U.S. patent application number 11/418367 was filed with the patent office on 2006-09-07 for nitrosylation of protein sh groups and amino acid residues as a therapeutic modality.
This patent application is currently assigned to Brigham and Women's Hospital, Brigham and Women's Hospital. Invention is credited to Joseph Loscalzo, Daniel Simon, David Singel, Jonathan Stamler.
Application Number | 20060198831 11/418367 |
Document ID | / |
Family ID | 27121187 |
Filed Date | 2006-09-07 |
United States Patent
Application |
20060198831 |
Kind Code |
A1 |
Stamler; Jonathan ; et
al. |
September 7, 2006 |
Nitrosylation of protein SH groups and amino acid residues as a
therapeutic modality
Abstract
Nitrosylation of proteins and amino acid groups enables
selective regulation of protein function, and also endows the
proteins and amino acids with additional smooth muscle relaxant and
platelet inhibitory capabilities. Thus, the invention relates to
novel compounds achieved by nitrosylation of protein thiols. Such
compounds include: S-nitroso-t-PA, S-nitroso-cathepsin;
S-nitroso-lipoprotein; and S-nitroso-immunoglobulin. The invention
also relates to therapeutic use of S-nitroso-protein compounds for
regulating protein function, cellular metabolism and effecting
vasodilation, platelet inhibition, relaxation of non-vascular
smooth muscle, and increasing blood oxygen transport by hemoglobin
and myoglobin. The compounds are also used to deliver nitric oxide
in its most bioactive form in order to achieve the effects
described above, or for in vitro nitrosylation of molecules present
in the body. The invention also relates to the nitrosylation of
oxygen, carbon and nitrogen moieties present on proteins and amino
acids, and the use thereof to achieve the above physiological
effects.
Inventors: |
Stamler; Jonathan; (Chapel
Hill, NC) ; Loscalzo; Joseph; (Dover, MA) ;
Simon; Daniel; (Waban, MA) ; Singel; David;
(Arlington, MA) |
Correspondence
Address: |
EDWARD D GRIEFF;HALE & DORR LLP
1455 PENNSYLVANIA AVE, NW
WASHINGTON
DC
20004
US
|
Assignee: |
Brigham and Women's
Hospital
Boston
MA
|
Family ID: |
27121187 |
Appl. No.: |
11/418367 |
Filed: |
May 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10216865 |
Aug 13, 2002 |
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11418367 |
May 5, 2006 |
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08437884 |
May 9, 1995 |
6562344 |
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10216865 |
Aug 13, 2002 |
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08287830 |
Aug 9, 1994 |
5593876 |
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08437884 |
May 9, 1995 |
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08198854 |
Feb 17, 1994 |
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08287830 |
Aug 9, 1994 |
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07943835 |
Sep 14, 1992 |
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08198854 |
Feb 17, 1994 |
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07791668 |
Nov 14, 1991 |
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07943835 |
Sep 14, 1992 |
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Current U.S.
Class: |
424/94.1 ;
435/183; 514/12.1; 514/15.1; 514/15.2; 514/19.3; 514/20.6; 514/7.4;
530/359; 530/363 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 9/00 20180101; C07K 14/4702 20130101; C12Y 304/21069 20130101;
A61P 11/00 20180101; C07K 14/3153 20130101; C12N 9/6459 20130101;
C07K 14/76 20130101; C07K 14/765 20130101; C07K 14/805 20130101;
C07K 16/00 20130101; C07K 1/1077 20130101; C12N 9/6472
20130101 |
Class at
Publication: |
424/094.1 ;
514/012; 530/363; 530/359; 435/183 |
International
Class: |
A61K 38/43 20060101
A61K038/43; A61K 38/38 20060101 A61K038/38; A61K 38/17 20060101
A61K038/17; C12N 9/00 20060101 C12N009/00; C07K 14/775 20060101
C07K014/775; C07K 14/765 20060101 C07K014/765 |
Goverment Interests
[0003] This invention was made with government support under
RO1-HL40411, HL43344 and RR04870, awarded by The National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A method for causing vasodilation or relaxing non-vascular
smooth muscle comprising administering to an animal in need thereof
a therapeutically effective amount of a pharmaceutical composition
comprising an S-nitroso-enzyme, an S-nitroso-lipoprotein, an
S-nitroso-immunoglobulin, an S-nitroso-hemoglobin or an
S-nitroso-albumin.
2. The method of claim 2, wherein the smooth muscle comprises
airway smooth muscle.
3. A method for treating a cardiovascular disorder or a respiratory
disorder comprising administering to an animal in need thereof a
therapeutically effective amount of a pharmaceutical composition
comprising an S-nitroso-enzyme, an S-nitroso-lipoprotein, an
S-nitroso-immunoglobulin, an S-nitroso-hemoglobin or an
S-nitroso-albumin.
4. A method for lysing a blood clot comprising administrating to an
animal in need thereof a therapeutically effective amount of a
pharmaceutical composition comprising an S-nitroso-tissue-type
plasminogen activator, an S-nitroso-streptokinase, or an
S-nitroso-urokinase.
5. A method for regulating protein or amino acid function or for
preventing cellular uptake of one or more proteins comprising
administering a therapeutic amount of a nitrosylating compound to
an animal in need thereof.
6. The method of claim 5, wherein the nitrosylating compound is
nitroglycerin, a nitrosothiol, or nitric oxide.
7. A method for regulating the function of proteins in which a
thiol is bound to a methyl group comprising: removing the methyl
groups from the thiol by selective de-methylation; and reacting the
free thiol group with a nitrosylating agent.
8. A method for regulating the function of a protein which lacks a
free thiol group comprising: adding a thiol group to the protein by
chemical or genetic engineering means; and reacting the thiol group
with a nitrosylating agent.
9. A method for regulating cellular function comprising
S-nitrosylating a protein, wherein the protein a cellular component
or affects cellular function.
10. The method of claim 9, wherein the protein is a cell receptor,
G-protein, a target protein, histone, a protein involved in cell
proliferation, a protein involved in inhibition of proliferation, a
protein involved in cellular repair, an immune modulator, a protein
with a cytostatic function or a protein with a cytotoxic
function.
11. A method for delivering nitric oxide to one or more specific
targeted sites in a body comprising administering to an animal in
need thereof a therapeutically effective amount of a pharmaceutical
composition comprising an S-nitroso-enzyme, an
S-nitroso-lipoprotein, an S-nitroso-immunoglobulin, an
S-nitroso-hemoglobin or an S-nitroso-albumin.
12. A method for inhibiting platelet function comprising
administering to an animal in need thereof a therapeutically
effective amount of a pharmaceutical composition comprising an
S-nitroso-lipoprotein, an S-nitroso-immunoglobulin, or an
S-nitroso-hemoglobin.
13. The method of claim 12, wherein the pharmaceutical composition
further comprises a pharmaceutically acceptable carrier.
14. The method of claim 12, wherein the S-nitroso-lipoprotein has
at least one nitrogen monoxide moiety bonded at a sulfur atom, an
oxygen atom or a nitrogen atom on the lipoprotein.
15. The method of claim 14, wherein the lipoprotein is a
chylomicron, a chylomicron remnant particle, a very low-density
lipoprotein, an intermediate-density lipoprotein, a low-density
lipoprotein, a high density lipoprotein or a lipoprotein (a).
16. The method of claim 12, wherein the S-nitroso-hemoglobin has at
least one nitrogen monoxide moiety bonded at a sulfur atom, an
oxygen atom or a nitrogen atom on the hemoglobin.
17. The method of claim 16, wherein the hemoglobin is
myoglobin.
18. The method of claim 12, wherein the S-nitroso-immunoglobulin
has at least one nitrogen monoxide moiety bonded at a sulfur atom,
an oxygen atom or a nitrogen atom on the immunoglobulin.
19. The method of claim 18, wherein the immunoglobulin molecule is
IgG, IgM, IgA, IgD or IgE.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/216,865 filed Aug. 13, 2002, which is a continuation of U.S.
application Ser. No. 08/437,884 filed May 9, 1995, issued as U.S.
Pat. No. 6,562,344, which is a continuation of U.S. application
Ser. No. 08/287,830 filed Aug. 9, 1994, issued as U.S. Pat. No.
5,593,876, which is a continuation of U.S. application Ser. No.
08/198,854 filed Feb. 17, 1994, abandoned, which is a continuation
of U.S. application Ser. No. 07/943,835 filed Sep. 14, 1992,
abandoned, which is a continuation-in-part of U.S. application Ser.
No. 07/791,668, filed Nov. 14, 1991, abandoned.
[0002] This application is related to U.S. Pat. Nos. 5,863,890,
6,291,424, 6,583,113 and 7,033,999 and U.S. application Ser. No.
11/349,178 filed Feb. 8, 2006.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] This invention relates to nitrosylation of proteins and
amino acids as a therapeutic modality. In particular, the invention
relates to S-nitroso-protein compounds and their use as a means to
selectively regulate specific protein functions, to selectively
regulate cellular function, to endow the protein with new smooth
muscle relaxant and platelet inhibitory properties and to provide
targeted delivery of nitric oxide to specific bodily sites.
[0006] Additionally, the invention relates to nitrosylation of
additional sites such as oxygen, carbon and nitrogen, present on
proteins and amino acids, as a means to achieve the above
physiological effects. The therapeutic effects may be achieved by
the administration of nitrosylated proteins and amino acids as
pharmaceutical compositions, or by nitrosylation of proteins and
amino acids in vivo through the administration of a nitrosylating
agent, perhaps in the form of a pharmaceutical composition.
[0007] 2. Brief Description of the Background Art
[0008] The reaction between low molecular weight thiols, such as
cysteine, homocysteine, and N-acetylcysteine, and nitric oxide (NO)
has been studied in biological systems. NO has been shown to induce
relaxation of vascular smooth muscle, and inhibition of platelet
aggregation, through activation of guanylate cyclase and elevation
of cyclic GMP levels. Evidence exists that low molecular weight
thiols react readily with NO to form S-nitrosothiols, which are
significantly more stable than NO itself, and act as potent
vasodilators and platelet inhibitors. These adducts have also been
proposed as biologically active intermediates in the metabolism of
organic nitrates (Ignarro et al., J. Pharmacol. Exp. Ther. 218:739
(1981); Mellion, et al., Mol. Pharmacol. 23:653 (1983); Loscalzo,
et al, J. Clin. Invest. 76:966 (1985)).
[0009] Many proteins of physiological significance possess
intramolecular thiols in the form of cysteine residues. These thiol
groups are often of critical importance in the functional
properties of such proteins. These sulfhydryl groups are highly
specialized and utilized extensively in physiological processes
such as metabolic regulation, structural stabilization, transfer of
reducing equivalents, detoxification pathways and enzyme catalysis
(Gilbert, H. F., "Molecular and Cellular Aspects of Thiol-Disulfide
Exchange", Advances in Enzymology, A. Miester, J. Wiley & Sons,
Eds. New York 1990, pages 69-172.)
[0010] Thiols are also present on those proteins the function of
which is to transport and deliver specific molecules to particular
bodily tissues. For example, lipoproteins are globular particles of
high molecular weight that transport non-polar lipids through the
plasma. These proteins contain thiols in the region of the protein
which controls cellular uptake of the lipoprotein (Mahley et al.
JAMA 265:78-83 (1991)). Hyper-liproteinemias, resulting from
excessive lipoprotein (and thus, lipid) uptake, cause
life-threatening diseases such as atherosclerosis and
pancreatitis.
[0011] The thiol contained in hemoglobin regulates the affinity of
hemoglobin for oxygen, and thus has a critical role in the delivery
of oxygen to bodily tissues. The reaction between the free NO
radical occurs at the iron-binding site of hemoglobin, and not the
thiol. As a result, methemoglobin is generated, which impairs
oxygen-hemoglobin binding, and thus, oxygen transport. Other
proteins such as thrombolytic agents, immunoglobulins, and albumin,
possess free thiol groups that are important in regulating protein
function.
[0012] Protein thiols may, under certain pathophysiological
conditions, cause a protein to exert a detrimental effect. For
example, cathepsin, a sulfhydryl enzyme involved in the breakdown
of cellular constituents, is critically dependent upon sulfhydryl
groups for proteolytic activity. However, uncontrolled proteolysis
caused by this enzyme leads to tissue damage; specifically lung
damage caused by smoking.
[0013] The reaction between NO and the thiols of intact protein
molecules has previously been studied only to a very limited
extent. There is some evidence for the reaction between proteins
and nitro(so)-containing compounds in vivo. Investigators have
observed that the denitrification of nitroglycerin in plasma is
catalyzed by the thiol of albumin (Chong et al., Drug Met. and
Disp. 18:61 (1990), and these authors suggest an analogy between
this mechanism and the thiol-dependent enzymatic denitrification of
nitroglycerin with glutathione S-transferase in a reaction which
generates thionitrates (Keene et al., JBC 251:6183 (1976)). In
addition, hemoproteins have been shown to catalyze denitrification
of nitroglycerin, and to react by way of thiol groups with certain
nitroso-compounds as part of the hypothesized detoxification
pathway for arylhydroxylamines (Bennett et al., J. Pharmacol. Exp.
Ther. 237:629 (1986); Umemoto et al., Biochem. Biophys. Res.
Commun. 151:1326 (1988)). The chemical identity of intermediates in
these reactions is not known.
[0014] Nitrosylation of amino acids can also be accomplished at
sites other than the thiol group. Tyrosine, an aromatic amino acid,
which is prevalent in proteins, peptides, and other chemical
compounds, contains a phenolic ring, hydroxyl group, and amino
group. It is generally known that nitration of phenol yields
ortho-nitrophenyl and para-nitrophenyl C-nitrosylation products.
Nitrosylation of tyrosine, using nitrous acid, has been shown to
yield C-nitrosylated tyrosine (Reeve, R. M., Histochem. Cytochem.
16(3): 191-8 (1968)), and it has been suggested that this process
produces O-nitroso-tyrosine as a preliminary product which then
rearranges into the C-nitrosylated product. (Baliga, B. T. Org.
Chem. 35(6):2031-2032 (1970); Bonnett et al., J. C. S. Perkin
Trans. I; 2261-2264 (1975)).
[0015] The chemistry of amino acid side chains, such as those found
on tyrosine and other aromatic amino acids, has a critical role in
ensuring proper enzymatic function within the body. In addition,
the hydroxyl group of tyrosine plays a central role in a variety of
cell regulatory functions, with phosphorylation of tyrosine being
one such critical cell regulatory event. In addition to possessing
bioactive side chains, these aromatic amino acids serve as
precursors to numerous important biomolecules such as hormones,
vitamins, coenzymes, and neurotransmitters.
[0016] The current state of the art lacks chemical methods for
modifying the activity and regulating the intermediary cellular
metabolism of the amino acids and proteins which play a critical
role in biological systems. Moreover, the ability to regulate
protein function by nitrosylation was, prior to the present
invention, unappreciated in the art.
[0017] It is appreciated in the art that, as a result of their
increased molecular weight and tertiary structure, protein
molecules differ significantly from low molecular weight thiols.
Furthermore, because of these differences, it would not be expected
that protein thiols could be successfully nitrosylated in the same
manner as low molecular weight thiols, or that, if nitrosylated,
they would react in the same manner. Furthermore, it would be
equally unexpected that nitrosylation of additional sites such as
oxygen, carbon and nitrogen would provide a means for regulation of
protein function.
[0018] Because of the great importance of diverse proteins and
amino acids in all biological systems, it would be extremely
desirable to have a method for achieving selective regulation of
protein and amino acid function. There are virtually unlimited
situations in which the ability to regulate amino acid or protein
function by nitrosylation would be of tremendous therapeutic
significance. Examples of ways in which regulation or modification
of function could be achieved would be the following: (1) to
enhance or prolong the beneficial properties of the protein or
amino acid; (2) to imbue the protein or amino acid with additional
beneficial properties; (3) to eliminate detrimental properties of a
protein or amino acid; and (4) to alter the metabolism or uptake of
proteins or amino acids in physiological systems.
[0019] The present invention represents a novel method for
achieving these therapeutically significant objectives by
regulation of protein and amino acid function with either of the
following methods: (1) administration of particular nitrosylated
proteins or amino acids to a patient; and (2) nitrosylation of a
protein or amino acid in vivo by the administration of a
nitrosylating agent to a patient. In addition, the invention
represents the discovery of exemplary S-nitroso-proteins and amino
acids of great biological and pharmacological utility.
SUMMARY OF THE INVENTION
[0020] This invention is based on the discovery by the inventors
that nitrosylating thiols, as well as oxygen, carbon and nitrogen
present on proteins and amino acids provides a means for achieving
selective regulation of protein and amino acid function. This
concept can be employed to generate S-nitroso protein compounds, as
well as other nitrosylated proteins and amino acids, which possess
specific properties, and can be directly administered to a patient.
In the alternative, the invention provides a means for in vivo
regulation of protein or amino acid function by nitrosylation. The
invention is therefore directed to novel S-nitroso-proteins and the
therapeutic uses thereof, as well as the nitrosylation of proteins
in vivo, as a therapeutic modality. The invention is also directed
to nitrosylation of oxygen, carbon and nitrogen sites of proteins
and amino acids, as a therapeutic modality.
[0021] In particular, this invention is directed to compounds
comprising an S-nitroso-enzyme. Enzymes contained in this compound
include tissue-type plasminogen activator, streptokinase, urokinase
and cathepsin.
[0022] This invention is also directed to compounds comprising
S-nitroso-lipoprotein. Lipoproteins which may be contained in the
compound include chylomicrons, chylomicron remnant particles, very
low-density lipoprotein (VLDL), intermediate-density lipoprotein
(IDL), low-density lipoprotein (LDL) high-density lipoprotein (HDL)
and lipoprotein (a).
[0023] This invention is also directed to compounds comprising
S-nitroso immunoglobulin. Immunoglobulins contained in this
compound include IgG, IgM, IgA, IgD, and IgE.
[0024] The invention is also directed to the compound
S-nitroso-hemoglobin.
[0025] The invention is also directed to the compound
S-nitroso-myoglobin.
[0026] The invention is also directed to pharmaceutical
compositions containing the compounds of the invention, together
with a pharmaceutically acceptable carrier.
[0027] The invention is also directed to a method for regulating
oxygen delivery to bodily sites by administering pharmaceutical
compositions containing S-nitroso-hemoglobin and
S-nitroso-myoglobin.
[0028] The invention also relates to methods for effecting
vasodilation, platelet inhibition, and thrombolysis; and for
treating cardiovascular disorders, comprising administering the
pharmaceutical compositions of the invention to an animal.
[0029] This invention is also directed to a method for effecting
platelet inhibition, comprising administering a pharmaceutical
composition comprised of S-nitroso-albumin. An additional
embodiment of the invention comprises the method for causing
relaxation of airway smooth muscle and for the treatment or
prevention of respiratory disorders, comprising administering a
pharmaceutical composition containing S-nitroso-albumin.
[0030] This invention also is directed to a method for causing
vasodilation, platelet inhibition and thrombolysis, comprising
administering a nitrosylating agent to an animal.
[0031] This invention also is directed to a method for regulation
of protein function in vivo, comprising administering a
nitrosylating agent to an animal.
[0032] The invention is directed to a method for preventing the
uptake of a protein by cells, comprising administering a
nitrosylating agent to a patient.
[0033] The invention is also directed to a method for causing
relaxation of non-vascular smooth muscle, comprising administering
the pharmaceutical compositions of the invention to an animal.
[0034] The invention is also directed to a method for regulating
the function of proteins in which the thiol is bound to a methyl
group, comprising the steps of removing the methyl groups from the
protein by selective de-methylation, and reacting the free thiol
group with a nitrosylating agent.
[0035] The invention is also directed to a method for regulating
the function of a protein which lacks a free thiol group,
comprising the steps of adding a thiol group to the protein, and
reacting the thiol group with a nitrosylating agent.
[0036] The invention is also directed to a method for regulating
cellular function, comprising the S-nitrosylation of a protein
which is cellular component or which affects cellular function.
[0037] The invention is also directed to a method for delivering
nitric oxide to specific, targeted sites in the body comprising
administering an effective amount of the pharmaceutical
compositions of the invention to an animal.
[0038] The invention is also directed to a method for inhibiting
platelet function, comprising the nitrosylation of a protein or
amino acid at other sites, in addition to thiol groups, which are
present on said protein or amino acid.
[0039] The invention is also directed to a method for causing
vasodilation, comprising the nitrosylation of a protein or amino
acid at other sites, in addition to thiol groups, which are present
on said protein or amino acid.
[0040] The invention is also directed to a method for relaxing
smooth muscle, comprising the nitrosylation of a protein or amino
acid at other sites, in addition to thiol groups, which are present
on said protein or amino acid.
[0041] The invention is also directed to a method for regulating
cellular function, comprising the nitrosylation of a protein or
amino acid at other sites, in addition to thiol groups, which are
present on said protein or amino acid.
[0042] The invention is also directed to a method for delivery of
nitric oxide to specific, targeted sites in the body, comprising
the nitrosylation of a protein or amino acid at other sites, in
addition to thiol groups, which are present on said protein or
amino acid.
[0043] The sites which are nitrosylated are selected from the group
consisting of oxygen, carbon and nitrogen.
[0044] The invention is also directed to a method for inhibiting
platelet function, comprising administering a pharmaceutical
composition comprised of a compound selected from the group
consisting of any S-nitroso-protein.
[0045] The invention is also directed to a method for causing
vasodilation, comprising administering a pharmaceutical composition
comprised of a compound selected from the group consisting of any
S-nitroso-protein.
[0046] The invention is also directed to a method for treatment or
prevention of cardiovascular disorders, comprising administering a
pharmaceutical composition comprised of a compound selected from
the group consisting of any S-nitroso-protein.
[0047] The invention is directed to a method for relaxing
non-vascular smooth muscle, comprising administering a
pharmaceutical composition comprised of a compound selected from
the group consisting of any S-nitroso-protein.
[0048] The invention is also directed to a method for treatment or
prevention of respiratory disorders, comprising administering a
pharmaceutical composition comprised of a compound selected from
the group consisting of any S-nitroso-protein.
[0049] The invention is also directed to a method for delivering
nitric oxide to specific, targeted sites in the body, comprising
administering a pharmaceutical composition comprised of a compound
selected from the group consisting of any S-nitroso-protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1A is the ultraviolet absorption spectrum of S--NO-t-PA
(15 .mu.M) relative to unmodified t-PA.
[0051] FIG. 1B is the chemical shift of S--[.sup.15N]O-t-PA (35
.mu.M) at 751 ppm relative to nitrite using [.sup.15N]NMR.
[0052] FIG. 2 is the determination of S--NO bond formation in the
synthesis of S--NO-t-PA.
[0053] FIG. 3 is the [.sup.15N]-NMR spectrum of [.sup.15N]-labeled
S-nitroso-BSA.
[0054] FIG. 4 shows the concentration-dependent binding of t-PA and
S--NO-tPA to fibrinogen-coated wells.
[0055] FIG. 5A is the double reciprocal plot 1/v versus 1/s for
t-PA and S--NO-t-PA generated against the chromogenic substrate
S2288. Results are expressed as mean.+-.S.D. (n=3).
[0056] FIG. 5B are the curves for activation of glu-plasminogen
(0.1-10 .mu.M) by t-PA and S--NO-t-PA, generated using the
plasmin-specific chromogenic substrate S2251. Results are expressed
as mean.+-.S.D. (n=3).
[0057] FIG. 6 shows the fibrinogen stimulation of enzymatic
activity of t-PA (clear bars) and S--NO-t-PA (hatched bars),
compared in the coupled enzyme assay at concentrations of 0.1 .mu.M
and 1.0 .mu.M of plasminogen.
[0058] FIG. 7 shows increases in intracellular platelet cyclic GMP
caused by S--NO-t-PA.
[0059] FIGS. 8A-C show the inhibition of platelet aggregation by
S--NO-t-PA.
[0060] FIGS. 9A-C are a comparison of S--NO-t-PA-induced
vasorelaxation caused by (A) t-PA (150 nM), (B) S--NO-t-PA (150
nM), and (C) S--NO-t-PA (150 nM).
[0061] FIG. 10 is a dose-dependent relaxation of vascular smooth
muscle and inhibition of platelet aggregation caused by
S-nitroso-BSA (S--NO-BSA).
[0062] FIG. 11A shows illustrative tracings comparing the platelet
inhibitory effects of (a) S--NO-BSA; (b) NaNO.sub.2; (c) BSA; (d)
iodoacetamide-treated BSA exposed to NO generated from acidified
NaNO.sub.2.
[0063] FIGS. 11BA, BB, BC, BD, BE are illustrative tracings
comparing the vasodilatory effects of (a) BSA (1.4 .mu.M); (b)
iodoacetamide-treated BSA treated with NO generated from acidified
NaNO.sub.2 as described in FIG. 3a; (c) S--NO-BSA (1.4 .mu.M) after
platelets were pretreated with 1 .mu.M methylene blue for ten
minutes; (d) S--NO-BSA (1.4 .mu.M).
[0064] FIG. 12 shows the coronary blood flow in anesthetized dogs
following infusion of S-nitroso-BSA.
[0065] FIG. 13 shows the duration of increased coronary blood flow
following infusion of S-nitroso-BSA.
[0066] FIG. 14 shows coronary vasodilation following infusion of
S-nitroso-BSA.
[0067] FIG. 15 shows a dose-dependent vasodilatory response caused
by S-nitroso-cathepsin.
[0068] FIG. 16 shows tracings of dose-dependent inhibition of
platelet aggregation caused by S-nitroso-LDL.
[0069] FIG. 17 shows representative tracings of vessel relaxation
caused by S-nitroso-LDL.
[0070] FIG. 18 shows tracings of dose-dependent inhibition of
platelet aggregation caused by S-nitroso-immunoglobulin.
[0071] FIG. 19 shows representative tracings of vessel relaxation
caused by S-nitroso-immunoglobulin.
[0072] FIG. 20 shows the concentration-dependent relaxation of
airway smooth muscle caused by S--NO-BSA.
[0073] FIGS. 21A-E show the nitrosylation of L-tyrosine as (A)
[.sup.15N]-NMR spectrum; (B) [.sup.1H]-NMR spectrum; (C) FTIR
spectrum; (D) UV spectrum for 1.8 mM of tyrosine; and (E) UV
spectrum for 34 mM of tyrosine.
[0074] FIG. 22 show the [.sup.15N]-NMR spectrum for the
nitrosylation of L-phenylalanine.
[0075] FIGS. 23A-E are the UV spectrum for nitrosylation of
tryptophan after a reaction time of 5 minutes, 10 minutes, 15
minutes, 30 minutes and 60 minutes, respectively.
[0076] FIG. 24 shows the [.sup.15N] NMR for nitrosylated bovine
serum albumin.
[0077] FIGS. 25A-C show the UV spectrum for time-dependent NO
loading of BSA after a reaction time of 1 minutes, 5 minutes and 30
minutes, respectively.
[0078] FIG. 26 shows the absorbance v. nanometers for the
nitrosylation of t-PA.
[0079] FIG. 27 shows the vasodilatory effects of NO-loaded BSA.
[0080] FIG. 28 shows a spectrum for the S-nitrosylation of
hemoglobin.
[0081] FIG. 29 shows the UV spectrum of hemoglobin incubated with
S-nitroso-N-acetylcysteine.
[0082] FIG. 30 shows the reaction of nitric oxide at the
iron-binding site of hemoglobin.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0083] The invention is based on the discovery by the inventors
that nitrosylation of proteins and amino acids provides a means by
which protein and amino acid function may be selectively regulated,
modified or enhanced.
[0084] The term "nitrosylation" refers to the addition of NO to a
thiol group (SH), oxygen, carbon or nitrogen by chemical means. The
source of NO may be endogenous NO or endothelium-derived relaxing
factor, or other nitrosylating agents, such as nitroglycerin,
nitroprusside, nitrosothiols, nitrous acid or any other related
compound.
[0085] The term "regulated" means effective control of the activity
of a protein or amino acid, in a selective manner so as to cause
the protein or amino acid to exert a desired physiological
effect.
[0086] The term "modified" means to effectively alter the activity
of a protein or amino acid in a selective manner, so as to cause
the protein or amino acid to exert a desired physiological effect.
The term "enhanced" means to alter effectively the activity of a
protein or amino acid in a selective manner, so as to cause an
increase or improvement in the activity of the protein or amino
acid, or endow the protein or amino acid with additional
capabilities.
[0087] The term "activity" refers to any action exerted by the
protein or amino acid which results in a physiological effect.
[0088] The inventors have investigated the reaction of NO with
protein thiols and have demonstrated that a variety of proteins of
biological significance and relative abundance can be
S-nitrosylated. S-nitrosylation of proteins endows these molecules
with potent and long-lasting NO-like effects of vasodilation and
platelet inhibition, mediated by guanylate cyclase activation, and
also provides a means for achieving selective regulation of
particular protein functions.
[0089] To develop the S-nitroso-protein compounds of the invention,
certain thiol-containing proteins which are representative of
various functional classes were nitrosylated. Such proteins include
enzymes, such as tissue-type plasminogen activator (t-PA) and
cathepsin B; transport proteins, such as lipoproteins, hemoglobin,
and serum albumin; and biologically protective proteins, such as
immunoglobulins.
[0090] The data demonstrate that 1) NO can react with thiol groups
in proteins to form S-nitrosothiols; 2) this reaction occurs under
physiologic conditions; 3) these compounds are biologically active,
exhibiting vasodilatory and anti-platelet properties that are
independent of their method of synthesis; 4) the long chemical
half-lives of S-nitroso-proteins vis-a-vis the half life of NO is
reflected in their different relaxation kinetics:
S-nitroso-proteins, through activation of guanylate cyclase, is
fully consistent with that of other nitroso compounds; although the
possibility of other mechanisms by which S--NO proteins can produce
biologic effects cannot be excluded, such as the transfer of NO to
another protein thiol, the function of which is thereby modulated.
(Craven et al. J. Biol. Chem. 253:8433 (1978); Katsuki et al. J.
Cyc. Nuc. Prot. Phos. Res. 3:23 (1977); Osborne et al., J. Clin.
Invest. 83:465 (1989)).
[0091] One embodiment of the invention relates to S-nitroso-enzyme
compounds, derived from nitrosylation of enzymatic proteins.
[0092] A particular aspect of this embodiment relates to the
compound, S nitroso-t-PA (S--NO-t-PA), derived from the
nitrosylation of tissue-type plasminogen activator (t-PA).
[0093] Acute occlusive events are precipitated by thrombogenic
stimuli and alterations in flow dynamics within the vessel.
Platelet activation, augmented local vasoconstriction, and
recruitment of the coagulation system each plays a major role in
the subsequent development of a thrombus (Marder et al., New Engl.
J. Med. 318:1512, 1520 (1988)). t-PA is one of the products
secreted by blood vessel endothelium, which specifically
counteracts these thrombogenic mechanisms. t-PA, a serine protease,
converts plasminogen to plasmin on fibrin and platelet thrombi,
which in turn induces fibrinolysis and platelet disaggregation.
Loscalzo et al., New Engl. J. Med. 319(14):925-931 (1989); Loscalzo
et al., J. Clin. Invest. 79:1749-1755 (1987).
[0094] Attempts have been made to improve the thrombolytic efficacy
and pharmacological properties of plasminogen activators, such as
t-PA. In light of the role of platelets in clot formation and in
reocclusive vascular events, one major focus has involved the use
of ancillary antiplatelet therapy. Some success has been achieved
with aspirin (ISIS-2 Lancer 2:349-360 (1988)), and other benefits
are reported for several newer antiplatelet compounds (Gold, H. K.
New Engl. J. Med. 323:1483-1485 (1990)). Attempts have also been
made to improve the functional properties of the plasminogen
activator itself through site-directed mutagenesis and synthesis of
hybrid molecules and biochemical conjugates (Runge et al.,
Circulation 79:217-224 (1989); Vaughan et al., Trends Cardiovasc.
Med. Jan/Feb: 1050-1738 (1991)).
[0095] Motivated by the need for a plasminogen activator with
improved thrombolytic efficacy and anti-thrombogenic properties,
the inventors discovered that nitrosylation of t-PA creates a new
molecule (S--NO-t-PA) which has improved thrombolytic capability,
(e.g., the enzymatic activity of the enzyme is enhanced) as well as
vasodilatory and platelet inhibitory effect. The inventors
demonstrated that S-nitrosylation significantly enhances the
bioactivity of t-PA, without impairing the catalytic efficiency or
other domain specific functional properties of the enzyme.
[0096] In particular, S-nitrosylation of t-PA at the free cysteine,
cys 83, confers upon the enzyme potent antiplatelet and
vasodilatory properties, without adversely affecting its catalytic
efficiency or the stimulation of this activity by fibrin(ogen). In
addition, the S-nitrosothiol group does not appear to alter the
specific binding of t-PA to fibrin(ogen) or the interaction of t-PA
with its physiological serine protease inhibitor, PAI-1. The
proteolytic activity, fibrin(ogen)-binding properties and regions
for interaction with PAI-1 reside in several functional domains of
the molecule that are linearly separate from the probable site of
S-nitrosylation in the growth factor domain (cys 83). Thus,
chemical modification of t-PA by NO does not markedly alter
functional properties of t-PA residing in other domains. In
addition, S-nitrosylation enhances the catalytic efficiency of t-PA
against plasminogen, and increases its stimulation by
fibrinogen.
[0097] NO is highly labile and undergoes rapid inactivation in the
plasma and cellular milieu. This suggests that the reaction between
NO and the protein thiol provides a means of stabilizing NO in a
form in which its bioactivity is preserved. Specifically,
S--NO-t-PA is a stable molecule under physiologic conditions and,
much like NO, is capable of vasodilation and platelet inhibition
mediated by cyclic GMP. Stabilizing NO in this uniquely bioactive
form creates a molecule with intrinsic vasodilatory, antiplatelet,
and fibrinolytic properties, which enable it to counteract each of
the major thrombogenic mechanisms.
[0098] Another aspect of this embodiment relates to the
administration of S--NO-t-PA as a therapeutic agent to an animal
for the treatment and prevention of thrombosis. Current
thrombolytic strategies are based on the understanding of the
endogenous mechanisms by which the endothelium protects against
thrombogenic tendencies. In particular, platelet inhibition and
nitrovasodilation are frequently used concomitant therapies with
which to enhance reperfusion by plasminogen activators as well as
to prevent re-thrombosis (Gold, H. K. New Engl. J. Med.
323:1483-1485 (1990); (Marder et al., New Engl. J. Med.
318:1512-1520 (1988)).
[0099] Administration of S--NO-t-PA to a patient in need thereof
provides a means for achieving "fibrin-selective" thrombolysis,
while simultaneously attenuating the residual thrombogenicity
resulting from simultaneous platelet activation and thrombin
generation during thrombolysis. Furthermore, by virtue of its
fibrin binding properties, S--NO-t-PA provides targeted delivery of
the antiplatelet effects of NO to the site of greatest platelet
activation, the actual fibrin-platelet thrombus. S--NO-t-PA has
therapeutic application in the treatment or prevention of
conditions which result from, or contribute to, thrombogenesis,
such as atherothrombosis, myocardial infarction, pulmonary embolism
or stroke.
[0100] In summary, S--NO-t-PA possesses unique properties that
facilitate dispersal of blood clots and prevent further
thrombogenesis. The discovery of this unique molecule provides new
insight into the endogenous mechanism(s) by which the endothelium
maintains vessel patency and offers a novel, and beneficial
pharmacologic approach to the dissolution of thrombi.
[0101] Another aspect of this embodiment relates to the compounds
derived from the nitrosylation of other thrombolytic agents, such
as streptokinase, urokinase, or a complex containing one or more
thrombolytic agents, such as streptokinase, urokinase, or t-PA.
These compounds may also be administered to an animal, in the same
manner as S--NO-t-PA for the treatment and prevention of
thrombosis.
[0102] An additional aspect of this embodiment relates to compounds
derived from the nitrosylation of other enzymes. One particular
compound is S--NO cathepsin, derived from the nitrosylation of
cathepsin B, a lysosomal cysteine protease. The inventors have
demonstrated that S--NO-cathepsin exerts a vasodilatory and
platelet inhibitory effect. Thus, this compound may be administered
as a therapeutic agent to an animal, to promote vasodilation and
platelet inhibition, and to treat or prevent cardiovascular
disorders.
[0103] Another embodiment of the invention relates to
S-nitroso-lipoprotein compounds derived from the nitrosylation of
lipoproteins. Such lipoproteins include chylomicrons, chylomicron
remnant particles, very low-density lipoprotein (VDL), low-density
lipoprotein (LDL), intermediate-density lipoprotein (IDL), and high
density lipoprotein (HDL) and lipoprotein (a). The inventors have
demonstrated that S-nitroso-lipoproteins exert vasodilatory and
platelet inhibitory effect. Thus, these compounds may be
administered as a therapeutic agent, to an animal, to promote
vasodilation and platelet inhibition, and to treat or prevent
cardiovascular disorders.
[0104] An additional embodiment of the invention involves the in
vivo nitrosylation of lipoproteins as a means for regulating
cellular uptake of lipoproteins. Consequently, nitrosylation
provides a means for regulating lipid uptake, and treating or
preventing disorders associated with hyperlipidemias, such as
atherosclerosis.
[0105] Another embodiment of the invention relates to the S-nitroso
immunoglobulin compounds derived from the nitrosylation of
immunoglobulins. Such immunoglobulins may include IgG, IgM, IgA,
IgD, or IgE. The inventors have demonstrated that these compounds
exert vasodilatory and platelet inhibitory effect. Thus, these
compounds may be administered as therapeutic agents, to an animal,
to promote vasodilation and platelet inhibition, and to treat or
prevent cardiovascular disorders. The half lives of these
compounds, in the order of one day, produce unique, long lasting
vasodilatory effects which are notably different from those of low
molecular weight nitroso-compounds.
[0106] An additional embodiment of the invention is the compound
S-nitroso hemoglobin, derived from the nitrosylation of hemoglobin.
This compound may be used as therapeutic agent to promote
vasodilation and platelet inhibition, and to treat or prevent
cardiovascular disorders.
[0107] As demonstrated by the inventors, S-nitrosylation of
hemoglobin increases its oxygen-binding capacity. Hemoglobin is a
globular protein, which binds reversibly to blood oxygen through
passive diffusion from entry of air into the lungs.
Hemoglobin-oxygen binding greatly increases the capacity of the
blood to transport oxygen to bodily tissues; thus, the binding
affinity between hemoglobin and oxygen is a critical factor in
determining the level of oxygen transport to the tissues. The thiol
group on the hemoglobin molecule regulates the affinity of
hemoglobin for oxygen. The inventors have demonstrated that some
S-nitrosothiols, such as S-nitroso-proteins do not react with the
iron-binding site of hemoglobin, as does NO., but instead, bind to
the thiol group. Thus, methemoglobin formation is prevented and
hemoglobin oxygen binding is unimpaired.
[0108] Furthermore, the inventors have also demonstrated that
S-nitrosylation of hemoglobin not only prevents impairment of
binding, but actually increases hemoglobin-oxygen binding.
Therefore, another embodiment of the invention involves the
administration of S--NO-hemoglobin or the in vivo nitrosylation of
hemoglobin, to increase the oxygen-carrying capacity of the blood,
and oxygen transport to bodily tissues. As a result, these
compounds may be useful in the treatment of disorders which are
associated with insufficient oxygen transport, or in clinical
situations in which increased oxygen transport is needed. Examples
of such clinical situations include, but are not limited to,
hypoxic disorders resulting from pneumothorax, airway obstruction,
paralysis or weakness of the respiratory muscles, inhibition of
respiratory centers by drug or other agents, or other instances of
decreased pulmonary ventilation. Additional clinical indications
include impaired alveolar gas diffusion such as occurs in
interstitial fibrosis, bronchiole constriction, pulmonary edema,
pneumonia, hemorrhage, drowning, anemias, arteriovenous shunts, and
carbon monoxide poisoning.
[0109] In addition, S--NO-hemoglobin may also be used to modulate
the delivery of carbon monoxide or nitric oxide (bound to
hemoglobin) to bodily tissues.
[0110] In addition, any thiol-containing heme proteins may be
nitrosylated and used to enhance the oxygen-carrying capacity of
the blood.
[0111] An additional embodiment of the invention is the compound
S-nitroso myoglobin, derived from the nitrosylation of myoglobin, a
protein which also transports oxygen. This compound may be used as
a therapeutic agent to promote vasodilation and platelet
inhibition, and to treat or prevent cardiovascular disorders.
[0112] Another embodiment of the invention relates to a method for
using S-nitroso-proteins as a means for providing targeted delivery
of NO. The term "targeted delivery" means that NO is purposefully
transported and delivered to a specific and intended bodily site.
In the same manner as S--NO-t-PA, S--NO-immunoglobulin can be
modified, by cationic modification of the heavy chain, to provide
targeted delivery of NO to the basement membrane of the glomerulus
in the kidney. Successful delivery of four NO molecules per
immunoglobulin have been directed to the kidney basement membrane
in this matter. Targeted delivery of NO provides a means for
achieving site-specific smooth muscle relaxation, or other
NO-mediated effects. In addition, delivery may be for the purpose
of nitrosylation of various molecules present in the body. For
example, S-nitroso-proteins would deliver NO, and thus nitrosylate
hemoglobin or myoglobin in order to increase oxygen binding.
[0113] A significant advantage of S-nitroso-proteins is that they
deliver NO in its most biologically relevant, and non-toxic form.
This is critical, because the pharmacological efficacy of NO
depends upon the form in which it is delivered. This is
particularly true in airways, where high levels of O.sub.2 and
O.sub.2 reactive species predispose to rapid inactivation of the NO
moiety. As demonstrated by the inventors, S-nitroso-proteins
deliver NO as the charged species, nitrosonium (NO.sup.+) or
nitroxyl (NO.sup.-), and not the uncharged NO radical (NO.). This
is important because the charged species behave in a very different
manner from NO. with respect to chemical reactivity.
[0114] In contrast to NO., nitrosonium and nitroxyl do not react
with O.sub.2 or O.sub.2 species, and are also resistant to
decomposition in the presence of redox metals. Consequently,
administration of NO equivalents does not result in the generation
of toxic by-products or the elimination of the active NO moiety. By
delivering nitrosonium or nitroxyl, S-nitroso-proteins provide a
means for achieving the smooth muscle relaxant and anti-platelet
effects of NO, and at the same time, alleviate significant adverse
effects previously associated with NO therapy.
[0115] Another embodiment of the invention relates to the
administration of S-nitroso-albumin as a therapeutic agent to
promote platelet inhibition, or to cause relaxation of airway
smooth muscle. The inventors have demonstrated that S-nitroso-BSA
exerts a platelet inhibitory effect, and also promotes long-acting
vasodilatory effect, which can be distinguished from that of NO or
the low molecular weight thiols.
[0116] The inventors have also demonstrated that S-nitroso-BSA
relaxes human airway smooth muscle. As discussed above, by
delivering NO in the form of charged NO equivalents, such as
nitrosonium, S-nitroso-proteins cause airway relaxation, and also
eliminate the adverse effects which occur with administration of
other NO species. Thus, S-nitroso-albumin may be administered for
the treatment or prevention of respiratory disorders including all
subsets of obstructive lung disease, such as emphysema, asthma,
bronchitis, fibrosis, excessive mucous secretion and lung disorders
resulting from post surgical complications. In addition these
compounds may be used as antioxidants, and thus, in the treatment
of diseases such as acute respiratory distress syndrome (ARDS).
[0117] Another embodiment of the invention relates to a method for
nitrosylation of those proteins which lack free thiols. The method
involves thiolating the protein by chemical means, such as
homocysteine thiolactone (Kendall, BBA 257:83 (1972)), followed by
nitrosylation in the same manner as the compounds discussed above.
Recombinant DNA methods may also be used to add or substitute
cysteine residues on a protein.
[0118] Another embodiment of the invention relates to a method for
nitrosylation of those proteins in which the thiol is blocked by a
methyl group. The method involves selective de-methylation of the
protein by chemical means, such as reacting with methyl
transferase, followed by nitrosylation in the same manner as the
compounds discussed above.
[0119] Another embodiment of the invention involves the use of
S-nitroso protein compounds, to relax non-vascular smooth muscle.
Types of smooth muscle include, but are not limited to, bronchial,
tracheal, uterine, fallopian tube, bladder, urethral, urethral,
corpus cavernosal, esophageal, duodenal, ileum, colon, Sphincter of
Oddi, pancreatic, or common bile duct.
[0120] An additional embodiment of the invention involves the in
vivo nitrosylation of protein thiols, by administration of a
nitrosylating agent as a pharmaceutical composition. In vivo
nitrosylation provides a means for achieving any of the
physiological effects discussed above, or for regulation of
additional protein functions.
[0121] In addition to thiol groups, proteins and amino acids
possess other sites which can be nitrosylated. For example, such
sites may include, but are not limited to, oxygen, nitrogen, and
carbon. Thus, an additional embodiment of the invention relates to
the nitrosylation of additional sites, such as oxygen, nitrogen,
and carbon which are present on proteins and amino acids, as a
means for achieving any of the physiological effects discussed
above, or for regulation of additional protein or amino acid
functions. The inventors have shown that aromatic amino acids, such
as tyrosine, phenylalanine and tryptophan can be nitrosylated at
the hydroxyl, and amino groups, as well as on the aromatic ring,
upon exposure to nitrosylating agents such as NaNO.sub.2, NOCl,
N.sub.2O.sub.3, N.sub.2O.sub.4 and NO.sup.+. Other amino acids,
such as serine and threonine may also be nitrosylated in the same
manner.
[0122] The ability to bind NO to a variety of different sites on an
amino acid or protein provides a greater concentration of NO, and
thus may enhance regulation of protein function, as well as other
NO-mediated effects such as smooth muscle relaxation and platelet
inhibition. Thus, another embodiment of the invention relates to
the use of amino acids and proteins which contain numerous NO
molecules, to regulate protein or amino acid function and to effect
smooth muscle relaxation and platelet inhibition. Additional
therapeutic uses of these compounds include the treatment or
prevention of such disorders as heart failure, myocardial
infarction, shock, renal failure, hepatorenal syndrome,
post-coronary bypass, gastrointestinal disease, vasospasm of any
organ bed, stroke or other neurological disease, and cancer.
[0123] Another embodiment of the invention relates to a method for
using these nitrosylated proteins and amino acids as a means for
providing targeted delivery of NO to specific and intended bodily
sites. These compounds have the capacity to deliver charged NO
equivalents. For example, alkyl nitrites having the formula X--CONO
and containing a beta-election withdrawing group would be able to
deliver these charged NO equivalents.
[0124] The hydroxyl group of tyrosine also plays a central role in
a variety of cell regulatory functions. For example,
phosphorylation of tyrosine is a critical cell regulatory event. In
addition, serine residues also provide phosphorylation sites. Thus,
a particular aspect of this embodiment relates to the nitrosylation
of amino acids such as tyrosine and serine, to regulate cellular
process such as, but not limited to, phosphorylation.
[0125] Another embodiment of the invention relates to the use of
O-nitrosylation of tyrosine residues on bovine serum albumin as a
method for achieving smooth muscle relaxation and platelet
inhibition.
[0126] Another embodiment of the invention relates to the
nitrosylation of t-PA at additional sites, such as oxygen. For
example, O-nitrosylation of t-PA, in addition to conferring
vasodilatory and platelet inhibitory properties, alters the
pharmokinetics of t-PA in such a way as to make it unavailable as a
substrate for its natural inhibitor, PA-I.
[0127] Another embodiment of the invention relates to the
administration of a pharmaceutical composition comprised of any
S-nitroso-protein, to inhibit platelet function, cause
vasodilation, relax smooth muscle, deliver nitric oxide to specific
targeted bodily sites, or for the treatment or prevention of
cardiovascular or respiratory disorders.
[0128] An additional application of the present invention relates
to the nitrosylation of additional compounds such as peptides,
neurotransmitters, pharmacologic agents and other chemical
compounds, as a therapeutic modality. For example, nitrosylation of
dopamine, a neurotransmitter improves the cardiac profile of the
drug, by enhancing afterload reduction and scavenging free
radicals, while simultaneously inhibiting platelets and preserving
renal blood flow. Nitrosylation of epinephrine and related
sympathomimetic drugs alters the half-life of the drug and affects
its .beta.-agonist selectivity.
[0129] The nitrosylated proteins and amino acids of the present
invention, or the nitrosylating agents may be administered by any
means that effect thrombolysis, vasodilation, platelet inhibition,
relaxation of non-vascular smooth muscle, other modification of
protein functions or treatment or prevention of cardiovascular
disorders, or any other disorder resulting from the particular
activity of a protein or amino acid. For example, administration
may be by intravenous, intraarterial, intramuscular, subcutaneous,
intraperitoneal, rectal, oral, transdermal or buccal routes.
[0130] According to the present invention, a "therapeutically
effective amount" of therapeutic composition is one which is
sufficient to achieve a desired biological effect. Generally, the
dosage needed to provide an effective amount of the composition, in
which can be adjusted by one of ordinary skill in the art, will
vary, depending on the age, health, condition, sex, weight, and
extent of disease, of the recipient. In addition, the dosage may
also depend upon the frequency of treatment, and the nature of the
effect desired.
[0131] Compositions within the scope of this invention include all
compositions wherein the S-nitroso-protein or the nitrosylating
agent is contained in an amount effective to achieve its intended
purpose. While individuals needs vary, determination of optimal
ranges of effective amounts of each component is within the skill
of the art. Typical dosage forms contain 1 to 100 mmol/kg of the
S-nitroso-protein. The dosage range for the nitrosylating agent
would depend upon the particular agent utilized, and would be able
to be determined by one of skill in the art.
[0132] In addition to the pharmacologically active compounds, the
new pharmaceutical preparations may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. Preferably,
the preparations, particularly those preparations which can be
administered orally and which can be used for the preferred type of
administration, such as tablets, dragees, and capsules, and also
preparations which can be administered rectally, such as
suppositories, as well as suitable solutions for administration by
injection or orally, contain preferably, about 0.01 to 5 percent,
preferably from about 0.1 to 0.5 percent of active compound(s),
together with the excipient.
[0133] The pharmaceutical preparations of the present invention are
manufactured in a manner which is itself known, for example, by
means of conventional mixing, granulating, dragee-making,
dissolving, or lyophilizing processes. Thus, pharmaceutical
preparations for oral use can be obtained by combining the active
compounds with solid excipients, optionally grinding the resulting
mixture and processing the mixture of granules, after adding
suitable auxiliaries, if desired or necessary, to obtain tablets or
dragee cores.
[0134] Suitable excipients are, in particular, fillers such as
sugars, for example lactose or sucrose, mannitol or sorbitol,
cellulose preparations and/or calcium phosphates, for example
tricalcium phosphate or calcium hydrogen phosphate, as well as
binders such as starch, paste, using, for example, maize starch,
wheat starch, rice starch, potato starch, gelatin, tragacanth,
methyl cellulose, hydroxypropylmethylcellulose, sodium
carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,
disintegrating agents may be added such as the above-mentioned
starches and also carboxymethylstarch, cross-linked polyvinyl
pyrrolidone, agar, or algenic acid or a salt thereof, such as
sodium alginate. Auxiliaries are, above all, flow-regulating agents
and lubricants, for example, silica, talc, stearic acid or salts
thereof, such as magnesium stearate or calcium stearate, and/or
polyethylene glycol. Dragee cores are provided with suitable
coatings which, if desired, are resistant to gastric juices. For
this purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
polyethylene glycol and/or titanium dioxide, lacquer solutions and
suitable organic solvents or solvent mixtures. In order to produce
coatings resistant to gastric juices, solutions of suitable
cellulose preparations such as acetylcellulose phthalate or
hydroxypropymethyl-cellulose phthalate, are used. Dye stuffs or
pigments may be added to the tablets or dragee coatings, for
example, for identification or in order to characterize
combinations of active compound doses.
[0135] Other pharmaceutical preparations which can be used orally
include push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a plasticizer such as glycerol or
sorbitol. The push-fit capsules can contain the active compounds in
the form of granules which may be mixed with fillers such as
lactose, binders such as starches, and/or lubricants such as
lactose, binders such as starches, and/or lubricants such as talc
or magnesium stearate and, optionally, stabilizers. In soft
capsules, the active compounds are preferably dissolved or
suspended in suitable liquids, such as fatty oils, or liquid
paraffin. In addition, stabilizers may be added.
[0136] Possible pharmaceutical preparations which can be used
rectally include, for example, suppositories, which consist of a
combination of the active compounds with a suppository base.
Suitable suppository bases are, for example, natural or synthetic
triglycerides, or paraffin hydrocarbons. In addition, it is also
possible to use gelatin rectal capsules which consist of a
combination of the active compounds with a base. Possible base
materials include, for example, liquid triglycerides, polyethylene
glycols, or paraffin hydrocarbons.
[0137] Suitable formulations for parenteral administration include
aqueous solutions of the active compounds in water-soluble form,
for example, water soluble salts. In addition, suspensions of the
active compounds as appropriate oily injection suspensions may be
administered. Suitable lipophilic solvents or vehicles include
fatty oils, for example, sesame oil, or synthetic fatty acid
esters, for example, ethyl oleate or triglycerides. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the
suspension may also contain stabilizers.
[0138] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention.
EXAMPLES
Example 1
Synthesis of S-Nitroso-t-PA
A. Nitrosylation of t-PA
[0139] 1. Materials
[0140] t-PA was kindly provided by Genentech, Inc. San Francisco,
Calif. Reactivated purified plasminogen activator inhibitor-1
(PAI-1) and a panel of six murine anti-t-PA monoclonal antibodies
were kindly provided by Dr. Douglas E. Vaughan. Horse-Radish
Peroxidase linked-sheep anti murine antibodies were purchased from
Amersham Corp., Arlington, Ill. Sodium nitrite was purchased from
Fisher Scientific, Fairlawn, N.J.
H-D-isoleucyl-L-prolyl-L-arginyl-p-nitroanilide (S2288) and
H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide (S2251) were purchased
from Kabi Vitrum, Stockholm, Sweden. Human fibrinogen purified of
plasminogen and von Willebrand factor, was obtained from Enzyme
Research Laboratories, South Bend, Ind. Epinephrine, ADP and
iodoacetamide were purchased from Sigma Chemical Co., St. Louis,
Mo. Bovine thrombin was obtained from ICN, ImmunoBiologicals
(Lisle, Ill.). Radioimmunoassay kits for the determination of cGMP
were purchased from New England Nuclear, Boston, Mass.
[0141] 2. Plasminogen Preparation
[0142] Glu-plasminogen was purified from fresh frozen plasma thawed
at 37.degree. C. using a modification of the method of Deutsch and
Mertz (Deutsch et al., Science 170:1095-1096 (1970), herein
incorporated by reference). Plasma was passed over a
lysine-Sepharose column and the column washed with 0.3 M sodium
phosphate, pH 7.4, 3 mM EDTA. Plasminogen was eluted from the
column with 0.2 M epsilon-aminocaproic acid, 3 mM EDTA, pH 7.4.
Contaminant plasmin was removed by passing the eluted column over
benzamidine sepharose 2B. The plasminogen obtained was subsequently
dialyzed before use against 10 mM sodium phosphate, pH 7.4, 0.15 M
NaCl.
[0143] 3. Thiol Derivatization
[0144] The free thiol of t-PA was carboxyamidated by exposure of
the enzyme to a 10-fold excess of iodoacetamide in the dark for one
hour at 37.degree. C. in 10 mM Tris, pH 7.4, 0.15 M NaCl TBS). t-PA
was then dialyzed extensively against 10 mM HCl in order to remove
excess iodoacetamide.
[0145] 4. Microcarrier Endothelial Cell Culture
[0146] Endothelial cells were isolated from bovine aorta by
established techniques (Schwartz, S. M. In Vitro 14:966-980 (1978),
herein incorporated by reference) and cultured on a microcarrier
system of negatively charged spherical plastic beads (Biosilon),
according to the method of Davies and colleagues (Davies et al., J.
Cell Biol. 101:871-879 (1985), herein incorporated by
reference).
[0147] 5. Nitrosylation
[0148] t-PA was first dialyzed against a large excess of 10 mM HCl
for 24 hours to remove excess L-arginine used to solubilize the
protein. t-PA was then exposed to NO.sub.X generated from equimolar
NaNO.sub.2 in 0.5 N HCl (acidified NaNO.sub.2) or in control
experiments, to 0.5 N HCl alone, for 30 minutes at 37.degree. C.
Solutions were titrated to pH 7.4 with equal volumes of 1.0 N NaOH
and Tris Buffered Saline (TBS), pH 7.4, 0.05 M L-arginine.
Dilutions were then made as necessary in TBS.
[0149] For comparative purposes, and to illustrate the potential
biological relevance of S--NO-t-PA, this compound was synthesized
with authentic EDRF in selected experiments. In this method, t-PA
was incubated with bovine aortic endothelial cells stimulated by
exposure to high shear forces to secrete EDRF, as we have
previously described (Stamiler et al., Cir. Res. 65:789 (1989),
herein incorporated by reference). Owing to the stability of the
S--NO bond in S--NO-t-PA under physiologic conditions
(t.sub.1/2>24 hours in TBS, pH 7.4, 20.degree. C.), samples were
stored at pH 7.4 on ice throughout the course of the
experiments.
[0150] S--NO-t-PA has also been synthesized by exposure of t-PA to
NO gas bubbled into buffered (TBS) solution of enzyme. This further
illustrates the potential for s-nitrosylation, by exposure of
proteins to a variety of oxides of nitrogen including NOCl,
N.sub.2O.sub.3, N.sub.2O.sub.4 and other nitroso-equivalents.
B. Confirmation of S--NO bond
[0151] 1. Methods
[0152] The formation of and stability of the S--NO bond was
confirmed by several published analytical methods.
[0153] In the first, NO displaced from S-nitrosothiol groups with
Hg.sup.2+ was assayed by diazotization of sulfanilamide and
subsequent coupling with the chromophore
N-(1-naphthyl-)ethylenediamine (Saville, B. Analyst 83:670-672
(1958), herein incorporated by reference). In the second, the
characteristic absorption spectrum of S-nitrosothiols in the range
of 320 nm-360 nm was detected (Stamiler et al., Proc. Natl. Acad.
Sci. USA in press (1991); Oac et al., Org. Prep. Proc. Int.
15(3):165-169 (1983)).
[0154] In the third, [.sup.15N] NMR was used. Measurements of
RS--NOs were made according to the method of Bonnett and colleagues
(Bonnett et al., JCS Perkins Trans. 1:2261-2264 (1975), herein
incorporated by reference). [.sup.15N]NMR spectra were recorded
with a Brucker 500 MHZ spectrometer, Billerica, Mass. Deuterium
lock was effected with [D].sub.2O and the spectra referenced to an
[.sup.15N] natural abundance spectrum of a saturated solution of
NaNO.sub.2 at 587 ppm. Spectra were recorded at 50.68 MHZ and the
nine transients of 16 k data points collected with a 30.degree.
pulse width and a 10-second relaxation delay. Data were multiplied
by a 2-Hz exponential line broadening factor before Fourier
transformation.
[0155] Confirmation of the above chemical evidence for protein
S-nitrosothiol synthesis was obtained by UV, NMR and IR
spectroscopy. Previous characterization of S-nitrosothiols,
revealed that they possses UV absorption maxima at 320-360 nm,
chemical shifts of approximately 750 ppm relative to nitrite
(Bonnett et al., JCS Perkins Trans. 1:2261-2264 (1975)), and IR
stretches at approximately 1160 cm.sup.-1 and 1170.sup.-1 cm.
(Loscalzo et al., JPET 249:726-729 (1989)).
[0156] 2. Results
[0157] In accordance with these observations, S--NO-t-PA exhibited
an absorption maximum at 322 nm (FIG. 1a), and a chemical shift at
751 ppm (relative to nitrite) (FIG. 1b); elimination of the
chemical shift was achieved by sample treatment with excess
HgCl.sub.2. In addition, the presence of two absorption bands at
1153 cm.sup.-1 and 1167 cm.sup.-1, is entirely consistent with the
formation of an S-nitrosothiol bond (Myers et al., Nature
345:161-163 (1990); Oac et al., Org. Prep. Proc. Int. 15(3):165-169
(1983); Bonnett et al., JCS Perkins Trans. 1:2261-2264 (1975). The
quantification of NO (Protein-NO+free NO.sub.x in the Saville
reaction, and the NMR results demonstrating a single chemical
shift, reveal that all NO bound to the protein exists in the form
of an S-nitrosothiol.
[0158] FIG. 2 illustrates the time-dependent formation of
S--NO-t-PA. Aliquots of the solution containing NaNO.sub.2 were
removed sequentially for determination of --S--NO bond formation
(Schwartz, S. M. In Vitro 14:966-980 (1978)). Results are expressed
as mean.+-.S.D. (n=3). By 30 minutes of exposure to acidified
NaNO.sub.2, S-nitrosylation is essentially complete; the
stoichiometry of S--NO-t-PA (mo/mol) is 0.0.+-.0.1 (n=3) at the
completion of the reaction as determined by the method of Saville
(Saville, B. Analyst 83:670-672 (1958)). Carboxyamidation of t-PA's
free thiol with iodoacetamide completely prevents S-nitrosothiol
formation as determined by this chemical method (Saville, B.
Analyst 83:670-672 (1958)).
[0159] FIG. 2 also illustrates the effect of acid treatment on the
amidolytic activity of t-PA. At different intervals, aliquots of
the enzyme exposed to 0.5 N HCl alone were neutralized, and
amidolytic activity was assayed using the chromogenic substrate
S2288. Results are expressed as mean.+-.S.D. (n=3), relative to
t-PA not treated with 0.5 N HCl. At 30 minutes, the duration of
exposure subsequently used for S-nitrosothiol synthesis, the
enzymatic activity of t-PA is largely preserved. Quantification of
S--NO-t-PA synthesis with authentic EDRF was similarly determined
by the method of Saville (Saville, B. Analyst 83:670-672
(1958)).
Example 2
Synthesis of S-Nitroso-BSA
A. Nitrosylation
[0160] In the first method, nitrosylation of BSA was accomplished
by incubating BSA (200 mg/ml with NO generated from equimolar
NaNO.sub.2 in 0.5N HCl (acidified NaNO.sub.2) for thirty minutes at
room temperature. Solutions were titrrated to pH 7.4 with equal
volumes of 1.0 N NaOH and Tris Buffered Saline (TBS), pH 7.4, 0.05
M L-arginine. Dilutions were then made as necessary in TBS.
[0161] In the second method, nitrosylation was achieved in
helium-deoxygenated solutions of 0.1 M sodium phosphate (pH 7.4) by
exposing the protein solution in dialysis tubing to authentic NO
gas bubbled into the dialysate for fifteen minutes. The proteins
were then dialyzed against a large excess of 0.01 M phosphate
buffer at pH 7.4 to remove excess oxides of nitrogen.
[0162] In the third method, proteins were incubated with bovine
aortic endothelial cells stimulated by exposure to high shear
forces to secrete EDRF, as in Example 1(A). As a corollary of this
method, proteins were also incubated directly with NO synthase
purified from bovine cerebellum (Bredt et al., Proc. Natl. Acad.
Sci. USA 87-682 (1990), herein incorporated by reference) in the
presence of the substrate L-arginine and cofactors required for
enzyme activity (Ca.sup.++, calmodulin, and NADPH).
B. Confirmation of S-nitroso-protein formation
[0163] The formation and stability of the S-nitroso-protein was
confirmed by several published analytical methods. NO displaced
from S-nitrosothiol groups with Hg.sup.2+, was assayed by
diazotization of sulfanilamide and subsequent coupling with the
chromophore N-(1-naphthyl-ethylenediamine (Mellion et al., Mol.
Pharmacol. 23:653 (1983); Saville, B. Analyst 83:670 (1958)). The
stoichiometries of S--NO-BSA determined by these chemical methods
is shown in Table 1.
[0164] Confirmatory evidence for S-nitrosothiol bond formation in
proteins was obtained by spectrophotometry; S-nitrosothiols possess
dual absorption maxima at 320-360 nm and at approximately 550 nm
(Oae et al., Organic Prep. and Proc. Int. 15:165 (1983); Ignarro et
al., J. Pharmacol. Exp. Ther. 218:739 (1981); Mellion et al., Mol.
Pharmacol. 23:653 (1983); Loscalzo, J., Clin. Invest. 76:966
(1985)).
[0165] As one additional, more specific measure of protein
S-nitrosylation, [.sup.15N]-NMR spectroscopy was used. BSA was
S-nitrosylated with Na[.sup.15N]O.sub.2 and the [.sup.15N]-NMR
spectrum of the resulting species recorded in FIG. 3. FIG. 3
demonstrates the [.sup.15N]-NMR Spectrum of [.sup.15N]-labeled
S-nitroso-BSA. The chemical shift for S-nitroso-BSA was 703.97,
which falls into the same range as other S-nitrosothiols (e.g.,
S-nitroso-L-cysteine) prepared under similar conditions (Bonnett et
al., J. Chem. Soc. Perldns Trans. 1:2261 (1975)). The spectrum was
recorded at 50.68 MHZ and the nine transients of 16K data points
were collected with a 30.degree. pulse width and a 2.5-sec
relaxation delay. Data were multiplied by a 2-Hz exponential line
broadening factor before Fourier transformation. The region of 590
to 810 ppm is displayed.
Example 3
Synthesis of S-Nitroso-Cathepsin B
[0166] Nitrosylation of cathepsin, and determination of
S-nitrosothiol formation, was accomplished according to the methods
described in Example 2. The stoichiometry of S-nitrosothiol/protein
molecules for cathepsin is shown in Table 1.
Example 4
Synthesis of S-Nitroso-Lippoprotein
[0167] Synthesis was accomplished by nitrosylating purified
low-density-lipoprotein (LDL) according to the methods described in
Example 2. Confirmation of S-nitroso-protein formation was verified
according to the methods of Example 2. The stoichiometry of
S-nitrosothiol/protein molecules for LDL is shown in Table 1.
Example 5
Synthesis of S-Nitroso-Immunoglobulin
[0168] Synthesis was accomplished by nitrosylating purified gamma
globulin (Sigma) according to the methods described in Example 2.
Confirmation of S-nitroso-protein formation was verified according
to the methods of Example 2. The stoichiometry of
S-nitrosothiol/protein molecules for immunoglobulin is shown in
Table 1. TABLE-US-00001 TABLE I S-NITROSO-PROTEIN SYNTHESIS
--S--NO/protein (mol/mol) Bovine Serum Albumin 0.85 .+-. 0.04 t-PA
0.88 .+-. 0.06 Cathepsin B 0.90 .+-. 0.02 Human plasma 0.87 .+-.
0.02 Immunoglobulin 0.35 .+-. 0.01 Lipoprotein (LDL) 1.80 Legend
The stoichiometries for the individual --S--NO/protein molar ratios
are given in the table and represent the mean .+-. SEM of 3 to 6
determinations.
Example 6
Demonstration of Thrombolytic, Anti-Platelet and Vasodilatory
Effect of S--NO-t-PA
A. Thrombolysis
[0169] 1. Fibrinogen Binding
[0170] The binding of t-PA and S--NO-t-PA to fibrinogen was
measured using polystyrene microliter wells (flat-bottom, high
binding 96-well EIA plates, cat. #3590, Costar, Cambridge, Mass.).
Wells were coated with fibrinogen (0.08 ug/ul) and the remaining
binding sites with 2% bovine serum albumin. Quantification of t-PA
binding was determined using a Horse-Radish Peroxidase linked sheep
antimurine antibody in a colorimetric assay in the presence of
O-phenylenediamine, 0.014% H.sub.2O.sub.2. Color change was
measured spectrophotometrically with a Dynatech MR500 Card Reader
(Dynatech, Chantilly, Va.) at 490 nm.
[0171] Binding of t-PA is reversible and specific, and saturates at
1500-3000 nM; at saturation, 18 ng of t-PA are bound per well (0105
moles t-PA per mole of fibrinogen) with an estimated K.sub.D in the
range of 15-650 nM. Binding of t-PA and S--NO-t-PA was quantified
by ELISA over the concentration range of 150-1500 nM using a
mixture containing six murine monoclonal anti-t-PA antibodies.
[0172] a. Comparison of t-PA and S--NO-t-PA
[0173] The binding of t-PA to fibrin(ogen) accounts for the
relative "fibrin specificity" of the enzyme as compared to certain
other plasminogen activators (Loscalzo et al., New Engl. J. Med.
319(14):925-931 (1989); Vaughan et al., Trends Cardiovasc. Med.
Jan/Feb: 1050-1738 (1991)). The effect of S-nitrosylation on this
functional property of the enzyme was therefore assessed. The
binding isotherms for t-PA and its S-nitrosylated derivatives were
not significantly different from each other by two-way ANOVA.
Therefore, these data were subjected to a single best-curve-fit
binding isotherm (FIG. 4). From a Scatchard analysis, the estimated
apparent D.sub.D of S--NO-t-PA for surface-bound fibrinogen is 450
nm, which falls well within the reported range for t-PA (Ranby, M.
Biochim. Biophysica Acta 704:461-469 (1982)).
[0174] 2. Measurement of Enzymatic Activity
[0175] The amidolytic activities of t-PA and its S-nitrosylated
derivative were measured using the relatively specific chromogenic
substrate, S2288. Substrate hydrolysis was measured
spectrophotometrically at 405 nm with a Gilford Response UV/Vis
Spectrophotometer (CIBA-Corning, Oberlin, Ohio). Activity was
measured at 25.degree. C. in TBS using substrate concentrations
varying from 0.1-2.0 mM and t-PA at a concentration of 100 nM.
Kinetic parameters were determined from initial rates by double
reciprocal plot analysis. The assessment of inhibition of t-PA and
S--NO-t-PA enzymatic activity by PAI-1 was made at an enzyme
concentration of 10 nM and a molar ratio of t-PA to active PAI-1 of
1.0. The degree of inhibition was determined relative to the
initial rates in the absence of the inhibitor.
[0176] In the coupled enzyme assay, t-PA and S--NO-t-PA activities
were assayed using the native substrate S2251. In selected
experiments, fibrinogen stimulation of enzymatic activity was
assessed at a fibrinogen concentrations of 1 mg./ml. Substrate
hydrolysis was measured spectrophotometrically with a Dynatech MR
5000 Card Reader (Dynatech, Chantilly, Va.) in TBS, pH 7.4, at
25.degree. C. Initial reaction velocity was determined from the
slope of the plot of absorbance (at 405 nm)/time vs. time (Ranby,
M. Biochim. Biophysica Acta 704:461-469 (1982)) using
glu-plasminogen concentrations ranging from 0.1-10 .mu.M at an
S2251 concentrations of 0.8 mM. Kinetic parameters were determined
from initial rates by double reciprocal plot analysis.
[0177] a. Comparison of t-PA and S--NO-t-PA
[0178] The amidolytic activity of t-PA and S--NO-t-PA were first
compared against the chromogenic substrate S2288. From a double
reciprocal plot analysis it is evident that the kinetic parameters
(K.sub.m and V.sub.max) and the catalytic efficiency
(K.sub.cat/K.sub.m) of these molecules are essentially identical,
as shown in FIG. 5a. The values of these kinetic constants are
provided in Table 2.
[0179] The effect of S-nitrosylation on the ability of t-PA to
activate its physiologic substrate, plasminogen, was assessed in
the coupled enzyme assay in the presence and absence of fibrinogen.
As seen in the Lineweaver-Burke plot (FIG. 5b) and from the derived
kinetic parameters (Table 2), S--NO-t-PA has a K.sub.m for
substrate similar to "wild type" t-PA. However, S--NO-t-PA has a
slightly, but significantly, greater V.sub.max yielding a catalytic
efficiency that is 23% greater than that of native t-PA.
[0180] 3. Discussion
[0181] Both fibrin and fibrinogen increase the rate of activation
of plasminogen by t-PA. The enhanced enzymatic activity of t-PA is
the result of its ability to bind directly fibrin(ogen), which
brings about a conformational change either in t-PA or plasminogen
that promotes the interaction of t-PA with its substrate (Loscalzo
et al., New Engl. J. Med. 319(14):925-931 (1989)).
[0182] The consequences of S-nitrosylation on these important
functional properties of t-PA were therefore studied in a
comparative analysis with t-PA in the coupled enzyme assay. The
results, summarized in FIG. 6, indicate that S--NO-t-PA binds to
fibrinogen; that as a result of this binding its enzymatic activity
is enhanced; and that in the presence of physiologic (1 .mu.M)
plasminogen concentrations, the degree of stimulation is equivalent
to that of "wild type" t-PA. At lower plasminogen concentrations
(0.1 .mu.M), fibrinogen stimulation of S--NO-t-PA was 3.5-fold
greater than t-PA (1 .mu.M) (p<0.05). Absolute rates of
plasminogen activation were again slightly greater for S--NO-t-PA
(vida supra).
[0183] t-PA is rapidly inhibited by its cognate plasma serpin,
PAI-1 (Loscalzo et al., New Engl. J. Med. 319(14):925-931 (1989);
Vaughan et al., Trends Cardiovasc. Med. Jan/Feb: 1050-1738 (1991)).
By serving as a pseudo substrate, PAI-1 reacts stoichiometrically
with t-PA to form an inactive complex. PAI-1 was equally effective
at inhibiting the hydrolytic activity of t-PA and S--NO-t-PA in the
direct chromogenic assay with S2288 (n-3; P-NS). Thus,
S-nitrosylation of t-PA does not appear to alter its interaction
with PAI-1.
B. Platelet Inhibition
[0184] 1. Preparation of Platelets
[0185] Venous blood, anticoagulated with 1-mM trisodium citrate,
was obtained from volunteers who had not consumed acetylsalicylic
acid for at least ten days. Platelet-rich plasma (PRP) was prepared
by centrifugation at 150 g for ten minutes at 25.degree. C.
Platelet counts were determined with a Coulter counter (model ZM;
Coulter Electronics, Hialeah, Fla.).
[0186] 2. Platelet Gel-Filtration and Aggregation
[0187] Platelets were gel-filtered on a 4.times.10 cm column of
Sepharose 2B in Tyrode's Hepes buffer as described previously
(Hawiger et al., Nature 2831:195-198 (1980), herein incorporated by
reference). Platelets were typically suspended at a concentration
of 1.5.times.10.sup.8/ml and were used within 30 minutes of
preparation. Platelet aggregation was monitored using a standard
nephelometric technique (Born, et al., J. Physiol. 168:178-195
(1963), herein incorporated by reference), in which 0.3-ml aliquots
of gel-filtered platelets were incubated at 37.degree. C. and
stirred at 1000 rpm in a PAP-4 aggregometer (Biodata, Hatboro,
Pa.). Gel-filtered platelets were preincubated with t-PA or
S--NO-t-PA for up to 45 minutes and aggregations induced with 5
.mu.M ADP or 0.025 U/ml thrombin.
[0188] Aggregations were quantified by measuring the maximal rate
or extent of light transmittance and expressed as a normalized
value relative to control aggregations.
[0189] 3. Cyclic Nucleotide Assays
[0190] The antiplatelet actions of S-nitrosothiols are mediated by
cyclic GMP. Measurements of cGMP were performed by
radioimmunoassay. Gel-filtered platelets were pre-incubated for 180
seconds with S--NO-t-PA (9 .mu.M), and related controls. Reactions
were terminated by the addition of 10% trichloracetic acid.
Acetylation of samples with acetic anhydride was used to increase
the sensitivity of the assay.
[0191] S--NO-t-PA incubated with platelets for 180 seconds, induced
an 85% increase in intracellular cyclic GMP above basal levels in
the presence of t-PA (p<0.01). The elevation in intracellular
platelet cGMP induced by S--NO-t-PA was entirely prevented by
preincubation of platelets with the guanylate cyclase inhibitor
methylene blue (10 .mu.M for ten minutes (n=3) (FIG. 7).
[0192] 4. Discussion
[0193] The effects of S--NO-t-PA were studied in a gel-filtered
platelet preparation. In these experiments, NO.sub.x generated for
NaNO.sub.2 had no significant effect on the extent of platelet
aggregation (tracing not shown). Mean results for inhibition by
S--NO-t-PA are presented in Table 4.
[0194] FIG. 8 illustrates platelet inhibition induced by S--NO-t-PA
(333 nM) synthesized with EDRF. In these experiments, t-PA was
exposed to endothelial cells stimulated to secrete EDRF for 15
minutes after which the formation for S--NO-t-PA was verified by
method for Saville (Saville, B. Analyst 83:670-672 (1958)).
S--NO-t-PA was then preincubated with platelets for ten minutes
prior to induction of aggregation with 5 .mu.M ADP. In the absence
of t-PA, effluent from endothelial cells stimulated to secrete EDRF
had no significant effect on platelet aggregation. S--NO-t-PA
inhibited platelet aggregation to 5 .mu.M ADP in a dose-dependent
manner, with 50.+-.16% (mean.+-.S.D.) inhibition in rate and extent
of aggregation observed at 1.4 .mu.M S--NO-t-PA (n=4; p<0.001
vs. control). Inhibition of platelet aggregation induced by ADP (5
.mu.M) or thrombin (0.024 U/ml) was demonstrable at concentrations
of S--NO-t-PA in the pharmacologic range of 15-150 nM, as shown in
the illustrative tracings of FIGS. 8(a) and (b) and in Table 4. In
further support of the potential biological relevance for RS--NOs,
and the comparable bioactivity of S--NO-t-PA irrespective of its
method of synthesis, inhibition of platelet aggregation by
S--NO-t-PA (333 nM) synthesized with authentic EDRF is illustrated
in FIG. 8(c).
C. Vasodilation
[0195] 1. Preparation of Blood Vessels
[0196] New Zealand White female rabbits weighing 3-4 kg were
anesthetized with 30 mg/kg IV sodium pentobarbital. Descending
thoracic aortae were isolated and placed immediately in a c-old
physiologic salt solution (Kreb's) (mM): NaCl, 118; CKI, 4.7;
CaCl.sub.2, 2.5; MgSO.sub.4, 1.2; KH.sub.2PO.sub.4, 1.2;
NaHCO.sub.3, 12.5; and D-glucose, 11.0. The vessels were cleaned of
adherent connective tissue, and the endothelium removed by gentle
rubbing with a cotton tipped applicator inserted into the lumen,
after which the vessel was cut into 5 mm rings. The rings were
mounted on stirrups and connected to transducers (model FF03C Grass
Instruments, Quincy, Mass.) by which changes in isometric tension
were recorded.
[0197] 2. Bioassay
[0198] Samples were added to a standard bioassay in which vessel
rings were suspended in glass chambers containing seven ml of
oxygenated Kreb's buffer, in a standard bioassay (Cook et al., Am.
J. Physiol. 28:H804 (1989), herein incorporated by reference).
Sustained contractions, to 2 gm tension, were induced with 1 .mu.M
epinephrine, after which the effects of t-PA and S--NO-t-PA were
tested. In certain experiments the guanylate cyclase inhibitor,
methylene blue, was preincubated with vessel rings for 15 minutes
prior to initiation of contractions.
[0199] 3. Vascular Relaxations
[0200] As shown in the illustrative tracings of FIG. 9, S--NO-t-PA,
at pharmacologic concentrations, induces relaxations that are
unmatched by equimolar amounts of the reactant protein-thiol or NO
alone. Furthermore, consistent with the mechanism of other
nitro(so)-vasodilators, relaxations were attenuated by the
guanylate cyclase inhibitor, methylene blue. Table 3 depicts the
effect of S--NO-t-PA on vessel relaxation for several such
experiments. TABLE-US-00002 TABLE 2 Kinetic Parameters of S2288
Hydrolysis and GLU-Plasminogen (S2251) Activation By t-PA and
S--NO-t-PA K.sub.m k.sub.cat k.sub.cat/K.sub.m (.mu.M) (sec.sup.-1)
(sec.sup.-1 - M.sup.-1) S228 t-PA 280 0.52 0.0019 S--NO-t-PA 295
0.52 0.0019 S2252 t-PA 3.5 0.200 0.056 S--NO-t-PA 3.8 0.262
0.069
[0201] TABLE-US-00003 TABLE 3 VESSEL RELAXATION % Relaxation t-PA
(150 nM) 2.5 .+-. 4 NO (150 nM) 1.0 .+-. 1.7 S--NO-t-PA (150 nM) 20
.+-. 7* Means results (.+-.S.D.; n = 4) of vessel relaxation
induced by S--NO-t-PA, and the comparable relaxation induced by
equivalent concentrations of NO (generated from acidified
NaNO.sub.2) a t-PA. *Relaxations to S--NO-t-PA were significantly
greater than those induced by NaNO.sub.2 or t-PA, as shown in this
table for equal concentrations.
[0202] TABLE-US-00004 TABLE 4 PLATELET INHIBITION % Normalized
Extent Aggregation Thrombin ADP (5 .mu.M) (0.024 U/ml) t-PA (150
.mu.M) 1.06 .+-. 0.24 0.90 .+-. 0.15 S--NO-t-PA (150 .mu.M) 0.77
.+-. 0.28.dagger. 0.73 .+-. 0.28* Mean results (.+-.S.D.; n =
13-17) of platelet inhibition mediated by S--NO-t-PA to both
AD-induced platelet aggregation. NO generated from NaNO.sub.2 (150
nM) had no significant effect on platelet inhibition in these
experiments (0.98 .+-. 0.11, n = 5). *p < 0.025 compared with
t-PA; .dagger.p < 0.01 compared with t-PA.
Statistics
[0203] Determination of statistical significance was analyzed using
a nonpaired t-test or two-way analysis of variance (ANOVA) followed
by a Newman 20Keul's comparison.
Example 7
Demonstration of Platelet Inhibitory and Vasodilatory Effect of
S-Nitroso-BSA
A. Platelet Inhibition
[0204] The effect of S-nitroso-BSA on platelet aggregation was
studied, using a gel-filtered platelet preparation, as previously
described (Hawiger et al., Nature 2831:195 (1980)) and suspended at
150,000 platelets/ul in HEPES buffer, pH 7.35. S--NO-BSA was
incubated with platelets for ten minutes at 37.degree. C. in a
PAP-4 aggregometer (BioData, Hatboro, Pa.), after which
aggregations were induced with 5 .mu.M ADP. Aggregations were
quantified by measuring the extent of change of light transmittance
and expressed as a normalized value relative to control
aggregations.
[0205] In control experiments, neither NaNO.sub.2 at concentrations
up to 15 .mu.M nor the effluent from cells stimulated to secrete
EDRF in the absence of BSA had any significant effect on either
vessel tone or platelet aggregation. All non-nitrosylated proteins
studied had no significant effect on platelet aggregation at any
concentration tested.
[0206] Dose-dependent inhibition of ADP-induced platelet
aggregation was observed over the range of 150 nM to 15 .mu.M
S-nitroso-protein. A nitrosylated protein plasma fraction was even
more potent, manifesting inhibition at estimated --S--NO
concentrations of 150 pM. S-nitroso-proteins synthesized with
acidified NaNO.sub.2, with NO gas, or by exposure to bovine aortic
endothelial cells stimulated to secrete EDRF were essentially
equipotent, as shown for S-nitroso-BSA in FIG. 10. Furthermore, the
platelet inhibitory effect of S-nitroso-BSA (1.4 .mu.M) was
confirmed both in platelet-rich plasma and in whole blood (using
impedance aggregometry in this latter case) (Chong et al., Drug
Met. and Disp. 18:61 (1990) herein incorporated by reference).
[0207] Representative mean data and illustrative aggregation
tracings for S-nitroso-BSA are provided in FIGS. 10 and 11a,
respectively. Carboxyamidation of protein thiols with iodoacetamide
or pretreatment of platelets with the guanylate cyclase inhibitor
methylene blue abolished the antiplatelet effects of
S-nitroso-proteins (FIG. 11a). In addition, the half-life of the
antiplatelet effects correlated with that for vascular smooth
muscle relaxation.
B. Vasodilation
[0208] 1. Methods
[0209] The vasodilatory actions of S-nitroso-BSA were examined in a
standard bioassay containing endothelium-denuded rabbit aortic
strips in Kreb's buffer, pH 7.5, at 37.degree., as described in
Example 6.
[0210] 2. Results
[0211] Dose-dependent relaxations were observed over the range of
15 nM to 15 .mu.M S-nitroso-proteins, and representative mean data
for S-nitroso-BSA are provided in FIG. 10. S-nitroso-proteins
synthesized with acidified NaNO.sub.2, with NO gas, or by exposure
to bovine aortic endothelial cells stimulated to secrete EDRF were
essentially equipotent; this is again exemplified for S-nitroso-BSA
in FIG. 10. The relaxation response to S-nitroso-BSA proteins
differed from that generally ascribed to EDRF, authentic NO, and
the relatively labile low molecular weight biological
S-nitrosothiols, all of which are characterized by rapid, transient
relaxations. In marked contrast, S-nitroso-BSA induced a less
rapid, but much more persistent, relaxation response (FIG. 11b),
thus confirming that it acts as a long-acting vasodilator.
[0212] Furthermore, BSA incubated with NO synthase in the presence
of cofactors required for enzyme activity (calmodulin, NADPH,
Ca.sup.++) showed an L-arginine-dependent ability to induce
persistent vasorelaxation characteristic of S-nitroso-proteins.
[0213] The half-life of S-nitroso-BSA as determined in the bioassay
corresponded with chemical measurements of half-life and is
approximately twenty-four hours. This half-life is significantly
longer than the half-lives of low molecular weight S-nitrosothiols
and suggests that the temporal profile of the relaxation response
for S-nitrosothiols correlates with the lability of the S--NO
bond.
[0214] Blockade of protein thiols by carboxyamidation with
iodoacetamide prevented S-nitrosothiol formation as determined
chemically, and rendered the proteins exposed to NO or EDRF
biologically inactive (FIG. 11b). Consonant with the mechanism of
other nitro(so)-vasodilators (Ignarro, L. J. Cinc. Res. 65:1
(1989)), relaxations were abolished by methylene blue, an inhibitor
of guanylate cyclase (FIG. 11a). This mechanism was confirmed by
showing that S-nitroso-BSA (18 .mu.M) induces 3.5-fold increases
(n=2) in cyclic GMP over basal levels relative to BSA alone in
cultured RFL-6 lung fibroblasts containing a soluble guanylate
cyclase exquisitely sensitive to NO (Forstermann et al., Mol.
Pharmacol. 38:7 (1990)). Stimulation of guanylate cyclase by
S-nitroso-BSA was attenuated by methylene blue.
[0215] FIG. 10 demonstrates the dose-dependent relaxation of
vascular smooth muscle and inhibition of platelet aggregation with
S-nitroso-BSA (S--NO-BSA). Dose-effect curves for vessel relaxation
(.box-solid.-.box-solid.) and platelet inhibition
(.circle-solid.-.circle-solid.) were generated with S--NO-BSA
synthesized with equimolar NO generated from acidified NaNO.sub.2
as described in the text and then neutralized to pH 7.4. Data are
presented as mean.+-.SEM (n=6-18). The open symbols represent
experiments, in the vessel (.quadrature.) and platelet
(.largecircle.) bioassays, in which S--NO-BSA was synthesized by
exposure of BSA to bovine aortic endothelial cells stimulated to
secrete EDRF. These data are presented as mean.+-.SEM (n=3-8), with
the X-axis error bars indicating the variance in the concentration
of S--NO-BSA generated from EDRF and the Y-axis error bars
indicating the variance in the bioassay response.
[0216] In vessel experiments, relaxations to S--NO-BSA are
expressed as percent of tone induced by 1.0 .mu.M
norepinephrine.
[0217] Infusion of S--NO-BSA into anesthetized dogs, according to
standard methods known in the art, resulted in prolonged decreases
in blood pressure, unmatched by low molecular weight
S-nitrosothiols. In addition, this compound increased coronary
flow, thus preserving myocardial blood flow.
[0218] In a canine model of subtotal coronary artery occlusion,
S--NO-BSA inhibited platelet-dependent cyclic thrombus formation
and significantly prolonged bleeding times. These extremely potent,
but reversible anti-platelet properties in vivo are unmatched by
classic nitrates. As well, the improvement in coronary blood flow
contrasts markedly with the clinically used nitroso-compound,
nitroprusside, which has deleterious effects on coronary flow. As
shown in FIGS. 12-14, the constellation of anti-platelet effect,
long duration of action, and increased coronary blood flow, is
unmatched by other nitroso-compounds. Thus, S-nitroso-proteins have
very unique hemodynamic and bioactive profiles.
Example 8
Demonstration of the Vasodilatory Effect of S-Nitroso-Cathepsin
[0219] The effect of S--NO-cathepsin was studied according to the
methods described in Example 7a. Results obtained demonstrated that
S--NO-cathepsin, at a concentration of 150 nM-1.5 .mu.M, inhibits
platelet aggregation.
[0220] The effect of S--NO-cathepsin on vasodilation was studied
according to the methods described in Example 7b. As shown in the
illustrative tracings of FIG. 15, S--NO-cathepsin, at a
concentration of 150 nM 1.5 .mu.M induces vessel relaxation which
is unmatched by equimolar amounts of non-nitrosylated
cathepsin.
Example 9
Demonstration of the Platelet Inhibitory and Vasodilatory Effect of
S-Nitroso-Lipoprotein
[0221] The effect of S--NO-LDL on platelet aggregation was studied
according to the methods described in Example 7a. Aggregations were
quantified by measuring the extent of change of light
transmittance, and expressed as a normalized value relative to
control aggregations. As shown the illustrative tracings of FIG.
16, inhibition of platelet aggregation is demonstrable at a
concentration of 1 .mu.M S--NO-LDL.
[0222] The effect of S--NO-LDL on vasodilation was studied
according to the methods described in Example 7b. As shown in FIG.
17, S--NO-LDL induces vessel relaxation which is unmatched by
equimolar amounts of non-nitrosylated LDL.
Example 10
Demonstration of the Platelet Inhibitory and Vasodilatory Effect of
S-Nitroso-Immunoglobulin
[0223] The effect of S--NO-Ig on platelet aggregation was studied
according to the methods described in Example 7a. Aggregations were
quantified by measuring the extent of change of light
transmittance, and expressed as a normalized value relative to
control aggregations. As shown in FIG. 18, inhibition of platelet
aggregation is demonstrable at concentrations of S--NO-Ig in the
pharmacologic range of 150 nM-1.5 .mu.M.
[0224] The effect of S--NO-Ig on vasodilation was studied according
to the methods described in Example 7b. As shown in FIG. 19,
S--NO-Ig, at concentrations in the range of 150 nM-1.5 .mu.M,
induces relaxation which is unmatched by equimolar amounts of
immunoglobulin alone.
Example 11
Relaxation of Airway Smooth Muscle Caused by S-Nitroso-BSA
[0225] 1. Materials
[0226] Glutathione, L-cysteine, DL-homocysteine, D-penicillin,
hemoglobin (bovine), methylene blue and Medium 199 sets were
purchased from Sigma Chemical Co., St. Louis, Mo. N-acetylcysteine
was obtained from Aldrich Chemical Co., Milwaukee, Wis. Captopril
was kindly provided by Dr. Victor Dzau. Sodium nitrite, histamine
and methacholine were purchased from Fisher Scientific, Fairlawn,
N.J. Leukotriene D.sub.4 was purchased from Anaquest, BOC Inc.,
Madison, Wis. Antibiotic/antimycotic mixture (10,000 U/ml
penicillin G sodium, 10,000 mg/ml, streptomycin sulfate, 25 mg/ml
amphotericin B) was purchased from Gibco laboratories, Grand
Island, N.Y. Radioimmunoassay kits for the determination of cyclic
GMP were purchased from New England Nuclear, Boston, Mass.
[0227] 2. Preparation of Airways
[0228] Male Hartley guinea pigs (500-600 g) were anesthetized by
inhalation of enflurane to achieve a surgical plane of anesthesia.
The trachea were excised and placed in Kreb's-Henseleit buffer
(mM); NaCl 118, KCl 5.4, NaH.sub.2PO.sub.4 1.01, glucose 11.1,
NaHCO.sub.3 25.0, MgSO.sub.4 0.69, CaCl 2.32, pH 7.4. The airways
were then dissected free from surrounding fat and connective tissue
and cut into rings 2-4 mm in diameter. The trachea rings were
placed in sterile Medium 199 containing 1% antibiotic/antimycotic
mixture in an atmosphere of 5% CO.sub.2, 45% O.sub.2, 55% N.sub.2
and kept for up to 48 hours in tissue culture. The experiments were
also performed on human airways isolated by the same method.
[0229] 3. Bioassay
[0230] Trachea rings were mounted on stirrups and connected to
transducers (model FTO3C Grass), by which changes in isometric
tension were measured. Rings were then suspended in 10 cc of
oxygenated (95% O.sub.2, 5% CO.sub.2 buffer. Airway rings were
equilibrated for 60 minutes under a load of 1 gm and then primed
twice by exposure to 100 .mu.M methacholine. The rings were
contracted with various agonists at concentrations determined to
generate 50% (.+-.16% S.D.) of maximum tone, after which the effect
of S--NO-BSA was assessed. In selected experiments, relaxation
responses were determined in the presence of hemoglobin, or after
rings had been preexposed to methylene blue for 30 minutes.
[0231] 4. Results
[0232] As shown in FIG. 20, S--NO-BSA is a potent airway smooth
muscle relaxant, producing 50% relaxation at a concentration of
0.011 .mu.M and over 75% relaxation at a concentration of 10
.mu.M.
Example 12
Inhibition of Enzymatic Activity of Cathepsin B by
Nitrosylation
[0233] The enzymatic activity of S--NO-cathepsin B was measured
against the chromogenic substrate, S2251 at pH 5, in sodium acetate
buffer. S-nitrosylation resulted in a loss of enzymatic
activity.
Example 13
Nitrosylation of Aromatic Amino Acids
[0234] 1. Methods
[0235] a. Preparation of Nitroso-Tyrosine
[0236] 50 mmol of L-tyrosine (Sigma Chemical company; St. Louis,
Mo.) were dissolved into 0.5 ml of distilled water. 250 mmol of
NaI.sup.5NO.sub.2 (sodium N-[15] nitrite: MSD Isotopes, Merck
Scientific; Rahway, N.J.) were dissolved into 0.5 mL of 1 N HCL
(Fisher Scientific; Fair Lawn, N.J.) and transferred immediately to
the aqueous tyrosine solution with agitation by Vortex stirrer.
Solution was capped and allowed to sit at room temperature for 30
minutes.
[0237] NMR measurements were made as follows:
[0238] (a) .sup.15N-NMR: D.sub.2O was added and measurements were
taken immediately;
[0239] (b) .sup.1H-NMR: After .sup.15N-NMR was completed, solution
was removed and placed into a small round-bottom flask and water
was removed in vacuo. D.sub.2O was added to the dry off-white solid
(this time as a solvent) and measurements were run immediately;
[0240] (c) Infrared Spectroscopy: Fourier Transform Infrared
Spectroscopy (FFIR) samples were prepared through removal of water
(as in (b)) and subsequent creation of a Nujol Mull using mineral
oil.
[0241] (d) Ultraviolet and Visible Spectroscopy (UV-Vis): Samples
for UV Vis examination were used as per above prep without further
modification. Samples were referenced to distilled water.
[0242] b. Nitrosylation of Phenylalanine, Tyrosine, and L-Boc-Tyr
(Et)-OH.
[0243] 50 mmol of L-phenylalanine, L-tyrosine (Sigma Chemical
Company; St. Louis, Mo.), or L-boc-tyr(Et)-OH (Bachem Bioscientific
Incorporated; Philadelphia, Pa.) were dissolved into 0.5 ml of
distilled water. 250 mmol of Na.sup.15NO.sub.2 (sodium N-[15]
nitrite) were dissolved into 0.5 ml of 1 N HCl (aq.) and
transferred immediately to the aqueous amino acid solution with
agitation by Vortex stirrer. Solution was capped and allowed to sit
at room temperature for 30 minutes. .sup.15N-NMR and .sup.1H-NMR
were performed as per nitroso-tyrosine above. Standard reference of
L-tyrosine for FTIR was prepared as a Nujol Mull of pure
crystalline L-tyrosine.
[0244] c. Nitrosylation of Tryptophan
[0245] 1.7 mM of tryptophan were reacted with equimolar NaNO.sub.2
in 0.5 N HCl for time periods of 5, 10, 15 and 60 minutes at
25.degree. C.
[0246] 2. Results
[0247] a. 15N-NMR Data
[0248] All NMR [.sup.15N and .sup.1H] were run on two Bruker AM-500
MgHz spectrometers. Nitrosylation of tyrosine at pH 0.3 gives
signals at approximately 730 ppm and 630 ppm relative to saturated
sodium N-[15] nitrite aqueous solution referenced at 587 ppm.sup.12
(.sup.15NO.sub.2) (FIG. 21a.). A signal at 353 ppm (aqueous
NO.sup.12) was also observed. Nitrosylation of phenylalanine under
the same conditions gave the signal at approximately 630 ppm but
not the 730 ppm signal despite repeated attempts (FIG. 22).
Nitrosylation of phenylalanine also yielded signals at 587 ppm
(excess, unprotonated nitrite) and 353 ppm. Nitrosylation of
O-blocked tyrosine model, boc-tyr(Et)-OH, also yielded a signal at
approximately 630 ppm; and others, at 587 ppm and 353 ppm. Small
signals in the range 450495 ppm were observed for the tyrosine
models, phe and boc-tyr(Et)-OH.
[0249] b. .sup.1H-NMR Data
[0250] To further characterize the nitrosylation of the phenolic
functionality of L-tyr to the exclusion of C-nitrosylation,
proton-NMR was performed on nitrosylated tyrosine; modification of
L-tyr at the phenolic-OH would not appear in proton-NMR because of
proton exchange with the deuterated solvent (D.sub.2O). Examination
of the spectra showed the classic doublet of doublets at low field,
which is characteristic of para-disubstituted benzene, thus
excluding aromatic proton substitution (FIG. 21b). This, and other
values in the spectra were characteristic of unmodified L-tyr.
[0251] c. FTIR Data
[0252] All FTIR were run on a Nicolet 5ZDX FT-IR Spectrometer. FTIR
of a Nujol Mull of L-tyrosine showed a very characteristic and
well-documented alcoholic stretch in the spectra due to the
phenolic-OH (FIG. 1d. inlaid). This spectrum lacked any signal(s)
at the 1680-1610 cm.sup.-1 range that coincides with the O--N.dbd.O
stretch (not shown). FTIR of nitrosylated L-tyrosine showed no
evidence of alcoholic-OH stretches and contained two small bands in
the range of 1680-1610 cm.sup.-1 that could possibly account for
the expected O--N.dbd.O stretch (Wade, L. G., Organic Chemistry
(1st Ed.) Prentice-Hall Inc., Englewood Cliffs, N.J.: 1987. p.
1334) (FIG. 21c.).
[0253] d. UV-Vis Data
[0254] All UV-Vis spectroscopy was performed using a Gilford
Response UV-Vis Spectrophotometer (CIBA-Corning, Oberlin, Ohio).
Treatment of L-tyrosine with aqueous sodium nitrite at pH 0.3 (0.5N
HCl) resulted in a yellow solution with an absorption maximum at
361 nm. This result is similar to, but differs from previously
reported results with nitrosated L-tyrosine. Ortho-ring substituted
I-nitro-tyrosine (Sigma) absorbs at 356 nm at pH 0.3.
[0255] Treatment of phenylalanine with sodium nitrite at pH 0.3
gives a rapidly changing UV spectrum with a peak increasing in
wavelength from 318 nm at 5 min. to a maximum unchanging peak at
527 nm by 30 min.
[0256] FIG. 23(a-e) demonstrates time-dependent nitrosylation of
tryptophan. The data is suggestive of trosylation of both the
aromatic ring and amino groups.
Example 14
Nitrosylation of BSA
[0257] BSA, at 200 mg/ml, was loaded at a ratio of 20:1 with NO in
0.5 N HCl for 30 minutes at room temperature. As shown in FIG. 24,
the 726 ppm peak indicates O-nitrosation of the tyrosine residues
on BSA. FIG. 24 also provides evidence for the nitrosation of
several other functional groups on BSA. The data are also
suggestive of ring nitrosation and amine nitrosation (600 ppm peak)
as well.
[0258] Time-dependent NO loading of BSA was performed by exposing
BSA (200 mg/ml) in phosphate buffer (10 mM, pH 7.4) to NO gas
bubbled into the BSA solution, for 1, 5 and 30 minute time periods.
FIG. 25 provides UV spectrum data which indicates NO loading of
BSA.
Example 15
Nitrosylation of t-PA: NO Loading
[0259] t-PA at 10 mg/ml was exposed 10:1 to excess NaNO.sub.2 in
0.5 N HCl. FIG. 26 shows NO-loading of t-PA.
Example 16
Vasodilatory Effect of NO-Loaded BSA
[0260] BSA was loaded with NO according to the method described in
Example 14.
[0261] Vasodilatory effect was studied in a rabbit aorta bioassay,
according to the methods described in Example 6C. As shown in FIG.
27, increasing concentrations of NO resulted in an increase in
vessel relaxation induced by the resultant NO-BSA.
Example 17
Guanylate Cyclase Inhibitors Do Not Inhibit S-nitroso-Protein
Induced Relaxation in Human Airways
[0262] The effect of guanylate cyclase inhibitors upon
S-nitroso-protein induced airway relaxation and cGMP increase was
assessed, using the previously described bioassay and cyclic
nucleotide assay procedures. The bronchodilatory effect of
S-nitroso-albumin was examined in human airways (5-12 mm outer
diameter). Concentration-response relationships for rings
contracted with methacholine (7 .mu.M) resulted in IC50 values of
22 .mu.M, approximately two orders of magnitude greater than
theophylline.
[0263] S-nitroso-albumin (100 .mu.M) induced increases over control
airway cGMP levels. However, S-nitroso-albumin-induced airway
relaxation was not significantly inhibited by methylene blue
(10.sup.4) or LY83583 (5.times.10.sup.-5). Similarly, hemoglobin
(100 .mu.M) had little effect on S-nitroso-albumin-induced
relaxation (P=NS).
[0264] These results demonstrate that the mechanism by which
S-nitroso protein cause airway relaxation is not due solely to
increases in cGMP. Thus, S-nitroso-proteins cause airway relaxation
through both an increase in cyclic GMP, as well as a
cGMP-independent pathway. In doing so, they provide a means for
achieving combination therapy by maximizing the synergistic effect
of two separate mechanisms.
Example 18
S-nitroso-Proteins Resist Decomposition in the Presence of Redox
Metals
[0265] The stability of S-nitroso-albumin in the presence of oxygen
and redox metals was assessed. When subjected to conditions
consisting of 95% O.sub.2, pH 7.4, the half life of this compound
was shown to be on the order of hours, and significantly greater
than that of NO, or NO., which, under similar conditions, are on
the order of seconds.
[0266] In addition, S-nitroso-protein stability was assessed in the
presence of various redox metals or chelating agents.
S-nitroso-albumin was resistant to decomposition when Cu.sup.+,
Fe.sup.2+, or Cu.sup.2+ (50 .mu.M) or defuroxamine or EDTA (10
.mu.M) were added. Thus, these experiments demonstrate that, unlike
NO., S-nitroso-proteins are not rapidly inactivated in the presence
of oxygen, nor do they decompose in the presence of redox
metals.
Example 19
S-Nitrosylation of Hemoglobin Increases Hemoglobin-Oxygen
Binding
[0267] Additional experiments were conducted to evaluate the
reaction between S-nitrosothiols and hemoglobin. S-nitrosylation of
hemoglobin was accomplished by reacting 12.5 .mu.M hemoglobin with
12.5 .mu.M for 5 and 20 minute intervals (pH 6.9). S-nitrosylation
was verified, using standard methods for detection of
S-nitrosothiols (Saville, Analyst 83:670-672 (1958)). The Saville
method, which assays free NO.sub.x in solution, involves a
diazotization reaction with sulfanilamide and subsequent coupling
with the chromophore N-(1-naphthyl)ethylenediamine. The specificity
for S nitrosothiols derives from assay determinations performed in
the presence and absence of HgCl.sub.2, the latter reagent
catalyzing the hydrolysis of the S--NO bond. Confirmatory evidence
for S-nitrosothiol bond formation was obtained by
spectrophotometry, demonstrated by the absorption maximum of 450
nm, as shown in FIG. 28. This was demonstrated using NO.sup.+
equivalents in the form of SNOAC.
[0268] As demonstrated by FIG. 29, the UV spectrum of hemoglobin
incubated with SNOAC shows no reaction at the redox metal
(iron-binding site) of hemoglobin, over 15 minutes. For the
purposes of comparison, equimolar concentrations of hemoglobin and
NaNO.sub.2 were reacted in 0.5 N HCl, to form nitrosyl-hemoglobin,
and the UV spectrum was obtained. As shown in FIG. 30, NO reacted
instantaneously with the redox metal site on hemoglobin. The fact
that the S-nitrosothiol did not react with the redox metal site of
hemoglobin, but with its thiol group instead, indicates that the
reactive NO species donated by the S-nitrosothiol is nitrosonium or
nitroxyl.
[0269] S-nitrosylation of hemoglobin does not result in the
formation of methemoglobin and consequent impairment in
hemoglobin-oxygen binding. Furthermore, an additional experiment
demonstrated that S-nitrosylation of hemoglobin causes a leftward
shift in the hemoglobin-oxygen association curve, indicating an
increase in oxygen binding. Thus, the reaction between
S-nitrosothiols and hemoglobin not only eliminates the inhibition
of oxygen binding which occurs from the reaction with uncharged NO
and generation of methemoglobin, but it actually increases oxygen
binding.
[0270] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the invention and without undue experimentation.
[0271] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the inventions
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth as follows in the scope of the appended
claims.
* * * * *