INTRODUCTION:

Blood is a special type of fluid connective tissue composed of white blood cells, red cells, platelets, and plasma. It has a variety of functions in the body. In this regards, currently, artificial blood products are only designed to replace the function of red blood cells. It might even be better to call the products being developed now, oxygen carriers instead of artificial blood. (Kirschman.2005)

The ‘holy grail’ of blood research, the man-made blood would be free of infections that have blighted natural supplies and could be given to almost everyone regardless of blood group. The hope comes from Edinburgh and Bristol university researchers who have, for the first time made thousands of millions of red blood cells from stem cells ‘master cells’ seen as a repair kit for the body taken from bone marrow.

Large scale trials would follow, but the blood could be in routine use in a decade. Within 20 years, it may be possible to produce two million pints of artificial blood a year which would enough to satisfy the nation’s medical needs. Any embryonic stem cells used would be taken from four or five day old embryos left over from IVF treatment and donated to the research project.

Artificial Blood: What is it?

Since the seventeenth century, blood transfusions have been attempted to offset blood loss from trauma and childbirth or as a therapeutic modality during leeching or bloodletting. Until the identification of isoagglutinating antibodies however, transfusions were fraught with significant early complications. These early complications sparked interest in using hemoglobin as an oxygen carrier in plasma. Early trials of these solutions proved disastrous as well with significant immediate complications resulting from infusions of stroma-free human hemoglobin solutions. These complications were most often acute renal failure thought to be the result of direct hemoglobin nephrotoxicity. (Caron 2001) Artificial blood is a product made o ac as a substitute for blood for transportation of oxygen and carbon di oxide through the body the most promising blood products are perfluorocarbon and hemoglobin based oxygen carriers.

In 1966, experiments with mice suggested a new type of blood substitute, perfluorochemicals (PFC). These are long chain polymers similar to Teflon. It was found that mice could survive even after being immersed in PFC. This gave scientists the idea to use PFC as a blood thinner. In 1968, the idea was tested on rats. The rat's blood was completely removed and replaced with a PFC emulsion. The animals lived for a few hours and recovered fully after their blood was replaced.

However, the established blood bank system worked so well, research on blood substitutes waned. It received renewed interest when the shortcomings of the blood bank system were discovered during the Vietnam conflict. This prompted some researchers to begin looking for hemoglobin solutions and other synthetic oxygen carriers. Research in this area was further fueled in 1986 when it was discovered that HIV and hepatitis could be transmitted via blood transfusions. (Robb,1998)

Artificial blood developed for the battlefield:

US scientists working for the experimental arm of the Pentagon have developed artificial blood for use in transfusions for wounded soldiers in battlefields. The blood is made from hematopoietic stem cells from discarded human umbilical cords, which are turned into large quantities of red blood cells by a method called "blood pharming" that mimics the functions of bone marrow. Pharming is a method of using genetically engineered plants or animals to create medically useful substances in large quantities. Using this process, the cells from one umbilical cord can produce about 20 units of blood, which is enough for over three transfusions for injured soldiers on the field.

The blood is being manufactured for the Defense Advanced Research Projects Agency (DARPA) by a company in Ohio, Arteriocyte, which has already submitted samples of O-negative blood to the US Food and Drug Administration (FDA) for evaluation and safety testing. The company received funding of $1.95 million in 2008 to find a way of making large quantities of artificial blood. Don Brown of Arteriocyte said the method works but the production needs to be scaled up to produce enough blood.

Risk versus Benefit:

New testing and screening procedures have rendered the donor blood supply increasingly safe. For example, the risk of transfusion associated HIV infection is now estimated to be as low as 1 per 835,000 transfused patients . Similarly, the risk of transfusion associated infection with hepatitis C virus (HCV) is between 1 per 300,000 and 1 per 600,000, compared with an incidence of 1 per 103,000 in the early 1990s before a test for HCV became available. As the safety of the donor blood supply continues to improve, there must be careful consideration of the advantages of blood substitutes over donated blood, particularly given that new blood substitutes potentially carry unknown risks. Yet a shortage of donor blood for transfusion still advocates for the development of readily available blood substitutes. Most important, the intravascular dwell times of blood substitutes need to be increased, the cost of these products needs to be competitive, and difficulties with obtaining and processing sufficient amounts of these compounds must be overcome.

IDEAL BLOOD SUBSTITUTE:

a. Requires no cross-matching or compatibility testing

b. Be suitable for long-term storage (preferably at room temperature)

c. Be able to survive in the circulation for several weeks (the intravascular “dwell” time) before being cleared by the kidney Be free of side effects

d. Be free of pathogens

e. Not only transport but also effectively deliver oxygen to tissues

f. Increased availability that would rival that of donated blood, even surpass it

g. Oxygen carrying capacity, equaling or surpassing that of biological blood

h. Volume expansion

i. Long shelf life

j. Cost efficient

k. Universal compatibility: elimination of cross matching

Potential clinical applications of oxygen carrying solutions:

1. Therapy

(a) Blood substitutes: hemorrhagic shock; hemorrhage (war, surgery); anemia.

(b) Whole-body rinse out: acute drug intoxication; acute hepatic failure.

(c) Local ischemia: acute Myocardial infarction (MI); evolving Myocardial infarction (MI); cardiac failure; brain infarction; acute arterial thrombosis and embolism; Percutaneous transluminal coronary angioplasty( PTCA)of coronary artery.

(d) General ischemia: gas embolism, CO intoxication; High altitude cerebral edema (HACO)

(e) Aid for organ recovery: acute renal failure, acute hepatic failure, acute pancreatitis.

(f) Infectious disease: anaerobic and aerobic disease

(g) Adjuvant therapy: tumour radiotherapy; chemotherapy

2 Perfusional protection of organs during surgery – cardiopulmonary bypass, deep hypothermia, circulatory arrest, cardioplegia

3. Preservation of donor organs.

4. Drug carrier - drug-conjugated haemoglobin and perfluorochemicals.

5. Contrast agent - (Perfluoro-octylbromide)

Non-Clinical Applications:

1. Culture medium

2. Chemical examination - oxygen sensor; standard solution for oxygen calibrator

3. Bioreactor

Paradoxical Utilisations (of high-oxygen affinity):

1. Oxygen absorbent

2. Oxygen pulse therapy for malignant tumour in combination with radiotherapy or chemotherapy.

BLOOD PHARMING:

Another potential route to blood substitutes is to grow human red blood cells from cells extracted from umbilical cords a technique called blood pharming. One biotechnology firm exploring this approach is Arteriocyte, based in Cleveland, US. 'We have demonstrated proof of concept that we can take umbilical cord cells, expand them and differentiate them [into red blood cells] in useful volumes,' says Adam Sorkin, Arteriocyte's director of research and development. The first step is to isolate haematopoietic stem cells from an umbilical cord and then culture those on Nanex technology, which is a polymer nanofibre substrate that expands the stem cells much more rapidly than normal cell culture techniques, which will result in 10 days we get a 300 to 350-fold increase in cells and over a month the scientist can get hundreds of thousands, or a million-fold increase.'

The second stage is to recreate the differentiation process that occurs normally in the body, invitro. 'Using a special cocktail of growth factors that we change over the course of several weeks, the scientists gradually drive most of these stem cells to form red blood cells that are functionally indistinguishable from the red blood cells you would produce natively.' Finally, the cells are filtered and put in plasma or fluid before they can be transfused directly into the body. The end goal is to carry out the complete process onsite in remote areas such as in hospitals, in war zones.

HEMOPURE:

Although the FDA is currently wary of these products, a handful 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 the research and development at OPK Biotech, explains: 'an important distinction between Hemopure and any other haemoglobin-based oxygen carrier is that Hemopure is room temperature stable as a liquid for at least 3 years.'

To make Hemopure 'we take bovine blood and, using filtration and chromatography, purify the haemoglobin so it is 99.9 per cent pure protein,' explains Light. But sadly it isn't as simple as just dissolving the haemoglobin in a fluid and transfusing it into the patient. Native haemoglobin is tetrameric and dissociates into dimers in the body that are excreted through the kidneys with a half life of about 30 minutes. 'This is not a very effective therapy,' Light points out. 'You have to modify the haemoglobin so it doesn't dissociate.' A number of different approaches have been taken to stop this dissociation. 'What we do is polymerise with glutaraldehyde [pentane-1,5-dial] to make large polymers of haemoglobin that have a half life of between 18 and 24 hours in the body,' Light says. The polymerised haemoglobin is then put into a physiological buffer, 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. The advantage of the haemoglobin being in the plasma is that the oxygen it carries is closer to the sides of blood vessels. Oxygen binding and release in the plasma is more efficient than in the red cell, says Light, so Hemopure releases oxygen more quickly than donor blood once it gets to the target tissue. At end of its life, Hemopure is broken down by the normal biological pathway for haemoglobin. Hemopure is currently approved for use in South Africa, and has undergone some early clinical trials in the US. Production was stopped, however, when Biopure went bankrupt - and OPK Biotech is currently focusing on getting production back online. Referring to the FDA's concerns over these products, Light predicts that they will be approved overseas before approval in the US. The firm's parent company, OPK, is Russian and, according to Light, 'OPK have a great interest in bringing this product to Russia.'

MP4:

Another, slightly different, product making its way through clinical trials in Europe is Sangart's MP4. It has just completed a Phase II trauma study.The haemoglobin for MP4 comes from out-of-date human blood, rather than the bovine blood used for Hemopure and to prevent tetramer toxicity and dissociation, Sangart takes the alternative approach of coating the haemoglobin with polyethylene glycol (PEG). '2-Iminothiolane is added to haemoglobin to increase the number of thiol sites on the molecule. And then PEG is able to attach to these thiol sites,' Levy explains.

As well as coating the molecule, PEG binds next to haemoglobin's oxygen binding site - altering the way oxygen attaches and detaches from the molecule. 'It very much increases the affinity of oxygen, so that oxygen is held on very, very tightly and only released if it reaches the level in the tissues where there is an oxygen lack,' as reported by many workers and this is one of the key selling points of MP4.

PERFLUOROCARBONS (PFBOCs):

PFBOCs achieve oxygen delivery by using organic chemicals with high gas solubility. The perfluorinated carbons are chemically and biologically inert but are able to dissolve a large amount of gas. One of the problems with perfluorocarbons is that they are an oil-like fluid that does not mix well with water and cannot carry water-soluble salts and metabolic substrates. In order to be used as a red cell substitute, perfluorocarbons are mixed with fluids such as albumin or in physiologic electrolyte solutions. Today, most PFBOCs are mixtures of perfluorocarbons with emulsifying agents. Emulsifying agents are substances that help stabilize two seemingly unblendable things. PFBOCs utilize Puronic-68, egg yolk phospholipids and triglycerides as emulsifying agents. Green Cross Corp. in Osaka Japan developed the first PFBOC with a mixture of perfluorodecalin and perfluorotripropylamine using egg yolk phospholipids and Pluronic-68 as emulsifying agents. Their product was called Fluosol-DA. The problem with Fluosal-DA was that they dissolve less oxygen than pure liquids. It could only deliver 0.4 mL oxygen per 100 mL. In order to meet metabolic oxygen demand, the patients would have had to breathe in gas that was 100% oxygen which would lead to adverse effects due to oxygen toxicity.

More recently, Alliance Corp developed a mixture of perfluorooctyl bromide and egg yolk phospholipids as the emulsifying agent. Oxygent, their product, could deliver up to 1.3 mL oxygen per 100 mL, but this is still much lower than normal blood which could deliver blood 5 mL oxygen per 100 mL. The advantage of Oxygent over Fluosal-DA is that it has a longer circulation time, and it can be excreted from the body in 4 days compared to Fluosal –DA which could take months. (Winslow,1995)

Figure:1 Perfluorocarbons (PFBOCs) (www.Artificial Blood_ A Current Review _ Pharmainfo.net.mht)

Perfluorocarbon emulsions (PFCE) are one of the two major classes of oxygen therapeutics currently on the market. They are composed of liquid perfluorocarbon emulsified in water and salt. Due to PFC's inability to remain mixed with aqueous solutions, they must be prepared as emulsions before being used in patients. The PFCE particles are spherical, averaging about .2 microns in diameter, with a perfluorocarbon core and a thin egg yolk phospholipids surfactant as a coating.

PFC is a biologically inert material that can dissolve about 50 times more oxygen than blood plasma. They are relatively inexpensive to produce and can be made devoid of any biological materials. This eliminates the real possibility of spreading an infectious disease via a blood transfusion. From a technological standpoint, they have two significant hurdles to overcome before they can be utilized as artificial blood. First, they are not soluble in water, which means to get them to work they must be combined with emulsifiers—fatty compounds called lipids that are able to suspend tiny particles of perfluorochemicals in the blood. Second, they have the ability to carry much less oxygen than hemoglobin-based products. This means that significantly more PFC must be used. The Federal Drug Administration (FDA) has approved one product of this type for use, but it has not been commercially successful because the amount needed to provide a benefit is too high. Improved PFC emulsions are being developed but have yet to reach the market.

Perfluorocarbon Emulsions:

In 1989, Fluosol ®, a PFCE produced by Green Cross Corp. of Osaka,Japan , became the first of its kind to receive FDA approval. It was approved for use in coronary balloon angioplasty procedures. Unfortunat ely, due to a number of problems with the product such as low PFC content (20%/voume) and the necessity for a labor-intensive preparation among others, Fluosol was discontinued in 1994.

Figure: 2 blood vessels (www.Artificial Blood_ A Current Review _Pharmainfo.net.mht)

After the initial excitement regarding Fluosol, subsequent small studies demonstrated no benefit from Fluosol infusions in patients with profound anemia. With colloid solutions as a comparator, Fluosol did not improve indirect measures of oxygenation. However, Fluosol continued to be available for infusion as an oxygen carrier during high-risk percutaneous transluminal angioplasty procedures until early 1993, when the Food and Drug Administration rescinded approval for this indication for the emulsion.

New emulsions have been developed which utilize emulsifying agents similar to the primary compound. In particular, perflubron (perfluorooctyl bromide) has been developed as a stable emulsion safe for intravenous infusion by the addition of small amounts of perfluorodecyl bromide as an emulsifying agent; the emulsion is then buffered with egg yolk phospholipids. The resulting emulsion has a calculated oxygen carrying capacity which is approximately three fold the amount of oxygen carrying capacity of the earlier Fluosol solutions.

Perflubron oxygen carrying capacity is directly related to the oxygen partial pressure (Figure 2). In this regard, perflubron oxygen delivery is predictable; direct diffusion of oxygen is the mechanism by which oxygen is off-loaded to peripheral tissues. Theoretically, oxygen delivered by diffusion may be more available, and more readily off-loaded from the bloodstream, than hemoglobin-delivered oxygen. However, no data has been produced which support this premise.

First generation perfluorocarbons:

The first commercially available PFC was Fluosol - DA 20% (Green Cross, Osaka, Japan). This used Pluronic F-68, as an emulsifying agent, and was able to maintain a balance between the oxygen carrying capacity and tissue retention. It comprised two PFCs, perfluorodecalin (PFD) and perfluorotrypropylamine (FTPA). PFD was the primary component and oxygen carrier, whereas FTPA was to provide the much needed stability. Each of the two components had different halflives, with PFD’s being only 3 to 6 hours due to its rapid clearance. FTPA, on the other hand, persisted in the tissue for months.

Second generation perfluorocarbons:

Fluosol had paved the path for further refinement of PFCs. The desirable characteristics in the second generation PFC, as advocated by Reiss and Le Blane1982 were large oxygen-dissolving capacity,faster excretion and less tissue retention,lack of significant side effects, increasing purity, large scale production and availability.

The three candidates chosen as per these criteria were PFD, perfluorooctyl bromide (PFOB) and bis (perfluorobutyl) ethylene. PFOB, known in its emulsion as Oxygent, was favoured for clinical trials because of its stability and high excretion rate. Linear PFCs like PFOB dissolve oxygen better than cyclic ones like PFD. The oxygen solubility is inversely proportional to the molecular weight and directly proportional to the number of fluorine atoms. 90% weight/volume emulsions of PFOB have the capacity to contain four times the amount of oxygen as compared to a first generation PFC. PFCs being chemically inert, are not metabolised but are removed from the circulation, within 4-12 hours, by the reticuloendothelial system. They are stored in the liver and spleen and subsequently exhaled through the lung. Reducing the particle size or increasing the concentration leads to increased tissue deposition and subsequent tissue damage (Reiss,2005 ).

Benefits:

The general benefits of HBOCs over transfused red blood cells are summarized below:

1. no prior planning

2. faster and better oxygen distribution

3. ready to use

4. no waste

5. no equipment

6. long shelf life

7. no refrigeration

8. Universally compatible

9. Immediately offloads oxygen

10. No 2,3-DPG

11. Can be use by Jehovah's Witnesses

Challenges:

The challenges associated with the development of HBOC can be categorized into the following

Availability:

Ironically while one of the primary reasons to develop oxygen carriers is to have a readily available solution to ease the projected future shortages in blood supply, some approaches to the development of HBOCs face a similar supply challenge. It was estimated that over 70,000 kg of Hb would be needed to replace 20% of RBC transfusion in the United States. This presents a significant challenge to human HBOC products. While production of human Hb by recombinant DNA could be a possible solution, it remains unclear whether the technology could produce such massive quantities of Hb for future demand. A study has estimated that a population of 100,000 transgenic pigs would be required to stably produce up to 50% of human Hb.

Short half-life:

Outside a red blood cell, Hb dissociates into 32 kDa dimmers and are freely filtered by the glomerulus resulting in severe renal toxicity. Current HBOC products have chemically or genetically cross-linked Hb chains resulting in 128 kDa or larger molecules that are not readily filtered by the glomerulus, thus possessing a greatly increased half-life.

Increased oxygen affinity:

Hb in the plasma has a much higher affinity for O 2 than it does within the context of a red blood cell. The increased affinity for O2 is due to lack of 2,3-diphosphoglycerate (DPG) in the plasma. Consequently the HbO2 dissociation curve shifts to the left, making such a high-affinity Hb not an ideal oxygen delivery substance. However, chemically cross-linking the Hb structure has the net effect of decreasing O 2 affinity and optimizing intracellular oxygen delivery.

Vasoactive properties:

One of the major challenges facing the development of HBOCs is their effects on vasoactive properties. The theories regarding the mechanism of action of the vasoconstrictive effect

1. Nitric oxide scavenging by Hb

2. Excess O 2 delivery to the peripheral tissues

3. Direct effect on peripheral nerves

4. The oxidative properties of HB

Soluble Hb, unlike Hb in RBCs, interacts with NO to form metHb and NO-Hb. NO by definition is a potent endothelial vasorelaxant that inhibits the conversion of proendothelin to the vasoconstrictor endothelin. In the prevailing theory on vasoactive properties, the decrease in NO concentration due to its reaction with Hb is responsible for vasoconstriction. Alternative theories suggest that too much O 2 is delivered causing an autoregulatory vasoconstrictor reflex. Yet another theory argues that oxidation of soluble Hb can result in heme loss, free radical formation, loss of reactive iron, and oxidation of lipids. Such reaction and products result in endothelial stress causing vasoconstriction

Problems:

Perfluorocarbons are inert biologically. The molecules are sequestered in the reticuloendothelial system, particularly in the Kupffer cells of the liver and macrophages, and subsequently released back into the plasma as a dissolved gas. The perfluorocarbon gas is then exhaled unchanged and non-metabolized via the lungs. While previous perfluorocarbons had a significant amount of retention in the reticuloendothelial system, current generation perfluorocarbons such as perflubron have a retention time of approximately one week. This allows effective elimination of perfluorocarbons from the liver and spleen without the potential for significant organ dysfunction. However, despite the inert nature of perfluorocarbons, sequestration in the reticuloendothelial system may result in subtle consequences. Platelet count is known to decrease, presumably due to opsonization of platelets by the perfluorocarbon and subsequent sequestration and elimination by the reticuloendothelial system. Sufficient perfluorocarbon may also overwhelm the reticuloendothelial system, resulting in potential infectious or other complications; however, this is only a theoretical concern, as no increase in infectious complications has been noted in early clinical trials.

The retention of perfluorocarbons does pose an additional problem with respect to dosage. Perfluorocarbons are relatively evanescent in the plasma, with a half life of approximately 3-4 hours in the plasma phase. The reticuloendothelial system, however, has an approximate 3-5 day retention phase prior to exhalation of the perfluorocarbon. Therefore, although extremely short-lived in the plasma phase, additional dosing of perfluorocarbons may not be possible for several half-lives of the tissue-reticuloendothelial terminal elimination, i.e., one to two weeks. Thus perfluorocarbons become a single dose drug, with limitation of dosage due to the capacity of the reticuloendothelial system to handle the plasma elimination phase. At present, this limitation of dosing is theoretical, as no clinical data exist to discern whether perfluorocarbon redosing results in serious adverse effects; future studies and newer generation emulsions will address this issue.

The dependence of perfluorocarbons on Henry's Law of partial pressures allows the potential for increased oxygen availability. This fact of oxygen delivery also limits the effective use of perfluorocarbons to situations when the partial pressure of oxygen is supranormal, i.e., when the partial alveolar oxygen tension approaches 400 mmHg or greater. This is impossible to attain without supplemental oxygen administration; an effective partial oxygen pressure may be impossible with any maneuvers at altitude. Even in the presence of supplemental oxygen and controlled ventilation, patients with significant pulmonary disease may be unable to reach partial pressures of oxygen to allow perfluorocarbon to function as an effective oxygen carrier (Teicher, 1993)

Current Status Of Pfce Based Product:

Current PFCE products are referred to as second generation PFCE's and are marketed as oxygen therapeutics for patients at risk of acute hypoxia resulting from transient anemia, blood loss or ischemia. They use different PFC's and surfactants than the previous products. The PFCE particles travel in the plasma near the vessel walls and between RBC's. The largest plasma gaps between RBC's exist in microcirculation so as a result, PFCE's provide the most benefit to smaller vessels. Also, when there are local areas of vasoconstriction or blockages of the vessels, some plasma can still pass through and deliver PFCE's/oxygen to the tissues. Initially, they were created in order to avoid or reduce the need for blood transfusions in the treatment of trauma patients but as time went on, they began to be used in cardiovascular, orthopedic, urologic and other general surgeries. (Tremper et al.,1980)

Alliance Pharmaceutical Corp., with the help of Johnson and Johnson, is currently working for FDA approval of Oxygent. Oxygent is a second generation PFCE with a median particle diameter of 16-.18 microns, an optimal storage temperature of 2-8 °C, and a PFC content of 60%/voume. Alliance is hoping to initiate further Phase III studies involving general surgery. Alliance has also patented a procedure called Augmented Acute Normovolemic Hemodilution (Augmented-ANH), which is a technique that will further decrease the need for blood transfusions in moderate and high blood loss surgical procedures. Immediately prior to surgery, several pints of blood are removed from the patient and Oxygent is used to replace the oxygen-carrying capacity of the missing blood and following surgery, the removed blood is reinfused.

Recently, the European Agency for the Evaluation of Medical Products (EMEA) recommended that Alliance pursue an indication for Oxygent that does not require direct comparison to allogeneic blood transfusions. Since the incidence of serious adverse side effects associated with blood transfusions are so low, it would be difficult to show that Oxygent is safe or safer than allogeneic blood. Also recently, Alliance and its subsidiary PFC Therapeutics LLC began working with Double-Crane Pharmaceutical CO., Ltd. And LEO Pharma A/S to develop and market Oxygent in the People's Republic of China, Europe and Canada.

HAEMOGLOBIN-BASED OXYGEN CARRIERS (HBOCS):

HBOCs are oxygen carriers that use purified human, animal or recombinant hemoglobin. The first major clinical study with purified hemoglobin resulted in nephrotoxicites, poisonous effects on the kidney. The hemoglobin used was found to have ethrocyte membrane stromal lipids as well as bacterial endotoxins. To side-step these problems, stroma-free hemoglobin were developed that also was free of endotoxins, and it did reduce nephrotoxicity but new problems arose. The stroma-free hemoglobin was found to have too high of an affinity for oxygen which affected oxygen being delivered to tissue. In recent days, many new processes have been developed that fix these problems.

MODIFIED HAEMOGLOBIN:

Polymerised Haemoglobin:

Initially, glutaraldehyde (a polymerising reagent for proteins) was used to develop PLP-conjugated polymerised Hb with an adequate oxygen affinity .Since the number of molecules remained less, the colloidal osmotic pressure could be kept low (20 Torr, [Hb] = 15 gm/dl), thus enabling its transfusion as a solution with high Hb concentration. It had a circulation half-life of 24 hours and could be stored for more than 1 year. The drawbacks were related to the distribution of the molecular weight, uncertainty of the position of reacted sites, and high viscosity of the solution. Transient renal tubular vacuolation after multiple injections have also been reported. These problems were solved by using chromatographic separation techniques. Initial trials were conducted on adult healthy males in the United States, Guatemala and in Zaire. In 1992, the phase I clinical testing of a modified solution was carried out by Northfield Laboratories after approval of the FDA. The maximum dosage tried was 0.6 gm/Kg (63 gm maximum). No abnormality such as vasoconstriction was noticed. In the phase 1 trial using polymerised bovine Hb, dosing of upto 0.2 gm/kg caused a rise in blood pressure and a decline in heart rate, resulting in suspension of the trial Currently, soluble, cell-free, o-raffinose cross-linked and oligomerised human Hb (O-r-poly-Hb)(Hemolink, Hemosol, Canada) (molecular weight ranging from 32- >500 kDa) is in a phase III clinical trial for peri-operative use in cardiac and orthopaedic surgery (at doses from 25g (250 ml)/100 g (1000 ml) [2,8]. Its affinity for oxygen appears lower than normal blood and an n (Hill

coefficient) value of about 1 indicates a very low degree of co-operativity. Probably related to the low O2 affinity value and to the high molecular weight, O-r-poly-Hb has been shown to induce lesser haemodynamic perturbations than other first generation modified Hbs. (Caron, 2001)

Stabilized Hemoglobin (Tetrameric Hemoglobin):

One way to fix the problem that stroma-free hemoglobin has a high affinity towards oxygen is to use functional DPG analogs such as pyridoxal-5’-phospoate that attach to the DPG pocket. DPG stands for 2,3-diphosphoglycerate which is a substance made in red blood cells that helps control the movement of oxygen to body tissue. The more DPG in the cell the more oxygen delivered to the tissue. Lesser the DPG,lesser is the oxygen delivered to tissues.

Pridoxylated stroma-free hemoglobin nearly has normal oxygen affinity (p50 = 22-24 mmHg), but is dissociable into αß-dimers that are excreted. Hemoglobin can also be stabilized with an α-specific crosslinker bis-fumarate that produces cross-linked hemoglobin. The cross-linked hemoglobin demonstrated an oxygen affinity of p50 = 30mmHg and had a longer circulation time. To increase the circulation time of HBOCs, it is possible to make stabilized hemoglobin can undergo intermolecular cross-linking with bi- or poly-functional groups. This process is also known as polymerization. Pyridoxal-5’-phosphate, a DPG analog, can be polymerized with glutaraldehyde to increase intravascular circulation half-time of over 30 hours in adult baboons.

A process to increase circulation time that uses ring-opened raffinose (o-raffinose) as a cross-linker has also eliminated the stabilization process that is need prior to cross-linking. The o-raffinose HBOC delivers 4.3 mL of oxygen per deciliter which is close to the normal oxygen delivery capacity. Due to the limited availability of human stroma-free hemoglobin, low oxygen affinity bovine hemoglobin has been utilized as a starting material. Bovine hemoglobin has a natural low affinity to oxygen and is not DPG dependent. The major deterrent to using bovine hemoglobin is the potential of transmission of animal-borne diseases such as bovine spongiform encephalopathy (BSE).

Conjugated Hemoglobin:

To further increase the circulation time, hemoglobin can be linked to a macromolecule to increase its size. Human or bovine hemoglobin that is conjugated with polyethylene glycol is protected from renal excretion. The polyethylene glycol hemoglobin has a larger molecular size and has a higher viscosity.

Recently, a product called Hemospan has been developed by introducing additional surface thiols with iminothiolane onto the hemoglobin. This process usually adds about 5 additional thiols, and it is then linked to polyethylene glycol-5000. Hemospan then requires no more purification steps. One would think Hemospan would not work, for it has a lower hemoglobin concentration, higher viscosity, higher oxygen affinity and higher colloidal oncotic pressure than most other HBOCs in development. Hemospan did demonstrate an improvement in microcirculatory blood flow and tissue oxygenation in animal studies, but it is not known if it improves perfusion in the microcirculation of the critical organs. The increased viscosity could also increase the cardiac workload of the patient.

Figure:3 Hemoglobin Vesicles (Hemoglobin encapsulated, embedded and coated vesicles) (www.Artificial Blood_ A Current Review _ Pharmainfo.net.mht)

Hemoglobin and red cell enzymes encapsulated in nanometer size biodegradable polymer vesicles have been developed. The advantage of the encapsulated hemoglobin over lipid vesicles is that these polymer vesicles could be permeable to glucose and other molecules that are needed to reduce methemoglobin. Methemoglobin is a particular type of hemoglobin that is useless for carrying oxygen and delivering it to tissues. The nanocapsules could maintain hemoglobin concentration at 15 g/dL and normal p50 (oxygen affinity).

The earliest types of encapsulated hemoglobin had a short circulation half-life and formed methemoglobin. The circulation half-life was improved by surface changes using negative surface charges, sialic acid analogs or polyethylene-glycol. These modifications improved the half-life to over 24 hours. Methemoglobin formation was reduced by reducing enzymes such as methemoglobin reductase system. In animal studies, results were mostly successful, but complement activation occurred in rats and pigs. Hemoglobin aquosomes were developed by coating hemoglobin molecules on the sugar coated hydroxyapatite nanoparticles. In rats, no undesirable changes were reported.

Recombinant/transgenic Hemoglobin:

With improvements in technology, native or modified hemoglobin can be produced from microorganisms such as E. coli or yeast, transgenic plants or animals. Recombinant human hemoglobin was produced in E. coli and S. cerevisiae using an expression vector containing two mutant human globin genes. One was a low oxygen affinity mutant, and the other fused α-globins. These recombinant hemoglobin products advanced to clinical trials, but it was stopped due to vasoconstriction and other harmful effects. Transgenic mice and pigs have been used to produce human hemoglobin. Human α and β globin gene constructs are injected into fertilized eggs. The embryo is then developed in a surrogate mother. The problem is that these red blood cells contain the animal’s own hemoglobin as well as human hemoglobin.

Artificial Blood: What is it?

Figure:1 Perfluorocarbons (PFBOCs) (www.Artificial Blood_ A Current Review _ Pharmainfo.net.mht)

Benefits:

Problems:

Current Status Of Pfce Based Product:

COMPANIES:

1. Synthetic Blood International, Inc.

2. Sanguine Corp.

3. Perftoran

4. OxyVita

5. Hemolink

THE FUTURE: