the point o~ introduction of the neutralizing 2cid 6Dlution. The ~iltration process is maintained until the pH has dropped to a range o~ about ~.4 to 8.0 pH units in the reactor. At this time the introduction i~ terminated and ~he introduction of ~he ~ross-linking solution i6 made.
~ uitable cro~s-llnking agent~ are disclosed in U.S.
Patent 4,001,200 to ~on~en ~t ~1. The preferred-class of c.ross-linking agents are those having aldehyde functionality, most Preferably, r~
., ~ , . _ a ~r~

dialdehydes, with glutaraldehye being the cross-linking agent of choice.
Where glutaraldehyde i6 used, the glutaraldehyde is added, typically at a rate of about 100 milliliters per hour.
The glutaraldehyde solu~i~n is typically prep~red by thawing a high purity speciication glutarald~hyde (stored~at -20C
to 4C) in a ~hort, typically 2-5 minute, time ~rame. This solution, which preferably has about a 25% concentration of glutaraldehyds, is then added to pyrogen free water, the proportions of which make up a solution which is preferably about 5 milliliters of a 25% solution diluted intD 100 milliliters of pyrogen-free water. The solution is added at the rate specified above to the reactor and reaction mixture.
The monitoring of cross-linking solution and its effects on cross-linking (polymerization) is done by gel permeation chromatography. The gel permeation chromatography requires the use of a 300 Angstrom pore size hydrophilic packing material column with resolution capability of over 24,000 plates per meter. A typical column is available ~rom Waters Associates; a typical packing material is Waters Protein Pak 300SW. ~he eluting chromatogram as recorded is integrated over the time of peak elution and quantitated against the skarting materialO Preferably, a cross-linking percentage of 50~ to 70% is achieved. This number is determined by the percentage of material eluting from the column which is less than 600,000 molecular weight ~Daltons) and greater than 68,000 molecular weight (Daltons).

H. Xembrane Concentration Once greater than 50-55% cross-linking, as calculated by gel permeation chromatography, has been achieved, the 601ution is ready ~or 100,000 molecular weight membrane concentration. During this membrane filtration the tangen-tial flow of the reaction mixture is passed over the membrane with a permeation of material which is 68,000 or less through the membrane system. This is performed until about a 25 reduction in fluid has been achieved.
At the point when cross-linking i5 deemed to be com-plete, a ~uenching solution, i.e., a solution of pyr~gen-free lysine, pH 7, i8 added. The concentration of the lysine solution is 1 gram per liter. This lysine solution is added to quench the polymerization reaction of glutaraldehyde with hemoglobin and to complex with excess glutaraldehyde. It is also believed that this material will fix to unpolymerized glutaraldehyde bound to hemoglo~in molecules. At the completion of this addition, molecular weight distribution is determined and found to have been stabilized as measured by gel permeation chromatography. Filtration is then started to remove excess lysine, excess glutaraldehyde and any other molecular weight species which is below 100,000 M.W.
The gel permeation chromatogram of the initial non-cross-linked hemoglobin solution exhibits molecular weight sizes from 16,000 to 68,000 Daltons, with the largest amount at 68,000 Daltons. After filtration, there is some, not more than 50%, 68,000 Dalton hemoglobin and the appearance of material under 68,000 Dalton molecular weight is not detec-table. Filtration of the material after cross-linking al50 provides opportunity to balance the electrolytes and pH of the solution and thereby give a balanced physiologic solution for injection.
At the completion of this filtration process the material is removed from the system and bagged ready for freezing. At the completion of all processes and during the bagging, a sample of material is withdrawn for testing.

-4~-II. THE PRODVCT

Typically, the product has the ~ollowing characteris-tics. The molecular weight distribution of the materlal has greater than 90% o the material in the range of 68,000 Daltons to 5~0,000 Daltonæ. Osmolarity as mea~ured by ~reezing point depression is typically from 220 to 320 milliosmoles per liter of solution. The electrophoretic pattern exhibited on gel electrophoresis shows bands in 68,000 to 500,000 molecular weight range. The final hemo-globin content can be adjusted to 5 to 25, preferably 9 to 13 grams per deciliter and the methemoglobin level is under 20%, preferably under 10%. The ion concentrations of sodium chloride and potassium are nontoxic to the animal or to the species to be tested. Thin layer chromatography developed for the detection of phospholipids exhibit a clear plate upon developing by iodine staining. Phospholipids as determined by phosphoric acid reduction are non-detectable, with less than one nanomole per milliliter as the limit of detection.
Gas chromatography is used as a quantitative measure for ~ree glutaraldehyde. With detection o~ 1 part per million by gas chromatography as the limit, no glutaraldehyde may be detected. No protein other than hemoglobin is present as determined by gel chromatography and iso-electric ~ocusing techniques.
The solution generally has less than 0.01 endotoxin units per ml as measured by LAL (limulus amoebocytic lysate) assay with a .01 ~o .1 ~ensitivity scale, and is pyrogen-frea by all testing. Rabbit studies have been performed on this material which exhihits the same characteristics as would be exhibited by a pyrogen free material in that no ~ever is exhibited by the rabbits. This material does not produce any abnormal endotoxin response and other ~actors in ~he rabbits being tested as it related to hemorrhagic conditions done on Ir~ ,?~, ~

a control group of rabbits which were then supplied with a pure plasma fraction of lJ3 volume. It should be noted that in all cases, there was some elevated levels of enzymes and some histopathology that demonstrated changes in the organs.
Nost of these changes however, were deemed to be reversible and were, as mentioned earlier, similar to those fo~nd due to hemorrhagic conditions and replacement by a pure plasma protein fraction. This can be translated to a lack of endotoxin response and other factors in higher animals. The purity as monitored by high per~ormance liquid chromatography using an ion exchange capacity for separation exhibits four discrete peaks which upon quantitation are consistent between batches regardless of the molecular weight distribution characterized by gel permeation chromatography. The substance produced exhibits life sustaining capabilities in oxygen transport as demonstrated by P50 values of 20 to 28 mm of mercury. Further, importantly, the hemogloblin solutions of this invention demonstrate less clinically signi~icant vaso-constrictive properti~s than those demonstrated by other prior art cross-linked hemoglobin solutions. The material further exhibits properties of increased cellular appearance of red blood cells in various mammalian species, and does not cause any o~ the following abnormal and detrimental chemical and physiologic functions in vivo~ does not activate complement; (2) does not cause hemorrhagic disorders;
(3) does not cause abnormal platelet function or aggregation;
(4) does not cause abnormal prothrombin times (PT~; (5) does not cause abnormal partial thromboplastin times; (6) does not interfere with blood typing or cross-matching; (7) is non-toxic to the kidneys in 3.5 grams per kilogram per body weight or 8 grams per deciliter circulating blood volume;
(8) exhibits circulating persistence o~ at least seven days;
and (9) acts as a stimulus to accelerated erythropoiesis.

~ 3 ~ 3 --4~--The material is typically as characteri ed in the Table below.

'3 f,' CHARACTERIZATION OF TYPICAL BLOOD SUBSTITUTE
OF THIS INVENTION

STE~ILIT~ Stexile by standard culture technique NON-DETECTABLE NDOTOXIN LE~EL ~ 0.01 EU/ml ~ample when te~ted by L~L
and compared against standard curve that ranges in sensitivity from 0.01 EUJml to 0.125 EU/ml.
BAG CONTENTS Na 120~ 20 milliequivalent Cl lI5~ 20 milliequivalent K 4.0~ 1 milliequivalent Hemoglobin 11 grams ~2 per deciliter Lysine < 1 gram/liter Glutaraldehyde -- none detectable Tris <~.5 gram/liter Pyrogen Free H20 volume -- 450-500 ml Met Hemoglobin < 10%
Phospholipid < 1 nanomole/ml ~emoglobin molecular weight distribution:
% greater than 68,000 - at least 50%
% greater than 500,000 - 8% +2%
Osmolarity by freezing point depression 220-320 milliosmoles per liter of solution CONTAINER Fenwal Bag Code 4R2023 600 ml sterile, nonpyrogenic fluid path Supplied by Fenwal Laboratories STABILITY -20C no change for greater than 8 months 4C 5 days with methemoglobin level under I0%

III. UTILITY

The blood substitute o~ the pre~ent invention may be utilized in a manner 6imilar to the suggested prior art blood substitutes and blood expanders. Thus the blood substitute may be used for replacing blood lost by acute hemor~hage, to replace blood loss occurring during surgical operations, in resuscitation procedures after accidental blood loss, ~or supplying oxygen, and generally to maintain blood volume in related conditions. As a plasma expander, the blood substi-tute may be utilized in volume deficiency shock, an alleviant in anaphylactic and allergic shock, for replacing plasma lost after burns, and as a result of diarrhea.
The blood substitute of the present invention may be utilized as such for all mammalian species, but is particu-larly useful in the treatment of humans. The blood substi-tute of the present invention is miscible with recipient blood and its components, is substantially non-toxicj non-antigenic, non-pyrogenic, and, especially~ substantially free of endotoxins and other cell-bound and cell-free proteins. Its colloid-oncotic properties make the product especiall~ useful for maintaining the levPl of the blood and plasma in the management of disease states as well. Further, the material i5 extremely valuable in that it may be used without an accompanying risk of transmission of disease.
Further, it is believed that the blood substitute of the present invention is devoid of the immunologic problems that are associated with the administration of whole blood, and does not cause any of the ~ollowing abnormal and detrimental chemical and physiologic functions in vivo: (1) does not activate complement; (2~ does not cause hemorrhagic dis-orders; (3) does not cause abnormal platelet function or aggregation; (~) does not cause abnormal prothrombin times ~PT); (5) does not cause abnormal partial thromboplastin - ~9 -times; (6) does not interfere with blood typing or cross matching; (7) is non toxic to the kidneys in 3.5 grams per kilogram per body weight or 8 grams per deciliter circulating blood Yolume; ( ~ ) exhibits circulating persistence of at least seven days; and (9) act~ a~ a stimulus to accelerated erythropoiesis. The blood substitute o~ the prese~t inven-tion may be administered using techniques o~ administr~tion which are conventional in the art, as disclosed in lood Transfusion, by ~lustis.
As a blood expander, the blood ~ubstitute o~ the present invention may be mixed with water-soluble physiologically acceptable polymeric plasma substitutes such as polyethylene oxide, polyacrylamide, polyvinyl pyrrolidone, polyvinyl alcohol, and ethylene oxide-propylene glycol condensate. The material may also be mixed with colloidal plasma-like substitutes and blood plasma expanders such as linear polysaccharides, including dextrans having a molecular weight of 401000 to 70,000, gum arabic pectins, balanced ~luid gelatin, and hydroxyethyl starch.
Additionally, the blood substitute of the present invention maybe used as an artifical oxygen exchange solution in conventional oxygenators. When used for assisting the circulation in ill patients, oxygenators are widely used to mechanically oxygenate venous blood extracorporeally, utilizing one or more pumps for maintaining circulation and for perfusion of oxygen by the exchange of gases between blood in an isolated vascular bed and oxygen across an oxygenation membrane.

J;' EXAMPLES

E~;L

THE BLOOD PRODUCTION PROCESS

In the following Example, the equipment identified in parenthesis is further identified in the reference list which follows. Figures lA-lH are a flow sheet ~or the process of this Example, with the equipment reference numbers o~ the Example corresponding to the Figure reference numbers.

A. Blood Collection The startiny point was approximately 5 gallons of individual collection of e~ythrocyte (red cells) source, from slaughtered cows. Because of its ready availability the bovine blood obtained from slaughter houses is the preferred erythrocyte source.
Collection trochars were used to extract the blood in a sterile manner; the animal hide i5 cut, peeled back and the trochars then inserted in the animals major vessels close to the heart. The introduction of bacteria was avoided and the maintenance of pyrogen-free or low-pyrogen level material was accamplished using individual containers of 25 liters that were precharged with depyrogenated sodium citrate as anti-coagulant (0~5 liters~. The collected blood was capped off immediately to avoid exposure to the environment. Upon completion of the collection process, the material was chilled to about 4C to limit bacterial growth. There was no pooling of blood from different cows at this time. The blood was ehecked for pyrogens and sterility to ensure that (1) no one cow was sick or (2) that no contamination occurred during collection. The blood was transpoxted in a refriger-ated conditiDn from the ~laughter house to the process plant.

B. Red Cell Separation Blood was pumped using Feed Pump (P-301) from collection containers at 2.5 to 6 liters per minute, to the Separation Centrifuge (CT-301) which was run at 15,000 to 18,000 rpm.
The ef~luent ~rom the centrifuge (~eparated red cells) was collected under sterile conditions in a 114 liter Dilution Tank (T-301). At this point the effluent was pooled and wa~
no longer treated on an individual animal basis. Sterile nitrogen was applied at positive pressure to the centrifuge to exclude bacteria.

C. Red Cell Clarification The red cells were diluted in Dilution Tank (T 301) using Water~For-Injection (WFI) which had been maintained at 4C. The red cells were pumped by the Clarification Feed Pump ~P-302) at less than 005 liters per minute from the Dilution Tank (T-301) to the Clarification Centrifug~
(CT-302) which was run at 15,000 to 18,000 rpm~ This clarification step resulted in the separation of all cell debris from the liberated hemoglobin solution which flowed by gravity to the Serile Holding Tank (T-302).

D. Microporous Filtration The hemoglobin was pumped from the Sterile Holding Tank (T-302) by the Stage I Microfilter Feed Pump (P-401) at 5 liters per minute through the Stage I Microfilter (F-401~
~.45 micron). The retentate from the ~ilter was recycled to the Sterile Holding Tank (T-302). The fil~rate was pumped at ,~ 3 ~ J ~J ~.J' ._~

O.5 liters per minute by the Stage I ~icrofiltrate Pump (P-flO2) into the Stage I ~icrofiltrate Tank (T-402). Tank T-402 used in thi~ microporous filtration stage was a glass vessel of 100 liter capacity and is equipped with Water-For-Injection (WFI~ and Caustic ~lu~hing connections, with sterile vent filters. The microporous filters ars~of plate and frame construction (such as a Millipore Pellicon Cassette type) and were operated such that the average transmembrane pressure (ATP) was carefully maintained to between 1 and 2 pounds per square inch. While the tangential flow of the material across the mem~rane was 2 to 5 liters per minute, the flux through the membrane was on the order of 0.1 to 0.2 liters per minute. This operational rate was maintained to eliminate cellular debris from building up on the membrane.
When the concentration of the solution tangential to the membrane decreased to less than 10~ of the initial solution, the remaining zolution was discarded. (Alternatively, it is re-diluted with Water-For-Injection to achieve a high yield of hemoglobin from the system.) The solution was now ready for the molecular separations which followed~

E. Ultrafiltration Hemoglobin was pumped from the Stage I Microfiltrate Tank (T-402) through 100,000 molecular weight Stage Ultrafilters (F-501A & B) using the Stage I Ultrafilter Feed Pump (P-501). The retentate from the filters was recycled to the Microfiltrate Tank (T-402) and the filtrate was ~orwarded to the Stage I Ultrafiltrate Tank ~T-501). The flow rate on the retentate side was 5 liters per minute. The filtrate flow rate was 0.2 liters per minute. Memoglobin was then pumped ~rom the Stage I Ultra~iltrate Tank (T-501) using the Stage II Ultrafilter Fee~ Pump (P-502) through the Stage II
(~0,000 D) Ultrafilters (~-502A & B). The retentate either recycled to the Stage I Ultrafiltrate ~ank (T 501) or forwarded to the Stage II Vltrafiltrate Tank (T~502). The filtrate was sent to waste. The tanks were glass vessels of 100 liter capacity with sterile vent filters and are provided with connections ~or washing wikh WFI and Caustic. Connec-tions were provided beneath th2 Stage II Ultrafiltrate Tank (T-502) for 1ushing the downstream system with WFI and Caustic.

F. Chromatography The chromatography stage was automated based on a TI-530 software package. Key variables were protected by a lock ensuring repeatability of the process operations~ Hemoglobin was pumped from the Stage II Ultrafiltrate Tank (T-502) and injected onto the GDT Columns (C-601A-D) at approximately 1 liter per minute for a period of ~ minute utilizing the ~DT
Feed Pump (P~601). After injection of hemoglobin the gradient or variable composition flow was started and injected onto the column using Gradient Pump (P-602). The CQmposition of the gradient flow ~as established utilizing computer-controlled proportioning valves. Release of phospholipids took place prior to the elution of the hemo-globin, with endotoxins eluting after the hemoglobin peak of interest had been collected. Typically, the first portion of the eluting hemoglobin was discarded to waste. Then the collection of effluent was begun and continued until the peak or the response had been reduced to 20-10~ of its peak ampli-tude. This constituted the fraction which was collected and the fraction of interest for purification. The gradient continued to run after hemoglobin collection to remove contaminants rom the column prior to commencement of the wash cycle. The wash cycle through Wash Pump (P-603) was comprised of a wash utilizing firstly Tris/NaCl, then WFI, -5~-then Tris, which re-equilibrated the GDT column prior to the injection/elution cycle, The hemoglobin was collected in the GDT Tank (T -601), a 100 glass vessel, equipped with WFI and Caustic flushing connections and ~terile vent Pilter. This solution is tetrameric in nature with over 99.9% in the 68,000 Dalton range as measured by native gel electro-phoresis, and high performanc~ liguid chro~atography. The material is pyrogen-free and has a methemoglobin level below 2%. The concentration is 0.2 grams per deciliter before concentration and can be concentrated to 20 grams per deciliter.

G. Polymerization (Cross-linking) Reaction Material was pumped ~rom the GDT Tank (T-601~ by the Stage I Cross-Link Pump ~P-803~ through the 10,000 molecular weight Staqe I Cross-Link Filters. The retentate was recycled to the DGT Tank (T-601). The filtrate was ~ent to waste. This step was continued until a concentration of 7 to lO grams per deciliter was obtained and methemoglobin level was less than 1.5%. This material was bagged and frozen or pumped directly by the Stage II Fermenter Feed Pump (P-904) to the Stage II Cross-Link Fermenter (FR-902). Frozen material may be thawed and fed by gravity directly to the fermenter (FR-9023.
The processed material was pumped by the Stage II
Cross-Linked Pump (P-605) through lO,000 molecular weight Staye II Cross-Link Filter (F-904). The retentate was recycled to the Stage II Cross-~ink Fermenter (FR-902). The filtrate was ~ent to waste. The inside of the fermenter was maintained in a low oxygen environment by drawing a vacuum and blanketing with argon. The reactor system volume was maintained constant by ~imultaneously adding a sterile pyrogen-Pree bu~fer (pH 8.9-9.1) to the reactor through a depyrogenating membrane filter. This buffer was comprised of sodium, chloride, and potassium, The pH i6 ad3usted with HCl and Tris base. The cross-linking agent (glutaraldehyde) was then added to th2 reactor.

H. Membrane Concentration once greater than 50-55% cross-linking, as calculated by gel permeation chromatography, had been achieved, the material was pumped by the Stage II Cross-Link Pump ~P-904) through the 100,000 molecular weight Stage II Cross-Link Filter (F-905). The retentate was recycled to the Stage II
Cross-Link Fermenter (FR-902), until about a 25% reduction is ~luid volume had been attained. Electroly~es and pH were adjusted during the filtration stage to give a balanced physiologic ~olution ~or injection.
The material wa~ then flowed by gravity to the bag ~illing machine. Product was bagged for freezer storage.
Analysis of three separate batches o~ material produced by the above process, but eli~linating optional step E produced cross-linked hemoglobin solutions having the properties as set forth in the "RESULTS" section of Example IV.

~EFERENCE LIST

P-301 ALBIN SLP 107 P51 Bl Sanitary Lobe-Rotary Pump P-302 Same as P-301 P-402 Same as P-301 P-403 Cole Parmer ~asterflex ~odel 7019 Peristaltic Pump P-501 Same as P-301 P-502 Same as P-301 P-601 Same as P-301 P-602 Same as P 301 P-603 Same as P-301 P-905 Albin SLP 110 P51 Bl Sanitary ~obe-Rotary Pump T-301 25 Gallon 316L Stainless Steel, Elestro-Polished Interior Fab. by Thermo Electron Wisconsin, Inc.
T-302 Same as T-301 T-401 0-I/Schott 100~ Cylindrical Glass Yessel (GER
100) T-501 Same as P-401 T-502 Same as P-401 T-601 Same as P-401 CT-301 Sharples Model A-16 Type M-3500-520 2HHY CT-302 SAME

F-401 Stainless steel housing with sanitary pipe conn~c~
tions, fitted with millipore 5 sq. ft. filtration cassettes F-402 Same as F-401 F-501Af Same as F-401 F-502A/ Same as F-401 50~B
F-904 Same as F-401 F--905 same as F-401 C-601A-D Resistoflex 6" stainless steel TFE lined pipe and flanges R-902 3 liter applicon fermenter (H/D--2). Agitation:
2-6 blade paddle impellors (1.5 cm X 1 cm paddles) 3 cm and 14 cm from the t~nk bottom, and 4 baf~les.

J

Bag Filling Machine Model F-400-X Table Top Filling Hachine, Cozzi Machine Company.

EX~MPLE II~

~OLECULAR WEIGHT DISTRI~UTIQN
This study was undertaken to determine molecular weight distribution of final product. Hem~globin particles with M.W. more than 1,000,000 might cause some clinical problems in human and animals. One ul of ~inal product (~0 ug of protein) was diluted by 50, and this 50 ul was injected into Hewlett-Packard HPLC System. Water Data 740 Module Station was us~d to integrate results.
Since 1980 the classical gel filtration techniqu2 employing soft and semirigid organic gels for protein characterization and purification has received pro~ressively greater competition from high-performance size-exclusion chromatography (HPSEC~. The breakthrough of HPSEC i~
associated with the development of highly efficient buffer-compatible columns operating at elevated back pressure. The columns are packed with rigid hydrophilic porous silica gel particles of pre-determined pore size distribution and a derivatized protein-compatible surface. The proteins elute in the se~uence of decreasing molecule weight and size.
Four pyrogen ~re~ batches of material with Hb concentra-tions below were used in the testing:

Batch No. Hb ~ET Hb 2261 10.3 3.4 2271 10.4 3.8 2311 11.3 7.2 2341 9.0 3.5 r r~J ~i ~

The following protein standards were used:
1. Blue dextran M~W. 2,000~000 2. A1dQ1aSe M.W. 158,000 3. Bovine Albumin M.W. 67,000 4. Ovalbumin - M.W. 45,000 5. Ferritin M.W. 540,000 TEST SYSTEM
Columns: One Protein-Pak, 300 sw, Waters Associates Buffer: O.lM k pH 7.8 Flow rate: 1 ml/min.
HPLC System: Hewlett Packard, 1090 liquid Chromatograph 280 nm Detector filter 740 Water Data Module Station Statistical,Anal~sis Data was integrated using 740 Water Data Module Sta-tion. Before adding glutaraldehyde Hb solution ~50 ul~ was injected and retention time 9.699 was assumed for Hb with 68,000 M.W.
Retention time for blue dextran was around 4.8 min.
Retention time for Hb solution 4.959 corresponds to M.W. o~
more than 1,000,000.

Results Distribution of Molecular Weight:

Wave length:254 Flow rate:l ml/min ~ 3 ~

Retention Time 2261 2271 2311 2341 4.959 9.4% 7.8% ~Og% 8.5%

5.~65 8.6% 11.07% 5.2% ~ 3.7%

7.362 2g.3% 41.9% 33.3% 33.5%

8.259 17.8% 15.43% 18.27% 17.5%

9.699 34.9% 23.8% 34.33% 36.8%

CONCLUSIONS

All 4 batches o~ material showed very consistent distribution of molecular weight from batch to batch. The bQst Batch was 2271 with 23.8% non-cross-linked material and 7.8% particles with high molecular weight.
The % of cross-linked material ~or the other 3 batches is:

% % Particles With High Batch No. Non-X-LinkQd Molecular Wei~ht 2261 34.9 9,4 2311 34.3 8.9 2341 36.8 8.5 At the ~ame time percenk of particles with high molecular weight is 9.4%, 8.9% and B.5%.

~c~ r ' - so -EXAMPLE I I I

ENDVTOXIN CONCENTRATION DETERMINATION
The detection of endotoxin concentration within a cross-linked blood sample was tested by using the Limulus Amebocyte Lysate (LAL) Assay test. The L~L has been~obtainPd from the extracts of the amebocytes of the horseshoe crab.
The sample was tested to be positive or negative and was determined as such against an end-point reacti~n made by a series of dilutions of a reference endotoxin. A standard regression curve was made from colorimetric readings from the above-mentioned dilutions and the endotoxin content was determined from the interpolation from the curve.
On January 18, 1980 (38 FR 1404), the FDA announced that Limulus Amebocyte Lysate derived from amebocytes of the horseshoe crab is now a biologic product and can be used in place of rabbits. LAL has proved to be a sensitive indicator of bacterial endotoxin or pyrogens within the product.
Because of its hiqh sensitivity in detecting endotoxins, products can be prev~nted from being administered to humans which could cause fever, shock and death if found too high in pyrogens.

Test and Control Articles Four pol~merized blood samples were tested using LAL
assay test and found to ~e less than 0.01 endotoxin units per ml.

~ 3 3~ r Batch No. Eu/ml 2271 <~

1311 ~OoOl 2341 ~0.01 Materials:

1. Glass test tubes depyrogenated by baking in a 180C
oven f~r no less than 4 hours, pre~erably 24 hours 2. Limulus ~mebocyte Lysate, Lot #372, Spectrozyme sub-strate (Cape Cod~ Associates) 3. Acetic acid - 50% solution, H20 used for injection, diluted sample of polymerized blood.

EXAMPLE IV

ACUTE TOXICITY DETERMINATION
Three batches of the blood substitute of the present invention (Hb-I, Hb-TI and Hb-III) referred to as hemoglobin below, and human plasma protein fraction (PPF), in a con-trolled study, were used to replace one-third of estimated blood volume in 4 groups of 6 rabbits each.
This study was undertaken to evaluate the acute toxicity o~ the blood ~ubstitute of the present invention ~or intra-venous administration in rabbits. The study is hased in terms of ~1) mortality, (2) morbidity, (3) pathophysiologic changes affecting the vital organs, (4) pathologic changes q ~, IJ, (gross), and ~5) pathologic changes (micr~s~opic). The study is designed to compare the effects of 3 batche~ of the blood substitute (Hb~I, ~b~ I, a~d ~b-III) to those o~ human Plasma protein Fractions (PP~) following replacem~nt of 1/3 esti-mated blood volume in 4 groups of rabbit~.

~ XPERIMENTAL MODEL

New Zealand male rabbits of 4.0 Kg body w~igh~; sedated with chlorpromazine, 5 mg/Kg body weight I.M.; restrained, Instrumented with: (a) urinary catheter (b) arterial line (on~ ~ar~s.
artery) (c) venous lin~ (one ear~s margin-al vein) (d~ needle-electrodes for ECG
(e) thermoprobe (subcutaneous) ~12i~ u ,~

EXPERINENTAL PROTOCOL

Sedation 1 Instrumentation Tl (baseline) @ and Bleeding 2 Infusion of blood substitute T2 (15 minutes post-infusion) @ and *

3 @
T3 tl hour post-infusion) @ and *
Q

@

@

@
@

T4 ~3 hours post-infusion) @ and *; cannulae and electrodes removed; Animal returned to the cage 24 T5 (24 hours post-in~usion~ @ and * necropsy 0 = measurement of hemodynamic parameters * = blood and uxine samples ~ 3 ~ ~ r ~64-Anesthesia. The animals were sedated with chlorproma-zine 0.5 mg/kg body weight injected I.M., xestrain~.d in a metal rabbit-holder and allowed to spontaneously breathe room air. Body temperature was maintained by placing the animal on an electric heating pad.
Instrumentation. Plastic cannulae (22-gau~e3 were inserted into both ears' central arteries (one connected to a pressure transducer to monitor the arterial blood pressure and the other used for removal and sampling of arterial blood) and the ear vein~ for the infusion of hemoglobin solution. Needle electrodes wPre placed in the limbs to monitor the electrocardiogram. A catheter was inserted into the urinary bladder ~or the measurement of urinary output and the collection of urine samples. ~ temperature probe was inserted subcu$aneously to monitor body temperatureO
Procedure. Arterial blood was removed in the amount of 20 ml/kg body weight (approximately 1/3 of estimated blood volume) and this amount was immediately replaced with an equal amount of hemoglobin solution. All blood subse~uently removed for laboratory testing was replaced with hemoglobin, 1:1 v:v. The animal was closely observed for a period of 3 hours (time necessary for the completion of the pyrogenic ty test). During this interval, additional doses of chlorproma-zine were administered as necessary to maintain the animal sedated, and an intravenous infusion of 5% dextros~ in 1/4 normal saline was administered, 15 ml/Kg body weight/hour, to replace water losses.
Electrocardiogram, blood pressure and body temperature were monitored continuously and recorded at 15-minute intervals. Urinary output was recorded at 30-minute inter-vals. Blood samples were taken at baseline, and 15 minutes, 1 hour and 3 hours afker completion o~ the hemoglobin infusionO All monitoring lines were then disconnected and the animal was returned to its cage, where it was allowed ~65-water "ad libitum". Additional blood samples were taken at 6, 12 and 24 hours~ with the animal again restrained in the rabbit-holder and using the ear arteries. After 24 hours, the animal was killed with an overdose of pentobarbital and a complete necropsy was carried ou~. Special attention was paid to the possible presence of hemoglobin pigmen~t in the body cavities, including the anterior chamber of the eye.
Sections were taken of all organs for histologic examination.
The following tests were carried out on each blood sample:
1) Complete CBC, including platelet count (Coulter Counter), 2) PTT (MLA 700); fibrinogen; fibrin split products 3) Electrolytes (sodium, potassium, chloride and bicarbonate) (ASTRA Apparatus), 4) Alakaline P-ase, LDH, SGOT and SGPT (ASTRA Appara-tus) 5) BUN and creatinine (ASTRA ~pparatus) 6) Osmolarity (Vapor pressure) 7) Plasma hemoglobin concentration (Benzidine) 8) Arterial Blood Gases (IL pH/Blood Gas Analyzer) 9) Total Hb, Oxy-Hb, Co-Hb, Met-Hb and 02 Content (IL
282 Co-oximeter) 10) P50 (Aminco Hem-O-Scan) Urine was tested for:

1) hemoglobin concentration (Benzidine) 2) creatînine (ASTRA Apparatus) 3) sodium and potassium (ASTRA Apparatus) By the timed collection of urine and the determination of plasma and urine creatinine, a Creatinine Clearance Test was carried out at the 3-hour interval post-hemoglobin infusion.

The data will be tabulated as shown in the following tables.
~ a~a Evaluation. The data obtained ~rom the 6 animals in each group was tabulated as ~ean Values ~ Standard Errors. The ~tatistical significance o~ changes related to time was evaluated by analysis 4f variance (Table III).
Comparisons between the various groups of animals was made using Student~s T-test for paired data (Table IV). The raw data for compiling Tables III and IV is presented in Tables I
and Il.

METHODS
(A) Mortality (B~ Morbiditv Attention was paid to the development of the following manifestations:
1 = anaphylactic ~hoc~
2 = seizures or development of neurologic deficits 3 = bronchospas~ or pulmonary edema (immediate effects~
4 = fever 5 = hemoglobinuria 6 = hyphema 7 = lack of normal activity 8 = depression of normal functions (eating and drink-ing) at 24 hours (C) _PathoRhysioloqic chan~es These changes were studied according to the following scheme:
1. Clinical observations:
a. body wei~ht b. body temperature c. heart rate d. arrhythmias e. blood pressure (systolic) P. blood pressure (diastolic) g. urinary output d '~ ~,0 J

. La~oratory data refl ctinq~Ee~_ atory function a. arterial blood p~
b. PaO2 c. PaC02 d. P50 3. Hematoloqy (Coulter~;
a. hematocrit b. hemo~lobin c. WBC
d. platelets 4. Coaqulation:
a. fibrinogen b. fibrin split products c. P.T.
Liver function:
-a. total bilirubin b. SGOT
c. LDH
d. SGPT
6. Renal function:
a. ~UN
b. serum creatinine c. ~erum electrolytes d. serum osmolality (D) Gross Patholoay At necropsy, attention was focused on hemoglobinextravasation: into the anterior chamber of the eye, the pericardium, the pleurae and the peritoneum.
Heart, lunys, liver, spleen and kidneys were examined for gross signs of edema, congestion, hemorrhage and infarc-tio~.

-6~-(E) Histopatholo~y Section of heart, lungs, liver, spleen and kidneys wer~
processed for, and examined by, light microscopy.
A method of grading the hi~topathologic changes was developed in order to perform a statistical analys~s of ths data.
The main change seen in the heart was represented by focal areas of myocardial contracture. Each ~ocal area found on a transverse section of the left ventricle was assigned a 1 + grade.
;n the lunqs, pathology was also patchy. The main changes were represented by interstitial edema and cell infiltration ("interstitial pneumonia"~. Grading was carried out from an overall picture of tissue involvement, a~ well as from the severity o~ changes o~served in each in~olved area, developing a scale o~ l-to-4.
In the liver, the main alterations were represent d by congestion and by c~ntrolobular vacuolization. The~e changes were graded on a scale of l-to-~ on the basis o~ both, the number of lobules involved and the axtent o~ vacuolization starting from the centrolobular venule.
In the s~l~en, conges~ion was the main finding.
In the kidney, no glomerular alteration and no tubular necrosis or blockage by pigment casts was found. The main alteration was represented by a vacuolization of the tubular epithelium, starting at the subcap~ular area and extending from ~here toward the corticomedullary junction.
The degree of this extension was graded on a scale of l-to-4.

STATISTICAL ~NALYSIS

Analysi~ of the data was carried out using two tests:

_~9_ (a) Analysis of variance to study changes occurring in each group of Animals at Yarious time intervals; and (b) Student's t-test ~or paired data to study the changes occurring at each time interval in the various yroups of animals.
2esults of ~tatistical analyses performed are presented in Tables III and IV.

- 70~ 6 ~BLE a 1. ~ODY ~IEIG~
t ~ B.L T - 2Cl~mln ~ - 3~1hr i. ~ 3hr T ~ ~24hrs3 ~V 1 ~ ~ ~ ~ ~ 0 ~ ~ CI ~ ID _ II ~ ~ D ~ C ~ _ ~ _ ~
~ ~SD ~q ~SD M ~SD ~ 4SD ~ SD
P~ . ~166~262.4 ~ ~ ~ 309.~~ 2~2-~
--_. __ .__ __ 11b ~1~ 4350~125~8 . .. ~510~125.0 4366~ .5 ~ __ , 9~b JI~150~236.2 o ~ 42B5-226.8 4150~211~9 =-- _ . 4D92~84.0 4D14~51. D
2. BODY 7EMPERATURE ~C~
~PF t 3B.7~D.42 38.5~0.44 38.5~.46 38.5~0.47 38.6~0.45 ~ . _~ _~_ Hb I ~ 38 . 8~0 . 3d 38 . 840 . 38 38 . 9~0 . 46 38 . 9~ 9 . 58 39 . O ~ O . ~8 ~ _ ~
llb 1~ ~ 39.1 0.û9 38.~ 37 38.9~0.63 38.9~0.69 38.9~D.18 ~__ __~ __ Hb I~I ~ 3~.4~.4a 39.0~$ SO 39.1~0.46 38.9~0.68 39.1~D.~l 3. HEART RATE r~eDt5tmln;
PPF ~ 2~0~21.9 ~00~13.2Q 195~21.23~ 197+16.43~ lg6~5.03 H~ I , 230~50~7 212~8.49~ 218~8.97~ 225 7.63 b 222~1~.13~
_ ~ _~_ tlb II ~ 220~1~.71 200~11.60 199~15.60~ 203~18.97 293~9.42 ~ ----~
Hb lII ~ 211~24.00 190~33,00 196~33.00 213_39-0~ b 2D2~10,00 4. ~LOOD PR~SSURE lSYSTOLïC) tnm Hg~
P~F 100~6.32 95.B_14.06 9~10.DO i 96.7t5.~ 96~4.18 _ __ ~ __ ~ _._ Hb 1 ~ 104.5~14.~ 113.3~12.13C 109.5~10.22~ 108.3~10-27C 108.3~10.27C
~ . _ _ ~_ Hb 1~ ~ 95.8tS.33 111,6 15.72~C 1~5.8+11.33 106.7~12.13 105~8.16 ~ __ . . -. ~ . ~
Hb ~ 9703~0.21 95.2~7.00 102.3~9.59 90.~J.~6 88.3~6.20 LOOD ~RESSURE ~DIAS'rOLIC) I~mn Hg~
_.~.. _,__.. _____.. _.. .. ___._.. _.. ~. ,_.__.. ,_.. __ ____,.. ~,.. ~.. ~ ....... .. .
~PF ~ ~ ~'! l7 60.8 14.63 60.8~9 70 6z.s-6.89 65.Dt_~.03 Hb ~ ~ ~D.~+15.91 80.0~14.71 ~ 77.5414.92c 77.5~13.96 74.2~12.72 __. _ _ Ht~ ¢0.0~8.16 ~s.~15.11~e 73.3~Il.05~ 70.6~9.75 67.s~6.i2 t~b ~II3 8 64.2~3.43 69.2~.32 73.B+7.53~ 63.3_7.~0 61.~6.20 ~ 71 ~ ~L 3 ~
6. URII~ARY OUTPU~ ~m1~30 ~n.~ T~LE I (cont.) _~ t8 L ~o 0~_ _2 ll~mln3 _~ 3 ll h~3 'r 4 ~3hrs~ '7 5 ~24~hrs~
ll ~SD M ~SD M ~SD Pl ~D M ~5D
~PF 2.60~0.49 O.O~O.OOq'~* 3.00~0.61 3.lo+o.6l 3.00~0.53 __ __ __ __ llb 2 ~ 3 11~ 29 ~. ~0~ 00~ 6 . 66~ 2~b ~ ~ 66~1 . 59b~c ~ tO . 9S~- ~ ~ -~
Hb Il 3O00+0~57 0.0~0.00~ 5.~ 59~b 5.10~.641' 4.25+1.46 _ _, ~____ ___ Hb ~II ~.66~1.99c 6.51~4.B3~a 1~.80_2.9G*~t2 11.30~4.20~*a 5.40~1.26b ... ~10~. ~... 0~ ~ DOO~ ~ -- 0_~ ____~0 ~ __ ___________ 7. AR~ERaAL 8LOOD FtH 1UN~S~
~ .~. . ~ 0 ~.. ~ .~0 . ~ ~ ~ ~ 0 0~ ~ ~
PPF ~ 7.33~0.04 7.34~0.D5 7.33~0.03 7.33~0.03 7~39_0.13 ~ ~ __ Hb ~ 7.43~0.10 7.36_0.13 7.37~0.11 7.37~0.1D 7.38~0.06 Hb 1~ 7.36+0.03 7.36~0.03 7.34~0.12 7.42~0.11 7.36~D.04 ~Ib 111 3 7.2~+0. 03 7. 30~D. 02 7. 28~0. OS 7 .26~0. 06 7. 31~ O. 03 ____ _ _ 1 ~ _ ~ D _~ ~ _ __~__ _ _ O__ _ ~_D_________~ ~__0~_ _C_O_ 0______0__~__~.
. PdO;2 ~mrt Hg~ .
t 69.9+8.29 7D.8tll.72 62.0~4.6?~ 59~2~ 90 6~.2~5.88 ~--~ ~
Hb ~ ~ 74.1+16.7 67.5~8.54 73.7~10.2C 71.5~11.3~ 60.6~7.78 ~__ ~ ~
Hb II ~ 63.8~2.12 69.2~6.14 B1.6~9.~ 79.~5-~8~a 76.9~8.77 _--Hb lII ~ 63.3~12.2 76.3~14.4 70.7 7.30 ~ 88.1~18.3~b ~9.9~13.0 9. ~aC02 ~n~ Hg~
PPF 29 . 8~3 . 24 31. 2~2 . 77 33 . 7~2 . 98~ 34 . 8~2 .11~ 31 . 9~5 .15 ~ _ Hb ~ ~ 29.~3.08 33.0t5.27 34.4~4.59~ 32.SI5.33 32.5~.12 llb I~ i 2B.8~5.37 31.0~ 31.9+3.~17 3~.0~.30c 31.2~2.~1 c~ ~ __ __ ~
Hb II~ ~ 32.8~2.5~ 27.6+0.62~c 29.9~2.50c 33.0~3.49 32.411.45 ~ ............. ..__ ___.____2,.___ ~ ~ _.. __ .. 0_.. _.. _ _ _ .
10. IBL~OD P50 ~mmHg~
~ _0- ._....... _.~.,_ _____.___._,,_ _.___.. __.. _~__ _~ _ ~
PPF ~ 34.0~0.61 33.7 1.7~ 33.8~3.01 34.0~1.80 34.C~1 80 ~ __ . ., . ~ __ ~ I j ~ , ,.. ~ _ .......... _.. _ ,~ a9.7l0.~3'~ _0_~0.47~ 32.7~0.62 33.~
Hb III ~ 32.4+1.45 27.7~1.43~d~3 29~5~1.29~b 30.8~1.24c 31.~0.41c __ __.__ L~ ~ ~__o~ _ ___________-- ___ _o_ ~

-- 7 2 -- ~L 3 ~
. 1. COLLDID OSMDTIC PRESSURE (COP~ tmnHg~ TA~LE I ~csnt.~
T - 1 ~B.L.3 T - 2 ~lSm~n~. ~ ~ 3 ~1 hr~ . 7 - 4 ~3hrs~ ~ ;24 hrs ~1 ~SD M ~SD M ~SD M ~SD M ~SD
~ _ _ _ _ _ _ _ _ o _ _ ~ o _ ~ _ _ o ~ ~ _ u> _ ~ ~
PPF 18.2~0.53 19.2~0.06~ l9.~+0.t7~ 18.8 0.60 la.3~D 59 ___ __ __ __ Hb I ~
__ ~ __ __ ~
Hb ~I ~ 17.9~0.49 19.5~1~.55~ 19.5~1.17~ 19.~1.21 18.5~0.63 ~_~_ ~ __, Hb îII ¦ 17.5+0.07 19.~0.77~. 29.5~1.29~ 18.5~ 59~ 17.9+0.38~
~ O~ ~00~ 0_~ --~O__~D______ 0~ _ ~
12. PLAS~ Hb t9/dl3 ~ _ _~______o______ _.________~___ ~.. _.~__~______________ ____ PPF ~ a.oo+o.oo o.oo+~.oo ~.oo~o.os o.oo~o.oo o.~+o.oo ~ . , . . .... .. --~ ~ ------~ l i Hb II ' o.oo+o,oo 3.~2~0.71~ 3.50 0.68~a 3.10~0.40~a 2.10+0.
~__ ~ ~
Hb ~I2 0,00~0.00 3.16iO.7B~ 2.70~0.79~ 7.60+0.70't~ 2.10~0.156~-_._.. 0___.. ____.. ,__,____ _~ .. _.. _~ .. _ ~.______,.. _.. _ .~.~
13. ~IBC ~x 109~
PP; ! 6 ~7~2 11 6.18~ 6 7.2SA2,15 6.5B_1.S3 6.55 1.20 Hb ~ , q.86 0.~5 3.~6~û.43~ 5.~a~ .1 .53 6.4a~ . 60 ~ 1 .2 Ht I I4 . 88~0 . 87 4 . 7û+1 . 94 5 . 05~1 . 29 4 . 78 0 . 6 9c6 . 22~ 5 Hb I~I, 5.83~1.38 3.1~ 0.84~a 2.93~1.06~b 4.22~2.38 11.5~6.84 ___.____. ~________.. 0.. _, ~___~_____,_.. _.. ~ __.___.. __.. ~_.. _ _ __ .
14. RBC tx 191~
~a~_~___________o__~____~_~o_o________~___ _________~___~ ~
rP F ~D 12 3 . 75+0 . 42-'~ 3 . 90 ~ 0 . 27~ 3 . B0 ~ D . 39~ 3 . 7 9~ 0 . 3 2 -llb 1 . ~~1.86~0.23 3.78~0.30~ 3.133~0,30~ 3,79~0.331'~ 3.96'0.~4~
~_ __ __ ~_ IHb II ~5.~2~0.33 3.~6~0.19~ 3.86~0.~7**~ 3.87~0.2~ 4.18~û.6~*~
~ ~ ___ __ Hb ~ I ~ 85, ~9+0 . 55 ~ . 44~û . 33~c ~ ~ 47+0 . 37~e 4 ,1 8+D . 63~ 3 . 87~0 . 39~
o~______~ ~ ~ ~ _~__o__~______ _______~___o .. ~
115. H~MO~LOBIN 19/dl~
PPF g 11.6+0.t2 8.q~0.93~ 8.S+0.59~*~ 1~.6tO.72~* 8.7~û.~2*
.... . ., ... ,. . ... " _ _ __ _~ _ Hb ~ j 11.340.75 ~ Q.30 ~ lt.2+0.69 a 0.7+û.~2 ~ 11.1~u.23 3 Hb ~II , 1~.6+0.85 11.2~u.68 a 11.0~0.54 3 l0.6~û.30 ~ 10 2~3.~0 __ ~ __ _ Hb IlI i 12.1~1.25 12.1+1.00 o 12.0~û.g6 ~ 1.3~1.04 D 10~0 0.91~

}` ( v ~

~6. tlEMATO~RlT ~X~ TABLE I ~c~.
____~_______________ .___ .. ~ _ ~ ~___~_~.. ,__0,,. .__ ______~_--~__ ;B.L.3 7 2 ~lim~n3. T ~ 3 ~ hr~ '1 - 4 ~3 hrs~ ~ ~ 5 ~24 hrs~.
_ _ _ _ ~ 0 ,, 0 ~ ~ _ _ _ ~ _ _ ~ . . _ 0 ~ ~ . _ . _ _ , p~ ~SD M ~SD M ~SD M ~SD M ~SD
PPF~ 33.7~2.8~ 24.6 2.~ 25.5~1.77~ 24.9 2.01~ 2409 1~98 __ __~ ~
~ Y. y .l~ 25.5'2.32''a Z5,6-1.64-'- 25.5-1.t4~'~ 2~ 2.~1' U~ U -y 23 24.7tl t4-~l 24.~D.B2~ 24.8-1.25-~' 26.8l4.07 Hb IIIi 35.4~2.69 2B.4~2.24~b 28.S~2.25~c ~7.4~2~30~*~~5.3~.84 17. PLATELFrS ~x îo9~ .
____ _____0_~_~____ ______ ~ _~_____~__ ~ 0~ A O__~O~___~C.~_U. ~ ~ _ PPF ~ 388_~4.5 300~45.6~ 336~51.8~ 314~39.8~ 376~36.2 ~ __ __ ~ _ __ .
Hb ~ ~ 363+4B.~ 248418.7~*~c 236~43.5~b 256~24.9~c 367~111.2 _ _. ___ ~ ___ .
241t7~.9-^ 263t76.9-~ Z92~69 8~ 35~69.2 Hb ~ 461~63.0 264~111.0~ 281 g7.~ 274~71.0~ 402~1~3-0 18. ~IBRINOGEN ~m9ld PPF , 226~31.8 165~19.7~ lBO~ll.S~ 191421.~ 334~32 ~ ~_ ~ __ ~
Hb ~ ' 23~35.3 147~25.5~ 140+23.a~b 139 25.7~b 291~172.?
_ ~_ ~_ ___ Hb Il , 240~83.4 142~28.9~ 131 28.2~b 125~19.3~b 294 6.3 Hb 3II i 234439.Q 152~43.~ 13P~3a.0~U~ 126~36.0~ 35~149.0 ~ _~____~ _________O_ ______w____ __ __ ___ _~
19. FIBRIN SPLIT PROD. ~cg/mlJ ~0-~ Omcg/ml, lw~10<40mcg/ml, 2~a40mcg/ml~
~ __~_ ~ ______ ____. _ ~ _ O~.___ r._ PP~ , 9.0?~0.0 0.90 0.0 O.~S 0.37 O.OO~O.û 0.00~0.0 Hb I ~ ~.16~0.37 1.0~0.57~b 1.16~0.68~c 1.16~0.~7~b 0.7~0.42 ~ ~_ _~
Hb 1 I ~ 0 . 0~0 ~ O . 50~0 . 50~ 0 . 66~0 . 74 O . 50_û . 50 O . 50~0 . 50 Hb ~lI 8 O.oo~o.O 0.00~0l0 ~.00+0.0 0.16~0.37 0.16+0037 ~ ~ ._____._____ _____O____~.__ __________.. ___ 20. PROTH~OMBIN TlME Csec~
PPF ~ 1~.8~2.tl 11~2~ 9.2~+2.~0 9.~1.00 8.0~0.32A
~__ ____ __ __ Hb ~ ~ lD.7~1.32 11.1~2.33 11.5~2.64 12.2~2.72 lD.D 1.27c_ _ __ __ _ Hb ~I ~ 9.6~0.~2 9.4~0.~1c 10.6~2.21 9.~iD.36 E.4~0.48-~ ~ __ Hb lIl ~ llD.1~1.72 11.8~2 52 11.2+2.15 11.0~1.36 9 3~2 54 ~ 0_._., ______.. ____.. _ _______.. __~_ _.__~ ~ ..

~ 3 ~ 2 ~

21. ~O~AL BILIRU8IN Cnigld1~ TA~LE I (cont~
B - ~lo ~ 15mln;l ,T_ _3 l01,,hr~ T~ 4 ~3 Ihrs _T_ _50~24hrs3 ' M ~D M SD M ~SD M ~SD M SD
PP~ 0.33~0.05 ~ o.oa 0.23~ 05 .23~0.09 0.2~--D.11 ~ __ __~
_ ~_ __ __ _~
Hb ~lI ' 0.17+0.E~4 Sl.13~.04 0.33 0.26 .17~0.090.63-~0.20 ... .. __ _ Hb III 0.18tO.13 0.17~0.10 0.2B+0.20 0.32~0.260.42+D.19 ~ _~____~_____~______~__ __~ ~ ~ __~___________ 22. SGOT (~qST) ~ L~
~ 0~ ~ ~O~DO ______________ _O~ _~0_____~__0_O~
~PF 39.6~4.96 38.0'4.53~ 29.7~5.85~ 30.0-~3.~ 69.7~7.49~
~ ___ ~
Hb I , 28.3~1J.32 30.0~8.16 35.0~5.00 5.1 8.37108.3157.6~
_ _ __ __ __ Hb J~ , 39.5+16.3 38.3~2.35 36.6~4 ~0 8.7~18.7c38.0~8.36a Hb ~II ' 41.~3~15.~ 38.3~8.40 37.5~4.7~ 40.~7 10~4g3~305.9~a ~ ~ _____D_~_____ ~ ____ ~___ ___ _ _ .
23. L D ~ EIu/L~ .
~ ~ _~_~ ~C ____~_~______~ ~_____________ PPF 69 . 041~ . 0 50 . 8~5 . 83~ 60 . Q~l 4 . 0 ~2 . 649 . 63~ 135 . 0~3; . 5~
~--_ _ ~.= __ ~
Hb ~ 66.0+34.1 60.B~19.2 78.0t6.00c 112+32.9~ 73.3~33.k ~ __ ~ ~
llb II , 61.0~39.3 69.6+15.5 67.~10.2 70.6+24.5 109.2~17.9 Hb 81I , 52.'7t12.3 46.0~19.5 44.7~8.1a 58.0~9.2? 9270D~477~c __OD__~ _ __<~__~_______ __~_____~____ _______~___~ .~_ ___~_____ 24. SGPT (ALI ~ [uIIL~
~ .,____________. .______.. ______. .___.. ____._~_ ____________, ~ P F 4 6 . 2 8 . 6 7 31 . 5_7 . 9 9 3 3 . 2 8 . ~ 2 2 . 5 8 . 4 8 6 0 . 5 5 . 4 4 ___ __ ~ __ _ 4 8 . 6 9 . 9 2 4 3 . 3 2 . 3 S 4 6 . !5 2 . 3 5 t 1 . 0~ 1 3 . 5 4 8 . 8 1 6 . 8 _ ~
Hb 3~ 46.3~0.2 ~18.a~25.2 40.~4 08 9.0`~7.34 43.2~23.1 ~ ., --. . ~
Hb III i 41.2~11.6 43.15~10.6 37.0~9~27 41.2t4.13 191.5t119'~c_3~ ......... .. _.. _.--o~ _____________ 25. IB U N 1 sn91dl~
0_~____. .. ___.. ____.__.. _,_____,_____. .___________0_.. _________0___. _____.. _____.
PP~ Tl6.2~2.13 16.2+3.37 14.5+2.43 14.2~2.40 13.5~2.0R
. . , ~ -- ~-- ~ - .
Hb 13 21.0~2.~2e 21.l~2.8~C 21.5~2.93c 21.~3.33c 20.6~4.ql b ~ _ ~ ~ _ Hb ~l 22.~5.50C 22.B~5.3ge 22O3~4.8~ c 20.8~.07~ 21 .0~2.gl b _ . _ ~ .
Hb III:1~.0~1.52 17.2~1.6~ 17.3;;1.4Bc 16.2~1.34 20.~1~4.B9b _ ~_~_~ ___ ____ _ _~_ _~__ ___ _~ _ ~__~_____~_ _ ~ _ ~

2~. SERUM CR~ATININE ~mg~dl~ ~LlE 31 ~cont.~ fJ~, ~ 1' D 'I ~B. L ~ 2 El~m~n~ lr - 3 ¦;1 hr~ ~ - 4 ~3 hrs3 T ~ 5 ~24 hrs~ I
__0.. ~,._ ._,0 __.___0_ _~.__ _______ _ _ ~ ..... , ~ .
M ~ SD ~ ~SD M ~SD M ~SD M ~SD
~PF j1.25b~.18 1.23~D.18 .1.05~0.70 1.0? 1.16 1.10~0.12 Hb 1 ~3~û.14 2.26~1~.05 1~85~û.91c l.RB~D.~4b 1.~4 0.46c __ ~ __ ~ _ Hb 11 ~1O50~0~30 1.31~0.63 1.24~D.57 ~.64~0.~k 1.32~9.~2 - ~ - - - -Hb I~ i 1.38~1.17 2.1~1.. 113 2.32~0.90e 2.6~1.35~ ~ 0.42t 27. SERUM SODIUM ~q/1]
~ . 9 _ _ ~
PP~ 137.~2.19 139.0~2.B6 140.544.~8 141.044.31 145.0~ .25~2* _ __ ~ ~
Hb ~ ~,137.2_2.26 134.843.23c 139.5~70.7 136.fi~3.72 140.~i4.31c ._ ~
Hb ~l ,134.842.11 131.3~4.18c 133.041.73b 135.~8.21 740.0~4.0~c ., ~ ~ ~
~Ib lll ~130.0~3.50c 129.045.10b 128.ô~5.10a 129.B44.00b 138.0t3.5~c .______._____.._ 0,_.___ ______.. __._0~__ __0__________ ~ ________________ 28. SERUM PO~ASSIUM rmEq/1 PPF i 3.45~D.26 3.30~0.30 3.50~0.27 3.27 0.2a 4.35 0.35 _~ ~__ ___ _~ .
Hb I ~ 3O71~0.48 3.78~D.35c 4.2341.64 3.5û+0.43 84~0.32 ___ ~ __~
3.42~0.34 3.45~0.26 3.~3~0.39 ~.32+0.17~1 __ Hb lII ', 3.6040.41 3.33+0.43 3.1540.29 3.23~0.~7 3.60 0.79 ~ ~ ~ _ ~ 00 . ~ , ~ . ~ ~ .,~ . . ~ . ~ . 0 00 .~ .
29. SERuM CHLORID~ tmEq/1~
~___~___~_~_~_~_9~0 _____0___~_~_____~____________ ~___~
PPF ~101.2~2.71 tO4.742.50~ 107.~3.56~ 110.Q~2.53~* 104.7 2.7 _~ __ . ~
Hb I alO5.1~4.56 105O~3-34 105.8~4.22 105.2~3.34c 105.6~3.20 ~ ~ __ _____ Hb I I i 102 . 3~2 . ~5 103 . 6~2 . 81 103 . 8~1 . 95. 106 . 3~6 . 79 103 . 7~2 .16 ~Ib lII I 97~0~3O60 95.8~5.50 98.0~3 50b 97.5~6.00b 102.0~5.60 30, S8RUM 8~CARB. ~Eq/1~
PPF 8 22.2~2.64 22.7~2.80 23.B~1.60 24.3~1.37 26.2~.92 _~ _ __ .
Hb X ~ 22.6~1.59 22.2~2.67 22.4~2.1322.0~2.00c 22.8+1.47 __ ___ ___ .

~b 11 t 23.5~2.50 22.5~2.69 22.5~2.21 29.2~2067~ 22.2~2.0 _ _ ~ , Hb 11~ ~ 22.3+1.90 19.5~2.50 18.~3.25tb 20.0+2.40b 20.6~2.~0 a_~ o~__~__~_ ~_~ _ ~ _ ____~_~______ _~

- 7h - 1 3 ~ 2 ~
SE~UM OSMOLAL5TY ~n~s~kgJ T~LE I ~cont,~
~_ _l'_ D2_~ m'5n T ~ 3~ hr ~ 4 ~3~hrs~ ~ 5 ~24hrs~;
~ M ~SD ~ SD M ~SD ~ SD M s~SD
PPF .~ 304.4_11.3 299.5~11.3 290.~8.65~ 292.8~9.91 293,7~3.9?1 ~ ~ __ ____ __ _ _ Hb ~ ~ 300.0~5.38 299.5~6.20 296.5~5.~Q 295.2~6,B8 296.2~5.11 ~ . __ _ Hb lI ' 309.0~tB.8 299.~7.34 300.2~11O0 29496~10.5 9797.2~5.35 ~ __ ~ ~ _ Hb ~It 301.8~8.80 281.~6.80~ 279.6~8.20*~ 279.0~6 50~6 284.6~6.~0~
~ ~ ~ ~ ~ 0 _ O ~ W D ~10 ~ ~ O W O W _ W ~ ~ D ID _ CI~

: ARITHMETIC MEAN
~SD : STANDARD VEVIATION
~IFFERENCES BE~EEN ~ 0 T~Z, ~-3, ~-4~ T-S ~signific~nt difference P~O.O~l - signi~icant difference P~O.Ol ~ ~ slgnlficant ~ifference P~.05 DIFFER}NCES BET~EEN THE GROUPS: PPF-Hb~
PPF-HbII
~PF-HblII : a ~ slgnificant difference Pco.o~l ~ - significant difference P<~.Ol c ~ s~gnificanS difference P<~.O;
n ~ 6 rabbits TAE~L~

1. HISTDPATHOLOGY ~after 24 h~)urs~

710Wt7 ~ l.UN~S 8 1~IDN~Y5 ~, LIYER 3 SPL~EN
i ~ ~SD ~ M ~-~D t 114 ~SD ~ SD 3, M ~SD
PPF ', 11.00+0.~0 ~ 1.40~0.~8 ~3 1.60+0.48 ', 1.40~û.4B 3 1,.00 Q.OO
Hb X ~ 1.50~0.50 ~ 2.00~0.57 g 1.~3~D.36 ', 1.500~,0.76 ', 7.00~0.00 Hb II ~, 1.66+0.74 ~o 1.83~0.37 ', o~.l6lD~37 'o 1.83~0.68 . 1.00~0.00 Hb llI ~ 1.33+0-46 ~ 1-83+0.37 ~ 2~66~0.46~ 2.00~û.57 3 1.00~0.00 M : ARI ïHME~IC MEAN
~SD: S~ANDARD DEVI~ION
DIFFE~ENCES B~WEEN THE GROUP~; PPF-HbI
PPF-HbI I
PPF-HbIgl ~signifkant difference P~.O'`~
- s~nifi cant differencE P<O.Ol s~gnificant differ~r.ce P~.O~
n ~ 6 rabbi~s - 78 .

1. BODY ~ I GHT ~grn~ ll ~LE 1~ ~con8.
_.. :~r ~ ~ a ~ 0 ~~3.. ~T~: 4. ~ ~0~ .~.: 7 9~
~SD M~SD M~SD iP~+SD 1~SD M~a F ~ ~166+262 .~ , . 4260~309 . 8 4170~292 .0o ~ ~ __ ~
- ~b 3V ~4117~14B.2 . , 4185tl47.0 2. ~ODY lrEMPERA~URE j~C~
~PF ~, 38.7~0.~2 38.5+0.4438.5~0.4~ 38.5-~0.4~ 38.6~0.
o_ . __ . . .... __ ~ __ llb IY 39.0~0.12 38.9~û.34 38.B~ 2 38.8~0.44 39.0l0.17 ~ ~ _ ~ o ~ _ o _ ~ ~ ~ _ o ~ _ ~ o _ _ _ _ _ .
3. IHEAR~ E C8e~t5/m1n~
~ __________~______ ,___~__~ ~_ __ __ O___ _~O__~__~_____D~_O~
PPF g220_21.9 ~0~13.20 ~95t21.23~ 197~16.43~ 198~ 3~ ¦ ~
_ __ ~ ~ ~ ~
H~ lV ~232~8.97 191~20.0a*~ ~97~15.96~ 189~ 20.49~ 207~17.g4~
.___,___________0_. ~_~__.~ .. ___00~
4. BLOOD ~RESSlJRE l5Y~OLlC) ~mn)lg~
~ ~ _______~_____ ____n~__~__o_~_~o_~o___~
~PF . ~100~6.32 95.~14.06 9~10.00 96.7 5.18 96~4 18 ¦ -_--___~ _ _ ~
llb IV, 88 3.26~ 92.0~4.47 89.6 10.02 90.3+4.67 88~0 OOc . ~LODD ~RE5SURE ~DIAS~LIC) ~mr.Hg~
~PF g60.8t9.17 6û.~14.1i3 6û.1~9.~0 B2.5+S 89 S5.0~.0B ¦ ~D
- - - ~ - - -Hb ~V ,61.0~3.60 69.~3.5a~ 66.~+~.60 67.7~6.15 66.0~0,00 _ O~___~_______.__.__ .__ ______.._ .__..___ ,___, ~ _ __~
6. URINARY ou~PU'r lml/30 ~n,~
~ _____~ __~-__ ______________~_____~_________0____0_~____~7~_~_~_0 ~P~ ~2.60~ 9 O.O~Q.~ 3.00~0.~1 3.1.9~û.63 3.0û~.53 __ ~
Hb IY a,5.16~0.89b ~.5B+S.ûO~ 12.86~6.49~'a 1~.00~8.69 ~ ~.67~2.21~a 7~ ~R~ERIAL ~LOOD pH ~UNI7S~
~PF ~7.33~0.04 ~.34~.0~ ¦ 7.33+0.03 ~ 7.33~û.03 ¦7.39~0.13 Hb ~V ~7.31~0.01 ~.32~0.01 ~ 7.29~0.~3 ¦ 7.30~.C2 ¦7.32+0.01 . PaO2 tmm H9~
.----.--0~ .. v._.~.. _,~.. ~.. ~.__.__.. _.. ___.v.___.. _~___,,,._, .,,0.. _,,_.. , ----PP~ 86~.9l8.~9 1 ~0.~ 1~.72 1 62.0~ 67~ 1 59~2 ~b ~V ~6~.8_4.53 1 71.5~6.69 1 74.4~9.66c l 65.6~.75 l64 3~2 15 _ O~C---_o~__..___~._._.._~_~1~_~0_O_._O_____.,.,,,_.,.._.._,_.._ ~. 3 ~ ~ J ''~

9. PaC~2 ~ 19 ~ TAI~L llX ~amt.) ~D_ _ _ _ _ q _ _--O o~ ~ OO _Oo O_ ~ ~ D _ ~III~O O D ~ ~ _ _ ~
9 ._,T~ g ~ _ ~To~_3 _o_~ T_D~ 4 o _T_ _5~ -_ T 7 ! M~+5D ~D i~SD M~SD M~SD . M~SD
IPPF ~ ~9.8~3.24 31.2~2.77 33.7~2098* 34.~2.11~ 31.9~5.15 ~
__ __ __ Hb 311 ~ 28.~4.09 31.~4.89 31.2~4 87 3103~4.44 29.5 3.46 ..
~ __~_________ ~ ~ _ _~ 0____ __~
lC. ~LODD P50 ~H9~
3~P~ .61 33.7~1.75 33.1~3.1Et 3~ B0 34.0~1.8~ _ ~ _ ~ __ __~
Hb IV o 32.9l1.09 28.2~2.05~, 29.2~2.11 C 30.B~1.46~ 31.3~0.47 _ ~o~__~_~__~___ ~ __~ ~______o~ ___o_~__~o__ ___o___~____ ____ _______.
'~1. COLLOID OSMO~IC ~RESSUR~ (COP) ~nanH9~
~ ~___~________________o____~___________.
PPF i 18.2+0.S319.2~D.06~ 19.3+0.77~ 18.8~0.~0 lE.3~0.59 ..
~ _ _ _ _ ~
Hb IV, 17.û~0.24 19.2~D.36~ l9.û~0 38~'t lP~.5~û.3~ 1~ 0.41~ _ _ ._.._,_._.._.... _~ o__ ,_...._____.._ _~
LASMA Hb ~g/dl3 ~___oo~________~__o~___~___________~___~______~_______o_____ ~ ~
~PF 0.~0'090 O.Oû+0 00 O.OO~O.OD û.00~0.00 O.~0; 0.00 _ ~ ~__ ~
llb 2V o O-OOTO-OO 3,~3+û.24~ ~.46 0.31~a 3.03~0.32~ 1.90~0.22~
~.. ________________ ___ __o_~>___~ ._____________ ___ _______ ,_~ . .. _ _ _ _ 13. ~IBC t x lo9 ~
~ ~ ~ ~ ~ o ~ ~ ~ ~ ~ ~ ~ v ~ ~ ~ , . .
PPF 6.27~2.13 6.48+1.16 7.25 )2.15 6.58~1.53 6.~1.20 ,, ,,, .. ~... __ ~ ___ __ Hb IY 5.48~D.8~ 3.B8~D.B~b 6.2t~2.5D 9.00~3.43 12.1~ 4.92 5.~2~1.92 ~4. ~C Cx 10''~
o____o~______________._________________________________________..________ ____..__ ____.
~P~ i 5.10~D.42 3.~5+D.42*~ 3.90~0.27~ 3.80lD.39~ 3.79~0 32~* _ ~ ~-----~
Hb ~V 9 5.49+0.30 4.~2+0.34~ 4.06~0.39~ 4.1~+0.37~ 3.9~û.36~ 3.8~0.42 o~ _~_ _________ ~ , ~___~________ ~o____________ _ ~
15. HEMOGLOBIN ~g/dl~
o_____________o___o~t~______ ----------t~--------~PF 3 11~6~0.82 ~ 8.4~0.93~** 3a.8~o.sg~ 8.6~û.72~ 8.7~û.72~ ¦
tlb IV, 12.~0.~58 I 12.3~0.54~ I11.8~0.68a L11.8~û.84a LlD.~O.90~b L 9.~û.9^~"~
~ ~o____~o_o__~o~l~ _,.~.__._.__._ _._.__..____ _______,_____ __ _ . __ 16. iHEMA-rOCRI~ tx~
t t Illb ~Y ~ 37.2~1.56C I 27.9~2.18't~l27.6~2.B4~ 2B.1~2.24"~l26.9~2.18~ 29.0~2.54~~

17. I~L~ELET5 l~ XlO ~ ~AI~LE 1 o~~ _ ~ _3~__o~ __7~ _4~ 7_'_5~ T~ _7 5 e~SD ~+SD I~SD I~SD M~D M~SD
PPF S388~54.5300~45.6~ 336~51.8~ 314~39.8~ 376~36.2 ~ __ __ __ __ ~b ~Y i453~.5 285~5201~ 309 42.~* 324 49,~* 354~3,~ 4~0~17~.
18. ~IBRINûGEN [mg/dl3 ~______--____________ __O_________ _____~_ ~__0 ~ D~ ~ .______________ ~p~ 1226~31~8 lt5~19.7~ 180+11.5** 191~21.5~ ~l34~32.D~ ..
__ __ ~ __ Nb ~V '217~33.3 114l29.8~b 117~34.9 1~ 102~29.6~*b 391~33.2~ 50û~1~3.1~
~ .. _=. ._~_________ _____5.___,__ __0___._____0 _,_.. _.. _~_0.. ._0____ __~__ .
19. FIBRI~I SPLI~ PRDD. ~mC9/m1~ tO~<1OmC9~m1~ 1Y~1O~4OmC9Im1~ 2~40mcg~ml~
_______--_____.___~__ ~ .. ~_ 0____________ _~_~,__,_~___ __._____~_~__ PP~ 'o.oo~o.oo o-oo~o-~o 10.16+0.37 -~- o-o~-o~ .D
_ .. ......... _ __ ~__ Hb IV ,0.00~0.00 1.87iD.47 all.83~0.37~ 1.83~û.37~a~ n.7s~a 43~a~ ~
~ ___._________ ________~_0_ _~ _.. _.. ,0__ .. .~.. _ __.. _.____.
20. PRûTHROMSIN'rIME aSec~
~ _~ ~ ~ ~ _ ~
PP~ ',lC.~2.11 11.2~ 4 ~.2~2.0~ 9.40tl.~0 8.00~0~3 _ ~_ _ _--!Hb 3Y ,ll.Q~1.41 12.9l1.9~ 15.5~2.83~c 12.6~2.29c 8.10l0.10~ :3.02 D.O~
---- --___0~__ ~______ ~ ~ ~_______~_~. ______~_____, .,_ __0.~____. .
21. ~D~AL BILtRUBIN ~mg~dl~
0~ _ ~_~D~ ~_ _ D _ __~_~ ~_____ __ _ __ __o_~__~o___o~__ ~__ ' __ _ ~ _~ _ Il~ __ __ _ .
~P~ ~'û.33~0.0~ ~.20~D oR 0.23~0.05 ~.23~0.û9 ~.27~0.11 1 -- ~ - ~ -----Hb ~ 0.17~0.16 0.28~0.07 0.78~D.54 0.38~0.41 0.53~0.~ l) 4C D.ll*
22. 3~0T ~AST) ~Iu~L~
__~_~.00~ D~ _~ __________~O_ ___OO_______ ~ ~ __ __ __ __ __ _ _ PP~ ~39.6~4.~6 38.5tO.43~ ~9.7~.BS~ 30.0J3.~S~ 69.7~7,q9~ _ _ llb.lY ~34.8~9019 ~l.Q~4.89b 52.0_17.2C 62.0i27~8t, 80.0~137.9~b 58.2121.
~ _ ~____0_~.___.,_ ______.______ __________.. _ __ __ _ 23. L D H ~IulL~
~___._.. ,___~,___ 0___.. ____~ .. _~ t t ~PF _ ~,63.0~1200 _ T 50.8~5.83~ 65.~14.0 5~ = ~

Hb IV ~64.5~15.1 l 43.3~1~.9 60.0~40.t 86.0~49.27428.8 336't ¦180~12~.5 ~ ____.-______ ~ __0._~
24. SGP~ ~ALt3 1U~IL~
PPF ~46.2~8.67 ~ 31.5~7.99~ 33.2~B.~2~J 3Z 5 3 ~ ~
tlb IV ,4â.7~12.2 ¦ 3~.2~4.20 ¦ 48.0~17.2 ¦ ~0.0~6.32~ ~191~109.4~c ~58.7~16.7 _._.__...__..._____._~__,_,_..____~__._______.._,.,,_~,_,_,_____, _._.____.___. .________,_.

2~ mg1dl~~A,BLE 11 (con~.) _, ~ T~ ~ ,3~ T, _4~ ., r, ,~ , 2 ~SD~+~D ~+SD ~SD M~Sa ~l~SD
~PF ~16.2 2.13 ~6.2 3.37 14O~ 2~3 14~2 2~4EI 13~5-~2~08 ~ -I
_ __ ~~ ~
Hb IY ,~.15.2~3.13 16.3~3.25 15.8~ .~7 16.6~4.53 21D.4~3.49c 115.2~1.46 26. S~RUM C~EA'rItJINE ~mg/dl~ -~PF ~ 1.25~0.18 1.23~0.18 l .OS~.10 1.07~1.116 1.10~0.12 ., ~ _ ~ __ __ ~
Hb IV Ll.42~0.31 1~83~0.62 1.70~0.60c 1.. 36~0.54 1.~0~0.37 L~.26~D.l~
27. SERUM SODIIJM ~mEq/l 3 ~PF 3137.0~2.19 139.0 2.B6 ~40.S 4.08 141.0~4.31 145.0~.2 . _ _ __ __ .
Hb lY ' 138.6l3.39 l 135.0+3.07 135.8 2.03 1138.2~2.67 143.0~4.00 I143.0~0.0D
28. SERUM PO~ASSIUM ~mEq/l~
PPF 3.4~0.26 ¦ 3.30~0.30 3.50~0.27 ¦ 3.27+D.28 4.35~0.35 -------~--~
Hb IY ' 3.32~0.13 3.31~0.17 3.55~0.19 3.680~,0.30~ 3.25l0.Q5b 4.3~D.~O~-~
~ __O~_______~___ __--___ __ _0~
29. SERUM l:HLORIDF ~m~q/lJ
_ _ _ _ _ 0 . ~ , . . _, 0 _ _ _ _ . .. _ . .. ~ _ _ . _ _ _ PPF ~lDl.2t2.71 104.7~2.50~ 107.5~3.56~ ¦ llD.0~2.53~104.7~2.7~ ..
_ _~ ~__ Hb lV ,104.2~2.47 I104.6~4.34 lD5-0~-7t 1 105.6~3.85 lD6.5 2.5D I lO~ D
~ . _ _ ., . _ _ . _ _ _ ~
30. SERUM BIEARB. ~mEqJl~ .
o__r_ ___~____ ~ _ __~ ._~ ...... ~ _______ PPF 22.2~2.64 22.7'~2.80 23.~ 60 1 24.3~1.37 26.2~4~g2 _ _ _ __ _~
Hb ~V a 23.2~2.79 22.0~2.08 22.8 3.07 1 21~SI3.20 ~2.0~0.OD 24.0~0.00 ~ . _ 0 _ _ _ . ., _ _ _ _ .. _ . _ _ _ _ . ~
31. SERUM OSMOLALI~ ~mDsm~kg~
O~ .~_ 0~ ~ .~ ___._. .__~____............ __.. __,___. __ _ ~PF ' 304.4+11.3 1-299.5~11.3 290 5~8.65~29Z. ~9.91 293.7~3.21- _ _ _ ___ Hb lY ;290.5~2.96 I289.2~5.15 288.~g.06 291.0 6.22 293.5~0 SO 285.0~0.00 ~DDO~OI~ ~9~"~" _~_~__________~___~_~__ ~____________ __~___~____ ARlTHMETJC MEAN
~IFFERENCS BETWEEN ~-1 to ~-2, ~-3, ~-4, ~nd ~-S~ $~gn1~ic nt djffference P~Do 011 ~ - s~9nific~nt difference P~0.05 D3~FERENCES BE~EEN ~HE 6ROUPS: ~PF ~ Hb IY ~ ~gnifiC~n~ difference PcD Dl c ~ significant ~ifference ~<~.05 n ~ 6 r~bi~s ~ 3 ~ 2 - ~2 -SAE~L . m 1~ ~Iys~s of Yaria~ee ~ d on Dif~eflC~ ~ SC~S2S
4 gTOUp~ ~aly2cd 4 ~S
l~ra~cte~ ~a~li~ ~aia~ L~:

g~
Body Wei~ht ~ NS NS
Body Temper~ e ~ NS NS
L~l~e =~
Hean ~Itc ~-~P~ NS ~JS NS
3ystolio ~lood pr~ssure ~-- 0.017 ~S NS 0.03S
c~a.o74) Di~stolic ~lood ~ressure ~ S NS NS
Arrhythmi~s ~ S NS ~S ., Urinary ~u~put 5mll30 ~i~ --~O.D01 0,006 0.03S ~'S
. 2 ~S 0.~13 ~'S ~ (P=0.09) 3 Y~i. 2 ~â ~S ~S ~'S
4~3.2 ~:O.Onl~0.001 0.006 ~S (P0.09) ~a~LL~
Aner~al 131ood ~H ~-.NS NS P~S ~S (0.~9) P~ ~ 0.035 0.006 ~S (0.~74) î vs 2 NS ~S NS NS
3Y~ a ~s 0.006 0.014 ~S tO.OS5) ~ ~$. 2 NS ~ 0.002 ~S
~eo~ .~Ø0040.011 ~S ~S
IS ~ NS ~IS
3 ~s. 2 ~NS NS ~S NS
Y~. 2 0.006 0.006 NS ~IS

~5=DL~ TAleLE 113 ~cor Tl ~2 ~3 11~ T5 l~aramctgr 1~1~ ~o ~: ~hQ~ 2~h Blo~d P~so ~'S ....

WBC ~0.002 0.002 NS NS
~ ~rs. 2 1~.045 ~S ~S ~S
3 vs. 2 NS NS ~S ~S
~ ~s. a ~.ool ~o.ool NS NS
RBC --- 'hS ~S ~ ~S
~emoglobin -~).C)r)l ~.001 0.003 ~S
I ~s. 2 ~0.001 60.001 ~.~01 0.û2~
3 ~s. 2 ~.001 0.001 O.D04 ~IS
4 ~s. a ~o.ool eO.ûO~ 2 a~'~
H~mocrit Pl~telets -~0.030 0.037 0.010 ~S
1 ~5. 2 ~'S ~ ~S ~'S
3 ~l5. 2 0.03g 0.033 O.D~9 ~S
. 4 YS. 2 0.~0~ 0~ 0.00~ ~S

Fibrirl~en -~ ~3S ~S ~J5 ~S
~sothsmba~ ~i~e ~as NS '~S ~S

0.001 . ~ ~IS ~.043 1 ~5. ~ 60.001~15 (û.OS2) ~S ~'S
3 ~. 2 ~.0~1 ~IS ~S ~S
4 ~. 2 ~.001 N5 . NS 0.016 NS tO.087) ~IS ~0.0~0) DL~ TA~LE 111 tconl.
2~3 T~ SS
p~rame~er 9~a~ia~ ~hai~ ~Q~ ~ 2~a~

~;Pr D~ S N~
I Y~. 2 N~~.001 NS NS
3 ~ NS~.003 NS ~S
V3. 2 N~i~.001 ~NS !INS
~L~
s Seram ~rcatinine ~~IS ~0.035)NS ~0.069) NS
Sen~m s~diu~ ~ ~ ~S NS
Serum ~o~assium o N~ ~ XS ~'S
Serum ~hloride NS ~S 3~S NS
Serum bicarbona~e NS 0.033 0.044 ~S
~ ~rs. 2 ~S NS NS ~S
3 vs. 2 ~ iS O.Oû9 ~S
4 vs. 2 0.043 0.004 O.D30 ~S
Semm osmolan~y ~ iS ~ NS ~'S

-- 8 5 ~
TABLE ~ - St~ldents t-tes~ For Paircd Data ~ame 2 r~e 3 T~me ~ T~e 5 5 ~;rL base~i~e ~ b~seli~e 3 hrbaseline 24 ~ asel;s~e ~ea~e tn) ~eafl~sc ~ nea~ ~se S~ 2~n ! sc gE~
lBody ~vei~h e~ses 0.12~p.02 16 O.Dl ~.02 î~
~o~tr~l o.n9+0.02 6 O.û1~0.07 NS ~S
l~ody 1te~peraturc e~scs ~0.22~0.07 18~0.14~0.1S î8 ~.19~0.1418 -13~0.~0 î6 control~-0.23 0.13 ~~O.a8~0.î5 ~ DO.18_0.09 6 O.ûOiO.00 4 ~S NS D~ NS
Hean ~ra~c t~s~s ~18.7~S.~ J ~-25.9~6.1 ~ 8 -6.~9.î ~ 8 -6.~ 1 6~ontrols-23.0~9.2 ~-25.~10.8 5.a2.0~ïl.7 5 ~6.3~11.4 4 NS NS XS 2~'S
Sys~olic bloDd ~ressurc cases ~.5~3.2 ~ ~~6.7~3.0 13~2.7~3.3 18 ~6.5~'.9 13 eoa~rols~.2~3.3 6 -5.0~3.4 6o3.3~3.3 6 G4.0~.9 5 NS ~=0.060) O.OS0 ~S ~S
Diastolic blDsd ~KSSUre cascs ~10.0~2.9 ~8 9.9~2.B 1~5.Q~3.0 18 ~.2:~.6 ~3 eontrols 0.0:~.9 6 0.0 ' 2.6 6 ~ 3.3 6 2.0_4.~ 3 NS tP~076) NS ~-0.063) ~JS ~5 ~rrhy~hm i ~
~ses ~ û.S8~0.1~ ~7 0.06~ 61~ 3 eo~tsols 0~0 6 ~;~P 6 0 ' O 6 0;~
~s ~as ~ ~s Urirlary OUtpUl ~mll30 ~
eascs ~ a.6 ~ ~ 3.7~0.S ~ 83.4~0.8 1~ ~1.5~0.3 ~ 3 eontrols ~2.9~0.2 ~ 0.1~0.l ~0.3^t0.8 4 0.1~0.1 4 ~S (P~.074~ ~.001 ~0.00l ~.003 !llo~t ~
c~;e~ 0.02~0.02 ~ O.C4~0.03 ~B-0.0~0.03 ~18-0.02~0.07 IS
contJols 0.01;~.01 6 0.00_0.~2 60.00~0.02 1~0.08~0.06 4 ~S :I~IS NS 0.030 - 86 ~ `J
Slude~ts t-test F~r ~a~ncd Da~a 11A~LF IY ~c~t me 2 ~c 3 ~ me 5 aselin~ 1 ~r~baseline 3 ~r-bas~line a4 hr- b~seline ~ca~c (~ ~eaa~c (~ ea~e (~) ~ca~e ~) ease~ 4.0~3.5 ~.3~.3.9 1~12.6~.~ 3.9~3.0 eo~trol~0.9 S.6 ~.044.3 6olO.8~S.~ 6~0.8 ~.6 4 NS 0.035 0.017 NS
~aCOa eases 0.1~1.3 11~1.6+1.2 181.~; 1.2 18 1.7:~.1 lS
controls1.~ ~.2 6 3.9*1.2 65.0*3.9 ff 1.~ 1.2 4 NS ~S ~S NS
Blo~ ~50 cascs -4.4~0.6 IB-2.11~0.7 ~ 0.6 ~~o ~ata for eo~trols-0.3~.6 3 ~.2~1.3 3O.O~O.iS 3 cDnu~ls 0.~4 ~S
P
eas~s0.10~0.01 ~0O.a9~0.01 ~00.07~0.01 ~0O.D3~0.Ql ~
controlsO.OS_0.004 30.06 ~.01 30.03~0.01 30.00 0.003 3 0.007 NS ~S 0.030 cas~s~1.4~0.3 a~oO.6 o.~ ~.6 18 3.6 1.4 13 controlsO.l~.S 60.9~ 0.5 60.2~P.6 6 0.8l~0.
0.03~ ~s ~ =0.09~) g~ses -1.2~ .2~0.1 ~11.3~P.î 18 ~ p.2 ~13 col~tJ~ls ~ 0.2 18 ~.2~0.2 6 ol.3~0.2 6 NS ~S 3dS ~as Hem o~lob;n cases ~.2~.2 1~ 0.3~0.2 B 8~0.8 ~ 0.3 ~ 8 ~1.2~0.4 3 3 6~Dtrols-3.2~0.S 6 ~ 0.4 6-3.1~0.~. 6 ~2.6 tO,2~ 4 ~0.001 ~tO.OOI ~0.001 ~S QP=0.~99) Hematocrit ease$ ~1~.0 ~ 0.7 18~7.8~0.7 ~.2~0.8 18 -~.6~1.3 ~ 3 cD~rols~9.~.4 l~~8.2~1.1 6~.3~1.3 ~ ~7.4~.9 ~S NS NS ?~
lPlasel~t3 ~s~s ~î~t ~18 ~ IS2~î6 ~8 ~3~4 ~8 .36t~ 13 c~lrol$ -~8 14 6~3 ~34 6 -74 2S 6 ~12*30 4 -- 8 7 ~ h $tu~ t~ Dr ~d 1 ~L~ c~nt.
;r~me ~ ~lmc 3 ~e ~ 5 zscli.nc 1 ~r b2sclinc ~ l~r-bascline . 2~ br~ b~seline a~ean ~c (~ De~n se CD) a~ça~ se (~ ~aean~e ~n) ~.~35 iD.~08 ~.~2 IF;~rino~e~
~as~3 ~89~1~ 18 ~102tlO ~ 08~13 979~i36 a3 eo~u~ 61~19 6 o46~28 6~52~11 $ 91~21 ~S 0.029 9.037 ~S
~rothro~nbin ~De ~3es O.S~.6 16 î.0~0.6 ~ 60.7~.4 16 ~ 0.6 13 contr~s O.S~ 0.9 6 -1.5 0.4 6oO.7~1.0 5 ~2.4~0.7 ~S 0.0~4 NS ~'S
L~
~osal B,~o dala S~OT
eases64.2~5.1 50.7~27.8 ~361.3~t3.3 ~S 236.9~8D.0 13 contJols -9.4~3.7 -~0.2~7.6 5 -8.~5.6 ~ 21.7~20.7 3 0~ i~.05~ 0.0~ 1 0.021 LDH ~
æ~se~IP.34 ~.26 ~1~0.44~.29 17~.6~ ~.27 17 0.85 0.41 ~IS
controls -0.28~0.21 6 ~0.16 p.21 6 ~0.04~0.21 6 0.54~0.32 4 ~S ~JS ~S ~S
SGPr leasc5rt~9 ~5~ ) 48~4~4 913~2~39~ 3 6~1~6~33~
con~rols ~.5~.3 6 ~6.8~.4 6 -7.5+65 6 22.5~ 16.4 4 ~S ~0.0~1 ~S ~'S
~L~
~UN
case~ 0.4*0.~ 18 0.2~0.4 1$ ~0.6~0.8 ~ ~ 1.8-~1.6 ~ 3 ~ortt~ 0.7 6 ~.7~0.3 ~~2.0 ~ 6 ~2.0~.
~IS 0.016 NS NS
Seru~n gre3d~ine IgJIS~S 0.65~0.33 ~ 4 0.~ 871.~ ~p.47 ~7 ~.S6~0.28 14 eoatrol~ ~0.0~-~0.03 ~ -0.20~0.03 6 ~0.18*p.07 6 ~0.13 ~.0S
~S ~O.OS9) NS ~P-0.076~ 0.023 o.o27 - 88 - ~ 3 ~
$r~dc~ cst ~or Paired Da~
TABLE IY ~Conl.
~e 2 ~lme 3 T~e ~ ~a~e 5 ~;~ b~line 1 ~base~e 3 brba3el;ne 24 ~r- l~asc~ine sC t~ ~ea~s~ ~e~s~ ~) DDe~ c ~n) ~2.7 ~ 0.8 ~ 0.6:~ .5 ~ ~ 5.4~ 4 go~rol~ 2.~ 6 3.t~9.5 6 4.5~1.9 6 7.~ 0.3 s ~as ~P~0.089~ ~IS
Semm ~O~SS;
6ase~ ~.a4~.1t 1~ 3~ 8 -~.23:~.1S ~8 0.~4:~.23 t~
eontr~I3 ~0.1S ' 0.04 ~0.05~Øû3 6 oO.18 ~.14 6 1.03 ~0.~8 NS NS NS ~S ~=0.067) Ser~ ehlonde e~se~ 0.0~1.3 ~30.8~.9 ~~.2~ 18 2.~1~1.6 14 e~ntrols 3.3 ~ 0.9 6 6.3~1.5 68.8~0.9 6 ~.5~1.9 NS 0.007 Q.002 21S
Serum ~ic~r~on~e ~a~ .7:~.7 ~ ~-2.0~0.~ 1 8-2.~ 0.8 a ~-~ .4, 0.9 1 4 eontrols 0.5~0.3 61.7 ~1.2 62.2 0.9 6 4.3~1.9 4 0.007 D.010 0.004 0 010 Sen~ s~ol~rity es ollO.1~4.2 a7~11.4~.0 â~4.S~4.1 ~7 -12.7~4 14 conarsl~ ~1.5~2.9 ~~10.5~6.7 ~o7.S~10.6 4-19.0~6.0 2 ~L~
~eAn seore n $' ~alue ~leart eases 1.44 ~i 8 ~S
e~ntrols ~ 6 21.~ 3 23.~ ~
lLUD~S
ellsgs ~.~9 ~a ~s ~o.~7) 3~7) ~14(77) ~ 53 ~ 8 consrol~ ~.58 6~67) 2(33) 0(0) 0 6 Y
gases ~.7S 18 ~NS
eomrols 1.92 6 3.~ o~
~Ridney~
~se~ a33 as ID.002 2(~t62) 4t~ (6)18 æontrol~ ~.33 6 ~(83~1(17) 0 0 6 ~plgc~
~ase5 ~.00 9 NS
eoDtrols 1.~3 ~, DATA ANALYSIS

RESUL~S

The three batches o~ hemoylobin here studied were:

Hb-I = Group 1 Hb-II = Group 3 Hb III = Group 4 The batches were characterized as ~ollows:

Hb-I Hb-II Hb-III
1. Hemogobin, g/dl 14.0 13.0 10.0 2. Oxyhemoglobin 90.3 91.2 98.6 3. Carboxyhemoglobin1.6 0.9 1.7 4. Methemoglobin 8.8 10.6 2.7 5. Oxygen Vol. ~ 17.5 16.2 13.6 6. pH 7.5 6.55 7.0 7~ Sodium, mEq/L 118.5 102.3 119.2 8. Pota~sium, mEq/L3.88 4.~6 2.44 9. Chloridet mEq/L118.0 117.3 120.9 10. Osmolarity, mOsm/Kg 244 236 242 11. Endotoxins, EU/ml~0.01<0.01 <0.01 120 Molecular wt.
between 68,000-500,000 85% 80% 90%

~ 3 ~ 2 ~ J

13. Phospholipid analysis by TLC
silica gel plate developed in iodine vapor clear clear clear These batches were compared to human Plasma Protein Fraction ~Plasma-Plex--Armour Pharmaeutical Company). (Group 2 Control Group) (A~ Mortality None of the animals in the 4 groups (6 rabbits in each group) died by the end of the 24 hour observation period.

(B) Clinical Si~ns During the first 3 hours following the administration of hemoglobin, the only clinical sign was hemoglobinuria. At 24 hours, all animals appeared normal; i.e., with normal degrees of activity and eating and drinking normally. By that time, the hemoglobinuria had subsided.
No change occurred in body weight and temperature.

Gross Pa _olo~y None of the animals presented at necropsy with hemoglo-bin extravasation. All the organs appeared grossly normal, with the exception of the liver, which appeared congested in about half the animals.

Histopatholoqy Heart: Focal areas of myocardinal "contracture" were found in the PPF group, as well as in the hemoglobin groups.
The severity of the change was graded as 1-~ in the PPF group, and respectively 1.5~, 1.7~ and 1.25~ in the Groups 1, 3 and 4. The di~ference was not statistically signi~icant.

:~ 3 ~ ~;4 ~ J' Lun~s: Areas of interstitial edema, congestion and cellular infiltration were found in all groups, includiny the PPF group. The severity of changes was graded 1.4+ for the PPF group, and respectively 2+, l.B+ and I.8+ Por Groups 1, 3 and 4. The difference, again, was not ~tati~tically signifi-cant.
Liver: Changes found in the liver were more uniform than those observed in the other organs. Centrolobular vacuolization was graded as 1.4+ in the PPF group, and respectively 1.5+, 1.8+ and 2~ in Groups 1, 3 and 4. The difference was not significant.
Kidneys: No glomerular alteration and no acut~ tubular necrosis or blockage by pigment casts were found. Tubular epithelial vacuolization was uniformly found in the subcap-sular area. Extension from here toward the cortico-medullary junction was graded as 1.6+ for the PP~ group, and respPc-tively 1.8+, 2.15+ and 2.7~ for Groups 1, 3 and ~. The difference between PP~ and the hemoglobin groups was signifi-cant only for Group 3.

DISCUSSION AND CONCLUSION

Both the chemi~al and histopathologic changes observed in this study were mild-to-moderate and theoretically reversible. The determination of such reversibility is currently being investigated with the period of observation being extended from 24 hours to 1 week.

EXAMPLE V

EXCHANGE TRANSFUSION OF RABBITS
Utilizing the experimental protocol set out in detail in Example IV, three groups of rabbits were hemorrhage-tran~-fused. on~ group of ~ix rabbits (Group A) had one-third of ~ 3 ~

the estimated blood volume replaced with a hemoglobin solution containing 1-2 endotoxin units per ml, One group of four rabbits (Group B) had one-third of the estimated blood volume replaced with 5% PPF. ~nother group of six rabbits (Group C) had one-third of the estimated blood volume replaced with a hemoglobin solution of the present ~vention, this solution characterized as follows:

~ C~ ;'DJ ~

Rml 1 Run 2 RL~n 3 ~v~rage 1 . Hen~glcbin, g/dl 11. 6, 11. 6; 11. 4; 11. 5 2~ Oxy-hEwoglcbin, % 90~3; gO~2; 90ol; 90~2 3~ ~arboKy-Hb, % 0~1; 0~3; 0~5; 0~3 4~ ~t-~b~ % 9~; 9~5; 3~7t~ 9~6 5. Oxygen Vol. %14.7; 14.6; 14~4; 14~6 6~ pH, Uhits 7~1~0; 7~161; 7~168; 7~156 7~ ~ ~ Tbrr 1~1; 10~; 10~2; 11~6 8~ P02~ Tbrr 147~5; 147~2; 147~0 147~2 9~ P50~ T~rr 28~0; 28~0 10. ~olloid Osmatic ~ressu~, Ibrr20.7; 21.0; 20.9 20.9 11. Sc~li~n, niEg,/L 114 . 6 113 ~9 115.1 114 . 5 12 . P~assimn, Eq/L 3 . 72; 3 . 63; 3~ 70; 3 . 68 13. Chloride, mEq/L 111.0; 208~4; 107~2; 108~9 14~ Phosphorus, ~g % 0~097 O~Og7 0~97 0~097 15~ Endokoxins, EV/ml 0029 0~lg 0~23 0~23 16. ~!ho6Eholipid~s, ~ absent 17~ Polymerization, b~ colun~ 85g6 above tetx~meric fo~m.
~hy s f~\ ,r~
_9~, _ Compari~on~ for each group of one-third transfused rabbits during the twenty-four hour period ~ollowing trans-fusion was made wi~h regard to platelet numbers, white blood cell numbers, serum fibrinogen levels, prothrombin levels, and serum creatinine levels. The collected data a~pears in Figures 2-6 respectively. In each of Figures 2-6, the triangles represent mean values (+ ~tandard error3 of the Group A rabbits, the squares represent the mean values (~
standard error) for the Group B rabbits, and the circles represent the means value (+ standard error) ~or the Group C
rabbits.
A comparison o~ the data represented by Figures 2-6 demonstrates the cross-linked hemoglobin solution of this invention caused no mortality and no clinically important signsO Using an analysis of variance, at 24 hours post infusion there were no significant differences among the groups except for a slight increase in systolic pressure and an elevation of SGOT. The elPvation of systolic blood pressure w~s consid~red clinically unimportant because it occurred within the clinically acceptable range ~20mm Hg).
The SGOT elevation was considersd spurious because of colorimetric interference by plasma hemoglobin. Although the test groups exhibited transient hemoglobinuria, no signifi-cant increase in BUN or serum creatinine was detected.
Similar histopathologic changes were found in both the P~F
and the cross-linked hemoglobin solution of the present invention groups. These alterations were considered to be non~pecific and appeared to be reversi~le in nature.

EXAMPLE VI

~XCHANGE TR~N5FUSION IN DOGS
Preliminary studies on exchange trans~usions in dogs were performed on beagles and mongrel hounds with total ~lood volume exchanses ranging between 25% ~ 75%~ Dogs one, two and three are beagles weighing approximately ten kilograms each, and dogs number ~our, ~ive and si~ are ~ongrel hounds weighing approximately 20 kilograms each.
Dog number ~one (Figure 8~ is a beagle whose initial hematocrit of 24% indicated that he was anemic prior to transfusion. The anemia is of an undetermined type. This dog underwent a 40% exchange transfusion. Initial re~ponse was characterized by a rapid rise in hematocrit so that after 1.5 hours following exchange transfusion the hematocrit was measured at 28~. Subsequently, the hematocrit rose above 36%
by ~he second post transfusion day and remained in the range of 40% (102 days following transfusion). There was also a sustained rise in the hemoglobin which initially represented both intracellular hemoglobin and free plasma hemoglobin as a result of the test infusion.
Chem 20 profiles obtained in a serial fashion, in gen~ral, failed to reveal any significant abnormalities althouqh there was an indication of increased liver enzyme levels during the first nine days following in~usion.
Interpretation of these results is difficult because of interference producPd by free hemoglobin in solution with standardized automated means o~ measuring liver enzymesO
Boston dog number two (Figure 9) is a beagle who underwent a 25% exchange transfusion also without adverse clinical effects. The initial hematocrit was appropriakely depressed after the exchange transfusion and rapidly rose to exceed the resting level of 37% after 8 hours. The elevated hematocrit level was sustained through the 92nd post trans-fusion day and is con~irmed by appropriate parallel increases in the RBC count indicating increased red cell production.
Similar liver enzyme changes were noted in the Creatanine and appeared to be slightly elevated above resting levels. The remaining Chem 20 values did not change significantly.

Boston dog numb~x three (Figure 10~ is a beagle who underwent a 33% exchange transfusion. A similar rise above resting hematocrit levels was noted af er the first day and this was sustained through the 78th post transfusion day.
The liver en~ym0s showed a slightly differant picture in this animal. Although the LDH appeared to have a ~ransient elevation during the first two days, the LDH levels were normal thereafter. In contrast, the SGOT and SGPT values appeared to be moderately elevated over the resting levels for weeks after the initial transfusion. Cli~ically the animal appeared to have no adverse effects. However, the serum creatanine levels were slightly elevated following the infusion.
Dog number four (Figure 11~ is a mongrel weighing approximately 17 kilograms and was u~ed as a control animalO
A 33% hemorrhage was induced and the extracted blood volume was replaced with an equal amount of 5% human albumin. This was followed by the hematocrit level returning to normal and slightly exceeding the resting value for the ensuing 81 days served.
Dog number ~ive (Figure 12) is a mongrel hound weighing approximately 20 kilograms who underwent a 75% exchange transfusion with a signi~icant decrease in hematocrit followed by an increase in hematocrit exceeding the resting level on the 7th post transfusion day. The animal ~ustained a hematocrit which exceeded the resting level for the ensuing 43 days following the transfusion.
The liver enzymes showed a transi~nt rise in LDH and SGOT which gradually return0d to the normal range. There was a single observation of an increase in SGPT which may have been aberrant data.
Dog number six (Figure 13) is a mongrel hound weighing approximately 20 kilograms who underwent a 75% exchange ~ J

transfusion with similar findings as that found in dog number fiveO
All of the do~s tested appeared to be well clinically and experienced a rapid return to normal behavioral activity immediately after the acute e~fects of the anaesthesia subsided. There do not appear to be long term adverse effects observed in any of the tested animals.

Sl~ Y

In a prelimina~y study of acute toxicity and efficacy of the cross-linked hemoglobin solution of the invention, five test dogs received single exchange transfusions replacing 25-75% of ~alculated blood ~olume and one control dog underwent a 33% exchanye replacement with 5% albumin.
There was no mortality or clinical morbidity in any animal acutely or during an ~xtended observation up to twelYe weeks. All test dogs exhibi~ed normal activity imm~diately after replacement and experienced a rapid return of RBC
parameters to the normal range within two weeks. Test animal chemistry pr~files remained within normal limits except for a transient elevation of liver enzymes, a finding also observed in the control dog (Dog #4). ~rterial blood gas results on samples taken during and immediately after the exchange ~ransfusions indicated that normal P02 values were maintained in all animals including the two dogs which underwent 75%
exchanges.
Dogs #5 and #6 both received exchange transfusions of approximately 75% of the blood volume (calculated by body wei~ht). Dog #5 underwent a 50~ blood volume hemorrhage ~ollowed by immediate replacement with 5% albumin solution as a ~ir6t step in the exchange transfusion. This was followed by a rapid removal of another 50% of the blood volume at which time Dog #5 became dyspnic manifested by a sudden ~ 3 :~. t.~

increase in respiratory rate ~rom 14 per minute to 38 per minute associated with agonal breathing pattern. Because of the obvious clinical distress exhibited by Dog #5 at this time an equal amount of the cross-linked hemoglobin solution of the invention solution was rapidly infused to restore the blood volume to normal. During the in~usion o~ t~e cross-linked hemoglobin solution of ~he invention the respiratory rate returned to 14 associated with a cessation o~ the labored breathing pattern. Blood gas determinations prior to the first hemorrhage, aPter the first replacement and after the second replacement indicated that the P02 remained within the normal range.
Dog #6 also underwent a 75% exchange transfusion similar to Dog #~ but this time replacing the first 50% hemorrhage with an equal volume of the cross-linked hemoglobin solution of the invention and the second 50% hemorrhage with an equal volume composed o~ the cross-linked hemoglobin solution of the invention and 5% albumin mixed in equal parts. After the ~irst hemorrhage ~nd replacement, there were no signs o~
respiratory distress and there were no signs of respiratory distress during or after the second hemorrhage and replace-ment.
The art~rial blood gas determinations during and after the exchange transfusion indicated a normal P02 was main-tained throughout the exchange.
These data suggest that the cross-linked hemoglobin solution of the invention has both a volume expansion and an oxygen transport function. The response to a single trans-fusion o~ the cross-linked hemoglobin solution o~ the invention did not appear to be associated with abnormal clinical behavior or abnormal chemistry o~ hematologic para-meters, although a transient rise in liver enz~nes was seen in both the test dogs and the control.

~2~

EXAMPLE VII

The immunogenicity of the hemoglobin solution produced in accordance with the protocol o~ Example I and having the propertiss as characterized above (Invention Hemoglobin solution) was tested in primates, subjected to three hemor-rhage-transfusions of 1/3 the calculated blood volume.
Six Coebus monkeys, of 4 Kg body weight, were sedated with Ketamine, 15 mg/Kg body weight intramuscularly, and restrained. Sterile cannulae were inserted percutaneously into one femoral artery and vein. Blood was removed from the artery in an amount corresponding to 2% of body weight in kilograms (approximately 1/3 of blood volume). The Invention Hemoglobin solution was infused through the vein over a period of 30 minutes. Blood samples (2.5 ml~ were taken (1) before the removal of blood, (2~ 1 hour after the infusion of Invention Hemoglobin, (3) daily for 1 week, (4) weekly for 1 month, (5) monthly for three months. The sera were tested for the presence o~ antibodi~s, using Ouch~erlony's test The same experiment was carried out after 3 and ~ months. ~hus, each animal underwent 3 Inven-tion Hemoglobin infusions at three-month intervals~
All animals survived the cycle of three hemorrhage-transfusions. No sign of toxicity was noted (all animals appeared normal)O The Ouchterlony test resulted consistently negative for all sera, in all animals.

EXAMPLE VIII

MATERIAL PERSISTANCE IN THE CIRCULATION
Tbis etudy was undertaken to demonstrate the unique vascular per~istance of the hemoglobin product of the invention. ~ince the early days of hemoglobin research, it ~1 3~2~
--100 ~

has been stated that hemoglobin is only maintained briefly in the circula~ion. The new technique an~ unigue product not only works effectively but is maintained in the circulation.
To define the molecular weight o~ the product, we have developed data that characteri~es the unique hemoglobin-based temporary blood suhstitute of this invention. ~We haYe measured the time elimination of the material in d~g sera and have been able to characterize the blood substitute as having the following characteri~tic6 by testing protocol. In the following examples, the hemoglobin used on the test animals is the hemoglobin product produced in accordance with Example I and is rPferred to as Invention Hemoglobin.

1. Determination of molecular weight distribution_of ~emoalobin in doq sera Beagle dog sera which had bPen obtained during efficacy trials of isovolemic exchange down to 5%
hematocrit with Invention Hemoglobin were checked ~oncerning their hemoglobin-molecular weight distribution by gel permeation chromatography (HP
1090 A). Changes in molecular weight distribution with time after Invention Hemoglobin application were as follows~

1.1 Analytical Conditions:

~IPLC-Device: HP 1090 A
Integrator: ~P 3392 Detector: Diode array-UV-V/S/
(Hewlett Packard) GPC-column: TSK G 3.000 SW 300 mm x 7.5 mm Eluent: 0.1 n K2HP04 (pH 7.0) ~ 3 11 2 ~

Detection wavelengths: 260 nm (marker proteins) ~
405 nm (hemoglobin in dog sera) Results These results are tabulated in the following Table V and then graphically represented in Figure 14. It is ea~ily seen that vascular persistance is maintained for much longer than the originally reported 24-hour period demonstrated in ~ther solutions.
.
Table V
Evaluation of Bea~le_ oq Sera ~.Hb Peak No. *1 *2 *3 *4 Dog serum, 3.14 6.62 5.4 0 value 3 hr 20 min S.8 129.11 184.45 239.78 719.35 24 h 5170.11188.34 230.87 461.74 48 h 4.5511.98236.29 249.42 249.42 96 h 3.8910.38165.00 172.50 127.50 120 h 2.81,058.8180.Q0 80.00 40.00 144 h 1.51,837.0060.00 60.00 15.00 168 h 0.71,455.1742.50 45.00 5.00 216 h 0.0811.1817.74 18.47 240 h 0.097.2519.34 22.97 ~94 0.16.52 55.05 25.35 The numbers in each column represent the peak height in centimeters times the ~ull scale (in millivolts).

~ ~ 3 ~

Due to the elimination of hemoglobin the concentration decreases ~ontinuously (see Table V). Therefore, it had been necessary to work with increasing amplifications ("full scale" (mV)~ of the integrator system. In order to be able to compare the chromatographic peaks of different~samples, peaX areas or peak weights had to be recorded. This was carried ouk as ~ollowso The peak heights were multiplied by the amplification "full scale". The resulting values could be shown to be linear regarding hemoglobin concentation; this has been veri~ied by adding Invention Hemoglobin to dog sera in hemoglobin concentrations from 1% to 7%. By plotting values log "peak height X full scale'l vs. sampling time o~ sera (up to ~94 h (Table V)), the kinetics shown in Fiyure 14 can be obtained. Peak No. 1, representing the part of hemoglobin molecular weight distribution with the highest molecular waight, shows the highest retention time value, as is expected from theory. Half-life values cannot be calculated from this curve because it does not reveal an ideal exponen-tial function.
For the hemoglobin-components, represent2d by peaks ~, 3, and 4, respectively, the following intravascular half-life values were found:
peak 2: ca.84 h peak 3: ca.68 h peak 4: ca. 24 h Decreasing half-life values, i.e., shorter intravascular retention times with decreasing molecular weight~ confirm theoretical expectations.
Twenty-four hours after Invention Hemoglobin infusion, the molecular weight distribution in serum and in urine were examined. This comparison shows that exclusively peak No. 4, standing for the 68~000-Hb-component, appears in the urine after that time.

:L3~2~
-~03-The amount of the Hb-component (peak No. 4) in percent of the total lnvention Hemoglobln molecular weight distribu-tion can be calcul ted by integration o~ the peak areas ~see the fQllowing Table VI).

Table VI

PeaX No. 4 Area Sample(~ o~ Total Distributionl Invention Hemoglobin 48.0 Serum 3 h 20 min44.0 24 h 38.9 48 h 14.6 The best representation of elimination from sera is demontrated by the hal~life evaluation curves in Figure 14.
In this figure, which is a graphical representation of the data of Table V, the data is a representation in percent of total Invention Hemoglobin remaining in each subgroup molecular weight percentage.

~312~9 --lo~--EXAMPLE IX

SHEEP EXPERIMENTS

Obiective The objective o~ this ~tudy was to determine the ef~icacy of Invention Remoglobin ~olution infusion in splenectomized sheep through repeated exchange transfusion and removal of red blood cell6, and lowering of hematocrit to approximately 5~. By this testing design, the potential efficacy of Invention Hemoglobin ~olutions may be demon-strated by survivability in the a~sence o~ red blood content sufficient to sustain li~e. The nature, degree, and duration of therapeutic effect was also assessed for each animal in this study.

Test Substan~ce Invention ~emoglobin solution as described above and as produced by Example I.

Test System Purebred or mixed-braed sheep weighing 2000 to 25.0 ~g were used for this ~tudy. Animals were obtained ~rom a commercial colony whose anim~ls have been certified ~or general health prior to the study and were known to be sero-negative ~or Q-fever. The sheep were vaccinated for common viral and bacterial pathogens: tested and treated for endo-and ect~parasites; and otherwise treated to minimize the effects o~ any diseases which could create a variable.
Animals were housed individually in pens bedded with shav-ings~ fed guaranteed sheep ration and had continuous access to potable water. Environmental parameters were maintained at 70F~3F, 45% RH+ and 12 hr/12 hr light cycle. The animal room was cperated as a conventional animal ro~m, but techni-cians wore gloves, lab coats, and gowns when performing procedures.

Instrume_ ation At least two weeXs prior to study, sheep were fasted for 24 hours, pre-anesthetized with 0.2 mg I.M. atropine sul~ate, anesthetized with 4% halothane by mask, intubated and maintained with approximately 2% halothane. Splenectomy was then performed using aseptic technique through a ventral midline approach. The ~pleen was injected with ~:1000 epinephrine during this procedure to mobilize any stored ~BCs. Following splenectomy, bone marrow and liver biopsies were taken. Animals were then allowed to recover for 14 days and monitored for continued good health. Hematology values were compared befor~ and after splenectomy to determine any possible deleterious effects from the pre-study surgery procedure~
In order to conserve the test product Invention ~amo-globin, the initial exchanges were made with Ringer's lactate solution until a hematocrit of approximately 20% was achiev-ed. Then additional exchanges were made using Invention Hemoglobin until the residual hematocrit was less than 5%.
For this study, 7 sheep which were screened for general good health and pretreated for diseases were selected. Animals were uniquely identi~ied for this study. In addition, 6 control ~heep were tested in a similar way with HespanTM
(hydroxyethyl starch solution) alone. During this study, one sheep was randomly selected for testing on each of eiqht testing days. The sheep were fasted for 24 hours prior to the study, and water was withheld approximately 16 hours.
Sheep were weighed, premedicated with 0.2 mg I.M. atropine sulfate and anesthetized with ~% halothane administered by ~acemask. When suitably anesthetized, the sheep were intuba~ed ancl maintained on approximately 2% halothane.

:~ 3 ~ t~
--lo~--Anesthesia was titrated to Stage III, Plane 2-3 and main-tained. The femoral artery was dissected using aseptic techniques and 15-gauge catheters were placed in the vessel.
Using similar techniques, the ~ugular wa~ catheterized with a large bore Swan Ganz catheter. The animal was then placed in a metabolic cage and was allowed to recoYer for two hours.
Baseline blood pressur~, measured by arterial catheter transducer, and blood samples were obtained uaing the implanted catheters. Twenty cc~s of blood were removed each time for analysis of hematology, chemistry, and blvod gas determinations. Using an isovolumetric methsd, the blood volume was replaced in each animal, as follows:

Procedure 1. Approximately 400 to 600 ml of the calculated circulatory volume (CV) was withdrawn ~rom the femoral artery catheter. Blood pressure recordings were taken again and after shock was produced (BP
of 60/40) saline was infused with Ringer's lactate at 5 to 10 ml/minute to replace the whole blood (equal volume). Blood pressure recordings and samples were again made following this infusion and the animal was allowed to stabilize.
2. After the stabilization period, blood pressures were recorded, ~ollowed by a second removal of another 400-600 ml of blood~ ~ollowing this blood withdrawal, blood pressure values, samples, and clinical evaluation for signs of shock were performed for approximately 10 minutes.
3. After the 10 minute evaluation period, Invention Hemoglobin solution was administered at an infusion rate of 10 ml/minute. Blood pressure, samples, and clinical signs were monitored for 10 minutes to ~3~L2~`~9 -107~

identify conditions which would represent thera-p-utic ef~ects.
. Steps 2 and 3 were repeated until the hematocrit was reduced to below 5%. Animals were observed continually and clinical ~ignæ were recorded.
5. Each animal was monitored so as to check for any signs associated with hemorrhage ~nd tr~atment ~sr a period of 2 hour~. A terminal blood sample of ~0 cc~ was taken for hematology, chemistry, and blood gas analysis before removing the catheters and closing the catheterization sites sterilely under light halothane anesthesia.
6. Changes in urinary output were monitored in a ~etabolic cage and recorded thr~ugout the hemor-rhage treatment and recovery periods. Blood gas values were also measured during each phase while arterial catheter was in place.
7. When animals recovered ~rom anesthesia, they were returned to their cages and given ~upportive therapy as necessary indicated by their clinical condition. Comprehensive clincal monitoring and continued upportive therapy waR provided for 21 days.
At the beginniny of the study, baseline blood samples were drawn ~rom the jugular vein and 2~-hour urine samples were collected for urinalysis. At daily intervals and at the conclusion of the study, comparative blood samples were drawn, urine was collected and the ophthalmological examina tions were repeated. on Day 14, the sheep were anesthetized as previously described and repeated liver and bone marrow biopsies were performed. Animals that died were evaluated by gross pathological and histopathological evaluations. The spectrum o~ tests performsd were:

~ 3 ~

Blood Gases Hematoloqy Clinical Chamistry (arterial) Platelet Count LDG pH
Di~ferential WBC Alk Phos 2 ~aturation Hemoglobin BUN 2 content Hemataocrit Creatinine REBC Morphology Bilirubin (I and Reticulocyte Count D) Sodium Potassium Chloride Calcium Phosphorous Total Protein Albumin Globulin A/G/Ratio Glucose Cholesterol Osmolarity Carbon Dioxide Triglycerides Iron Iron Building Capacity Urinalysis Speci~ic ~ravity pH
Protein Glucos~
Ketones Hemoglobin Hemoglobin Polymer Creatinine/Sedi-ment Exam Aqueous Humor Hemoglobin RBC Serum and Hemoglobin Bovine Hemoglobin Antibody ~3~l2~

CSF Polymerized Hemoglobin Hemoglobin Ferritin RBC Heptiglobin ross and Histo-Coagulation Tests logic Patholoqy PT Lungs Heart PTT Liver (biopsies Ridneys (L and R) included) Brain Fibrinogen Eyes Adrenals (L and R~
Thyroid S
Bone Marrow (biopsies included) ~3~2~

Hemodynamics Cardiac Output (thermal dilu-tion~
Arterial Pressure Central Venous Pressure Pulmonary Artery Wedge Pressure All data from observations and tests were recorded on specific history, observation and testing report forms.

STATISTICAL ANALYSIS

Seven shsep were tested using the efficacy protocol.
Each sheep has baseline blood samples and 24-hour urine samples performed. These data were obtained at daily intervals during the follow-up period. For each day of follow-up, statistical comparisons were performed using the paired test comparing each follow-up measurement with the corresponding baseline measurement. Of particular interest is a comparison of blood and measurements immediately after the completion of all exchange transfusions with the corres-ponding baseline measurements.

REFERENCES

1. Blood Policy and Technolo~y (Washington, D.C.: U.S.
Congress, Office Technology Assessment, OTA-H-260, January, pp. 133-150, 1985).

2. 'IBlood Groups and Blood Transfusion." In: The Merck Veterinary Manual. 5 ed. Merck Company, Inc., Rahway, NJ, pp. 42-49, 1979.

3. Xolata, R.J., Burrows, C.F~ and Soma, L.R., "Shock:
Pathophysiology and Management." In: rurrent Veteri~
nary Therapy in Small ~n mal Practice. 7th ed., W.B.
Saunders Company, Philadelphia, pp. 32-48, 1980.

PRELIMINARY RESVLTS OF THE SHEEP EFFICACY PROTOCOL are included in this example. The raw data regarding hemodynamic parameters is presented in tabulated Porm in Tables VII (Test Animals) and VIII (Control Animals). The graphic presenta-tion o~ the blood gas and hemodynamic data in Figures 15-19 show that the test animals maintained arterial and venous oxygen contents well above the levels achieved in the controls which received no oxygen-carrying volume replacement during exchange transfusions. All test animals survived the exchange.

In contrast, all six o~ the control animals did not survive the acut exchange transfusion. Six of the control animals showed deteriorating cardiac output as arterial and mixed venous oxy~en contents, and hematocrits, ~ell below 10%.

In Figure 18, the contribution oP the Invention Hemo-globin as compared to the sheep hematocrit is compared at decreasing hematocrit levels. Figure 18 demonstrates that at the end of the exchanges when the residual hematocrit i5 below 5% that approximately 80-90~ of the ar~erial oxygen content is contributed by the Invention Hemoylobin as compared to approximately 10-20% which is contributed by both the liquid phase oP th~ blood and the remaining red blood cells in the test animals.

Both the test group and the control sheep breathed room air at all times during the experiment. Both the test group ~3~2~9 and the control group showed an identical decline in arterial oxygen c~ntent during the first exchange which lowered their respective groups from the baseline Aematocrit down to the approximately 20% hematocrit levels. From this point onl the test group received Invention Hemoglobin in exchange for the native sheep red blood cells. At this point, th~ figures demonstrate that the oxygen content is well maintained in the animals where bovine hemoglobin i~ used to replace the removed blood. In the case of the controls, there is decrease in the venous oxygen contents which continues to parallel the decline in hematocrit since HespanTM does not carry a significant amount of oxygen except as a dissolved gas.
The association of ~xygen delivery with the infused bovine hemoglobin is demonstrated in Figure 19 which clearly shows ~he departure of the declining oxygen delivery which is at first associated with the declining hematocrit in the initial exchange and which then increases in association with the increasing concentration of bovine hemoglobin which is used to replace the sheep red blood cells during the ex-change~

CONCLUSIONS

This study clearly demonstrates the efficacy of Inven-tion Hemoglobin as an oxygen transport solution. The blood gas data and hemodynamic data are consistent with the survivability of the test animals in contrast to the control animals, none of which survived the acute exchange trans-fusion. Since the residual hematocrits in all of the test animals were less than 5% and, in some cases, between 1 and 2%, ~tudy clearly demonstrates Invention Hemoglobin con tributed significantly to adequate oxygen transport in the test animals. ~urthermore, all of the test animals survived on a long-term basis without lntensive care or increased inspired oxygen.

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DOG EXPERIMENTS

OBJECTIVE

Inven~ion Hemoglobin was tested for efficacy, tolerabil-ity, and side effects on beagle dogs. The e~perimental arrangement was intended to answer the question of whether the preparation studied was able to assume the oxYgen transport function and the volume replacement in case of severe losses of endogenous blood, and how rapidly the hemoglobin supplied is excreted from the body~ The toler-ability and the occurrence of unexpectad side effects was also determined secondarily.
There is an indication for an 2 transportation solution only when the remaining amount of endogenous hemoglobin is no longer able to satisfy the 2 requirement of the tissue.
From this it follows that the anemia of the experimental animals must be so severe that at least detectable injuries occur from the 2 deficiency, and at the same time, the administration of the hemoglobin solution demonstrably prevents these injuries.

TEST SUBSTANCE

Invention ~emoglobin solution as described above and as produced by the process of Example I.

EXPERIMENTAL MODEL

7 purebred beagle dogs were used for study who had been splenectomized approximately 3 weeks before the beginning of the study.

2~9 INSTRUMENTATION

After premedication and the initiation of anesthesia, an isovolemic hemodilution was carried out through peristaltic pumps in a controlled manner on 4 animals with ~nvention Hemoglobin solution up to a residual hematocrit of 5%, and on 3 control animals with a hydroxyethyl starch solution (HES) whose ionic composition and colloid osmotic pressure were equivalent to those of the Invention Hemoglobin solution. A
number of parameters were determined during the exchange transfusion and the follow-up period of 10 days.

DOG TEST__ESULTS

In this study, 4 test dogs received ex~hange tran~fusion with Invention Hemoglobin under general anesthesia over a 3-hour period with measurements approximately every 10 minutes.
All test dogs survived the acute exchange transfusion with residual hematocrits below 5~. The hemodynamic and blood gas analysis revealed that all test dogs were well oxygenated at the end of the procedure in contrast to the 3 control dogs which did not survive the exchange transfusion with the hydroxyethyl starch solution. The 3 control dogs had evidence of inadequate oxygenation associated with the decreasing hematocrit levels. Figures 20 24 demonstrate the differences in the test and control groups for selected parameters measured.

CONCLUSION

The preliminary results of this efficacy study demon-strate that Invention Hemoglobin solution contributes to normal oxygenation of test dogs which are severely depleted ~ 3 ~
î18-of red blood cell mass. In cont:rast to the control dogs which could nc~t survive with very low residual hematocrit levels, the test animals all survived the acute exchange transfusion. The conclusion of this study is that it is evident that Invention Hemoglobin ~olution transports oxygen under extreme conditions of severe red blood cell los`s.

Having now fully described the invention, it will be readily apparent to one skilled in the art that many changes and modifications may be made thereto without departing fxom the spirit or scope thereof.

Claims (18)

1. A hemoglobin solution for transporting and releasing oxygen to living cells, comprising hemoglobin suspended in an aqueous medium, said hemoglobin solution being substantially free of red blood cell stroma and having an endotoxin level which is reduced to a level which does not cause complement activation upon in vivo administration to a human being.

2. A hemoglobin solution of claim 1 wherein the endotoxin level is less than about 0.5 endotoxin units per milliliter (EU/ml) determined by a chromogenic Limulus Amebocytic Lysate (LAL) assay against an end-point reaction made by a series of dilutions of a reference endotoxin solution and thereafter interpolating from a standard regression curve made from colorometric readings from said dilutions.

3. A hemoglobin solution of Claim 2 wherein said hemoglobin comprises bovine hemoglobin.

4. A hemoglobin solution of Claim 3 wherein said hemoglobin is crosslinked.

5. A blood-substitute comprising the hemoglobin solution of Claim 4.

6. A plasma-expander comprising the hemoglobin solution of Claim 4.

7. A resuscitation fluid comprising the hemoglobin solution of Claim 4.

8. A blood-substitute comprising an aqueous solution of crosslinked bovine hemoglobin, said blood substitute being substantially free of cell stroma, non-hemoglobin proteins and pyrogens and having an endotoxin level of less than about 0.5 endotoxin units per milliliter (EU/ml) determined by a chromatographic Limulus Amebocytic Lysate (LAL) assay against an end-point reaction made by a series of dilu-tions of a reference endotoxin solution and thereafter interpolating from a standard regression curve made form colorometric read-ings from said dilutions.

9. A blood-substitute comprising an aqueous solution of hemoglobin derived from bovine red blood cells, said hemoglobin being crosslinked to a sufficient degree to provide polyhemo-globin with at least about 50% of said poly-hemoglobin of greater than tetrameric form, said blood-substitute:
a) having physiologic conditions in the range of from about 18 to about 36 mmHg;
b) being substantially free of red blood cell stroma and non-hemoglobin proteins; and c) having an endotoxin level of less than about 0.5 endotoxin units per milliliter (EU/ml) determined by a chromogenic Limulus Amebocytic Lysate (LAL) assay against an end-point reaction made by a series of dilutions of a reference endo-toxin solution and thereafter interpolat-ing from a standard regression curve made from colorometric readings from said dilutions.

10. A blood-substitute of claim 9 having an endotoxin level of less than about 0.02 EU/ml.

11. A blood-substitute comprising an aqueous solution of crosslinked hemoglobin, said hemoglobin being derived from bovine red blood cells, said blood-substitute characterized by:
a) an endotoxin concentration of less than about 0.02 endotoxin units per milliliter (EU/ml) determined by a chromogenic Limulus Amebocytic Lysate (LAL) assay against an end-point reaction made by a series of dilutions of a reference endo-toxin solution and thereafter interpolat-ing from a standard regression curve made from colorometric readings from said dilutions;
b) a phospholipid concentration of less than about 1 nanogram per milliliter;
c) a hemoglobin molecular weight distribution of greater than about 90% in the range of 68,000-500,000 daltons;
d) an osmolarity as measured by freezing point depression in the range of 180-320 milliosmoles per liter;
e) a hemoglobin content of from bout 5 to about 25 grams per deciliter;

f) a methemoglobin content of less than about 20%, by weight;
g) a P50 in the range of about 18-36 mm Hg;
h) an intravascular half-life of at least about four days;
i) being substantially free of red blood cell stroma, non-hemoglobin proteins and pyrogens.

12. A mammalian blood-substitute comprising cross-linked hemoglobin suspended in an aqueous medium, said hemoglobin having been derived from mammalian blood cells, said blood-sub-stitute having: crosslinked hemoglobin having a molecular weigh distribution of greater than about 90% in the range of about 68,000 to 500,000 daltons; an osmolarity as measured by freezing point depression in the range of about 180-320 milliosmoles per liter of solution; a final hemoglobin content of from about 9 to about 13 grams per deciliter; a methemoglobin content of less than about 10%, by weight;
physiologic levels of sodium chloride and potassium chloride; less than about one nano-mole of phospholipid per milliliter; less than about one part per million of crosslinking agent; a P 50 in the range of about 18-36 mm Hg;
an intravascular half-life of at least about four days; and an endotoxin level of less than about 0.5 endotoxin units per milliliter (EU/ml) determined by a chromogenic Limulus Amebocytic Lysate (LAL) assay against an end-point reaction made by a series of dilu-tions of a reference endotoxin solution and thereafter interpolating from a standard regression curve made from colormetric read-ings from said dilutions.

13. A blood -substitute of claim 12 further charac-terized by the fact that, in vivo, it:
a) does not activate complement;
b) does not cause hemorrhagic disorders;
c) does not cause abnormal platelet function or aggregation;
d) does not cause abnormal prothrombin times (PT);
e) does not cause abnormal partial thrombo-plastin times;
f) does not interfere with blood typing or cross-matching;
g) is non-toxic to the kidneys of normal healthy mammals at concentrations of 3.5 grams per kilogram body weight and 8 grams per deciliter circulating blood volume;
h) exhibits circulating persistence of at least seven days; and i) acts as a stimulus to accelerated erythro-poiesis.

14.A method for producing a hemoglobin solution substantially free of endotoxins by performing the following sequence of steps under substan-tially endotoxin free conditions:

a) disrupting the red blood cell fraction of bovine blood without prior washing to produce a hemoglobin-containing fraction, b) clarifying said hemoglobin-containing solution to produce substantially stromal-free hemoglobin-containing solu-tion:
c) microporously filtering said substantially stromal-free hemoglobin-containing fiolu-tion to produce a partially sterilized, stromal-free, hemoglobin-containing solution;
d) ultrafiltering said partially sterilized, stromal-free hemoglobin-containing solu-tion to produce a size-separated, par-tially sterilized, stromal-free, hemo-globin-csntaining solution:
e) chromatographically treating said size-separated, partially-sterilized, stromal-free, hemoglobin containing solution to produce a substantially phospholipid-free hemoglobin: and f) eluting said substantially phospholipid free hemoglobin to produce a substantially phospholipid-free substantially endotoxin-free hemoglobin solution.

15, A method of Claim 14 including the additional steps of crosslinking hemoglobin in said substantially phospholipid-free, substantially endotoxin-free hemoglobin solution to produce a semi-synthetic blood substitute.

16. A hemoglobin solution produced by the method of Claim 14

17. A blood-substitute produced by the method of Claim 15.

18. A hemoglobin solution for transporting and releasing oxygen to living cells, comprising bovine hemoglobin in an aqueous pharmaceutically-acceptable carrier medium, said hemoglobin solution being substantially free of red blood cell stroma and having an endotoxin level of less than about 0.5 endotoxin units per milliliter (EU/ml) determined by a chromogenic Limulus Amebocytic Lysate (LAL) assay against an end-point reaction made by a series of dilutions of a reference endotoxin solution and thereafter interpolating from a standard regression curve made from colorometric readings from said dilutions.