Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1993-12-15
1995-01-10
Wityshyn, Michael G.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
A61K 3514
Patent
active
053808240
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for the preparation of a pyridoxylated modified hemoglobin. More particularly, the invention relates to such a method which is easier and less cumbersome to carry out than the methods known from the prior art.
Hemoglobin is the oxygen transporting protein of the red blood cells and makes up about 30 percent of the cell. The protein comprises four units, two alpha and two beta units, which are bonded together to a tetramer inside the cell. Inside the red cell, the hemoglobin is kept as a tetramer of two .alpha.-chains and two .beta.-chains. When free in plasma, hemoglobin dissociates and two dimers (.alpha., .beta.) are bound to haptoglobin. Free hemoglobin in plasma will soon leave the circulation with a half-life of about 3 hours.
The oxygen affinity is modulated by pH, CO.sub.2 concentration and the compound: 2,3-DPG (2,3-diphosphoglycerate) which is only available inside the red cell. Outside the cell the oxygen affinity of hemoglobin is high and therefore the ability to transfer oxygen to the tissue is low. Antigens which are bound to the cell wall surrounding the hemoglobin determine such factors as blood type, Rh factor and others. The cell wall residues obtained after lysis is called stroma.
Red blood cell substitutes are currently under development for use as oxygen transporting fluids. In "Blood substitues", Eds: Thomas M S Chang & Robert P Geyer; Marcel Dekker Inc. N.Y. 1989 (ISBN 0-847-8027-2) the present situation has been summarized.
It has been known for a long time that hemoglobin outside the cell has oxygen-transporting properties and can be given to patients regardless of their blood types. However, hemoglobin dissociates in the body into two alpha-beta units, which give rise to kidney dysfunction. Although this dysfunction is reversible, it can be very serious for patients who are already in a weakened state. Other side effects have also been known to occur.
W R Amberson (Biol Rev 12 p 48 (1937)) used red cell hemolysates as a blood substitute. It was nephrotoxic. The adverse effect was suggested by Rabiner et al (J Exp Med 126 p 1127 (1967)) to depend on the presence of stroma residues. However, even stroma-free hemoglobin solution had effects on the kidneys and was shown to give a transient decrease in creatinine clearance and urinary volume (G S Moss et al; Surg Gynecol Obstet 142 p 357 (1976); De Venuto et al; J Lab Clin Med 89 p 509 (1977) and Savitsky et al; Clin Pharm Ther 23 p 73 (1978)).
Different types of modifications of the hemoglobin molecule have been described in Methods in Enzymology Vol 76 (Hemoglobins); Editors S P Colowick, N O Kaplan, Academic Press N.Y. (1981). Benesch et al; Biochemistry Vol 11, No 19 p 3576-3582 (1972) described the modification most commonly used to decrease the oxygen affinity, by the incorporation of pyridoxal-5'-phosphate. This stabilizes the hemoglobin molecule in a configuration similar to the hemoglobin-DPG (diphosphoglycerate) complex inside the red cell. Lately the bispyridoxal tetraphosphate has been used for this type of modification (P E Keipert, A J Adenican, S Kwong & R E Benesch, Transfusion 29, p 768-773 (1989)).
Other compounds, e.g. inositol-hexaphosphate, can also be used for the modification of hemoglobin to obtain a product with lower oxygen affinity.
In U.S. Pat. Nos. 4,001,200; 4,001,401, 4,053,590 and 4,061,736, Bonsen et al have shown different routes to increase the molecular weight of hemoglobin and thus further stabilize the structure in order to increase the apparent half-life of the blood substitute based on hemoglobin.
A further problem in the administration of hemoglobin preparations lies in the absolute requirement that these preparations be free from microorganisms and viruses. Especially, it has been shown that viruses may be transmitted from blood donors to recipients.
If viruses in the hemoglobin are to be inactivated, the substantially cell-free hemoglobin solution is heated at a temperature from 45.degree. to 85.degree. C. while it is maintained in its deoxy form. This
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Eketorp Rainer
Marschall Robert
Pharmacia Aktiebolag
Sayala C.
Wityshyn Michael G.
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