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WO1996030006A1 - Substituts sanguins nitrosyles - Google Patents

Substituts sanguins nitrosyles Download PDF

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Publication number
WO1996030006A1
WO1996030006A1 PCT/US1996/003866 US9603866W WO9630006A1 WO 1996030006 A1 WO1996030006 A1 WO 1996030006A1 US 9603866 W US9603866 W US 9603866W WO 9630006 A1 WO9630006 A1 WO 9630006A1
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Prior art keywords
nitroso
protein
hemoglobin
nitrosylation
compound
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PCT/US1996/003866
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English (en)
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WO1996030006A9 (fr
Inventor
Jonathan Stamler
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Brigham And Women's Hospital
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Priority to AU53682/96A priority Critical patent/AU5368296A/en
Publication of WO1996030006A1 publication Critical patent/WO1996030006A1/fr
Publication of WO1996030006A9 publication Critical patent/WO1996030006A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/42—Haemoglobins; Myoglobins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)

Definitions

  • hemoglobins and other oxygen delivery systems that consume or scavenge nitric oxide thereby cause vasoconstriction which hinders oxygen delivery and thereby raises blood pressure.
  • Overcoming these limitations would end a lengthy quest for an alternative to red blood cell transfusions and satisfy a strong medical need.
  • Cell-free blood substitute compositions sought to be used for treating hemorrhage and preserving organs and tissue for transplantation have predominantly been preparations of heme-containing proteins.
  • Numerous investigators and foreign and domestic companies have sought to produce isolated human, bovine and other hemoglobin and modified hemoglobins for use in cell-free blood substitute preparations. Native hemoglobin has been isolated from blood and used in such preparations.
  • Modified hemoglobins have been prepared and used for such cell-free preparations.
  • cross-linked polymerized hemoglobins see U.S. Patent No. 5,194,590
  • cross-linked polymerized pyridoxylated hemoglobin U.S. Patent No. 5,194,590 and 4,826,811
  • Multimeric hemoglobin-like proteins based on pseudo-tetramer containing pseudo-dimer polypeptides with globin-like domains have been used in order to prolong hemoglobin half life (see WO 93/09143).
  • Variants such as conjugates of hemoglobin with other drugs are also used (see WO 93/088422).
  • Di-alpha- globin-like polypeptide and di-beta-globin-like polypeptides connected into a single chain and incorporating heme have been used to prepare a human hemoglobin-like protein (see WO 90/13645).
  • Hemoglobin mutants having lower O-affinity have been prepared by recombinant techniques and used as blood substitutes (see WO 88/09179).
  • Hemoglobin and heme- containing protein based blood substitute compositions are disclosed, inter alia , in U.S. Patent Nos. 5,217,648; 5,194,590; 5,061,688; 4, 826, 811;5,281,579; 5,128,452; 5,248,766; 5,041,615; 4,861,867; 4,831,012; 5,296,465; 5,084,558; 5,295,944; 4,780,210; 4, 925,574;5,264,555 and numerous others; and in PCT publication nos.
  • cell-free blood substitute compositions are oxygen carrying emulsion compositions of fluorocarbon particles. Typical particle sizes are 0.05 to 0.3 mu.
  • Perflurohydrocarbons are well known oxygen carriers for artificial blood and in vitro perfusion fluids.
  • the perflurocarbon compounds used can generally be of about 3-15 carbon atoms, particularly mixtures of them.
  • Examples include perfluoro-decalin, - methyldecalin, -(3-5C) alkylcyclohexanes, -(5-7C) alkytertrahydrofurans, -(4-6C) alkyltetrahydropyrans and (9- 11C) alkane ⁇ , pe r f 1uoro ethy 1 a a antane , perfluorodi ethyladamantane, perfluoroethylmethylada antane, perfluorodiethylada anatane and perfluoroadamantane.
  • compositions can also use perfluorotertiaryamines.
  • compositions can further include nonionic surfactants, (e.g., a polyoxyethylene - polyoxypropylene copolymer) phospholipids, polymeric e ulsifiers and fatty acids.
  • nonionic surfactants e.g., a polyoxyethylene - polyoxypropylene copolymer
  • phospholipids e.g., a polyoxyethylene - polyoxypropylene copolymer
  • polymeric e ulsifiers e.g., a polymeric e ulsifiers and fatty acids.
  • This invention relates to nitrosylation of blood substitutes, particularly including heme proteins such as hemoglobin as a therapeutic modality.
  • the invention also relates to 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.
  • An additional aspect of the invention is blood substitute compositions comprising nitrosylated proteins and other constituents. These blood substitute compositions are preferably cell free. Principally useful in such compositions are various forms of nitrosylated hemoglobin and modified hemoglobins.
  • nitrosylated blood substitutes have a vasorelaxant activity which is directly opposite to the vasoconstrictive properties of the non-nitrosylation forms.
  • the administration of the nitrosylated forms effect vasodilation both by adding the administered nitric oxide to the recipient but also by not reducing through scavenging, the constitutive levels.
  • the invention relates to nitrosylation of sites such as sulfhydryl (thiol), 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, such as in the form of a pharmaceutical composition.
  • the invention provides a method for inhibiting the vasoconstrictive and nitric oxide depleting effects of hemoglobin and heme-containing based blood substitute compositions by the concurrent systemic administration of nitric oxide or a compound which donates, releases or transfers nitric oxide.
  • Such administration can, for example, be by inhalation or intravenously, separately or in composition with the blood substitute.
  • NO has been shown to exert a wide variety of biological effects. In the context of this application, vasodilation, bronchodilation and inhibition of intestinal and sphincteric motility are noteworthy. Moreover, NO has also been implicated in the pathogenesis of inflammation, cellular dysfunction and toxicity when produced in settings of oxidant stress. These divergent effects in different biological systems are achieved via a rich redox and additive chemistry of the molecule. Both covalent modifications of proteins as well as oxidation events that do not involve attachment of the NO group, have been adopted as signalling mechanisms.
  • NO reacts with 0 2 ", 0 2 , and transition metals.
  • OONO peroxynitrite
  • NO. and metal-NO adducts respectively - support additional nitrosative and oxidative reactions with thiol groups.
  • thiol and metal-containing proteins are the major target sites for NO. These targets include plasma proteins (infra), membrane-associated signalling proteins, ion channels and receptors.
  • enzymes containing either thiols or metals are known to be NO-responsive.
  • NO activates the prototypic heme containing enzyme guanylyl cyclase, which elicits many target cell responses, including smooth muscle relaxation.
  • Heme proteins also serve in the biometabolism of NO. For example, inactivation of NO in the blood, and the tissues it perfuses is achieved by interactions with hemoglobin. Unlike CO and 0 2 , nitric oxide binds effectively to both ferric (III) and ferrous (II) heme. The ratio of rates of NO uptake and release for Fe(II)- hemoglobin is five-six orders of magnitude greater than that of 0 2 , largely due to the marked differences in ligand dissociation rates ( ⁇ 10 "5 s' 1 for NO and - 20 s' 1 for 0 2 ). The Fe(III) NO-heme adduct exhibits more rapid loss of NO. No is also inactivated rapidly by O 2 .hemoglobin, forming methemoglobin and nitrate.
  • NO + , NO*, and NO The distinctive properties and differential reactivities of these species is critical to the elucidation of the biological actions of "nitric oxide” .
  • NO* shows preferential reactivity towards metal centers
  • species with N0 + character target sulfhydryl groups, forming S-nitrosothiols (RS-NO).
  • RS-NO S-nitrosothiols
  • hemoglobin contains a highly reactive 393 SH group (pK of 5.5), making it susceptible to S-nitrosylation. Since the NO group possesses NO + character it is well shielded from the heme center of the molecule (which binds NO").
  • Thiols are also present on those proteins the function of which is to transport and deliver specific molecules to particular bodily tissues.
  • lipoproteins are globular particles of high molecular weight that transport nonpolar lipid ⁇ through the plasma. These proteins contain thiols in the region of the protein which controls cellular uptake of the lipoprotein (Mahley el al . JAMA 265:78-83 (1991)). Hyper-lipoproteinemias, resulting from excessive lipoprotein (and thus, lipid) uptake, cause life-threatening diseases such as atherosclerosis and pancreatitis.
  • 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.
  • 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.
  • Protein thiols may, under certain pathophysiological conditions, cause a protein to exert a detrimental effect.
  • cathepsin a sulfhydryl enzyme involved in the breakdown of cellular constituents, is critically dependent upon sulfhydryl groups for proteolytic activity.
  • uncontrolled proteolysis caused by this enzyme leads to tissue damage; specifically lung damage caused by smoking.
  • 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.
  • 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 .
  • synthesis of polynitrosated peptides and proteins can be achieved in several ways.
  • Mono S- nitrosylation is best achieved by incubating peptides and proteins (in deionized water in an equimolar concentration of acidified nitrite (final concentration 0.5 N HCL) for a period of 1-30 minutes. The incubation time depends on the efficiency of nitrosation and the tolerance of the protein.
  • Nitrosation can also be achieved with a variety of other nitrosating agents including compounds such as S- nitrosocysteines, S-nitrosoglutathione and related alkyl nitrites. These compounds are used when the peptide or protein does not tolerate harsh acidic conditions.
  • the peptide or protein is reduced in 100-1000 molar excess dithiothreitol for 30-60 minutes. This exposes intramolecular thiols.
  • the peptide or protein is separated from dithiothreitol by gel filtration (G-25).
  • the protein is then exposed to increasing concentrations of acidified nitrite (0.5 N HCI) in relative excess over protein.
  • acidified nitrite 0.5 N HCI
  • the protein can be treated with thiolating agent such as homocysteine thiolactone. This tends to add homocystine groups to exposed amine residues in proteins.
  • the derivatized protein can then be S-nitrosated by exposure to acidified nitrite. Exposure to incerasing concentrations of nitrite with complementary measurements of Saville can be used to ascertain when S-nitrosation is maximal.
  • thiol groups can be quantified on the protein using standard methodologies and then the protein treated with a stoichiometric concentration of acidified nitrite (0.5 N HCI) .
  • nucleophilic functional groups other than thiol
  • Polynitrosation of nucleophilic functional groups can be achieved when proteins are incubated with excess acidified nitrite.
  • the order of protein reactivity is tyrosine followed by amines on residues such as trytophan. Amide linkages are probably less reactive.
  • NO groups can be added to proteins by simply incubating the protein with high excess acidified nitrite. For example, exposure of albumin to 1000 fold excess nitrite leads to approximately 200 moles of NO/mole protein.
  • nitrosation can be achieved by exposure to authentic nitric oxide gas under anaerobic conditions.
  • nitrosation proteins should be incubated in at least 5 atmospheres of NO gas for several hours. Incubation time is protein specific. This can lead to NO attachment to a variety of protein bases. Best characterized reactions involve primary amines. This mechanism provides a pathway to sustain N-nitrosation reactions without deamination. Specifically, exposure to acidified nitrite would otherwise lead to deamination of primary amines whereas this method leads to formation of N-hydroxynitrosamines with potent bioactivity. Similar substitutions at other basic centers also occur.
  • amino acid side chains such as those found on tyrosine and other aromatic amino acids
  • tyrosine and other aromatic amino acids have a critical role in ensuring proper enzymatic function within the body.
  • 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.
  • these aromatic amino acids serve as precursors to numerous important biomolecules such as hormones, vitamins, coenzymes, and neurotransmitters.
  • 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.
  • the present invention represents a novel method for achieving these therapeuticaliy 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.
  • the invention represents the discovery of exemplary S-nitroso-proteins and amino acids of great biological and pharmacological utility.
  • 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.
  • 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.
  • this invention is directed to compounds comprising enzymes, nitrosylated thrombolytic agents, particularly nitrosylated thrombolytic enzymes.
  • enzymes include tissue-type plasminogen activator, streptokinase, urokinase and cathepsin and others known or yet to be discovered.
  • This invention is also directed to compounds comprising S-nitroso-lipoprotein.
  • Lipoproteins which may be contained in the compound include chylo icrons, chylomicron remnant particles, very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) high-density lipoprotein (HDL) and lipoprotein (a).
  • This invention is also directed to compounds comprising S-nitroso-immunoglobulin.
  • Immunoglobulins contained in this compound include IgG, IgM, IgA, IgD, IgE.
  • This invention is also directed to compounds comprising a blood substitute which is directly or indirectly linked to an NO or N0 2 group.
  • the blood substitute is, in one preferred embodiment, a heme protein, particularly a hemoglobin such as human or bovine hemoglobin.
  • the blood substitute compound of the invention also includes modified hemoglobins described in the background literature which have been nitrosylated by direct or indirect linking thereto of an NO or N0 2 group.
  • This NO or N0 2 group can be, for example, attached at a site consisting of an S-nitroso, N-nitroso, O- nitroso and C-nitroso moiety.
  • the invention is also directed to the compound S-nitroso-hemoglobin.
  • the invention is also directed to the compound S-nitroso-myoglobin.
  • the invention is also directed to pharmaceutical compositions containing the compounds of the invention, together with a pharmaceutically acceptable carrier.
  • the invention is particularly directed to blood substitute compositions comprising the nitrosylated derivative compounds of blood substitute compounds identified above.
  • the composition preferably includes nitrosylated heme proteins particularly nitrosylated hemoglobin and modified hemoglobins whether of human or animal origin. Also contemplated are heme containing proteins such as hemoglobin which are made by recombinant methods or are synthesized chemically.
  • the invention also contemplates a composition containing the nitrosylated blood substitute compounds of the invention and which further include an additional component selected from the group consisting of a nonionic surfactant, a phospholipid, an emulsifier and a fatty acid.
  • the invention also provides a blood substitute composition
  • a blood substitute composition comprising an emulsion of perfluorocarbon particles in a pharmaceutically acceptable carrier and which further comprises nitric oxide or a compound capable of donating, releasing or transferring nitric oxide.
  • This compound is preferably a nitrosothiol.
  • 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.
  • 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.
  • 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.
  • This invention also is directed to a method for causing vasodilation, platelet inhibition and thrombolysis, comprising administering a nitrosylating agent to an animal.
  • This invention also is directed to a method for regulation of protein function in vivo , comprising administering a nitrosylating agent to an animal.
  • the invention is also directed to a method for preventing the uptake of a protein by cells, comprising administering a nitrosylating agent to a patient.
  • 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.
  • 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.
  • 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.
  • the invention is also directed to a method for regulating cellular function, comprising the S-nitrosylation of a protein which is a cellular component or which affects a cellular function.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the other sites which are nitrosylated are selected from the group consisting of oxygen, carbon and nitrogen.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 7 Increases in intracellular platelet cyclic GMP, caused by S-NO-t-PA (Example 6.B.3).
  • FIG. 1 Representative tracings of vessel relaxation and platelet inhibition caused by S-nitroso-BSA (S-NO-BSA).
  • FIG. 14 Coronary vasodilation, following infusion of S-nitroso-BSA.
  • Figure 17 Representative tracings of vessel relaxation caused by S-nitroso-LDL.
  • Figure 18 Tracings of dose-dependent inhibition of platelet aggregation caused by S-nitroso-immunoglobulin.
  • Figure 19 Representative tracings of vessel relaxation caused by S-nitroso-immunoglobulin.
  • Figure 20 Concentration-dependent relaxation of airway smooth muscle caused by S-NO-BSA.
  • 2Id UV spectrum for 1.8 mM of tyrosine.
  • 21e UV spectrum for 34 mM of tyrosine.
  • FIG. 25 UV spectrum for time-dependent NO loading of BSA.
  • 25a 1 minute reaction time.
  • 25b 5 minute reaction time.
  • 25c 30 minute reaction time.
  • FIG. 27 Vasodilatory effects of NO-loaded BSA.
  • Figure 28 S-nitrosylation of hemoglobin.
  • Figure 29 UV spectrum of hemoglobin incubated with S-nitroBO-N-acetylcysteine.
  • Figure 30 Reaction of nitric oxide at the iron-binding site of hemoglobin.
  • FIG 31 UV spectrum of S-NO-hemoglobin (Hb) .
  • Hb S-NO-hemoglobin
  • Spectra shows that oxygen binding to the heme site is largely unaffected at S-NO/Hb stoichiometrie ⁇ (0.05 and 0.37) that are lacking in smooth muscle contractile activity (see Figure 2) .
  • Higher ratios of S-NO/Hb 1.59 are associated with methemoglobin formation.
  • Curves for oxy-Fe(II) -hemoglobin (dithionite-treated) andmethemoglobin (K 3 Fe(CN) -treated) are shown for comparison.
  • FIG 32 Reversal of hemoglobin (Hb) -induced contraction of aortic rings by S-nitrosylation. Hemoglobin is shown to constrict vessels in a dose-dependent manner (•) . S-NO-Hb, with a stoichiometry of 0.1 S-NO ⁇ Hb, entirely prevents the constrictor response (O) . Increasing the S-NO-Hb ⁇ Hb stoichiometry to 1 converts hemoglobin into a potent vasodilator (+) .
  • FIG 33 Endogenous levels of S-NO-hemoglobin in rat artery, rat vein and human vein. Rat arterial blood was obtained by direct cardiac puncture, rat venous blood from jugular vein and human blood by venipuncture, according to approved protocols.
  • 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.
  • nitroso-proteins 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 0 2 and 0 2 reactive species predispose to rapid inactivation of the NO moiety.
  • nitroso-proteins deliver NO as the charged species, nitrosonium (NO + ) or nitroxyl (NO), and not the uncharged NO radical (N0«). This is important because the charged species behave in a very different manner from NO» with respect to chemical reactivity.
  • nitrosonium and nitroxyl do not react with 0, or 0 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mutant refers to any structurally modified protein or polypeptide that retains the same physiological activity of interest, as the parent protein or polypeptide, whether to a greater or lesser extent. Other properties or advantages may be present in such mutant, variant or fragment compound, such as increased half-life or resistance to natural inhibitors of the parent protein. Mutation can refer, for example, to changes in one or more amino acids in the primary sequence. Variants can refer for example, to posttranslational modifications, such as glycosylation, conformational contraint, or the addition of lipid moieties. Fragments can refer to polypeptides that retain some, all or more of the desired physiological property of the parent protein while not having one or more amino acids or sequences thereof present in the parent protein.
  • substantially homologous refers to amino acid sequences of hemoglobins, modified hemoglobins and other heme-containing proteins, means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between reference and subject sequences.
  • sequences having greater than 90 percent homology, equivalent biological activity, and equivalent expression characteristics are considered substantially homologous.
  • truncation of the mature sequence should be disregarded. Sequences having lesser degrees of homology, comparable bioactivity, and equivalent expression characteristics are considered equivalents.
  • Other embodiments include fusion protein products that exhibit similar oxygen carrying activities. Further embodiments include such proteins that are chemically synthesized as well as any proteins or fragments thereof that are substantially homologous.
  • activity refers to any action exerted by the protein or amino acid which results in a physiological effect.
  • 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.
  • proteins which are representative of various functional classes were nitrosylated.
  • 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.
  • One aspect of the invention relates to nitrosylated enzyme compounds derived from nitrosylation of enzymatic proteins.
  • a particular embodiment of this aspect relates to the compound, S-nitroso-t-PA (S-NO-t-PA), derived from the nitrosylation of tissue-type plasminogen activator (t-PA).
  • S-NO-t-PA S-nitroso-t-PA
  • t-PA tissue-type plasminogen activator
  • 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.
  • 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.
  • S-NO-t-PA a new molecule which has improved thrombolytic capability, (e.g., the enzymatic activity of the enzyme is enhanced) as well as vasodilatory and platelet inhibitory effect.
  • S-nitrosylation significantly enhances the bioactivity of t-PA, without impairing the catalytic efficiency or other domain-specific functional properties of the enzyme.
  • 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) .
  • 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).
  • 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.
  • 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.
  • Another embodiment of this aspect 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.
  • platelet inhibition and nitrovasodilation are frequently used concomitant therapies with which to enhance reperfusion by plasminogen activators as well as to prevent rethrombosis (Gold, H.K. New Engl . J. Med. , 323:1483-1485 (1990); (Marder et aJ. , New Engl . J. Med. , 318:1512-1520 (1988) ) .
  • S-NO-t-PA 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.
  • thrombogenesis such as atherothrombosis, myocardial infarction, pulmonary embolism or stroke.
  • 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.
  • Another embodiment of this aspect relates to compounds obtained by 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.
  • An additional aspect of this invention 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 lysosonml cysteine protease.
  • 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.
  • nitroso-lipoprotein compounds derived from the nitrosylation of lipoproteins.
  • 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).
  • VDL very low-density lipoprotein
  • LDL low-density lipoprotein
  • IDL intermediate-density lipoprotein
  • HDL high density lipoprotein
  • S-nitroso-lipoproteins exert vasodilatory and platelet inhibitory effect.
  • 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.
  • An additional aspect 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 hyperlipide ias, such as atherosclerosis.
  • i munoglobulins may include IgG, IgM, IgA, IgD, or IgE.
  • IgG immunoglobulin
  • IgM immunoglobulin
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin E
  • the inventors have demonstrated that these compounds exert vasodilatory and platelet inhibitory effect.
  • 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.
  • nitrosylated blood substitutes particularly nitrosylated cell-free blood substitutes such as those containing the compound 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.
  • 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.
  • 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.
  • 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 nitroso-hemoglobin or the in vivo nitrosylation of hemoglobin, to increase the oxygen-carrying capacity of the blood, and oxygen transport to bodily tissues.
  • these compounds are also 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.
  • nitroso-hemoglobin may also be used to modulate the delivery of carbon monoxide or nitric oxide (bound to hemoglobin) to bodily tissues.
  • any thiol-containing heme proteins may be nitrosylated and used to enhance the oxygen-carrying capacity of the blood.
  • An additional aspect of the invention is the use of blood substitute compositions in accordance with the invention for maintaining and perfusing transplant organ or tissue materials such that they can be maintained for duration upon necessary to transport them or to culture expand them particularly when they are largely comprised of progenitor cells.
  • Such profusion of organ and tissue maintenance compositions are in liquid form and constitute the compounds of the invention in physiologically acceptable carriers.
  • An additional aspect of the invention is 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.
  • Another aspect of the invention relates to a method for using S-nitroso-proteins as a means for providing targeted delivery of NO.
  • targeted delivery means that NO is purposefully transported and delivered to a specific and intended bodily site.
  • SNO-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.
  • delivery may be for the purpose of nitrosylation of various molecules present in the body.
  • S-nitroso-proteins would deliver NO, and thus nitrosylate hemoglobin or myoglobin in order to increase oxygen binding.
  • Another aspect of the invention relates to the administration of nitroso-albumin as a therapeutic agent to promote platelet inhibition, or to cause relaxation of airway smooth muscle.
  • 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.
  • S-nitroso-BSA relaxes human airway smooth muscle.
  • S-nitroso-proteins cause airway relaxation, and also eliminate the adverse effects which occur with administration of other NO species.
  • 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.
  • these compounds may be used as antioxidants, and thus, in the treatment of diseases such as acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • Another aspect 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.
  • Another aspect 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.
  • 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, ileu , colon, Sphincter of Oddi, pancreatic, or common bile duct.
  • Another aspect 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.
  • Proteins and amino acids possess other sites In addition to thiol groups, in addition to thiol groups, which can be nitrosylated.
  • sites may include, but are not limited to, oxygen, nitrogen, and carbon.
  • the invention also 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.
  • 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 NaN0 2/ N0C1, N 2 0 3 , N 2 0 4 and N0 + .
  • 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, the invention provides for the nitrosylation of amino acids such as tyrosine and serine, to regulate cellular process such as, but not limited to, phosphorylation.
  • 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.
  • Other amino acids, such as serine and threonine may also be nitrosylated in the same manner.
  • 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, yocardial infarction, shock, renal failure, hepatorenal syndrome, post-coronary bypass, gastrointestinal disease, vasospasm of any organ bed, stroke or other neurological disease, and cancer.
  • 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.
  • alkyl nitrites having the formula X-CONO and containing a beta-election withdrawing group would be able to deliver these charged NO equivalents.
  • Another aspect 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.
  • 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.
  • additional compounds such as peptides, neurotransmitters, pharmacologic agents and other chemical compounds, as a therapeutic modality.
  • 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 0-agonist selectivity.
  • nitrosylated proteins and amino acids of the present invention 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.
  • administration may be by intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, rectal, oral, transdermal or buccal routes.
  • a "therapeuticaliy effective amount" of therapeutic composition is one which is sufficient to achieve a desired biological effect.
  • the dosage needed to provide an effective amount of the composition 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.
  • 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.
  • 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.
  • 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.
  • compositions 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.
  • 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.
  • 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, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.
  • 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, ⁇ tearic 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.
  • 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.
  • 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.
  • 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.
  • the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin.
  • stabilizers may be added.
  • 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.
  • gelatin rectal capsules which consist of a combination of the active compounds with a base.
  • Possible base materials include, for example, liquid triglycerides, polyethylene glycois, or paraffin hydrocarbons.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • 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.
  • the suspension may also contain stabilizers.
  • Preferred fatty acids generally contain from 4-36 carbon atoms and preferably contains at least 12 carbon atoms, most preferably 12 to 22. In some cases this carbon chain is fully saturated and unbranched, while others contain one or more double bonds. They can have saturated, unsaturated, branched or straight chain hydrocarbon chains. A few contain 3-carbon rings or hydroxyl groups. The compounds are not surface active. They are poorly soluble in water and the longer the acid chain and the fewer the double bonds, the lower the solubility in water. The carboxylic acid group is polar and ionized at neutral pH. This accounts for the slight solubility of short-chain acids in water.
  • Examples of such acids are those ranging from C, 6 to C 22 with up to three unsaturated bonds (also branching) .
  • Examples of saturated straight chain acids are n-dodecanoic acid, n-tetradecanoic acid, n-hexadecanoic acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, montanic acid and melissic acid.
  • unsaturated monoolefinic straight chain monocarboxylic acids examples of these are oleic acid, gadoleic acid and erucic acid.
  • Also useful are unsaturated (polyolefinic) straight chain monocaboxyic acids. Examples of these are linoleic acid, ricinoleic acid, linolenic acid, arachidonic acid and behenolic acid.
  • composition or preparation of the invention can further include a surfactant, or a mixture of two or more surfactants.
  • a surfactant is an amphiphilic molecule consisting of a hydrophobic tail and a hydrophilic head. These molecules possess distinct regions of both hydrophilic and hydrophobic character.
  • the hydrophobic tail can be a hydrocarbon or fluorocarbon chain of 8 to 18 carbon atoms. They are long chain molecules such as, for example, soaps or detergents.
  • Surfactants accumulate at the hydrophilic/ hydrophobic (water/oil) interface and lower the surface tension.
  • Surface active agents or surfactants are long chain molecules, such as soaps and detergents, which accumulate at the hydrophilic/hydrophobic(water/oil) interface and lower the surface tension at the interface.
  • the hydrophobic composition or other component of the preparation includes a surface-active agent, such as a surfactant, it is usually present in amounts of about 0.05% to 50.0% weight/weight of the hydrophobic composition with a preferred range of 1.0% to 3.0% (w/w) .
  • Preferred surfactants include, for example, the Tween(polyoxyethylene sorbate) family of surfactants(ICI, Wilmington DE), the Span(sorbitan long chain carboxylic acid esters) family of surfactants(ICI) , the Pluronic(ethylene or propylene oxide block copolymers) family of surfactants(BASF, Parsippany NJ), the Labrasol, Labrafil and Labrafac(each polyglycolyzed glycerides) families of surfactants(Gappe Fosse, St.
  • sorbitan esters of oleate, stearate, laurate or other long chain carboxylic acids poloxamers (polyethylene- polypropylene glycol block copolymers), other sorbitan or sucrose long chain carboxylic acid esters, mono and diglycerides, PEG derivatives of caprylic/capric triglycerides and mixtures thereof.
  • t-PA Nitrosylation of t-PA 1.
  • Materials t-PA was kindly provided by Genentech, Inc. San Francisco, CA.
  • 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 antimurine antibodies were purchased from Amersham Corp., Arlington, II.
  • Sodium nitrite was purchased from Fisher Scientific, Fairlawn, NJ.
  • 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 Vitru , Sweden. Human fibrinogen purified of plasminogen and von Willebrand factor, was obtained from Enzyme Research Laboratories, South Bend, IN. Epinephrine, ADP and iodoacetamide were purchased from Sigma Chemical Co., St. Louis, MO. Bovine thrombin was obtained from ICN, immunoBiologicals (Lisle, IL) . Radioimmunoassay kits for the determination of cGMP were purchased from New England Nuclear, Boston, MA.
  • Glu-plasminogen was purified from fresh frozen plasma thawed at 37°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.
  • t-PA 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°C in 10 mM Tris, pH 7.4, 0.15 M NaCl (TBS). t-PA was then dialyzed extensively against 10 mM HCI in order to remove excess iodoacetamide.
  • Microcarrier Endothelial Cell Culture 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).
  • Nitrosylation t-PA was first dialyzed against a large excess of 10 mM HCI for 24 hours to remove excess L-arginine used to solubilize the protein. t-PA was then exposed to NO. generated from equimolar NaN0 2 in 0.5 N HCI (acidified NaN0 2 ) or in control experiments, to 0.5 N HCI alone, for 30 minutes at 37°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.
  • TBS Tris Buffered Saline
  • 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 NOC1, N 2 0 3 , N 2 0 4 , and other nitroso-equivalents.
  • [ IS N] 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).
  • [ I5 N]NMR spectra were recorded with a Brucker 500 MHZ spectrometer, Billerica, MA. Deuterium lock was effected with [D] 2 0 and the spectra referenced to an [ l5 N] natural abundance spectrum of a saturated solution of NaN0 2 at 587 ppm. Spectra were recorded at 50.68 MHZ and the nine transients of 16k data points collected with a 30° pulse width and a 10-second relaxation delay. Data were multiplied by a 2-Hz exponential line broadening factor before Fourier transformation.
  • S-NO-t-PA exhibited an absorption maximum at 322 nm ( Figure la), and a chemical shift at 751 ppm (relative to nitrite) (Figure lb); elimination of the chemical shift was achieved by sample treatment with excess HgCl 2 .
  • the presence of two absorption bands at 1153 cm -1 and 1167 cm 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 el al .
  • Figure 2 illustrates the time-dependent formation of S-NO-t-PA.
  • Figure 2 also illustrates the effect of acid treatment on the amidolytic activity of t-PA.
  • the duration of exposure subsequently used for S-nitrosodiiol synthesis the enzymatic activity of t-PA is largely preserved.
  • Quantification of S-NO-t-PA ⁇ ynthe ⁇ i ⁇ with authentic EDRF was similarly determined by the method of Saville (Saville, B. Analyst 83:670-672 (1958)).
  • nitrosylation of BSA was accomplished by incubating BSA (200 mg/ml with NO generated from equimolar NaN0 2 in 0.5N HCI (acidified NaN0 2 ) for thirty minutes at room temperature. 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.
  • 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.
  • proteins were incubated with bovine aortic endothelial cells stimulated by exposure to high shear forces to secrete EDRF, as in Example 1(A).
  • proteins were also incubated directly with NO synthase purified from bovine cerebellum (Bredt et al . , Proc . et al . , Proc . Natl . Acad. Sci . USA 87:682 (1990), herein incorporated by reference) in the presence of the substrate Larginine and cofactors required for enzyme activity (Ca-', calmodulin, and NADPH) .
  • Bovine Serum Albumin 0.85 ⁇ 0.04 t-PA 0.88 ⁇ 0.06
  • LDL Lipoprotein
  • t-PA and S-NO-t-PA were measured using polystyrene microliter wells (flat-bottom, high binding 96-well EIA plates, cat. #3590, Costar, Cambridge, MA). Wells were coated with fibrinogen (0.08 ⁇ g/ ⁇ l) 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 0-phenylenediamine, 0.014% H 2 0 2 . Color change was measured spectrophotometrically with a Dynatech MR500 Card Reader (Dynatech, Chantilly, VA) at 490 nm.
  • Dynatech MR500 Card Reader Dynatech MR500 Card Reader
  • 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 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.
  • 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 n with a Gilford Response UV/Vis Spectrophotometer (CIBA-Corning, Oberlin, OH). Activity was measured at 25°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.
  • t-PA and S-NO-t-PA activities were assayed using the native substrate S2251.
  • fibrinogen stimulation of enzymatic activity was assessed at a fibrinogen concentration of 1 mg/ml.
  • Substrate hydrolysis was mea ⁇ ured spechtrophotometrically with a Dynatech MR 5000 Card Reader (Dynatech, Chantilly, VA) in TBS, pH 7.4, at 25°C.
  • Initial reaction velocity was determined from the slope of the plot of absorbance (at 405 nm)/time vs. time (Ranby, M. , Biochem. Biophysic Acta 704:461-469 (1982)).
  • S-NO-t-PA has a K,- for sub ⁇ trate ⁇ imilar to "wild type" t-PA.
  • S-NO-t-PA has a ⁇ lightly, but significantly, greater v tn - yielding a catalytic efficiency that is 23% greater than that of native t-PA.
  • 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)).
  • 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)).
  • PAI-1 By serving as a pseudosubstrate, PAI-1 reacts ⁇ toichiometrically 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.
  • Aggregations were quantified by measuring the maximal rate or extent of light transmittance and expressed as a normalized value relative to control aggregations.
  • 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 ⁇ M), and related controls. Reactions were terminated by the addition of 10% trichloracetic acid. Acetylation of sample ⁇ with acetic anhydride wa ⁇ used to increase the sensitivity of the assay.
  • S-NO-t-PA was then preincubated with platelets for ten minutes prior to induction of aggregation with 5 ⁇ M ADP.
  • effluent from endothelial cells stimulated to secrete EDRF had no significant effect on platelet aggregation.
  • 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 cold physiologic salt solution (Kreb's) (mM): NaCl, 118; CK1, 4.7; CaCl 2 , 2.5; MgS0 4 , 1.2; KH 2 P0 4 , 1.2; NaHCO j , 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 FT03C Grass Instruments, Quincy, MA) by which changes in isometric tension were recorded.
  • S-NO-t-PA As shown in the illustrative tracings of Figure 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.
  • S-nitroso-BSA 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/ ⁇ l in HEPES buffer, pH 7.35.
  • S-NO-BSA was incubated with platelets for ten minutes at 37°C in a PAP-4 aggregometer (BioData, Hatboro, PA), after which aggregations were induced with 5 ⁇ M ADP. Aggregations were quantified by measuring the extent of change of light transmittance and expressed as a normalized value relative to control aggregations.
  • S-nitroso-protein ⁇ ⁇ ynthesized with acidified NaN0 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 Figure 10.
  • 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.
  • S-nitroso-BSA induced a les ⁇ rapid, but much more persistent, relaxation response (Figure lib), thus confirming that it acts as a long-acting vasodilator.
  • BSA incubated with NO synthase in the presence of cofactors required for enzyme activity showed an L-arginine-dependent ability to induce persistent vasorelaxation characteristic of S-nitroso-proteins.
  • 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 liability of the S-NO bond.
  • 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 (Figure lib). Consonant with the mechanism of other nitro(so)-vasodilators (Ignarro, L.J.
  • Figure 10 demonstrates the dose-dependent relaxation of vascular smooth muscle and inhibition of platelet aggregation with S-nitroso-BSA (S-NO-BSA).
  • S-NO-BSA S-nitroso-BSA
  • the open symbols represent experiments, in the vessel (Q) and platelet (O) bioassays, in which S-NO-BSA was synthesized by exposure of BSA to bovine aortic endothelial cells stimulated to secrete EDRF.
  • S-NO-cathepsin 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 Figure 12, S-NO-cathepsin, at a concentration of 150 nM - 1.5 ⁇ M induces vessel relaxation which is ummatched by equimolar amounts of non-nitrosylated cathepsin. E «ffir ⁇ pl-r ?
  • S-NO-LDL 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 Figure 13, inhibition of platelet aggregation is demonstrable at a concentration of 1 ⁇ M S-NO-LDL.
  • S-NO-LDL The effect of S-NO-LDL on vasodilation was studied according to the methods described in Example 7b. As shown in Figure 14, S-NO-LDL induces vessel relaxation which is unmatched by equimolar amounts of non-nitrosylated LDL.
  • S-NO-Ig The effect of S-NO-Ig on platelet aggregation was studied according to the method ⁇ 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 Figure 15, inhibition of platelet aggregation is demonstrable at concentrations of S-NO-Ig in the pharmacologic range of 150 nM - 1.5 ⁇ M.
  • S-NO-Ig The effect of S-NO-Ig on vasodilation was studied according to the methods described in Example 7b. As shown in Figure 15, S-NO-Ig, at concentrations in the range of 150 nM - 1.5 ⁇ M, induces relaxation which is unmatched by equimolar amounts of immunoglobulin alone. Example 11 Relaxation of Airway Smooth Muscle By S-Nitroso-BSA
  • 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, WI.
  • Captopril was kindly provided by Dr. Victor Dzau.
  • Sodium nitrite, histamine and methacholine were purchased from Fisher Scientific, Fairlawn, N.J. Leukotriene D. was purchased from Anaquest, BOC Inc., Madi ⁇ on, WI.
  • 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, NY. Radioimmunoassay kits for the determination of cyclic GMP were purchased from New England Nuclear, Boston, MA.
  • the trachea rings were, placed in sterile Medium 199 containing 1% antibiotic/antimycotic mixture in an atmosphere of 5% C0 2 , 45% 0 2 , 55% N 2 and kept for up to 48 hours in tissue culture.
  • the experiments were also performed on human airways isolated by the same method.
  • Trachea rings were mounted on stirrups and connected to transducers (Model FT03C Grass), by which changes in isometric tension were measured. Rings were then suspended in 10 cc of oxygenated (95% 0 2 , 5 % C0 2 ) buffer. Airway rings were equilibrated for 60 minutes under a load of 1 gm and then primed twice by exposure to 100 ⁇ 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.
  • S-NO-BSA is a potent airway smooth muscle relaxant, producing 50% relaxation at a concentration of 0.01 ⁇ M and over 75% reaxation at a concentration of 10 ⁇ M.
  • 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.
  • L-tyrosine 50 mmol of L-tyrosine (Sigma Chemical Company; St. Louis, MO) were dissolved into 0.5 ml of distilled water. 250 mmol of Na 15 N0 2 (sodium N-[15]nitrite: MSD Isotopes, Merck Scientific; Rahway, NJ) were dissolved into 0.5 mL of 1 N HCI (Fisher Scientific; Fair Lawn, NJ) and transferred immediately to the aqueous tyrosine solution with agitation by Vortex stirrer. The solution wa ⁇ capped and allowed to sit at room temperature for 30 minutes.
  • Infrared Spectroscopy Fourier Transform Infrared Spectroscopy (FTIR) samples were prepared through removal of water (as in (b)) and subsequent creation of a Nujol Mull using mineral oil.
  • FTIR Fourier Transform Infrared Spectroscopy
  • UV-Vis Ultraviolet and Visible Spectroscopy
  • Nitrosylation of phenylalanine also yielded signals at 587 ppm (excess, unprotonated nitrite) and 353 ppm.
  • Nitrosylation of 0-blocked tyrosine model, boc-tyr(Et)-OH also yielded a signal at approximately 630 ppm; and other ⁇ at 587 ppm and 353 ppm.
  • Small signals in the range 450-495 ppm were observed for the tyrosine models, phe and boc-tyr(E+)-OH.
  • Figure 23(a-e) demonstrates time-dependent nitrosylation of tryptophan. The data is suggestive of nitrosylation of both the aromatic ring and amino groups.
  • BSA at 200 mg/ml, was loaded at a ratio of 20:1 with NO in 0.5 N HCI for 30 minutes at room temperature.
  • the 726 ppm peak indicates O-nitrosation of the tyrosine residues on BSA.
  • Figure 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.
  • 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.
  • Figure 25 provides UV spectrum data which indicates NO loading of BSA.
  • Example 15 Nitrosylation of t-PA: NO Loading t-PA at 10 mg/ml was exposed 10:1 to excess NaN0 2 in 0.5 N HCI.
  • Figure 26 shows NO-loading of t-PA.
  • Example 14 Vasodilatory effect was studied in a rabbit aorta bioassay, according to the methods described in Example 6C. As shown in Figure 27, increasing concentrations of NO resulted in an increase in vessel relaxation induced by the resultant NO-BSA.
  • S-nitroso-albumin 100 ⁇ M
  • S-nitroso-albumin-induced airway relaxation was not significantly inhibited by methylene blue (10"*) or LY83583 (5 x 10 s ).
  • S-nitroso-albumin The stability of S-nitroso-albumin in the presence of oxygen and redox metals was assessed. When subjected to conditions consisting of 95% 0 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 N0 2 , which, under similar conditions, are on the order of second ⁇ .
  • 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 + , Fe 2 , or Cu 2+ (50 ⁇ M) or defuroxamine or EDTA (10 ⁇ 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.
  • S-nitrosylation of hemoglobin was accomplished by reacting 12.5 ⁇ M hemoglobin with 12.5 ⁇ M acidified NaN0 2 or an RS-NO compound such as S-nitro ⁇ o-N-acetylcy ⁇ teine, S-nitroso- glutathione or S-nitroso-cysteine 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.
  • the UV spectrum of hemoglobin incubated with SNOAC shows no reaction at the redox metal (iron-binding site) of hemoglobin, over 15 minutes.
  • equimolar concentrations of hemoglobin and NaN0 2 were reacted in 0.5 N HCI, to form nitrosyl-hemoglobin, and the UV spectrum was obtained.
  • NO reacted instantaneously with the redox metal site on hemoglobin.
  • Hemoglobin is S-nitrosated by incubation with an alkylnitrite (e.g., amyl nitrite or tert-butyl nitrite) or a thionitrite (e.g., S-nitroso-glutathione, S-nitroso- penicillamine, S-nitroso-cy ⁇ teine and S-nitroso-N- acetylcy ⁇ teine) for period ⁇ of one minute to one hour.
  • the pH is optimized within the range of 6.5 to 7.5 to achieve stoichiometric S-nitrosylation.
  • the reactions are preferably performed anaerobically. It may also be desired to perform the above procedure in saturating carbon monoxide solutions and under increased atmospheric pressures (i.e., 5 atmospheres) to prevent interaction of NO with the heme site.
  • Figure 31 shows the UV spectrum of S-NO-hemoglobin (Hb).
  • the spectra show that oxygen binding to the heme site is largely unaffected at S-NO/Hb stoichiometries (0.05 and 0.37) that are lacking in smooth muscle contractile activity. Higher ratios of S-NO/Hb (1.59) are associated with methemoglobin formation. Curves for oxy-Fe(II)-hemoglobin (dithionite-treated) and methemoglobin (K 3 Fe(CN)-treated) are shown for comparison.
  • Synthesis of S-NO-hemoglobin is monitored by three complementary methods, two of which were developed in this laboratory: photolysis-chemiluminescence and capillary electrophoresis methodologies.
  • the ligand binding to heme is determined spectrophotometrically, using published extinction coefficients.
  • the structure of S-nitrosylated hemoglobin is then further characterized using SDS, polyacrylamide gel electrophoresis and isoelectric focusing immunoelectrophoresis.
  • the photolysis-chemiluminescence technique can be used to measure both protein and low molecular weight RS-NO.
  • the sample protein is introduced directly or as a chromatographic effluent from an attached FPLC/HPLC (for separation and identification of protein and amino acid RS- NO, respectively) into a photolysis cell where it is irradiated with a 200 watt mercury vapor lamp, designed to result in complete photolysis of the S-N bond. NO is then carried in a stream of helium towards the reaction chamber which yields the chemiluminescence ⁇ ignal.
  • HgCl 2 which selectively displaces NO from thiol groups. We have confirmed that metal-nitrosyl complexes are not affected by this treatment.
  • the capillary electrophoresis method (U.S. Patent No. 5,346,599) offer the added advantage of being able to separate hemoglobin variants as well as various RS-NO from their respective thiols and disulfide ⁇ .
  • Figure 32 shows reversal of hemoglobin (Hb)-induced contraction of aortic rings by S-nitrosylation.
  • Hemoglobin is shown to constrict vessels in a dose-dependent manner (•) .
  • S-NO-Hb with a stoichiometry of 0.1 S-NO ⁇ Hb, entirely prevents the constrictor response (O) .
  • Increasing the S-NO- stoichiometry to 1 converts hemoglobin into a potent vasodilator ( H ⁇ ) .
  • H ⁇ potent vasodilator
  • hemoglobin based blood substitutes involve an , understanding of oxygen binding capability.
  • oxygen affinity i.e., 2, 3, DPG, pH and Co 2
  • alkylation of the 093 SH group(s) of hemoglobin shifts the 0 2 hemoglobin association curve leftward.
  • Measurement of highly precise oxygen binding curves is performed using standard tonometry, an Imai cell and/or a thin-layer Gill cell modified with an oxygen electrode.
  • a thin layer dual-beam method is used for studies of oxygen binding by intact red blood cells, with or without added cell-free hemoglobins.
  • a modified Hemoscan American Instruments Co. ) operated in a discontinuous mode to avoid dynamic error.
  • Rabbits are routinely used in the laboratory for assessment of hemodynamic responses to pharmacological agents.
  • New Zealand white rabbits were anesthetized with ketamine (50 mg/kg i.m.) and sodium barbital (5-10 mg/kg i.v.) after which the femoral artery was cannulated to allow continuous measurement of blood pres ⁇ ure. Mean arterial pressure was then measured in response to intravenous bolus injections of S-NO hemoglobin (1 nmol/kg/min to 1 ⁇ m/kg/min) as well as continuous infusions.
  • the kinetics of S-NO-hemoglobin decomposition in vivo was measured by photolysis-chemiluminescence, as previously described. Blood samples, anticoagulated with 0.13 M trisodium citrate, were obtained for analysis at 5 minutes intervals after injection. The red cells were lysed in deionized water, passed across a G25 column at 4°C to remove glutathione, and the S-NO-Hb content was then measured directly and is shown in Figure 33.
  • Figure 33 shows endogenous levels of S-NO-hemoglobin in rat artery, rat vein and human vein.
  • Rat arterial blood was obtained by direct cardiac puncture, rat venous blood from the jugular vein and human blood by venipuncture, according to approved protocols.

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Abstract

La présente invention décrit des substituts sanguins qui sont directement ou indirectement liés à un groupe NO ou NO2, tout particulièrement à des protéines contenant des hèmes. L'invention décrit également des compositions pharmaceutiques comprenant lesdits substituts et des porteurs pharmaceutiques ainsi que leur utilisation en tant qu'agents thérapeutiques.
PCT/US1996/003866 1995-03-24 1996-03-25 Substituts sanguins nitrosyles WO1996030006A1 (fr)

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US6124255A (en) * 1997-01-17 2000-09-26 Baxter Aktiengesellschaft Preparation comprising thiol-group-containing proteins
US6153186A (en) * 1995-09-15 2000-11-28 Duke University Medical Center Red blood cells loaded with S-nitrosothiol and uses therefor
US6197745B1 (en) 1995-09-15 2001-03-06 Duke University Methods for producing nitrosated hemoglobins and therapeutic uses therefor
EP1029551A3 (fr) * 1999-02-08 2001-03-07 Hokkaido University Complexe d'un metabolite de monoxyde d'azote - polyoxyalkylène-hemoglobine
US6627738B2 (en) 1995-09-15 2003-09-30 Duke University No-modified hemoglobins and uses therefor
US6855691B1 (en) 1995-09-15 2005-02-15 Duke University Methods for producing and using S-nitrosohemoglobins
US6911427B1 (en) 1995-09-15 2005-06-28 Duke University No-modified hemoglobins and uses therefore
US6916471B2 (en) 1995-09-15 2005-07-12 Duke University Red blood cells loaded with S-nitrosothiol and uses therefor
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AU2003295697B2 (en) * 2002-12-12 2009-12-24 Duke University Forming iron nitrosyl hemoglobin
US8551536B2 (en) 2007-08-31 2013-10-08 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Nitrite and nitrite-metheme therapy to detoxify stroma-free hemoglobin based blood substitutes
US8927030B2 (en) 2003-07-09 2015-01-06 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of nitrite salts for the treatment of cardiovascular conditions

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US6884773B1 (en) 1995-09-15 2005-04-26 Duke University Modified hemoglobins, including nitrosylhemoglobins, and uses thereof
US7538193B2 (en) 1995-09-15 2009-05-26 Duke University NO-modified hemoglobins and uses therefor
US6153186A (en) * 1995-09-15 2000-11-28 Duke University Medical Center Red blood cells loaded with S-nitrosothiol and uses therefor
US6197745B1 (en) 1995-09-15 2001-03-06 Duke University Methods for producing nitrosated hemoglobins and therapeutic uses therefor
US7202340B2 (en) 1995-09-15 2007-04-10 Duke University No-modified hemoglobins and uses therefor
US6203789B1 (en) 1995-09-15 2001-03-20 Duke University Red blood cells loaded with S-nitrosothiol and uses therefor
US6916471B2 (en) 1995-09-15 2005-07-12 Duke University Red blood cells loaded with S-nitrosothiol and uses therefor
US6627738B2 (en) 1995-09-15 2003-09-30 Duke University No-modified hemoglobins and uses therefor
US6911427B1 (en) 1995-09-15 2005-06-28 Duke University No-modified hemoglobins and uses therefore
US6855691B1 (en) 1995-09-15 2005-02-15 Duke University Methods for producing and using S-nitrosohemoglobins
US6358918B1 (en) 1997-01-17 2002-03-19 Baxter Aktiengesellschaft Preparation comprising thiol-group-containing proteins
US6124255A (en) * 1997-01-17 2000-09-26 Baxter Aktiengesellschaft Preparation comprising thiol-group-containing proteins
WO1998034955A1 (fr) * 1997-02-06 1998-08-13 Duke University Medical Center Hemoglobines no-modifiees et leurs utilisations
US6667292B1 (en) * 1999-02-08 2003-12-23 Hokkaido University Nitric monoxide metabolite-polyoxyalkylene-hemoglobin complex
EP1029551A3 (fr) * 1999-02-08 2001-03-07 Hokkaido University Complexe d'un metabolite de monoxyde d'azote - polyoxyalkylène-hemoglobine
AU2003295697B2 (en) * 2002-12-12 2009-12-24 Duke University Forming iron nitrosyl hemoglobin
US8075922B2 (en) * 2002-12-12 2011-12-13 Montana State University-Bozeman Method of co-infusing a deoxygenated hemoglobin containing blood substitute and inorganic nitrite
US8927030B2 (en) 2003-07-09 2015-01-06 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of nitrite salts for the treatment of cardiovascular conditions
US9387224B2 (en) 2003-07-09 2016-07-12 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Treatment of specific cardiovascular conditions with nitrite
US9675637B2 (en) 2003-07-09 2017-06-13 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of nitrite salts for the treatment of cardiovascular conditions
US9700578B2 (en) 2003-07-09 2017-07-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of nitrite salts for the treatment of cardiovascular conditions
US7166577B2 (en) 2003-12-26 2007-01-23 Nipro Corporation Albumin having enhanced antimicrobial activity
WO2006096774A3 (fr) * 2005-03-07 2007-04-12 Sangart Inc Composition et methodes de diffusion de monoxyde de carbone (co) et de monoxyde d'azote (no) dans un tissu en utilisant des proteines heme comme supports
US9138464B2 (en) 2005-03-07 2015-09-22 Robert M. Winslow MalPEG Hb conjugate-containing compositions for delivering nitric oxide (NO) to cells
US9138463B2 (en) 2005-03-07 2015-09-22 Robert M. Winslow MalPEG-Hb conjugate-containing compositions for delivering carbon monoxide (CO) to cells
US8551536B2 (en) 2007-08-31 2013-10-08 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Nitrite and nitrite-metheme therapy to detoxify stroma-free hemoglobin based blood substitutes

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