USE OF OXYGENT, A PERFLUOROCHEMICAL-BASED OXYGEN CARRIER,
AS AN ALTERNATIVE TO INTRAOPERATIVE BLOOD TRANSFUSION.
Peter E. Keipert, Ph.D.
Alliance Pharmaceutical Corp.,
3040 Science Park Road, San Diego, CA, 92121
ABSTRACT
OxygentT is a stable concentrated perfluorochemical (PFC) emulsion being developed for use as a temporary oxygen carrier. In this application, PFC emulsions can be used to augment oxygen delivery during acute blood loss and thereby provide a margin of safety during hemodilution and surgical anemia. PFCs simply dissolve oxygen in direct proportion to its partial pressure. The oxygen transported by a PFC emulsion is present in the plasma compartment and is therefore easily extracted and consumed by the tissues. Preclinical and clinical studies have demonstrated that a relatively low dose (1.35 g PFC/kg) of Oxygent can support oxygen delivery despite ongoing blood loss. Clinical safety studies in 57 healthy, conscious volunteers and in 30 anesthetized surgical patients have been completed. In these studies, there were no hemodynamic changes or vasoconstriction and cardiac output increased normally in response to hemodilution. Two transient side effects were observed, but only in the high dose (1.8 g PFC/kg) group: a 1-1.5 deg C increase in body temperature (at 4-6 hours), and a moderate decrease in platelet count (mean nadir ~130,000/æL by 2-3 days) without any bleeding complications. Oxygent is presently being evaluated as an alternative to allogeneic blood transfusion in patients undergoing medium- to high-blood-loss surgical procedures.
INTRODUCTION
Perfluorochemical (PFC) technology has been under development due to the intrinsic ability of these synthetic liquids to dissolve significant quantities of gases such as oxygen and carbon dioxide. This was proven dramatically almost 30 years ago by Dr. Leland Clark's famous "liquid-breathing mouse" experiment. (See Reference 1) PFCs consist primarily of carbon and fluorine atoms and are extremely inert chemically and biochemically. Because PFCs are not miscible with water, they must be emulsified with a surfactant to create aqueous-based PFC emulsions for intravenous use. The ability of PFC emulsions to replace red cells and serve as an oxygen carrier was first demonstrated in "bloodless rats" by Dr. Geyer in 1968. (See Reference 2)
Unfortunately, the PFCs used in these early emulsions were not readily excreted from the body, resulting in prolonged tissue retention.
The first successful development of an injectable PFC-based commercial product was achieved by the Green Cross Corporation in Japan, when they made Fluosol-DA, a dilute (20% w/v) emulsion based on F-decalin and F-tripropylamine emulsified with Pluronic F-68 and egg yolk lecithin. (See Reference 3) Limitations of this first generation product included the need for frozen storage of the stem emulsion and subsequent mixing with two annex solutions prior to use; short product stability (8 hours) after reconstitution; and several side effects (e.g., leukocyte inhibition, complement activation) which were primarily caused by the Pluronic surfactant. (See Reference 4) During the last 10 years research efforts have developed emulsions with improved characteristics compared to Fluosol. One such effort has focused on a versatile PFC, perfluorooctyl bromide (C8F17Br), which has been given the generic name, perflubron. The use of perflubron has enabled development of Oxygent (Alliance Pharmaceutical Corp., San Diego), a concentrated stable emulsion using egg yolk lecithin as the surfactant.(See Reference 5) A potential clinical indication for this oxygen-carrying drug is to combine its use with autologous blood collection techniques during medium- to high-blood-loss surgical procedures, as a means to reduce or eliminate the need for allogeneic (donor) blood.
Blood Transfusion Practice
Risks of Allogeneic Blood
It is estimated that 11 million units of blood are transfused each year in the United States(See Reference 6) with the greatest amount (i.e., approximately two-thirds of the transfused units) being utilized during surgical procedures as a means to prevent the anemic hypoxia caused by acute blood loss. The remaining units are used mainly for treating disease-related chronic anemia or emergency trauma conditions. The shelf life for donated blood is only six weeks from the time it is collected, which adds significantly to the logistical problems of maintaining a safe and adequate blood supply.
The public perception of blood supply safety changed dramatically in the mid-1980s, due to the recognition that transmission of HIV could occur via allogeneic blood transfusions. Since then, improvements in both blood screening and testing procedures have significantly decreased the risk of viral disease transmission. However, it is clear from recent articles in MONEY magazine (May 1994) (See Reference 7) and U.S. News (June 1994) (See Reference 8) that the public's perception of this risk has not been reduced to the same degree. Patients are well informed and aware of the complications and risks associated with blood transfusion (see Table 1) and clinicians have
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Table 1. Transfusion Risks per Unit of Blood in the USA*
CytomegalovirusCommon Fever, chills, urticaria1:25 to 1:100 Hepatitis C1:3,000 to 1:5,000 Hemolytic transfusion1 : 6,000 HTLV I/II1 : 50,000 Fatal hemolytic transfusion1 : 100,000 Hepatitis B1 : 200,000 HIV (AIDS)1 : 41,000 to 1 : 225,000 Immune suppressionDifficult to quantify
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* Based on Welsh et al. (1992), (See Reference 9) Estafanous et al. (1994), (See Reference 10) and Klein (1994) (See Reference 11)
a strong desire to avoid patient exposure to allogeneic blood whenever possible. A recently published Clinical Guideline concluded that, "...a physician contemplating giving a transfusion to an anemic patient on an elective basis should discuss all risks and benefits with the patient and regard elective transfusion with allogeneic blood as an outcome to be avoided." (See Reference 12)
Autologous Blood Strategies
There are three autologous blood transfusion strategies available currently that can be employed, independently or in combination, to reduce exposure to donor blood. These include preoperative autologous donation (PAD), acute normovolemic hemodilution (ANH), and intra- or post-operative autologous blood salvage (ABS) using red cell recovery devices. Each technique, however, has some limitations and when used exclusively often does not provide sufficient autologous blood. (See Reference 13)
Preoperative autologous donation (PAD) is a technique whereby patients, in the 6 weeks prior to their elective surgery, may predeposit several units of their autologous (own) blood to be stored for later use during their surgery. PAD has become widely used in certain elective surgical procedures (e.g., orthopedic and urologic) due to its proven ability to increase total red blood cell mass and thereby reduce the need for intraoperative allogeneic blood transfusions. PAD is not always feasible, however, because the patient may not have enough lead time to donate sufficient blood, may not be healthy enough, or may not have any access to an established PAD program. Moreover, as with allogeneic blood, PAD carries with it the risk that the unit may be mislabeled and/or transfused to the wrong recipient since it must be stored by the blood bank until it is needed for surgery. Despite the obvious advantages and potential of PAD, this alternative is not without increased costs, (See Reference 14) and overall, total enrollment in autologous predonation at regional blood centers throughout the U.S. has reached only ~6% of all blood collected.(See Reference 15)
A related technique, acute normovolemic hemodilution (ANH), entails the collection of a portion of the patient's blood (e.g., 2-4 units) in the operating room just prior to surgery. As it is withdrawn, the blood is replaced with a crystalloid and/or colloid plasma expander to maintain constant circulating blood volume. The result of ANH is a diminished net loss of red cells in surgery, since the shed blood is more dilute. The patient's whole blood is re-infused during the surgery, if required, or at the end of surgery to raise the hematocrit to a safe level. In addition to greater patient convenience, ANH offers the potential for lower cost, as well as elimination of clerical errors and blood processing problems that can occur with stored blood, since the blood collected by ANH stays in the operating room with the patient. Another important advantage of the fresh blood collected during ANH is the preservation of fully functional platelets, active clotting factors and fresh red cells free of physiologic changes (i.e., normal P50 and pH), all of which are important for optimal oxygen delivery and hemostasis after surgery. Intraoperative ANH is presently limited in its application due, in part, to organizational logistics and the amount of blood that can be collected safely (since the plasma expanders administered do not replace the oxygen-carrying capacity of the withdrawn red cells).
Autologous blood salvage (ABS) techniques involve suctioning some of the blood lost during active bleeding in surgery and subsequently reinfusing the recovered red cells into the patient. Blood salvage devices are currently used in a significant percentage (approximately 25%) of all medium- to high-blood-loss surgeries in the United States, and their use is continuing to increase. The major risks associated with ABS include reinfusion of hemolyzed blood, coagulopathy, bacterial contamination, and infusion of air/particle emboli.16 The cell processing devices used for performing ABS are expensive, therefore making this technique cost-effective only when recovering the equivalent of at least 3 units of blood. (See Reference 17)
Limitations of Autologous Techniques
Avoidance of allogeneic blood depends primarily on the individual patient's ability to predonate sufficient autologous blood. To a large extent, this is related to the patient's basal hematocrit level and general health. In a recent study it was shown that even when the total number of PAD units requested by the surgeon was collected, up to 15% of the patients required allogeneic blood. (See Reference 18) Recent studies indicate that blood conservation strategies must generate the equivalent of four or more units to avoid allogeneic blood exposure in many orthopedic and coronary bypass graft procedures. (See Reference 19) Based on the published data from autologous blood collection programs, it is clear that avoidance of allogeneic blood in surgical patients is highly dependent upon the use of multiple autologous techniques.
Figure 1. Schematic diagram of a combined approach to reduce or avoid allogeneic blood transfusions in elective surgery, using Oxygent as an adjunct to ANH. Oxygent is administered instead of blood when transfusion is being considered. Reinfusion of the fresh autologous blood can therefore occur toward the end of surgery once hemostasis has been achieved, allowing the patient to leave surgery without being exposed to allogeneic blood.
COMBINING OXYGENT WITH HEMODILUTION
While the misnomer "blood substitute" continues to be popular when discussing both PFC-based and Hb-based oxygen-carrying drugs, neither of these products are intended to replace whole blood, but rather to temporarily perform the gas-transport properties of red cells. Both Hb solutions and PFC emulsions have a limited blood half-life (hours) which is substantially less than that of newly formed red cells (~120 days). In addition, both product types have demonstrated dose-dependent side effects in animal studies as well as in early-phase human clinical trials which will limit the maximum clinical dose that can be administered safely. Thus, these oxygen-carrying agents can be viewed as temporary "anti-hypoxic" drugs designed to maintain oxygenation of tissues.
A novel approach has been developed to combine the use of Oxygent with autologous blood techniques. The optimal use of Oxygent will be as an adjunct to ANH during medium- to high-blood-loss surgeries. It is anticipated that the administration of Oxygent will increase safety during ANH by maintaining and possibly improving tissue oxygenation.20 In doing so, the use of Oxygent could safely allow for surgery at lower Hb levels and would reduce or eliminate the need for intraoperative blood transfusions.21
A schematic of this combined approach using ANH and Oxygent is shown in Figure 1. Initially, the patient will undergo ANH just prior to surgery to collect 2 to 4 units of fresh autologous whole blood. By starting surgery at a reduced hematocrit (e.g., 27-30%), fewer red cells will be lost during subsequent bleeding. Instead of initiating transfusion with blood, a single dose of Oxygent (e.g., 1.35 g PFC/kg) is administered while the concentration of inspired oxygen gas is maintained at 100% to maximize the amount of dissolved oxygen being delivered from the PFC and the plasma. During subsequent surgical bleeding, the patient is carefully monitored for any signs indicating that a blood transfusion might be required. The fresh autologous blood is returned to the patient toward the end of surgery when hemostasis has been achieved (or intraoperatively, if required), to reach a safe postoperative hematocrit without the need for allogeneic blood.
In the future, in situations requiring lengthy surgical procedures with extensive blood loss, avoidance of allogeneic blood will depend on the combined use of multiple autologous conservation techniques, i.e., PAD prior to surgery (if feasible), erythropoietin therapy to accelerate the regeneration of red cells during PAD, ANH to collect fresh autologous blood, administration of Oxygent (in place of intraoperative transfusion) to maintain tissue oxygenation during subsequent surgical bleeding, and intraoperative ABS when appropriate. By using Oxygent with multiple autologous approaches, surgery can proceed safely at lower hematocrits, and a maximum quantity of autologous whole blood can be collected and reinfused at the end of surgery to raise the hematocrit to a safe level. If a PAD unit remains available, it can be reserved for postoperative use in those surgeries where continued drainage from the wound is common.
Oxygen Transport By Perfluorochemicals
Oxygen molecules are dissolved in PFC particles as they pass, in the blood, through the lungs. These oxygen-loaded PFC particles travel through the blood to the capillaries where tissue oxygen concentrations are low. At that point, oxygen is exchanged for carbon dioxide molecules. PFCs exchange gases more rapidly and more completely than do red cells because they load and unload gases by simple diffusion. When the particles return to the lungs, the carbon dioxide is expired and the PFC particles take on new oxygen molecules.
Oxygen delivery to tissues by PFC emulsions differs fundamentally from that of Hb in red cells. Hb has a fixed capacity to bind 4 molecules of oxygen per tetramer; therefore, oxygen transport is directly related to the Hb concentration and requires dissociation of the oxygen from the heme prior to transfer to tissues. By contrast, oxygen physically dissolves in PFCs, which results in a more rapid loading and unloading versus Hb.22 More importantly, the oxygen-carrying capacity of a PFC emulsion is directly proportional to the partial pressure of oxygen (i.e., according to Henry's Law) and can be maximized simply by elevating the inspired Figure 2. Oxygen dissociation curves for whole blood and for a 60% w/v perflubron emulsion (Oxygent). Arrows indicate preferential extraction of the dissolved oxygen from the perfluorocarbon compared to hemoglobin.
concentration of oxygen. Finally, the dissolved oxygen carried by the PFC is present in the plasma compartment (i.e., does not require diffusion across the red cell membrane), thereby increasing the concentration gradient and facilitating diffusion of oxygen to the tissues.
Comparing efficacy between a PFC emulsion and blood requires a consideration of the efficiency of oxygen extraction from a 3-compartment system comprising red cells, plasma, and the oxygen carrier. As shown in Figure 2, only 20-30% of the oxygen carried by Hb is actually extracted and consumed under normal conditions, while extraction of oxygen from the PFC and also the plasma can be >90%. In addition to the necessity for elevated arterial PO2, cardiac output is also critical in determining the contribution of PFC-dissolved O2 to total oxygen consumption (VO2). The normal physiological response to hemodilution is an increase in cardiac output primarily due to the stroke volume increase which occurs because of the reduction in blood viscosity (i.e., greater venous return due to a lower resistance to flow). The higher the cardiac output, the greater the delivery of dissolved oxygen from the PFC emulsion. An oxygen carrier that does not affect vascular resistance or interfere with the normal increase in cardiac output will therefore offer the greatest contribution to VO2 when used in conjunction with hemodilution. Thus, oxygen delivery from the PFC is based on its linear oxygen solubility, the contribution of virtually all of its oxygen to tissue VO2, and the additional benefit derived from an increased cardiac output during normovolemic hemodilution.
Computer Modeling of PFC-Based Oxygen Transport
It is generally accepted that mixed venous PO2 (PvO2) obtained via a pulmonary artery catheter is, at present, the best available overall indicator of whole body oxygenation status that can be monitored in the surgical patient.23 An oxygen transport computer model was developed to predict the PvO2 during the normal physiological responses to both ANH and volume-compensated surgical blood loss.24 Inputs include those that determine blood oxygen content (e.g., total Hb, blood PO2) and the position and shape of the oxyhemoglobin dissociation curve (i.e., Kelman constants, PCO2, pH, temperature); those determining oxygen delivery (e.g., cardiac output, cardiac output response to ANH, rate of bleeding) and oxygen consumption (VO2 - measured or calculated); and those related to the properties of the oxygen carrier being administered (e.g., dose, solubility for O2, blood half-life). When the arterial and venous blood gases, Hb concentrations, and whole body VO2 are entered, the oxygen transport and VO2 variables can be determined for each of the three compartments, i.e., the individual contributions from red cells, the plasma, and the oxygen carrier (either Hb- or PFC-based).
Output from the computer model demonstrates how the addition of an oxygen carrier would influence PvO2 during surgical bleeding, and thereby allows a calculation of the amount of blood that may be lost before transfusion becomes necessary. The accuracy of PvO2 predictions by the computer model has been validated using clinical data from hemodiluted patients and data from animal studies.25 The computer program has demonstrated that a single dose of Oxygent (1.35 g PFC/kg) would be able to support tissue oxygenation (i.e., based on keeping the PvO2 at or above predosing levels) while permitting significant additional blood loss (i.e., 2800 mL) which would lower total Hb levels from about 8.0 to 4.5 g/dL (model assumptions include: PaO2 = 500 torr, VO2 = 3.0 mL/min/kg at 37C, surgical blood loss = 2.0 L/hour, and a cardiac output increase of 0.5 L/min/g Hb decrease in a 70 kg adult under anesthesia).
Preclinical Efficacy Model
An anesthetized dog model was designed to mimic preoperative ANH and also progressive surgical anemia due to intraoperative volume-compensated blood loss.26 Purpose-bred beagles (n = 27) were maintained under isoflurane anesthesia, mechanically ventilated, and instrumented with EKG leads, venous, arterial, pulmonary artery, and bladder catheters. All blood vessels to the spleen were ligated via a midline abdominal incision to prevent release of sequestered red cells. Hemodynamics, blood gases, total hemoglobin (Hb), and blood oxygen contents were measured. Isovolemic hemodilution with Hespanr (6% hydroxyethyl starch solution, Dupont) in a 1:1 ratio (vol:vol) to shed blood.
Figure 3. Changes in PvO2 in hemodiluted dogs after receiving a dose (1.35 g PFC/kg) of Oxygent at a Hb = 8.0 g/dL or an equal volume of Plasma-Lyte (Controls). Dogs were subjected to a rapid, volume-compensated bleed (0.75 mL/kg/min) to a final Hb = 2.0 g/dL while breathing 100% O2. Dashed line indicates O2-breathing baseline level.
was used to lower the Hb to a target of 8.0 g/dL. The FiO2 was then increased to 1.0 to establish an oxygen-breathing mixed venous PO2 baseline (PvO2BL). After injection of a single dose of Oxygent (1.35 g PFC/kg) or an equivalent volume of Plasma-Lyte in controls, all dogs were subjected to a rapid volume-compensated bleed (0.75 mL/kg/min) under normovolemic conditions (i.e., Hespan was given to maintain pulmonary wedge pressure) to a final Hb of 2.0 g/dL. Hemodynamic and oxygen transport variables were measured at 15 minute intervals.
There were no physiologically relevant changes in mean arterial pressures, heart rate, or wedge pressures following injection of Oxygent. In response to the reduction in blood viscosity caused by removal of red cells, however, cardiac outputs increased similarly in both groups while left ventricular stroke work index remained unchanged. Total VO2 was maintained closer to baseline levels in the treated dogs despite the severe anemia induced by progressive volume-compensated bleeding. The PvO2 increase predicted by the computer model after giving Oxygent was also observed in this dog study.27 After infusion of Oxygent, mean PvO2 levels increased significantly by ~15 mmHg above the pretreatment PvO2BL, compared to only a 2 mmHg increase in the controls (see Figure 3). The sustained, elevated PvO2 levels permitted the treated dogs to withstand significantly greater blood loss (almost 70 mL/kg) compared to controls (only 10 mL/kg) before mean PvO2 levels had decreased to the PvO2BL levels. Thus, PvO2 levels provided a means to monitor the effect of the PFC-delivered oxygen on global tissue oxygenation.
CLINICAL STUDIES
To date, in excess of 145 human subjects have been tested with perflubron emulsions in various European and U.S. clinical studies. Clinical testing of Oxygent in the U.S. has included completion of a saline-controlled Phase I dose escalation safety study in 57 conscious healthy volunteers; and a multi-center randomized, Ringer's lactate-controlled dose escalation Phase I/II safety study in 30 consenting adult surgical patients undergoing low-blood-loss elective surgery under general anesthesia.28 There were no clinically meaningful effects on blood chemistry, coagulation values, hematology (except platelet count), liver function, renal function, and pulmonary function tests in either of these studies. The only relevant findings in the surgical patients (but observed only at the highest dose studied, i.e., 1.8 g PFC/kg), included a transient febrile response (onset at ~ 4 hrs post-dosing; peak temperature of 38 to 39C at 6 to 8 hrs post-dosing; full resolution by 12 to 14 hrs) and a moderate reduction in platelet count (mean nadir ~130,000/æL on day 3; return to normal by 7 days). There were no bleeding problems observed. Patients in the control group experienced similar responses of a slightly lesser magnitude. Thus, there were no clinically meaningful safety findings that would preclude the continued testing of Oxygent in surgical patients.
Preliminary data from a single-dose Phase II efficacy study29 in patients undergoing high-blood-loss surgical procedures demonstrated increases in mean PvO2 levels that remained elevated above predosing levels (achieved on 100% oxygen-breathing) while ongoing blood loss caused total Hb to decrease by about 2 g/dL (i.e., from about 8.3 to 6.5 g/dL) prior to reaching the point where transfusion with blood was deemed necessary. Thus, these data suggest that the single dose (0.9 g PFC/kg) of Oxygent was equivalent to at least one unit of blood.
CONCLUSION
Using PvO2 as a measurable index of global tissue oxygenation, it has been possible to demonstrate that Oxygent can temporarily sustain oxygenation during blood loss-induced surgical anemia. Efficacy of a PFC-based oxygen carrier is due to preferential extraction of the PFC-dissolved O2 from the plasma compartment (i.e., >90%) versus about 20-30% extraction of O2 from Hb inside red cells. Thus, ANH supplemented with Oxygent (@FiO2=1.0), has the potential to increase the safety of both ANH and surgery at lower Hb levels. The net saving will be a reduction or elimination in the need for allogeneic blood since fewer red cells would be lost during active surgical bleeding. In addition, more of the fresh autologous blood would be available for reinfusion once hemostasis has been achieved, thereby returning the patient to a safe hematocrit using only autologous blood. By combining this use of ANH and Oxygent with multiple autologous techniques including PAD and salvage during surgery, it may be possible to completely avoid exposure to allogeneic blood in most medium-to-high-blood-loss surgical procedures.
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