Innovation

Blood Relatives: Artificial Oxygen Carriers

between Promise and Concern

Chandra Shekhar

Massive bleeding during childbirth kills

more than 100,000 women worldwide

each year. Blood loss kills a vast num-

ber of accident victims before they can

reach a trauma center. Loss of blood is

also blamed for 50% of deaths on the

battlefield. Prompttransfusion ofblood

could prevent most of these deaths.

Unfortunately, since the life-giving fluid

has to be kept refrigerated and needs

to be typed and cross-matched before

use, it is rarely available in ambulances

or in combat situations. And with a

growing population accompanied by

a decline in blood collection, even the

best-equipped hospitals are not im-

mune to shortages. Any catastrophic

event such as an earthquake or a

terrorist attack could rapidly exhaust

available supplies.

Blood shortages in developing coun-

triesareparticularlyacute,accordingto

the World Health Organization. These

nations account for more than 80% of

the world’s population, yet collect less

than 35% of the global blood supply.

Safety standards there are often lower

aswell.TheUnitedStatesandotherde-

veloped countries have come a long

way since the 1980s, when thousands

of hemophiliacs died from receiving

HIV-tainted blood products. But more

than 70 developing countries still

don’t test all their donated blood for

HIV, hepatitis, or syphilis. ‘‘Blood is in

shortsupply,andthebloodthatisavail-

able is often unsafe,’’ says Harvey

Klein, who heads the department of

transfusion medicine at the National In-

stitutes of Health (NIH). ‘‘A substitute

that doesn’t need typing and doesn’t

transmit disease would be invaluable.’’

These considerations give new ur-

gency to the search for a blood sub-

stitute—or more correctly, an artificial

oxygen carrier. Scientists have known

since the 19th century that a protein

in red blood cells (RBCs), hemoglobin,

is essential for oxygen transport. Early

attemptsin the1930stotransfuseaso-

lution of purified cell-free hemoglobin

largely failed. It was only in the 1950s

that scientists discovered the protein’s

molecular structure: a tetramer con-

sisting of four polypeptide chains,

each equipped with a heme unit that

can bind to an oxygen molecule. With

about 250 million of these oxygen-hun-

gry molecules packed into each of the

25 trillion RBCs in a human body, he-

moglobin is the key to an amazingly

effective system that delivers the right

amountofoxygentotissuesat alllevels

of activity.

Outside the RBC, however, the he-

moglobinmoleculeislesswellbehaved:

it enters blood vessel walls, causing

over-oxygenation and reduction of the

nitric oxide level that maintains blood

vesselwallsinarelaxedstate.Theresult

is vasoconstriction—a narrowing of

arteries and capillaries that causes

blood pressure to shoot up. Free hemo-

globin also generates reactive oxygen

species such as hydrogen peroxide

and superoxide ions that damage cells

and tissues. Furthermore, hemoglobin

molecules outside RBCs rapidly break

down into dimers highly toxic to the kid-

ney, as was observed in the first human

safety trial of free hemoglobin in 1978.

To make a more stable compound,

a common strategy is to crosslink re-

active amino groups that lie on the he-

moglobin molecule’s surface. Cross-

links within the molecule can prevent

it from breaking down into toxic di-

mers, but not from getting into blood

vessel walls. Products based on this

approach, such as Baxter’s HemAs-

sist, have fared poorly in human trials

and have largely been abandoned.

Crosslinks across molecules, on the

other hand, result in large polyhemo-

globin aggregates that don’t infiltrate

vascular walls as easily. Examples of

these so-called second-generation

oxygen carriers are Northfield Labora-

tories’ PolyHeme and Hemosol’s He-

molink, both based on human hemo-

globin, and Biopure’s Hemopure,

based on bovine hemoglobin. All three

products have shown mixed results in

human trials; while they reduce the

need for blood transfusion, they seem

to increase the risk of cardiovascular

and other problems. ‘‘The good news

is that they deliver oxygen effectively,’’

says Klein. ‘‘But they show disturbing

evidence of toxicity.’’

In addition to posing safety con-

cerns, the products are challenging

to manufacture because they require

extraction, purification, and modifica-

tion of hemoglobin. Further, the raw

material in most cases is either cow’s

blood, which might raise disease con-

cerns, or expired human blood, which

is dwindling in supply.

Fluorocarbon emulsionsarea poten-

tial alternative to hemoglobin. Devel-

oped initially during the Manhattan

Project as an inert buffer material for

handling corrosive uranium isotopes,

fluorocarbons turned out to be good

solventsforoxygen.Inadramaticdem-

onstration, scientists showed in 1966

that mice immersed in an oxygen-satu-

rated fluorocarbon liquid could survive

for up to ten minutes. As oxygen car-

riers, fluorocarbons offer several ad-

vantages: they are nontoxic, use easily

available raw materials, and are simple

to manufacture. ‘‘We just put a handful

In a dramatic demonstration, scientists showed in 1966 that

mice immersed in an oxygen-saturated fluorocarbon liquid

could survive for up to ten minutes.

Chemistry & Biology 14, October 2007 ª2007 Elsevier Ltd All rights reserved 1

of chemicals together and make it into

an emulsion,’’ says Thomas Drees,

president and CEO of Sanguine, a fluo-

rocarbon manufacturer. ‘‘The process

is not capital or labor intensive.’’

Althoughlesstoxicthanhemoglobin,

fluorocarbons are not problem free.

They can provoke the complement

system, affect platelet function, and

potentially cause strokes. And unlike

hemoglobin-based products, which

can deliver oxygen very effectively un-

der physiologic conditions, fluorocar-

bon-based carriers require the patient

to breathe oxygen-enriched air. ‘‘The

bulk of evidence suggeststhat a hemo-

globin-based carrier is more likely to

come to market,’’ says Klein.

Both classes of product have shown

mixed results in clinical trials. HemAs-

sist showed promise in early studies,

but in Phase III trials it seemed to

increase mortality rates. Baxter sus-

pended a Phase III trial of the product

in 1998 because of adverse outcomes.

Hemolink fared similarly: although one

Phase III trial demonstrated its effec-

tiveness in reducing blood transfusion

needs, another Phase IIb trial of the

product was suspended in 2003 be-

cause of increased incidence of heart

problems. A Phase III trial completed

in Europe in 2002 showed that Oxy-

gent, a fluorocarbon-based carrier

made by Alliance, could effectively

reduce blood transfusion needs during

surgery. However, another Phase III

trial of this product in the U.S. ended

early in 2001 because of an apparent

increase in the risk of stroke. In a Phase

III trial of Hemopure completed in

2002, nearly 60% of surgery patients

getting the product did not need any

blood transfusions at all. However,

safety concerns caused FDA to reject

Biopure’s 2006 proposal for a Phase

III trial of the product for hemorrhagic

shock. (At present Hemopure is ap-

proved in South Africa; in the U.S,

a low-grade version is available for

veterinary use.) And in a recently com-

pleted Phase III trial by Northfield Lab-

oratories, trauma victims infused with

PolyHeme, which performed well in

earlier, smaller human studies, fared

worse than controls who got saline fol-

lowed by blood. Such setbacks have

landed many of the manufacturers in

serious financial difficulty.

Many of these failures can be attrib-

uted to poor study design, says Jona-

than Jahr, an anesthesiologist at the

University of California, Los Angeles,

who has led five clinical trials of oxy-

gen carriers. Jahr points out that no

pathway currently exists for indepen-

dent investigators to obtain and study

these products, and he urges the Food

and Drug Administration (FDA) and the

NIH to rectify this situation. ‘‘Other-

wise, companies rush into clinical trials

without a product that was validated in

the first place,’’ he says.

Some companies express frustra-

tion with regulatory policies. ‘‘Safety

standards kept creeping up as you

went towards a trial,’’ says David Bell,

vice president of drug development

at Hemosol, referring to the suspended

2003 Hemolink clinical trial. ‘‘You

started to move away from the true

benefit of the product and started fo-

cusing on the risks.’’ For instance, reg-

ulators may require a product de-

signed to resuscitate trauma victims

in the field, where blood is typically

unavailable, to be at least as safe as

blood,sinceitcouldpotentiallybeused

‘‘off-label’’ in a different setting. Such

requirements are a challenge for the in-

dustry, agrees Jahr. ‘‘A head-to-head

comparison with blood is the worst-

case scenario for an oxygen carrier,’’

he says. Regulatory officials, however,

argue that their task is complicated by

thescientific complexity of oxygencar-

riers. Inherent product toxicities and

lack of adequately predictive animal

models make it ‘‘difficult to judge the

relative benefits and risks of proposed

trials,’’ says Jay Epstein, director of the

Office of Blood Research and Review

at the FDA.

While existing oxygen carriers await

regulatory approval, some companies

are making ‘‘third-generation’’ prod-

ucts that modify hemoglobin in new

ways. Examples are Hemosol’s HRC

101, which attaches a starch polymer

to the hemoglobin molecule; Sangart’s

Hemospan, which affixes strands of

polyethylene glycol to the molecule;

and Oxyvita’s Oxyvita, a polymerized

hemoglobin made without crosslinks.

While HRC 101 and Oxyvita are in the

preclinical stage, Hemospan is in

Phase III trials in Europe for use during

surgery. According to the manufac-

turers, preliminary results are promis-

ing: the new products are less toxic

and don’t constrict blood vessels as

much. ‘‘We’ve tuned the oxygen re-

lease parameters so that the capillaries

are open and flowing and oxygen is

delivered to tissue,’’ says Robert Win-

slow, Sangart’s president and CEO.

What will be the commercial poten-

tial for an oxygen carrier, once it is

licensed? That depends on the appli-

cation, says Eugene Trogan, biotech-

nology analyst for the investment

banking firm Morgan Joseph. For situ-

ations where blood is unavailable,

such as field resuscitation, Trogan es-

timates the U.S. market to be about

$150 million per year. But if the prod-

uct replaces blood in the 1.8 million

transfusions that are given each year,

the market could exceed a billion dol-

lars, he says. A longer shelf life and

the lack of a typing/cross-matching re-

quirement would, in theory, make such

a product attractive. To access this

larger market, however, the product

would need to be at least as safe as

blood. It would also need to last for

a reasonably long time in the human cir-

culation; current products, unfortu-

nately, work only for a few days,

whereas a RBC circulates for nearly

four months. ‘‘This field has a tremen-

dous potential in the marketplace,’’

says Trogan. ‘‘The problem is that it

hasbeenmiredwithclinicaldifficulties.’’

Leading oxygen carrier manufac-

turers have spent $150–$500 million

in research and development, Trogan

estimates. Given this outlay, the prod-

ucts may cost $500–$1000 per unit, 2–

4 times more than blood. The price tag

may deter their use where they are

most needed: in the developing world,

where blood is often either unavailable

or unsafe. ‘‘If you can’t afford to test for

HIV, you probably can’t afford to buy

a blood substitute,’’ says Klein.

Despite past setbacks, manufac-

turers of oxygen therapeutics remain,

well, sanguine. They are confident that

their products will soon surmount the

scientific and regulatory hurdles and

succeed commercially. ‘‘It’s a very ex-

citing time,’’ says Winslow. ‘‘I think we

are getting close to the finish line here.’’