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ARTIFICIAL CELLS, BLOOD SUBSTITUTES, AND BIOTECHNOLOGY

Vol. 32, No. 3, pp. 353374, 2004

Review of Hemoglobin-Induced

Myocardial Lesions

Kenneth Burhop,* Donovan Gordon, and Timothy Estep

Baxter Healthcare Corporation, Deerfield, Illinois, USA

ABSTRACT

Over 100 preclinical studies in several small and large animal species

were performed to evaluate the safety and efficacy of diaspirin cross-

linked hemoglobin (DCLHb; Baxter Healthcare Corp.) as an oxygen

therapeutic. During the preclinical evaluation of DCLHb, myocar-

dial lesions were observed following the administration of DCLHb

to certain species. These lesions were characterized as minimal to

moderate, focal-to-multifocal myocardial degeneration and/or necro-

sis that were scored using a severity scale of minimal to marked

in relative severity. The lesions were typically observed 2448 h after

single topload infusions of DCLHb into rhesus monkeys or pigs

at doses as low as 200 or 700 mg/kg, respectively. Dogs, sheep, and

rats did not develop these lesions after single-dose administrations

*Correspondence: Dr. Kenneth Burhop, Vice President Project Management

R&D, Medication, Delivery, Baxter Healthcare Corporation, DF3-2W,

One Baxter Parkway, Deerfield, Illinois 60015, USA; E-mail: ken_burhop@

baxter.com.

353

DOI: 10.1081/LABB-200027429 1073-1199 (Print); 1532-4184 (Online)

Copyright &2004 by Marcel Dekker, Inc. www.dekker.com

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of DCLHb. The left ventricular myocardium, typically near the base

of or including the papillary muscles, was the most severely affected

region, followed by the intraventricular septum and the right

ventricle. The left and right atria were usually not affected. In a

study in rhesus monkeys, morphometric analysis revealed that these

lesions comprised less than 3% of the total myocardium. Although

increases in serum enzyme activities (AST, CK, LDH) were observed

after infusion of DCLHb, myocardial-related isoenzymes did not

increase. ECG analysis and echocardiography were not altered by

these lesions, and there was no observable adverse effect on myo-

cardial function. Polymerization of DCLHb reduced, but did not

eliminate, the incidence and severity of the lesions. However, infusion

of hemoglobin solutions with reduced reaction rates with nitric

oxide (NO) resulted in a significant decrease in lesion incidenceand severity, while administration of L-NAME, an NO synthase

inhibitor, resulted in the appearance of lesions that were indis-

tinguishable from those induced by hemoglobin, suggesting that

reduction in normal NO levels is an important mechanistic factor.

Overall, the presence of myocardial lesions represents a histopatho-

logic finding that must be considered during the preclinical testing

and development of new HBOCs.

INTRODUCTION

The quest by medical researchers for a safe intravenous acellular

oxygen-carrying solution has continued for over 60 years (Amberson,

1937). This search has been driven by multiple factors, including the

risk posed by blood-borne pathogens, the necessity to match the blood

type of the donor with the recipient, the short shelf-life of stored blood,

and the immunosuppressive effects that may follow blood transfusion

(Linden and Bianco, 2001). As a result of these concerns, several

modified hemoglobin solutions are currently being developed as oxygen-

carrying resuscitation solutions.

Diaspirin crosslinked hemoglobin (DCLHb, Baxter Healthcare

Corp.) is a modified human hemoglobin solution produced by reacting

deoxygenated human hemoglobin with the crosslinking agent, bis(3,5-

dibromosalicyl) fumarate (DBBF), to form a stabilized tetramer that is

covalently linked between the alpha globin chains (Chatterjee et al., 1986;Synder et al., 1987; Walder et al., 1979; Zaugg et al., 1980). During the

initial toxicological evaluations of DCLHb, single dose studies were

performed in several standard animal species. In studies in rats and dogs,

no adverse effects of DCLHb on the heart were observed with doses up

354 Burhop, Gordon, and Estep
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to 40 mL/kg (4000mg DCLHb/kg). Dose escalation and repeat dosing

studies with DCLHb were subsequently conducted in cynomolgus

monkeys. During the microscopic examination of heart tissues in these

studies, myocardial degeneration and/or necrosis of mild-to-moderate

severity was observed in animals treated with moderate to high doses of

DCLHb.

After discovery of these lesions, a variety of different experiments

were performed to better understand their etiology, pathogenesis, and

clinical significance. The initial objective of this work was to develop

a relevant, sensitive, and reproducible animal screening model in which

heart lesions similar to those seen in primates could be produced in

response to hemoglobin administration. Another objective was to

fully characterize the myocardial lesion in those species in which thispathology was observed. Finally, the mechanism of lesion development

was studied and interactions designed to mitigate lesion development

were assessed. The purpose of this review is to summarize the results

obtained from this body of work.

OVERVIEW

Animal Model Development

Although originally found in cynomolgous monkeys, and subse-

quently observed in African green monkeys, both of these species were

significantly less sensitive than rhesus monkeys to the development

of myocardial lesions (Fig. 1). Cynomolgus monkeys infused with

2000 mg/kg of DCLHb typically developed lesions of myocardial dege-

neration and/or necrosis graded as minimal in severity (1.0 on a scale of

04) with an incidence of 67%. In contrast, rhesus monkeys developed

more severe heart lesions at relatively low doses of DCLHb.

While these data demonstrate that primates are very sensitive to the

development of this lesion, the use of primates for screening purposes

was not practical. Therefore, experiments were performed to identify a

more cost effective model that would allow rapid and reproducible

screening for identifying potential mechanisms and/or co-medicaments

with the least number of animal use issues. This goal was challenging

since heart lesions had not been observed in experiments performedwith dogs, sheep, or rats following single infusions of DCLHb, implying

that the no-effects doses in these species were greater than 4000 mg/kg.

Heart lesions with a similar appearance could be produced in rabbits,

however 10-fold higher doses of DCLHb (>3000 mg/kg) were required to

Review of Hemoglobin-Induced Myocardial Lesions 355
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produce a comparable incidence and severity to that seen in rhesus

monkeys. Moreover, in rabbits there was a significant background

incidence of degenerative, necrotic, and inflammatory heart lesions in

untreated, sham-treated, and control rabbits. The DCLHb induced heart

lesions in rabbits also typically had a more pronounced inflammatory

component (myocarditis) consisting of interstitial infiltrations of hetero-

phil leukocytes and mononuclear cells resembling lymphocytes and

macrophages. Inflammation was sometimes present in the absence of

discernible myofiber degeneration or necrosis. Due to these differences,

development of this animal model was not continued.

On the other hand, it was found that swine were a very good model

because they consistently developed heart lesions after infusion of

moderate doses of hemoglobin solutions with a low level of background

incidence. In addition, swine are generally recognized as a good species

for studying the effects of agents on the cardiovascular system and are

also a species that reproduces the hemodynamic responses observed in

humans after the infusion of DCLHb with respect to increases in mean

arterial blood pressure. Therefore, it was decided to further develop the

swine model for cardiac lesion development. With a no-effects dose less

than 700 mg/kg, swine appeared to be more sensitive than cynomolgusmonkeys and almost as sensitive as rhesus monkeys (Fig. 2).

To ensure comparability between experiments, the swine model used

for evaluation of heart lesions was standardized. All experiments utilized

young crossbred domestic swine that weighed between 10 and 20 kg.

Figure 1. Dose response characteristics of heart lesion development in primates

48 h after a single infusion of DCLHb.

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Approximately 24 h prior to dosing animals were anaesthetized and

chronic catheters were placed in the jugular vein, and sometimes in the

carotid or femoral artery, for infusion of test and control articles and

blood sampling for clinical chemistry analysis. Test or control solutions

were infused intravenously using an infusion pump at a constant rate of

1 mL/kg/min. When assessing the effect of interventions, a standardized

DCLHb dose of 2000 mg/kg was typically infused. This dose was

found to be the best compromise between minimizing volume load and

consistently producing lesions. As a treated control, human serum

albumin (HSA) that was oncotically matched to each test article was

infused into separate swine at the same rate and volume. Blood samples

were routinely taken at baseline, immediately postinfusion, and at 8, 24,

and 48 h postinfusion of test or control article for the measurement of

various enzyme levels. Clinical observations were performed throughout

the experiments. At approximately 48 h postdosing, the animals were

euthanized, a complete necropsy examination was performed, and varioustissues, including the heart, were taken for histopathologic evaluation.

The heart specimens routinely examined consisted of the atria, left

and right ventricular free wall, and inter-ventricular septum, includ-

ing associated papillary muscle. As noted in more detail below, 48 h

Figure 2. Incidence and dose response of myocardial degeneration and necrosis

in various species.

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postdosing was determined to be the optimal time for sacrifice because

this interval allowed enough time for the lesion to develop, but yet was

not so long after the initial insult that damaged myocardial cells might

already be removed and cleared by repair processes in the body.

Lesion Characterization

Once sensitive and reproducible animal models were identified,

the hemoglobin-induced myocardial lesions were characterized in more

detail. In primates, myocardial lesions observed after hemoglobin

infusion consist of a minimal to moderate myocardial degeneration

characterized by cytoplasmic swelling and vacuolization of myofibers,occurring primarily in the left ventricle and/or septum. The lesions are

usually focal or multifocal in distribution, sometimes only involving a

few cells, although occasionally they may be locally extensive. Often the

degeneration is associated with foci of coagulative myofiber necrosis

that display a homogeneous to granular eosinophilic staining cytoplasm.

Enlargement of the nuclei (karyomegaly) of myocytes and minimal to

mild interstitial fibrosis are also frequently associated with the degene-

rative lesions. The karyomegaly is interpreted as a reactive change. In

some animals, a mild lymphocytic inflammatory infiltrate is also present.

Similar to the lesions observed in primates, the lesions found in

swine were described as myocardial degeneration and/or necrosis, with

a focal to multifocal distribution, and of minimal to moderate severity.

The myocardial degeneration was characterized by focal cytoplasmic

swelling and slight hypereosinophilia of myofibers, whereas necrosis

was evident as areas of moderate to marked homogeneous to granular

eosinophilic cytoplasmic staining with shrinkage (pyknosis), fragmenta-

tion (karyorrhexis) or lysis (karyolysis) of the nuclei. Necrotic areas were

usually associated with a mononuclear inflammatory infiltrate consisting

of macrophages and a lesser number of lymphocytes. Mineralization

of cellular debris could also be observed occasionally at some sites of

necrosis. Shown in Fig. 3 are microscopic changes involving the left ven-

tricle of swine illustrating typical lesions of necrosis classified as minimal

or moderate following a single IV infusion of 2000 mg/kg DCLHb.

To quantify the characteristics of the cardiac lesions using anatomic

pathology, both incidence and severity parameters were utilized. Incidencewas defined as the number of hearts that exhibited any evidence of lesion

formation divided by the total number of hearts examined (e.g., 2/4).

Severity was a measure of lesion intensity and extent that was scored by

the evaluating pathologist on an ascending scale of 04. Lesions of

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Grade 1 were considered minimal, Grade 2 were considered mild, Grade 3

were moderate, and Grade 4 lesions were severe. In a given group of tissue

specimens, an overall average severity score was calculated by summing

the severity grades for each affected heart and dividing by the total

number of hearts evaluated in that group.

By combining data from several studies, the variation of lesion

incidence and severity after the administration of single doses of DCLHb

was defined in rhesus monkeys (Table 1). Above the no-effect level of100 mg/kg, a dose-response relationship was observed, with a 100%

incidence and maximization of the average lesion severity at a score

of approximately 2.3 at 700mg/kg. At substantially higher doses, no

significant increase in the severity of the lesion was observed. To examine

Figure 3. Photomicrographs of H&E stained sections of myocardium fromthe left ventricle of different swine following infusion of 2000mg/kg of

DCLHb, illustrating typical heart lesions of different severity. (nec necrosis).

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the severity of the lesions quantitatively, a morphometry study was con-

ducted in rhesus monkeys after the infusion of 2000 mg/kg of DCLHb.

In this study, five animals were infused intravenously with 20 mL/kg

of a 10 g% DCLHb solution at a rate of 1.0 mL/kg/min, the animals

were sacrificed, seven days postinfusion, and the heart tissue collected,

fixed, sectioned, and examined by morphometric analysis. Myocardial

lesions were observed in four out of five monkeys. In the affected

hearts, the mean fraction of tissue with degeneration or necrosis was

1.26% (range 0.152.92%) (Fig. 4). The most sensitive tissue was the

left ventricular papillary muscle, followed by the left ventricular free wall

and inter-ventricular septum. The right ventricle was sometimes affected,

but to a much lesser degree. The atria were almost never affected.

In this experiment, as well as in many subsequent experiments in

a number of different animal species, an attempt was made to identify

a clinical pathology marker for myocardial injury that would allow

for more rapid and sequential monitoring of lesion development.

Unfortunately, to date no statistically significant increases in typical

markers of myocardial injury, such as the myocardial isoenzyme of

creatine kinase (CK-MB) or the lactic dehydrogenase isoenzyme LDH-1,

were observed following infusion of large and repeated doses of DCLHb,

even into sensitive species such as the rhesus monkey. These studies

were evaluated by an outside expert investigator that utilized specific

monkey immunoassays for analysis of CK-MB activity. The small

percentage of myocardium involved in the monkeys is consistent with

the observation that no significant levels of cardiac specific enzymes wereidentified in the plasma after hemoglobin infusion. In fact, to date no

surrogate marker of myocardial injury has been identified.

The self-limiting nature of the severity of the lesion is also

apparent in the data accumulated in a 28-day repeat dose toxicity study

Table 1. Incidence and severity of heart lesions

in primates.

Dose

(mg/kg) Incidence

Average

severity score

50 0/5 0

100 0/5 0

200 1/5 0.6

350 3/5 1.2

700 5/5 2.0

2000 14/15 2.3

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in rhesus monkeys. This study investigated the toxicity of DCLHb in

awake rhesus monkeys following daily infusions of DCLHb for 28 days,

with doses ranging from 1000 to 4000 mg/kg/day. The planned sacri-

fice intervals were either Day 29 (after receiving 28 doses) or Day 64,

with the latter providing 28 days of daily dosing followed by a recovery

period. Although some animals received cumulative doses as large as

112,000 mg/kg of DCLHb, the severity of the lesions (Table 2) was no

greater than that seen in the earlier single dose studies. Even more

intriguing was the observation that many animals had no histologic

evidence of myocardial lesions following infusion of DCLHb at cumu-lative doses substantially greater than the 100 mg/kg no-effects dose in

single dose studies. For example, only three of seven monkeys dosed

with 2000 mg DCLHb per kg and examined at Day 29 had histological

evidence of myocardial lesions, despite the fact that they each received

Figure 4. Percent of heart tissue with degeneration and/or necrosis 7 days

following infusion of 2000 mg/kg of DCLHb into rhesus monkeys.

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Tabl

e2.

Incidenceofmyocardialdegenerationvs.

Doseinrhesusmonkeysduring

a28dayrepeatdosestudy.

Doselevel(mg/kg)

1000mg/kg(10mL/kg)

2000mg/kg(20mL/kg)

4000mg/kg(40mL/kg)

Earlydeath

sacrificea

bofanimalswithlesions

0

2(2)b

(2-mild)

8(2)b

(2-minimal,

3-mild,

3-moderate)

bTotalofa

nimalsexamined

2

3

8

Cumulativedose(mg/kg)

Day29sacrifice

28,0

00mg/kg(280mL/kg)

56,0

00mg/kg(560mL/kg)

1,12,0

00mg/kg(1120mL/kg)


2(1.5

)b

3(1.3

)b

2(2)b

(1-minimal,

1-m

ild)

(2-minimal,

1mild)

(mild)

bTotalofa

nimalsexamined

7

7

3

Day64sacrifice


0

0

0

bTotalofa

nimalsexamined

3

2c

1

Note:TheD

CLHbwasformulatedataconcentrationof100mg/mL.

a

Unfortun

ately,someanimalsdiedduringthisstudyofothercauses(additionalstudiesstr

onglysuggestedfluidoverloadasthe

causeofdeath),butyet,theirheartsandothertissu

eswereexamined.

bAveragenumericalscore

1

minimal,

2

mild,

3

moderate,4

severe.

cEvidenceof

minimalfocalmyocardialfibrosiswhic

hmayhavebeenrelatedtomyocardial

degeneration.

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a cumulative dose of 56,000mg/kg of hemoglobin. This is in direct

contrast to the results in monkeys that received a single 2000 mg/kg

infusion (Table 1), in which the incidence of heart lesions was 14/15

monkeys. The explanation for this difference in response is not known

with certainty, but may reflect the competency of the cardiac tissue repair

process.

An advantage of utilizing swine in the study of cardiac lesions was

the ability to perform chronic experiments in unanaesthetized animals,

which allowed for easier and more thorough examination of cardiovas-

cular function. This permitted assessment of the functional consequences

of cardiac lesion development utilizing electrocardiography (ECG). To

do so objectively, cardiac function in swine infused with DCLHb or HSA

control solutions was compared. DCLHb (2000 mg/kg) or an oncoticallymatched HSA solution was infused into swine at a rate of 1 mL/kg/min.

Cardiac function was assessed preinfusion, and 24 and 48h post-

infusion, by ECG analysis performed by a veterinary cardiologist who

was blinded to treatment. The cardiac index and selected clinical

chemistry parameters were also measured. At 48 h postinfusion, cardiac

tissue was evaluated microscopically. Heart lesions were observed in all

six DCLHb treated pigs with an overall pathology score of 2.7, while no

lesions were observed in animals infused with HSA. In DCLHb treated

pigs, serum aspartate transaminase (AST) concentrations increased

from a baseline of 28 2 to 126 8 IU/mL at 48 h postinfusion, and

total serum creatine kinase (CK) concentrations increased from a

baseline of 20 2 to 675 SU/mL. These increases were typical and

representative of the response seen following infusion of DCLHb into

swine. Yet, none of the animals exhibited disturbances in cardiac rhythm

or conduction, although minor changes in T-wave morphology and

polarity were observed in both groups. No clinically significant effect

on cardiac function by DCLHb could be discerned in this study.

To assess the time course of lesion development, tissues collected

from animals sacrificed at different time intervals were examined micro-

scopically. From these examinations, it was concluded that the degene-

rative myocardial changes appeared as early as 16h postinfusion.

Electron microscopy was required to detect the changes at early time

points. While some degenerative cells became necrotic, others apparently

recovered their normal appearance and function. Necrotic tissue

was ultimately removed and subsequently replaced, in part, by fibrousconnective tissue. Another component of the recovery process was the

enlargement of myocytes adjacent to affected areas, which probably

represented a physiologic hypertrophy caused by increased functional

demand on the unaffected cells. Morphologic evidence of muscle fiber

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regeneration was also evident in swine. The long-term consequence of

myocardial lesion development was the loss (necrosis) of a small fraction

of the myocytes originally present that were replaced by proliferation of

connective tissue and possible regeneration of muscle cells.

Co-medicament Experiments

Using the standardized swine model, the mechanism of hemoglobin-

induced heart lesion formation and possible methods for mitigation of

this process were extensively examined. In considering these experiments,

it should be noted that the standardized DCLHb dose of 2000 mg/kg

produced heart lesions in 96% of the treated animals with an averageseverity score of approximately 2 (n 105, Table 3). Occasionally, lesions

were seen in animals that were infused with HSA, but the incidence

and severity was extremely low. Background lesions were not routinely

seen in normal, untreated swine. For the purposes of this manuscript,

experimental results will be summarized.

One set of experiments was designed to assess whether there

was a specific contaminant in DCLHb that was responsible for the

cardiac pathology (Table 4). Infusion of DCLHb that was subjected to an

Table 4. Incidence and severity of heart lesions in pigs infused with DCLHb

and co-medicaments.

Treatment variation

Hb variations

DCLHb dose

(mg/kg) Incidence %

Mean

severity

Chromatographically purified 2000 4/4 100 2.5

Human SFHb 2000 4/4 100 1.3

Porcine SFHb 2000 5/6 83 2.3

CyanometDCLHb 2000 3/4 75 1.3

Table 3. Heart lesions following infusion of DCLHb (reference range in swine).

Treatmentvariation

DCLHb dose(mg/kg) Incidence %

Meanseverity

Cumulative DCLHb 2000 101/105 96 2.1

HSA (8 g/dL) 1600 3/27 11 0.1

Sham 0 0/17 0 0

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additional chromatographic purification step produced the same results

as the standard DCLHb solution, suggesting that contamination was not

responsible for causing the lesion. Additionally, infusion of purified,

uncross-linked, human stroma-free hemoglobin (SFHb) produced the

same heart lesion as DCLHb with the same incidence, albeit with a

slightly reduced severity. The reduced severity was probably due to the

considerably shorter circulating half-life of unmodified hemoglobin

compared to DCLHb due to the rapid excretion of the SFHb through

the kidney. This would be expected to somewhat reduce the direct

exposure of the heart to the SFHb.

Furthermore, infusion of purified swine SFHb into pigs caused the

same heart lesion as that seen following infusion of DCLHb or human

SFHb, suggesting that this phenomenon is probably a more general

property of acellular hemoglobins. These experiments also demonstrated

that myocardial lesions were not due to infusion of a human protein

into a nonhuman species. Finally, in order to investigate if heart lesion

formation could be related to the reduced heme component of DCLHb,

the effect of conversion of the heme to the cyanomet form was examined.

It was found that conversion to the cyanomet form had no significant

effect on the incidence and/or severity of the heart lesion, although

this result may have been compromised by in vivo conversion of

cyanometHb to reduced Hb.

To gain further insight into the potential mechanism of heart lesion

formation, as well as to identify potential interventions that would be

clinically useful, the effect of co-administration of many different agentswith varying pharmacologic actions was assessed. In addition, the impact

of variations in the hemoglobin administration protocol were evaluated.

In the typical experiment, the standardized swine testing protocol was

utilized with the key independent variable being the comedicament

or specific protocol variation. In some cases, a variety of dosing regimens

or administration protocols were evaluated with each agent. The primary

endpoint in each case was histologic evaluation of the hearts as quanti-

fied by the myocardial lesion incidence and overall severity score.

Comedicaments and protocol variables that were examined include the

following:

Antihypertensives: Nicardipine, adenosine, phenoxybenzamine, pro-pranolol, verapamil, captopril, ATP-MgCl2, metroprolol, halothane,

sodium nitroprusside, l-arginine.

Anticoagulants: Aspirin, dipyridamole, heparin.

Anti-inflammatory: Dexamethasone, ibuprofen, benadryl.

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Antioxidants: Taurine, vitamin E, selenium, ascorbate, OTC (l-2-

oxothizolidine-4-carboxylic acid), MPG (N-2-mercaptopropionyl

glycine), oxypurinol, mannitol, lactobionate, carnitine, allopurinol,

lipoic acid.

Iron binding: Deferoxamine.

Protocol variations: Topload (hypervolemic) infusion; differing levels

of isovolemic exchange transfusion; predosing with hemoglobin;

dosing of hemoglobin in hemorrhage/resuscitation protocols; animal

source; animal gender; effect of splenectomy, hydration state, or

anesthesia; effect of catecholamine depletion before hemoglobin

administration.

After extensive testing, no effective comedicament was identified,nor was any definitive mechanism of action elucidated in this series of

experiments. Likewise, the administration protocol did not seem critical,

as similar lesions were observed if the hemoglobin was administered as

a volume load, by exchange/transfusion, or to hemorrhaged animals.

EFFECTS OF HEMOGLOBIN MODIFICATION

Polymerization

To assess the potential effect of the molecular size of the hemoglobin

molecule on the generation of heart lesions, several experiments were

performed with different DCLHb derivatives. In one study, DCLHb

was treated with gluteraldehyde to create a polydisperse family of hemo-

globin polymers. This solution was then diafiltered against a membrane

having a nominal 300,000 Dalton molecular weight cut-off. The resulting

retentate solution was essentially free of unpolymerized hemoglobin

tetramers, while the filtrate was enriched in this molecular weight frac-

tion. After diafiltration into the same electrolyte vehicle, these two

solutions were infused into swine. The lesion incidence and overall

severity scores were lower in animals that received the polymerized

DCLHb retentate (2/5 and 0.5, respectively) compared to those animals

treated with filtrate (5/5, 2.6). Similar results were obtained when

DCLHb was polymerized with bifunctional polyethylene glycol basedreagents. In most cases, both the incidence and severity of the heart

lesions could be reduced, but not completely eliminated, by increasing

the molecular size of the DCLHb. These data imply that the size of the

hemoglobin molecule does have an influence on the generation of heart

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lesions, but that the lesions could not be completely eliminated in

sensitive species simply by polymerization.

Genetic Modifications to Inhibit Hemoglobin

Reaction with Nitric Oxide

As part of the investigation into possible mechanisms of cardiac

lesion development, the potential role of nitric oxide was investigated.

Native hemoglobin interacts very strongly with nitric oxide (NO), a

ubiquitous and potent chemical messenger found throughout the body.

In vivo, nitric oxide scavenging by hemoglobin occurs primarily via

two rapid reactions: the oxidative reaction of NO with oxyhemoglobin

to produce nitrate and methemoglobin, and NO binding to deoxy-

hemoglobin to form a stable complex (Patel, 2000). Both reactions likely

contribute to in vivo NO scavenging, with the relative significance

depending on local abundances of oxy- and deoxyhemoglobin. There is

also evidence that this scavenging of NO may be associated with some of

the adverse outcomes observed with the first generation hemoglobins.

For example, studies in rats have clearly demonstrated that increases

in mean arterial pressure observed immediately after hemoglobin

infusion correlate directly with the rate of NO scavenging; as the NO

scavenging is decreased, the pressor response is decreased (Doherty et al.,

1988). More recently, it has been reported that the chronic inhibition

of nitric oxide production by L-NAME causes myocardial infarction inrats (Moreno et al., 1997; Ono et al., 1999).

L-NAME is an inhibitor of the enzyme nitric oxide synthase that

produces NO. Infusion of L-NAME into swine resulted in heart lesions

similar in incidence, severity, and appearance to the lesions observed after

infusion of DCLHb (Table 5).

To systematically investigate the role of hemoglobin/NO interac-

tions, a series of genetically altered hemoglobins were produced using

Table 5. Incidence and severity of heart lesions in pigs infused with DCLHb and

various co-medicaments.

Treatment variation

{Co-med}

DCLHb dose

(mg/kg) Incidence %

Mean

severity

L-NAME alone 100 mg/kg 1/1 100 2.0

L-NAME alone 40 mg/kg 5/5 100 1.5

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recombinant technology. These hemoglobins were specifically designedto exhibit varying rates of reaction with NO. Recombinant hemoglobins

with NO scavenging properties similar to those of native human hemo-

globin (e.g., rHb1.1 produced by Somatogen) produced heart lesions

with the same incidence and severity as those seen with DCLHb. In

contrast, recombinantly produced hemoglobin solutions that contained

heme-pocket modifications that reduced the rate of nitric oxide interac-

tion exhibited a reduced rate of heart lesion formation after infusion into

swine (Table 6). More specifically, a hemoglobin variant with a 25-fold

decrease in nitric oxide reactivity produced no detectable heart lesions in

swine. This variant was internally crosslinked by recombinant techniques,

and was very similar to rHb1.1 or DCLHb with respect to molecular

weight, oxygen affinity, and oxygen binding kinetics.

As a result of these promising results in swine, this same hemoglobin

variant was subsequently tested in rhesus monkeys. In contrast to the

results in swine, myocardial lesions were observed in all of the test

animals following infusion into monkeys, although the lesion severity

was substantially reduced. This led to exploration of the effect of the

combination of polymerization and a reduced rate of NO interaction

on heart lesion development. To do so, an intramolecularly cross-linked

hemoglobin with reduced NO reactivity was polymerized and deriva-

tized with a bifunctional polyethylene glycol reagent. This new material,

designated as rHb2.0 for Injection, was evaluated in both single dose

and repeat dose studies in rhesus monkeys, as well as in swine and rats. In

a single dose toxicity study in rhesus monkeys, no cardiac lesions were

observed in animals that were sacrificed 48 h after receiving a single doseof either 500 (n 8), 1000 (n 8), or 2000mg/kg (n8) of rHb2.0. In a

separate group of monkeys that were sacrificed two weeks after dosing,

there was also no evidence of myocardial lesions. In a repeat dose study

in which rhesus monkeys received every other day infusions of either 1000

Table 6. Incidence and severity of heart lesions in pigs infused with various

hemoglobin solutions.

Treatment

agent

KNO

(mM1S1)

MW

(kD)

Incidence

(%)

Overall

severity

HSA 64 3/27 (11) 0.1

DCLHb 60 64 35/37 (95) 1.9

rHb1.1 60 64 4/4 (100) 2.0

NO Mutant Hb 2 64 0/4 (0) 0

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or 2000 mg/kg of rHb2.0 for Injection (10 animals per dose group) for a

total of seven infusions over 13 days, only one animal in the high dose

group exhibited a myocardial lesion, and it was focal and of minimal

severity. Moreover, according to the reviewing pathologist, this lesion

was of uncertain association with study drug administration since a back-

ground lesion of similar appearance is sometimes observed in monkeys.

None of the other monkeys examined at the 48 h sacrifice interval, or

in the recovery group sacrificed 14 days after receiving the seventh

dose, had any evidence of myocardial lesions. In total, only one of 56

monkeys receiving rHb2.0 for Injection exhibited any finding of myofiber

degeneration or necrosis. These data suggest a major role for nitric

oxide depletion in the mechanism of myocardial lesion development.

In a swine cardiovascular function/safety study in which the heartswere examined histologically, there were no cardiac lesions observed in

swine infused with 2000 mg/kg of rHb2.0 for Injection. However, animals

receiving DCLHb as a positive control exhibited cardiac lesions with

a similar incidence and severity to those observed in previous studies.

In contrast to first generation hemoglobin solutions, rHb2.0 for

Injection did produce observable cardiac changes in rats in a single dose

rat toxicity study. However, both the incidence and severity of these

lesions did not appear to follow a dose-response relationship and none

of the rats in the recovery group, sacrificed 14 days after dosing ( n 10/

group), had evidence of myocardial lesions. Interestingly, myocardial

lesions attributable to rHb2.0 were not observed in a hemorrhage/

resuscitation study performed in rats nor in a separate diabetic rat study.

The difference and significance of these findings in rats between the first

and second generation HBOCs is not known, although previous testing

would suggest that the results in swine and primates are more relevant

to man.

DISCUSSION

Over the past decade there has been a substantial effort by Baxter

researchers to understand the mechanism of heart lesion formation

following the intravenous infusion of hemoglobin solutions (Burhop

and Estep, 2001). Characterization of the lesion suggests that there is

significant variation among species with respect to susceptibility to thedevelopment of this pathology, with swine and primates being the most

susceptible, and dogs and rodents being relatively insensitive. In addition,

only a small percentage of heart muscle cells appears to be affected in

even the most sensitive species, since the fraction of necrotic cells plateaus

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an average molecular weight of several hundred thousand Daltons, and

mutation of heme pocket amino acids to reduce the rate of heme

interaction with nitric oxide, result in a reduction in the incidence and/or

severity of cardiac lesion formation. It is believed that polymerization

acts to reduce the rate of hemoglobin extravasation into heart tissue

and thereby lowers the hemoglobin concentration near sensitive cells,

while modification to reduce the rate of interaction with nitric oxide

results in a reduced rate of NO scavenging which has a salutory effect

on lesion development. Moreover, these two modifications appear to be

at least somewhat additive in that the lowest incidence of heart lesion

development in rhesus was achieved with hemoglobin molecules that

were both polymerized and altered to reduce the inherent rate of NO

scavenging. On the basis of these observations, an hypothesis can begenerated for the mechanism by which hemoglobin induces the formation

of cardiac lesions. The pertinent facts are that:

. Hemoglobin scavenges nitric oxide.

. Infusion of nitric oxide inhibitors can produce myocardial lesions.

. The papillary muscle of the left ventricle is the most sensitive

myocardial tissue with respect to the adverse effects of hemo-

globin infusion and nitric oxide inhibition.

. The left ventricular papillary muscle is one of the highest oxygen

consuming tissues in the body.

. Infusion of hemoglobin into sensitive species, such as pigs

and monkeys, produces significant increases in blood pressure

and thereby an increase in after-load on the heart. This results

in increasing myocardial oxygen demand that can result in a

localized tissue hypoxia.

. Polymerization of hemoglobin, which can slow down, but not

completely eliminate, extravasation of hemoglobin from the

vascular space, reduces both the severity and incidence of the

myocardial lesions.

. Recent data suggest that inhibition of nitric oxide synthesis

increases mitochondrial oxygen consumption and may also affect

Ca hemostasis (Arstall and Kelly, 1999; Bernstein et al., 1996;

Boveris et al., 2000; Henry and Guissani, 1999; Shen et al., 1994;

Zhao et al., 1999).

When considered as a whole, these facts suggest that infusion of

hemoglobin leads to enhanced oxygen consumption throughout the

body as a consequence of a reduction in tissue levels of nitric oxide.

In the heart, especially in the papillary muscle, there is an increase

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in mitochondrial oxygen consumption due not only to the decrease in

NO levels, but also as a consequence of the increased after-load due

to peripheral vasoconstriction. As a result, oxygen demand may exceed

oxygen supply in the most sensitive cells in the heart, leading to

microscopic areas of hypoxia, cell injury, and ultimately death. Likewise,

as a result of interactions between hemoglobin and NO, there may be

alterations in calcium hemostasis that may ultimately lead to myocardial

cell degeneration and necrosis (i.e., produce contraction band necrosis).

The inflammatory response that is seen in conjunction with the necrosis

is likely a secondary event that represents removal, by macrophages, of

necrotic myocardial cells.

It is important to note that to date no evidence of a hemoglobin-

induced myocardial lesion has been observed in man. Furthermore,there have been no increases seen in enzymatic markers of myocardial

injury such as CK-MB or troponin-I in any of the human clinical

trials conducted with DCLHb. However, the detection of hemoglobin

induced heart lesions in humans is confounded by the fact that patients

treated with hemoglobin therapeutics have myocardial damage from

other causes. It is therefore unclear whether the lesions observed in

swine or primates occur in man. Nevertheless, the presence of myocardial

lesions represents a histopathologic finding that must be considered

during the testing and development of new hemoglobin therapeutics

and confirmation of the basic mechanism of lesion development

would be helpful in estimating the potential clinical relevance of this

finding.

ACKNOWLEDGMENTS

The authors would like to thank the host of technical staff across

a variety of research and development groups at Baxter who made

significant contributions to this research. Without all of their help, this

work would not have been possible. The expert advice and scientific input

of outside consultants such as Dr. Robert Jennings from Duke University

is also greatly appreciated.

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