Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
  • A61M1/02—Blood transfusion apparatus

Definitions

• 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).
• 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
• immunologic reaction i.e., transfusion reactions, immunosuppresion, graft versus host reaction.
• Other drawbacks of using allogeneic blood transfusions include no universal
• Autologous blood the patient's own blood
• 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 The withdrawn blood can then be used during surgery (perioperatively) or during the recuperation time after the surgery has been completed (postoperatively).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 is another technique that is used to reduce exposure to autologous blood.
• blood is withdrawn from the patient at the time of or just prior to surgery.
• the volume of blood that is withdrawn from the patient is then replaced with an equal volume of a non-oxygen carrying crystalloid or colloid solution.
• the blood that was withdrawn from the patient is then re-infused at the end of surgery or during the recuperation period if needed.
• 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.
• 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.
• replacement of withdrawn blood with an oxygen-carrying fluid could increase that amount of blood that could be withdrawn during predonation.
• hemoglobin 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).
• hemoglobin particularly recombinant hemoglobin, could be useful as a blood volume expander and /or oxygen carrier during predonation or ANH under ordinary anesthetic practice.
• rHbl.l 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.
• 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.
• the present invention relates to a method for facilitating autologous blood donation by a patient, comprising:
• 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.
• removing and storing a portion of patient's blood occurs less than 72 hours prior to the patient undergoing the loss of blood.
• the cell-free hemoglobin is non-erythrocyte derived, and is especially recombinant hemoglobin, particularly rHbl.l.
• the autologous blood donation is predonation.
• the autologous blood donation is perioperative.
• the present invention relates to a method for treating oxygen debt comprising administering therapeutically effective amount of cell-free hemoglobin to treat oxygen debt.
• 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.
• FIG. 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—).
• 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.
• hemodilution or "acute normovolemic hemodilution”
• 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.
• predonation may be improved and have expanded use in one or more ways.
• 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.
• 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.
• predonation may be able to occur more frequently or with less time between multiple predonation events.
• Perioperative isovolemic dilution 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).
• 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.”
• 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.
• 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.
• 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).
• 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 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.
• 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.
• 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.
• 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.
• 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") .
• simultaneous with or subsequent to the removal of blood for possible use in autologous donation there is administered to the patient cell-free hemoglobin.
• 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 (P5 0 ) 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.
• 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.
• 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.
• cell-free hemoglobin can be administered to reduce the hematocrit level as described above and decrease blood viscosity while preserving oxygen delivery.
• 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.
• 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.
• 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).
• carbohydrates for example, reducing carbohydrates such as glucose, maltose, lactose or non- reducing carbohydrates such as sucrose, trehalose, raffinose, mannitol, isosucrose or stachyose
• alcohols or poly alcohols such as polyethylene glycols, propylene glycols, dextrans, or polyols.
• compositions of the invention can also contain 0.005 - 1% of one or more surfactants and 0-200 ⁇ M of one or more chelating agents (for example, ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis ( ⁇ - aminoethyl ether) _V,N,N',N'-tetraacetic acid (EGTA), o-phenanthroline, diethylamine triamine pentaacetic acid (DTPA also known as pentaacetic acid) and the like).
• EDTA ethylenediamine tetraacetic acid
• EGTA ethylene glycol-bis ( ⁇ - aminoethyl ether) _V,N,N',N'-tetraacetic acid (EGTA), o-phenanthroline, diethylamine triamine pentaacetic acid (DTPA also known as pentaacetic acid) and the like.
• EDTA ethylenediamine tetraacetic acid
• 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 ⁇ M of one or more chelating agents.
• the composition contains 0 - 150 mM NaCl, 0 - 10 mM sodium phosphate, and 0.01 - 0.1% surfactant, and 0-50 ⁇ M of one or more chelating agents, pH 6.6 - 7.8.
• 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 ⁇ M EDTA, pH 6.8 - 7.6.
• 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.
• anti- oxidants e.g. ascorbate or salts thereof, alpha tocopherol
• anti-bacterial agents e.g. albumin or polyethylene glycols
• oncotic pressure agents e.g. albumin or polyethylene glycols
• iron chelating agents such as, for example, desferroxamine, and other
• 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.
• compositions of the instant invention can be used for the treatment of oxygen debt.
• 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.
• albumin 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.
• 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).
• 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.
• the viscosity of blood is lowered as a result of the administration of cell-free hemoglobin.
• 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).
• 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.
• administration of hemoglobin solutions may result in increased oxygenation of tissues as a result of both increased diffusive delivery of O 2 and reduction of blood viscosity.
• 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.
• slaughter houses produce very large quantities of hemoglobin-containing cells.
• those creatures may be specifically bred for this purpose in order to supply the needed blood.
• 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.
• 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.
• 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.
• 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.
• all these hemoglobins must be cell-free hemoglobins, that is they must be substantially free of the starting material cellular components.
• 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.
• the patient may or may not undergo a loss of blood.
• loss of blood may occur as a result of many types of trauma but typically occurs as a result of surgery.
• 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.
• 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.
• breathing gas enriched with oxygen can be administered to patients during the practice of the present invention.
• 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).
• kits for hemoaugmentation are also contemplated by the present invention.
• a kit would include the hemoglobin solution used for hemoaugmentation, and in addition, supplies necessary for this procedure.
• 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.
• 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.
• 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.
• NMR Nuclear Magnetic Resonance spectroscopy
• 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.
• PCr phosphocreatine
• Pi orthophosphate
• ATP nucleotide triphosphates
• ATP adenosine triphosphate
• 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).
• 31 P 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.
• HSA human serum albumin
• 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 31 P 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 31 P 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.
• 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.
• pH pK + log[( ⁇ - ⁇ m in)/( ⁇ ma ⁇ - ⁇ )]
• 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.
• 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.
• 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 ⁇ er se to supply oxygen to the tissues; thus improvements in ti /_ may lead to enhanced survival times.
• 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.
• the phosphorus metabolism was slightly affected but the animal remained alive.
• 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 ⁇ 2 consumption ventilator and the femoral artery was also instrumented for frequent intermittent or continuous measurement of Pa ⁇ 2, PaC ⁇ 2, pH and base excess.
• 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.
• 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.
• 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.
• rHbl.l 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.
• 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.
• HSA human serum albumin
• 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.
• 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.
• 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.
• pancreatic or hepatobiliary tissues 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.
• rHbl.l 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.
• complement activation was assayed by measuring total hemolytic complement (CH50) and complement split products from both the classic and alternate complement pathways.
• CH50 total hemolytic complement
• rHbl.l did not affect either pathway of the complement system in this 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.
• 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.
• data concerning potential hemodynamic and cardiovascular effects of rHbl.l resuscitation were obtained during preclinical toxicology studies of rHb 1.1.
• rHbl.l 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.
• GI gastrointestinal
• 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 sy ⁇ iptoms.
• 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.
• 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.
• 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.
• Control 3 1000 PSS 1000 PSS 500 PSS 0 0 2000 a
• One PSS control will be randomized with each rHbl.l dose group.
• 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).
• at least 2 units of blood are harvested, but the hemodilution can be used to harvest as many as 4 units of blood.
• Normovolemia is maintained with 2000 mL normal saline exchange.
• a third unit of blood is removed and replaced with rHbl.l (500 mL) or normal saline (1000 mL).
• a third unit is removed and replaced with 25 g (500 mL) rHbl.l or normal saline (1000 mL).
• a fourth unit is removed and replaced with another 25 g rHbl.l (500 mL) or normal saline (1000 mL).

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