WO1996028197A1 - Method for facilitating autologous blood donation and treating oxygen debt - Google Patents

Abstract

The present invention relates to a method for facilitating autologous donation. A portion of a patient's blood is removed and then an oxygen delivery enhancing amount of a cell-free hemoglobin is administered. The invention further relates to a method for treating oxygen debt by administration of an oxygen delivery enhancing amount of cell-free hemoglobin.

Description

METHOD FOR FACILITATING AUTOLOGOUS BLOOD DONATION AND TREATING OXYGEN DEBT

BACKGROUND OF THE INVENTION

During surgery, many patients lose blood. In clinical practice, patients suffering acute losses of only moderate amounts of blood may require only replacement of the volume that is lost. More severe blood loss, however, may require both volume replacement and replacement of oxygen carrying capacity. In situations involving massive blood loss, it may be necessary to replace other blood components, such as platelets and clotting factors, by transfusing whole blood.

Many surgeries can result in the need for blood transfusion due to blood loss. It is estimated that about 9 million units of blood are transfused in connection with surgical procedures each year. It is generally accepted that postoperative recovery can be accelerated if hemoglobin concentrations are not allowed to fall below 10 g/dL, which is the generally accepted indication for transfusion (Zauder, Anesth. Clin. North Amer. 8:471-80, 1990). Allogeneic blood transfusions (transfusions of blood collected from donors, not the patient) impose inherent risks to the recipient of the transfusion including: (1) infectious disease transmission (i.e., human immunodeficiency virus (HTV), non-A and non- 13 hepatitis, hepatitis B, Yersinia enter ocolitica, human T-cell leukemia virus 1 and 2, cytomegalovirus) and (2) immunologic reaction (i.e., transfusion reactions, immunosuppresion, graft versus host reaction). Other drawbacks of using allogeneic blood transfusions include no universal compatibility, limited availability and limited stability (shelf life of 42 days or less; cannot be frozen).

These risks and limitations have highlighted the desirability of using autologous blood (the patient's own blood) for transfusions. Autologous blood for transfusion can be collected by pre-donation of blood prior to surgery. Predonation typically involves withdrawal of several units of a patient's blood during the six weeks or so prior to surgery. The withdrawn blood can then be used during surgery (perioperatively) or during the recuperation time after the surgery has been completed (postoperatively).

Acute normovolemic hemodilution (ANH) is another technique that is used to reduce exposure to autologous blood. For this procedure, blood is withdrawn from the patient at the time of or just prior to surgery. The volume of blood that is withdrawn from the patient is then replaced with an equal volume of a non-oxygen carrying crystalloid or colloid solution. Finally, the blood that was withdrawn from the patient is then re-infused at the end of surgery or during the recuperation period if needed.

In addition to providing allogeneic blood units for transfusion, the ANH process may improve the outcome of some surgical procedures because the viscosity of the patient's blood is reduced due to dilution of the blood with the crystalloid or colloid solution. It appears that the basic mechanisms that compensate for most of the decreased oxygen capacity of the diluted blood are a rise in cardiac output and increased organ blood flow, both of which may be beneficial, and both of which appear to result from the reduced viscosity of blood at lower hematocrits (Messmer et al., Eur. Surg. Res. 18: 254-263, 1986).

The major limitation associated with both ANH and predonation is the limitation on the amount of blood that can be removed from a patient without compromising the oxygen carrying capacity of the patient. In other words, the donation or removal of too much blood can compromise the oxygen carrying capacity of the blood, i.e., sufficient blood can be lost to result in oxygen deficit or oxygen debt in the patient. Therefore, the use of an appropriate oxygen carrying compound in a replacement fluid could permit additional amounts of autologous blood to be donated in ANH procedures. In addition, replacement of withdrawn blood with an oxygen-carrying fluid could increase that amount of blood that could be withdrawn during predonation.

ANH procedures using oxygen carrying perfluorocarbons as the replacement fluid have been utilized in specific limited circumstances (Roth et al., U.S. Patent 5,344,393). However, these procedures require the administration of breathing gas enriched with 50% - 100% oxygen. High oxygen concentrations in the breathing gas can lead to complications due to oxygen toxicity (Beisbarth et al., in Advances in Blood Substitute Research, Bolin, Geyer and Nemo (eds), Alan R. Liss, Inc., New York, pp 373-380, 1983). Therefore, the use of a oxygen carrying molecules other than perfluorocarbons would be more compatible with presently accepted anesthetic practices as there is no need to increase oxygen in the breathing gas to enhance oxygen delivery by the oxygen carrying molecule. Such a candidate for an oxygen carrying replacement fluid is hemoglobin, which has been proposed as a blood substitute to replace lost blood (Hoffman and Nagai, U.S. Patent 5,028,588), and as a hemodiluent in acute normovolemic hemodilution when used in conjunction with a breathing gas of at least 50% oxygen (Roth et al., U.S. Patent 5,344,393). However, there is no teaching in the prior art that hemoglobin, particularly recombinant hemoglobin, could be useful as a blood volume expander and /or oxygen carrier during predonation or ANH under ordinary anesthetic practice.

A number of extracellular hemoglobins have been proposed for use as oxygen delivery vehicles and other uses (drug delivery vehicles, vasopressor agents, iron sources, etc.). Recombinant human hemoglobin (e.g., rHbl.l, Looker et al, Nature, 356: 258-260 (1992)) is a novel hemoglobin-based oxygen carrier (HBOC) whose safety and pharmacokinetics has been assessed in animals and normal adult males. Because rHbl.l is a genetically-engineered, red blood cell-free

HBOC, derived from fermentation rather than from whole blood, it may eliminate or minimize the risks and limitations associated with blood transfusions. The fact that rHbl.l has volume replacement characteristics and oxygen transport properties makes it a potential versatile replacement fluid for patients who have lost blood through trauma, surgery or blood donation or for patients who are suffering from oxygen debt, whatever the cause. Administration of rHbl.l will replace a portion of the oxygen transport capacity lost during predonation or ANH.

SUMMARY OF THE INVENTION

The present invention relates to a method for facilitating autologous blood donation by a patient, comprising:

(a) removing a portion of the patient's blood; (b) administering an oxygen delivery enhancing amount of cell-free hemoglobin; and

(c) optionally administering a breathing gas, wherein said breathing gas is not enriched with oxygen.

In a further aspect of the invention, the blood that is removed from the patient can be stored. The method can further comprise the step of readministering said stored blood to said patient. In another embodiment of the invention, removing and storing a portion of patient's blood occurs less than 72 hours prior to the patient undergoing the loss of blood. In a further embodiment, the cell-free hemoglobin is non-erythrocyte derived, and is especially recombinant hemoglobin, particularly rHbl.l.

In a still futher embodiment of the invention the autologous blood donation is predonation. In another aspect of the invention, the autologous blood donation is perioperative.

In another embodiment of the invention, the present invention relates to a method for treating oxygen debt comprising administering therapeutically effective amount of cell-free hemoglobin to treat oxygen debt. In a further aspect of the invention, the cell-free hemoglobin is non- erythrocyte derived, and is especially recombinant hemoglobin, particularly rHbl.l.

The present invention also contemplates a kit comprising cell-free hemoglobin and associated supplies.

DESCRIPTION OF THE FIGURES

Figure 1. Oxygen debt as a function of time. Dogs were first bled and then 120% of the blood volume removed was replaced with either recombinant hemoglobin ( rHbl.l; -X~) or colloid followed by autologous blood (control; —A—).

DETAILED DESCRIPTION OF THE INVENTION

In general, the present invention provides a method for facilitating autologous blood donation by replacement of all or part of the removed blood with a cell-free hemoglobin capable of binding and releasing oxygen to tissues. Therefore, distinctive from hemodilution, the present invention replaces removed blood not only with the lost volume to provide possible benefits of fewer red blood cells in the blood (e.g., higher cardiac output), but also with some or all of the oxygen delivery capacity lost due to the removal of the blood. As described herein, this distinction provides certain advantages, especially when large or immediate autologous blood donation would be beneficial or oxygen debt would be a likely result.

Whereas the replacement of removed blood by a volume expander is often referred to as "hemodilution" or "acute normovolemic hemodilution", the present invention is more aptly described as "acute normovolemic hemosupport", "hemosupport", "acute normovolemic hemoaugmentation", "hemoaugmentation", "perioperative isovolemic substitution", or "acute normovolemic oxygenation."

Predeposit requires that the surgery be planned in advance. Blood is donated by the patient during the weeks and months prior to surgery and then stored for subsequent administration to the patient during or after surgery. Blood donation of 300-400 ml units are typically obtained at 2-7 day intervals, with the last collection more than 72 hours prior to surgery. The blood may be stored in the liquid state as whole blood, or it may be separated into red cells and plasma that can be frozen to preserve labile components. With the present invention, predonation may be improved and have expanded use in one or more ways. First, the present invention may allow a patient to donate more blood than is usually donated because part of the oxygen carrying capability of the blood that has been removed is replaced by cell-free hemoglobin at the time of donation. Second, blood may be autologously predonated by the patient closer to the time when blood loss is likely to occur, i.e., less than 72 hours prior to surgery. This method could be particularly useful in the event of emergencies such as unscheduled surgeries where predonation by typical techniques is not presently possible. Third, predonation may be able to occur more frequently or with less time between multiple predonation events.

Perioperative isovolemic dilution (ANH) is the process of collecting blood immediately before or during surgery with the concomitant replacement by a sufficient volume of blood volume expander, particularly a crystalloid or colloid solution. This practice decreases blood viscosity during surgery, thereby reducing the work load on the heart and increasing microcirculation. The blood that is removed from the patient is then stored for possible readministration to the patient during or after surgery.

The amount of blood to be removed during an acute normovolemic hemodilution procedure, and the desired resultant residual hemoglobin level in the patient, can be readily determined by one of skill in the art and will depend on multiple factors. These factors include the procedure to be performed, the condition of the patient, the need for reduced blood viscosity, the minimally safe hemoglobin content for the patient in the estimation of the skilled artisan, the estimated amount of blood that will be needed for future readministration to the patient and the like. For example, patients undergoing coronary bypass surgery have been hemodiluted to hematocrits of 15% (Mathru, M. and M. Rooney, Problems Crit. Care (USA), 400-410, 1991).

After removal of some of the blood, or simultaneously with the removal, a crystalloid or colloid plasma expander (or both) is administered to the patient to maintain blood volume at a desired value, for example, about the blood volume prior to removal of any blood. If the perioperative blood volume expander includes hemoglobin capable of binding and releasing oxygen, the procedure of perioperative isovolemic dilution ceases to be a dilution of the oxygen carrying capacity of the patient's blood and becomes "perioperative isovolemic substitution."

In the present invention, procedures resulting in the collection of autologous blood, particularly predeposit and perioperative isovolemic substitution, are used to obtain blood for possible later transfusion into a patient facing a loss of blood. Methods for blood cell salvage, including intraoperative, postoperative and autotransfusion are described in US Patent No. 5,344,393, which is incorporated herein by reference.

A patient facing a loss of blood is one who is facing or is likely to face a situation where the patient may lose sufficient blood such as to significantly compromise the ability of the blood to deliver adequate oxygen to tissues. Such situations include planned scheduled surgeries as well as emergency unscheduled surgeries and trauma. In the case of trauma and emergency unscheduled surgery, perioperative isovolemic substitution would allow donation of blood up to essentially the beginning of the surgical procedure or even during the surgical procedure. A compromise in the ability of the blood to deliver adequate oxygen to tissues is referred to as oxygen debt or oxygen deficit. Oxygen debt can occur when the oxygen consumption needs of the body, or any tissue of the body, exceeds the ability of the body to provide oxygen. Oxygen debt can be measured, for example, as a decrease in consumption of inhaled oxygen. This decrease reflects the reduced capacity of the blood to bind oxygen at the lungs and deliver it to metabolizing tissues. Oxygen deficit has also been defined as the accumulating difference over time between the oxygen demand (equal to the stable V02 at baseline) and the actual V02. V02 in turn is defined as the inhaled oxygen consumption, i.e. the difference in oxygen content between inhaled and expired gas, and can be derived by solution of the Fick equation: V02 = Cardiac output (mis of blood /min) * Arterio- Venous oxygen content difference (mis 02 /ml of blood). The deficit or debt therefore is the integral of the decrease in V02 below the demand over a given period of time (Siegel, Amer. Assoc. Clin. Chem. 36(8B): 1585, 1990).

In the methods of the instant invention, cell-free hemoglobin has been shown to reverse oxygen debt, more rapidly than red blood cell transfusion. Therefore, the present invention is also useful to prevent and treat the symptoms of oxygen debt which are often associated with blood loss, particularly a large volume of blood loss.

The amount of blood that is typically predonated by a patient for later re-administration (acute predonation) is on the order of two units. Removal of more blood at any one time may result in a compromise in the ability of the blood to adequately oxygenate tissues, and thus multiple predonations with sufficient recovery time between each predonation may be required to bank sufficient autologous blood prior to a medical procedure. However, typically no more than twelve units are collected during extended predonation because of limitations in storage of collected blood and logistics of scheduling medical procedures and donations. Therefore, it may not be possible to collect sufficient blood over a long enough time to meet the requirements of autologous transfusion at the time of a surgery. If there is not sufficient autologous blood to meet a patient's need, then the patient may be exposed to allogeneic blood units. If, however, some or all of the blood removed is replaced with cell-free hemoglobin which is capable of binding and releasing oxygen, the portion of blood removed at one time from the patient and stored for later use can be increased, or the time of recovery between donations can be decreased. Thus both the amount collected at any one time donation can be increased, as can the total amount of blood collected during an extended predonation protocol by using the method of the instant invention. Furthermore, this invention need not be limited to only those patients who face loss of blood and are thus storing autologous blood units; the methods of the invention can be used to increase the amount of blood donated by any donor for transfusion to any patient in need of such transfusion (allogeneic transfusion). Therefore, in addition to utility for acute normovolemic hemoaugmentation, the present invention can be used to allow donation of more blood than would otherwise be possible or recommended ("hyperdonation") .

In the practice of the present invention, simultaneous with or subsequent to the removal of blood for possible use in autologous donation, there is administered to the patient cell-free hemoglobin. Preferably, the removal of blood and administration of the cell-free hemoglobin are performed sequentially or subsequently to one another, but it is contemplated that the simultaneous removal of blood and administration of cell-free hemoglobin may be beneficial in some medical situations. For example, in the case of trauma and emergency unscheduled surgery it may be necessary to perform simultaneous removal of blood and administration of cell-free hemoglobin because of severe bleeding or the necessity to immediately initiate a surgical procedure.

The removal and storage of a patient's predonated blood can be accomplished using any well known methods of blood donation and storage. The administration of the cell-free hemoglobin is typically in the form of an infusion, particularly an intravenous infusion. The dosage of cell- free hemoglobin can be readily determined by the skilled practitioner and depends on, among other factors, the amount of liquid required by the patient, the infusion rate, the volume of blood removed, the amount and oxygen carrying capacity (P5₀) of the cell-free hemoglobin and the total amount of liquid to be infused. The amount of cell-free hemoglobin administered is preferably a sufficient quantity to replace some or all of the oxygen-delivery lost as a result of the removal of blood. The amount of cell-free hemoglobin to be administered can replace all or a portion the volume of blood removed, for example from about 10% of the volume of blood removed to about 150% of the volume of blood removed, preferably from about 50% of the volume of blood removed to about 150% of the volume of blood removed. Alternatively, the amount of cell-free hemoglobin that can be administered according to the methods of the instant invention can replace all or a portion of the oxygen delivery capacity lost as a result of the removal of blood; a 1:1 replacement of the oxygen delivery capacity of the lost blood volume is not necessary. At a minimum, however, sufficient oxygen delivery capacity in the form of hemoglobin must be infused to avoid a significant compromise in the ability of the blood to deliver adequate oxygen to tissues (to prevent or to treat oxygen debt) or to facilitate hyperdonation. The optimal dosage of cell-free hemoglobin used for hyperdonation, hemoaugmentation or to prevent or treat oxygen debt can be determined by skilled practitioners. Such optimal dosage will depend on, for example, the underlying medical condition, the characteristics of the individual patient, the predonation schedule, autologous transfusion requirements and the like.

As another alternative, cell-free hemoglobin can be administered to reduce the hematocrit level as described above and decrease blood viscosity while preserving oxygen delivery. Typically, infusion rates for cell-free hemoglobin range from a controlled flow to essentially gravitational flow, at rates ranging from about 1 ml /kg /hour to about 75 ml/kg/hour. Suitable rates include from about 7 ml/kg/hour to about 30 ml /kg /hour. The cell-free hemoglobin of the methods of the present invention used for facilitating autologous blood donation or treating oxygen debt can comprise a physiologically and/or pharmaceutically and/or therapeutically effective amount of hemoglobin as the active ingredient alone or in combination with other active or inert agents. For example, a parenteral therapeutic composition can comprise a sterile isotonic saline solution containing between 0.001% and 50% (w/v) hemoglobin. Suitable compositions can also include 0 - 200 mM of one or more buffers (for example, acetate, phosphate, citrate, bicarbonate, or Good's buffers). Salts such as sodium chloride, potassium chloride, sodium acetate, calcium chloride, magnesium chloride can also be included in the compositions of the invention at concentrations of 0-2 M. In addition, the compositions of the invention can include 0-2 M of one or more carbohydrates (for example, reducing carbohydrates such as glucose, maltose, lactose or non- reducing carbohydrates such as sucrose, trehalose, raffinose, mannitol, isosucrose or stachyose) and 0-2 M of one or more alcohols or poly alcohols (such as polyethylene glycols, propylene glycols, dextrans, or polyols). The compositions of the invention can also contain 0.005 - 1% of one or more surfactants and 0-200 μM of one or more chelating agents (for example, ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis (β- aminoethyl ether) _V,N,N',N'-tetraacetic acid (EGTA), o-phenanthroline, diethylamine triamine pentaacetic acid (DTPA also known as pentaacetic acid) and the like). The compositions of the invention can also be at about pH 6.5 - 9.5.

In another embodiment, the composition contains 0 - 300 mM of one or more salts, for example chloride salts, 0-100 mM of one or more non-reducing sugars, 0-10 mM anti-oxidants, 0-100 mM of one or more buffers, 0.01 - 0.5% of one or more surfactants, and 0-150 μM of one or more chelating agents. In a still further embodiment, the composition contains 0 - 150 mM NaCl, 0 - 10 mM sodium phosphate, and 0.01 - 0.1% surfactant, and 0-50 μM of one or more chelating agents, pH 6.6 - 7.8. Another suitable the hemoglobin-containing composition includes 5 mM sodium phosphate, 100-150 mM NaCl, 0.025% to 0.1% polysorbate 80, 2 mM sodium ascorbate, and 25 μM EDTA, pH 6.8 - 7.6.

Other components can be added if desired. For example 0 - 5 mM reducing agents such as, for example, dithionite, ferrous salts, sodium borohydride, sodium cyanoborohydride and ascorbate can be added to the composition. Additional additives to the formulation can include anti- oxidants (e.g. ascorbate or salts thereof, alpha tocopherol), anti-bacterial agents, oncotic pressure agents (e.g. albumin or polyethylene glycols), iron chelating agents such as, for example, desferroxamine, and other formulation acceptable salts, sugars and excipients known to those of skill in the art, the selection of which depends upon the particular purpose to be achieved and the properties of such additives which can be readily determined.

The compositions of the present invention can be formulated by any method known in the art. Such formulation methods include, for example, simple mixing, sequential addition, emulsification, diafiltration and the like.

Such compositions of the instant invention can be used for the treatment of oxygen debt. In addition, such compositions can be used to treat hemorrhage, whether voluntary, as in hemodilution or hemoaugmentation procedures, or involuntary, such as trauma. The formulations of the instant invention can be used not only to increase oxygen delivery to tissues as described above, but also as simple volume expanders that provide oncotic pressure due to the presence of the large hemoglobin protein molecule. That portion of the osmotic pressure exerted by macromolecules, such as proteins, is the colloid osmotic pressure or oncotic pressure. The oncotic pressure of the intravascular plasma is higher than the oncotic pressure of the interstitial fluid. Plasma oncotic pressure is a key force in keeping water in the intravascular space, and thus maintaining intravascular volume. Because administered cell- free hemoglobin will circulate as a soluble plasma protein, it has the potential to maintain and expand intravascular volume by exerting colloid osmotic pressure effects. It counter balances the hydrostatic pressure within the microvasculature which tends to push water out of the intravascular space. Oncotic pressure is proportional to the molar concentration of capillary-impermeable macromolecules. Normally, plasma albumin is responsible for 70-80% of the plasma oncotic pressure. The colloid osmotic pressure of a 5% solution of hemoglobin is similar to that of 5% human serum albumin when measured on a Wescor 4420 Colloid Osmometer. Since the molecular weight of albumin is 66,500 daltons and the molecular weight of hemoglobin is 64,600 daltons, they will have a similar molarities and thus, similar oncotic pressure. Albumin is commonly formulated in 25 g doses usually in a volume of about 500 ml. Therefore, the administration of 25 g of hemoglobin in a similar volume could have volume expansion characteristics similar to the administration of 25 g of albumin.

A physiologically and /or pharmaceutically and /or therapeutically effective amount of the hemoglobin of the present invention is that amount of hemoglobin that is capable of binding oxygen in the lungs of the patient and releasing sufficient oxygen in the tissues to prevent the ill effects of oxygen deprivation (hypoxia) in tissues. Whether a hemoglobin will be useful for binding and releasing oxygen to the tissues can be determined by its oxygen equilibrium binding curve (OEC- typically characterized by the P50 value and Hill coefficient [n]) as well as other factors described below. Suitable methods for measuring the OEC are described in Hoffman and Nagai, US Patent 5,028,588, herein incorporated by reference. The amount of oxygen delivered to a tissue will be determined by multiple factors, including, for example, the OEC of the cell-free hemoglobin(s), the concentration of cell-free hemoglobin in a given composition, the amount of cell-free hemoglobin administered to the patient, the half-life in the patient of the cell-free hemoglobin, and the partial pressure of oxygen in the arteries as well as the partial pressure of oxygen in the target tissues. For example, a low affinity hemoglobin, as described in US Patent 5,028,588, can be used according to the methods of the present invention. A low affinity hemoglobin can deliver oxygen better than an equal amount of hemoglobin bound in red blood cells, and thus lower dosages (relative to, for example, the amount of hemoglobin that would be contained in a red blood cell transfusion) can be utilized (see Examples 1- 3). In contrast, a higher affinity hemoglobin might be used, and thus would required administration of a higher dosage (amount of hemoglobin /kg body weight) to achieve the same amount of oxygen delivery. A higher affinity hemoglobin can be used to release greater oxygen at tissues experiencing greater hypoxia, such as tumors.

Note that according to the present invention, the viscosity of blood is lowered as a result of the administration of cell-free hemoglobin. In general, decreases in blood viscosity which occur with "acute normovolemic hemosupport", "hemosupport", "acute normovolemic hemoaugmentation", "hemoaugmentation", "perioperative isovolemic substitution", or "acute normovolemic oxygenation", have been shown to increase mean tissue PO2 in various organs (Messmer et al., Res. Exp. Med. 159: 152-56, 1973). In addition, oxygenation of tissues may be enhanced by administration of cell-free hemoglobin because diffusion of the oxygen from the oxygen delivery vehicle (cell-free hemoglobin) involves only the disassociation of the oxygen from the hemoglobin and not diffusion of oxygen through a red blood cell membrane. Without being bound by theory, administration of hemoglobin solutions may result in increased oxygenation of tissues as a result of both increased diffusive delivery of O₂ and reduction of blood viscosity. Note that in the case of hemoaugmentation, oxygen is available not only from red blood cells but also from the hemoglobin dissolved in the plasma itself. Cell-free hemoglobin is hemoglobin that is not substantially bound in cells and does not contain a substantial amount of intact cells or cellular debris. Hemoglobin-containing cells (e.g. erythrocytes) suitable as starting material for the cell-free hemoglobin solution are readily available from a number of sources. Such sources include but not limited to outdated human red blood cells, bovine red blood cells. Non-erythrocyte systems used to express hemoglobin, and thus provide hemoglobin containing cell include, without limitation, bacterial, yeast, plant, and mammalian cells.

For example, slaughter houses produce very large quantities of hemoglobin-containing cells. In addition, if a particular species or breed of animal produces a hemoglobin-containing cell especially suitable for a particular use, those creatures may be specifically bred for this purpose in order to supply the needed blood. Also, transgenic animals may be produced that can express a recombinant mutant, non-mutant or transgenic hemoglobin red blood cells and their progenitors. Human blood banks must discard human blood, including hemoglobin-containing cells, after a certain expiration date. Such discarded blood can also serve as a starting material for the present invention. Purification of hemoglobin from any source can be accomplished using purification techniques which are known in the art. For example, hemoglobin can be isolated and purified from outdated human red blood cells by hemolysis of erythrocytes followed by chromatography (Bonhard, K., et al, U.S. Patent 4,439,357; Tayot, J.L. et al, EP Publication 0 132 178; Hsia, J.C, EP Patent 0 231 236 Bl), filtration (Rabiner, S.F. et al. T. Exp. Med. 126: 1127-1142, 1967; Kothe, N. and Eichentopf, B. U.S. Patent 4,562,715), heating (Estep, T.N., PCT application number PCT/US89/01489, Estep, T.N., U.S. Patent 4,861,867), precipitation (Simmonds, R.S and Owen, W.P., U.S. Patent 4,401,652; Tye, R.W., U.S. Patent 4,473,494) or combinations of these techniques (Rausch, C.W. and Feola, M., EP 0277289 Bl). Recombinant hemoglobins produced in transgenic animals have been purified by chromatofocusing (Townes, T.M. and McCune, PCT publication PCT/US/09624) while those produced in yeast and bacteria have been purified by ion exchange chromatography as well as immobilized metal affinity chromatography (Hoffman, S.J and Nagai, K. in U.S. Patent 5,028,588, Hoffman, et al, WO 90/13645 and Milne et al., WO 95/14038, all herein incorporated by reference).

Hemoglobins derived from natural and recombinant sources have been chemically modified to prevent dissociation and /or improve oxygen carrying characteristics by a variety of techniques. Any of these techniques may be used to prepare hemoglobin suitable for the methods of the present invention. Examples of such modifications are found in Iwashita, Y., et al, U.S. Patent 4,412,989, Iwashita, Y. and Ajisaka, K., U.S. Patent 4,301,144, Iwashita, K., et al, U.S. Patent 4,670,417, Nicolau, Y.-C, U.S. Patent 4,321,259, Nicolau, Y.-C. and Gersonde, K., U.S. Patent 4,473,563, Wong, J.T., U.S. Patent 4,710,488, Wong, J.T.F., U.S. Patent 4,650,786, Bonhard, K., et al, U.S. Patent 4,336,248, Walder, J.A., U.S. Patent 4,598,064, Walder, J.A., U.S. Patent 4,600,531 and Ajisaka, K. and Iwashita, Y., U.S. Patent 4,377,512 among others. Generally, these chemical modifications of hemoglobin involve chemically altering or reacting one or more amino acid residues of the hemoglobin molecule with a reagent that either chemically links the alpha /beta dimers or modifies the steric transformations of the hemoglobin by, for example, binding in the diphosphoglycerate binding site, or links the dimers and modifies the oxygen binding characteristics at the same time. Modifications such as chemical polymerization of globin chains such as described in co-pending application of Anderson et al., WO 93/09143, herein incorporated by reference, glycosylation, and pegylation, and /or encapsulation in a liposome or cell membranes are also contemplated. However, all these hemoglobins must be cell-free hemoglobins, that is they must be substantially free of the starting material cellular components.

Genetically modified hemoglobins that have been modified to stabilize hemoglobin against dimerization or to alter oxygen affinity are also suitable for the methods of this invention. A particularly suitable hemoglobin is recombinantly derived hemoglobin, such as hemoglobin produced in E. coli containing at least a mutation to stabilize against the formation of dimers, preferably hemoglobin produced in E. coli containing at least a mutation to stabilize against the formation of dimers and a mutation to alter oxygen affinity (designated rHbl.l) described in copending patent publication number WO 90/13645 of Hoffman et al. purified by the methods of Milne et al., patent publication number WO 95/14038.

Following administration of cell-free hemoglobin, the patient may or may not undergo a loss of blood. Such loss of blood may occur as a result of many types of trauma but typically occurs as a result of surgery. Depending on the amount of blood lost during this post administration event, some of the patient's blood may be readministered. The methods of readministering blood are well known and the amount of blood readministered, if any, can be determined by the skilled practitioner. At any time after the removal of a portion of a patient's blood, particularly during the loss of blood and /or after administration of the cell-free hemoglobin, it is sometimes recommended that a breathing gas with an enhanced oxygen content be administered to the patient. A higher concentration of oxygen in the breathing gas would increase the partial pressure of oxygen in the lungs and may improve the oxygen binding of some hemoglobins, especially those hemoglobins with a significantly lower oxygen affinity than hemoglobin inside a red blood cell. Therefore, although it is not required, the present invention contemplates the administration of a breathing gas enriched with oxygen to a patient undergoing a loss of blood as contemplated by this invention. The breathing gas can be enriched with any amount of oxygen higher than about the 20% found in air up to essentially 100% oxygen.

Although there may be advantages of administering breathing gas enriched with oxygen to patients during the practice of the present invention, potential detrimental effects can occur from inhaling breathing gas enriched with oxygen, particularly more than 50% oxygen. Some of the detrimental effects of inhaling a breathing gas with an enhanced oxygen content include pulmonary edema and endothelial tissue damage (Harper, Principles and Methods in Toxicology, 3rd Ed., Raven Press, page 883). These effects can be further enhanced when such a hyper- oxygenated breathing gas is used in conjunction with anesthetics. Therefore, it is preferable that breathing gas administered to the patient be less than 50% oxygen and more preferably about 20% (ambient air oxygen content).

A kit for hemoaugmentation is also contemplated by the present invention. Such a kit would include the hemoglobin solution used for hemoaugmentation, and in addition, supplies necessary for this procedure. These supplies can include, for example, reagents, tubing, bags, containers, filters and the like.

The present invention is useful to facilitate autologous donation of blood, and is especially useful in being able to allow donation of more blood than would otherwise be possible or recommended ("hyperdonation"). The invention is also useful for recuperation from oxygen debt more rapidly than by transfusion alone.

For example, the present invention may allow a patient to donate more blood than is usually donated because part of the oxygen carrying capability of the predonated blood is replaced with cell-free hemoglobin. Also, blood may be predonated by the patient closer to the time when blood loss is likely to occur, i.e., less than 72 hours prior to surgery. This method could be particularly useful in the event of emergency, unscheduled surgeries where predonation by typical techniques is not presently possible. Additionally, predonation may be able to occur more frequently or with less time between multiple predonation events. The present invention is also useful in that it does not require that the patient undergoing hemoaugmentation inhale breathing gas that is high in oxygen content, thereby allowing autologous blood donation in patients where inhaling such breathing gas with enhanced oxygen content may be detrimental. The present invention is also useful in preventing and treating the symptoms associated with oxygen debt that often occurs in conjunction with blood loss, particularly blood loss involving large volumes of blood. EXAMPLES

The following examples are provided by way of describing specific embodiments of the present invention without intending to limit the scope of the invention in any way.

Example 1 Demonstration of Oxygen Delivery to Tissues by a 5g/dL Cell-free Hemoglobin Solution

³¹P Nuclear Magnetic Resonance spectroscopy (NMR) is a sensitive, non-invasive probe of oxygen transport to, and the bioenergetic status of, cells tissues and organs (Taylor et al., Proc Soc. Magn. Reson. Med. 4: 292, 1985; Bittl et al, Biochemistry, 26: 6083-6090, 1987; Rosenberg et al., Ann. Emerg. Med. 18: 341-347 (1989); Masson and Quistorff, Biochemistry 31: 7488-7493, 1992; Blum et al., Ann. Surgery 83-88, July 1986; Icenogle et al., Proc. Soc. Magn. Reson. Med. 5: 899-900, 1986; Kushmerick et al., Adv. Exp. Med. Biol. 159: 303-325. 1982: Murray et al., Anesthesiology 67: 649- 653, 1987; Katz et al., Am. T. Physiol. 255: H189-H196, 1988; Marcovitz et al. Am. Heart T. 124: 1205-1212. 1992: Martin et al.. Am. T. Surgery 164: 132-139, 1992; Thompson et al., Quart. T. Med. 85: 897-899, 1992)- With 31p NMR, the amounts of phosphate compounds [phosphocreatine (PCr), orthophosphate (Pi), and nucleotide triphosphates (mainly adenosine triphosphate (ATP)] involved in oxidative energy metabolism in tissues can be non-invasively monitored. For example, Pi is the low-energy degradation product of phosphorus metabolism which accumulates during hypoxia or ischemia, while ATP and particularly PCr decrease during hypoxia or ischemia (Taylor et al, supra; Blum et al., supra; Icenogle et al., supra; Marcovitz et al., supra; Martin et al, supra).

³¹P NMR spectroscopy was applied in real time to monitor the rat gut prior to, during, and after isovolemic exchange transfusions to determine the efficacy of oxygen delivery of a recombinant human hemoglobin (rHbl.l; Hoffman et al., Proc. Nat. Acad. Sci. USA 87: 8521- 8525, 1990) with respect to the entire range of its function as an alternative to whole blood. Controls for these experiments were rats that underwent exchange transfusion with a solution containing human serum albumin (HSA) and no oxygen carrier, and rats having undergone only sham carmulation and no exchange.

Sprague-Dawley rats (weighing 283-552 g) of either sex were cannulated via the femoral artery and vein using silastic tubing (0.012 in. ID, 0.025 in. OD). Blood samples were removed periodically from the arterial catheter for hematocrit determination (40%-57% for controls). Recombinant human hemoglobin was frozen, stored at -70° F and thawed just prior to use as a 5% (w/v) solution in 5 mM phosphate buffered saline. Animals were anesthetized with nembutal (50 mg/kg), weighed, and placed on a heating pad at 38°C into the 31 cm bore of a horizontal 1.9 T magnet. The cannulae were flushed with heparinized saline and then connected to a peristaltic pump set to a speed of ~1 mL/min. Either the rHbl.l or the HSA was pumped into the venous cannula, and blood was removed and its volume measured through the arterial cannula until (-45 min.) the hematocrit became too low to reliably measure (<3%); then the pump was stopped.

Baseline ³¹P NMR spectra from the liver, gut, abdominal- musculature and diaphragm were acquired at 32.5 MHz in 5-10 min blocks for up to one hour after carmulation and prior to isovolemic exchange using a 30 mm diameter surface coil. The animal's blood was then replaced with either rHbl.l or HSA and the ³¹P NMR spectrum of the target organs followed for 4-6 hours. The animals were weighed before and after the isovolemic exchange and were found to have maintained fluid balance within 2% during the exchange process.

The free induction decays following a 60 μs rf pulse (-200° at the surface of the coil) were collected into 2K data points with a 2 kHz sweep (61.54 ppm) and a recycle time of 2 sec. This recycle time attenuated the PCr signal somewhat because it has a spin-lattice relaxation time (Ti) on the order of 2 sec (Bittl et al., supra). The time-domain data from the spectrometer's VAX computer were transferred to a Sim SPARC-2 workstation and converted to NMRi (Syracuse, New York) format, apodized with a 10 Hz filter, Fourier- transformed, phased and baseline corrected. The peak areas, positions and widths were determined for all the resolvable peaks by fitting the signals to Lorentzians using NMRi software. The width of the signals varied from 30 Hz for PCr to -60 Hz for the β-phosphate of ATP. Chemical shifts are reported relative to PCr at δ = -2.35 ppm. The pH was determined from the chemical shift difference (ppm) between PCr and PI according to the equations:

pH = pK + log[(Δ-Δ_min)/(Δ_maχ-Δ)]

where pK = 6.75, Δ_min = 3.27 ppm, and Δ_maχ = 5.69 ppm. The normal pH for whole rat blood (Bittl et al., supra) is 7.38 ± 0.11. This method gave a resting pH of the normal human forearm muscles of 7.03 in accord with established findings (Kushmerick et al, Adv. Exp. Med. Biol. 159: 303- 325, 1982).

Signals from PCr and ATP were observed in the baseline ³¹P NMR spectrum. From the chemical shift of the small Pi signal ( δ = 2.93 ppm) a pH of 7.44 + 0.10 was calculated, a value which is close to that reported for whole blood {vide supra) and is in agreement with that reported (Bittl et al., supra) for tissue in the abdomen (7.35 + 0.11). The standard errors in the chemical shift (and hence the pH) measurements were found to be + 0.10 ppm (n = 21) in baseline spectra.

In order to positively determine that we could monitor changes in the ³¹P NMR spectra associated with a reduction and elimination of oxygen delivery, all the blood of rats (n=3) was exchanged at a rate of 1.2 mL/min with HSA. The exponential exchange process (tι ₂ - 8 min) was terminated and the ³¹P NMR spectrum was monitored for an additional 40 min. The animals entered respiratory arrest at 43 + 11 min (or about 5 half-times) after exchange initiation. The ³¹P NMR spectrum of the animal taken 80 minutes after the start of the exchange was dominated by an increased Pi (>800%) signal along with a 2-fold drop in the PCr signal relative to baseline levels .

Major changes occurred at hematocrits less than -25%, where an exponential increase in Pj was observed as the hematocrit dropped to zero. As the animal's hematocrit was lowered from normal there was a <10% drop in the average high-energy phosphate (as PCr) ³¹P NMR signals and a modest <50% rise in Pi until an apparent critical hematocrit of -25% was reached. At this critical hematocrit, the high-energy phosphate versus hematocrit data show an apparent change of slope. Linear fits for hematocrits above 25% (h>25%) gave a slope of 0.017 + 0.094 and an intercept, 101.3 + 3.1%, while for hematocrits less than 25%, the slope and intercept were 2.12 + 0.14 and 50.6 ± 4.5%, respectively. The fact that the slope determined for h > 25% was not significantly different from zero indicates that rats have considerable reserve capacity to withstand hemodilution; up to a ~ 2-fold reduction in hematocrit. Note that below the approximate critical hematocrit, the slope of the line was significantly different from zero. The critical hematocrit in this rat model, he, was determined from the intersection of the two linear fits described above as h = 26.0 ± 2.0%.

As the hematocrit dropped and oxygenation of the tissues declined, the tissue became acidotic from the accumulation of lactic acid. The average pH prior to the exchange (h = 57%) was 7.58 + 0.12, and this fell to below 6.8 as the hematocrit approached zero. Again, there was little change in pH for a for hematocrits between 57% and -25%, and a marked drop below -25%. Thus the change in pH with hematocrit can be used as an independent estimate of critical hematocrit by fitting two lines to the data and calculating the intersection as described above. Thus, for hematocrits between -25% and 57%, the equation for the line was pH = (2.05 ± 3.16) x lO"³ h + (7.32 ± 0.14), and for hematocrits less than -25%, the equation for the line was pH = (2.06 ± 0.55) x 10"² h + (6.85 ± 0.03). The calculated critical hematocrit was then 25.3 ± 12.4%, a value which was essentially identical to that found above, although the data scatter render this estimate less reliable.

HSA exchange transfusion produced a useful model of fatal tissue hypoxia which we have compared to exchange transfusion with a buffered solution of 5% rHbl.l. The four rats which underwent the exchange transfusion procedure with rHbl.l survived an average of 7.8 times longer than with HSA (337 + 52 min, versus 43 + 11 min, respectively).

The ³¹P NMR spectra of these rats remained stable at control values for >4 hours post-exchange. There was no time-dependent drop in the high-energy phosphates and no concomitant rise in Pi in contrast to the behavior observed with HSA exchange transfusion. Linear fits to the time- dependent rHbl.l exchange data showed only that the slope of PCr data was significantly different from zero ( Pi slope = (-3.9 + 10.8) x 10"3

Integral-Units /min). Note that the PCr shows a small increase with time with rHbl.l exchange, in contrast to the decline seen with HSA.

The average pH of 7.35 ± 0.15 (n = 68) from all of the rHbl.l- exchange rats is consistent with normal pH (Bittl et al., supra). There was no significant change in the pH with time, either during or after the exchange of blood with rHbl.l, up to the animal's death. The slope of the line fitted to the time-dependent pH data and the total pH change were found to be -6.71 ± 17.29 x 10^"5 min"¹, and ΔpH = 0.08 ± 0.07, respectively. Neither the slope nor the total pH change over the entire range of hematocrits from 0 to 57% were significantly different from zero.

A feature of pH regulation and tissue metabolism shown by the HSA data is the relationship between the hydrolysis of high energy phosphates and pH; the hydrolysis of ATP and PCr generates protons and the orthophosphate anion. As tissue becomes hypoxic, there is little oxygen available for electron transport and NADH (and NADPH) production. Lactate accumulates as the reducing power of the cytoplasm decreases. This failure in acid-base balance is manifested in a correlation between pH and orthophosphate generation. Such a relationship was observed when rats were exchange-transfused with HSA, but not when the rats were exchanged with rHbl.l. The rHbl.l data in this case cluster about the average pH, indicating that rHbl.l supports normal tissue/blood pH regulation. This behavior is in marked contrast to the decrease in pH seen as the hematocrit fell below 25% when the animal's blood was replaced with HSA.

The death of the animals may have been due to rHbl.l clearance from the circulation (ti /₂ -107 min) (Vlahakes et al., Euro T. Cardio- Thoracic Surgery 3: 353-354, 1989; Hess et al, T. Appl. Physiol. 70: 1639- 1644, 1991; Hoffman et al., Proc. Natl. Acad. Sci. (USA) 87: 8521-8525, 1990; Looker et al., Nature 356: 258-260, 1992; Shen et al., Proc. Natl. Acad. Sci. (USA) 90: 8108-8112, 1993) and the resulting tissue hypoxia, rather than from a failure of the rHbl.l γer se to supply oxygen to the tissues; thus improvements in ti /_ may lead to enhanced survival times.

These experiments demonstrate the efficacy of rHbl.l in vivo in the extreme situation of total blood replacement, and make it clear that cell- free hemoglobin sustains vital energy-producing functions of tissues at levels which are indistinguishable from those f ound when whole blood is present, even though the hemoglobin (rHbl.l) concentration used (5 g/dL) was only 1/3 that of normal blood (-15 g/dL).

Example 2 Demonstration of Oxygen Delivery to Tissues by Hemoglobin - 3 g/dL solution

Rats were treated as describe in Example 1, except that a 3 g/dL solution of cell-free hemoglobin was used for exchange transfusion rather than a 5 g/dL solution. Eight rats were exchange transfused. For [Hb]_rb > 10, the mean value of the high energy phosphate concentration [HEP] was 100.00% (± 1.6%, n=9 observations), while for [Hb]_rb_c < 1.0, the mean value of [HEP] was 91.0% (±3.8%, n=6 observations), which is significantly smaller than the [HEP] at the start of the exchange (α =0.025). Thus, at a total [Hb] of 3 g/dL in the blood, the phosphorus metabolism was slightly affected but the animal remained alive. Indeed, the [HEP] determined at a total [Hb] of 3 g/dL with rHbl.l 91.0% (±3.8%, n=6) was significantly higher (α =0.05) than that found with erythrocytes during human serum albumin exchange (78.5% ± 4.6%, n=5).

Example 3 Use of Human Recombinant Hemoglobin Solution to Resuscitate Dogs From Hypovolemic Shock

These experiments compared the effectiveness of resuscitation with a cell-free hemoglobin solution (rHbl.l) to standard resuscitation with a crystalloid and shed whole blood. The acute effects were assessed by the use of continuous intra-arterial base deficit measurement in combination with continuous measurement of oxygen consumption (using the delta trac metabolic monitor), which was used as a precise guide to the use of fluid volume.

A canine model of oxygen debt based on hypovolemic shock was utilized. The anesthetized animal was intubated and instrumented for measure of arterial blood pressure, central venous pressure, pulmonary artery pressures and cardiac output (by the thermal-dilution and /or Cardiogreen dye method). The endotracheal intubation tube was connected to a Delta Trac θ2 consumption ventilator and the femoral artery was also instrumented for frequent intermittent or continuous measurement of Paθ2, PaCθ2, pH and base excess. Following a period of stabilization to assess the baseline level of oxygen consumption under these conditions, the animal preparation was bled to a level of oxygen debt which approximates the mean LD5₀ oxygen debt (114 ml/kg) as previously determined by extensive studies in this preparation (Dunham et al., Crit. Care Med. 19(2): 231-243, 1991). This level of oxygen debt was achieved within 60 minutes, thus there was a standard rate of O2 debt formation as well as a standardized final oxygen debt. Following baseline measurements taken after achievement of the target oxygen debt, resuscitation by one of two methods was initiated. The rate of return and overshoot of the oxygen consumption, the rate of reduction of the base deficit, and the fall in lactic acid were monitored during and after the resuscitation procedures.

The two methods of resuscitation that were compared were as follows:

1. Control resuscitation. Resuscitation was performed with a crystalloid- colloid solution equal to 60% of the shed blood volume in the first 20 minutes after resuscitation was initiated, followed by return of 60% of the total volume of shed blood (re-infusion of autologous blood). This procedure mimics standard clinical resuscitation which generally involves an initial crystalloid-colloid resuscitation followed by the transfusion of whole blood as soon as it is obtained from the blood bank.

2. Hemoglobin solution (rHbl.l) resuscitation. Resuscitation with an initial volume of rHbl.l equal to 60% of the hemoglobin removed by hemorrhage (approximately 75 to 108 g of rPIbl.l depending on dog weight) plus a volume of crystalloid-colloid so that the total volume of resuscitation fluid was equal to that used in (1) above. After an initial study with the full percentage of rHbl.l, the use of smaller quantities of rHbl.l with the same total replacement volume can be evaluated to determine whether lower levels of circulating hemoglobin can be as effective as higher hemoglobin levels for the maintenance of critical levels of oxygen delivery.

In each of these groups, the rate of return of oxygen consumption to baseline levels and the magnitude of the overshoot and its duration were precisely quantified. In addition, the rate of reduction of the base deficit was followed in order to see how closely it corresponded to the rate of reduction of volume (initially crystalloid-colloid solutions and then shed blood). Those animals surviving the LD5₀ oxygen debt level determined from the control resuscitation group were followed for 7 days post-shock to monitor for evidence of residual organ toxicity using biochemical determinations of liver function and renal function. At the end of the 7 days, the surviving animals were anesthetized and tissues removed for histologic study of liver, lung, and kidney. The animals were then sacrificed while under anesthesia. Both light histology and electron- microscopic studies of representative tissues were carried out in order to determine the presence or persistence of ultra-structural injuries which might reflect ischemic and re-perfusion injury. In this way, the rate of recovery of oxygen consumption and the reduction in oxygen debt and base deficit produced by hemoglobin solution resuscitation versus the control resuscitation were related not only to the percentage of survival, but also to the presence or absence of post-recovery organ failure sequelae. These experiments demonstrated that resuscitation with rHbl.l, in a dog model of hypovolemic shock, produces a more rapid and essentially complete repayment of the oxygen debt created by blood loss. Traditional resuscitation with crystalloid and autologous blood repaid only 73% of oxygen debt at 140 minutes post-initiation of hypovolemia. In contrast, resuscitation with rHbl.l repaid 95% of oxygen debt at 140 minutes (Figure 1) and nearly 100 percent of oxygen debt at 182 minutes. Thus, rHbl.l results in a more rapid and more complete repayment of oxygen debt relative to blood transfusion. As has been shown, this rapid restoration of normal oxygen consumption is crucial to survival (Siegel, Clin. Chem. 36: 1585, 1990).

Example 4 Preclinical Toxicology Studies of Hemoglobin

The safety of rHbl.l was assessed in five animal studies and two in vitro studies using human cells. These include a pilot toxicology study in dogs, a pivotal single-dose toxicology study in stressed dogs, a single-dose hemodynamic study in severely stressed anesthetized dogs, a cardiovascular study in anesthetized dogs, a gastrointestinal study in rats and in vitro studies of rHbl.l effects on human complement activation and human neutrophil function.

No serious treatment-related effects were observed following the administration of rHbl.l at doses up to 30% blood volume resuscitation (1.1 g/kg) in the pivotal single-dose toxicology study in stressed dogs. Results showed only limited, reversible effects including transient neutropenia and elevation of AST and CK. No major organ systems were affected, no histopathologic changes were observed and no hemoglobin was detected in the urine.

Hemodynamic and cardiovascular parameters were measured in the single-dose hemodynamic study in severely stressed anesthetized dogs before and after volume resuscitation. Hypovolemic shock was induced by removal of 50% of the animals' blood and was maintained for 30 minutes before resuscitation with rHbl.l, 5% human serum albumin (HSA) or autologous blood. Heart rate, cardiac output, systemic and pulmonary blood pressures and vascular resistance were determined. While significant changes in these parameters were observed in response to the induced hypovolemic stress and resuscitation, no apparent differences were observed in either the magnitude or the time course of cardiovascular and hemodynamic parameters between animals resuscitated with rHbl.l and those resuscitated with autologous blood. This study demonstrated that rHbl.l did not cause adverse cardiovascular or hemodynamic effects when used to resuscitate severely hypovolemic dogs.

A study to evaluate cardiovascular effects was completed following the administration of rHbl.l in anesthetized dogs. The dogs in this study were anesthetized with halothane, instrumented for cardiopulmonary monitoring and subjected to a 50% isovolemic hemodilution with rHbl.l. While rHbl.l produced mild systemic hypertension, pulmonary and coronary hypertension were not observed in this study, nor did heart rate change significantly after rHbl.l hemodilution. rHbl.l had no effects on myocardial function or coronary hemodynamics.

Following the observation of gastrointestinal adverse events in clinical studies, a study was conducted in rats to specifically observe any potential pancreatic or hepatobiliary effects of rHbl.l. In addition to its wide use in the study of heme catabolism, this animal was selected as it is a common model to study pancreatitis by various agents. In the rat, pancreatitis can be determined by the measurement of serum amylase and lipase as well as pancreatic wet/ dry weight ratio which is a sensitive measure of pancreatic edema. The study was designed to examine the potential of rHbl.l to cause pancreatic effects after single doses of rHbl.l ranging from 0.35 g/kg to 2.85 g/kg (approximately 10% and 80% of the blood volume, respectively) bolus intravenous top load. Serial serum amylase and lipase determinations were normal from 2 to 48 hours post administration of rHbl.l. There were no changes in the pancreatic wet/dry ratios at hours 24 and 48 post rHbl.l administration. Reversible mild elevation in ALT and AST levels were observed in 3 of 12 rats dosed with a 2.85 g/kg rHbl.l top load. Gross pathological examination of the abdominal organs revealed no significant abnormalities. Examination of the abdominal histopathology revealed no effects in the pancreatic or hepatobiliary tissues. Mild to moderate effects were seen in the kidneys of rHbl.l -treated rats, which is likely attributable to administration as a large volume top-load. Significant pancreatic or hepatobiliary injury was not induced in this rat model following a high volume top load of rHbl.l at a dose approximately 20 fold higher than the largest clinical dose given to date.

The potential influence of rHbl.l on the human immune system was determined in both an in vitro complement activation assay and in vitro neutrophil assay. In the first study, complement activation was assayed by measuring total hemolytic complement (CH50) and complement split products from both the classic and alternate complement pathways. rHbl.l did not affect either pathway of the complement system in this study. In the second study, the potential of rHbl.l to activate, inhibit or directly damage human neutrophils was investigated. rHbl.l had no effects on any parameter measured, granulocyte chemotaxis, adhesion or viability.

Example 5 Preclinical Pharmacology Studies of rHbl.l

A clinically effective oxygen carrier and volume expanding agent should remain localized in the plasma to exert oncotic pressure and to transport oxygen from the lungs to the tissues and should not be rapidly eliminated after injection. Pharmacokinetic studies were performed in dogs after single doses of rHbl.l to determine whether rHbl.l has a clinically useful half-life and remained confined to the intravascular space after administration. In addition, data concerning potential hemodynamic and cardiovascular effects of rHbl.l resuscitation were obtained during preclinical toxicology studies of rHb 1.1.

The pharmacokinetic study in dogs demonstrated that the plasma half-life of rHbl.l after the infusion of 1.8 g/kg was 11.6 ± 2.5 hours. Peak concentrations of rHbl.l in dogs were 30 ± 2.7 mg/mL, observed at the end of a 30 minute rHbl.l infusion. The volume of distribution of rHbl.l in dogs (60.6 ± 2.0 mL/kg) was almost identical to plasma volume, indicating that rHbl.l remained almost entirely confined to the intravascular space after administration.

Example 6 Clinical Studies of rHbl.l in Awake Volunteers

A total of 76 male subjects have been dosed with rHbl.l, where the highest dose level administered was 0.32 g/kg (25.5 g total dose) infused at 3.75 mL/kg/hr. The majority of the subjects have received greater than or equal to 0.15 g/kg of rHbl.l. Dose escalation studies were designed to assess the safety and pharmacokinetics of a single intravenous infusion of rHbl.l in normal human male volunteers. Another study was implemented to evaluate mild to moderate gastrointestinal (GI) adverse events seen at the higher doses and to determine if a specific therapeutic intervention could attenuate or resolve the symptoms. Another study was designed to further evaluate and quantitate the GI events by conducting esophageal manometry following the infusion of rHbl.l.

No serious adverse events have been observed during or following the intravenous administration of rHbl.l in any of these studies. In addition, many of the adverse events and toxicities historically associated with the infusion of cell-free hemoglobin solutions have not been seen. Specifically, there was no evidence of renal dysfunction or appreciable renal clearance of rHbl.l, and no evidence of changes in hepatic or pulmonary toxicity or effector system activation. However, there were mild increases in blood pressure in some subjects during the rHbl.l infusion. In addition, mild to moderate adverse events occurred in these studies after completion of the rHbl.l infusion. The predominant adverse event in the first study was a pyrogenic response. In the later studies at higher doses, GI adverse events were observed.

The pyrogenic response observed was manifested by mild to moderate symptoms of fever, chills, myalgias, headache, and transient mild neutrophilia. These symptoms resolved spontaneously or after administration of ibuprofen. A modification was made in the product manufacturing process which resulted in an improvement in product purity. Subjects in subsequent studies who received higher doses of rHbl.l produced after this manufacturing change, occasionally developed mild fever and neutrophilia, but rarely the other syπiptoms.

In studies that were conducted at higher doses, the majority of the subjects in the 0.15 and 0.18 g/kg dose groups developed mild to moderate GI adverse events. These events typically developed 1-3 hours post infusion and consisted of dysphagia, mid-epigastric discomfort and occasionally nausea, vomiting, abdominal cramps and /or diarrhea. These events, while not serious, limited the dose escalation in these normal volunteers. These findings were consistent with the diagnosis of generalized GI dysmotility, particularly lower esophageal sphincter spasm as manifested by the nature of the epigastric discomfort and dysphagia. This diagnosis was corroborated in 1 subject by a gastrografin esophagram and observations from human and animal GI manometry studies. Retrospective review of a previous study indicated that 2 of 16 subjects reported similar although mild adverse events.

The GI phenomena may be related to smooth muscle contraction and GI dysmotility. Therefore, several interventions were assessed to prevent, attenuate or resolve the subjects' symptoms. Of those evaluated to date, glucagon, nifedipine and naloxone were somewhat useful, though none were consistently therapeutic. When administered prophylactically, terbutaline sulfate was the most useful and allowed dose escalation. These studies demonstrated post-infusion laboratory abnormalities. Occasional increases in bilirubin and transaminases, though not clinically significant, have occurred. Several subjects showed mild transient increases in serum amylase and lipase values, which resolved spontaneously within 12-24 hours. Abdominal examinations were benign. These findings have not correlated with the reported abdominal complaints, though abdominal complaints have been seen in some of these subjects. The laboratory abnormalities did not appear to correlate with any demographic data, time of observation or with serum hemoglobin levels.

Numerous other adverse events were observed less frequently in these studies. For example, mild transient urticaria was seen after the infusion of rHbl.l in approximately 9% of the subjects. It resolved spontaneously or with antihistamine treatment and none of the subjects who developed urticaria developed any associated cardiovascular findings, bronchospasm or evidence of anaphylaxis.

One subject who received 0.15 g/kg of rHbl.l developed mild bronchospasm approximately 15 minutes after the start of infusion. The infusion was terminated and wheezing resolved immediately after he received epinephrine and diphenhydramine (Benadryl). He had no associated urticaria or cardiovascular compromise and symptoms resolved without clinical sequelae. No further such events have been observed.

Transient asymptomatic cardiac conduction defects were observed after the infusion of rHbl.l in 3 of 76 subjects. All occurred in subjects receiving 0.15 g/kg of rHbl.l. One subject developed multiple episodes of type I second degree AV heart block and one brief episode of type II second degree AV block after the rHbl.l infusion. He remained asymptomatic without cardiovascular compromise. The conduction defect resolved spontaneously without treatment. A full evaluation was negative, though some mild rhythm changes, normal for his age group, were observed by 24 hr ECG monitoring. Two other subjects developed transient SA node slowing with isorhythmic AV dissociation and a junctional escape rhythm. One subject also demonstrated this AV dissociation before receiving rHbl.l. These subjects were asymptomatic and the conduction defect resolved spontaneously. The etiology of these effects remains unclear.

Example 7 Clinical Study of rHbl.l used Intra-Operatively in Anesthetized Patients

The initial safety evaluation of rHbl.l in has been studied in elective surgery patients under general anesthesia. Patients enrolled in the study received rHbl.l or normal saline as a volume control at doses ranging from 0.075 g/kg (5.25 g for a 70 kg individual) to 0.35 g/kg (24.5 g for a 70 kg individual) infused at 3.75 mL/kg/hr. In the 14 patients dosed to date, no serious adverse events have been observed. Most of the patients, including the controls, have developed mild to moderate post¬ operative nausea and vomiting. In addition, four patients in the rHbl.l group have developed mild hypertension requiring treatment. No gastrointestinal adverse events consistent with symptoms observed in the unanesthetized volunteer studies have yet been observed. Example 8 Clinical Study of rHbl.l used as an Exchange Solution in ANH

In addition to the above study, rHbl.l has been evaluated as an exchange solution during acute normovolemic hemodilution. During this procedure up to 2 units are exchanged with physiologic saline solution/rHbl.l or a physiologic saline solution alone. Three dose groups of 12.5 g, 25 g, and 50 g with 3 rHbl.l patients per group have been or will be evaluated as shown in Table 1. In addition, there were 3 physiologic saline solution control patients. The patients given rHbl.l to date have not shown any clinically serious adverse effects.

TABLE 1 EXCHANGE FLUID VOLUMES

Total

A 3 1000 1000 PSS 250 rHbl.l 250 12.5 500 PSS^b

B 3 1000 PSS 1000 PSS 500 rHbl.l 500 25 1000

C 3 1000 PSS 500 PSS 500 rHbl.l 1000 50 2000

500 rHbl.l

Control³ 3 1000 PSS 1000 PSS 500 PSS 0 0 2000 ^aOne PSS control will be randomized with each rHbl.l dose group. ''Physiologic saline solution

Example 9 Use of Recombinant Human Hemoglobin (rHbl.l) For Acute

Normovolemic Hemodilution In Patients Undergoing Cardiopulmonary

Bypass Surgery

The objectives of the study are to determine the safety of rHbl.l administered as a hemodilution solution prior to and during cardiopulmonary bypass surgery (CPB) and to evaluate the effect of rHbl.l administration as part of the hemodiluent requirement on changes in hemodynamic parameters, fluid requirements and transfusion requirements during and after surgery. Patients are randomized to receive one of two doses 25 g or 50 g (500 mL or 1000 mL, respectively) of intravenous rPIbl.l or 1000 mL normal saline (as a volume control). During this study at least 2 units of blood are harvested, but the hemodilution can be used to harvest as many as 4 units of blood. After induction of anesthesia and the pre-infusion procedures are completed, 2 units of blood are removed. Normovolemia is maintained with 2000 mL normal saline exchange. In the 25 g dose group, a third unit of blood is removed and replaced with rHbl.l (500 mL) or normal saline (1000 mL). In the 50 g dose group, a third unit is removed and replaced with 25 g (500 mL) rHbl.l or normal saline (1000 mL). If possible, a fourth unit is removed and replaced with another 25 g rHbl.l (500 mL) or normal saline (1000 mL).

The patients that are given rHbl.l when compared to those patients who do not receive rHbl.l, have less need for allogeneic blood transfusion and /or autologous blood transfusion. The foregoing description of the invention is exemplary for purposes of illustration and explanation. It will be apparent to those skilled in the art that changes and modifications will be possible without departing from the spirit and the scope of the invention. It is intended that the following claims be interpreted to embrace all such changes and modifications.

Claims

WHAT IS CLAIMED IS:

1. A method for facilitating autologous blood donation by a patient, comprising:

(a) removing a portion of the patient's blood to result in a removed blood portion;

(b) administering an oxygen delivery enhancing amount of cell-free hemoglobin; and

(c) optionally administering a breathing gas, wherein said breathing gas has a concentration of less than 50% oxygen.

2. The method of claim 1 further comprising the step of storing said removed blood portion.

3. The method of claim 2 further comprising the step of readministering said stored blood to said patient.

4. The method of claim 1 wherein said removing of patient's blood is less than 72 hours prior to said patient undergoing said loss of blood.

5. The method of claim 1 wherein the cell-free hemoglobin is non- erythrocyte derived hemoglobin.

6. The method of claim 1 wherein the cell-free hemoglobin is recombinant hemoglobin.

7. The method of claim 6 wherein the recombinant hemoglobin is rHbl.l.

8. The method of claim 1 wherein the autologous blood donation is predonation.

9. The method of claim 1 wherein the autologous blood donation is perioperative.

10. The method of claim 1 wherein said breathing gas is not enriched with oxygen.

11. A method for treating oxygen debt comprising administering a therapeutically effective amount of a cell-free hemoglobin to treat oxygen debt.

12. The method of claim 11 wherein said cell-free hemoglobin non- erythrocyte derived hemoglobin.

13. The method of claim 11 wherein the cell-free hemoglobin is recombinant hemoglobin.

14. The method of claim 13 wherein the recombinant hemoglobin is rHbl.l.

15. A kit comprising cell-free hemoglobin and associated supplies for use with autologous blood donation or treating oxygen debt.