Use of nitric oxide scavengers to modulate inflammation and matrix metalloproteinase activity

TECHNICAL FIELD

[0002] The present invention relates to modulating inflammation, or improving hemodynamic function, associated with trauma caused by surgery, through the administration of metal complexes, such as ruthenium nitric oxide scavengers. Further, the present invention relates to modulating matrix metalloproteinase activity also by administering such metal complexes. Modulation of the surgical trauma or the metalloproteinase activity reduces tissue damage or causes a protective effect against organ damage.

BACKGROUND OF THE INVENTION

[0003] Coronary artery bypass grafting (CABG) is an invasive surgical procedure undertaken to redirect or “bypass” blood around a discrete point(s) within a clogged artery supplying the heart. These arteries can become clogged through the build-up of fat, cholesterol, or other substances, leading to a narrowing or blockage referred to as an atherosclerotic plaque. CABG is a frequently performed operation undertaken throughout the world, with an estimated 800,000 cardiac surgeries worldwide reported in 1997 (American College of Cardiology).

[0004] Without CABG surgery, atherosclerosis can impede the flow of blood through the vessels supplying the heart, resulting in myocardial infarcts or heart attacks. During the operation, healthy segments of blood vessels (both arteries and veins) from other regions of the body, for example the saphenous veins from the leg, are used in this operation. For a majority of the CABG surgery performed, a heart-lung machine is used, causing blood to be exposed to an extra-corporeal (outside of the body) circuit. The surgical procedure involves grafting the healthy blood vessels onto the coronary vessel before the point of damage and re-directing the flow of blood to other regions of the heart and circulatory system.

[0005] Due to the invasiveness of the pulmonary bypass procedure, CABG is associated with inducing systemic inflammation, by releasing a host of inflammatory mediators from macrophages, including interleukin-6 (IL-6) and tumor necrosis factor (TNF) (Lahat et al. Clin. Exp. Immunol. 89:255-260, 1992), where both IL-6 and TNF in turn may induce the inducible form of nitric oxide synthase (iNOS) activity, one of a family of enzymes responsible for the production of nitric oxide (Cunha et al. Immunology. 81:211-215, 1994); and matrix metalloproteinases (MMPs), including MMP-2 and MMP-9. The inducible iNOS class of enzyme produces nitric oxide (NO) in high amounts and at a much higher rate. More importantly, elevated levels of nitric oxide can be cytotoxic and will breakdown in the presence of oxygen radicals to form the toxic peroxynitrite (ONOO−) radical. There is evidence suggesting that tissue and organ damage, particularly to the brain, lung, and heart, are associated with CABG surgery and are the result of inflammatory processes leading to the production of nitric oxide and its toxic metabolites.

[0006] Furthermore, MMPs are known to play a role in tissue re-modeling after a cardiac bypass surgery, and are also implicated in inflammatory diseases, such as rheumatoid arthritis, are a causative contributory factor in joint erosion, and cancer where they play a role in metastasis. Thus, by inhibiting iNOS and MMP activity and/or removing excess concentration of nitric oxide, organ damage, i.e., to the brain, lung, and heart, which are complications associated with the systemic inflammatory response following CABG, could be prevented.

[0007] There exists a need for a treatment or prevention of complications described above, and the present invention addresses these needs by providing such methods by administering metal complex nitric oxide scavenger compounds to patients, thereby affording organ protection following CABG and following inflammatory challenges, e.g., ischemia reperfusion injury, surgical trauma, and circulatory shock.

SUMMARY OF THE INVENTION

[0008] The invention provides a method of modulating inflammation associated with trauma, such as caused by surgery, which comprises administering to a patient in need of such modulation an effective amount of a neutral, anionic, or cationic metal complex of Formula I

[Ma(XbL)cYdZe]n± Formula I,

[0009] where:

[0010] M is a ruthenium ion;

[0011] X is cation or a mixture of cations;

[0012] L is a ligand, or mixture of ligands each containing at least two different donor atoms selected from nitrogen, oxygen and sulphur donor atoms;

[0013] Y is a ligand, or a mixture of the same or different ligands each containing at least one donor atom, which donor atom is selected from nitrogen, oxygen, sulphur, carbon, and phosphorus, provided that the ligand is not a sulphoxide group, 1,10-phenanthroline or substituted 1,10-phenanthroline, substituted 2,2-bypyridine or dihydrophenazine; and

[0014] Z is a halide or pseudohalide ion or a mixture of halide ions and pseudohalide ions;

[0015] a=1-3; b=0-12; c=0-18; d=0-18; e=0-18; and n=1-10;

[0016] provided that at least one of c, d and e is 1 or more;

[0017] and where c is 0; b is also 0;

[0018] and where a is 1; c, d and e are not greater than 9 in total;

[0019] and where a is 3; c, d and e are not greater than 18 in total;

[0020] and where d is 3, Y is not a basic heterocyclic compound containing at least one nitrogen atom.

[0021] Also provided is a method of modulating matrix metalloproteinase activity which comprises administering to a patient in need of such modulation an effective amount of a neutral, anionic, or cationic metal complex of the Formula I

[Ma(XbL)cYdZe]n± Formula I,

[0022] where:

[0023] M is a ruthenium ion;

[0024] X is cation or a mixture of cations;

[0025] L is a ligand, or mixture of ligands each containing at least two different donor atoms selected from nitrogen, oxygen and sulphur donor atoms;

[0026] Y is a ligand, or a mixture of the same or different ligands each containing at least one donor atom, which donor atom is selected from nitrogen, oxygen, sulphur, carbon, and phosphorus, provided that the ligand is not a sulphoxide group, 1,10-phenanthroline or substituted 1,10-phenanthroline, substituted 2,2-bypyridine or dihydrophenazine; and

[0027] Z is a halide or pseudohalide ion or a mixture of halide ions and pseudohalide ions;

[0028] a=1-3; b=0-12; c=0-18; d=0-18; e=0-18; and n=1-10;

[0029] provided that at least one of c, d and e is 1 or more;

[0030] and where c is 0; b is also 0;

[0031] and where a is 1; c, d and e are not greater than 9 in total;

[0032] and where a is 3; c, d and e are not greater than 18 in total;

[0033] and where d is 3, Y is not a basic heterocyclic compound containing at least one nitrogen atom.

[0034] Further provided by this invention is a method of improving vascular or haemodynamic function associated with trauma which comprises administering to a patient in need of such improvement an effective amount of a neutral, anionic, or cationic metal complex of Formula I

[Ma(XbL)cYdZe]n± Formula I,

[0035] where:

[0036] M is a ruthenium ion;

[0037] X is cation or a mixture of cations;

[0038] L is a ligand, or mixture of ligands each containing at least two different donor atoms selected from nitrogen, oxygen and sulphur donor atoms;

[0039] Y is a ligand, or a mixture of the same or different ligands each containing at least one donor atom, which donor atom is selected from nitrogen, oxygen, sulphur, carbon, and phosphorus, provided that the ligand is not a sulphoxide group, 1,10-phenanthroline or substituted 1,10-phenanthroline, substituted 2,2-bypyridine or dihydrophenazine; and

[0040] Z is a halide or pseudohalide ion or a mixture of halide ions and pseudohalide ions;

[0041] a=1-3; b=0-12; c=0-18; d=0-18; e=0-18; and n=1-10;

[0042] provided that at least one of c, d and e is 1 or more;

[0043] and where c is 0; b is also 0;

[0044] and where a is 1; c, d and e are not greater than 9 in total;

[0045] and where a is 3; c, d and e are not greater than 18 in total;

[0046] and where d is 3, Y is not a basic heterocyclic compound containing at least one nitrogen atom.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047]
FIG. 1. Illustrates the protocol followed by four groups of dogs. Animals were randomized to receive cardiopulmonary surgery (CPB-PLAC or CPB-SCV) or to serve as controls (CTRL-PLAC or CTRL-SCV). (CPB=cardiac pulmonary bypass; PLAC=placebo; SCV=scavenger; CTRL=control.)

[0048]

FIGS. 2 and 3

a
and b. Illustrate mean±SD (standard deviation) values of Ca2+-dependent and Ca2+-independent NOS activity. Values are shown for left atrium, left ventricle. Lung and brain. * represents a significant difference (p<0.05) between Bypass (black bars) and Control (white bars) groups. Note that Ca2+-independent NOS activity is markedly increased in the Bypass group.

[0049]

FIGS. 4 and 5

a
and b. Illustrate mean±SD values of matrix metalloprotease MMP activity. Values are shown for left atrium, left ventricle, lung, and brain. *represents a significant difference (p<0.05) between Bypass (black bars) and Control (white bars) groups.

DISCLOSURE OF THE INVENTION

[0050] Nitric oxide plays a varied and vital role in the human body. For example, NO plays a vital role in the control of blood pressure: it acts as a neurotransmitter; and it plays a role in inhibition of platelet aggregation (important in thrombosis or blockages of the blood vessels) and in cytostasis (important in fighting of tumors). Overproduction of NO however, has been implicated in a number of disease states, including vascular/pressor diseases, such as septic shock, post-ischemic cerebral damage, migraine and dialysis induced renal hypotension; immunopathologic diseases, such as hepatic damage in inflammation and sepsis allograft rejection, graft versus host diseases, diabetes and wound healing; neurodegenerative diseases such as cerebral ischemia, trauma, chronic epilepsy, Alzheimer's disease, Huntington's disease, and AIDS dementia complex; and side effects of treatment such as restenosis following angioplastic treatment and secondary hypotension following cytokine therapy. Pharmacological modulation of nitric oxide or other reactive oxygen species in any of these disease states is beneficial.

[0051] One above-mentioned disease relating to overproduction of NO is septic shock. This is precipitated by local septicemia or endotoxaemia, (high local levels of bacterial endotoxins). The result is activation of macrophages, lymphocytes, endothelial cells and other cell types capable of producing NO further mediated by cytokine production by these cells. The activated macrophages produce excess NO which causes vasodilatation of the blood vessels, and results in local vascular damage and vascular collapse. This destruction of vascular integrity may be so great that it leads to the collapse of hemodynamic homeostasis, the end result being death.

[0052] Current ideas for pharmacological modulation of nitric oxide in such diseases are based on dealing with the mediators of septic shock such as cytokines, endotoxins and platelet activating factor (PAF). The approaches include use of antibodies to cytokines such as tumour necrosis factor (TNF) receptor antagonists such as interleukin I (IL-1) antibodies to lipopolysaccharide (the endotoxins produced by Gram negative bacteria) and PAF antagonists. All such approaches while challenging a factor mediating septic shock do not attempt to deal with the etiology or cause of the disease. Recent advances in understanding of NO have lead to the proposal that inhibitors of the NO synthase enzyme such as NG-monomethy-L-arginine (L-NMMA) may be useful in the treatment of septic shock and other NO overproduction related to diseases since they inhibit NO production. While these inhibitors have shown some utility in animal models and preliminary clinical studies they have the disadvantage of undesirably inhibiting total NO synthesis in the body.

[0053] The present invention provides methods for modulating inflammation associated with trauma comprising administering to a patient in need of such modulation an effective amount of a neutral, anionic or cationic metal complex of Formula I. Typically, the inflammation is the result of organ or tissue trauma associated with surgery, such as cardiac bypass surgery, cardiac valve surgery, chronic congestive heart failure, heart transplantation, ischemic reperfusion, angioplasty and the like.

[0054] Modulation of the inflammation reduces the tissue damage and/or causes a protective effect against the impairment of an organ function, wherein the organ is the brain, lung, or cardiac muscle. CABG negatively affects cognitive function, including short- and long-term cognitive decline, as well as reduced levels of overall cognitive functioning.

[0055] A moderate degree of lung dysfunction is common after CABG surgery, which may contribute to morbidity and mortality. The inflammatory response induced by cardiac surgery leads to activation of complement and influx of granulocytes into the pulmonary vasculature, with increased adherence of neutrophils. Modulation of the inflammatory response by scavenging NO may ameliorate this lung dysfunction.

[0056] Following CABG surgery there is a reversible diastolic dysfunction or a reversible depression of systolic function. Nitric oxide has been implicated in cardiac impairment following reperfusion injury, an event occurring post surgery. Increased NO production and interaction with Reactive Oxygen Species (ROS) lead to peroxynitrite production (ONOO−). Scavenging of NO could therefore attenuate the production of peroxynitrite. Also NO can depress cardiac muscle contractility thus impairing cardiac function. Increased MMP activity has also been associated with a decrease in cardiac function. The modulation of MMP activity therefore could improve cardiac function after CABG surgery. Scavenging of NO could therefore reduce cardiac dysfunction seen after CABG surgery.

[0057] Also provided by the present invention are methods for modulating MMPs utilizing a metal complex of Formula I. MMPs are a family of zinc-containing enzymes that are involved in the remodeling and degradation of extracellular matrix proteins. (See A. Zask, et al., Current Pharmaceutical Design, (1996), 2, 624-661.) The natural substrates for MMPs are a family of proteins, including collagen, gelatin, laminin, fibronectin and elastin, located in the extracellular matrix (ECM). (See D. E. Levy, et al., “Chapter Ten: Matrix Metalloproteinase Inhibitor Drugs”, Emerging Drugs, (1997), 2, 205-230; and D. Celentano, et al., J. Clin. Pharmacol., (1997), 37, 991-1000.)

[0058] The aberrant regulation of MMP production has been implicated in pathological tissue breakdown in numerous disease processes. Increased levels of MMPs are associated with cardiovascular diseases, including, but not limited to, smooth muscle cell migration in restenosis, and plaque formation and rupture in atherosclerosis. MMPs are implicated in angiogenesis, vascular diseases, autoimmune diseases, such as multiple sclerosis (MS), and the like, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), osteoporosis, renal disease, tissue ulceration, retinopathy, corneal ulceration, psoriasis, periodontal disease, abnormal wound healing, rupture of fetal membranes, tumor growth, and tumor metastasis.

[0059] Additional inflammatory diseases associated with MMPs include, but are not limited to, systemic lupus erythematosis, acute transplantation/graft rejection, myasthenia gravis, progressive systemic sclerosis, multiple myeloma, atopic dermatitis, hyperimmunoglobin E, hepatitis B antigen, negative chronic active hepatitis, Hashimoto's thyroiditis, Familial Mediterranean fever, Grave's disease, autoimmune hemolytic anemia, and primary biliary cirrhosis.

[0060] MMPs have similar domain structures with four major regions: an N-terminal leader sequence involved in secretion, a prodomain that inhibits the enzymatic activity, a catalytic domain, a hemopexin domain involved in substrate specificity and enzyme activity. MMP subtypes include: MMP-1, MMP-8, MMP-13, MMP-7, MMP-3, MMP-10, MMP-11, MMP-12, MMP-2, MMP-9, MMP-14, MMP-15, MMP-16 and MMP-17. Typically the MMP family is subdivided based upon substrate preferences as: collagenases, gelatinases, stromelysins, and integral membrane proteins.

[0061] The metal complexes of the present invention can be utilized in conjunction with other therapies, such that the dosage required for the additional therapy is reduced. For example, in the instance of a vascular disease treatment or therapy, the dosage could be reduced. Alternatively, the amount of fluid introduced also can be reduced. The applicable vasoconstrictor or fluid is administered in an ICU setting to restore or maintain blood pressure.

[0062] The present invention also provides methods of improving vascular or hemodynamic function associated with surgical trauma by administering a metal complex of Formula I. These functions relate to circulation of the blood and include, but are not limited to, blood vessel integrity, reversal of NO mediated hypotension, maintenance of vascular tone, maintenance of blood pressure, reversal of hyporesponsiveness to vasoconstrictors, reduction of vascular leak (blood vessel integrity), and improvement in the regulation of microcirculation through organs including (but not restricted to) heart, lung, kidney, and liver.

[0063] Some metal complexes are known in pharmaceutical compositions for the treatment of diseases of the human body. For example certain complexes of platinum and ruthenium have been used or indicated in the treatment of cancer. This invention provides for the use of a neutral anionic or cationic metal complex having at least one site for coordination with NO of Formula I

[Ma(XbL)cYdZe]n± Formula I

[0064] in the manufacture of a medicament for the attenuation of NO levels and other reactive oxygen species when implicated in disease,

[0065] where:

[0066] M is a metal ion or a mixture of metal ions;

[0067] X is a cation or a mixture of cations;

[0068] L is a ligand, or mixture of ligands each containing at least two different donor atoms selected from the elements of Group IV, Group V or Group VI of the Periodic Table;

[0069] Y is a ligand or a mixture of the same or different ligands each containing at least one donor atom or more than one donor atom which donor atom is selected from the elements of Group IV, Group V or Group VI of the Periodic Table; and

[0070] Z is a halide or pseudohalide ion or a mixture of halide ions and pseudohalide ions;

[0071] a=1-3; b=0-12; c=0-18; d=0-18; e=0-18; and n=0-10; provided that at least one of c, d and e is 1 or more;

[0072] and where c is 0: b is also 0;

[0073] and where a is 1: c, d and e are not greater than 9;

[0074] and where a is 2: c, d and e are not greater than 12. By “complex” in this specification is meant a neutral complex or anionic or cationic species.

[0075] The term “Group” which is used herein is to be understood as a vertical column of the periodic table in which elements of each Group have similar physical and chemical properties. The definition of the Periodic Table is that credited to Mendeleev; Chamber Dictionary of Science and Technology, 1974 Published by W & R Chambers Ltd.

[0076] This invention may also be stated as providing a method of attenuation of reactive oxygen species when implicated in diseases of the human body. Thus the invention comprises administering a pharmaceutical composition containing a neutral, anionic or cationic metal complex of Formula I.

[0077] This invention may also provide for the use of a neutral anionic or cationic metal complex of formula I in the manufacture of a medicament for the treatment of diseases in which reactive oxygen species are overproduced.

[0078] This invention may also be stated as providing a method of attenuation of nitric oxide when implicated in diseases of the human body. Thus the invention comprises administering a pharmaceutical composition containing a neutral, anionic or cationic metal complex of Formula I. This invention may also be stated as providing a method of treatment of diseases of the human body resultant of overproduction of NO in the human body comprising administering a pharmaceutical composition containing a neutral anionic or cationic metal complex of Formula I. Where the Formula I represents an anionic species a cation will also be present. Where Formula I represent a cationic species an anion will also be present. The metal complexes may be hydrated. Preferably M is a first, second or third row transition metal ion. For example M may be an Rh, Ru, Os, Mn, Co, Cr or Re ion, and is preferably an Rh, Ru or Os ion.

[0079] Suitably M is in an oxidation state III. When the metal ion for example ruthenium is in oxidation state III, the rate at which it binds with NO is significantly faster than when it is in oxidation state II.

[0080] X may be any cation, such as mono-, di- or tri-valent cation. Suitable cations may be H+, K+, Na+, NH4+ or Ca2+. Conveniently X may be H+, K+, or Na +.

[0081] Preferably L is a ligand, such as ethylenediamine-N,N′-diacetic acid (edda), ethylenediaminetetraacetic acid (edta), nitrilotriacetic acid (nta), dipicolinic acid (dipic), picolinic acid (pic), diethylenetri-aminepentaacetic acid (dtpa), thiobis(ethylenenitrilo)tetraacetic acid (tedta), dithioethanebis(ethylenenitrilo)tetraacetic acid (dtedta), and N-(2-hydroxyethyl) ethylenediamine-triacetic acid (hedtra).

[0082] Preferably Y is a ligand containing nitrogen, oxygen, sulphur, carbon or phosphorus donor groups. Suitable nitrogen donor groups may be for example ammine, amine, nitrile and nitride or derivations thereof. Suitable oxygen donor groups may be for example carboxylic acid, ester or derivations thereof, water, oxide, sulphoxide, hydroxide, acetate, lactate, propionate, oxalate and maltolate. Suitable sulphur donor groups may be for example sulphoxide, dialkysulphide, dithiocarbamate or dithiophosphate. Suitable carbon donor groups may be for example carbon monoxide or isocyanide. Suitable phosphorus donor groups may be for example trialkylphosphine.

[0083] Z may be any halide and is preferably chloride, bromide or iodide. Most conveniently, Z is chloride.

[0084] Examples of metal complexes for use according to the present invention include optionally hydrated ruthenium complexes of Formula II

[Ru(H0-6LII)1-3Y0-2Cl0-4](0-4)± Formula II

[0085] where LII is a polydentate aminocarboxylate ligand, for example edta, nta, dipic, pic, edda, tropolone, dtpa, hedtra, tedta or dtedta or diamide of edta or dtpa (or an amide or ester derivative thereof) or a mixture of any of these and Y is as defined above and may for example be selected from: acetylacetone (acac); a β-diketonate; water; dimethylsulphoxide (dmso); carboxylate; bidentate carboxylate; catechol; kojic acid; maltol; hydroxide; tropolone; malonic acid; oxalic acid; 2.3-dihydroxynaphthalene; squaric acid; acetate; a sulphate and a glycolate. The skilled artisan will be able to substitute other known ligands at Y and which will fall within the scope of the inventions.

[0086] Preparative methods of tedta, dtedta and diamide of edta and dtpa are described in the following references respectively:

[0087] P Tse & J E Powell, Inorg Chem, (1985), 24, 2727;

[0088] G Schwartzenbach, H Senner, G Anderegg, Helv Chim Acta (1957), 40, 1886;

[0089] M S Konings, W C Dow, D B Love, K N Raymond, S C Quay and S M Rocklage, Inorg Chem (1990), 29, 1488-1491; and

[0090] P N Turowski, S J Rodgers, R C Scarrow and K N Raymond, Inorg Chem (1988), 27, 474-481.

[0091] Where the complex of Formula II is an anion, a cation will be required. For example the complexes of Formula II are present in

[0092] K[Ru(Hedta)Cl]2H2O

[0093] [Ru(H2edta)(acac)]

[0094] K[Ru(hedtra)Cl]H2O

[0095] K[Ru(dipic)2]H2O

[0096] (H2pic)[RuCl2(pic)2](Hpic)H2O

[0097] K[Ru(H2edta)Cl2]H2O

[0098] K[Ru(Hnta)2]½H2O

[0099] K[Ru(H2dtpa)Cl]H2O

[0100] [Ru(Hhedtra)acac]H2O

[0101] [Ru(Hhedtra)trop] or

[0102] [Ru(H3dtpa)Cl].

[0103] Complexes of Formula H have not to the best of our knowledge been previously indicated in any pharmaceutical composition. Therefore the present invention also provides a pharmaceutical composition containing an optionally hydrated ruthenium complex of Formula II.

[0104] Further examples of metal complexes for use according to the present invention include optionally hydrated complexes of Formula III,

[M1-3Y1-18Cl10-18](0-6)± Formula III

[0105] where Y is a sulphur donor ligand. For example, such complex is present in

[0106] [Ru(mtc)3] (mtc=4-morpolinecarbodithoic acid) or Ru(S2CNCH2CH2NMeCH2CH2)3½H2O.

[0107] Complexes of Formula III in which Y is a sulphur donor ligand have not to the best of our knowledge been previously indicated in any pharmaceutical composition. Therefore, the present invention also provides a pharmaceutical composition containing an optionally hydrated complex of Formula III when Y is a sulphur donor ligand.

[0108] Yet further examples of metal complexes for use according to the present invention include optionally hydrated complexes of ruthenium of Formula IIIa

[MIII1-3YIII1-18Cl0-18](0-6)± Formula III

[0109] where MIII is ruthenium and YIII is an oxygen-donor ligand such as acetate, lactate, water, oxide, propionate (COEt), oxalate (ox), or maltolate (maltol) or a combination of these. For example complexes of Formula III or IIIa are present in

[0110] [Ru3O(OAc)6](OAc)

[0111] [Ru3O(lac)6](lac)

[0112] [Ru2(OAc)4]NO3

[0113] [Ru2(OCOEt)4]NO3

[0114] K3[Ru(ox)3]

[0115] [Ru2(OAc)4]Cl or

[0116] [Ru(maltol)3].

[0117] Some complexes of Formula III have not to the best of our knowledge been previously indicated in any pharmaceutical composition. Therefore the present invention also provides a pharmaceutical composition containing an optionally hydrated complex of ruthenium of Formula III wherein MIII is ruthenium and YIII is an oxygen-donor ligand selected from the group acetate, lactate, oxide, propionate and maltolate.

[0118] Further examples of metal complexes for use according to the present invention include optionally hydrated complexes of ruthenium of Formula IV

[RuYIV1-9Cl1-9](0-4)± Formula IV

[0119] where YIV is a nitrogen-donor ligand such as: ammine; ethylenediamine (en); pyridine (py); 1,10-phenanthroline (phen): 2,2-bipyridine (bipy) or 1,4,8,11-tetraazacyclotetradecane (cyclam); 2,3,7,8,12,13,17,18-octaethylporphyrin (oep); or a combination of these. For example complexes of Formula IV are present in

[0120] [Ru(HN3)5Cl]Cl2

[0121] trans-[RuCl2(py)4]

[0122] K[Ru(phen)Cl4]

[0123] [Ru(cyclam)Cl2]Cl

[0124] K[Ru(bipy)Cl4]

[0125] [Ru(NH3)6]Cl3 or

[0126] [Ru(NH3)4Cl2]Cl.

[0127] Some complexes of Formula IV have not to the best of our knowledge been previously indicated in any pharmaceutical composition. Therefore the present invention also provides a pharmaceutical composition containing an optionally hydrated complex of ruthenium of Formula IV wherein YIV is a nitrogen-donor ligand selected from the group en, py, phen, bipy, cyclam and oep. Derivations of these ligands can be prepared by a skilled artisan and which will fall within the scope of the inventions.

[0128] Still further examples of metal complexes for use according to the present invention include optionally hydrated complexes of ruthenium or osmium of general Formula V

[M1-3YV1-18Cl0-18](0-6)± Formula V

[0129] where YV is a combination of donor ligands such as are described hereinabove, for example ammine, dmso, oxalate, bipy, acac and acetonitrile (MeCN). Complexes of Formula V are present in for example

[0130] [Ru(NH3)(dmso)2Cl3]

[0131] cis-[Ru(dmso)4Cl2]

[0132] cis-[Ru(NH3)(dmso)3Cl2]

[0133] [Ru(dmso)3Cl3]

[0134] [Os(ox)(bipy)2]H2O or

[0135] [Ru(acac)2(MeCN)2]CF3SO3.

[0136] The complex ions of the latter two compounds above have not to the best of our knowledge been previously indicated in any pharmaceutical composition. Therefore the present invention also provides a pharmaceutical composition containing an optionally hydrated complex of formula [Os(ox)(bipy)2]; and further a pharmaceutical composition containing an optionally hydrated complex of formula [Ru(acac)2(MeCN)2]+.

[0137] The complexes of the present invention may be included as an active component in a pharmaceutical composition containing an optionally hydrated complex of any of Formulae I-V, in admixture with a pharmaceutically acceptable carrier or diluent. Said pharmaceutical composition may be formulated according to well known principles, and may be in the form of a solution or suspension for parenteral administration in single or repeat doses or be in capsule, tablet, dragee, or other solid composition or as a solution or suspension for oral administration, or formulated into pessaries or suppositories, or sustained release forms of any of the above. The solution or suspension may be administered by a single or repeat bolus injection or continuous infusion, or any other desired schedule. Suitable diluents, carriers, excipients and other components are known. Said pharmaceutical composition may contain dosages determined in accordance with conventional pharmacological methods, suitable to provide active complexes in the dosage range in humans of 1 mg to 10 g per day. Actual required dosage is largely dependent on where in the body there is the excess concentration of NO or other reactive oxygen species and for how long overproduction continues or attenuation of the levels of NO or reactive oxygen species, where such reactive oxygen species is implicated in disease, is required.

[0138] The model of CABG used in this invention differs in some aspects from the human heart model. For this invention, the use of the dog to represent the human model is described, where the results are representative of human CABG procedure. Dogs, unlike human, do not have pre-existing heart disease. Intraoperative bypass grafting was not performed and aortic flow was occluded using an internal rather than an external cross-clamp. However, it has been previously shown that sham bypass can activate inflammatory pathways (Mayers et al. J. Crit. Care. 11:189-197 (1996)). Therefore, the control animals were anesthetized but not subject to a sham bypass procedure or re-infusion of blood collecting within the mediastinum.

EXAMPLES

[0139] Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

Example A

[0140] Animal Preparation

[0141] These studies were carried out with the approval of the University of Saskatchewan Animal Care Committee and comply with published National Institutes of Health guidelines. Mongrel dogs (weight 20-25 kg) were anesthetized with pentobarbital (15 mg/kg), incubated and mechanically ventilated (Harvard Model #607). Anesthesia was maintained by continuous infusion of pentobarbital (1.0 mgrkg/hr), morphine (0.1 mg/kg/hr), and vecuronium (0.1 mg/kg/hr). A pulmonary artery catheter was used to measure mean pulmonary artery pressure (PPA), pulmonary capillary wedge pressure (PCWP), mean right atrial pressure (PRA), and cardiac output (CO). Mean systemic artery pressure (PSA) and arterial blood gas sampling were obtained through a femoral artery catheter.

[0142] The CPB animal preparation has been previously described (Mayers et al., J. Thoracic Cardiovasc. Surg. 117:1009-1016, 1999). Briefly, the heart was exposed via midline sternotomy. Following heparinization (5,000 U i.v. followed by 1,000 units/hr i.v.), catheters were placed in the left atrium and right atria to complete the bypass circuit. A balloon angioplasty catheter (#6 F), positioned proximal to the aortic valve via the right internal carotid artery, acted to internally cross-clamp the aorta when inflated. CPB was initiated using a membrane oxygenator (Capiox Hollow Fiber Oxygenator) and a blood pump (Sarns Model 5000 Console) at 100 ml/kg flow. Cold (7-8° C.), antegrade cardioplegia solution was delivered and then the aorta was occluded by inflation of the angioplasty balloon. Blood cardioplegia (BCD4 Sharely) was continuously administered to eliminate electrical activity. The animals were cooled (24° C.) and the aortic occlusion maintained for a further 50 minutes. Then the angioplasty balloon was deflated, mechanical ventilation resumed and the animals warmed over 30 minutes. Therefore, CPB lasted for a total of 90 minutes. PSA was prospectively maintained at >60 mmHg by first increasing PRA to 15 mmHg by fluid administration and then infusing phenylephrine. The animals were maintained for 4 hours post-CPB.

[0143] Experimental Groups

[0144] Dogs were randomized to receive CPB (n=12) or act as controls (n=12). Dogs receiving CPB were then randomized to receive a continuous infusion of an NO scavenger [Ru(H3dtpa)]Cl (CPB-SCV group; n=6) or to receive a placebo (CPB-PLAC group; n=6). The NO scavenger (6221 AnorMED) was continuously infused (128 mg/kg/hr) starting prior to the sternotomy and ending 30 minutes following termination of CPB (see FIG. 1). This dose was chosen based upon pilot data from endotoxin treated (0.2 mg/kg) dogs to simulate a state of NO excess (data not shown). Arterial pH was maintained between 7.3 and 7.45 by NaHCO3 as needed.

[0145] The control dogs followed a similar protocol but did not have a sternotomy or placement of CPB catheters. At the conclusion of the studies all animals were sacrificed by barbiturate overdose. Biopsies (0.5 cm2) from left ventricle, left atrium, lung (right lower lobe) and brain (cerebral cortex) were obtained, frozen in liquid nitrogen, and later analyzed (see below).

[0146] Measurements and Calculations

[0147] After catheter insertions, baseline blood samples were obtained for routine hematology, biochemistry and blood gases (see Table 1). Hemodynamics measurements including SVR, fluid requirements, CO, PSA, PCWP, and PRA were repeated at 1 and 4 hours following bypass and as needed to maintain our predetermined hemodynamic goals post CPB. Neutrophil expression of CD18 was assessed with flow cytometry (FACScan) as previously described (Mayers et al., J. Crit. Care. 1996; 11:189-197). Briefly, after cells were prepared they were reacted with an irrelevant, isotype-matched FITC-conjugated rat monoclonal antibody (MCA1125F)(Serotech Ltd) or FITC-conjugated rat anti-human CD18 antibody (MCA503F; Serotech) that cross-reacted with canine. Flow cytometric analysis was restricted to neutrophils based upon forward angle and right angle light scatter. Neutrophil CD18 expression was determined as the percentage of cells with a fluorescence exceeding that of cells reacted with the irrelevant, isotype-matched control.

[0148] NOS activity was measured in homogenized tissue samples as previously described (Radomski et al., Cardiovasc Res. 1993; 27:1380-1382). Briefly, tissue aliquots were incubated with L[U14-C]-arginine (Amersham) for 20 min in the appropriate buffer. NOS-dependent L-citrulline formation, expressed as pmol/min/mg protein, was used as an index of enzyme activity. A Ca2+-chelating agent (EGTA, 1 mM) was used to differentiate between Ca2+-dependent and -independent NOS activities of enzymes. Tissue MMPs activity was measured by zymography as previously described (Sawicki et al., Nature 1997; 386:616-618) using 7% SDS-PAGE with copolymerized gelatin as a substrate.

[0149] Statistics

[0150] Data were compared between period and groups with a one way or two-way analysis of variance as appropriate and when the F statistic showed a significant difference, a SNK (Student-Newman-Keuls test) multiple comparison test was used to determine specific group and period differences. We prospectively limited the possible number of comparisons to limit required correction factor. Spearman correlation was used to assess the interaction between CD1 8 expression and NOS or MMP activity. A p value less than 0.05 was considered to show a significant difference. All values are shown as means±SD.

RESULTS

[0151] Hemodynamics and Gas Exchange

[0152] PSA and CO decreased while systemic vascular resistance (SVR) increased over time in the CPB groups (Table 1). To achieve our predefined hemodynamic endpoints (PSA>60 mm Hg and PRA<15 mm Hg), 3 of 6 CPB-PLAC animals required a phenylephrine compared to 0 of 6 the CPB-SCV animals (p<0.05). Total fluid administration was reduced in the NO scavenger groups compared to the placebo groups.

[0153] Pooled CK values (Table 2) in the CPB-PLAC group were increased over the CPB-SCV group (2654±6225 U vs 476±681 U; p<0.005). Comparing baseline values to 4 hr post CPB, PO2 decreased in the CPB-SCV group and CPB-PLAC group and intrapulmonary shunt increased (19%±10 versus 18%±14 respectively; p>0.05). Neutrophils, as apercentage of WBC, increased from Baseline to 4 hr post CPB in the CPB-PLAC group (66%±7 to 83%±5; p=0.03). CD18 expression at 4 hr post CPB was lower in CPB-SCV group compared to the CPB-PLAC group (p<0.05).

[0154] NOS and MMP Activities

[0155] Ca2+-dependent NOS activities were similar between all groups in heart, brain and lung tissues. Ca2+-independent NOS activities were elevated (p<0.05) in the both CPB groups compared to both CTRL groups (FIG. 2 and FIG. 3). Brain Ca2+-independent NOS activity was significantly reduced (p<0.05) in the CPB-SCV group compared with CPB-PLAC group but was still increased compared with the groups that did not receive CPB (p<0.05).

[0156] MMP-2 and MMP-9 activities were significantly (p<0.05) elevated in the CPB-PLAC group compared to CRTL-PLAC group in heart and lung (FIG. 4). Only MMP-2 was significantly (p<0.05) elevated in brain in the CPB-PLAC group compared to the CRTL-PLAC group (FIG. 5). MMP activity was significantly reduced in the CPB-SCAV group compared to the CPB-PLAC group (p<0.05). MMP activity, except brain MMP-9, was positively correlated to CD18 peak expression (rho range from 0.46 to 0.73; p<0.005). NOS activity was not correlated to CD18 peak expression (p>0.05).

1TABLE 1Selected hemodynamic values.Baseline1 Hr Post4 Hr PostPSA(mm Hg)CTRL-PLAC123 ± 15 112 ± 15 109 ± 12 CTRL-SCV106 ± 16 114 ± 14 113 ± 17 CPB-PLAC125 ± 11 98 ± 14*101 ± 18*CPB-SCV142 ± 9 89 ± 13*95 ± 4*PRA(mm Hg)CTRL-PLAC6 ± 27 ± 28 ± 2CTRL-SCV5 ± 17 ± 26 ± 2CPB-PLAC5 ± 16 ± 16 ± 1CPB-SCV6 ± 18 ± 27 ± 2PCWP(mm Hg)CTRL-PLAC8 ± 28 ± 19 ± 1CTRL-SCV6 ± 17 ± 16 ± 1CPB-PLAC8 ± 29 ± 28 ± 2CPB-SCV9 ± 29 ± 19 ± 3SVR(dyne/sec/cm5)CTRL-PLAC6503 ± 15175461 ± 11916347 ± 1025CTRL-SCV8403 ± 13609452 ± 2264 11143 ± 2130*#CPB-PLAC8112 ± 22516898 ± 132311672 ± 918* CPB-SCV9593 ± 27137227 ± 206211406 ± 2812*CO(L/min)CTRL-PLAC1.5 ± 0.41.6 ± 0.61.4 ± 0.2CTRL-SCV1.0 ± 0.31.0 ± 0.20.8 ± 0.2CPB-PLAC1.3 ± 0.41.1 ± 0.3 0.7 ± 0.2*CPB-SCV1.2 ± 0.51.0 ± 0.2 0.7 ± 0.1*FluidRequirements(ml)CTRL-PLAC——2655 ± 82+ CTRL-SCAV——892 ± 159CPB-PLAC——1617 ± 254+CPB-SCV——983 ± 134Where PSA = mean systemic blood pressure, PRA = mean right atrial pressure, PCWP = pulmonary capillary wedge pressure, SVR = systemic vascular resistance (calculated as SVR = Psa-Pra/CO *80), CO = cardiac output. Note *denotes a significant difference (p < 0.05) compared to other periods within same group, #denotes a significant difference (p < 0.05) compared to the same period in the placebo group (CTRL-PLAC or CPB-PLAC), +denotes a significant difference (p < 0.05) comparing pooled groups (CTRL-PLAC vs CTRL-SCV or CPB-PLAC vs CPB-SCV)

[0157]

2

TABLE 2

Selected biochemical and hematological values in all four groups.

Baseline
1 Hr Post
4 Hr Post

Hemoglobin

(gm/dL)

CTRL-PLAC
10.9 ± 1.3
9.9 ± 1.5
9.0 ± 1.2

CTRL-SCV
11.7 ± 0.7
11.4 ± 0.8
11.9 ± 0.8*#

CPB-PLAC
12.5 ± 0.8
6.7 ± 0.4*
7.9 ± 0.6*

CPB-SCV
10.4 ± 3.4
7.3 ± 0.7*
7.6 ± 1.1*

WBC

(109 cells)

CTRL-PLAC
8.1 ± 2.0
7.2 ± 1.2
7.7 ± 0.8

CTRL-SCV
6.6 ± 2.3
7.7 ± 2.6
8.6 ± 2.2

CPB-PLAC
5.3 ± 1.5
5.3 ± 1.7
6.9 ± 2.7

CPB-SCV
6.0 ± 1.4
5.4 ± 1.7
6.7 ± 1.7

PO2

(mm Hg)

CTRL-PLAC
569 ± 20
557 ± 58
589 ± 23

CTRL-SCV
558 ± 28
564 ± 42
566 ± 24

CPB-PLAC
438 ± 63
273 ± 168*
276 ± 135*

CPB-SCV
442 ± 52
303 ± 144*
305 ± 121*

PCO2

(mm Hg)

CTRL-PLAC
39 ± 5
41 ± 3
40 ± 2

CTRL-SCV
32 ± 10
34 ± 7
34 ± 8

CPB-PLAC
36 ± 7
36 ± 4
39 ± 5

CPB-SCV
25 ± 8
34 ± 8
30 ± 6

CD18

expression

(units)

CTRL-PLAC
65 ± 11
71 ± 8
66 ± 7

CTRL-SCV
53 ± 25
51 ± 31
35 ± 26*#

CPB-PLAC
69 ± 22
78 ± 15
81 ± 11

CPB-SCV
52 ± 25
54 ± 33
38 ± 27#

CK

CTRL-PLAC
87 ± 42
71 ± 69
74 ± 58

CTRL-SCV
97 ± 20
132 ± 105
88 ± 32

CPB-PLAC
118 ± 30
2858 ± 4976
4986 ± 8475

CPB-SCV
44 ± 34
434 ± 513
948 ± 800

Where

WBC = white blood cell corrected for hemodilution effect,

CD18 expression = peak fluorescent intensity of neutrophils for CD18 (see text).

Note

*denotes a significant difference (p < 0.05) compared to other periods within same group,

#denotes a significant difference (p < 0.05) compared to the same period in the placebo group (CTRL-PLAC or CPB-PLAC),

+denotes a significant difference (p < 0.05) comparing pooled groups (CTRL-PLAC vs CTRL-SCV or CPB-PLAC vs CPB-SCV).

PO2 = partial pressure of oxygen (this is a measure of the dissolved oxygen concentration),

PCO2 = partial pressure of carbon dioxide (this is a measure of the dissolved carbon dioxide concentration), and

CK = creatine kinase.

[0158] A Ruthenium NO scavenger complex (AMD6221) [Ru(H3dtpa)Cl)] was administered to dogs undergoing cardiopulmonary bypass. Control, untreated animals required both fluid resuscitation and vasoconstrictors to maintain blood pressure. In addition there was upregulation of iNOS and MMPs in lung, cardiac and brain tissue, and increased levels of circulating neutrophils. In AMD6221 dogs there was a reduced requirement for both fluid resuscitation and vasoconstrictors, which is compatible with previously published data on AMD6245 [Ru(Hedta)H2O] in septic shock. There was also a reduction in creatine kinase levels in treated animals. These indicators point to an improvement in cardiac function with treatment. MMPs are implicated in cardiac dysfunction, so lowering of MMPs in all tissues provides an organ protective effect of the NO scavenger in the heart, lung and brain. NO is also a mediator of tissue damage and therefore lowering NO levels also will contribute to the protective effect. The reduction in creatine kinase CK levels also indicates a protective effect towards brain and muscle.

[0159] A number of commercially available compounds, and compounds prepared by routes known in the literature, containing the complexes of the present invention were tested in vitro, in vitro cell culture, and ex-vivo in order to determine ability to coordinate with NO. The complexes tested were as follows:

3TABLE 3Literature ReferenceExampleCompoundfor Preparation1K[Ru(Hedta)Cl]2H2OA A Diamantis & J VDubrawski, Inorg. Chem,(1981), 20, 1142-502[Ru(H2edta)(acac)]A A Diamantis & J VDubrawski, Inorg. Chem.,(1983), 22, 1934-363K[Ru(hedtra)Cl]H2OH C Bajaj & R van Eldik,Inorg. Chem. (1982), 28,1980-34K[Ru(dipic)2]H2ON H Williams & J KYandell, Aust. J. Chem.(1983), 36 (12), 2377-23865(H2pic)[RuCl2(pic)2](Hpic)H2OJ D Gilbert, D Rose &G Wilkinson, J. Chem. Soc.(A), (1970), 2765-96K[Ru(H2edta)Cl2]H2OA A Diamantis & J VDubrawski, Inorg. Chem.(1981), 20, 1142-507K[Ru(Hnta)2]½H2OM M Taqui Khan,A Kumar & Z Shirin,J. Chem. Research (M),(1986), 1001-10098K[Ru(H2dtpa)Cl]H2OM M Taqui Khan,A Kumar & Z Shirin,J. Chem. Research (M).(1986), 1001-10099[Ru3O(lac)6](lac)A Spencer & G Wilkinson,J. Chem. Soc. DaltonTrans, (1972), 1570-7710[Ru3O(OAc)6](OAc)A Spencer & G Wilkinson,J. Chem. Soc. DaltonTrans. (1972), 1570-7711[Ru2(OAc)4]NO3M Mukaida, T Nomura & TIshimori, Bull. Chem. Soc.Japan, (1972), 45, 2143-712[Ru2(OCOEt)4]NO3A Bino, F A Cotton & T RFelthouse, Inorg. Chem.(1979), 18, 2599-260413K3[Ru(ox)3]C M Che, S S Kwong, C KPoon, T F Lai & T C WMak Inorg. Chem. (1985),24, 1359-6314[Ru2(OAc)4]ClR W Mitchell, A Spencer &G WilkinsonJ. Chem. Soc. DaltonTrans., (1973), 846-5415[Ru(NH3)5Cl]Cl2A D Allen, F Bottomley,R O Harris, V P Reinsalu &C V SenoffJ. Amer. Chem. Soc.(1967), 89, 5595-559916[Ru(en)3]I3T J Meyer & H TaubeInorg. Chem. (1968), 7,2369-237917K[RuCl4(phen)]H2OB R James & R SMcMillanInorg. Nucl. Chem. Lett.(1975), 11 (12) 837-918[Ru(cyclam)Cl2]ClP K Chan, D A Isabirye &C K PoonInorg. Chem. (1975), 14,2579-8019K[RuCl4(bipy)]B R James & R SMcMillanInorg. Nucl. Chem. Lett.(1975), 11 (12), 837-920[RuCl3(dmso)2(NH3)]Patent: InternationalPublication NoWO 91/1355321[Ru(NH3)6]Cl3Matthey Catalogue Sales:Cat No [190245]22cis-[RuCl2(dmso)4]E A Alessio, G Mestroni,G Nardin, W M Attia,M Calligaris, G Sava &S ZorgetInorg. Chem. (1988), 27,4099-410623cis-[RuCl2(dmso)3(NH3)]M Henn, E Alessio,G Mestroni, M Calligaris &W M AttiaInorg. Chim, Acta, (1991),187, 39-5024[RuCl3(dmso)3]E Alessio, G Balducci,M Calligaris, G Costa,W M Attia & G MestroniInorg. Chem. (1991), 30,609-61825[Ru(mtc)3]A R Hendrickson, J MHope R L MartinJ. Chem. Soc. DaltonTrans. (1976), 20, 2032-926[Ru(maltol)3]W P Griffith & S J GreavesPolyhedron, (1988), 7 (19),1973-927[Ru(acac)2MeCN)2]CF3SO3Y Kasahara, T HoshinoK. Shimizu & G P SatoChem. Lett. (1990), 3,381-428K2[RuCl5(H2O)]Matthey Catalogue Sales:Cat No [190094]29[Os(ox)(bipy)2].H2OD A Buckingham, F PDwyer, H A Goodwin &A M Sargeson Aust. J.Chem. (1964), 325-336G M Bryant, J E Fergusson& H K J PowellAust. J. Chem. (1971),24 (2), 257-7330[Ru(NH3)4Cl2]ClS D Pell, M M Sherban,V Tramintano & M JClarkeInorg Synth, (1989), 26,65.31[Ru(Hedtra)(dppm)]M M Taqui Khan,K Venkatasubramanian,Z Shirin, M M BhadbhadeJ Chem Soc Dalt Trans(1992), 885-89032Ru(oep)PhM Ke, S J Rettig, B RJames and D DolphinJ Chem Soc ChemCommun (1987), 1110

[0160] A number of new compounds were prepared according to the following protocols. The first four compounds are examples of ruthenium complexes of formula [Ru(H0-6LII)1-3Y O-2Cl0-4](0-4)± (Formula II), the subsequent two are examples of [M1-3Y1-8Cl0-18](0-6)± (Formula III).

[0161] Preparation of [Ru(Hhedtra)acac].H2O

[0162] Excess acetylacetone (1 cm3) was added to an aqueous solution (5 cm3) of K[Ru(hedtra)Cl] (0.5 g). The solution colour changed to violet. The mixture was warmed for 20 minutes then left to stand at room temperature for 20 minutes. The violet solution was extracted with chloroform (20 cm3). The extraction was repeated twice more. A violet product precipitated from the aqueous fraction. The product was filtered, washed in acetone and dried in vacuo, yield 0.1 g (18%).

[0163] AnaL Calc. for C15H25O1ON2Ru: C, 36.43; H, 5.11; N, 5.70. Found: C, 36.16; H, 5.42; N, 5.61%.

[0164] Preparation of [Ru(Hhedtra)trop]2H2O.

[0165] A three-fold excess of tropolone (0.78 g) dissolved in 50:50 water/absolute ethanol (5 cm3) was added to a warm aqueous solution of K[Ru(hedtra)Cl] (10 cm3). The mixture was heated for 1 hour. On cooling, the dark green mixture was extracted with 3×2O cm3 portions of dichloromethane. On standing, a dark green product precipitated from the aqueous fraction. The product was filtered, washed with water (1 cm2), either and dried in vacuo, yield 0.4 g (36%).

[0166] Anal. Calc. for C17H22N2O9Ru.2H2O: C,38.13; H, 4.86; N 5.23. Found C 38.55; H, 4.67; N 5.28%.

[0167] Preparation of [Ru(H3dtpa)Cl]

[0168] K2[RuCI5H2O].×H2O (1 g) was suspended in HClO4 (15 cm3, 1 mM) and diethylenetrianiinepentaacetic acid (1.05 g) added. The reaction mixture was heated under reflux for 1.5 hours forming a yellow/brown solution. On cooling a yellow product crystallised which was collected by filtration, washed with 90% absolute ethanol/water, diethyl ether and dried in vacuo, yield 0.75 g, 53%.

[0169] Anal. calcd. for C14H21N3010ClRu: C, 31.85; H, 3.98; N, 7.96; Cl, 6.73. Found: C, 29.77; H, 3.81; N, 7.36; Cl, 6.64.

[0170] Preparation of K[RuHHBEDCl]3H2O

[0171] 0.41 g of K2[RuCl5]×H2O was dissolved in water (20 ml). To this solution was added 1 equivalent (0.39 g) of N,N′di (2-hydroxy-benzyl)ethylene-diamine N,N-diacetic acid (hbed) dissolved in water (50 ml) with KOH (0.12 g) and MEOH (1 ml.). This mixture was heated at reflux for 90 minutes. Upon cooling a dark, insoluble precipitate formed. This material was removed by filtration and the resulting red-violet solution was taken to dryness by rotary vaporation. Trituration with water and washing with acetone yielded 90 mg of a dark solid.

[0172] AnaL calcd. for C18H22N2O9RuClK: C, 36.89; H, 3.96; N, 4.78; Cl, 6.04. Found: C, 37.09; H, 4.23; N, 4.92; Cl, 6.28.

[0173] Preparation of Ru(S2CNCH2CH2NMeCH2CH2),½H2O

[0174] Me4N[S2CNCH2CH2NMeCH2CH2] was made by the same standard method and crystallised from methanol-ether in 71% yield.

[0175] RuCl3×H2O, 0.50 g, 2.15 mmol was refluxed in 30 ml of methanol for 10 minutes and cooled. 1.87 g, 7.50 mmol of Me4N[S2CNCH2CH2NMeCH2CH2] was added and the mixture refluxed for 16 hours. After cooling 0.72 g of crude product was filtered off, dissolved in dichloromethane and filtered. The filtrate was loaded into 15 cc of basic alumina and eluted with dichloromethane. Removal of solvent and crystallisation from dichloromethane with ether by vapour-phase diffusion gave 0.51 g 0.80 mmol, 37% of brown-black crystals, Ru(S2CNCH2CH2NMeCH2CH2)3½H2O.

[0176] Analysis for C18H34N6O5RuS6: Calc: C, 34.00; H, 5.39; N, 13.22; S, 30.25. Found: C, 34.21; H, 5.47; N, 13.12; S, 30.36.

[0177] Preparation of Ru[S2P(OC2H2OC2H4OMe)2]3

[0178] Ru[S2P(OC2H2OC2H4OMe)2]3 was made by standard method and crystallised from methanol in 76% yield.

[0179] RuCl3×H2O, 1.00 g, 4.30 mmol was refluxed in 50 ml of 0.1 N HCl with 1 ml of ethanol for 20 minutes and cooled. To this solution was added 5.28 g (excess) K[S2P(OC2H4OC2H4OME)2] and the mixture stirred at 30′ C. for 1 hour. The reaction mixture was extracted with dichloromethane and the solvent removed. The residue was extracted with ether-hexane and solvents removed. This residue was crystallised from 25 ml of hot ether by cooling to −20° C. giving 2.98 of red crystals. 2.41 g of the crude product was purified by chromatography on 60 cc of silica gel with 5% ethanol in ether. The first band was collected, reduced to dryness and crystallised from ether by cooling to −20° C . The yield of red crystals, Ru(S2P{OC2H4OC2OMe}2)3, was 2.16 g, 56%.

[0180] Analysis for C30H66O18P3RuS6: Calc: C, 32.72; H, 6.04; S, 17.47. Found: C, 32.68; H, 6.08; S, 17.16.

[0181] In the in vitro tests, which were carried out in an atmosphere of argon, each compound (1×1O4 moles) was dissolved in double-distilled deionized and deoxygenated water. The resulting solution was placed in a three-necked pear-shaped flask and stirred by a magnetic stirrer at constant speed of 1000 rpm, at a constant temperature in the range 20° C.-24° C. A manometer was attached to the flask, purified, dried nitric oxide gas (known volume in the range 3-5 cm3) was introduced via a septum, using a gas syringe, at atmospheric pressure into the headspace above the reaction solution. The pressure within the flask was recorded periodically over a period of one hour.

[0182] A control experiment was carried out according to the above but without any complex present. The recorded pressures in association with the results of the control experiment were analyzed in order to determine the rate of NO uptake as a function of time for e each compound tested.

[0183] On completion of each in vitro test, the reaction solution was freeze-dried. An infrared spectrum of the freeze-dried product provided information on metal-NO bond formation.

[0184] In the in vitro cell culture tests, murine (RAW264) macrophage cell lines, which can be induced to produce nitric oxide, were seeded, 106 cells/well, onto 24 well culture plates of 2 ml volume per well, in Eagles modified minimal essential medium (MEM) plus 10% foetal bovine serum without phenol red.

[0185] The cells were activated to produce nitric oxide, with 10, μg/ml lipopolysaccharide and 100 units/ml interferony γ for 18 hours. Concurrently, test compounds made up in MEM were added at non-cytotoxic concentrations.

[0186] Control cells as above, which were activated to produce nitric oxide as above, but to which no test compound was added, were used as a measure of the amount of nitric oxide produced by the cells during the tests.

[0187] Background nitric oxide was assessed by measurement of nitrate and nitrite in cells which were not activated.

[0188] Cell viability was confirmed by Trypan blue dye exclusion at the end of the incubation period.

[0189] Nitric oxide was determined by measurement of nitrate and nitrite in the cell supernatant. These anions are the stable end-products of reactions of NO in solution. Such reactions may or may not be catalysed in biological systems. The sum of nitrite and nitrate concentrations gives the total NO production. Nitrite was determined using the Griess reaction in which nitrite reacts with 1% sulphanilamide in 5% H3PO4/O.1% naphthylethylenedianine dihydrochloride to form a chromophore absorbing at 540 nm. Nitrate was determined by reducing nitrate to nitrite with a bacterial nitrate reductase from Pseudomonas oleovorans and then measuring nitrite with the Griess reaction. In the absence of test compounds nitrite concentration plus nitrate concentration is equal to total nitric oxide production. The effect of test compounds on available nitric oxide (measured as nitrite+nitrate) was determined. The reduction in available nitric oxide compared with the control level may be taken as an indication of the degree of binding of NO by the test compounds.

[0190] In the ex vivo tests, segments of rat tail artery (0.8-1.5 cm) were dissected free from normotensive adult Wistar rats. The arteries were internally perfused with Krebs solution (mM: NaCl 118, KCl 4.7, NaHCO3 25, NaH2PO4 1.15, CaCl2 2.5, MgCl2 1.1, glucose 5.6 and gassed with 95% O2/5% CO2 to maintain a pH of 7.4) in a constant flow perfusion apparatus. A differential pressure transducer located upstream of the vessel detected changes in back pressure. The rat tail artery preparation was pre-contracted with 6.5 μM phenylephrine to give a physiologically normal pressure of 100-120 mm Hg. The pre-contracted vessels were then perfused with the test compound. The arteries were perfused with Krebs solution between applications of test compound to wash out the test compound.

[0191] Pressure changes in the system served to indicate artery vasoconstriction. The vasoconstriction is a direct result of the removal of endogenous nitric oxide (edrf) from the endothelial cells of the rat tail artery.

[0192] Results

[0193] The results of the in vitro, in vitro cell culture and ex-vivo tests were as follows:

[0194] In vitro Tests

Example I

K[Ru(Hedta)Cl]2H2O

[0195] A pressure decrease indicated binding of NO to the metal compound.

[0196] The IR spectrum showed a peak at 1897 cm−1, indicating the presence of a RU—NO bond.

Example 2

[Ru(H2edta)(acac)]

[0197] The IR spectrum showed a peak at 1896 cm−1, indicating the presence of a RU—NO bond.

Example 3

K[Ru(hedtra)Cl]H2O

[0198] A pressure decrease indicated binding of NO to the metal compound.

[0199] The IR spectrum showed a peak at 1889 cm−1, indicating the presence of a RU—NO bond.

Example 4

K[Ru(dipic)2]H2O

[0200] A pressure decrease indicated binding of NO to the metal compound.

[0201] The IR spectrum showed a peak at 1915 cm−1, indicating the presence of a RU—NO bond.

Example 5

(H2pic)[RuCl2(pic)2](Hpic)H2O

[0202] The IR spectrum showed a peak at 1888 cm−1, indicating the presence of a RU—NO bond.

Example 6

K[Ru(H2edta)Cl2]H2O

[0203] A pressure decrease indicated binding of NO to the metal compound.

[0204] The IR spectrum showed a peak at 1896 cm−1, indicating the presence of a RU—NO bond.

Example 7

K[Ru(Hnta)2]½H2O

[0205] A pressure decrease indicated binding of NO to the metal compound.

[0206] The IR spectrum showed a peak at 1889 cm−1, indicating the presence of a RU—NO bond.

Example 8

K[Ru(H2dtpa)Cl]H2O

[0207] A pressure decrease indicated binding of NO to the metal compound.

[0208] The IR spectrum showed a peak at 1905 cm−1, indicating the presence of a RU—NO bond.

Example 9

[Ru3O(lac)6](lac)

[0209] The IR spectrum showed a peak at 1884 cm−1, indicating the presence of a RU—NO bond.

Example 10

[RU3O(OAc)6](OAc)

[0210] The IR spectrum showed a peak at 1877 cm−1, indicating the presence of a RU—NO bond.

Example 11

[RU2(OAc)4]NO3

[0211] The IR spectrum showed a peak at 1891 cm−1, indicating the presence of a RU—NO bond.

Example 12

[Ru(OCOEt)4]NO3

[0212] The IR spectrum showed a peak at 1891 cm−1, indicating the presence of a RU—NO bond.

Example 13

K3[R-(ox)3]

[0213] The IR spectrum showed a peak at 1889 cm−1, indicating the presence of a RU—NO bond.

Example 14

[Ru2(OAc)4]Cl

[0214] The IR spectrum showed a peak at 1895 cm−1, indicating the presence of a RU—NO bond.

Example 15

[Ru(NH3)5Cl]Cl2

[0215] The IR spectrum showed two peaks at 1909 cm−1 and 1928 cm−1, indicating the presence of a RU—NO bond.

Example 16

[Ru(en)3]I3

[0216] The IR spectrum showed a peak at 1906 cm−1, indicating the presence of a RU—NO bond.

Example 17

K[RuCl4(phen)]H2O

[0217] The IR spectrum showed a peak at 1904 cm−1, indicating the presence of a RU—NO bond.

Example 18

[Ru(cyclam)Cl2]Cl

[0218] The IR spectrum showed a peak at 1895 cm−1, indicating the presence of a RU—NO bond.

Example 19

K[RuCl4(bipy)]

[0219] The IR spectrum showed a peak at 1885 cm−1, indicating the presence of a RU—NO bond.

Example 20

[RuCl3(dmso)2(NH3)]

[0220] The IR spectrum showed a peak at 1889 cm−1, indicating the presence of a RU—NO bond.

Example 21

[Ru(NH3)6]Cl3

[0221] The IR spectrum showed a peak at 1910 cm−1, indicating the presence of a RU—NO bond.

Example 22

cis-[RuCl2(dmso)4]

[0222] The IR spectrum showed a peak at 1881 cm−1, indicating the presence of a RU—NO bond.

Example 23

cis-[RuCl2(dmso)3(NH3)]

[0223] The IR spectrum showed a peak at 1893 cm−1, indicating the presence of a RU—NO bond.

Example 24

[RuCl3(dMso)3]

[0224] The IR spectrum showed a peak at 1880 cm−1, indicating the presence of a RU—NO bond.

Example 25

[Ru(mtc)3]

[0225] The IR spectrum showed a peak at 1862 cm−1, indicating the presence of a RU—NO bond.

Example 26

[Ru(maltol)3]

[0226] The IR spectrum showed a peak at 1866 cm−1, indicating the presence of a RU—NO bond.

Example 27

[Ru(acac)2(MeCN)2]CF3SO3

[0227] The IR spectrum showed a peak at 1899 cm−1, indicating the presence of a RU—NO bond.

Example 28

K2[RuCl5(H2O)]

[0228] The IR spectrum showed a peak at 1903 cm−1, indicating the presence of a RU—NO bond.

Example 29

[Os(ox)(bipy)2]H2O

[0229] The IR spectrum showed a peak at 1894 cm−1, indicating the presence of a Os—NO bond.

[0230] In vitro Cell Culture Tests

[0231] Results are shown in Table 4.

Example 1

K[Ru(Hedta)Cl]2H2O

[0232] Available nitric oxide was reduced in a dose-dependent fashion with a maximum reduction of 75% at a concentration of 100 μM.

Example 2

[Ru(H2edta)(acac)]

[0233] Available nitric oxide was reduced by 82% at 100 μM test compound.

Example 3

K[Ru(Hedtra)Cl]H2O

[0234] Available nitric oxide was reduced by 42% at 100 μM.

Example 6

K[Ru(H2edta)Cl2]H2O

[0235] Available nitric oxide was reduced by 77% at 100 μM test compound.

Example 14

[RU2(OAc)4]Cl

[0236] Available nitric oxide was reduced by 47% at 100 μM.

Example 15

[Ru(NH3)5Cl]Cl2

[0237] Available nitric oxide was reduced by 86% at 100 μM test compound.

Example 26

[Ru(maltolato)3]

[0238] Available nitric oxide was reduced by 71% at 100 μM.

4TABLE 4% Decrease of AvailableNitric OxideExample 127μM1250μM23100μM75Example 2100μM82Example 3100μM42Example 6100μM77Example 14100μM47Example 15100μM86Example 26100μM71

[0239] Ex-vivo Tests

Example 2

[0240] Application of test compound resulted in a dose-dependent vasoconstriction at 10 μM and 100 μM. This effect was reversible by washout with Krebs solution.

Example 3

[0241] Application of test compound resulted in a dose-dependent vasoconstriction at 10 μM and 100 μM. This effect was reversible by washout with Krebs solution.

Example 14

[0242] Application of test compound resulted in a dose-dependent vasoconstriction at 10 μM and 100 μM. This effect was reversible by washout with Krebs solution.

Example 15

[0243] Application of test compound resulted in a dose-dependent vasoconstriction at 10 μM and 100 μM. This effect was reversible by washout with Krebs solution.

Example 26

[0244] Application of test compound resulted in a dose-dependent vasoconstriction at 10 μM and 100 μM and 1000 μM. This effect was reversible by washout with Krebs solution.

5TABLE 5% VasoconstrictionExample 210μM20100μM69Example 310μM17100μM59Example 1410μM11100μM40Example 1510μM16100μM86Example 2610μM10100μM181000μM25

[0245] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be incorporated within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

[0246] Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these documents.

	Number	Date	Country
Parent	09175028	Oct 1998	US
Child	09802523	Mar 2001	US
Parent	08602814	Feb 1996	US
Child	09175028	Oct 1998	US

	Number	Date	Country
Parent	09802523	Mar 2001	US
Child	10017764	Dec 2001	US

Use of nitric oxide scavengers to modulate inflammation and matrix metalloproteinase activity

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