Repetitive Administration of Hemoglobin Raffimer in Experimental Models and Clinical Applications

Repetitive Administration of

Hemoglobin Raffimer in Experimental Models and Clinical Applications

Hae Won Kim

, George Biro

, and A. Gerson Greenburg

Summary. Hemoglobin (Hb)-raffimer (Hemolink) is an oxygen therapeutic based on human Hb that is intramolecularly and intermolecularly crosslinked with o-raffinose. Hb-raffimer appears to be free of severe toxicity, as shown in a variety of topload/repeat dose preclinical animal studies. In addition, Hb- raffimer does not appear to be immunogenic. Limited information is avail- able on the acute and chronic effects of high volume infusion in animals and on responses to repeated dosing in human subjects. Hb-based oxygen thera- peutics have not been tested in a wide variety of possible indications. In con- clusion, Hb-raffimer and other oxygen therapeutics could be potentially life saving in certain clinical situations. Current and future clinical trials will eval- uate expanded indications and dosing regimens.

Key words. Hemoglobin (Hb), Hb-raffimer, Oxygen therapeutic, Repeat dose, Clinical trial

Introduction

Oxygen therapeutics are therapeutic agents designed to deliver oxygen (O

) to tissues where normal O

supply is impaired or compromised due to hem- orrhage or impaired blood flow. Current candidate oxygen therapeutics are mainly based on hemoglobin (Hb) or synthetic perfluorochemicals.

Hemoglobin-based oxygen therapeutics are chemically modified human- or animal-derived Hbs or genetically “engineered” Hbs designed to improve on properties of unmodified stroma free hemoglobin solution obtained from

53

1

Brown University and The Miriam Hospital, 164 Summit Avenue, Providence, RI 02906, USA

2

Hemosol, Inc., Missisauga, Ontario, Canada

lysed red cells. Improving intravascular circulation time is another element of the design criteria.

Hb-raffimer (Hemolink) is an oxygen therapeutic based on human Hb that is intramolecularly and intermolecularly crosslinked with oxidized raffinose (o-raffinose) [1]. Hb-raffimer has a lower O

affinity (higher P50) and viscos- ity than native red cells, a physicochemical characteristic considered desir- able in an oxygen therapeutic. Hb-raffimer has been extensively tested in preclinical animal studies without evidence of significant toxicities. In Phase I and II clinical trials with Hb-raffimer, no serious adverse effects have been revealed [2,3] although some mild undesired effects (e.g., transient hyper- tension, dysphagia and abdominal discomfort) have been observed in some patients [4,5]. In a Phase III trial in 148 subjects in a number of Canadian centers, the safety and efficacy of Hb-raffimer was evaluated in patients undergoing coronary artery bypass grafting (CABG) operations.

Characteristics of Hb-raffimer

Hb-raffimer is a highly purified, pasteurized, solution of human HbA

stabi- lized and covalently crosslinked with o-raffinose. Hb-raffimer is formulated as an iso-oncotic solution in Ringer’s Lactate for Injection United States Phar- macopeia (USP) (Table 1). The o-raffinose crosslinking method consistently yields a distribution of molecular weight species in the range of 64–500 kDa.

Hb-raffimer contains 10 g Hb/dl; >90% in ferrous deoxy state.

Mechanism of Action of Hb-raffimer

The goal of oxygen therapeutics in an acute anemic condition is normaliza- tion of tissue perfusion and improved oxygen kinetics. Optimal tissue perfu-

Table 1. Characteristics of Hb-raffimer solution (Hemolink) Modification Crosslinking with o-raffinose

Hb Source Outdated human red cells

Hb concentration 10 g/dl (<7% metHb) Colloidal osmotic pressure 25 mmHg

Hb molecular weight distribution <64 kD: <5%

64 –500 kD: >90%

>500 kD: <3%

Electrolyte base Ringer’s lactate

pH 7 .5

P50 52 mmHg

Hill Coefficient (n) 1 .0

Sterilility (USP XXII) Pass

Endotoxin <0.006 EU/ml

Viscosity 1 .14 (cP)

sion is the end result of complex interactions of many physiologic regulatory systems. An adequate supply of oxygen is required for normal tissue metab- olism and to maintain homeostasis. How does this oxygen therapeutic work?

There are many elements to consider including oxygen carrying capacity, oxygen delivery kinetics, colloids and electrolytes in solution, viscosity, effects on hemodynamics and the vascular system, leukocyte activation/deactivation and erythrocyte clumping. Three important characteristics of Hb-raffimer are: acellularity, oxygen delivery capacity, and the shape of the oxyhemoglo- bin dissociation curve (OHDC) (Fig. 1). The acellular nature of Hb-raffimer allows improved perfusion of capillary beds blocked by leukocytes or ery- throcyte thrombi that impede blood flow. Reduced viscosity lowers blood flow resistance and oxygen in the plasma phase enhances the diffusing surface to tissue cells. An increased oxygen carrying capacity of blood enhances oxygen delivery generally and the rightshifted OHDC enables nearly as much oxygen offloading across the standard arterio-venous oxygen gradient as whole blood of similar Hb concentration. All of these factors work synergistically to effect improved tissue perfusion and acellular oxygen supply. The improved deliv- ery of oxygen to tissues is an important mechanism of action of this oxygen therapeutic.

Fig. 1. Oxyhemoglobin dissociation curves for blood and Hb-raffimer

Preclinical Topload/Repeat Dose Studies

Certain clinical conditions may require multiple administrations of Hb-based oxygen therapeutics to the same subject. Repeated dosing regimens render a greater potential for toxicity than a single dose treatment because they usually result in higher blood concentration and more protracted exposure. They may also stress or overwhelm the normal clearance/metabolic mechanisms (liver, spleen, kidneys, etc.), causing accumulation of potentially toxic Hb breakdown products. Lastly, they are more likely to reveal toxic reactions that occur rarely when administered in “normal” or single doses. To test safety of repeated doses, rats and dogs were subjected to topload repeated intravenous infusions of Hb-raffimer [6].

In rats with single 5% topload, Hb-raffimer did not cause any deaths or toxic effects on the major body organs or systems. All animals showed normal activity and positive body weight gain. There were no significant detrimental effects on hematology, biochemistry, or histology. No significant immune response was noted at this dose. In studies of 14 day daily repeated dosing (iv infusion) of 5, 10, 15, or 30 ml/kg/day, dose related slowing of weight gain was observed. In addition, histopathology revealed foamy histiocytes, foamy sinu- soidal cells with pigment in the liver, and pigment accumulation in the renal cortical tubular epithelia cells. Gross pathology revealed carcass discoloration and dark pigmentation of the kidneys. The carcass discoloration was no longer apparent after day 29, i.e., after 14 days of treatment free recovery.

There were no deaths in any of these studies.

A similar 14-day toploading repeat dose study in dogs with 10 ml/kg/day resulted in no deaths [6]. Increased aspartate aminotransferase (AST), crea- tine phophokinase, amylase, total bilirubin, dark yellow/amber coloration of the urine and “bilirubin” stained cells in the urine were observed. The histo- pathological effects were largely reversed during the 14-day recovery period.

In studies with 20 and 30 ml/kg/day, on each of fourteen consecutive days, reduction in the rate of body weight gain and feed intake was noted. There was increased pigmentation in the skin, eyes, and mucous membranes. Red cell counts and hematocrit decreased slightly but plasma hemoglobin con- centration increased. Phagocytic leukocyte count was increased and bilirubin was significantly elevated. Discoloration and increased liver and kidney weights were reversed during the 14 day treatment free recovery period. At the end of recovery, there was an elevated plasma iron level with reduced iron binding capacity. No deaths resulted in any of these studies.

The repeated dosing subjected the animals to prolonged exposure to

unphysiologically high concentrations of Hb-raffimer in the blood. The

cumulative total doses were many times those anticipated in the clinical use

of this oxygen therapeutic, and amounted to several times the animals’ total

red cell and Hb mass. Nevertheless, the treatment was tolerated well in these models. The biochemical and histological findings were consistent with serious loads on Hb catabolic mechanisms. Yet, no evidence of serious organ toxicity was observed in these studies.

Preclinical developmental studies are designed to fulfill two primary objec- tives. First, preclinical studies produce data that support and validate the effectiveness of a therapeutic candidate for its intended clinical use. Second, preclinical studies are also conducted to test potential toxicity (safety) of a therapeutic candidate. Toxicity is generally assessed in terms of changes in physiologic parameters, blood and urine chemistries, and organ/tissue func- tion, histopathology and other relevant indicators. Because many, and often unknown, differences in genetics and physiology between animal models and human patients, results from preclinical efficacy and safety studies do not always translate into efficacy and safety in actual human patients although they are generally good predictors.

To be clinically useful, oxygen therapeutics must be free of serious toxic side effects. Therefore, one key question for the pre-clinical programs is:

can preclinical studies predict serious adverse effects in the human subjects?

Results of the completed preclinical studies indicate that Hb-raffimer appears to be safe in a variety of models. Using the laboratory to identify the under- lying mechanisms of clinical observations is an interesting exercise in uncov- ering physiologic processes that may be involved in human response. In retrospect some of the issues may be addressed but animals cannot tell us about their clinical conditions such as abdominal pain, discomfort, etc.

However, they provide clues by not eating or slowed rate of weight gain which could indicate some gastrointestinal and other organ functional impairment.

Did we find it looking backwards? There were hints and suggestions, but not conclusive data.

Clinical Studies

The primary purpose of clinical studies are to demonstrate that oxygen ther-

apeutics are safe and effective as described in the U.S. Food and Drug Admin-

istration Points-to-Consider guidelines [7,8]. Results from preclinical and

clinical studies conducted over the last 20 years have enhanced our under-

standing of various safety issues such as renal toxicity, immunogenicity, and

hypertensive and other effects of oxygen therapeutics. Demonstration of clin-

ical efficacy, however, turned out to be more difficult since clinical endpoints

have not been well defined [9]. Some proposed endpoints include mortality,

transfusion avoidance and various laboratory and clinical indicators of organ

function. Currently, transfusion avoidance appears to be a popular efficacy

endpoint adopted in most oxygen therapeutics clinical trials regardless of indicated use. However, demonstration of clinical efficacy is contingent upon an indicated use. Therefore, clinical endpoints of oxygen therapeutics should be defined as distinctively as possible depending on indicated use (e.g., hemorrhagic shock resuscitation, ischemic rescue, transfusion alternative, pre/peri-operative hemodilution, adjuvant to cancer therapy, etc). More work needs to be done in this area.

To date, Hb-raffimer has been tested in over 500 human subjects in various clinical protocols including normal subjects and coronary artery bypass grafting (CABG), orthopedic surgery, and chronic renal failure patients.

Safety in Healthy Volunteers (Phase I Clinical Trial)

Safety of Hb-raffimer was evaluated in a Phase I clinical trial of 42 normal healthy volunteers, of which 33 received Hb-raffimer [2]. Hb-raffimer was administered intravenously in doses ranging from 0.025 to 0.6 g/kg or an equivalent volume of Ringer’s lactate. The subjects were monitored for 3 days and followed for up to 6 weeks. Pre- and post-infusion cardiovascular, pulmonary and other major organ functions were assessed along with clini- cal laboratory measurements. Hb-raffimer administration was well tolerated in healthy volunteers with no evidence of serious organ dysfunction. At doses

>0.4 g/kg, some subjects complained of moderate to severe abdominal pain which was relieved by smooth muscle relaxants. Hb-raffimer elicited a mod- erate dose-dependent transient elevation of mean arterial blood pressure which, at a 0.1 g/kg dose, plateaued approximately 14% above the pretreat- ment value. In these subjects, there was a concomitant bradycardia without ECG abnormalities. Dose dependent elevations of serum bilirubin and LDH values were noted. In some subjects, elevated AST, ALT, creatine kinase and serum amylase were noted. All other clinical chemistry and hematologic parameters were within the normal range. In these subjects, plasma T

of Hb-raffimer ranged from 1.6 h at 0.25 ml/kg to 15.6 hours at 5 ml/kg [10]. After 1 .0 ml/kg dose, T

was 6.3 hours for the oligomeric fraction (>64 kD) and 2 .6 hours for tetrameric (64 kD) fraction. At 5.0 ml/kg, T1/2 was 18.6 h for oligomers, 7.1 h for the tetrameric fraction and 2.6 h for the fraction which appeared as dimer on SDS-PAGE.

Phase II and III Clinical Trials

Hb-raffimer has been tested in patients undergoing routine coronary artery

bypass grafting (CABG) operation and orthopedic surgery, or in patients with

certain other conditions (Table 2). In Phase II and III clinical trials of patients

undergoing elective CABG operations, Hb-raffimer was well tolerated and

appears to reduce allogeneic blood transfusion in this group as median

volume of allogeneic red cells transfused was significantly lower than patients treated with control (6% hetastarch) [11,12].

In a similar study of randomized double blind study of post-operative cardiac patients, polymerized bovine hemoglobin (HBOC-201) has been shown to moderately but significantly reduce the need for red cell transfusion [13]. HBOC-201 group required a mean of 1.7 units of red cells while blood transfusion group received a mean of 2.2 units of red cell units.

Anti-Hb-raffimer antibodies were detected in 16%–20% of subjects treated with Hb-raffimer compared to 1.4% in control subjects (G. Adamson, 2002, Personal communication). There was no apparent correlation of antibody development to clinical response or adverse events. In in vitro studies, anti- bodies from subjects receiving Hb-raffimer did not bind to normal RBCs and did not cause red cell disruption. The potential for pathological effects is unknown.

Compassionate Use Cases

As of December 2002, 21 subjects were approved by regulatory authorities for treatment with Hb-raffimer for compassionate use. Of these, 16 subjects received Hb-raffimer (Table 3). In these subjects, nadir RBC Hb ranged from 1 .1 to 5.1 g/dl and Hb-raffimer was administered in doses from 500 to 5000 ml. Of those treated, 8 of 16 subjects survived. In compassionate use cases, treatments were largely uncontrolled, thus, determination of adverse events is difficult. Elevated enzymes (amylase, lipase, bilirubin) were noted in these cases. There were incidences of transient elevation of blood pressure, abdominal pain and jaundice in some patients.

Other Potential Indications/Benefits and Issues

Solid hypoxic tumors are often resistant to radiation and chemotherapy.

Because Hb-raffimer and other acellular oxygen therapeutics have low vis- cosity, they may provide conditions for improved perfusion in solid tumors,

Table 2. Hb-raffimer completed clinical trials

Clinical setting Total no. subjects Dose ranges

Phase I safety 42 0 .25–6.0 ml/kg

Orthopedic surgery 16 1 .5–6.0 ml/kg

Orthopedic surgery 40 25 –125 g

Primary CABG 60 25 –100 g

Primary CABG 60 25 –75 g

Chronic renal failure 29 5 –10 g

Primary CABG 299 75 g

CABG, coronary artery bypass graft.

thereby resulting in higher tumor oxygen tension. Therefore, administration of Hb-raffimer and certain other oxygen therapeutics prior to radiation/

chemotherapy may increase tumor sensitivity to these therapeutic modalities [14]. It appears that in some in-vitro systems exogenous Hb may accelerate erythropoiesis [15]. In addition, acelluar Hb based oxygen therapeutics may also be useful in treating malaria since it slowed P. falciparium growth in murine malaria [16].

Of note, it has been shown that Hb based oxygen therapeutics appear to interfere with certain, predominantly colorometric, clinical laboratory tests (e.g., serum creatinine) [17]. Therefore, when interpreting clinical laboratory test results of patients following administration of Hb based oxygen thera- peutics, caution should be exercised.

Conclusion

Hb-raffimer appears to be free of severe toxicity in a variety of topload and repeat dose preclinical animal studies. In addition, Hb-raffimer does not appear to be immunogenic in animals although antibodies have been detected, and repeated dosing with heterogeneic crosslinked Hb can result in anaphylaxis in some animals. Limited information is available on acute and chronic effects of high volume infusion in animals. In addition, Hb based oxygen therapeutics have not been tested in a wide variety of possible indi- Table 3. Compassionate use cases

Sex Age Indication Nadir Hb Hb-raffimer Outcome

g/dl dose (ml)

F 57 Burns 5 .1 4500 Survived

F 54 SLE 3 .7 2750 Died

M 61 UGI bleed 2 .7 2750 Survived

F 75 UGI bleed 3 .9 2500 Survived

M 19 GI bleed 2 .7 4500 Died

F 31 Hemolytic anemia 2 .7 2250 Died

F 14 ITP with bleeding 2 .6 500 Survived

F 29 Blood loss 2 .9 1250 Survived

M 43 Aortic dissection 1 .1 3000 Died

M 39 Multiple trauma 3 .0 3500 Died

F 53 Post-op bleeding 3 .2 5000 Survived

F 51 Pancreatitis 3 .2 500 Died

F 67 Uterine CA 3 .2 1000 Died

F 67 Ruptured esophagus 2 .4 2750 Died

F 20 Menstrual bleeding 3 .1 1250 Survived

M 54 GI bleed 2 .8 750 Survived

SLE, systemic lupus erythematosus; UGI, upper gastro-intestinal; GI, gastro-intestinal; ITP,

idiopathic thrombocytopenic purpura; CA, cancer.

cations. In conclusion, Hb-raffimer and other oxygen therapeutics could be potentially life saving in certain clinical situations. Current and future clini- cal trials will evaluate expanded indications and dosing regimens.

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