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biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
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Thus far, the only molecules that have found even a limited clinical application as an oxygen-carrying blood substitute are the fluorocarbons and haemoglobin.
Fluosol is a fluorocarbon emulsion of perfluorodecalin and perfluorotripropylamine. Such fluorocarbon emulsions can absorb, transport and release both oxygen and carbon dioxide. Although fluosol failed to show efficacy in the treatment of life-threatening anaemia, it is used as an effective oxygen carrier during coronary angioplasty procedures. A 20% emulsion is normally used, and can be administered regardless of blood type. It also will be free of any blood-borne pathogens, and is stable for years when stored at room temperature.
Haemoglobin displays the property of reversible oxygenation and, hence, is being investigated as a blood substitute. Human haemoglobin is a tetramer of molecular mass 64 kDa. It consists of two a-chains (141 amino acids) and two b-chains (146 amino acids), with each subunit displaying an associated haem prosthetic group. The interaction of the oxygen-binding haem moiety and the globulin constituent is important, as free haem is toxic and the apoglobin is insoluble.
Because of its structural characteristics, haemoglobin displays a non-linear oxygen saturation curve, with maximal oxygen saturation occurring in arterial blood. At tissue sites, the oxygen partial pressure is lower and much of the haemoglobin-bound oxygen is released. This process is aided in vivo by the effector molecule 2,3-diphosphoglycerate (2,3-DPG), which binds to deoxyhaemoglobin (in the presence of 2,3-DPG and at normal tissue oxygen partial pressures, over 20% of the bound oxygen is released from haemoglobin; in the absence of 2,3-DPG, this value falls to 2-4%). Native haemoglobin, when extracted from erythrocytes, is less attractive as a tissue oxygen delivery system, as free haemoglobin no longer binds 2,3-DPG. Furthermore, outside the erythrocyte (in which haemoglobin is present at very high concentrations), haemoglobin tends to disassociate into dimers. Attempts have been made to stabilize free haemoglobin and to modify it in order to reduce its oxygen affinity.
Pyridoxal 5'-phosphate (a derivative of pyridoxal; vitamin Âá) is similar in size and charge to 2,3-DPG. Covalent attachment of pyridoxal 5'-phosphate reduces the oxygen affinity of the haemoglobin molecule. Covalent attachment of benzene isothiocyanates to the amino termini of the four haemoglobin polypeptide chains, also yields derivatives which display lower oxygen affinity. These may prove worthy of clinical investigation.
In addition to altered oxygen-binding characteristics, free haemoglobin in plasma disassociates rapidly into ab dimers, which are in turn rapidly oxidized and cleared by the kidneys. Indeed, high plasma concentrations can result in kidney toxicity. Development of a
358 BIOPHARMACEUTICALS
clinically useful modified haemoglobin would have to circumvent this, probably by polymerization or micro-encapsulation of the molecules prior to their administration.
Haemoglobin conjugated to additional high molecular mass substances, such as dextrin and polyethylene glycol, are also generating clinical interest. When compared to native haemoglobin, such high molecular mass derivatives often display prolonged vascular retention (i.e. prolonged useful half-life), reduced antigenicity (if animal haemoglobin is used) and potent plasma volume expansion properties.
Polymerized haemoglobins (usually formed by crosslinking with gluteraldehyde) have also been generated. The aim again is to prolong product half-life by preventing disassociation into dimers or monomers. Additionally, such products often display increased thermal stability, facilitating a subsequent viral-inactivation heat step.
In addition to chemical crosslinking, recombinant DNA technology has been used to generate stable intact haemoglobin variants. ‘Dialpha’ haemoglobin, for example, is an engineered molecule in which two a-chains are fused together, head to tail. Co-expression in E. coli along with the b-gene results in the formation of a working, stable haemoglobin analogue. Haemoglobin molecules can be obtained from whole blood/red cell concentrates. Human haemoglobin a and b genes have been expressed in a wide variety of prokaryotic and eukaryotic systems, including transgenic plants and animals.
Although research continues in an effort to develop an effective haemoglobin-based red blood cell substitute, no suitable candidate has yet been developed that has gained widespread clinical acceptance.
HAEMOSTASIS
Blood plays various vital roles within the body and it is not surprising that a number of processes have evolved capable of effectively maintaining haemostasis — the rapid arrest of blood loss upon vascular damage, in order to maintain a relatively constant blood volume. In humans, three main mechanisms underline the haemostatic process:
• The congregation and clumping of blood platelets at the site of vascular injury, thus effectively plugging the site of blood leakage.
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