Future Generations of Red Blood Cell Substitutes
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Journal of Internal Medicine 2003; 253: 527–535 MINISYMPOSIUM Future generations of red blood cell substitutes T. M. S. CHANG From the Artificial Cells & Organs Research Center, Faculty of Medicine, McGill University, Montreal, Quebec, Canada Abstract. Chang TMS (Artificial Cells & Organs erties. New artificial red blood cells that are more like Research Center, McGill University, Montreal, RBC are being developed. One is based on haemo- Quebec, Canada). Future generations of red blood globin lipid vesicles. A more recent one is based on cell substitutes (Minisymposium). J Intern Med 2003; nano-dimension artificial red blood cells containing 253: 527–535. haemoglobin and RBC enzymes with membrane formed from composite copolymer of polyethylene Polyhaemoglobins (PolyHb) and perfluorochemicals glycol–polylactic acid. Their circulation time is double are in advanced phase III clinical trials and conju- that of PolyHb. gated haemoglobins in phase II clinical trial. New recombinant human haemoglobin with no vasoac- Keywords: antioxidant, artificial red blood cells, tivity is being developed. A soluble macromolecule of blood substitutes, microencapsulation, nanoen- PolyHb–catalase–superoxide dismutase is being capsulation, oxygen carriers. studied as an oxygen carrier with antioxidant prop- Introduction The first attempt at modified haemoglobin was to prepare artificial red blood cells encapsulating hae- Fluids for volume replacement have been in routine moglobin and red blood cell enzymes, but this proved clinical use for many years. When haematocrit falls to be an overambitious approach at that time [6, 7]. A below the critical level and fluid replacement alone is much simpler approach is based on polyhaemoglobin not enough, red blood cells or whole blood is now (PolyHb) which is prepared based on the use of the only next step. The present effort in the bifunctional agents to cross-link haemoglobin inter- development of blood substitutes is to produce a molecularlly [6, 7] including the use of glutaralde- substitute for red blood cells. Concentrated efforts to hyde to cross-link haemoglobin into soluble PolyHb develop blood substitutes for public use were only (Fig. 1) [8]. This type of cross-linking prevents the seriously started in the 1990s because of concerns breakdown of the haemoglobin into dimers that was a regarding HIV in donor blood. At present, red blood problem when unmodified stroma-free haemoglobin cells substitutes in phase III clinical trials are (SF-Hb) was used [9]. Promising results using PolyHb nothing more than oxygen carriers [1–5]. For are being gathered in phase III clinical trials involving example, the most commonly used first generation mostly perioperative uses where oxygen carriers are blood substitutes in phase III clinical trial consist of needed in addition to volume replacements [10–17]. Ringer-Lactate solution with an oxygen carrier in One type has been approved for routine use in South the form of modified haemoglobin (Hb) or perfluor- Africa [15]. Another oxygen carrier is based on the ochemicals. conjugation of haemoglobin molecules to polymers to Ó 2003 Blackwell Publishing Ltd 527 528 T. M. S. CHANG Principle first reported [27, 28]. Both of these have very good oxygen α1 α2 releasing characteristics (high P50). However, infu- Crosslinked polyHb α1 α2 - β1 β2 sion of 2 units or more in clinical trials can result in 1964 Chang - Diacid β1 β2 - α α vasoconstriction [26, 28]. One theory is that unlike 1971 Chang - Glutaraldehyde - 1 2 1980 Hsia - o-raffinose PolyHb and conjugated haemoglobin, the smaller β1 β2 single tetrameric haemoglobin molecules can cross Conjugated Hb the intercellular junction of endothelial cells lining 1964 Chang: polyamide the vessel walls and enter the interstitial space α1 α2 1968 Wong: dextran where they bind nitric oxide [10, 11]. When 2 units 1975 Sunder conjugated Hb β1 β2 or more are used, this can result in the removal of 1980 Iwashita: polyethylene glycol sufficient amount of nitric oxide to result in vaso- constriction and other smooth muscle effects. It Crosslinked tetrameric Hb α1 α2 1968 Bunn & Jandl - should be noted that PolyHb and conjugated hae- 1979 Walder et al: Diaspirin β1 β2 moglobin usually contain a small fraction of intra- molecularly cross-linked tetrameric haemoglobin. Recombinant human Hb The PolyHb containing <1% of tetrameric Hb did α1 α2 1990 Hoffman et al not result in vasoactivity when very large volumes nd β1 β2 1998 Doherty et al (2 generation) were infused in clinical trials. Polyhaemoglobin with <5% of tetramers results in slight vasoactivity when Fig. 1 Different types of modified haemoglobin. Updated, modified and reprinted with permission from: Chang, TMS, Blood Substi- large volumes were infused. Polyhaemoglobin with tutes. Karger Publisher, Basel, 1997, courtesy of copyright holder higher concentration of tetramer results in vasoac- [10]. tivity when lower volumes were infused. As mentioned above, when only intramolecularly form conjugated haemoglobin (Fig. 1) [7, 18–20]. cross-linked tetrameric haemoglobin was used, there Improved conjugated haemoglobins in phase II clin- was marked vasoactivity when small volumes were ical trials have been reported recently [21, 22]. Still used. This seems to support the possible role of another type of oxygen carrier in phase III clinical tetrameric haemoglobin in vasoacitivity [10, 11]. trial is based on perfluorochemicals [23, 24]. These Another possible contribution to vasoconstriction oxygen carriers are an intermediate step between has also been proposed related to the difference in volume replacement and red blood cells. While they the flow and oxygen release characteristics of work well when the conditions only require short- oxygen carriers in the microcirculation of oxygen term oxygen carriers, there are other conditions carriers as compared with RBC [3]. Second-genera- requiring more than just oxygen carriers. Further- tion recombinant human haemoglobin has recently more, progress in the field requires further basic been developed in which the addition of an amino fundamental knowledge. This paper concentrates on acid, tryptophan resulted in steric hindrance for the three areas: (1) modified haemoglobin, nitric oxide nitric oxide receptor site [29]. This new tetrameric and vasoactivity; (2) oxygen carriers with antioxidant recombinant human haemoglobin does not cause acitivity for those conditions with potentials for vasoconstriction when infused into animals. How- ischaemia–reperfusion injuries and (3) red blood cell ever, there was a change in the oxygen release substitutes that resemble more closely their biological characteristics (lower P50). This problem was solved counterpart. by replacing the distal histidine of the recombinant human haemoglobin with glutamine resulting in Vasoactivity and nitric oxide normal oxygen release characteristics. This new approach is now being actively developed. Cross-linking each haemoglobin molecule intra- molecularly can also prevent its breakdown into Conditions with potentials dimers [25, 26]. Another approach is the use of for ischaemia–reperfusion injury recombinant technology for Escherichia coli to pro- duce ‘fused’ single human haemoglobin molecules Red blood cells contain catalase (CAT), superoxide that do not break down into half molecules (Fig. 1) dismutase (SOD) and other enzymes. However, the Ó 2003 Blackwell Publishing Ltd Journal of Internal Medicine 253: 527–535 MINISYMPOSIUM: FUTURE BLOOD SUBSTITUTES 529 90 SF-Hb 0.8 PolyHb 80 SF-Hb + SOD + CAT * PolyHb Saline 70 0.6 PolyHb-SOD-CAT 60 Sham control 0.4 50 40 g per forebrain) 0.2 µ ( 30 3,4 DHBA production PolyHb-SOD-CAT Evan blue in brain tissue in brain blue Evan 20 0.0 10 0246810 Reperfusion time (min) 0 1 2 3 4 5 6 Fig. 2 Oxygen radicals in ischaemic intestinal reperfused with Time after reperfusion (h) PolyHb or PolyHb–SOD–catalase. Effects on oxygen radicals as measured by effluent 3,4-dihydroxybenzoate. Reprinted with Fig. 3 Effects of PolyHb–SOD–CAT on blood–brain barrier in permission from: S Razack, F D’Agnillo, and TMS Chang. Artificial ischaemia–reperfusion compared to other solutions. Reprinted Cells, Blood Substitutes and Immobilization Biotechnology, 25: with permission from Artificial Cells, Blood Substitutes and 181–192, 1997. Courtesy of Marcel Dekker Publisher Inc., NY. Immobilization Biotechnology, an International Journal, 30: 25– 42, 2002. Courtesy of Marcel Dekker Inc., NY. present first generation blood substitutes are only oxygen carriers with no enzyme activities [30]. 400 SF-Hb Lack of oxygen supply in severe haemorrhagic PolyHb 350 shock, stroke, myocardial infarction, organ trans- Saline 300 SF-Hb + SOD + CAT plantation and other conditions may result in PolyHb-SOD-CAT ischaemia. Ischaemia leads to alterations in meta- 250 Sham control bolic reactions producing hypoxanthine and activa- 200 ting the enzyme xanthine oxidase. The level of 150 hypoxanthine increases with the duration and severity of ischaemia. When the tissue is reperfused 100 with oxygen carrying fluid, xanthine oxidase con- 50 (mg of water per forebrain) (mg of water verts oxygen and hypoxanthine into superoxide. By content water Changes in brain 0 several mechanisms, superoxide results in the 0 123 4 5 6 formation of oxygen radicals that can cause tissue Time after reperfusion (h) injury. Superoxide dismutase and CAT in red blood Fig. 4 Effects of PolyHb–SOD–CAT on brain oedema in isch- cell converts superoxide into hydrogen peroxide that aemia–reperfusion compared to other oxygen carrying solutions. is