Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-02-26
2001-03-20
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C530S385000, C536S023100
Reexamination Certificate
active
06204009
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to mutant recombinant hemoglobins containing mutations around one or more of the heme pockets of the hemoglobin molecule. The invention relates particularly to mutant recombinant hemoglobins that have altered geometry or polarity around the distal portion of the heme pocket which results in mutant recombinant hemoglobins with reduced autooxidation rates, reduced or increased affinities for ligands or both reduced autooxidation and changed ligand affinity.
BACKGROUND OF THE INVENTION
Loss of blood often requires replacement of both the volume of fluid that is lost and the oxygen carrying capacity of that fluid This is typically accomplished by transfusing red blood cells, either as packed RBC's or as units of whole blood. However, it is not always possible, practical or desirable to transfuse a patient with donated blood. Human blood transfusions are associated with many risks and limitations, such as:
1) Infectious disease transmission (i.e., human immunodeficiency virus (HIV), non-A and non-B hepatitis, hepatitis B,
Yersinia enterocolitica,
cytomegalovirus, human T-cell leukemia virus 1)
2) Immunologic reaction (i.e., hemolytic transfusion reaction, immunosuppresion, graft versus host reaction)
3) Typing and cross-matching required prior to administration
4) Limited availability
5) Limited stability (shelf life of 42 days or less; cannot be frozen)
When human blood is not available or the risk of transfusion is too great, volume can be replaced utilizing plasma expanders such as colloid and crystalloid solutions but to date, none of the volume replacement therapies currently approved for human use can transport oxygen. In situations where replacement of lost blood is necessary and blood is not available for transfusion, a red blood cell substitute that can transport oxygen, such as a hemoglobin solution, is desirable. Administration of a hemoglobin solution can increase and/or maintain plasma volume and decrease blood viscosity in the same manner as conventional plasma expanders, but, in addition, a hemoglobin-based red blood cell substitute should be able to support adequate transport of oxygen from the lungs to peripheral tissues. Moreover, an oxygen-transporting hemoglobin-based solution may be used in most situations where red blood cells are currently utlized. For example, oxygen-transporting hemoglobin-based solution may be used to temporarily augment oxygen delivery during or after pre-donation of autologous blood prior to the return of the autologous blood to the patient.
To address this need, a number of red cell substitutes have been developed (Winslow, R. M.(1992)
Hemoglobin-based Red Cell Substitutes,
The Johns Hopkins University Press, Baltimore 242 pp). These substitutes include synthetic perfluorocarbon solutions, (Long, D. M. European Patent 0307087), stroma-free hemoglobin solutions, both chemically crosslinked and uncrosslinked, derived from a variety of mammalian red blood cells (Rausch, C. and Feola, M., U.S. Pat. Nos. 5,084,558 and 5,296,465; Sehgal, L. R., U.S. Pat. Nos. 4,826,811 and 5,194,590; Vlahakes, G. J. et al., (1990) J. Thorac. Cardiovas. Surg. 100: 379-388) and hemoglobins expressed in and purified from genetically engineered organisms (for example, non-erytocyte cells such as bacteria and yeast, Hoffman et al., WO 90/13645; bacteria, Fronticelli, C. et al., U.S. Pat. No. 5,239,061; yeast, De Angelo et al., WO 93/08831 and WO 91/16349; and transgenic mammals, Logan et al., WO 92/22646; Townes, T. M and McCune, S. L., WO 92/11283). These red blood cell substitutes have been designed to replace or augment the volume and the oxygen carrying capability of red blood cells.
The oxygen carrying portion of the red blood cell is the protein hemoglobin. Hemoglobin is a tetrameric protein molecule composed of two identical alpha globin subunits (&agr;
1
, &agr;
2
), two identical beta globin subunits (&bgr;
1
, &bgr;
2
) and four heme molecules. A heme molecule is incorporated into each of the alpha and beta globins to give alpha and beta subunits. Heme is a large macrocyclic organic molecule containing an iron atom; each heme can combine reversibly with one ligand molecule such as oxygen. In a hemoglobin tetramer, each alpha subunit is associated with a beta subunit to form two stable alpha/beta dimers, which in turn associate to form the tetramer (a homodimer). The subunits are noncovalently associated through Van der Waals forces, hydrogen bonds and salt bridges.
In the unliganded state (deoxygenated or “deoxy”) state, the four subunits form a quaternary structure known as “T” (for “tense”) state. During ligand binding, the &agr;
1
&bgr;
1
and &agr;
1
&bgr;
1
and &agr;
2
&bgr;
2
interfaces remain relatively fixed while the &agr;
1
&bgr;
2
and &agr;
2
&bgr;
1
interfaces exhibit considerable movement. When a ligand is bound to the hemoglobin molecule, the intersubunit distances are increased relative to the deoxygenated distances, and the molecule assumes the “relaxed” or “R” quaternary structure which is the thermodynamically stable form of the molecule when three or more ligands are bound to the heme.
Red blood cell replacement solutions have been administered to animals and humans and have exhibited certain adverse events upon administration. These adverse reactions may include hypertension due to vasoconstriction, renal failure, neurotoxicity, and liver toxicity (Winslow, R. M., ibid., Biro, G. P. et al., (1992) Biomat., Art. Cells & Immob. Biotech. 20: 1013-1020) and in the case of perfluorocarbons, hypertension, activation of the reticulo-endothelial system and complement activation (Reichelt, H. et al., (1992) in Blood Substitutes and Oxygen Carriers, T. M. Chang (ed.), pg. 769-772; Bentley, P. K. ibid, pp. 778-781). For hemoglobin based oxygen carriers, renal failure and renal toxicity is the result of the formation of hemoglobin &agr;/&bgr; dimers. The formation of dimers can be prevented by chemically crosslinkng (Sehgal, et al, U.S. Pat. Nos. 4,826,811 and 5,194,590; Walder, J. A. U.S. Reissue Pat. No. RE34271) or genetically linking (Hoffman, et al., WO 90/13645) the hemoglobin dimers so that the tetramer is prevented from dissociating.
However, prevention of dimer formation has not alleviated all of the adverse events associated with hemoglobin administration. Blood pressure changes upon administration of hemoglobin solutions have been attributed to vasoconstriction resulting from the binding of endothelium derived relaxing factor (EDRF) by hemoglobin (Spahn, D. R. et al., (1994) Anesth. Analg. 78: 1000-1021; Biro, G. P., (1992) Biomat., Art. Cells & Immob. Biotech., 20: 1013-1020; Vandegriff, K. D. (1992)
Biotechnology and Genetic Engineering Reviews,
Volume 10: 404-453 M. P. Tombs, Editor, Intercept Ltd., Andover, England). Endothelium derived relaxing factor has been identified as nitric oxide (NO) (Moncada, S. et al., (1991) Pharmacol. Rev. 43: 109-142 for review); both inducible and constitutive NO are primarily produced in the endothelium of the vasculature and act as local modulators of vascular tone. CO has also been implicated in blood pressure regulation since it can also activate guanylate cyclase (Snyder, S. H. and Bredt, D. S. (1992) Sci. American May, 68-77). Hemoglobin can bind both nitric oxide and carbon monoxide, thus preventing vascular relaxation and potentially leading to the hypertension sometimes observed upon administration of extracellular hemoglobin solutions. In addition to direct binding to deoxyhemoglobin, NO can also oxidize oxyhemoglobin producing peroxynitrite and methemoglobin. This reaction could also lower free concentrations of NO and lead to hypertension.
Some inflammatory responses are also mediated by nitric oxide (Vandegriff, ibid., Moncada, S., et al., ibid.). For example, nitric oxide produced by the endothelium inhibits platelet aggregation and as nitric oxide is bound by cell-free hemoglobin solutions, platelet aggregation may be increased. As platelets aggregate, they release potent vasoconstrictor compounds such as thromboxane A
2
an
Aitken Jacqueline F.
Mathews Antony J.
Nagai Kyoshi
Olson John S.
Baxter Biotech Technology Sarl
Carlson Karen Cochrane
Senniger Powers Leavitt & Roedel
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