Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1993-05-20
2001-08-14
Travers, Russell (Department: 1617)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
active
06274581
ABSTRACT:
TECHNICAL FIELD
This invention is directed to a novel method of inhibiting the induction of nitric oxide formation in biological systems by bacterial endotoxins and cytokines.
BACKGROUND OF THE INVENTION
For several decades nitroglycerin has been administered to humans as a vasodilating agent in the treatment of cardiovascular disease. Recently, it has been shown that nitroglycerin so administered is converted in the body to nitric oxide which is the pharmacologically active metabolite. Still more recently, nitric oxide has been shown to be formed enzymatically from arginine as a normal metabolite which is an important component of endothelium-derived relaxing factors (EDRFs). EDRFs are currently being intensively studied as participating in regulation of blood flow and vascular resistance. In addition to vascular endothelium, macrophages have also been shown to produce nitric oxide in the body which is a component of their cell killing and/or cytostatic function.
More recently it has been established that the enzyme forming nitric oxide from arginine, i.e., nitric oxide synthase, occurs in two distinct forms, namely a constitutive form and an inducible form. The constitutive form is present in normal endothelial cells, neurons and some other tissues. Formation of nitric oxide by the constitutive form in endothelial cells is thought to play a role in normal blood pressure regulation. The inducible form of nitric oxide synthase has been found to be present in activated macrophages and is induced in endothelial cells and vascular smooth muscle cells, for example, by various cytokines and/or microbial products. It is thought that in sepsis or cytokine-induced shock, overproduction of nitric oxide by the inducible form of nitric oxide synthase plays an important role in the observed life-threatening hypotension. Furthermore, it is thought that overproduction of nitric oxide by the inducible form of nitric oxide synthase is a basis for insensitivity to clinically used pressor agents such as &agr;
1
. adrenergic agonists in the treatment of septic or cytokine-induced shock patients.
Considerable research effort has been expended to discover inhibitors for nitric oxide synthase activity. Before the work described herein, said research effort has been directed at uncovering arginine antagonists to function to inhibit nitric oxide synthase activity. A problem with use of the arginine antagonists for this purpose is that the ones uncovered thus far block not only inducible nitric oxide synthase activity but also constitutive nitric oxide synthase activity; and any specificity of inhibition of any particular arginine antagonist for inducible nitric oxide synthase activity is not so high that it is possible to block hypotension-causing, pathological overproduction of nitric oxide (an inducible enzyme-mediated process) to a therapeutically adequate extent (i.e. so that clinically serious hypotension that would normally occur in sepsis or cytokine-induced shock is avoided or so that pressor agent sensitivity is restored), and, at the same time, not block the physiological nitric oxide synthesis which is thought to play a role in neural function and normal blood pressure regulation (constitutive enzyme-mediated processes) and thereby avoid the toxicity (e.g. neuronal toxicity and hypertension) associated with interfering with physiological nitric oxide synthesis.
SUMMARY OF THE INVENTION
The invention herein does not rely on arginine antagonists but rather uses a novel approach to selectively block the induction of nitric oxide synthesis by cytokines and/or microbial products (e.g. bacterial endotoxins) without blocking physiological (constitutive enzyme-mediated) nitric oxide production.
The invention herein draws on the recent discovery that tetrahydrobiopterin is a cofactor in the induction of nitric oxide synthesis (Kwon, N. C., et al, J. Biol. Chem. 264:20496-20501, 1989, and Tayeh, M. A., et al J. Biol Chem. 264:19654-19658, 1989).
The invention herein also draws on the discovery that cytokines, e.g. interferon-gamma, have been found to markedly increase tetrahydrobiopterin levels in various cells including macrophages (Werner, E., et al, Biochem. J 262:861-866, 1989; Kerler, F., et al, Experimental Cell Research 189, 151-156, 1990; Ziegler, I., et al, The Journal of Biological Chemistry, 265, No. 28, 17026-17030, 10/05/90), the discovery in the course of the invention herein that tetrahydrobiopterin synthesis is induced by bacterial endotoxins, the discoveries that tetrahydrobiopterin synthesis occurs via a guanosine triphosphate pathway and via a pterin salvage pathway (Nichol, C., et al, Ann. Rev. Biochem. 54, 729-764, 1985; Milstien, S., et al, Biochem. and Biophys. Res. Comm., 128, No. 3, 1099-1107, 1985; Kaufman, S., et al, J. Biol. Chem., 234, 2683-2688, 10/59; Kaufman, S., J. Biol. Chem., 242, 3934-3943, 9/10/67), and the discovery in the course of the invention that the continuous production of tetrahydrobiopterin, via a guanosine triphosphate pathway or a pterin salvage pathway, is not required for maintaining constitutive nitric oxide synthase activity over at least a period of hours.
It has been discovered herein that inhibiting the synthesis of tetrahydrobiopterin in vascular smooth muscle cells via the guanosine triphosphate pathway and/or the pterin salvage pathway selectively inhibits the induction of nitric oxide synthesis in said cells by bacterial endotoxins and cytokines, i.e. performs such inhibiting without affecting physiological constitutive enzyme-mediated nitric oxide synthesis. The inhibition of nitric oxide synthesis in smooth muscle cells in accordance with the invention is an unexpected result since it has been shown that macrophages which are “normal” , ie, are not included for nitric oxide synthesis, already contain enough tetrahydrobiopterin for a maximal rate of nitric oxide synthesis (see Kwon, N. C., et al, J. Biol. Chem. 264:20496-2501, 1989).
In a first embodiment the invention herein is directed at a method of inhibiting induced nitric oxide synthesis from arginine in vascular smooth muscle cells in a subject in need of said inhibition (e.g. for prophylaxis or treatment of systemic hypotension or to restore vascular contractile sensitivity to effects of pressor agents such as &agr;
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, adrenergic agents), said method comprising administering to said subject of a nitric oxide synthesis inhibiting therapeutically effective amount of (a) at least one guanosine triphosphate pathway tetrahydrobiopterin synthesis antagonist which is not a reduced pterin that is a substrate for the pterin salvage pathway or (b) at least one pterin salvage pathway tetrahydrobiopterin synthesis antagonist or of both (a) and (b).
In a second embodiment, the invention herein is directed at a method of inhibiting induced nitric oxide synthesis from arginine in vascular smooth muscle cells in a subject in need of said inhibition (e.g. for prophylaxis or treatment of systemic hypotension or to restore vascular contractile sensitivity to effects of pressor agents such as &agr;
1
, adrenergic agents), said method comprising administering to said subject of nitric oxide synthesis inhibiting therapeutically effective amounts of at least one guanosine triphosphate pathway tetrahydrobiopterin synthesis antagonist which is a reduced pterin that is a substrate for the pterin salvage pathway and of at least one pterin salvage pathway tetrahydrobiopterin synthesis antagonist.
The term “subject” is used herein to mean any mammal, including humans, where nitric oxide formation from arginine occurs. The methods herein for use on subjects contemplate prophylactic use as well as curative use in therapy of an existing condition. When the combination of guanosine triphosphate pathway tetrahydrobiopterin synthesis antagonist and pterin salvage pathway tetrahydrobiopterin synthesis antagonist is used, the amounts used of each should be such that the combination inhibits induced nitric oxide synthesis from arginine.
The guanosine triphosphate pathway (also referred to as the tetrahydropterin path
Cornell Research Foundation Inc.
Travers Russell
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