Blood substitutes Artificial blood Synthetic alternatives to donor blood have been stuck in development for decades. Nina Notman reports on recent promising progress

Human blood substitutes have been blood reserves, says Chang. More could be contaminated, companies in the pipeline since the 1980s. But, niche needs include patients, such In short started to make many different for a combination of scientific and as Jehovah’s Witnesses, who for  The HIV crisis in the types of blood substitutes, and political reasons, there are none religious reasons can not use donor 1980s triggered research unfortunately some don’t work and currently on the market in either blood, and patients who have into artificial blood some have adverse side effects.’ Europe or the US. There are a few developed immune responses to  Obtaining regulatory The concerns came to a head in blood substitutes still progressing donated blood. approval for these early 2008, when a meta-analysis through clinical trials, and the Aside from not being reliant on products has proved of findings from 16 clinical trials academic community is still actively donor blood supplies, other major difficult so far in Europe of five different products that had improving the products, also advantages of artificial blood include and the US been used on over 3500 patients known as oxygen therapeutics and not needing to check blood types  Commercial design was published in the Journal of haemoglobin-based oxygen carriers. before transfusion – artificial blood approaches include the American Medical Association. ‘No one was interested in this type is the universal blood group O leaving haemoglobin free The study, led by Charles of work until HIV came in the 1980s,’ negative. Artificial blood also avoids in plasma, and encasing Natanson, a senior scientist at the explains Thomas Chang from McGill shelf life issues – donor blood can it in artificial, polymeric US National Institutes of Health, University, Montreal, Canada, an be stored for a maximum of 42 days red blood cells revealed a threefold increase in early pioneer of the field, ‘and then before use, and starts to degrade in  The academic the risk of heart attacks in patients everyone started to look into making quality shortly after collection. community is working who received the substitutes, artificial blood substitutes.’ on bettering the basic compared with the control group He adds, ‘we have to be ready Outsmarting obstacles understanding of how who received donor blood (see for another HIV episode or The need and utility may be clear, but blood works, to further Chemistry World, June 2008, p13). similar when the blood might be the route to regulatory approval in improve the products ‘The paper by Natanson contaminated.’ It was HIV concerns Europe and the US has been tortuous combined the results of all the that led to South Africa becoming – with numerous hurdles seemingly clinical trials, of all the products, one of the few countries so far to intent on blocking the way. from all the companies, and lumped approve the use of a blood substitute The first clinical trials for them together,’ says Chang, ‘and – a product called Hemopure. these products – run by a number that was a disaster for this field.’ Fears of contaminated donor of different companies – had Some of the products had fewer blood supplies are just one reason disappointing results with some side effects than others, but for the interest in this area. Ongoing unwanted side effects, most because they were treated as one concerns about lack of donor blood seriously an increase in blood everyone started to worry and the supplies in remote locations are also pressure. Chang blames the results US Food and Drug Administration driving the field forward, with the on a rush to get a product to market (FDA) became reluctant to approve US army and navy funding much in the wake of the HIV crisis: ‘not anything, he says. of the research. You never know enough basic research was done Chang’s frustration is shared when there will be a disaster or prior to HIV – and as a result, when by many scientists working in the war when we won’t have sufficient it was realised that donor blood field, including John Olson, based 40 | Chemistry World | October 2010 www.chemistryworld.org TEK IMAGE / SCIENCE PHOTO LIBRARY LIBRARY PHOTO SCIENCE / IMAGE TEK www.chemistryworld.org Chemistry World | October 2010 | 41 Blood substitutes

a very consistent finding no matter ‘Like everything what type of patients you were looking at or which of the various OPK BIOTECH OPK in science, it is how you companies you looked at, he adds. package Hemopure Although the FDA is currently the data for wary of these products, a handful presenting’ of companies in the US are still working their way through the obstacles in the attempt to get a blood substitute to market. According to Olson, the best of the bunch is Hemopure – a blood substitute being developed by the oxygen therapeutics firm OPK Biotech. Hemopure was initially designed by the small start up company Biopure, which was brought by OPK Biotech when it went bankrupt last year (see Chemistry World, 14 May 2009, The haemoglobin in OPK bit.ly/4kdkEn). Biotech’s Hemopure ‘Hemopure is the one, in my is stabilised by opinion, that should be closest to polymerisation being approved for use when no donated blood is available,’ Olson at Rice University, Houston, US: Natanson, however, stands by his says. ‘It is the simplest and the ‘the Natanson article says every findings: ‘meta-analysis showed that cheapest technology.’ blood substitute is evil and causes if you put all the studies together As Rick Light, vice president of heart attacks. But like everything in there was a very common toxicity – research and development at OPK science, it is how you package the that is they appeared to cause heart Biotech, explains: ‘an important data for presenting.’ attacks and death.’ It appeared to be distinction between Hemopure and any other haemoglobin-based oxygen carrier is that Hemopure is Blood pharming room temperature stable as a liquid Another potential route to blood substitutes is to director of research and development. for at least 3 years.’ You just pull it off grow human red blood cells from cells extracted The first step is to isolate haematopoietic the shelf and use it immediately, he from umbilical cords – a technique called blood stem cells from an umbilical cord. ‘We then says. This stands it apart from other pharming. culture those on our Nanex technology, which is potential blood substitutes that One biotechnology firm exploring this a polymer nanofibre substrate that expands the need refrigeration, or are freeze- approach is Arteriocyte, based in Cleveland, US. stem cells much more rapidly than normal cell dried, meaning they have to be ‘We have demonstrated proof of concept that culture techniques,’ Sorkin explains. ‘In 10 days reconstituted with plasma. we can take umbilical cord cells, expand them we get a 300 to 350-fold increase in cells and To make Hemopure ‘we take and differentiate them [into red blood cells] in over a month we can get hundreds of thousands, bovine blood and, using filtration useful volumes,’ says Adam Sorkin, Arteriocyte’s or a million-fold increase.’ and chromatography, purify the The second stage is to recreate the haemoglobin so it is 99.9 per cent differentiation process that occurs normally pure protein,’ explains Light. in the body, in vitro. ‘Using a special cocktail of But sadly it isn’t as simple as just growth factors that we change over the course dissolving the haemoglobin in a fluid of several weeks, we gradually drive most of and transfusing it into the patient. these stem cells to form red blood cells that Native haemoglobin is tetrameric are functionally indistinguishable from the red and dissociates into dimers in the blood cells you would produce natively.’ body that are excreted through the Finally, the cells are filtered and put in plasma kidneys with a half life of about or fluid before they can be transfused directly 30 minutes. ‘Which is not a very into the body. effective therapy,’ Light points out. The end goal is to carry out the complete ‘You have to modify the haemoglobin process onsite in remote areas such as in so it doesn’t dissociate.’ hospitals in war zones. ‘Once it’s up and running, A number of different approaches the idea is that it will be producing universal have been taken to stop this donor blood on demand,’ says Sorkin. dissociation. ‘What we do is ‘What we are working on now is implementing polymerise with glutaraldehyde the bioreactor technology that will handle the [pentane-1,5-dial] to make large differentiation step. At that point we would polymers of haemoglobin that have ARTERIOCYTE move into [clinical] trials.’ Sorkin predicts that it a half life of between 18 and 24 hours Arteriocyte’s Nanex polymer nanofibre substrate will take another 5–10 years for this product to in the body,’ Light says. for rapid stem cell culturing reach the market. The polymerised haemoglobin is then put into a physiological buffer,

42 | Chemistry World | October 2010 www.chemistryworld.org which contains salts and lactates to maintain heart function. At this stage the product still contains about 30 per cent stabilised haemoglobin tetramers – and while they may not dissociate in this form, they are still problematic because they can enter blood vessel walls and scavenge nitric oxide. NO naturally causes the walls of blood vessels to relax, so if too much NO is scavenged, relaxation is prevented, making it more difficult for blood to flow, resulting in an increase in blood pressure. A final clean up stage removes most of the tetramer haemoglobin, significantly reducing the blood pressure effect, explains Light. Once transfused into the body, polymerised haemoglobin works in the same way as a red blood cell: it binds to oxygen in the lungs, carries it to the appropriate site in the body and then releases it. What makes it different to red blood cells is that the haemoglobin is free in the plasma –

rather than bound within the cell. SANGART The advantage of the haemoglobin being in the plasma is bankrupt – and OPK Biotech is Sangart’s MP4 consists ‘Our agent is given together with that the oxygen it carries is closer to currently focusing on getting of haemoglobin coated blood to improve the oxygen transfer the sides of blood vessels. Oxygen production back online. Referring with polyethylene glycol to tissues from red cells,’ explains binding and release in the plasma is to the FDA’s concerns over these Howard Levy, chief scientific officer more efficient than in the red cell, products, Light predicts that they for the San Diego-based firm. says Light, so Hemopure releases will be approved overseas before MP4 is transfused into patients in oxygen more quickly than donor approval in the US. The firm’s an approximately 1:19 MP4:donor blood once it gets to the target parent company, OPK, is Russian blood ratio. Explaining the need for tissue. At end of its life, Hemopure and, according to Light, ‘OPK have his product, Levy says: ‘the trouble is broken down by the normal a great interest in bringing this is [when] blood is stored for long biological pathway for haemoglobin. product to Russia.’ periods; beyond 3 days it undergoes Hemopure is currently approved changes that make it less efficient at for use in South Africa, and has MP4 delivering oxygen. undergone some early clinical trials Another, slightly different, product ‘MP4 first releases its own in the US. Production stopped, making its way through clinical oxygen, and is then able to take however, when Biopure went trials in Europe is Sangart’s MP4. oxygen from red cells and give it to tissues,’ he adds. Clinical trials in Europe with Artificial red blood cell this product are progressing well. The toxicity problems that two enzymes – catalase and ‘We have just completed a Phase plague blood substitutes are superoxide dismutase – that II trauma study,’ says Levy. ‘So we believed to originate from the remove toxic radicals in a are in the process of cleaning the

THOMAS CHANG THOMAS free tetrameric haemoglobin in further attempt to recreate database and will then proceed to the products, and an alternative conditions similar to the analyse the data.’ He adds: ‘we’re approach being considered is natural cell. looking for market launch at the end to encase the tetramers in an ‘The artificial cell is about of 2013 or the beginning of 2014.’ artificial container – in essence 80nm in diameter, 100 times The haemoglobin for MP4 mimicking the red blood cells. smaller that natural red cells,’ comes from out-of-date human One researcher working in says Chang. Initial animal blood, rather than the bovine this field is Thomas Chang from tests have been encouraging, blood used for Hemopure. And McGill University, Montreal, showing no adverse effect to prevent tetramer toxicity and Canada. ‘The artificial red on the function of the liver or dissociation, Sangart takes the blood cell contains a membrane spleen. The artificial cells also alternative approach of coating the or polymer [casing] around stay circulating in the body’s haemoglobin with polyethylene the haemoglobin,’ he explains. Chang pioneered artificial blood blood supply for twice as long as glycol (PEG). The casing is made from the polymerised haemoglobin ‘2-Iminothiolane is added to biodegradable polymers, and then water and carbon products. ‘It looks promising haemoglobin to increase the number such as poly(lactic acid), dioxide in the body. Inside his and we are continuing of thiol sites on the molecule. And that degrades into lactic acid ‘cell’, Chang has also included researching this,’ says Chang. then PEG is able to attach to these thiol sites,’ Levy explains.

www.chemistryworld.org Chemistry World | October 2010 | 43 Blood substitutes

[naturally present in the body] and creates toxic free radicals,’ says Cooper. ‘So what we’ve done is look at these electron transfer pathways and engineer around them.’ The team found that there are residues of the amino acid tyrosine within haemoglobin’s structure TEK IMAGE / SCIENCE PHOTO LIBRARY LIBRARY PHOTO SCIENCE / IMAGE TEK and adding more – using protein engineering – allows the body to better defend against free radical attack. Olson, whose Rice-based team is also looking at bioengineering haemoglobin, explains, ‘we can look at the molecule like an engineer looking at an internal combustion machine and say “how can I engineer this to work better?”’ Both Olson and Cooper grow their modified human haemoglobin in Escherichia coli. ‘It’s a great idea in principle; all you need is corn syrup, minerals and bacteria to have continuous production of haemoglobin using conventional manufacturing standards,’ says Olson. ‘We can change the programme for making haemoglobin by selectively changing amino acids, to try to get rid of any side effects or to optimise it for better transport,’ he adds. His team is currently making adaptations to improve shelf life and efficiency.

Weighing up costs The E. coli approach is appealing as it avoids the need for supplies of either human or bovine blood – but As well as coating the molecule, Blood banks can only there are no side effects at all in there is a huge issue: price. ‘No one PEG binds next to haemoglobin’s store donated blood for a future blood substitutes,’ says Chang has ever made E. coli technology as oxygen binding site – altering the maximum of 42 days at McGill University. Chris Cooper, cheaply as we would have to make way oxygen attaches and detaches an expert in haemoglobin at the this,’ Olson explains. A unit of donor from the molecule. ‘It very much University of Essex, UK, agrees: ‘The blood (60g) costs a hospital $300 increases the affinity of oxygen, so blood substitute field has in a sense (£194), meaning to match the price that oxygen is held on very, very gone back to the academics for the the product would need to be made tightly and only released if it reaches moment.’ for $6 a gram. ‘For recombinant the level in the tissues where there In the quest to find this next technology that’s not easy to do, we is an oxygen lack,’ Levy says. And generation blood substitute, Cooper are currently at $200–1000 a gram.’ this is one of the key selling points and Rice University’s Olson – The cost issue is not unique to the of MP4, according to Levy: that it among others – are studying the E. coli approach however, with both delivers oxygen exactly where it is fundamentals of how haemoglobin Sangart and OPK Biotech admitting needed in the body. works to figure out what causes the their technology is not cheap and increase in blood pressure. Adam Sorkin from Arteriocyte (see Academic interest The observation that tetramers box p42) admitting that ‘cost is one While it looks hopeful that these scavenge NO is a well known cause of the challenges we face as we scale blood substitutes may eventually of haemoglobin’s toxicity outside of up our process.’ make it to market in Europe and red blood cells, initially uncovered So with cost problems to add to the US, many others have failed by Olson, but more recently Cooper the ever growing list of hurdles blood during the development or clinical has suspected a multifactorial substitutes must jump before they trials phases – including Northfield ‘We have to look cause. In 2008, Cooper and his team make it to market – is the concept Laboratories’ Polyheme and published a paper in the Journal of doomed to fail? No, is the resounding Baxter’s Rhb2.0. seriously into Biological Chemistry demonstrating answer from the experts. ‘It just needs ‘We definitely have to look new generations that a previously unknown electron one product to get on the market and seriously into new generations of transfer pathway in haemoglobin is then there will be an obvious market blood cell substitutes to solve some of blood cell partly key to the toxicity problem. and others will take off,’ Cooper of the problems and ensure that substitutes’ ‘Haemoglobin reacts with peroxides says confidently. 44 | Chemistry World | October 2010 www.chemistryworld.org