Pharmacological Action

The pharmacodynamics of HB0C-201 is primarily associated with oxygen delivery and, although it is also a colloidal protein solution, HB0C-201 enhances oxygenation by promoting both

Hemopure stability: methemoglobin formation

Hemopure stability: methemoglobin formation

40

♦ H8C003 (40°C) ■ H8C005 (40°C) a H8C006 (40°C) - Specification

12 10

♦ H8C003 (RT) ■ H8C005 (RT) a H8C006 (RT) - Specification

12 10

co 6

15 20

Figure 36.2 Percentage methemoglobin (% metHb) as a function of time for three lots of HBOC-201 at 37°C (75* humidity). The specification is no more than 10 per cent. The solid lines are linear regression fits.

♦ H8C003 (2-8

°C)

■ H8C005 (2—E

°C)

a H8C006 (2-8

°C)

- Specification

Figure 36.2 Percentage methemoglobin (% metHb) as a function of time for three lots of HBOC-201 at 37°C (75* humidity). The specification is no more than 10 per cent. The solid lines are linear regression fits.

convective and diffusive oxygen transport. The pharmacodynamic profile includes:

• increased rate of oxygen uptake and release (demonstrated in vitro)

• increased tissue oxygenation associated with increased oxygen extraction ratio

• eliminated and/or reduced tissue hypoxia caused by vessel stenosis and occlusion

• efficacy of oxygen transport evidenced in animal models of tumor oxygenation, hemor-rhagic shock trauma, and veterinary use of Oxyglobin.

HBOC-201 promotes hemodynamic and metabolic stability, and has been shown to treat the signs and symptoms of anemia and to reduce tissue hypoxia in models of either extended cardiac arrest or myocardial infarction. Effects of earlier

HBOC solutions on smooth muscle-mediated responses in vivo appear to have been attenuated by the reduction of the low molecular weight hemoglobin components in newer formulations (HBOC-201).

Enhanced oxygen transport: in vitro oxygen flux studies

In contrast to the hemoglobin in RBCs, HBOC-201 is uniformly distributed in the plasma phase of blood, impacting fundamental oxygen transport processes. The low solubility of oxygen in the plasma phase of erythrocyte suspensions generates large diffusional resistance. However, when added to plasma containing RBCs, HBOC-201 reduces this resistance and facilitates the offloading of oxygen from erythrocytes (Page et ai, 1998a, 1998b). Furthermore, the lower oxygen

Normal blood flow Anemic blood flow Hemopure

Oxygen offloading to tissue bed

RBC -

If A ' • l^L ,

Capillary -,-. , , Tissue bed

Stenosis (constricted capillary)

Figure 36.3 Because HBOC-201 circulates freely in plasma, and is smaller, has lower viscosity (resistance to flow) and can more readily release oxygen to tissues than red blood cells, it can carry oxygen at low blood pressure and through constricted or partially blocked blood vessels to areas of the body that red blood cells cannot reach due to their larger size. See color plate 15.

affinity of HBOC-201 compared to native RBC hemoglobin increases the tendency to off-load oxygen to tissues (Figure 36.3).

Page et al. developed and validated a mathematical simulation model (1998a) based on experimental data from in vitro capillary studies (1998b). This model was used to analyze the oxygen flux of various HBOC solutions, RBCs, and mixtures of the two in model capillaries. The degree of saturation of hemoglobin by oxygen was examined as a function of the residence time in an artificial capillary. HBOC was more efficacious than RBC suspensions in uptake and release of oxygen. Increasing the HBOC content of mixtures of HBOC and RBCs, or HBOC alone, resulted in an increase in the efficacy of oxygen transport (off-loading). In mixtures of RBCs and HBOC, when HBOC content reached 50 per cent the mixture had the equivalent oxygen transport efficacy of HBOC solution alone. These data suggest that HBOC more effectively transports oxygen than RBCs and, when added to RBCs, can increase the efficacy of RBC oxygen transport. These observations may explain, in part, the ability of Biopure's HBOC formulations to increase oxygen extraction and tissue oxygenation.

Persistent increases in oxygen extraction in the presence of HBOC may be indicative of improved oxygenation compared to RBCs alone. The observed decrease in cardiac output following administration of HBOCs may be an autoregula-tory response to the increased efficiency of oxygen transport to tissues and increased oxygen extraction. These important properties of HBOCs are illustrated in this review of the published in vivo studies with the various HBOC formulations produced by Biopure.

PRECLINICAL STUDIES OF TISSUE OXYGENATION AND TRAUMA

The development of HBOC-201 has included completion of over 250 studies, many of which have been described in previous reviews (Light et al., 1998; Pearce and Gawryl, 1998). These studies demonstrated the efficacy of the various hemoglobin preparations in oxygenating tissues and stabilizing hemodynamics, sustaining life after complete exchange transfusion, resuscitation from hemorrhagic shock, hemodynamic support of animals with septic shock, and resuscitation following cardiac arrest, and characterized their effects on renal function. Furthermore, in assessing safety, these studies demonstrated that HBOC-201 produced few side effects: an increase in mean arterial blood pressure (MAP) (10-20 mmHg), gastrointestinal signs (vomiting, diarrhea), increases in serum enzyme activities (aspartate aminotransferase, lipase) not associated with clinical disease, and skin and/or scleral discoloration. A summary of the more recent animal studies is included below.

Tissue oxygenation

Several studies reviewed by Pearce and Gawryl (1998) directly demonstrated increased tissue oxygenation at higher oxygen extraction ratios and lower cardiac outputs after treatment with HBOC- 201 compared to RBCs.This hemodynamic phenomenon relates to the distribution of extracellular hemoglobin in the plasma phase of blood. More recent studies supporting these conclusions are described below.

Arterial stenosis and occlusion

The presence of HBOC in the plasma phase of blood results in a global distribution of oxygen-carrying hemoglobin in the circulation and contributes to the overall increase in oxygen extraction. Theoretically, the plasma phase distribution is of benefit when tissue injury or disease limit or prevent the flow of RBCs. Thus, the effect of HBOC in various animal models of partial to complete vessel occlusion has been studied to test this hypothesis.

The first study to demonstrate the potential benefit of HBOC-201 in partial vascular occlusion was that of Horn et al. (1997) in a canine model of arterial stenosis. Fourteen dogs underwent isovolemic hemodilution with lactated Ringer's (RL) to a hematocrit of 25 per cent, after which a 95 per cent artificial stenosis of the popliteal artery was experimentally induced. The animals were randomized to receive either 50 ml HBOC-201 or 200 ml 6 per cent hetastarch (HES). In both groups, oxygen delivery and oxygen consumption of the muscle decreased in parallel with decreasing blood flow following arterial stenosis. During stenosis, tissue PO2, as measured by an Eppendorf polarographic needle microelectrode, was decreased in both groups when compared with baseline (P< 0.001). Following treatment, tissue PO2 remained low in the HES group but returned nearly to baseline with HBOC-201 (P< 0.001). Similarly, the oxygen extraction ratio increased after infusion of HBOC-201 and was higher when compared with HES-treated animals (P< 0.05). With the exception of higher MAP and mean pulmonary artery pressure in HBOC-201 treated animals, hemodynamics did not differ between the two treatment groups. In contrast with HES infusion, administration of HBOC-201 resulted in the restoration of baseline skeletal-muscle tissue oxygen tensions during nearly complete arterial stenosis.This may be due to the ability of HBOC-bound oxygen to reach poststenotic tissues via reduced plasma flow by the 95 per cent occlusion, as well as increased oxygen extraction despite the RBC flow being blocked.

One of the more important potential applications of HBOC-201 in cardiovascular disease may be in the treatment of ischemia due to coronary vessel occlusion. Standl et al. (1999) investigated the effect of 0.6g/kg HBOC-201 or RL on tissue oxygenation in the heart of dogs during sustained normovolemic hemodilution (Hb ^ 7.5g/dl) and acute 90 per cent stenosis of the left anterior descending coronary artery (LAD). Cardiac muscle oxygenation was measured using a flexible microelectrode in the area supplied by the LAD. Following administration of RL or HBOC-201, before 90 per cent stenosis of the LAD, cardiac tissue oxygen levels distal to the occlusion site decreased from 21 ± 6mmHg to 7 ± 6mmHg in the RL treated group but were unchanged (18 ± 7 mmHg) in the HBOC-201 group. If HBOC-201 was administered after establishment of the stenosis, tissue oxygen levels were partially restored (23 ± 7 mmHg before stenosis and 15 ± 5 mmHg after stenosis). These results are similar to the earlier observations in canine muscle reported by Horn et al. (1997), where the presence of HBOC-201 in the plasma phase antagonized the ischemia that developed following 95 per cent vascular occlusion.

The beneficial effect of HBOC-201 may extend beyond the ability to perfuse and oxygenate tissue distal to partial occlusions. Strange et al. (2000) investigated the potential cardioprotective effects of HBOC-201 in a canine model of complete vascular occlusion of the LAD. HBOC-201 (equivalent to 10 per cent total blood volume) was infused just prior to a 90-minute complete occlusion of the coronary artery, after which reperfusion was established for 4.5 hours. Histological analysis of infarct size showed a greater than 55 per cent reduction as a result of HBOC-201 infusion compared with a vehicle control (0.9 per cent saline) (P< 0.01). Consistent with the decreased infarct size, neutrophil infiltration was also significantly (P< 0.01) reduced in the HBOC-201 group. Neutrophil accumulation into a previously ischemic zone is considered a hallmark of reperfusion injury following ischemia. Animals treated with HBOC-201 did not show the hemodynamic instability and arrhythmias that were characteristic of the control group and plasma levels of creatinine kinase andTroponin I were lower, consistent with the protective effect of HBOC-201 on cardiac muscle (P< 0.05).

The presence of HBOC-201 in the circulation compensates, in part, for the loss of adequate direct perfusion of cardiac muscle, and may involve enhanced transport of oxygen via collateral vessels.These data further suggest that HBOC-201 augmentation of oxygen transport may counteract the severe tissue hypoxia and injury that occur with complete vascular blockage.

Tumor oxygenation

Teicher and colleagues performed a series of studies to determine whether HBOCs increase PO2 in hypoxic tumors and, subsequently, if this enhanced tumor oxygenation would result in increased sensitivity to radiation and chemotherapy. Initial data (Teicher et al., 1991, 1992a, 1992b, 1993) were generated from studies performed with HS1-2 (a variant of H1S with 40 per cent rather than 50 per cent unpolymerized hemoglobin) and later confirmed in the same models with HBOC-201 (Robinson et al., 1995; Teicher et al., 1995a, 1995b). Tumor PO2 levels were measured directly in rats breathing either normal air (21% O2) or carbogen (95% O2/5% CO2) using a polarographic needle microelectrode. Tumor growth delay was also measured following chemotherapy or the combination of chemotherapy and fractionated radiation treatment. In the five studies in which either single dose radiation or fractionated radiation treatment over 5 days or both were employed, HBOC increased tumor oxygenation and enhanced the irradiation response measured in terms of tumor growth delay and survival time (Robinson et al., 1995; Teicher et al., 1991, 1992a, 1992b, 1995a, 1995b). The radiation response was seen in mammary adenocarcinoma 13672, 9L gliosarcoma and FSaIIC Lewis lung carcinoma (fibrosarcoma) (Robinson et al., 1995;Teicher et al., 1993, 1995b). The addition of carbogen breathing to treatment with HBOC resulted in an increase in tumor PO2 values and decreased the hypoxic fraction (per cent PO2 readings ^ 5 mmHg) of cells in tumors compared to HBOC alone (Robinson et al., 1995; Teicher et al., 1991, 1995a, 1995b). These changes were associated with increases in tumor growth delay and animal survival. Similar findings have been obtained when HBOC and HBOC/carbogen have been used in combination with alkylating chemotherapeutic agents (Teicher et al., 1991, 1992a, 1992b, 1995a).

Collectively, the results of these studies suggest that infusion of Biopure's HBOC formulations produced increased tumor PO2 levels and an associated decrease in the hypoxic fraction of tumors. In almost all treatment paradigms investigated, the increased tumor oxygenation corresponded with an increase in tumor growth delay. These studies provide further evidence of enhancement of tissue oxygenation by Biopure's HBOC formulations.

Trauma, hemorrhage and/or shock

Initial studies using hemodilution (Pearce and Gawryl, 1998) demonstrated the efficacy of various formulations of bovine HBOCs to satisfy oxygen demands under normotensive conditions without untoward effects. These early studies demonstrated that HBOC-201 could be used as an 'oxygen bridge', supplying oxygen needs until blood cells regenerated. The results of those studies provided the background for experiments assessing the use of HBOCs in the treatment of hypovolemic trauma with hypoperfusion.

Several studies (below) have assessed the efficacy of HBOC-201 in both acute and survival models of controlled or uncontrolled hemorrhage with severe organ injury and lethal whole body trauma, as well as acute and survival studies utilizing hypotensive resuscitation.

Controlled hemorrhage models

Studies with early HBOC formulations (Pearce and Gawryl, 1998) involved animal models of controlled hemorrhage and extended hypovolemia. A typical design for these models was the controlled removal of blood from a resected vessel or from an intravenous or intra-arterial catheter and maintenance of the resulting hypovolemic and hypotensive state for a predetermined time.These early studies demonstrated that resuscitation with HBOC restored hemodynamics, corrected acidosis to an extent comparable to RBCs, and supported survival equal to RBCs without causing any important acute adverse cardiopulmonary effects or organ dysfunction.

Uncontrolled hemorrhage models

Clinically, hemorrhage is often uncontrolled and associated with tissue injury. In standard prehospital care, normal saline or RL solution is administered to hypotensive trauma victims with the objective of delaying exsanguinating cardiac arrest. Several experiments have evaluated the use of HBOC-201 to treat severe, continuous, uncontrolled hemorrhage with associated liver injury modeling a patient with blunt abdominal trauma and uncontrolled hemorrhage in a pre-hospital setting.

Manning et al. (2000) developed a novel swine model of blunt-trauma liver injury with uncontrolled hemorrhage, shock and death. Liver injury was produced by partial resection of each of the four liver lobes. Nine minutes after the onset of bleeding, swine were randomized to receive approximately 10 ml/kg per minute of intravenous RL (n = 10) or HBOC-201 (n = 7) to obtain and maintain an MAP of 60mmHg or until hemodynamic collapse while bleeding continued for 2 hours. All animals were initially resuscitated successfully. However, the 2-hour survival was 1/10 with RL, and 7/7 with HBOC-201 (P = 0.0004). Nine swine treated with RL experienced cardiovascular collapse at 36 ± 10 minutes. Thirty minutes after resuscitation, lactate levels were significantly (P< 0.05) lower in HB0C-201 treated animals (12 ± 2mmol/l) versus controls (18 ± 3 mmol/l).The severity of this model is reflected by the low hematocrit levels (< 1 per cent) in the majority of treated and control animals. HB0C-201 infusion stabilized hemodynamics. The low volume requirement for HB0C-201 was reflected by the lower infusion rate for HB0C-201 treated animals (2.6 ml/kg per minute) versus the infusion rate for RL treated animals (10.8 ml/kg per minute) and is likely to prove useful in the pre-hospital-care environment, particularly when extreme transport times are required or in battlefield situations where resuscitative fluid availability is restricted by logistical constraints.

Katz et al. (2002) used the same exsanguinating liver injury model to assess survivability with HB0C-201. Swine underwent a liver crush, laceration, and 50 ml/kg initial blood loss. The liver continued to bleed at 3 ml/kg per minute during the resuscitation phase. Withholding fluid resuscitation (NF, n = 6) or resuscitation with HES (n = 8) were fatal in this model, while all HB0C-201 (n = 8) swine survived 24 hours and seven of eight survived 96 hours with good functional recovery. The investigators concluded that HB0C resuscitation during profuse liver bleeding and traumatic shock enhanced survival with good physiological outcome in swine.

Aggressive use of HB0C-201 during resuscitation from severe hemorrhage may have limitations, however. When large volumes of HB0C-201 were administered rapidly (6 ml/kg per minute to animals) with severe volume depletion in the study (Katz, 2000), blood pressure and pulmonary artery pressure increased. These changes did not compromise pulmonary function or survival, and future studies will assess their effect on the potential to increase bleeding.

Model of lethal whole body trauma

Severe trauma is often associated with intravascular coagulation, interstitial edema and release of toxic humoral mediators, leading to severe hypotension and multiple organ injury. Thus, Hayward and Lefer (1999) examined the effect of HB0C-201 infusions (5 per cent, 10 per cent, and

15 per cent of calculated blood volume) in a rat model of lethal traumatic shock. Anesthetized rats subjected to Noble-Collip drum trauma developed shock with marked hypotension, as well as splanchnic vascular endothelial dysfunction characterized by an impaired vasorelaxation response of the superior mesenteric artery (SMA) to endothelium-dependent vasodilators, and a four-fold increase in intestinal myeloperoxidase activity. Treatment of rats 10 minutes posttrauma with 10% HB0C-201 resulted in a two-fold increase (P< 0.05) in survival time from 108 ± 20 minutes in control animals to 228 ± 31 minutes in HB0C-201 animals. HB0C-201 also normalized MAP and produced a marked preservation of mesenteric vascular endothelial function. Treatment with HB0C-201 had no effect on neutrophil infiltration as indicated by an absence of change in ileal tissue content of the polymorphonuclear leukocyte enzyme, myeloperoxidase. Treatment with a vehicle control post-trauma did not result in any beneficial effects. These data suggest that infusion of HB0C-201 post-trauma normalizes systemic blood pressure and protects endothelial function.

Hypotensive resuscitation

The ability to provide adequate tissue oxygenation under conditions of low volume and pressure resuscitation can be of enormous value in battlefield or civilian casualty situations, particularly for far forward or remote locations where supplies are limited and delayed evacuation is expected. Several studies investigated the effects of HB0C-201 using animal models of low-volume or hypotensive resuscitation.

McNeil et al. (2001) evaluated the ability of HB0C-201 to restore tissue perfusion under conditions of hypotensive resuscitation in a porcine model of severe hemorrhage to a MAP of 30mmHg. Animals were maintained at a MAP of 35 mmHg for 45 minutes by continuous hemorrhage and subsequently resuscitated with HB0C-201, RL, or RL plus blood to a MAP of 60-80 mmHg. Animals were monitored for 4 hours post-resuscitation.

As demonstrated by measurements of base excess, pH and lactate levels, low-volume (as compared to LR or LR plus blood) and low-pressure resuscitation with HB0C-201 after controlled hemorrhage in swine provided sufficient tissue perfusion and oxygen delivery to reverse anaerobic metabolism in the presence of continued hypotension, hypovolemia and low cardiac

Table 36.2 Resuscitation volumes after hypotensive resuscitation with HBOC-201

Group

Resuscitation

volume (ml)

HBOC-201 @ 60 mmHg

463 ± 57

LR @ 80 mmHg

16 358 ± 2571

LR + blood @ 80 mmHg

4777 ± 260

Pvalue (ANOVA)

< 0.001

output (Table 36.2). Similar results were seen when this study was repeated (York et al., 2003) and animals allowed to survive. Hypotensive resuscitation with HBOC-201 provided adequate tissue oxygenation for survival in this porcine model of controlled hemorrhage.

HBOC-201 enhances oxygen tension and tissue oxygenation when traditional interpretation predicts otherwise. The potential adverse effects of hypovolemia, hypotension and low cardiac output are all compensated for by the oxygen transport characteristics of HBOC-201, as demonstrated by measures of blood lactate, pH, jejunal oximetry and physiologic outcome following resuscitation and sustained survival over several days. Sampson et al. (2003) compared 7.5% hypertonic saline (HS), hypertonic saline 7.5%/6% dextran-70 (HSD), 6% pentastarch, 6% HES, or HBOC-201 with RL and no resuscitation controls in a swine model of controlled hemorrhagic shock. After 45 minutes of shock, animals were resuscitated to and maintained at a MAP of 60mmHg for 4 hours. Death occurred in five of six animals in the no-resuscitation control group, six of six in the HS group, and one animal in the HSD group before completion of the study. HBOC-201 restored tissue oxygenation and reversed anaerobic metabolism at significantly lower volumes when compared to HTS, HSD, Pentastarch or HES.

Brain oxygenation during hemorrhagic shock

Outcomes after head injury, the leading cause of traumatic death in the US, are severely worsened in the presence of hypotension. HBOC-201 treatment of hypovolemia in patients with traumatic brain injury may lead to improved outcomes. Previous reports (Sadrzadeh et al., 1987; Regan and Panter, 1993, 1996) have suggested that hemoglobin solutions can be toxic to neurons in vivo and in vitro in culture. In some of these studies, many different preparations, from autologous blood to 'purified hemoglobin', were injected into the brain. Other studies focused on establishing the role of hemoglobin released from hemolyzed RBCs in the pathological vasospasm that follows subarachnoid hemorrhage rather than on examining the potential direct neurotoxic effects of hemoglobin.To what extent the reported effects of HBOC solutions relate to the vasoactivity of tetrameric hemoglobin, the direct neurotoxic effect of hemoglobin, or toxic effects of some other component of these preparations is not clear.

Two early studies by Waschke et al. (1993, 1994) demonstrated that near total replacement of blood by H1S satisfied the circulatory and metabolic demands of the normal rat brain. In a more recent study of brain tissue oxygenation, Manley et al. (2000) investigated the effects of resuscitation with HBOC-201 (in a swine model of pre-hos-pital small-volume resuscitation) on brain tissue oxygen tension, MAP and cardiac output (CO). MAP and CO decreased significantly with hemorrhage. Small volume resuscitation with HBOC-201 restored and maintained cerebral oxygenation, MAP, and CO following severe hemorrhagic shock in swine. In addition, in a double-blind study using a similar swine model of hemorrhagic shock, Manley and coworkers (2002) compared the effects of hypertonic saline dextran (HSD), RL, and HBOC-201 on brain oxygenation. Resuscitation with HBOC-201 provided more efficacious and durable improvement in brain oxygen and cerebral perfusion pressure. However, in the same model Knudson et al. (2003) failed to show improvement in liver or deltoid muscle oxygenation with HBOC-201 (and 100% O2) in comparison with RL or HSD (and 100% O2). MAP and systolic blood pressure were stabilized more effectively in HBOC-201 pigs, while cardiac output was highest in HSD-treated pigs.

These results, indicating that HBOC-201 provides adequate brain tissue oxygenation and promotes long-term survival, are supported by studies assessing the effects of HBOC-201 on neurons in cell culture. Ortegon et al. (2002) compared the effects of incubating HBOC-201 and purified human hemoglobin (hHb, Sigma) (0.02, 0.2, 2.0 and 6.5 g/dl) with rat fetal neural cells in culture for 24 hours. Neural cells exposed to HBOC-201 did not lyse, and maintained levels of proliferation and metabolism similar to controls. However, cultures exposed to hHb (^0.2 g/dl) demonstrated significantly decreased proliferation, decreased metabolic activity, and increased cell lysis when compared with controls (P< 0.05). Neural cells exposed to HBOC-201 in culture were able to continue sustained metabolic activity and normal proliferation with no evidence of neurolysis, suggesting that HBOC-201 does not display the toxic characteristics of hHb.

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