Red blood cells loaded with S-nitrosothiol and uses therefor

Details Red blood cells loaded with S-nitrosothiol and uses therefor Red blood cells loaded with S-nitrosothiol and uses therefor

1997-06-12

2001-03-20

Witz, Jean C. (Department: 1651)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Animal or plant cell

C424S093700, C435S372000

Reexamination Certificate

active

06203789

ABSTRACT:

BACKGROUND OF THE INVENTION
Interactions of hemoglobin (Hb) with small diffusible 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 functions, which arise in the lung and systemic microvasculature, are allosterically controlled. Such responsiveness to the environment: has not been known to apply in the case of NO. Specifically, it has been thought previously that NO does not modify the functional properties of Hb to any physiologically significant degree. Kinetic modeling 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 K
m
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 question is 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 can diffuse freely in and out of cells, unlike Hb. In particular, the NO group of RSNOs possesses nitrosonium (NO
+
) character that distinguishes it from NO itself. It is increasingly appreciated that RSNO's 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 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 (cyso&bgr;93) (Olson, J. S.,
Meth. in Enzym.
76:631-651 (1981); Antonini & Brunori,
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. Furthermore, under some conditions, reactions of NO with other components present in cells and in body fluids 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.
SUMMARY OF THE INVENTION
The invention relates to a method for the extracorporeal loading of red blood cells (RBCs) with a low molecular weight reagent which is capable of entering the red blood cells and causing S-nitrosylation at thiol groups. S-nitrosothiols such as S-nitrosocysteine, and related peptides can be used to load red blood cells, which can then be introduced into a mammal. The red blood cells thereby become carriers of NO. In this manner, medical conditions characterized by abnormal oxygen metabolism of tissues, oxygen-related toxicity, abnormal vascular tone, abnormal red blood cell adhesion, or abnormal O
2
delivery by red blood cells, can be treated.


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