Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1996-06-20
2001-03-06
Celsa, Bennett (Department: 1627)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S832000, C530S385000, C530S829000
Reexamination Certificate
active
06197745
ABSTRACT:
BACKGROUND OF THE INVENTION
Interactions of hemoglobin (Hb) with small diffusable ligands, such as O
2
, CO
2
and NO, are known to occur at its metal centers and amino termini. The O
2
/CO
2
delivery functionalities, which arise in the lung and systemic microvasculature, are allosterically controlled. Such responsiveness to the environment is not known to apply in the case of NO. Specifically, it is thought that Hb(Fe) is involve d in limiting NO's sphere of action (Lancaster, J. R.,
Proc. Natl. Acad. Sci. USA,
91:8137-8141 (1994); Wood and Garthwaite,
J. Neurophacmacol.,
33:1235-1244 (1994)), but that No does not modify the functional properties of Hb to any physiologically significant degree. Kinetic modeling based on this assumption, however, predicts that the vast majority of free NO in the vasculature should be scavenged by Hb (Lancaster 1994). Accordingly, the steady-state level of No may fall below the Y, for target enzymes such as guanylate cyclase (Lancaster 1994), if not in the unperturbed organism, then with oxidant stress such as that found in atherosclerosis. These considerations raise the fundamental question of how NO exerts its biological activity.
One answer to this paradox may be found in the propensity of nitric oxide to form S-nitrosothiols (RSNOs) (Gaston, B. et al.,
Proc. Natl. Acad. Sci. USA,
90:10957-10961 (1993)), which retain NO-like vasorelaxant activity (Stamler, J. S., et al.,
Proc. Natl. Acad. Sci, USA,
89:444-448 (1992)), but which are not subject to the diffusional constraints imposed by the high concentration of Hb in the blood. In particular, the NO group of RSNOs possesses nitrosonium (NO+) character that distinguishes it from NO itself. It is increasingly appreciated that RSNOs have the capacity to elicit certain functions that NO is incapable of (DeGroote, M. A. et al.,
Proc. Natl. Acad. Sci. USA,
92:6399-6403 (1995); Stamler, J. S., Cell, 78:931-936 (1994)). Moreover, consideration has been given to the possibility that —SNO groups in proteins may serve a signaling function, perhaps analagous to phosphorylation (Stamler, J. S. et al.,
Proc. Natl. Acad. Sci. USA,
89:444-448 (1992); Stamler, J. S. Cell, 78:931-926 (1994)). Although S-nitrosylation of proteins can regulate protein function (Stamler, J. S. et al.,
Proc. Natl. Acad Sci. USA,
89:444-448 (1992); Stamler, J. S.,
Cell,
78:931-936 (1994)), the identification of S-nitrosoproteins within cells—the sine qua non of a regulatory posttranslational modification —has heretofore not been demonstrated.
Hemoglobin is a tetramer comprised of two alpha and two beta subunits. In human Hb, each subunit contains one heme, while the beta (&bgr;) subunits also contain highly reactive SH groups (cys#93) (Olson, J. S.,
Methods in Enzymology
76:631-651 (1981); Antonini, E. & Brunori, M. In
Hemoglobin and Myoglobin in Their Reactions with Ligands,
American Elsevier Publishing Co., Inc., New York, pp. 29-31 (1971)). These cysteine residues are highly conserved among species although their function has remained elusive.
NO (nitric oxide) is a biological “messenger molecule” which decreases blood pressure and inhibits platelet function, among other functions. NO freely diffuses from endothelium to vascular smooth muscle and platelet and across neuronal synapses to evoke biological responses. Under some conditions, reactions of NO with other components present in cells and in serum can generate toxic intermediates and products at local concentrations in tissues which are effective at inhibiting the growth of infectious organisms. Thus, it can be seen that a method of administering an effective concentration of NO or biologically active forms thereof would be beneficial in certain medical disorders.
Platelet activation is an essential component of blood coagulation and thrombotic diathesis. Activation of platelets is also seen in hematologic disorders such as sickle cell disease, in which local thrombosis is thought to be central to the painful crisis. Inhibition of platelet aggregation is therefore an important therapeutic goal in heart attacks, stroke, peripheral vascular disease and shock (disseminated intravascular coagulation). Researchers have attempted to give artificial hemoglobins to enhance oxygen delivery in all of the above disease states. However, as recently pointed out by Olsen and coworkers, administration of underivatized hemoglobin leads to platelet activation at sites of vascular injury (Olsen S. B. et al.,
Circulation
93:327-332 (1996)). This major problem has led experts to conclude that cell-free underivatized hemoglobins may pose a significant risk in the patient with vascular disease or a clotting disorder (Marcus, A. J. and J. B. Broekman,
Circulation
93:208-209 (1996)). A new method of providing for an oxygen carrier and/or a method of inhibiting platelet activation would be of benefit to patients with vascular disease or who are otherwise at risk for thrombosis.
SUMMARY OF THE INVENTION
The invention relates to methods of forming SNO-Hb by reaction of Hb with S-nitrosothiol in procedures which avoid oxidation of the heme. The invention also includes methods of producing nitrosated (including nitrosylated at thiols or metals) and nitrated derivatives of hemoglobins in which the heme Fe may or may not be oxidized, depending on the steps of the method. The invention also relates to a method of therapy for a condition in which it is desired to oxygenate, to scavenge free radicals, or to release NO
+
groups to tissues. SNO-Hb in its various forms and combinations thereof (oxy, deoxy, met; specifically S-nitrosylated, or nitrosated or nitrated to various extents) can be administered to an animal or human in these methods. Thiols and/or NO donating agents can also be administered to enhance the transfer of NO
+
groups. Examples of conditions to be treated by nitrosated or nitrated forms of hemoglobin include ischemic injury, hypertension, angina, reperfusion injury and inflammation, and diseases characterized by thrombosis.
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CAPLUS No. 1988: 108985 to Sprokholt et al.
Lancaster, Jack R., Jr., “Simulation of the Diffusion and Reaction of Endogenously Produced Nitric Oxide,”Proc. Natl. Acad. Sci. USA,91:8137-8141 (1994).
Butler, Anthony R. et al., “No, Nitrosonium Ions, Nitroxide Ions, Nitrosothiols and Iron-Nitrosyls in Biology: A Chemist's Perspective,”TiPS,16:18-22 (1995).
Stamler, Jonathan S. et al., “S-Nitrosylation of Proteins with Nitric Oxide: Synthesis and Characterization of Biologically Active Compounds,”Proc. Natl. Acad. Sci. USA,89:444-448 (1992).
Langford, E.J. et al., “Inhibition of Platelet Activity by S-Nitrosoglutathione During Coronary Angioplasty,”The Lancet,344:1458-1460 (1994).
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Doyle, Michael P. et al., “Structural Effects in Alkyl Nitrite Oxidation of Human Hemoglobin,”Journal of Biological Chemistry.259(1):80-87 (1984).
Shah, N.S. et al., “Efficiacy of Inhaled Nitric Oxide in a Porcine Model of Adult Respiratory Distress Syndrome,”Archives of Surger
Celsa Bennett
Duke University
Hamilton Brook Smith & Reynolds P.C.
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