WO2007110755A1

WO2007110755A1 - Prophylaxis or treatment of cardiovascular inflammation

Info

Publication number: WO2007110755A1
Application number: PCT/IB2007/000778
Authority: WO
Inventors: Peter Lay; Trevor Hambley
Original assignee: Medical Therapies Ltd
Current assignee: Anagenics Ltd
Priority date: 2006-03-27
Filing date: 2007-03-27
Publication date: 2007-10-04
Anticipated expiration: 2008-09-27

Abstract

There is described, methods for the prophylaxis or treatment of cardiovascular inflammation in a mammal, comprising administering a therapeutically effective amount of a complex of a metal and a carboxylate, or a derivative of a carboxylate, having anti-inflammatory activity.

Description

PROPHYLAXIS OR TREATMENT OF CARDIOVASCULAR

INFLAMMATION

FIELD OF THE INVENTION

The invention relates to the prophylaxis or treatment of cardiovascular diseases and conditions. In particular, the present invention relates to a method for the prophylaxis or treatment of cardiovascular inflammation comprising the administration of a metal complex having anti-inflammatory activity.

BACKGROUND

Non-steroidal anti-inflammatory drugs (NSAIDs) are used in the treatment of a variety of inflammatory conditions. NSAIDs are, for example, used to treat inflammatory conditions such as rheumatoid arthritis, osteoarthritis, acute musculoskeletal disorders (such as tendonitis, sprains and strains), lower back pain (commonly referred to as lumbago), and inflammation, pain and edema following surgical or non-surgical procedures. However, many NSAIDs cause adverse side effects, particularly adverse gastrointestinal (GI) effects.

Indomethacin (IndoH) is a NSAID effective in treating inflammatory conditions in both humans and animals. The structure of indomethacin is as follows:

However, oral administration of indomethacin can cause ulcerations in the oesophagus, stomach, duodenum and intestines. In dogs, oral administration of indomethacin causes fatal gastrointestinal haemorrhaging. Other effects associated with oral administration of indomethacin include: (a) inhibition of platelet aggregation, (b) adverse cardiovascular effects (fluid retention and peripheral oedema), (c) ocular effects (corneal deposits and retinal disturbances), (d) central nervous system effects (headaches and dizziness), (e) masking of infections due to antipyretic properties, and (f) renal effects (as with other NSAIDs, there have been reports of acute interstitial nephritis with hematuria, proteinuria and, occasionally, nephrotic syndrome in patients receiving long-term administration of indomethacin). The literature further indicates that administration of indomethacin e.g. as a suppository or by topical application can have adverse effects. ("Anti-inflammatory activity of Indomethacin following topical application", Amico-Roxas, M., Mater, M., Caruso, A., Puglisi, G., Bernadini, R., Rinaldo, G., Eur. Rev. Med. Pharmacol. Sd., 1982, IV, 1999, 204). Such adverse effects have limited the use of indomethacin in the treatment of inflammation in humans and animals.

It has been reported that dinuclear metal complexes of indomethacin (i.e. complexes containing two metal coordination centres) cause less adverse gastrointestinal effects, and result in improved uptake of the drug, compared to the free indomethacin. The ligand N,iV-dimethylformamide (DMF) is toxic to humans and animals, and irritates the eyes, skin and respiratory system, causes nausea, vomiting and colic, liver damage, hepatomegaly, hypertension and dermatitis. However, the oral administration of the dinuclear copper(II) complex of indomethacin, bis(Λζ7V-dimethylformamide)tetrakis-μ- (O, O -Indo)dicopper(II) ([Cu₂(Indo)₄(DMF)₂]), has been found to cause less gastrointestinal toxicity than indomethacin. The mechanism of the reduced gastrointestinal toxicity has not been elucidated, but may be due to reduced interaction of the indomethacin with the COX-I enzyme in the gastrointestinal tract. Compositions containing this complex in the form of tablets or paste are sold under the name Cu- Algesic and have been used in veterinary practice in Australia, New Zealand, South

Africa and other countries.

The NSAID aspirin (acetylsalicylic acid) is widely used in low dosages to prevent cardiovascular events and is generally prescribed as a standard treatment for prophylaxis of cardiac disease in high-risk patients. However, the effect of aspirin is not consistent with a significant proportion of the population (up to 45%) being aspirin resistant ("Aspirin resistance: Definitions, mechanisms, prevalence, and clinical significance", Macchi, L., Sorel, N., Christiaens, L., Curr. Pharm. Des., 2006, 12, 251- 258). A recent study also suggested that while aspirin may be beneficial in patients with recent myocardial infarction or multiple vascular risk factors, it may actually worsen outcomes in patients with chronic heart failure ("Aspirin use in chronic heart failure: What should we recommend to the practitioner?", Massie, B.M, J. Am. Coll. Cardiol., 2005, 46, 963-966). The use of aspirin as a prophylactic also presents considerable uncertainty as to appropriate dosing with the need to strike a balance between the potential protective effect in preventing myocardial infarction and stroke against the side-effect of the risk of significant bleeding ("Aspirin to prevent heart attack and stroke: what's the right dose?", Dalen, J.E., Am. J. Med., 2006, 119, 198-202). Moreover, it has recently been reported that some of the side-effects of low-dose aspirin treatment such as upper gastrointestinal bleeding and associated mucosal abnormalities, can be confused with heart disease itself ("Upper gastrointestinal mucosal abnormalities and blood loss complicating low-dose aspirin and antithrombotic therapy", Taha, A.S., Angerson, WJ., Knill- Jones, R.P., Blatchford, O., Aliment. Pharmacol. Ther., 2006, 23, 489-495).

A further study relating to administering aspirin with the NSAID ibuprofen suggested any cardiovascular benefit of aspirin may be undermined ("Ibuprofen may abrogate the benefits of aspirin when used for secondary prevention of myocardial infarction", Hudson, M., Baron, M., Rahme, E., Pilote, L., J. Rheumatol., 2005, 32, 1589-93), although other studies have found no evidence for a reduced cardioprotective effect of aspirin with such concomitant NSAID use ("Current use of nonsteroidal antiinflammatory drugs and the risk of acute myocardial infarction", Fischer, L.M., Schlienger, R.G., Matter, CM., Jick, H., Meier, C.R., Pharmacotherapy, 2005, 25, 503- 510). Indomethacin has recently been studied in the treatment of strokes and heart attacks ("Neurogenesis in Rats After Focal Cerebral Ischemia is Enhanced by Indomethacin", Hoehn, B.D., Palmer, T.D., Steinberg, G.K., Stroke, 2005, 36, 2718- 2724), and is approved for use worldwide for the treatment of a number of neonatal cardiac conditions. For example, it is used for the prophylaxis of intraventricular haemorrhage in premature infants ("Prophylactic indomethacin for prevention of intraventricular haemorrhage in premature infants", Bandstra, E.S., Montalvo, B. M., Goldberg R.N., Pediatrics, 1988, 82, 533-42) and as a treatment for closing patent ductus arteriosus ("Effectiveness and Side Effects of an Escalating, Stepwise Approach to Indomethacin Treatment for Symptomatic Patent Ductus Arteriosus in Premature Infants Below 33 Weeks of Gestation", Sperandio, M., Beedgen, B., Fenegerg, R., Huppertz, C₅ Brussau, J., Poschl, J., and Linderkamp, O., Pediatrics, 2005, 116, 1361- 1366).

While some studies have indicated that indomethacin may be useful for preventing certain abdominal aortic aneurysms in rats ("Indomethacin prevents elastase-induced abdominal aortic aneurysms", J Surg Res, 1996, 63, 305-9), other studies have found that indomethacin does not limit aneurysm in a rat model of abdominal aortic aneurysm ("Suppression of experimental aortic aneurysms: comparison of inducible nitric oxide synthase and cyclooxygenase inhibitors", Armstrong, PJ., Franklin, D.P., Carey, DJ., Elmore, J.R., Ann. Vase. Surg., 2005, 19, 248-257). Similarly, while it has been suggested that the non-specific NSAID naproxen may be cardio-protective ("Nonsteroidal anti-inflammatory drug use and acute myocardial infarction", Rahme, E., Pilote, L., LeLorier, J., Arch Intern Med., 2002, 162, 1111-1115), other studies have found no cardio-protective effect of naproxen ("Non-steroidal anti-inflammatory drugs and risks of serious coronary heart disease: an observational cohort study", Ray, W.A., Stein, CM., Hall, K., Daugherty, J.R., Griffin, M.R., The Lancet, 2002, 359, 118-123). Recently, there have been reports of increased risk of significant adverse cardiovascular side-effects associated with the long term use use of COX-2 selective NSAIDs. Data, for example, have suggested that COX-2 inhibitors such as rofecoxib, celecoxib, valecoxib and parecoxib may be associated with an increased risk of thrombotic events ("Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention", Scott, D. et al, NEJM, 2005, 352, 1071-1080; "Complications of the COX-2 inhibitors parecoxib and valdecoxib after cardiac surgery", NEJM, 2005, 352, 1081-1091; "Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial", NEJM, 2005, 352, 1092- 1102). The literature further indicates that at least some transition metals may have a role in the development of cardiovascular disease at the molecular level, hi particular, both copper and zinc have been shown to accumulate in atherosclerotic plaques at a higher rate than in surrounding vascular tissue ("Relationship of calcium, magnesium, zinc and copper concentrations in the arterial wall and serum in atherosclerosis obliterans and aneurysm", Iskra, M., Patelski, J., Majewski, W., J. Trace Elem. Med. Biol., 1997, 11, 248-252) suggesting that they play a role in cardiovascular pathogenesis.

In addition, while copper-containing enzymes such as superoxide dismutase and caeruloplasmin are considered beneficial for the cardiovascular system, it has been suggested that free copper ions can catalyse an oxidative modification of low density lipoprotein, which is seen as a key event in atherogenesis ("The possible role of copper ions in atherogenesis: the Blue Janus", Ferns, G. A., Lamb, DJ., Taylor, A.,

Atherosclerosis, 1997, 133, 443-445). Zinc has also been implicated as a potential contributor to cardiovascular disease through its role in metal-containing proteins such as the matrix metalloproteinases, the inhibition of which has become a recent goal for cardiovascular drug development ("Matrix metalloproteinases: a therapeutic target in cardiovascular disease", Sierevogel, MJ., Pasterkamp, G., de Kleijn, D.P., Strauss, B.H., Curr. Pharm. Des., 2003, 9, 1033-1044). As a result, cardiovascular inflammatory related diseases have been treated by seeking to lower copper and zinc levels within the cardiovascular system or to reduce the risk of accumulation of these metals in the cardiovasculature. More efficacious drugs and unproved delivery modes are needed for the treatment of cardiovascular diseases and conditions.

SUMMARY OF THE INVENTION

In a first aspect of the present invention there is provided a method for the prophylaxis or treatment of a cardiovascular inflammation in a mammalian subject, comprising treating the subject with a therapeutically effective amount of a complex of a metal and a carboxylate, or derivative of a carboxylate, having anti-inflammatory activity, wherein the carboxylate or derivative is other than salicylate or a derivative of salicylate.

The carboxylate or the derivative of the carboxylate having anti-inflammatory activity will generally have such activity when administered to a human or animal. However, in some embodiments, the derivative of the carboxylate can be hydrolysed in vivo, and the hydrolysed compound may have anti-inflammatory activity. The derivative of the carboxylate can be selected from the group consisting of a hydroxamate, hydroximate, amide or ester. These derivatives have functional groups that bind to one or more metal(s) as a monodentate ligand, a chelate and/or a bridging ligand.

Accordingly, in another aspect of the present invention there is provided a method for the prophylaxis or treatment of cardiovascular inflammation in a mammalian subject, comprising treating the subject with a therapeutically effective amount of a complex of a metal and a carboxylate, or a hydroxamate, hydroximate, amide or ester, having anti-inflammatory activity.

The carboxylate having anti-inflammatory activity can be any deprotonated carboxylic acid compound having anti-inflammatory activity. The carboxylate having anti-inflammatory activity may, for example, be the deprotonated anionic form of any one of the following carboxylic acids NSAIDs:

Suprofen ((+)-α-methyl-4-(2-thienylcarbonyl)phenylacetic acid ("SupH"));

Tolmetin (l-memyl-5-(p-toluoyl)-lH-pyrrole-2-acetic acid ("ToIH")); Naproxen (6-meώoxy-α-methyl-2-naphthaleneacetic acid ("NapH")); Ibuprofen ((+)-α-methyl-4-(isopropyhnethyl)benzeneacetic acid ("IbuH")); Flufenamic Acid ((N-trifluoromethylphenyl)anthranilic acid ("FlufenH")); Niflumic Acid ((2-(3-trifluoromethyl)phenylamino)-3-pyridinecarboxylic acid

("NifH"));

Diclofenac (2-[(2,6-dichlorophenyl)amino]phenylacetic acid ("DicH"));

Indomethacin (l-(4-chlorobenzoyl)-5-methoxy-2-methyl-lH-indole-3-acetic acid ("IndoH")); Acemetacin (l-(4-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid carboxymethyl ester ("ACMH"))

Ketorolac ((±)-5-benzoyl-2,3-dihydro-lH-pyrrolizine-l-carboxylic acid, ("KetH") 2-amino-2-(hydroxymethyl)- 1,3 -propanediol); and Indobufen (4-(l ,3-dihydro-l-oxo-2H-isoindol-2-yl)-a-ethylbenzeneacetic acid ("Indob")) Indobufen, in addition to its anti-inflammatory effects, is used to prevent thrombosis in much the same way as aspirin, but suffers from the same problems as the other anti-inflammatory drugs in terms of GI toxicity at therapeutic doses, although the effects are not as severe as aspirin. (Endoscopic evaluation of the effects of indobufen and aspirin in healthy volunteers. Marzo A; Crestani S; Fumagalli I; Giusti A; Lowenthal D T, Am. J. Therapeutics (2004), 11, 98-102.

In this specification, the inclusion of the "H" at the end of an abbreviation for a carboxylate (e.g., any one of the carboxylic acids listed above) or eg., a hydroxamate, hydroximate, or amide, is used to refer to the uncharged form of the carboxylate or amide or the parent hydroxamic acid or its monodeprotonated hydroxamate form of the doubly deprotonated hydroximate. Accordingly, the abbreviation without the "H" is used to refer to the deprotonated anionic form. For example, "IndoH" refers to the uncharged form of indomethacin, and "Indo" is used to refer to the deprotonated anionic form of indomethacin. Similarly, "ACMH" refers to the uncharged form of acemetacin and "ACM" refers to the deprotonated anionic form.

The carboxylate, or hydroxamate, hydroximate, ester or amide derivative having anti-inflammatory activity can be any non-steroidal anti-inflammatory drug (NSAID). For example, the NSAID can be indomethacin (IndoH), or an ester derivative of indomethacin, such as acemetacin, ibuprofen, indobufen, diclofenac, vaproxen, or ketorolac, or a hydroxamate, hydroximate, or amide derivative of indomethacin or acemetacin or other NSAID. The terms hydroxamate or hydroximate are to be taken to mean the deprotonated and doubly deprotonated forms of the ligands. Further NSAIDs that can be utilized in the metal complexes (as can their derivatives) as described herein include: Carprofen (6-chloro-a-methyl-9H-carbazole-2-acetic acid);

Etodolac (1 ,8-diethyl-l ,3,4,9-tetrahydro-pyrano[3,4-b]indole-l -acetic acid);

Fentiazac (4-(4-chlorophenyl)-2-phenyl-5-thiazoleacetic acid);

Flurbiprofen (2-fluoro-a-methyl-[l,r-biphenyl]-4-acetic acid);

Ketoprofen (3-benzoyl-a-methylbenzeneacetic acid); Oxaprozin (4,5-diphenyl-2-oxazolepropanoic acid);

Pranoprofen (a-methyl-5H-[l]benzopyrano[2,3-b]pyridine-7-acetic acid); Sulindac ((lZ)-5-fluoro-2-methyl-l-[[4-(niethylsulfinyl)phenyl]methylene]- lH-indene-3 -acetic acid); and

Suxibuzone (butanedioic acid, l-[(4-butyl~3,5-dioxo-l,2-diphenyl-4- pyrazolidinyl)methyl] ester).

Acemetacin, 1 -(4-chlorobenzoyl)-5-methoxy-2-methylindole-3 -acetic acid carboxymethyl ester, is a glycolic acid ester of indomethacin. The structure of acemetacin is shown below as is the structure for ketorolac.

Acemetacin Indomethacin

Ketorolac The metal complex used in a method embodied by the invention may be any complex comprising at least one metal ion and at least one carboxylate, or hydroxamate, hydroximate, ester or amide derivative having anti-inflammatory activity. As will be apparent to a person skilled in the art, the carboxylate or hydroximate, hydroxamate, ester or amide derivative can be coordinated with the metal ion via the carboxylate, hydroximate, hydroxamate, amide and other groups attached to the amide or ester linkages, such as sugars, amino acids, peptides, chelates containing heterocycles, and other chelating ligands.

The metal complex may be a mononuclear, dinuclear, or trinuclear metal complex, or a metal complex having higher nuclearity, or an oligomeric or polymeric complex containing one or more metal centres and one or more carboxylates, or derivatives of a carboxylate (eg., hydroxamate, hydroximate, ester, and amide ligands) having anti-inflammatory activity.

Typically, the complex includes other ligands in addition to the carboxylate or carboxylate derivative(s) having anti-inflammatory activity. These other ligands can also have anti-inflammatory and/or anti-cancer activities.

In some embodiments, the complex is one of the following complexes (eg., see Copper Complexes of Non-steroidal Anti-inflammatory Drugs: An Opportunity yet to be Realized Weder, J. E.; Dillon, C. T.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; Biffin, J. R.; Regtop, H. L.; Davies, N. M. Coord. Chem. Rev. 2002, 232, 95-126).

Where ^a Suprofen = (+)-α-methyl-4-(2-thienyl-carbonyl)phenylacetic acid (SupH); ^b Tolmentin = l-methyl-5-(/?-toluoyl)-lH-pyrrole-2-acetic acid (ToIH);

⁰ DMSO = dimethylsulfoxide; ^d Naproxen = 6-methoxy-α-methyl-2-naphthaleneacetic acid (NapH); ^e Ibuprofen = (+)-α-methyl-4-(isopropylmethyl)benzeneacetic acid (IbuH);

^•^Metronidazole = 2-methyl-5-nitrobenzimidazole ^g Flufenamic Acid = (iV-trifluoromethylphenyl)anthranilic acid (FlufenH); ^h Niflumic Acid = 2-(3-trifluoromethyl)phenylamino)-3-pyridinecarboxylic acid (NifH);

¹ Indomethacin = l-(4-chlorobenzoyl)-5-methoxy-2-mefhyl-lH-indole-3-acetic acid (IndoH);

^J Diclofenac = 2-[(2,6-dichlorophenyl)amino]phenylacetic acid (DicH); and

M^a is a metal ion and in at least some enbodiments, a transition metal ion. M^a can, for example, be a copper ion. In other embodiments, the complex can be any one of the complexes referred to in the table above in which the metal ion is a transition metal ion other than copper (eg zinc, nickel, ruthenium, iron, cobalt ions, and preferably zinc or ruthenium). See for instance, Copper and Zinc Complexes as Anti- Inflammatory Drugs. Dillon, C. T.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; Weder, J. E.; Zhou, Q. in "Metal Ions and Their Complexes in Medication", Vol. 41 of 'Metal Ions in Biological Systems'; Sigel, A.; Sigel, H., Eds.; M. Defcker, Inc., New York & Basel, 2004, Ch. 8, pp 253-27; Syntheses and Characterization of Anti-inflammatory Dinuclear and Mononuclear Zinc Indomethacin Complexes. Crystal Structures of [Zn₂(Indomethacin)₄(L)₂] (L = N,N-dimethylacetamide, Pyridine, l-Methyl-2- pyrrolidinone) and [Zn(Indomethacin)₂(L₁)₂] (L₁ = Ethanol, Methanol). Zhou, Q.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; Turner, P.; Warwick, B.; Biffin, J. R.;

Regtop, H. L. Inorg. Chem. 2000, 39, 3742-3748; XAFS Studies of Anti-inflammatory Dinuclear and Mononuclear Zn(II) Complexes of Indomethacin. Zhou, Q.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A. Inorg. Chem. 2003, 42, 8557-8566; NMR Spectroscopic Characterization of Copper(II) and Zinc(II) Complexes of Indomethacin. Ramadan, S.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A. Inorg. Chem. 2004, 43, 2943- 2946. Zhou, Q, PhD Thesis, University of Sydney, 2001).

In some embodiments, the complex is a mononuclear, dinuclear, trinuclear or polynuclear complex of a metal (where each metal of the complex is independently selected) and a ligand of the formula L²:

wherein:

R¹ is H or halo (i.e. Cl, F, Br or I); R² is H; a C₁ to C₆ alkyl, an alkenyl or an alkynyl, where the C₁ to C₆ alkyl, alkenyl or alkynyl may be optionally substituted; or

wherein each R^2A is independently selected from the group consisting of H, C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted; R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN, -OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH₃, -SCH₃ or -CH₂CH₃ may be optionally substituted; and n is 1, 2, 3, 4 or 5. When R² is a C₁ to C₆ alkyl, an alkenyl or an alkynyl, the C₁ to C₆ alkyl, alkenyl or alkynyl may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂.

When R^2A is a C₁ to C₆ alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or an arylalkyl, the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂.

When R⁵ is -CH₃, -OCH_3, -SCH₃ or -CH₂CH₃, the -CH₃, -OCH₃, -SCH₃ or - CH₂CH₃ may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂.

The complex may be a complex of formula (1), (2) or (3):

[M(L²)₂L_m]^p (1)

where M is a divalent or trivalent metal ion,

L is a ligand as defined above: each L is independently selected and is a monodentate ligand; m is 1 or 2; and p is the charge of the complex;

[M₂(L²)₄L_m]^p (2)

where each M is independently selected and is a divalent or trivalent metal ion; L² is a ligand as defined above: each L is independently selected and is a monodentate ligand; m is 0, 1 or 2; and p is the charge of the complex;

[M'₃O(L²)₆L₃]^P (3)

where each M' is independently selected and is a trivalent or tetravalent metal ion;

L² is a ligand as defined above: each L is independently selected and is a monodentate ligand; and p is the charge of the complex. In formulas (1), (2) and (3), when R² is a C₁ to C₆ alkyl, an alkenyl or an alkynyl, the C₁ to C₆ alkyl, alkenyl or alkynyl may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂.

In formulas (1), (2) and (3), when R^2Ais a C₁ to C₆ alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or an arylalkyl, the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂.

In formulas (1), (2) and (3), when R⁵ is -CH₃, -OCH_3, -SCH₃ Or-CH₂CH₃, the -CH₃, -OCH₃, -SCH₃ or -CH₂CH₃ may be substituted with one or more substituents.

The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂. R¹ is typically H.

R³ is typically H.

R² is typically CH₃.

In at least some embodiments, L² is ACM. M may be any divalent or trivalent metal ion. M is preferably copper ion, zinc ion, cobalt ion, nickel ion, chromium ion, molybdenum ion, tungsten ion or ruthenium ion. More preferably, M is copper ion.

M' may be any trivalent or tetravalent metal ion. M' is preferably iron ion, vanadium ion, manganese ion, chromium ion or ruthenium ion, and more preferably iron ion or ruthenium ion.

The ligand L may be any monodentate ligand. L may be a charged or uncharged ligand. L may for example be water, an alcohol, ΛζiV-dimethylformamide (DMF), iV-methylpyrrolidone, dimethylsulfoxide or ΛyV-dimethylacetamide (DMA).

The complexes of formula (1), (2) or (3) may be dissolved in a solvent, or may be in the form of a solid. Crystals of a complex of formula (1), (2) or (3) may include solvents of crystallisation. Crystals of a complex of formula (1), (2) or (3) may also include waters of crystallisation.

When the complex of formula (1), (2) or (3) is charged, a counter ion will be present in crystals of the complex. In further embodiments, the complex is a mononuclear complex of the following foπnula (4):

IML^Uf (4)

where

M is a divalent or trivalent metal ion, L¹ is a ligand of the formula:

wherein:

wherein each R^2A is independently selected from the group consisting of H, C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted; R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN, -OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH₃, -SCH₃ or -CH₂CH₃ may be optionally substituted; and n is i, 2, 3, 4 or 5; each L is independently selected and is a monodentate ligand; m is 1 or 2; and p is the charge of the complex;

In some embodiments, the mononuclear complex of formula (4) is a complex of formula (4A):

[M(η²-L¹)₂L₂;|^P (4A) where

M is a divalent or trivalent metal ion;

"η²-L^1?' is a bidentate ligand of the formula L¹:

wherein:

R¹ is H or halo (i.e. Cl, F, Br or I);

R² is H; a C₁ to C₆ alkyl, an alkenyl or an alkynyl, where the C₁ to C₆ alkyl, alkenyl or alkynyl may be optionally substituted; or

wherein each R ,2A is independently selected from the group consisting of H, C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted;

R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN, -OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH_3, -SCH₃ or -CH₂CH₃ may be optionally substituted; n is 1, 2, 3, 4 or 5; each L is independently selected and is a monodentate ligand; and p is the charge of the complex.

In formulas (4) and (4A), when R is a C₁ to C₆ alkyl, an alkenyl or an alkynyl, the C₁ to C₆ alkyl, alkenyl or alkynyl may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂.

In formulas (4) and (4A), when R^2Ais a C₁ to C₆ alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or an arylalkyl, the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂.

In formulas (4) and (4A), when R⁵ is -CH₃, -OCH₃, -SCH₃ or -CH₂CH₃, the - CH₃, -OCH_3, -SCH₃ or -CH₂CH₃ may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂.

In formulas (4) and (4A), R¹ is typically H.

In formulas (4) and (4A), R³ is typically H.

In formulas (4) and (4A), R² is typically CH₃.

In formulas (4) and (4A), each R⁵ is typically halo (i.e. F, Cl, Br or I), and n is typically 1, 2 or 3.

In formulas (4) and (4A), L¹ may for example be Indo.

In formulas (4) and (4A), M may be any divalent or trivalent metal ion. M is preferably copper ion, zinc ion, cobalt ion, nickel ion, chromium ion, molybdenum ion, tungsten ion or ruthenium ion. More preferably, M is copper ion or zinc ion. In other embodiments, the complex is a dinuclear complex of the formula (5):

[M^μ-L^Uf (5)

wherein each M is independently selected and is a divalent or trivalent metal ion, μ-L¹ is a ligand of the formula L¹:

where

R¹ is H or halo (i.e. Cl, F, Br or I);

wherein each R^2A is independently selected from the group consisting of H, C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted;

R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN,

-OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH₃, -SCH₃ or -CH₂CH₃ may be optionally substituted; and n is 1, 2, 3, 4 or 5; each L is independently selected and is a monodentate ligand; m is O, 1 or 2; and p is the charge of the complex.

In formula (5), when R² is a C₁ to C₆ alkyl, an alkenyl or an alkynyl, the C₁ to C₆ alkyl, alkenyl or alkynyl may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂.

In formula (5), when R^2Ais a C₁ to C₆ alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or an arylalkyl, the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂.

In formula (5), when R⁵ is -CH₃, -OCH₃, -SCH₃ or -CH₂CH₃, the -CH₃, -OCH₃₎ -SCH₃ or -CH₂CH₃ may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂. In formula (5), R¹ is typically H.

In formula (5), R³ is typically H.

In formula (5), R² is typically CH₃.

In formula (5), L¹ may for example be Indo. In formula (5), M is any divalent or trivalent metal ion. M is typically selected from copper ion, zinc ion, cobalt ion, nickel ion, chromium ion, molybdenum ion, tungsten ion and ruthenium ion. Preferably, M is copper ion.

In formula (5), the ligand L may be any monodentate ligand. L may, for example, be water (OH₂), an alcohol, dimethylsulfoxide (DMSO), tetrahydrofuran (THF), or L may be a ligand containing a tertiary amide or cyclic tertiary amide.

In other embodiments, the complex is a trinuclear complex of the following formula (6):

[M'₃O(μ-L¹)₆L₃]^p (6)

where each M' is independently selected and is a trivalent or tetravalent metal ion; and μ-L¹, L and p are as defined above for formula (5).

In further embodiments, the complex is a trinuclear complex of the following formula (7):

[Zn₂M(L³)₆(L⁴)₂] (7) where:

M is a divalent metal ion;

L³ is a carboxylate having anti-inflammatory activity; L⁴ is an optionally substituted heterocyclic base comprising one or two heterocyclic rings independently having 5 or 6 ring members and 1 to 3 heteroatoms independently selected from N, O and S; wherein each L³ is independently selected; and each L⁴ is independently selected.

In the complex of formula (7), L³ can be a monodentate, bidentate or bridging ligand of formula L¹ or L² as follows:

wherein:

R¹ is H or halo (i.e. Cl, F, Br or I);

R is H; a C₁ to C₆ alkyl, an alkenyl or an alkynyl, where the C₁ to C₆ alkyl, alkenyl or alkynyl may be optionally substituted; or

wherein each R >2^A^A is independently selected from the group consisting of H, C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted; R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN, -OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH_3, -SCH₃ or -CH₂CH₃ may be optionally substituted; and n is i, 2, 3, 4 or 5. In the complex of formula (7), M can, for example, be selected from the group consisting of zinc ion, cobalt ion, nickel ion, magnesium ion, copper ion and calcium ion.

In some embodiments of the complex of formula (7), the heterocyclic base comprises one or more N atoms. In some embodiments, the heterocyclic base is optionally substituted. The heterocyclic base may, for example, be selected from the group consisting of isoquinolyl, quinolyl, piperidinyl, pyridinyl, 2-methylpyridinyl, imadazoyl, pyranyl, pyrrolyl, pyrimidinyl, indolyl, purinyl and quinolizinyl. Preferably, the heterocyclic base is quinolyl.

In other embodiments the metal complex is a complex of formula (8) as follows:

[M(L⁵)_m(L⁶)_nf (8) wherein

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁵ is independently selected and is a monodentate carboxylate, (preferably Indo, ACM or Keterolac) or an amide ligand (O or N bound) (preferably a derivative of Indo, ACM or Keterolac), having anti-inflammatory activity; each L⁶ is independently selected and is NH₃, a monodentate ligand, a polydentate ligand, or a macrocyclic ligand; m is 1, 2, 3 or 4 n is O, 1, 2, 3, 4 or 5; and p is the charge of the complex.

In some embodiments, the metal complex is a metal complex of formula (8 a) as follows:

[M(L⁵)_m(L⁶)_n]^p (8a) wherein

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁵ is independently selected and is NH₃, a monodentate ligand, a polydentate ligand, or a macrocyclic ligand; each L is independently selected and is a chelating derivative of a carboxylate such as a hydroximate, hydroxamate, hydrazine, ester, amino acid, peptide or sugar or amide chelating ligand (O or N bound), having anti-inflammatory activity; m is O, 1, 2, 3, 4, or 5; n is 1 , 2, 3 or 4; and p is the charge of the complex.

In other embodiments the metal complex is a complex of formula (9) as follows:

[M(L⁷)_ffi(L⁸)_n]^p (9) wherein

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁷ is independently selected and is NH₃, a monodentate ligand, a polydentate ligand, or a macrocyclic ligand; each L⁸ is independently selected and is a chelating derivative of a carboxylate such as a hydroximate, hydroxamate, amino acid, peptide or sugar, or amide ligand (O or N bound), having anti-inflammatory activity; m is O, 1, 2, 3, 4, or 5; n is 1, 2, 3 or 4; and p is the charge of the complex.

In other embodiments, metal complexes that can be used in methods embodied by the invention include metal complexes of formula (10) as follows:

[M_q(L¹UL²UL³)^ (10) wherein

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L¹ is independently selected and is NH₃, another monodentate ligand, a polydentate ligand, or a macrocyclic ligand; each L² is independently selected and is a chelating derivative of a carboxylate such as a hydroximate, hydroxamate, ester, amino acid, peptide or sugar or amide chelating ligand (O or N bound), having anti-inflammatory activity; each L is independently selected and is a bridging ligand, such as an oxo, hydroxo, carboxylate (including a NSAID), halide, or other bridging group, m is an integer from 0 to 5q; n is an integer from 1 to 2q; p is the charge of the complex; q is typically a number between 2 and 20 inclusive; and r is an integer from 1 to 60.

In still other embodiments, the metal complex is a complex of formula (11) as follows:

[M(L⁵)_o(L⁷)_m(L⁸)_n]^p (11) wherein

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁵ is independently selected and is a monodentate carboxylate or amide ligand (O or N bound), having anti-inflammatory activity; each L⁷ is independently selected and is a monodentate or a polydentate ligand; each L⁸ is independently selected and is a chelating derivative of a carboxylate, or amide ligand (O or N bound), having anti-inflammatory activity; o is 1, 2, 3, 4 or 5 m is 1, 2, 3 or 4; n is 1, 2, or 3; and p is the charge of the complex.

In other embodiments the metal complex is a complex of formula (12) as follows:

[M_q(L⁵)_m(L⁷)_n(L⁸)_rf (12) wherein M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁵ is independently selected and is a monodentate or bidentate carboxylate, or monodentate amide ligand (O or N bound), having anti-inflammatory activity; each L⁷ is independently selected and is a monodentate or a polydentate ligand; each L⁸ is independently selected and is a bridging ligand, such as an oxo, hydroxo, carboxylate (including a NSAID), halide, or other bridging group. m is an integer from 0 to 5q; n is an integer from 1 to 5q; p is the charge of the complex; q is typically an integer between 2 and 20 inclusive; and r is an integer from 1 to 60. One or more of the ligands L⁵ and L⁸ in any combination may also form dimeric, trimeric, tetrameric, oligomeric or polymeric complexes with one or more metal ions.

In all of the formula (I)-(12) or other complexes containing NSAID ligands and/or derivatives thereof useful in one or more embodiments of the present invention, the ancilliary ligands can be chosen from ligands that exert a separate anti-inflammatory activity.

In one or more embodiments of the invention, the metal complex may be administered to the mammalian subject alone or in combination with other anti- inflammatory drug(s) or treatments for cardiovascular inflammation. Such administration can be given to improve the response to the other anti-inflammatory drug(s) or treatment(s), or to reduce the dose of such other drug(s) to reduce toxic side- effects while maintaining a comparable efficacy.

It will also be understood the ligand(s) of the metal complex used in a method embodied by the invention may not have anti-inflammatory activity alone, the activity being provided by the combination of the metal and the ligand(s) in the complex.

Methods of the invention find broad application in the prophylaxis or treatment of cardiovascular diseases and conditions. Any cardiovascular disease or condition involving inflammation responsive to a metal-NSAID complex can be treated by a method as described herein. Hence, in another aspect of the invention there is provided a method for prophylaxis or treatment of a cardiovascular disease or condition in a mammalian subject, comprising administering to the subject a therapeutically effective amount of a metal complex and a carboxylate, or derivative of a cafboxylate, having antiinflammatory activity, wherein the carboxylate or derivative is other than salicylate or a derivative of salicylate.

The present invention also provides the use of a complex of a metal and a carboxylate, or a derivative of a carboxylate, having anti-inflammatory activity in the manufacture of a medicament for the prophylaxis or treatment of cardiovascular inflammation in a mammalian subject, the carboxylate or derivative being other than salicylate or a derivative of salicylate.

Methods embodied by the invention find application in the prophylaxis or treatment of cardiovascular diseases and conditions including, but not limited to, acute and chronic cardiovascular inflammation including as a result of surgery or other trauma, cardiovascular disease, angina pectoris, atheroma, atherosclerosis, arteriosclerosis, congestive heart failure, coronary heart disease, cardiomyopathy, myocardial infarction, stroke, ischeamic conditions, ischaemic cardiomyopathy, patent ductus arteriosus, high blood pressure, pulmonary hypertension peripheral artery disease, coronary artery disease, coronary artery spasm and pericarditis.

One of the issues associated with the use of non-selective NSAIDs such as indomethacin and their derivatives is gastrointestinal (GI) and renal activity. Metal complexes as described herein can be incorporated into formulations that minimize their decomposition by biological fluids, such as gastric acid, or to change the profile of absorption of the bioactives as exemplified in International Patent Application No. PCT/AU2005/000442, to reduce GI and/or renal toxicity while substantially maintaining or enhancing efficacy of the complexes. The use of all such formulations for administration of metal complexes as described herein is expressly encompassed. In one or more embodiments, chelating derivatives of NSAIDs can also enhance the stability of metal-NSAID complexes. This can result in one or more of:

(i) a reduction in GI toxicity by increasing the stability of the drugs in the

GI tract; (ii) slow release forms of the NSAIDs to improve efficacy and safety profiles;

(iii) water-soluble, slow release forms of the NSAIDs for intraveneous use; and/or (iv) the provision of chelates for delivering metal ions and other groups that are synergistic and/or enhance the efficacy of the parent NSAID in its mode of action.

The release of the NSAID from the ligand can be induced by hydrolysis of the ligand by cleaving the metal ligand bonds, and/or the ester or amide bonds; ligand substitution reactions; and/or redox catalysed substitution reactions. Once released, the NSAID derivative, the NSAID, and the metal may provide synergistic activities. For instance, the decomposition of the metal hydroximates/hydroximates can have multiple effects. As an example, a copper hydroxamate complex can exert anti-inflammatory activity by a combination of independent COX-2 inhibition (by both the parent NSAID and the NSAIDHAH₂), the release of NO from the NSAIDHAH₂, 5 -lipoxygenase inhibition by the hydroxamic acid, and the effects of Cu once the complex decomposes at a target site. Moreover, inert oxidation states of metals (e.g., Ru(III) or Co(III)) may substantially selectively target hypoxic regions such as is associated with many cardiovascular conditions. In at least some embodiments, metal ions, co-ligands and metal oxidation states of the metal complexes can be utilized to optimise the rate of release and/or hydrolysis of the NSAID-derivative (eg. at sites of hypoxia) to enable sufficient stability to target the disease site before the anti-inflammatory ligands of the metal complex are released. The use of metal NSAID complexes with a variety of different metal ions can allow allow a therapeutic regime to be tailored to the type of cardiovascular condition, its location, and for instance the severity of hypoxia at the intended site of action. For instance, labile and lipophilic metal complexes are particularly suitable for applications in which the formulation is administered to, or in the vicinity of, the site of action. For inert metal ions, and labile metal ions with chelating ligands, NS AID-ligand chelating derivatives can be tailored to target the tissue type, or co-ligands can be added to improve targeting. These complexes can also be provided in water-soluble form for optimized systemic delivery by intravenous injection or infusion.

Metal complexes used in at least some embodiments of the invention will have reduced toxicity associated side effects compared to ligand(s) in the metal complex (eg carboxylates, hydroxamates, hydroximates, esters and amides). In particular, the metal complex can have substantially less gastrointestinal and/or renal toxicity than the parent anti-inflammatory ligand. Moreover, by administering the metal complex in a pharmaceutical composition formulated to reduce dissociation of the metal from the complex to limit any toxicity normally associated with the free ligand, the metal complexes can be administered more safely at normal therapeutic doses, or in higher dosages (eg., for acute conditions) and/or over longer periods of time resulting in increased efficacy in treatment. Hence, advantageously, one or more methods embodied by the invention can provide an alternative prophylactic or therapeutic treatment for the treatment of cardiovascular diseases and conditions which avoid or reduce the adverse cardiovascular side-effects associated with the use of selective COX-2 NSAIDs.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in this specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed anywhere before the priority date of this application.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The features and advantages of methods of the present invention will become further apparent from the following detailed description of preferred embodiments.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 shows the chemical structure of [Cu2(Indo)₄(DMF)₂];

Figure 2 is a graph showing the extent of gastric ulceration measured in mm² in rats following administration of an equimolar Indo dose of IndoH or [Cu₂(Indo)₄(DMF)₂] in 2% w/w carboxymethylcellulose (CMC) compared with control rats treated with CMC only (n = 4; Mean±SEM). Ulceration was measured 3 h after dosing; Figure 3 is a graph showing gastric permeability following treatment of rats with equimolar Indo doses of IndoH or [CU₂(LKIO)₄(DMF)₂] in 2% w/w CMC compared with control rats treated with CMC only (n = 4; Mean±SEM). Permeability was determined by dosing the rats with the drug or the vehicle, then 0.5 mL of sucrose solution (0.5 g/mL) two hours later. The amount of sucrose in the urine was then determined in urine collected from 0-24 h after sucrose administration;

Figure 4 is a graph showing plasma haptoglobin following dosing of rats with equimolar Indo doses of IndoH and [Cu₂(Indo)₄(DMF)₂] in 2% w/w CMC compared with control rats treated with CMC only (n = 4; Mean±SEM). Plasma was obtained by cardiac puncture and the haptoglobin level was measured using a commercial kit (Dade Behring, Mannheim, Germany);

Figure 5 is a graph showing the extent of small intestinal ulceration following treatment of rats with equimolar Indo doses of IndoH or [Cu₂(Indo)₄(DMF)₂] in 2% w/w CMC compared with control rats treated with CMC only (n = 4; Mean+SEM). Ulceration was measured 24 h after dosing;

Figure 6 is a graph showing the extent of small intestine permeability as determined by the % ⁵¹Cr-EDTA excretion in urine collected for 0-24 h after dosing of rats with equimolar Indo doses of IndoH or [Cu₂(Indo)₄(DMF)₂] in 2% w/w CMC compared with control rats treated with CMC only (n = 4; Mean±SEM); Figure 7 is a graph showing the haemoglobin concentration in the caecum following treatment of rats with equimolar Indo doses of IndoH or [Cu₂(Indo)₄(DMF)₂] in 2% w/w CMC compared with control rats (no treatment) (n = 4, Mean±SEM);

Figure 8 is a graph showing the enteric bacteria numbers per gram of tissue following treatment of rats with equimolar Indo doses of IndoH or [Cu₂(Indo)₄(DMF)₂] in 2% w/w CMC compared with control rats (n = 4, Mean±SEM);

Figure 9 is a graph showing the extent of mitochondrial DNA damage in rat intestinal tissue after administration of single doses equimolar Indo doses of IndoH or [Cu₂(Indo)₄(DMF)₂] (n = 5, Mean±SEM);

Figure 10 is a graph showing N-acetyl-β-D-glucosaminidase (NAG) concentration in urine collected for 0-24 h after dosing of rats with equimolar Indo doses of IndoH or [Cu₂(Indo)₄(DMF)₂] in 2% w/w CMC compared with control rats (n = 4; Mean±SEM); Figure 11 is a graph showing the reduction in neutrophils (as a measure of reduction in acute coronary inflammation) compared to a saline control in a carotid arterial walls of a New Zealand White (NZW) rabbit model of arterial inflammation. The rabbits were infused with saline only (as an untreated control), saline plus lipid-free Al (ApoA-1, 8 mg/kg) as a positive control for an anti-inflammatory effect; or an oral gavage of an MCT organogel formulation containing IndoH (3 mg/kg), [Cu₂(Indo)₄(OH₂)₂] (1 or 3 mg/kg Indo equivalents) or [Cu(ACM)₂(OH₂)₂] (3 mg/kg Indo equivalents);

Figure 12 shows cross sections of arteries in the rabbit model of arterial inflammation showing neutrophil levels as the dark-stained regions following the treatment with drugs as outlined in Figure 11. The 1 and 3 after Cu-indo refer to doses of 1 or 3 mg/kg Indo equivalents, respectively;

Figure 13 is a graph showing endothelial V-CAM expression in the carotid arterial walls of a New Zealand White (NZW) rabbit model of arterial inflammation following treatment with undomodifϊed indomethacin (Indo; 3 mg/kg),

[Cu₂(Indo)₄(OH₂)₂] (Cu-Indo; 2 mg/kg and 3 mg/kg) , [Cu(ACM)₂(OH₂)₂] (Cu-ACM; 3 mg/kg) and [Zn(Indo)₂(OH₂)₂] (Zn-Indo; 3 mg/kg) compared to an untreated control group; and

Figure 14 is a graph showing endothelial I-CAM expression in the New Zealand White (NZW) rabbit model of arterial inflammation following the Indo, Cu-

Indo, Cu-ACM, Zn-Indo and Zn-ACM ([Zn(ACM)₂(OHb)₂] treatments.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION In this specification a reference to "copper indomethacin" or "Culndo" is a reference to [Cu₂(Indo)₄(DMF)₂]. Similarly, a reference to "Ibup" is a reference to Ibuprofen; a reference to "Im" is a reference to imidazole; a reference to "2-MeIm" is a reference to 2-methylimidazole; a reference to "Py" is a reference to pyridine; a reference to "3-pic" is a reference to 3-picoline; a reference to "4-pic" is a reference to 4-picoline; a reference to "Bim" is a reference to benzimidazole; a reference to

"IndoH" is a reference to indomethacin; a reference to "AcSHAH₂" is a reference to acetylsalicylhydroxamic acid; a reference to "SHAH₂" is a reference to salicylhydroxamic acid; a reference to "IndoHAEb" is a reference to indomethacin hydroxamic acid; a reference to "EtOAc" is a reference to ethyl-acetate; and the abbreviation "THF" is a reference to tetrahydrofuran; "AN" refers to acetonitrile; "Pyrro" refers to pyrrolidine; "DMA" refers to ΛζN-dimethylacetamide; "DMSO" refers to dimethylsulfoxide; "NMP" refers to iV-methylpyrrolidone; and "DMF" refers to ΛζiV-dimethylformamide.

In this specification, the term "halo" refers to fluoro, chloro, bromo or iodo. In this specification, the term "alkyl" used either alone or in a compound word such as "arylalkyl", refers to a straight chain, branched or mono- or polycyclic alkyl. Examples of straight chain and branched alkyl include methyl, ethyl, propyl, isø-propyl, butyl, zsø-butyl, sec-butyl, tert-buiyl, amyl, iso-amyl, sec-amyl, 1,2-dimethylpropyl, 1,1- dimethylpropyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1 ,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl. Examples of cyclic alkyl include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

In this specification, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic alkyl having 3 to 12 carbons.

In this specification, the term "alkenyl" refers to a straight chain, branched or cyclic alkenyl with one or more double bonds. Preferably the alkenyl is a C₂ to C₂₀ alkenyl, more preferably a C₂ to C₆ alkenyl. Examples of alkenyl include vinyl, allyl,

1-methylvinyl, butenyl, zsø-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methylcyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl, 1 ,4-pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,3-cyclohexadienyl, 1 ,4-cyclohexadienyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl and 1,3,5,7-cyclooctatetraenyl.

In this specification, the term "alkynyl" refers to a straight chain, branched or cyclic alkynyl with one or more triple bonds, preferably a C₂ to C₂o alkynyl, more preferably a C₂ to C₆ alkynyl. In this specification, the term "aryl" used either alone or in compound words such as "arylalkyl", refers to a radical of a single, polynuclear, conjugated or fused aromatic hydrocarbon or aromatic heterocyclic ring system. Examples of aryl include phenyl, naphthyl and furyl. When the aryl comprises a heterocyclic aromatic ring system, the aromatic heterocyclic ring system may contain 1 to 4 heteroatoms independently selected from N, O and S and may contain up to 9 carbon atoms in the ring. In this specification, the term "arylalkyl" refers to an alkyl substituted with an aryl group. An example of arylalkyl is benzyl.

In this specification, the term "bidentate ligand" refers to a ligand having two co-ordination bonds to a metal atom. Bidentate ligands include unsynimetric bidentate ligands with one weaker and one relatively stronger bond to the metal atom. In this specification, the term "monodentate ligand" refers to a ligand having a single co-ordination bond with a metal atom.

The present invention stems from the recognition that at least some metal complexes and in particular complexes of a metal and a carboxylate, hydroxamate, hydroximate, ester or amide having anti-inflammatory activity are effective in the prophylaxis or treatment of inflammation associated with cardiovascular diseases and conditions, and can be more effective in treating cardiovascular inflammation in terms of efficacy and/or safety than the carboxylate, hydroxamate, hydroximate, ester or amide having anti-inflammatory activity itself. For example, complexes of a metal and indomethacin may be more effective in the prophylaxis or treatment of cardiovascular inflammation, in terms of efficacy and/or safety, than indomethacin itself.

The metal complex used in the method of the invention may, for example, be any of the complexes set out in following Table 1. It will also be understood that copper ion may be substituted with another transition metal ion (eg zinc, nickel, or cobalt ions as described in Copper and Zinc Complexes as Anti-Inflammatory Drugs. Dillon, C. T.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; Weder, J. E.; Zhou, Q. in "Metal Ions and

Their Complexes in Medication", Vol. 41 of 'Metal Ions in Biological Systems'; Sigel, A.; Sigel, H., Eds.; M. Dekker, Inc., New York & Basel, 2004, Ch. 8, pp 253-27; Syntheses and Characterization of Anti-inflammatory Dinuclear and Mononuclear Zinc Indomethacin Complexes. Crystal Structures of [Zn₂(Indomethacin)₄(L)₂] (L = N,N- dimethylacetamide, Pyridine, l-Methyl-2-pyrrolidinone) and [Zn(Indomethacin)₂(L₁)₂]

(L₁ = Ethanol, Methanol). Zhou, Q.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; Turner, P.; Warwick, B.; Biffin, J. R.; Regtop, H. L. Inorg. Chem. 2000, 39, 3742-3748; XAFS Studies of Anti-inflammatory Dinuclear and Mononuclear Zn(II) Complexes of Indomethacin. Zhou, Q.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A. Inorg. Chem. 2003, 42, 8557-8566; NMR Spectroscopic Characterization of Copper(II) and Zinc(II) Complexes of Indomethacin. Ramadan, S.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A. Inorg. Chem. 2004, 43, 2943-2946. Zhou, Q, PhD Thesis, University of Sydney, 2001).

Where ^a Suprofen = (+)-α-methyl-4-(2-thienyl-carbonyl)phenylacetic acid (SupH); ^b Tolmentin = l-methyl-5-(p-toluoyl)-lH-pyrrole-2-acetic acid (ToIH); ^c DMSO = dimethylsulfoxide; ^d Naproxen = β-methoxy-α-methyl-l-naphthaleneacetic acid (NapH); ^e Ibuprofen = (+)-α-methyl-4-(isopropylmethyl)benzeneacetic acid (IbuH);

^Metronidazole = 2-methyl-5-nitrobenzimidazole ^s Flufenamic Acid = (TV-trifluoromethylphenyl)anthranilic acid (FlufenH); ^h Niflumic Acid = 2-((3-trifluoromethyl)phenylamino)-3-pyridinecarboxylic acid (NifH);

¹ Indomethacin = l-(4-chlorobenzoyl)-5-methoxy-2-methyl-lH-indole-3-acetic acid (IndoH);

⁷ Diclofenac = 2-[(2,6-dichlorophenyl)amino]phenylacetic acid (DicH).

Further suitable NSAIDs include: Carprofen (6-chloro-a-methyl-9H-carbazole-2-acetic acid);

Etodolac (l,8-diethyl-l,3,4,9-tetrahydro-pyrano[3,4-b]indole-l-acetic acid);

Fentiazac (4-(4-chlorophenyl)-2-phenyl-5-thiazoleacetic acid);

Flurbiprofen (2-fluoro-a-methyl-[l,r-biphenyl]-4-acetic acid);

Pranoprofen (a-methyl-5H-[l]benzopyrano[2,3-b]pyridine-7-acetic acid);

Sulindac ((I Z)-5-fluoro-2-methyl-l -[[4-(methylsulfinyl)phenyl]methylene]- lH-indene-3 -acetic acid); and Suxibuzone (butanedioic acid, l-[(4-butyl-3,5-dioxo-l,2-diphenyl-4- pyrazolidinyl)methyl] ester).

Metal complexes useful in methods embodied by the invention can be prepared by methods known in the art, or prepared by methods described below. Methods known in the art are described in, for example, United States Patent No. 5,466,824 or the paper: Anti-inflammatory Dinuclear Copper(II) Complexes with Indomethacin. Synthesis, Magnetism and EPR Spectroscopy; Crystal Structure of the ΛζiV-Dimethylformamide Adduct. Weder, J. E.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; MacLachlan, D.; Bramley, R.; Delfs, C. D.; Murray, K. S.; Moubaraki, B.; Warwick, B.; Biffin, J. R.; Regtop, H. L. Inorg. Chem. 1999, 38, 1736-1744.

The complex can be a complex comprising at least one metal ion and at least one hydroximate, hydroxamate, ester or amide derivative of a carboxylate NSAID having anti-inflammatory activity. A hydroxamic acid having anti-inflammatory activity can form hydroxamato or hydroximato complexes with a metal ion in the complex. An amide having anti-inflammatory activity can for example, also form chelates of deprotonated amides or amide monodentate complexes with a metal ion in the complex.

Inert metal complexes (eg. see formula 8) incorporating carboxylate ligands having inflammatory activity can be prepared by methods known in the art, or as described below. Such reactions include the substitution of a leaving group in an inert metal complex with a carboxylate group of an NSAID, or an amide group in a NSAID or an amide derivative of a NSAID. This is exemplified by Example 1 in the preparation of [Co(NH₃)₅(Indo)]X₂ as follows:

[M(Y)_m(L⁶)_n]^p + HiR⁶CO₂(H) → [M(L⁵)_m(L⁶)_n]^p + mY(H),

[M(Y)_m(L⁶)_n]^p + InR⁶CONR⁷R⁸ → [M(L⁵)_m(L⁶)_n]^p + mY,

where R⁶CO₂ ^" = NSAID = L⁵, M = Co(III), Cr(III), Ir(III), Os(III), Rh(III), Ru(III), Pt(IV), preferably Co(III) or Ru(III). In general, such complexes can be prepared by substitution of a weakly coordinated, trifluoromethanesulfonato, or solvent, or other such ligands, as described for example in, Introduction to Trifluoromethanesulfonates and Trifluoromethanesulfonato-0 Complexes. Dixon, N. E.; Lawrance, G. A.; Lay, P. A.; Sargeson, A. M.; Taube, H. "Inorganic Synthesis" Schreeve, J. M.; Ed.; Wiley: New York, NY, 1986; Vol. 24, 243-250.

In addition, embodiments of metal complexes of the invention can be prepared by reaction of a derivative OfR⁶COZ with a hydroxo or deprotonated amine ligand on the metal, for example:

[M(OH)_m(L⁶)_n]^p + mR⁶COZ → [M(L⁵)_m(L⁶)_n]^p + mZ

[M(NR⁷R⁸)_m(L⁶)_n]^p + KiR⁶COZ → [M(L⁵)_m(L⁶)_n]^p + mZ.

An example of these synthetic routes is described in: Song, R.; Kim, Kwan M.; Lee, S, S.; Sohn, Y. S. Electrophilic Substitution of (Diamine)tetrahydroxo- platinum(IV) with Carboxylic Anhydrides. Synthesis and Characterization of (Diamine)platinum(IV) Complexes of Mixed Carboxylates. Inorg. Chem. (2000), 39, 3567-3571.

It will also be understood that metal complexes embodied by the invention can be prepared by other methods including substitution reactions of non-NSAID ligands to give new complexes. Further derivatives of carboxylic acids that can be employed in metal complexes embodied by the invention also include esters of carboxylates having antiinflammatory activity, and amide derivatives of carboxylates that bind in a monodentate fashion to the metal. The esters and amides can contain heterocyclic groups or aliphatic or aromatic groups that contain other functional groups that bind to the inert metal in a monodentate fashion. A monodentate amide ligand can bind via O to the metal ion or deprotonate and bind via N to a metal ion in the complex, as described in: Fairlie, D. P.; Ilan, Y.; Taube, H. Oxygen versus Nitrogen Bonding of Carboxamides to Pentaammineruthenium(II/III) Inorg. Chem. (1997), 36, 1029-1037. Oxygen and nitrogen-bound forms of the amide complexes can be interconverted by a change pH or other means of protonation/deprotonation reactions, for example:

[M(OC(R⁶)NHR⁸)_m(L²)_n]^p + mX² → [M(NR⁸ (COR⁶))_m(L²)_n]^p-^m + mHX² In the above chemical equations, X is a conjugate base of a strong or a weak acid (eg., X can be a halide, oxyanion, carboxylate, sulfonate, etc.); X² is a conjugate base of a weak acid, examples of which include oxyanions, carboxylates, amines, and N- heterocycles; Y is a leaving group, examples of which include halo, alkylsulfonato, O- bound sulfoxides, O-bound amides, aldehydes, ketones, and nitrate ligands; and R COZ is an acyl halide, anhydride or ester derivative of a ΝSAID.

Metal complexes embodied by the invention can also be prepared by methods outlined in Example 1 below. In at least some forms, the complexes contain Indo, ACM, ketorolac or derivatives of Indo, ACM or ketorolac ligands as described above, or amide or ester derivatives of these or other NS AIDs. The functional groups of the ligands can themselves bind to the metal ion, and/or other ligating groups that are linked by these functionalities can bind to the metal.

In some embodiments, any R group (eg., alkyl, aryl etc) of an amide derivative can also contain donor functional group(s) that form a co-ordination bond with a metal ion. Such functional groups include, for instance, a carboxylate group of an amino acid or peptide derivative of a NSAID, or a RS^", thioether, phenol, amine, or N-heterocyclic side-chain of such an amino acid or peptide derivative. It will also be understood that a large variety of other functional groups would be suitable.

Hydroxamic acids having anti-inflammatory activity that may be utilised in the complexes used in the method of the invention include those of the type described in

International Patent Application No. WO 2004/000215 comprising a NS AID covalently linked by a linker to a hydroxamate. Hydroxamic acid derivatives of carboxylates having anti-inflammatory activity, such as Indo and esters of Indo (e.g. ACM), are particularly preferred. The equilibrium between a hydroxamato and the hydroximato complex may change with pH as shown below in Scheme 1. As such, the terms hydroxamato and hydroximato in the context of the present invention are interchangeable. In scheme 1, RCO₂H is the parent carboxylic acid with anti-inflammatory activity.

R = AIk or Ar hydroxamato hydroximato complex complex

Scheme 1

Amide derivatives of carboxylic acids having anti-inflammatory activites can be prepared as described in International Patent Application No. WO 95/04030, or modifications thereof. See, for instance, the indomethacin example below (Scheme 2). In this example, R may be a proton, alkyl or aryl group, in which case the ligand would be a monodentate O or N donor to the metal. Alternatively, R may contain one or more functional groups that could act as other donor groups to form a metal chelate. Suitable coupling reactions include those with amino acids to form a mixed amide/carboxylate donor set (or a mondentate or bidentate carboxylate donor only), or more complex donor bidentate sets with amino acids containing metal binding side-chains, e.g., cysteine, serine, methionine, histidine, tyrosine, etc. Other suitable R groups include amino sugar derivatives and glycoproteins that target tumour cells. The coupling reaction may also involve short chain peptides, which can act as chelating ligands, or other groups to give metal chelators with anti-inflammatory activities, as described in WO 95/04030.

Scheme 2

Ester derivatives of carboxylic acids having anti-inflammatory activites may be prepared by a variety of ester coupling reactions. See, for instance, the indomethacin example shown below (Scheme 3). In this example, R may be an alkyl or aryl group containing a substituent, which can act as a monodentate or polydentate ligand, e.g., a carboxylate group as in ACM, or an amino group (prepared from an aminoalcohol) with a monodentate or polydentate amine ligands or more complex donor bidentate sets with serine or short-chain peptides containing serine. Other suitable chelating groups that may be coupled and may also target tumours, include sugars and glycoproteins.

agent

Scheme 3

In some embodiments, the ligand having anti-inflammatory activity is an amide-containing NSAID that is not a carboxylate. NSAIDs that are not carboxylates include for instance oxicam NSAIDs such as piroxicam (4-hydroxy-2-methyl-N-2- pyridyl-2H-l ,2-benzothiazine-3-carboxamide-l ,1 -dioxide), tenoxicam (4-hydroxy-2- methyl-iV-2-pyridinyl-2H-thieno(2,3 -e)- 1 ,2-thiazine-3 -carboxamide- 1 , 1 -dioxide) and meloxicam.

In some embodiments, the complex is a complex of the formula (A):

[M(L⁴)_nL_m]^p (A)

where

M is a metal ion;

L⁴ is a hydroxamate or hydroximate having anti-inflammatory activity or a chelating amide (such as those containing an amino acid or peptide linkage as the chelate) having anti-inflammatory activity; each L is independently selected and is a monodentate or polydentate ligand; n is 1, 2 or 3; m is O, 1, 2, 3 or 4; and p is the charge of the complex. In some embodiments, in formula (A), M is a divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion.

Further suitable metal complexes of NSAIDs and NSAID ligands that may be utilised are described in for instance Weder, J. E.; Dillon, C. T.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; Biffin, J. R.; Regtop, H. L., Davies.; N. M. Coord. Chem. Rev. 2005, 232, 95-126 and Dillon, C. T.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; Weder, J. E.; Zhou, Q.; in "Metal Ions and Their Complexes in Medication", Vol. 41 of 'Metal Ions in Biological Systems': Sigel, A.; Sigel, H., Eds.; M. Dekker Inc., New York & Basel, 2004. Ch. 8, pp 253-277.

Metal complexes with the ligand L²

The complex used in the method of the present invention may be a mononuclear, dinuclear, trinuclear, oligomeric or polymeric complex containing the ligand L² as defined above. For example, the complex may be a complex of formula (1), (2), (3) or (8), as defined above. Complexes of formula (1) or (2) can be prepared by mixing a stoichiometric amount of a compound L H (where L is as defined above) and a divalent or trivalent metal salt, preferably a basic salt such as M(OAc)₂, in a solvent L (the solvent forming the ligand L in the resultant complex, or in the case of the aqua complex, adventitious water in the organic solvent). The mixture is then heated until precipitation occurs and it is then cooled and the solid is filtered off. Depending on the purity of the complex (as assessed by relevant spectroscopic or powder X-ray diffraction measurements), the product may need to be recrystallised until elemental, spectroscopic and/or diffraction methods demonstrate that the complex is of the required purity.

A similar procedure may also be performed in which L²H, a divalent or trivalent metal salt and the ligand L are added to a solvent more weakly coordinating than L or in which L is added or is adventitious water. The same procedures as discussed above may then be followed for the isolation and purification of the complex. Complexes of formula (3) can be prepared by the same procedures as those described above for the preparation of complexes of formula (1) or (2) using a trivalent or tetravalent metal salt. Such complexes are obtained by mixing L²H and a suitable metal salt in aqueous/organic solvent mixtures under basic conditions. Additional ligands L may be added to these solutions to precipitate the complexes.

Methods of preparation of complexes of formula (1), (2) and (3) are discussed in a co-pending International Patent Application filed 24 March 2006 in the name of Medical Therapies Limited entitled "Metal complexes" and claiming priority from Australian provisional patent application No. 2005901462, the contents of which is incorporated herein by cross-reference in its entirety.

The complexes of formula (1), (2) or (3) may be charged or neutral.

Complexes of formula (1) and (2) are neutral in charge if M is a divalent metal ion and all the ligands L are neutral ligands. However, in some embodiments, a complex of formula (1) or (2) may have a charge, for example, p may be 1^" or 2^~. Complexes of formula (3) have a charge of 1 if all the metal ions M' in the complex are trivalent metal ions and all the ligands L are neutral. However, in some embodiments, the complex of formula (3) may have a charge of 2^~, \^~, 0, or 2 .

Examples of complexes of formula (1) include [Cu(ACM)₂(DMF)₂], [Cu(ACM)₂(OH₂)₂], [Zn(ACM)₂(DMF)₂] and [Zn(ACM)₂(OH₂)₂]. Examples of complexes of formula (2) include [Ru₂(ACM)₄L]^p and

[Ru₂(ACM)₄L₂]^P (where Ru is Ru(II)), and [Ru₂(ACM)₄Lf (a Ru(II)/Ru(III) mixed- valence complex), where p is the charge of the complex.

In some embodiments, the complex of formula (2) is a complex of the formula (2A):

[M₂(L²)₄L₂]^P (2A)

wherein M, L², L and p are as defined above for formula (2).

An example of a complex of formula (2A) is [Cu₂(ACM)₄(DMF)₂]:

Mononuclear metal complexes with the ligand L¹ In some embodiments, the complex is a mononuclear complex of the following formula (4):

[M(L^l)₂L_mf (4)

as defined above.

In further embodiments, the mononuclear complex of formula (4) is a complex of formula (4A):

as defined above.

The complex of formula (4) may be charged or neutral. Preferred complexes include [Cu(Indo)₂(Im)₂], [Cu(ACM)₂(OH₂)₂], [Zn(Indo)₂(OH₂)₂].nH₂O and [Zn(ACM)₂(OH₂)₂]. The complex of formula (4) may be in solution, or may be in the form of a solid. Crystals of a complex of formula (4) may include solvents of crystallisation. Crystals of a complex of formula (4) may also include waters of crystallisation. If L is an anionic ligand, the complex of formula (4) will be charged (e.g., p is 1^~ or 2^~) and a solid of the complex of formula (4) will include cations that are counter ions to the anionic complexes. Such solids include solids having the following formulas: YtMCη'-L¹)^] (4B) and

wherein M, η²-L¹ and L are as defined above for formula (4A), Y is a counter ion having a 2 charge and Y is a counter ion having a 1 charge.

Complexes of formula (4A) include the complex [Cu(Indo)₂(Pyrro)₂], [Cu(Indo)₂(Im)₂], [Zn(Indo)₂(L)₂] where L = OH₂ or an alcohol, [Co(Indo)₂(OH₂)₂], [Co(Indo)₂(EtOH)₂] where EtOH is ethanol, or [Ni(Indo)₂(OH₂)] as described in Q. Zhou, PhD Thesis, University of Sydney, 2001. The present inventors found that Cu(II) complexes of formula (4) are formed when copper(II) indomethacin complexes are formed using strong donor ligands, as described in the Applicants a co-pending International Patent Application entitled "Copper complexes" filed 24 March 2006 and claiming priority from Australian Provisional Patent Application No. 2005901464, the contents of which are incorporated herein by cross-reference in its entirety.

Complexes of formula (4) where M is Cu(II) may, for example, be formed using the ligand pyrrolidine. Other ligands having a similar donor strength to, or a greater donor strength than, pyrrolidine can also form complexes of formula (4). In some embodiments, L is a ligand containing an iV-heterocyclic group. Ligands containing an JV-heterocyclic group include pyrrolidine, alkyl-substituted pyrrolidines, proline, proline derivatives, imidazole, imidazole derivatives such as substituted imidazoles or ligands containing an imidazole ring (e.g. benzimidazole), pyrrole, ligands containing pyrrole, nicotinamides and nicotinic acids. In some embodiments, L is an amine, e.g. NH₃ or an organic amine (e.g. diethylamine), an alcohol or an amide (e.g. diethylacetamide), or another ligand that is a strong donor such as triethylphosphate. L can be a solvent having a solvent donor number of about 30 or greater. Complexes of formula (4) may, for example, be prepared by direct reaction of the appropriate ratios of a compound of the formula L¹H where L¹ is as defined above and a copper salt such as copper(II) acetate in a solvent having a solvent donor number of about 30 or greater, the solvent forming the ligand L in the resulting complex.

Complexes of formula (4) may also be prepared by adding a solvent having a solvent donor number of about 30 or greater, or adding a ligand that is not a solvent but has a similar donor strength to a solvent having a solvent donor number of about 30 or greater, to a solution of the metal ion (e.g. Cu(II)) and L¹ in a weaker donor solvent.

Alternatively, complexes of formula (4) can be prepared by re-crystallisation of a dinuclear complex, such as [Cu₂(Indo)₄(DMF)2], in a solvent having a solvent donor number of about 30 or greater, such as pyrrolidine, or in a solvent containing a ligand that is a strong donor such as imidazole (Im).

The complexes of formula (4) are more lipophilic than indomethacin or other compounds of the formula L¹H and thus are more easily absorbed through membranes and taken up by tissues locally. The complexes of formula (4) are, therefore, expected to be more readily absorbed into cells than free indomethacin or other compounds of the formula L H when administered topically.

Dinuclear metal complexes with the ligand L¹ In other embodiments, the complex is a dinuclear complex of the formula (5):

as defined above.

Complexes of formula (5) may be charged or neutral in charge. When each M is a divalent metal ion and each L is a neutral ligand, the complex is not charged (i.e., p is 0).

In formula (5), the ligand L may be any monodentate ligand. The ligand L may be charged or uncharged. L may, for example, be water (OH₂), an alcohol, dimethylsulfoxide (DMSO), pyridine (Py), acetonitrile (AN), tetrahydrofuran (THF), or L may be a ligand containing a tertiary amide or cyclic tertiary amide. For example, L may be a molecule of a tertiary amide of the formula:

or a molecule of a cyclic tertiary amide (JV-substituted lactam) of the formula:

C = O

R₂

N - R₁

wherein R₁ is an alkyl having from 1 to 4 carbon atoms, and each R₁ may be the same or different, and R₂ is a cycloalkyl having from 2 to 7 carbon atoms.

The tertiary amide or cyclic tertiary amide may be, for example, ΛξJV-dimethylformamide, N, N-dimethylacetamide or 7V-methylpyrrolidone.

An example of a complex of formula (5) is a dinuclear complex of the formula (5A):

[M.Cμ-L¹)^]¹⁵ (5A)

where μ-L¹ is a ligand of the formula L¹:

as defined above for formula (5);

M is Cu or Zn; each L is independently selected and is a monodentate ligand; and p is the charge of the complex.

L¹ may for example be Indo. In a preferred embodiment, the complex is a dinuclear metal/indomethacin complex of the formula (5B):

[M₂(μ-Indo)₄Y₂] (5B) wherein M is Cu or Zn, Y is water (OH₂) or a ligand containing a tertiary amide or cyclic tertiary amide (such as DMF and DMA), or Y is selected from an alcohol, DMSO, pyridine, acetonitrile, or tetrahydrofuran.

Indomethacin is one of the most lipophilic NSAIDs and, again without being limited by theory, the present inventors believe that the binding of the ligand to a metal makes complexes of formula (5B) more lipophilic than IndoH and hence promotes the transport of M (i.e. Cu or Zn) and Indo into the vasculature (i.e. there is greater absorption of the complex than IndoH). In addition, the greatly reduced adverse effects of the complex compared with free indomethacin (such as gastrointestinal and particularly, the newly discovered reduction in renal effects on oral administration as described herein) may enable higher doses and, therefore, even higher efficacy than is possible with indomethacin alone.

Complexes of formula (5B) include, for example, [Cu₂(Indo)₄(DMF)₂], [Cu₂(UIdO)₄(DMA)₂], [Cu₂(Indo)₄(NMP)₂], [Cu₂(Indo)₄(DMSO)₂], [Cu₂(Indo)₄(THF)₂], [Cu₂(Indo)₄(Py)₂], [Cu₂(Indo)₄(AN)₂], [Cu₂(Indo)₄(OH₂)₂], [Cu₂(Ket)₄(OH₂)₂],

[Cu₂(Ket)₄(OHCH₃)₂], [Zn₂(Indo)₄(DMA)₂], [Zn₂(IMo)₄(NMP)₂] and [Zn₂(Indo)₄(Py)₂], wherein NMP is iV-rnethylpyrrolidone.

A preferred complex is [Cu₂(Indo)₄(OH₂)₂].nH2θ, wherein n is the number of waters of crystallisation. The number of waters of crystallisation will vary depending on the technique used to prepare the complex, and is typically from 1 to 5.

Complexes of formula (5) may be prepared by methods known in the art. For example, copper(II) and zinc(II) complexes with indomethacin may be prepared as described in United States Patent No. 5,466,824 or as generally described in the paper: Anti-inflammatory Dinuclear Copper(II) Complexes with Indomethacin. Synthesis, Magnetism and EPR Spectroscopy; Crystal Structure of the iV,iV-Dimethylformamide Adduct. Weder, J. E.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; MacLachlan, D.; Bramley, R.; Delfs, C. D.; Murray, K. S.; Moubaraki, B.; Warwick, B.; Biffin, J. R.; Regtop, H. L. Inorg. Chem. 1999, 38, 1736-1744; Preparation and Characterization of Dinuclear Copper-Indomethacin Anti-Inflammatory Drugs. Morgan, Y. R.; Turner, P.; Kennedy, B. J.; Hambley, T. W.; Lay, P. A.; Biffin, J. R.; Regtop, H. L.; Warwick, B.

Inorg. Chim. Acta 2001, 324, 150-161; and Syntheses and Characterization of Antiinflammatory Dinuclear and Mononuclear Zinc Indomethacin Complexes, Crystal Structures of [Zn₂(Indomethacin)₄(L₂)] (L = N,iV-dimethylacetamide, Pyridine, 1- Methyl-2-pyrrolidinone) and [Zn(Indomethacin)2(L₁)2] (L₁ = Ethanol, Methanol). Zhou Q.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; Turner, P; Warwick, B.; Biffin, J. R.; Regtop, H. L. Inorg. Chem. 2000, 399, 3742-3748.

Trinuclear metal complexes with the ligand L¹

[M'₃O(μ-L¹)₆L₃]^p (6)

Trinuclear metal complexes with the ligand L³ and L⁴

In some embodiments, the complex is a trinuclear complex of the following formula (7):

[Zn₂M(L³)₆(L⁴)₂] (7) where M is a divalent metal ion;

L³ is a carboxylate having anti-inflammatory activity; L⁴ is an optionally substituted heterocyclic base comprising one or two heterocyclic rings independently having 5 or 6 ring members and 1 to 3 heteroatoms independently selected from N, O and S; and wherein each L³ is independently selected; and each L⁴ is independently selected.

In the complex of formula (7), L³ may be a monodentate, bidentate or bridging ligand of formula L¹ or L² as follows:

wherein:

R¹ is H or halo (i.e. Cl, F, Br or I);

R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN, -OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH_3, -SCH₃ or -CH₂CH₃ may be optionally substituted, and n is 1, 2, 3, 4 or 5. In formula (7), when R² is a C₁ to C₆ alkyl, an alkenyl or an alkynyl, the C₁ to

C₆ alkyl, alkenyl or alkynyl may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂.

In formula (7), when R^2Ais a C₁ to C₆ alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or an arylalkyl, the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OH, -COOH and -NH₂.

In formula (7), when R⁵ is -CH₃, -OCH₃, -SCH₃ or -CH₂CH₃, the -CH₃, -OCH₃, -SCH₃ or -CH₂CH₃ may be substituted with one or more substituents. The one or more substituents may, for example, be independently selected from the group 0 consisting of halo, -OH, -COOH and -NH₂.

R¹ is typically H.

R³ is typically H.

R² is typically CH₃.

In some embodiments, L² is ACM. 5 In formula (7), M may be selected from the group consisting of zinc, cobalt, nickel, magnesium, copper and calcium.

In some embodiments of formula (7), the heterocyclic base comprises one or more N atoms. In some embodiments, the heterocyclic base is optionally substituted. The heterocyclic base may be selected from the group consisting of isoquinolyl, O quinolyl, piperidinyl, pyridinyl, 2-methylpyridinyl, imadazoyl, pyranyl, pyrrolyl, pyrimidinyl, indolyl, purinyl and quinolizinyl. Preferably, the heterocyclic base is quinolyl.

Further metal complexes that can be used in methods and compositions embodied by the invention include the metal complexes of following formulae (8) to 5 (10). Preferably, the ligands of these complexes comprise carboxylate ligands, or derivatives of carboxylates, having anti-inflammatory activity such as hydroximate, hydroxamate, amide, or ester derivatives having anti-inflammatory activity. Metal complexes of these formulae can for instance, be prepared by methods outlined in Example 1 of this application or by other suitable synthesis methods. The metal O complexes can, for instance, include ligands L¹ and L² as described above, ketorolac or other NSAID as described herein, or their hydroximate, hydroxamate, hydrazine, amide, or ester derivatives. In the complexes of formulae (8) to (12), the functional groups of the ligands can themselves bind to the metal ion, and/or other ligating groups that are linked by these functionalities can bind to the metal. The metal comlexes can be inert or labile complexes. As will be understood, the metal ion or metal ions of inert complexes have an inert oxidation state. In particular, in some embodiments, the metal complex is a complex of formula

(8):

[M(L⁵)_m(L⁶)_nf (8) where M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁵ is independently selected and is a monodentate carboxylate (preferably Indo, ACM or Keterolac) or an amide ligand (O or N bound) (preferably a derivative of Indo, ACM or Keterolac), having anti-inflammatory activity; each L⁶ is independently selected and is NH₃, a monodentate ligand, a polydentate ligand, or a macrocyclic ligand; m is 1, 2, 3 or 4 n is O, 1, 2, 3, 4 or 5; and p is the charge of the complex. In some embodiments of the complex of formula (8), L⁵ is NH₃ or a monodentate, polydentate, or macrocyclic amine ligand. Preferred complexes of formula (8) include tM(O₂CR⁶)_m(NR⁹R¹⁰R¹¹)(6-m)]^p where M is selected from Co(III), Cr(III), Ir(III), Os(III), Rh(III), Ru(III) or Pt(IV) and more preferably, Co(III) or Ru(III), and R⁶CO₂ ^" is an anti-inflammatory NSAID such as exemplified above, and R⁹, R¹⁰ and R¹¹ can independently be H or an optionally substituted aliphatic or aromatic group. In further embodiments of the complex of formula (8), L⁵ is NH₃ or a monodentate, polydentate, or macrocyclic amine ligand. Preferred complexes of formula (8) include: [M(L¹)_m(NR⁹R¹⁰R¹¹)_n]^p where L⁵ is independently chosen from a NSAID, R⁶CO₂ ^", R⁶CON(R⁷)^" or an amide (R⁶CONR⁷R⁸) or ester derivative (R¹COOR⁴) of a NSAID, (NR⁹R¹⁰R^{1 J}) is individually selected from monodentate or polydentate amine ligands, and M is selected from Ru(II), Co(III), Cr(III), Ir(III), Os(III), Rh(III), Ru(III) and Pt(IV) and more preferably, Ru(II), Co(III), and Ru(III); [M(L⁵)_m(OH_s)n]^p where L⁵ is independently selected from a NSAID, R¹CO₂ ^", R¹CON(R³)^" or an amide (R¹CONR²R³) or ester derivative (R¹COOR⁴) of a NSAID, s 0 is independently selected from 1 or 2, and and M is selected from Co(III), Cr(III), Ir(III), Os(III), Rh(III), Ru(III) and Pt(IV) and more preferably, Co(III) and Ru(III); [M(L⁵)_m(L₂)n] where L⁵ is independently selected from a NSAID, with formula R⁶CO₂ ^", R¹CON(R³)^" or an amide (R¹CONR²R³) or ester derivative (R¹COOR⁴) of a NSAID; and L⁶ is independently selected from halo, aqua, hydroxo, carboxylato, JV-heterocycle, 5 sulfoxide, amine, amide, amino acid, peptide and macrocyclic ligands; and R⁹, R¹⁰ and R¹¹ are independently selected from H, aliphatic, aromatic and heterocyclic substituents that may be optionally substituted with one or more amine groups that form coordination bond(s) of the metal complex.

Examples of metal complexes of formula (8) include O [Co(NH₃)₅(Indo)](CF₃SO₃)₂ (see Example 1 below) and [Co(NH₃)₅(ACM)](CF₃SO₃)₂.

In other embodiments, the metal complex is a complex of formula (8 a):

[M(L⁵)_m(L⁶)_n]^p (8a) where 5 M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁵ is independently selected and is NH₃, a monodentate ligand, a polydentate ligand, or a macrocyclic ligand; each L⁶ is independently selected and is a chelating derivative of a carboxylate O such as a hydroximate, hydroxamate, hydrazine, ester, amino acid, peptide or sugar, or a amide chelating ligand (O or N bound), having anti-inflammatory activity; m is 0, 1, 2, 3, 4, or 5; n is 1, 2, 3 or 4; and p is the charge of the complex.

Complexes of formula (8a) include: [M(L⁶)_n(NR⁹R¹⁰R^u)(_6-2m)]^p where each L⁶ is independently a bidentate derivative of an NSAID, each (NR⁹R¹⁰R¹¹) is independently a monodentate amine ligand or a polydentate amine ligand, and M is selected from

Co(III), Cr(III), Ir(III), Os(III), Rh(III), Ru(III) and Pt(IV) and more preferably, Co(III) and Ru(III); [M(L^(NR⁹R¹⁰R¹ %f where L⁶ is a tridentate derivative of an NSAID, each (NR⁹R¹⁰R¹¹) is independently a monodentate amine ligand or polydentate amine ligand, and M is selected from Co(III), Cr(III), Ir(III), Os(III), Rh(III), Ru(III) and Pt(IV) and 0 more preferably, Co(III), and Ru(III); [M(L^(NR⁹R¹⁰R^{1 !})₂]^p where L⁶ is a tetradentate derivative of an NSAID, (NR⁹R¹⁰R^π)₂ is two independently selected monodentate amine ligands or a bidentate amine ligand, and M is selected from Co(III), Cr(III), Ir(III), Os(III), Rh(III), Ru(III) and Pt(IV) and more preferably, Co(III) and Ru(III); [M(L^(NR⁹R¹⁰R^{1 x})f where L⁶ is a pentadentate derivative of an NSAID, and M is 5 selected from Co(III), Cr(III), Ir(III), Os(III), Rh(III), Ru(III) and Pt(IV) and more preferably, Co(III) and Ru(III); [M(L⁶)(NR⁹R¹⁰R^π)₂]^p where L⁶ is a bidentate derivative of an NSAID, (NR⁹R¹⁰R¹ ^₂ is two independently selected monodentate amine ligands or a bidentate amine ligand, and M is Cu(II), Pd(II), Pt(II) or Au(III) and more preferably Cu(II); [M(L^(NR⁹R¹⁰R¹¹)]⁺ where L⁶ is a tridentate derivative of an NSAID, and M is O Cu(II), Pd(II), Pt(II) or Au(III) and more preferably Cu(II); and R⁹, R¹⁰ and R^{1 J} can independently be H or an optionally substituted aliphatic or aromatic group.

Other complexes of formula (8a) include: [M(L⁶)₃]^P where L is a bidentate derivative of an NSAID, and M is selected from Cu(II), Zn(II), Co(III), Cr(III), Ga(III), Ir(III), Os(III), Rh(III), Ru(III) and Pt(IV) and more preferably, Cu(II), Zn(II), Co(III), 5 Ga(III) and Ru(III); [M(L⁶)₂]^P where L⁶ is a tridentate derivative of an NSAID, and M is selected from Cu(II), Zn(II), Co(III), Cr(III), Ga(III), Ir(III), Os(III), Rh(III), Ru(III) and Pt(IV) and more preferably, Cu(II), Zn(II), Co(III), Ga(III), and Ru(III); [M(L⁶)]^P where L⁶ is a sexidentate derivative of an NSAID, and M is selected from Cu(II), Zn(II), Co(III), Cr(III), Ga(III), Ir(III), Os(III), Rh(III), Ru(III) and Pt(IV) and more preferably, O Cu(II), Zn(II), Co(III), Ga(III) and Ru(III); and [M(L⁶)₂]^P where L⁶ is a bidentate derivative of an NSAID, and M is Cu(II), Ni(II), Pd(II), Pt(II) or Au(III) and more preferably Cu(II). Further complexes of formulae (8a) include: [M(L⁶)_n(OH_t)_(6-2m)]^p where L⁶ is a bidentate derivative of an NSAID, t is independently selected from 0, 1 or 2, and M is selected from Fe(II), Mn(II), Cu(II), Zn(II), Co(III), Cr(III), Fe(III), Ga(III), Fe(III), Ir(III), Mn(III), Os(III), Rh(III), Ru(III), Mn(IV), Pt(IV), Ti(IV), V(IV), V(V), Mo(VI), and W(VI) and more preferably, Fe(II), Cu(II), Mn(II), Zn(II), Co(III), Ga(III), Ru(III), Mn(III), Mn(IV), V(IV), V(V), and Mo(VI); [M(L⁶)(OH_t)₃]^p where L⁶ is a tridentate derivative of an NSAID, t is independently selected from 0, 1 or 2, and M is selected from Fe(II), Mn(II), Cu(II), Zn(II), Co(III), Cr(III), Fe(III), Ga(III), Fe(III), Ir(III), Mn(III), Os(III), Rh(III), Ru(III), Mn(IV), Pt(IV), Ti(IV), V(IV), V(V), Mo(VI), and W(VI) and more preferably, Fe(II), Cu(II), Mn(II), Zn(II), Co(III), Ga(III), Ru(III),

Mn(III), Mn(IV), V(IV), V(V), and Mo(VI); [M(L⁶)(OH_t)₂]^p where L⁶ is a tetradentate derivative of an NSAID, t is independently selected from 0, 1 or 2, and M is selected from Fe(II), Mn(II), Cu(II), Zn(II), Co(III), Cr(III), Fe(III), Ga(III), Fe(III), Ir(UI), Mn(III), Os(III), Rh(III), Ru(III), Mn(IV), Pt(IV), Ti(IV), V(IV), V(V), Mo(VI), and 5 W(VI) and more preferably, Fe(II), Cu(II), Mn(II), Zn(II), Co(III), Ga(III), Ru(III),

Mn(III), Mn(IV), V(IV), V(V), and Mo(VI); [M(L⁶)(OH_q)]^p where L⁶ is a pentadentate derivative of an NSAID, q is 0, 1 or 2, and M is selected from Fe(II), Mn(II), Cu(II), Zn(II), Co(III), Cr(III), Fe(III), Ga(III), Fe(III), Ir(III), Mn(III), Os(III), Rh(III), Ru(III), Mn(IV), Pt(IV), Ti(IV), V(IV), V(V), Mo(VI), and W(VI) and more preferably, Fe(II), O Cu(II), Mn(II), Zn(II), Co(III), Ga(III), Ru(III), Mn(III), Mn(IV), V(IV), V(V), and Mo(VI); and five-coordinate, [V(O)(L⁵)_(m-1)(L⁶)_n]^p.

In some embodiments, the metal complex is a complex of the formula (9):

[M(L⁷)_ffi(L⁸)_n]^p (9) 5 wherein

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁷ is independently selected and is NH₃, a monodentate ligand, a polydentate ligand, or a macrocyclic ligand; O each L⁸ is independently selected and is a chelating derivative of a carboxylate such as a hydroximate, hydroxamate, hydrazine, ester, amino acid, peptide or sugar, or amide chelating ligand (O or N bound), having anti-inflammatory activity; m is O, 1, 2, 3, 4, or 5; n is 1, 2, 3 or 4; and p is the charge of the complex.

In some embodiments of the metal complex of formula (9), L is NH₃ or a monodentate, polydentate, or macrocyclic amine ligand. Preferred complexes of formula (2) include [M(NR⁹R¹⁰R¹ ^^(Λf when L⁸ is abidentate ligand, [M(NR⁹R¹⁰R¹¹ML⁸)]¹⁵ when L⁸ is a tridentate ligand, and [M(NR⁹R¹⁰R¹ ^₂(L⁸)]¹⁵ when L⁸ is a tetradentate ligand, where M is selected from Co(III), Cr(III)₅Ga(III), Ir(III), Os(III), Rh(III), Ru(III), Ru(II) and Pt(IV) and more preferably, Co(III), Ga(III), Ru(III) and Ru(II), and R⁹, R¹⁰, and ^π are as defined for formula (8 or 8a) above.

Alternatively, the metal complex of formula (9) can be [M(NR⁹R¹⁰R¹ ^₂(L⁸)]¹⁵ where L⁸ is a bidentate ligand, [M(NR⁹R¹⁰R¹¹XL⁸)]¹³ where L⁸ is a tridentate ligand, and where M is Cu(II), Ni(II), Pd(II), Pt(II) or Au(III) and more preferably Cu(II), and R⁹, R¹⁰, R¹¹ and R⁵ are as defined for formula (8 or 8a) above. In other embodiments of formula (9) the metal complex is [M(L⁸)_n]^p. In this instance, when L is a bidentate ligand n is 3, or when L is a tridentate ligand n is 2, and wherein M is preferably Co(III), Cu(II), Zn(II), Ga(III), Ru(III), or Ru(II); or when L⁸ is a bidentate ligand n is 2, or when L⁸ is a tetradentate ligand n is 1, wherein M is preferably Cu(II), Ni(II), Pd(II), Pt(II) or Au(III) and more preferably Cu(II). R⁹, R¹⁰ and R¹¹ of formulae (8 a) and (9) can for instance be selected from aliphatic and aromatic groups consisting of substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl and heterocyclic groups. Examples of heterocyclic groups include heterocyclic bases comprising one or more N atoms. In some embodiments, the heterocyclic base is optionally substituted. The heterocyclic base may for example be selected from the group consisting of isoquinolyl, quinolyl, piperidinyl, pyridinyl, 2- methylpyridinyl, imadazoyl, pyranyl, pyrrolyl, pyrimidinyl, indolyl, purinyl and quinolizinyl.

Examples of metal complexes of formula (9) include [Cu(IndoHAH)(OH)], [Co(en)₂(IndoHA)]Cl₂, [Co(en)₂(IndoHA)](CF₃SO₃)₂, [V^vO(IndoHAH)(IndoHA)] -2MeOH-1.5H₂O, [Cr(IndoHA)₂(OH₂)₂](NO₃)-H₂O,

[Cu(Indo-Gly)(Im)₂] and [Ga(IndoHAH)₃] (see Example 1 below). In other embodiments, metal complexes that can be used in methods embodied by the invention include complexes of formula (10):

[Mq(L¹V(L²ML³),]¹⁵ (10) wherein

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L¹ is independently selected and is NH₃, another monodentate ligand, a polydentate ligand, or a macrocyclic ligand; each L is independently selected and is a chelating derivative of a carboxylate such as a hydroximate, hydroxamate, hydrazine, ester, amino acid, peptide or sugar, or amide chelating ligand (O or N bound), having anti-inflammatory activity; each L is independently selected and is a bridging ligand, such as an oxo, hydroxo, carboxylate (including a NSAID), halide, or other bridging group. m is an integer from 0 to 5 q; n is an integer from 1 to 2q; p is the charge of the complex; q is typically an integer between 2 and 20 inclusive; and r is an integer from 1 to 60. In still other embodiments, the metal complex can be a complex of the following formula (11):

[M(L⁵)o(L⁷)_m(L⁸)_n]^p (11) wherein M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁵ is independently selected and is a monodentate carboxylate or amide ligand (O or N bound), having anti-inflammatory activity; each L⁷ is independently selected and is a monodentate or a polydentate ligand; each L⁸ is independently selected and is a chelating derivative of a carboxylate, or amide ligand (O or N bound), having anti-inflammatory activity; o is 1, 2, 3, 4 or 5; m is 1, 2, 3 or 4; n is 1, 2, or 3; and p is the charge of the complex.

Examples of metal complexes of formula (11) for instance, include: [M(O₂CR^6A)(L⁷)_m(L⁸)_n]^p where, m is 0, 1 , 2, or 3, n is 1 or 2, R^6ACO₂ ^~ is a NSAID, R⁶CO₂ ^", or an ester derivative of an R⁶CO₂ ^" NSAID having a terminal carboxylate, at least one of L⁸ is independently selected from a hydroxamate or hydroximate derivative of a NSAID, an amino acid derivative of a NSAID, a peptide derivative of a NSAID, and an amine derivative of a NSAID, and M is Co(III), Rh(III), Ir(III), Cr(III), Ru(III) or Pt(IV), and preferably Co(III) or Ru(III); [M(O₂CR^6A)₂(L⁷)_m(L⁸)_nf where, m is 0, 1 or 2, n is 1 or 2, R^6ACO₂ ^~ is independently a NSAID, or an ester or amino acid derivative of an R CO₂ ^" NSAID having a terminal carboxylate, at least one of L is independently a hydroxamate or hydroximate derivative of a NSAID, an amino acid derivative of a NSAID, a peptide derivative of a NSAID, or an amine derivative of a NSAID, and M is Co(III), Rh(III), Ir(III), Cr(III), Ru(III) or Pt(IV), and preferably Co(III) or Ru(III); and [M(O₂CR^6A)₂(L⁸)]^P, where L⁸ is a peptide derivative of a NSAID or an amide derivative of an NSAID.

In other embodiments the metal complex is a complex of the following formula (12): [M_q(L⁵)_m(L⁷)_n(L⁸)_r]^p (12) wherein

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁵ is independently selected and is a monodentate or bidentate carboxylate, or monodentate amide ligand (O or N bound), having anti-inflammatory activity; each L⁷ is independently selected and is a monodentate or a polydentate ligand; each L⁸ is independently selected and is a bridging ligand, such as an oxo, hydroxo, carboxylate (including a NSAID), halide, or other bridging group. m is an integer from 0 to 5q; n is an integer from 1 to 5q; p is the charge of the complex; q is typically an integer between 2 and 20 inclusive; and r is an integer from 1 to 60.

One or more of the ligands L⁵ to L⁸ above in any combination may also form dimeric, trimeric, tetrameric, oligomeric or polymeric complexes with one or more metal ions.

Examples of complexes for formula (12) include [Ru₂(Indo)₄Cl], [Ru₂(Indo)₄(O₂CR^6A)] and [M₃ ^m(O)(μ-O₂CR^6A)₅(μ-L⁸)(L⁷)₃]⁺, (where M is independently selected from Cr(III), Co(III), Co(IV), Ru(III), Ru(IV), and preferably Co and Ru; R^6A is R⁶ or an ester-linked derivative of an amino acid ( e.g., serine or tyrosine), or a hydroxy acid derivative of R⁶CO₂H (i.e., R⁶COOR¹²), L³ = OH^~,

O(R¹³)^~, or O₂C(R¹³)^", L⁷ is a JV-heterocycle or aqua ligand, and R¹¹ is a H, alkyl or aryl substituent; and [(L⁵)_mM(μ-OCOR^1A)_r(μ-L⁸)_r-M(L⁷)_n]^p, where r = 1, 2, or 3, L³ = O^2~ or OH^" and r' = 0, 1, or 2.

Monodentate ligands which can be used in metal complexes described herein include monodentate ligand such as halo, aqua, hydroxo, oxo, CO, NO, amines, alcohols, amides, sulfoxides, JV-heterocylces, O-heterocycles, and iS-heterocycles. Polydentate acyclic ligands include amines, amino acids, peptides, alcohol sugars, hydroxyacids, polycarboxylates, iV-heterocylces, O-heterocycles, and 5-heterocycles, and functional groups that can form co-ordinate bonds with a metal ion. Polydentate macrocyclic ligands include amines, crown ethers, thioethers, macrocyclic peptides and amides, and ligands with combinations of these and other metal binding substituents.

Examples of bridging ligands that can be utilized in metal complexes as described herein include oxo, hydroxo, carboxylate (including carboxylate NSAIDs), halo and other bridging groups. Examples of aliphatic and aromatic groups that can be employed include substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl and heterocyclic groups. Examples of heterocyclic groups include heterocycles comprising one or more N, O and/or S atoms. In some embodiments, the heterocycle is optionally substituted. The heterocycle can for example be selected from the group consisting of isoquinolyl, quinolyl, piperidinyl, pyridinyl, 2-methylpyridinyl, imadazoyl, pyranyl, pyrrolyl, pyrimidinyl, indolyl, purinyl and quinolizinyl as described above. The metal ion of metal complexes that can be utilized in methods embodied by the invention include d-block, f-block, p-block and s-block metal ions. In some embodiments the metal M will be a divalent, trivalent, tetravalent, pentavalent or hexavalent d-block metal, preferably, Co(II), Cu(II), Fe(II), Mn(II), Ni(II), Pt(II), Ru(II), Zn(II), Au(III), Co(III), Cr(III), Fe(III), Mn(III), Ru(III), Mn(IV), Mo(IV), Pt(IV), Ru(IV), Ti(IV), V(IV), Mo(V), V(V), W(V), Mo(VI), or W(VI), or a trivalent or tetravalent ρ-block metal such as Ga(III), Bi(III) or Sn(IV).

Suitable methods for the synthesis of metal complexes are for instance, further described in: (Romakh, V. B.; Therrien, B.; Labat, G; Stoekli-Evans, H.; Shul'pin, G. B.; Suess-Fink, G. Dinuclear iron, ruthenium and cobalt complexes containing 1,4- dimethyl-l,4,7-triazacyclononane ligands as well as carboxylato and oxo or hydroxo bridges. Inorg. CHm. Acta (2006), 359, 3297-3305; Eberlin, M. N.; Tomazela, D. M.; Araki, K.; Alexiou, A. D. P.; Formiga, A. L. B.; Toma, H. E.; Nikolaou, S. Electrospray Ionization Tandem Mass Spectrometry of Polymetallic μ-Oxo- and Carboxylate-Bridged [Ru₃O(CH₃COO)₆(Py)₂(L)]^4" Complexes: Intrinsic Ligand (L) Affinities with Direct Access to Steric Effects. Organometallics (2006), 25, 3245-3250; Eremin, A. V.; Belyaev, A. N.; Simanova, S. A. Binuclear Ruthenium(III) μ-Oxocarboxylates of the Nonelectrolyte Type. Molecular Structure of the Complex [Ru^m ₂(μ-O)(μ- O₂CCF₃)₂Py₄(O₂CCF₃)₂]. (CHs)₂CO. Rus. J. Coord. Chem. (2005), 31, 761-767); Belyaev, A. N.; Simanova, S. A. Ru, Rh, and Ir Trinuclear Mixed- Valence Oxygen- Bridged Carboxylate Complexes. Rus. J. Coord. Chem. (2004), 30, 184-193) for carboxylate bridged complexes of formulae (1) or (2);

In some of the complexes in any of the formulae (1)-(12) one of the ancillary ligands can be CO, since Ru-CO complexes for promoting angiogenesis could be prepared for example by using methods similar to (Li Volti, G.; Sacerdoti, D.; Sangras,

B.; Vanella, A.; Mezentsev, A.; Scapagnini, G.; Falck, J. R.; Abraham, N. G. Carbon monoxide signaling in promoting angiogenesis in human microvessel endothelial cells. Antioxidants & Redox Signaling (2005), 7, 704-710).

Another desirable ligand is the NO ligand in complexes of Fe(II) or Ru(II) since these can release NO for vasodilation.

In particularly preferred embodiments of the invention, metal complexes of indomethacin, ibuprofen, naproxen, dichlofenec, ketorolac, and/or derivatives thereof having anti-inflammatory activity are utilised for the prophylaxis or the treatment of cancers as described herein. However, any suitable such NSAID or derivative thereof can also be utilized.

Further examples of ligands and metal complexes that can find application in methods and pharmaceutical compositions embodied by the invention as well as methods for their preparation are described in the Applicant's co-pending International Patent Applications PCT/AU2006/000402 and PCT/AU2006/000391, as well as the Applicant's International Patent Applications entitled "Anti-inflammatory metal complexes" and "Metal complexes having anti-inflammatory activity", both filed 26 March 2007, the contents of all of which are incorporated herein by cross-reference in their entirety.

As used herein, the term "therapeutically effective amount" means an amount effective to yield a desired therapeutic response, eg prophylaxis or treatment of a cardiovascular inflammation or a cardiovascular disease or condition as described herein. The metal can be administered alone or be co-administered in combination with one or more agents conventionally used in the treatment of cardiovascular asscociated diseases or conditions. By "co-administered" is meant simultaneous administration in the same formulation or a plurality of formulations by the same of different routes, or sequential administration by the same or different routes. By "sequential" administration is meant one is administered one after the other. As will be understood, the use of a metal complex in one or more methods embodied by the invention in combination with another anti-inflammatory agent may enhance the effectiveness of the other anti-inflammatory agent or allow the dosage of the other anti-inflammatory agent to be lowered to reduce toxic side effects of the other anti-inflammatory agent. The specific "therapeutically effective amount" of the metal complex utilised in a method embodied by the present invention will vary with such factors as the particular condition being treated, the physical condition age and weight of the human or animal, the type of animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific composition and complex employed. The dosage administered and route of administration will be at the discretion of the attending, clinician or veterinarian and will be determined in accordance with accepted medical or veterinary principles. For instance, a low dosage may initially be administered which is subsequently increased at each administration following evaluation of the response of the subject. Similarly, the frequency of administration can be determined in the same way, that is, by continuously monitoring the response of the subject and modifying the interval between dosages. It will also be understood that a plurality of different metal-NS AID complexes as described herein can be administered to the mammalian subject in the treatment of a cardiovascular disease or condition. The metal complexes can be selected to provide different anti-inflammatory activies, have different rates of release of anti-inflammatory ligands, and/or for instance be targeted to different tissues to provide complimentary anti-inflammatory activity.

The complex(es) can be administered to the mammalian subject systemically and/or applied directly to the site of cardiovascular inflammation such as during surgery. The complex can also be applied topically to the skin for diffusion into the body of the subject to the site of action, or by oral administration (including buccal or sublingual administration), or by suppository or any other mode of administration suitable for the particular disease or condition being treated.

The complex will generally be administered hi the form of a composition comprising the complex together with a pharmaceutically acceptable carrier. In particularly preferred embodiments the composition may be formulated as described in International Application No. PCT/AU2005/000442, the contents of which is incorporated herein by cross-reference in its entirety. As described in PCT/AU2005/000442, a formulation having a colloidal structure or which forms a colloidal structure post administration is particularly desirable for administration of metal complexes. Examples of suitable compositions having a colloidal structure or which form a colloidal structure upon, or following administration, are exemplified in PCT/AU2005/00042 and any suitable such formulations for the selected mode of administration can be utilised in methods embodied by the present invention. Formation of the colloidal structure can for instance occur when the composition contacts an aqueous biological fluid in the human or animal body, for example, on contact with an aqueous fluid in the digestive tract.

A composition has a colloidal structure if it comprises a colloidal system. A colloidal system is a system in which particles of a colloidal size of any nature (eg., solid as liquid or gas) are dispersed in a colloidal phase of a different composition or state. In particularly preferred embodiments, the composition comprises micelles in an aqueous carrier or is an oil-in- water emulsion, or forms micelles or an oil-in- water emulsion when the composition is administered to a human or animal body. Without wishing to be bound by theory, it is believed the colloidal structure protects the metal complex from interaction with acids or other compounds which would otherwise interact with the complex to cause the complex to dissociate. It is also believed the colloidal structure reduces the extent to which some compounds present in the composition are able to interact with the complex, e.g. during storage of the composition, that may cause the complex to dissociate. Similarly, when such a composition is administered to a subject, the colloidal structure may limit the extent to which some compounds that come into contact with the composition after it is administered are able to interact with the complex and which cause the complex to dissociate before it is absorbed. For example, for compositions administered orally, the colloidal structure may limit the extent to which compounds present in stomach acid are able to interact with the complex to cause the complex to dissociate before it is absorbed through the gastrointestinal tract. Similarly, for compositions administered by other routes, the colloidal structure may limit the extent to which compounds that come into contact with the composition after it is administered, e.g. strong chelators of Cu(II), such as peptides, or reductants of Cu(II), such as thiol-containing biomolecules, are able to interact with the complex to cause the complex to dissociate. As indicated above, some compositions may not have a colloidal structure but will be formulated such that when administered to a human or animal body by the intended route of administration, a colloidal structure is formed. For example, in some embodiments, the composition is immiscible with water, and is thus immiscible with aqueous biological fluids whereby a colloidal system is thereby formed.

Preferably, the colloidal structure is maintained for a sufficient time after administration of the composition for the majority, for example more than 70%, 80% or 90%, of the metal complex, to be absorbed by the body as a metal complex. Oils for use in the compositions include pharmaceutically acceptable vegetable or mineral oils. Suitable oils include, but are not limited to: triglycerides, particularly medium chain triglycerides, combinations of medium chain and long-chain triglycerides, combinations of triglycerides with fish oil; vegetable oils, such as, soya oil, safflower oil and sunflower oils; isopropyl myristate; and paraffins. Such oils are suitable for use in compositions for oral, injectable, or topical administration.

When the composition comprises micelles in an aqueous carrier, the composition will typically further comprise one or more surfactants for formation of the micelles. Any surfactants may be used that are capable of forming micelles in the aqueous carrier, are pharmaceutically acceptable when administered by the intended route of administration, and which substantially do not interact with the metal carboxylate complex to cause dissociation from the metal when the composition is stored in the absence of light. Suitable surfactants for use in compositions for oral or topical administration include, but are not limited to, the sorbitan fatty acid ester group of surfactants. Such surfactants comprise mono-, tri-, or partial esters of fatty acids such as oleic, lauric, pahnic and stearic acids, and include sorbitan trioleate (Span 85), sorbitan monooleate (Span 80), sorbitan tristearate (Span 65), sorbitan monostearate (Span 60), sorbitan monopalmitate (Span 40), and sorbitan monolaurate (Span 20).

Other suitable surfactants include the macrogol (polyoxyethylene) esters and ethers. These surfactants include, but are not limited to, the caster oil polyoxyethylene group of surfactants, such as Termul 1284 and caster oil ethoxylate. Additional suitable surfactants in this class include the Polyoxyethylene Sorbitan Fatty Acid Esters group of surfactants, including polyoxyethylene (20) sorbitan monolaurate (T ween 20), polyoxyethylene (4) sorbitan monolaurate (T ween 21), and polyoxyethylene (20) sorbitan monooleate (T ween 80).

Further surfactants that may be used include the block copolymers based on ethylene oxide and propylene oxide such Poloxamer 124 (Pluronic L44 NF), Poloxamer

188 (Pluronic F68 NF), Poloxamer 331(Pluronic LlOl NF), and Poloxamer 407 (Pluronic F 127 NF). Suitable surfactants also include the polyethylene glycol fatty acid esters (PEG esters) group of surfactants. Such surfactants comprise mono-, tri-, or partial esters of fatty acids such as oleic, lauric, palmic, oleic, and stearic acids, including but not limited to PEG 200 monolaurate, PEG 300 dilaurate, ethylene glycol distearate, PEG 300 monooleate, PEG 400 monooleate, PEG 350 monostearate, PEG 300 monostearate, PEG 400 monostearate, PEG 600 monostearate, PEG 1000 monostearate, PEG 1800 monostearate, PEG 6500 monostearate, PEG 400 mono-iso stearate, PEG 600 mono-iso-stearate, PEG 200 dilaurate, PEG 600 distearate, PEG 6000 distearate, PEG 200 distearate, PEG 300 distearate, and PEG 400 distearate.

Preferably, such compositions have more than 80%, preferably more than 90%, and more preferably more than 95%, of the total amount of the carboxylate, or hydroxamate, hydroximate, ester, or amide derivative having anti-inflammatory activity present in the composition as part of a metal complex. Preferably, also less than 10% of the carboxylate, or hydroxamate, hydroximate, ester or amide derivative complexed with the metal dissociates from the metal when the composition is stored for 12 months in the absence of light at room temperature (18⁰C to 25⁰C). The amount of the carboxylate, or hydoxamate, hydroximate, ester or amide remaining bound to the metal complex can be readily determined by a person skilled in the art using known methods such as EPR spectroscopy for complexes that give EPR signals or using more specialized experiments involving X-ray absorption spectroscopy for all complexes (e.g., XAFS Studies of Anti-inflammatory Dinuclear and Mononuclear Zn(II) Complexes of Indomethacin. Zhou, Q.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A. Inorg. Chem. 2003, 42, 8557-8566; Determination of the Structures of Antiinflammatory Copper(II) Dimers of Indomethacin by Multiple-Scattering Analyses of X-ray Absorption Fine Structure Weder, J.E.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; Foran, G. J.; Rich, A. M. Inorg. Chem. 2001, 40, 1295-1302; Three-Dimensional Structure Determination using Multiple-Scattering Analysis of XAFS: Applications to Metalloproteins and Coordination Chemistry. Levina, A.; Armstrong, R. S.; Lay, P. A. Coord. Chem. Rev. 2005, 249, 141-160).

Strong chelating ligands such as peptides, certain carboxylate donors, reductants such as vitamins C and E, thiolate groups such as glutathione- or cysteine- containing species, can cause metal carboxylate complexes to dissociate. Accordingly, the compositions preferably do not comprise, or are substantially free of, peptides, carboxylate donors, reductants and thiolate groups, apart from those in the derivatives of the NSAID utilised. Preferably, the composition is also not strongly acidic or basic as strong acids and bases can cause metal carboxylate complexes to dissociate. More generally, the metal-NSAID complex can be dissolved in the composition or can be present in the composition as a solid. The solid complex can be in the form of a crystal containing solvents of crystallisation and/or waters of crystallisation. When the complex is charged, the complex will be associated with a counter ion. As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the complex to a human or animal. The carrier can be liquid or solid and is selected with the intended manner of administration in mind. The carrier is "pharmaceutically acceptable" in the sense of being not biologically or otherwise undesirable, i.e., the carrier may be administered to a human or animal along with the complex without causing any or a substantial adverse reaction. For instance, the carrier can be a solvent or dispersion medium containing one or more of physiological saline, ethanol, polyol (e.g. glycerol, propylene glycol, liquid polyethylene glycol and the like), vegetable oils and mixtures thereof. The composition for use in the method of the invention may be suitable for oral, rectal, nasal, topical (including buccal and sublingual), ophthahnological, vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, or for administration respiratoraly, intratrachaely, nasopharanyngealy, intraoccularly, intrathecally, intranasally, by inhalation, infusion, or via IV group patch and by implant. With respect to the intravenous route, particularly suitable routes are via injection into blood vessels to be treated or which supply blood vessels or particular organs to be treated. Agents may also be delivered into cavities such as for example the pleural or peritoneal cavity.

The composition can also conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Such methods include the step of bringing into association the complex with the carrier. Typically, the carrier comprises two or more ingredients. In general, the composition of the present invention is prepared by uniformly and intimately bringing into association the complex with the carrier, and then, if necessary, shaping the product. The complex and the one or more components making up the carrier may be mixed in any order. However, it is preferred that the components are mixed in a manner that minimises the amount of the complex that dissociates during the preparation of the composition.

A composition for oral administration can be in the form of a viscous paste, an ingestible tablet, a capsule, a chewable composition, or any other form suitable for oral administration. If desired, the composition can be encapsulated in a hard or soft capsule

(e.g. gelatine) by techniques known in the art. Moreover, the metal complex can be provided in the form of buccal tablets, troches, elixirs, suspensions or syrups. Slow release formulations and formulations for facilitating passage through the environment of the stomach to the small intestines are also well known to the skilled addressee and are expressly encompassed by the invention.

A composition for oral use can for instance, also comprise one or more agents selected from the group of sweetening agents such as sucrose, lactose or saccharin, disintegrating agents such as corn starch, potato starch or alginic acid, lubricants such as magnesium stearate, flavouring agents, colouring agents and preserving agents e.g. such as sorbic acid, in order to produce pharmaceutically elegant and palatable preparations. A chewable composition can, for example, comprise the complex, one or more flavours, a base formulation, one or more preservatives, one or more pH modifiers, one or more desiccants and one or more fillers. As an example, for a chewable composition the base may comprise pre-gel starch, gelatine, flour and water. The composition may also comprise other components including phosphoric acid, salt, sugar, sorbitol and/or glycerol, sorbic acid and/or potassium sorbate, benzoic acid, propionic acid and maltodextrin. A chewable composition for an animal such as a dog for example, may comprise the complex, meat emulsion, an acidulate (e.g. phosphoric acid), one or more antifungal agents (e.g. benzoic acid and sorbic acid), sugar or sugar alcohol, and salt.

A composition for topical application may comprise the complex in a conventional oil-in- water emulsion, water-in-oil emulsion, or water-immiscible pharmaceutical carrier suitable for topical application. Such carriers include for example, lacrilube, cetomacrogol cream BP, wool fat ointment BP or emulsifying ointment BP. Such carriers are typically in the form of an emulsion or are immiscible with water.

An example of a composition for topical application to skin is a composition comprising 0.5-2% w/w of the complex in an emulsifying cream with chlorocresol (4- chloro-3-methylphenol) as a preservative, the emulsifying cream comprising:

cetomacrogol emulsifying wax 15 g liquid paraffin 1O g white soft paraffin 1O g chlorocresol 0.1 g propylene glycol 5 mL purified and cooled water to 100 g Another example of a topical composition for application to skin is a composition comprising 2% w/w of the complex in wool fat. This composition is immiscible with water. Compositions for parenteral administration include compositions in the form of sterile aqueous or non-aqueous suspensions and emulsions. The composition can also include one or more pharmaceutically active components in addition to the complex that have anti-inflammatory or other therapeutic activity. Such active components include conventionally used anti-inflammatory drugs. Typically, a metal complex will constitute about 0.025% to about 20% by weight of the composition, preferably about 0.025% to about 20% by weight of the composition, more preferably about 0.1% to about 20% by weight of the composition and most preferably, the complex constitutes about 0.1% to about 10% by weight of the composition. For some compositions such as topically acceptable compositions for application to the skin, a composition embodied by the invention may comprise the metal complex in an amount of about 1% by weight of the composition or less.

Suitable pharmaceutically acceptable carriers and formulations useful in the present invention may for instance be found in handbooks and texts well known to the skilled addressee, such as "Remington: The Science and Practice of Pharmacy (Mack Publishing Co., 1995)" and subsequent update versions thereof, the contents of which is incorporated herein in its entirety by reference.

The mammalian subject can be a human or an animal. The animal can, for example, be a companion animal such as a dog or cat, or a domestic animal such as a horse, pony, donkey, mule, camel, llama, alpaca, pig, cow or sheep, or a zoo animal. Suitable animals include members of the Orders Primates, Rodentia, Lagomorpha,

Cetacea, Carnivora, Perissodactyla and Artiodacty Ia. Typically, the subject will be a primate and more usually, a human being.

A number of embodiments of the present invention will now be described below by reference to the following non-limiting examples. EXAMPLE 1 Synthesis of metal complexes

1.1 Preparation of compounds

Metal complexes useful in one or more embodiments of methods of the invention were prepared as follows.

1.1.1 Metal carboxylate complexes

[Cu₂(Indo)₄(OH₂)₂]._nH2θ

Cu(II) acetate monohydrate (0.028 g, 0.140 mmol) in water (0.75 mL) was added drop wise to indomethacin (0.1 g, 0.28 mmol) dissolved in ethanol (1.75 mL) at room temperature. Warming the ethanol mildly (~40°C) helped solubilise the indomethacin before adding the Cu(II) acetate solution. On addition of the Cu(II) acetate monohydrate in water, bright green Culndo/aqua complex precipitated out of solution immediately. This precipitate was filtered, washed with water and dried. Spectroscopic analysis confirmed that it was the [Cu₂(Indo)₄(OH₂)₂].nH₂θ complex, and EPR spectroscopy showed it was >99% dimer.

The crystal size and colour of the Cu-aqua complex was checked with a light microscope. The crystals were found to be green in colour, with a star-like shape and 50-100 microns in diameter. This size was larger (by at least an order of magnitude) than crystals prepared by synthetic methods reported elsewhere.

[Cu(tndo)₂(Pyrro)₂]-2Pyrro H₂0

Crystals that consisted of pale blue plates were grown by recrystallisation of [Cu₂(Indo)₄(DMF)₂] in pyrrolidine as the solvent. Anal. Found: C, 59.91 ; H, 6.32; N, 7.84; Cu, 6.01%. CaIc. for CuC₅₄H₆₈Cl₂N₆O₉: C, 60.15; H, 6.36; N, 7.80; Cu, 5.84%. Spectroscopic analysis shows that the pale blue plates were crystals of [Cu(Indo)₂(Pyrro)₂]-2PyrroΗ₂O complex. [Cu(Indo)2(Iin)2] complex

Cu(OAc)₂-H₂O (0.108 g, 0.5408 mmol) in methanol (8 mL) and water (1 niL) was sonicated for 0.5 h to dissolve, Indomethacin (0.4 g, 1.118 mmol) and imidazole (0.076 g, 1.118 mmol) in methanol (12 mL) was stirred to dissolve. The solution of Cu²⁺ was added drop wise to the solution of indomethacin and imidazole at ~30 ⁰C with stirring, the mixture changed colour from green to dark blue. The solid was formed after 20 min. The solid was filtered and washed with 3 mL methanol once, and dried under N₂ at room temperature. Anal. Calcd. for C₄₄H₃₈Cl₂CuN₆O₈: C, 57.87; H, 4.19; N, 9.20; Cl₅ 7.76, Cu, 6.96%. Found: C, 58.18; H, 4.49; N, 8.99; Cu, 6.64%.

[Cu₂(IbUp)₄] complex

Ibuprofen (1.03 g, 5 mmole) was allowed to dissolve in a solution of potassium bicarbonate (0.55 g, 5.5 mmole) in 80 ml of water. To this stirring solution was slowly added a solution of copper sulfate pentahydrate (0.625 g, 2.5 mmole) in 5 ml of water. The mixture was allowed to stir for 30 minutes. The aquamarine precipitate which formed was collected, washed with water and ethanol, and then recrystallized from ether/dichloromethane (80 mL). The product was air-dried. Anal. Calcd. for CuC₂₆H₃₄O₄: C, 65.86; H, 7.24. Found: C, 65.37; H, 6.54%.

[Cu(IbUp)₂(Im)₂] complex

A solution of 0.089 g (1.30 mmol) of imidazole in methanol (15 mL) was added to 0.303 g (0.32 mmol) of Cu₂(Ibup)₄. The mixture was stirred at about 6O⁰C for 1 hr. The blue solution was filtered and left in the hood to evaporate. The blue precipitate that formed recrystallized from methanol and air dried. Anal. Calcd. for C₃₂H₄₂N₄O₄Cu: C, 63.98; H, 6.94; N, 9.18. Found: C, 63.16; H, 5.89; N, 9.08%.

[Cu(Ibup)₂(2-Mim)2] complex

A solution of 2-methylimidazole (0.096 g, 1.165 mmol) in methanol (15 mL) was added to Cu₂(IbUp)₄ (0.269 g, 0.284 mmol). The mixture was stirred at about 60°C for 1 hr. The blue solution was filtered and left in the hood to evaporate. The bluish purple precipitate that formed was washed with anhydrous diethyl ether and air dried. Anal. Calcd. for C₃₄H₄₆N₄O₄Cu: C, 64.00; H, 7.21; N, 8.89. Found: C, 63.25; H, 6.92; N₅ 9.46%.

[Ru₂Cl(Indo)₄] complex

[RuCl(O₂CCH₃)₄] was prepared as previously reported (R. W. Mitchell, A. Spencer and G. Wilkinson, J. Chem. Soc, Dalton Trans) 1973, 846-854). The [Ru₂Cl(O₂CCH₃)₄] (0.25 g, 0.48 mmol) was dissolved in methanol (70 niL) under N₂, a solution of IndoH (0.82 g, 2.3 mmol) in methanol (30 mL) was added, and the mixture was refluxed for 4 h under N₂. After cooling, the brown microcrystals were filtered off, washed with methanol, and dried in air at room temperature. Ru₂Cl(IndoH)₄, Anal. CaIc for C₇₆H₆₀Cl₅N₄O₁₆Ru₂: C, 54.83; H, 3.63, N, 3.37, Cl, 10.65, Ru, 12.14. Found: C, 53.70; H, 3.76, N, 3.26, Cl, 11.01, Ru, 12.06.

1.1.2 Metal hydroxymate complexes

IndoHA and its copper complex

Scheme 5: Synthetic routine for IndoHAH₂

A mixture of indomethacin (7.14 g, 20 mmol) and excess SOCl₂ (4.5 mL) in dry benzene (150 mL) was refluxed for 3 h in a 100 mL round-bottom flask fitted with a condenser and drying tube (using CaSO₄). The reaction was clarifying after refluxing. Excess SOCl₂, SO₂, and HCl were removed under partial vacuum at 0°C. The indomethacin acid chloride remaining was then reconstituted in anhydrous distilled tetrahydrofuran (THF). (See Meyer, G.A., Lostritto, R.T., Johnson, J.F., J. Appl. Polymer Sd. 1991, 42, 2247-2253 and Davaran, S., Entezami, A. A., J. Controlled Release 1997, 47, 41-49.)

A mixture of hydroxylamine hydrochloride (1.39 g, 0.02 mmol) and potassium carbonate (2.764 g, 0.02 mmol) was dissolved in water (4 mL), diethyl ether (200 mL) was added, and the resulting suspension was stirred for 10 min, then the suspension of indomethacin acid chloride (prepared above) in THF (120 mL) was added and the reaction mixture was allowed to come to room temperature (RT) and additionally stirred for 48 h. The suspension was then filtered and washed with additional anhydrous ether and then re-suspended in boiling THF (300 mL) and allowed to recrystallize at RT. The white crystals were collected by filtration and washed twice with diethyl ether to offer 5.6 g white product. Anal Calcd for C₁₉H₁₇ClN₂O₄: C, 61.21; H, 4.60; Cl, 9.51; N, 7.51. Found: C, 60.68; H, 4.77; Cl, 9.06; N, 7.07%. NMR (DMSO-J₆): d 10.65 (s, IH, NOH), 8.82 (bm, 1Η, -NHOΗ), 7.67 (m, 4Η, -C₆H₄Cl), 7.15-6.66 (m, 3Η, -C₆H₃OCH₃), 3.77 (s, 3H, -OCH₃), 3.37 (s, 2H, O=CCH₂-), 2.50 (s, 3Η, CCH₃).

Copper complex of IndoΗAΗ — [Cu(IndoΗAΗ)(OΗ)]

IndoHA (0.932 g, 2.5 mmol) in ethanol (15 mL) was added to Cu(OAc)₂-H₂O (0.25 g, 1.25 mmol) in ethanol (15 mL) and the mixture was left to stir overnight. The resulting green complex was filtrated, washed with ethanol and dried in vacuo. Anal. Calcd. for C₁₉H₁₇ClCuN₂O₅: C, 50.45; H, 3.79; N, 6.19; Cl, 7.84; Cu, 14.05. Found: C,

50.96; H, 3.52; N, 6.03; Cl, 8.05; Cu, 13.59.

Vanadium(V) complex of IndoHA

VOSO₄ SH₂O (50.6 mg, 0.200 mmol) and IndoHAH₂ (149 mg, 0.400 mmol) were dissolved in methanol (MeOH, HPLC grade, 5.0 mL). The solution immediately turned dark-red. This solution was added to ice-cold H₂O (Milli-Q grade, 50 mL), which led to the formation of a fine brown precipitate. The precipitate was isolated by centrifugation (5 min at 4000 g) and dissolved in a minimal volume of MeOH (-20 mL). The resultant solution (which was slightly cloudy) was filtered through a small-pore (No. 4) glass filter under vacuum. The volume of the filtrate was reduced by -2/3 under reduced pressure (at 40 ⁰C), which led to the formation of a red precipitate. After cooling the solution to -0 ⁰C, the precipitate was isolated on a glass filter (No. 4) under vacuum, and dried overnight under vacuum over silica gel. Yield, 48.9 mg (54.3% as calculated for [V^vO(IndoHAH)(IndoHA)]-2MeOH-1.5H₂O..

The most intense signal in the electrospray mass spectrum (solution in MeOH, ~1 mg rnL^'1): m/z -807.1 (corresponds to [V^VO(L)₂]\ according to the isotope distribution pattern). Characteristic signals in the IR spectrum (solid mixture with KBr, diffuse reflectance mode): 1686 cm^"1 (s) (C=O of the hydroxamato group); -3300 cm^'1 (br) and 1590 cm^"1 (m) (-NH of the hydroxamato group); 992 cm^"1 (m) (V=O). EPR spectrum (X-band, 22 ⁰C, solution in acetone, ~10 mg mL^"1): no signals (i.e., the product is V(V) rather than V(IV) complex). Calculated for

[V^vO(IndoHAH)(IndoHA)] -2MeOH- 1.5H₂O (C₄₀H₄₂Cl₂N₄O₁₂.₅V): C, 53.34%; H, 4.70%; N, 6.22%. Found: C, 54.05%; H, 4.46%; N, 6.22%.

The colour of the complex is strongly solvent-dependent: the solid compound is brown, solutions in methanol are orange-red, and solutions in tetrahydrofuran are dark- purple. Thus, it is most likely that the complex is six-coordinate, with a molecule of solvent as a ligand:

Vanadium(V) complex of IndoHA(H), [VOChKIoHAH)₂(OMe)] or [VO(IndoHA)(IndoHAH)(MeOH)]

It is not possible to determine whether there is a second deprotonation of one of the hydroxamic acids or a deprotonation of the MeOH has occurred since the complexes have identical microanalysis and mass spectra.

Synthesis of IndoHA

Scheme 6 To a stirred suspension of indomethacin (0.50 g, 1.40 mmol) in toluene (5 mL) was added thionyl chloride (1 mL, excess) and the mixture was heated to 80 ⁰C. The solid dissolved and the batch became darker in colour, turning green and then brown. After 1.5 hr, TLC (quench into methanol) showed that the reaction was complete. The majority of the solvent and acidic vapours (SOCl₂, HCl, SO₂) were removed in the fume hood under a stream of nitrogen. The remainder was then removed via rotary evaporator to give a dark green oil / foam. This was redissolved in THF (3 mL).

To a second vessel was charged NH₂-OH.HC1 (196 mg, 2.80 mmol) and K₂CO₃ (0.39 g, 2.80 mmol), followed by water (0.75 mL) and diethyl ether (12.5 mL) to give a mixture of two liquid phases (solids all dissolved). To this was added the THF solution of the acid chloride, over 2 minutes. A pale brown precipitate formed and the reaction was left to stir at room temperature. After 2 hr, TLC indicated that reaction was complete. Sat. NaHCO₃ (aq) (20 mL) and EtOAc (20 mL) were then added, these phases were separated, and the organic phase was washed with further sat. NaHCO₃ (aq) (20 mL). The combined aqueous phase was back-extracted with EtOAc (20 mL). The combined organic portions were washed with water (20 mL), dried (MgSO₄) and concentrated in vacuo to give an orange / yellow solid. This was recrystallised from EtOAc / hexane to give a tan coloured solid (0.28 g, 54%). The vanadium (V) complex was then synthesised using Indo-HA as follows.

V-lhdo-HA complex

Scheme 7 Indo-HA (300 mg, 0.81 mmol) was stirred in methanol (20 mL) to give a yellow slurry. To this was added a (pale blue) solution of VOSO₄.5H₂O (107 mg, 0.40 mmol) in methanol (10 mL). The batch immediately turned dark red and almost all the solid dissolved. More methanol (2 mL) was added to rinse in the vanadium solution. A small amount of solid remained out of solution, but after 20 minutes this had dissolved to give a complete dissolution. After 2 hr, the batch had formed a solid dark red precipitate. Oxygen was bubbled through the batch for 1 minute to ensure complete oxidation from V(IV) to V(V). There was no visual change with the oxygen bubbling. The batch was concentrated to reduce the volume to around half the original volume, prior to being cooled to 0 ⁰C and then filtered. The orange / red solid obtained was dried under high vacuum to give 251 mg. Anal. Calcd for CSgH₃₅N₄Cl₂O_1OV: C, 55.66; H, 4.19; N, 6.66; Cl, 8.43; 0, 19.01; V, 6.05. Found: C, 55.69; H, 4.22; N, 6.72, Cl, 8.53; O, 19.05; V, 6.08.

Chromium(III) complex of IndoHA

Cr(NO₃)₃-9H₂O (40.0 mg, 0.100 mmol) and IndoHA (149 mg, 0.400 mmol) were dissolved with stirring in MeOH (HPLC grade, 5.0 mL), and triethylorthoformate (2.0 mL; a water-withdrawing agent) was added. The solution was gently refluxed (-60 ⁰C) for 4 h (which led to a colour change from blue to dark-green), then left at 22 ⁰C overnight. The volume of the solution was reduced to ~1 mL under a stream of N₂. The concentrated solution was applied to a column (1.5x15 cm) of Sephadex LH-20, and eluted with MeOH. The fast-moving grey-green fraction was collected, evaporated to dryness under reduced pressure (40 ⁰C), and dried over silica gel under vacuum overnight. A smaller second fraction (yellow-green) was also collected, but it did not show the expected signals in the electrospray mass spectra (ESMS), and was not further analysed. Yield of the grey-green compound, 66.4 mg (72.6% based on the [Cr(LH)₂(OH₂)₂J(NO₃)-H₂O structure, MW = 913.6 Da, where LH₂ = IndoHA).

The m/z values for the most intense signals in ESMS (solution in MeOH, ~1 mg mL^"1) were as follows: +1165.9 (rel. abund. 100%; [Cr(LH)₂J⁺-LH₂ or [Cr(LH)₃J-H⁺); +857.5(99%; [Cr(LH)₂]⁺-2Me0H); +843.4 (98%; [Cr(LH)₂J⁺-MeOH-H₂O); +829.3

(85%; [Cr(LH)₂]⁺-2H₂O); +811.2 (32%; [Cr(LH)₂J⁺-H₂O); +794.0 (56%; [Cr(LH)₂J⁺); +1960.8 (60%; 2[Cr(LH₂)J⁺-LHl; -855.0 (100%; [Cr(LH)₂(OMe)₂J-); -917.9 (62%; [Cr(L)(LH)J-NO₃ ^"); -792.2 (55%; [CrL₂]^"); and -1649.8 (30%;

[Cr(LH)₂(OMe)₂P[Cr(L)(LH)]). All the assignments are in agreement with the isotope distribution patterns.

Characteristic signals in the IR spectrum (solid mixture with KBr, diffuse reflectance mode): 1686(s) (C=O of the hydroxamato group); ~3300(br) and 1595(m) (N-H of the hydroxamato group); ~3000(br) (H₂O). Calculated for [Cr(LH)₂(OH₂)₂](NO₃)-H₂O (C₃₈H₄₀Cl₂N₅O₁₄Cr): C, 49.95%; H, 4.41%; N, 7.66%. Found: C, 50.86%; H, 4.29%; N, 7.21%.

Ga(III)-IndoHAH complex

An acidic aqueous solution of Ga(III) (0.64 M) was prepared by partial dissolution of a piece of metallic Ga (99.99%, Fluka) in aqueous HCl (~5 M, ultra-pure, Merck), and the amount of dissolved Ga was determined by the mass difference. A portion of this solution (5.0 mM) was evaporated to dryness at 100 ⁰C, and the residue was dried under vacuum overnight and dissolved in anhydrous MeOH (5.0 mL), giving a solution Of GaCl₃ (0.64 M) in MeOH. This solution (0.78 mL, 0.050 mmol Ga) was added to a solution of IndoHA (55.8 mg, 0.15 mmol) in MeOH (~2 mL), followed by the addition of a solution of KOH in MeOH (1.5 mL of 0.10 M solution, 0.15 mmol KOH). The resultant solution was left at room temperature (RT) for 6 h (during which time a small amount of white precipitate formed), then filtered through a 0.20-μm membrane filter, and the filtrate was evaporated to dryness under a stream of N₂. The yellow oily residue was suspended in H₂O (~5 mL), leading to a yellow-white amorphous solid. This solid was separated by vacuum filtration, washed with H₂O, and dried under vacuum (yield of the dry solid, 45.5 mg). Repeated syntheses were carried out as described above, but either without the addition of KOH, or with the addition of excess KOH (0.45 mmol). All three syntheses led to yellow- white amorphous solids, which gave identical IR spectra. Therefore, only one product (from the above- described synthesis) was further characterised.

A comparison of ¹H NMR spectra of IndoHAH₂ and the Ga(III)-IndoHAH complex (in ^₆-DMSO) showed that all the signals of the parent ligand were split into two for the complex, but all the resultant signals were shifted compared with that of the ligand (i.e., there was no unreacted ligand left). Only one highly polar proton signal (~12 ppm), corresponding to the hydroxamato group, was observed in the spectrum of Ga(III)-IndoHAH, instead of two such signals for IndoHA (~9 and ~11 ppm). Thus, the NMR data indicate the formation of two Ga(III)-IndoHAH complexes (or geometric isomers) with singly deprotonated IndoHAH ligands. These results are consistent with the IR data, showing that the C=O signal at 1649 cm^"1 for IndoHAH₂ was replaced with two signals at 1684 and 1591 cm^'1. The main ESMS signal for Ga(III)-IndoHAH (~1 mM solution in DMF) was that at m/z = +883.7 (⁶⁹Ga), corresponding to [Ga(LH)₂J⁺-DMF (where LH₂ = IndoHAH₂). The Ga content in the complex, determined spectrophotometrically with 4-(2-pyridylazo)resorcine (PAR) after digestion of the complex with concentrated HNO₃, was 8.0 and 8.7% (for two parallel samples). An acidic aqueous solution of Ga(III) (0.64 M, see above) was used as a standard. The Ga content in the complex is close to that expected for [Ga(LH)₂(Cl)(OH₂)] (FW = 865.4, 8.0% Ga) or [Ga(LH)₂(OH₂)₂]Cl (FW = 883.4, 7.9% Ga), but not to that for [Ga(LH)₃] (FW = 1183.0, 5.9% Ga). In summary, the data the formation of a bis-ligated Ga(III) hydroxamato complex, [Ga(LH)₂(OH₂)₂]⁺

Cobalt-IndoHA complex Preparation of JrCfWs-[Co(Cn)₂Cl₂]Cl

CoCl₂-OH₂O (20.1 g) was dissolved in water (20 mL) in a beaker and ethylenediamine (en) (10% w/w) solution was added to it cautiously with stirring. The solution was then cooled in an ice-bath to 0-5 °C and 25 mL OfH₂O₂ (27.5%) was added dropwise with the additional funnel. After completion OfH₂O₂ addition the solution was warmed gently to about 60-70 ⁰C for 10 min, and 40 mL of cone. HCl was added to the solution with stirring and the solution was evaporated with stirring to two third of the original. After cooling the solution in an ice-bath, 30 mL of ethanol was added and further cooled 10 min, green crystals were filtered, washed with ethanol and dried in an oven at 110 ⁰C. Yield: 12.5 g

Cw-[Co(Cn)₂(OSO₂CF₃)] (CF₃SOs)₂ Nitrogen was bubbled through a solution of trans-[Co(en)₂Cl₂]Cl (8.0 g) in anhydrous CF₃SO₃H (36 mL) as it was heated at 90-100 °C for 3 h. After the solution was cooled, diethyl ether (0.15 L) was added slowly with vigorous stirring. The solid was filtered, triturated on the frit with ether (~60 mL) and dried in air. The product was ground in a mortar, boiled in chloroform (~80 mL) for 10 min to remove traces of CF₃SO₃H, filtered, washed with ether, and dried in vacuum over P₂O₅ to give a free- flowing purple powder (27 g, 94% yield). ¹HNMR (300 MHz): d 2.90 (3 H, trans NH₃), 3.16 (12 H, cw NH₃).

[Co(en)₂(IndoΗA)](CF₃Sθ3)2

To a solution of cw-[Co(en)₂(OSO₂CF₃)](CF₃SO₃)₂ (1.25 g, 2 mmol) in DMSO (10 mL) was slowly added a solution of oxamethacin (0.802 g, 2.2mmol) in DMSO (10 mL) and the resulting mixture was heated at 80 °C overnight. After cooling, the orange solution was slowly added to ice-cooled diethyl ether (400 mL) with vigorous stirring, the oily compounds solidified after decantation, stirring and sonication. To remove impurities, the orange colored compound was dissolved in the minimum volume of ethanol, filtered, and the filtrate was slowly added to ice-cold diethyl ether with vigorous stirring. The precipitate obtained was dried in a vacuum desiccator over P₂O₅. ¹H NMR (DMSO-^₆): d 11.84(s). 7.69-6.72(m), 5.02-4.17 (m), 3.79(s), 3.64(s), 3.32(s), 2.45(t), 2.26(s). ¹³C NMR (DMSO-^₆): 13.46, 23.80, 42.84, 42.98, 44.81, 45.07, 55.50, 102.01, 111.18, 111.53, 114.54, 122.79, 129.04, 130.37, 130.54, 131.25, 133.99, 136.15, 137.82, 155.60, 167.88, 168.59.

[Co(en)₂(IndoHA)]Cl2

[Co(en)₂(IndoHA)](CF₃SO₃)₂ was dissolved in minimum volume of acetone (HPLC grade), and was added to two equivalent OfEt₄NCl-SH₂O with stirring, after stirring 10 min, a pink coloured solid precipitated from the solution. The solid was removed by filtration, and re-dissolved in water, filtered and the insoluble solids discarded. The filtrate was then slowly added to ice-cold diethyl ether with vigorous stirring. The precipitate was filtered, washed with diethyl ether, and dried in desiccator under vacuum. ¹H NMR (DMSO-4): d 11.84(s, broad), 7.72-6.72(m, Ph-H), 5.05-4.39 (m), 3.79(d), 3.65(s), 3.33(s), 3.21(tetra), 2.25(s), 1.16. ¹³C NMR (DMSO-J₆): 7.41, 13.85, 24.20, 43.26, 43.37, 45.17, 45.43, 51.77, 55.89, 102.40, 111.57, 111.99, 114.92, 129.42, 130.75, 130.94, 131.64, 134.39, 136.50, 138.18, 155.98, 168.26, 168.91. Acemetacin-hydroxamic acid complex

HiO, THF

Scheme 8

To a suspension of acemetacin (1.40 g, 3.37 mmol) in benzene (14 ml) was added thionyl chloride (1 ml, excess). The batch was stirred and heated to reflux for 30 minutes. After this time the reflux condenser was exchanged for a distillation kit and the batch was distilled to remove HCl, SO, SO₂, and most of the benzene. The residue was then concentrated further on the rotary evaporator to obtain a pale orange solid, and the solid was redissolved in THF (15 ml).

Hydroxylamine HCl (0.47 g, 6.74 mmol) and potassium carbonate (0.93 g, 6.74 mmol) were dissolved in water (2 ml), diethyl ether (35 ml) was then added and the mixture was stirred. To this was added the acid chloride solution described above. After 16 hrs, EtOAc (40 ml) and sat. NaHCO₃ (aq) (40 ml) were added, and the two phases were separated. The organic phase was further washed with sat. NaHCO₃ (aq) (3 x 20 ml), dried (MgSO₄), and concentrated in vacuo to afford a yellow foam: 1.15 g obtained. This was recrystallised from EtOAc / hexane to afford 1.02 g as a pale yellow solid, 70% molar yield. m/z (negative electrospray): 429 (L^" for AcHA 3 "5,Cl), 100%, and 431 (L^' for AcHA ³⁷Cl), 34%. dH (300 MHz, OMSO-d₆) 10.73 (br s, 0.70 H), 10.67 (br s, 0.09 H), 10.22 (br s, 0.16 H), 9.22 (br s, 0.16 H), 8.97 (br s, 0.70 H), 8.84 (br s, 0.09 H), 7.70-7.63 (4H, m, C₆H₄Cl), 7.08 (IH, d, J2.4 Hz), 6.94 (IH, d, J9.0 Hz), 6.72 (IH, dd, J9.0, 2.4 Hz), 4.47 (2H, s, OCH₂), 3.87 (2H, s, O=CCH₂), 3.78 (3H₅ s, OCH₃), and 2.22 (3H, s, CCH₃). 1.1.3 Metal amine complexes with mononuclear ligands

[Co(NH₃)SaHdO)](CF₃SOs)₂

[Co(NH₃)₅(OSO₂CF₃)](CF₃SO₃)₂ (0.5 g, 0.84 mmol) was slowly added to excess indomethacin (1.0 g, 2.8 mmol) and triethylamine (0.283 g, 2.8 mmol), after stirring for 3 h at 80 ⁰C, the solution was cooled and then added to ice-cooled diethyl ether (400 mL) with vigorous stirring, the oily compounds solidified after decantation, stirring and sonication, alternatively. The solid was filtered off and washed twice with diethyl ether and an orange powder was obtained. The solid was then dissolved in a large amount of warm water, filtered, and the filtrate was evaporated on the rota-evaporator to a small volume and then left to crystallize. The crystals obtained were dried in a vacuum desiccator over P₂O₅. Anal. Calcd for C₃H₁₅N₅F₉O₉S₃Co: C₅ 5.67; H, 2.38; N, 11.03; S, 15.14. Found: C, 5.6; H, 2.5; N, 11.3; S, 14.9%. ¹H NMR (DMSO-J*): d 7.71-6.64 (m, Ph-H), 4.04 (s), 3.79(s), 3.65(s), 3.10 (s), 2.62(s), 2.24 (s). ¹³C NMR (DMSO-J₆): 14.20, 34.67, 56.42, 103.48, 112.15, 115.88, 116.79, 119.90, 124.14, 130.28, 132.16, 132.44, 132.93, 136.15, 136.22, 139.46, 157.38, 169.34, 183.11.

Rat paw oedema studies on this complex using the methodology described in Example 2, gave 30% reduction in inflammation and, remarkably, no gastric ulceration when dosed at 10 mg/kg Indo molar equivalent and dissolved into an MCT paste.

[Co(NΗ₃)₅(Indo)]Cl₂.0.5Et₂θ.0.5Η₂θ

[Co(NH₃)₅(Indo)](CF₃SO₃)₂ was dissolved in minimum volume of acetone (HPLC grade), and to the resultant solution was added to two equivalents of Et₄NCl-SH₂O with stirring. After stirring for 10 min, an orange coloured solid precipitated from the solution. After filtration, the solid was re-dissolved in the minimum volume of water, filtered again and the filtrate was then slowly added to ice- cold diethyl ether with vigorous stirring. The precipitate was filtered, washed with diethyl ether, dried in desiccator in vacuo (yield 63.7%). Anal. Calcd for C₂₁H₃₆N₆Cl₃O₅Co: C, 40.82; H, 5.87; N, 13.60; Cl, 17.21, Co, 9.54%. Found: C, 40.85; H, 5.68; N, 13.42; Cl, 16.92, Co, 9.56%. ¹³C NMR (D₂O): 13.18, 30.68, 33.48, 39.25,

56.18, 102.53, 110.89, 115.16, 115.53, 129.18, 130.98, 131.30, 131.66, 13.63, 136.36, 139.06, 155.66, 169.64, 183.71. Similar reactions were used to prepare [Ru(NH₃)₅(Indo)]X₂ complexes. 1.1.5 Metal amine complexes with amide derivatives of carboxylates Preparation of Indomethacin-glycine adduct

The method is based on that described in International Patent Application No. WO 95/04030, with the modification that 1-hydroxybenzotriazole (HOBt) was used in place of hydroxysuccinimide (HOSu). To a mixture of IndoH (3.97 g, 11.10 mmol) and HOBt (1.65 g, 12.21 mmol) was added acetonitrile (100 ml) and the resulting yellow suspension was stirred at room temperature. To this slurry was added N₅N'- dicyclohexylcarbodiimide (DCC) (2.52 g, 12.21 mmol). After a few minutes, the mixture became thick. Stirring was continued at room temperature. After 1 hr, a solution of glycine (0.92 g, 12.21 mmol) in water (10 ml) and triethylamine (1.7 ml, 1.23 g, 12.21 mmol) was added dropwise. Additional water (20 ml) was added to help improve the mobility of the mixture. After stirring at room temperature for 20 hours, water (100 ml) was added, and the mixture was acidified using dil. HCl (aq). EtOAc (30 ml) was then added and the batch was filtered to remove solid urea by-product. The aqueous and organic phases were separated and the aqueous was again extracted with EtOAc (30 ml). The combined organic phases were dried (MgSO₄) and concentrated in vacuo to give a yellow foam. This crude product was purified by column chromatography using silica and a gradient eluent system of DCM:MeOH (98:2, increasing the methanol content gradually to 3%, 5% then 10%). The fractions containing clean product were combined and concentrated in vacuo to give a pale yellow solid: 2.75 g (60%). Electrospray mass spec (negative ion): 413 (L^" for ³⁵Cl), 100%, and 415 (L^" for ³⁷Cl), 40%.

Scheme 9

[Cu(Indo-Gly)(Im)2] complex

Cu(OAc)₂-H₂O (0.108 g, 0.5408 mmol) in methanol (8 mL) and water (1 rnL) was sonicated for 0.5 h to dissolve. Indomethacin-glycine (0.464 g, 1.118 mmol) and imidazole (0.076 g, 1.118 mmol) in methanol (25 mL) was stirred to dissolve. A solution of Cu²⁺ was added dropwise to the solution of indomethacin-glycine and imidazole at ~30 °C with stirring and the mixture changed colour from green to dark blue. The solid was formed after 30 min and filtered and washed with methanol (3 mL) once, and dried under N₂ at room temperature. Yield 0.26 g (46.8 %). EPR confirmed that this compound is a monomer. Electrospray mass spectroscopy of [Cu(Indo- GIy)₂(Im)₂] in DMF shows the presence of the [Cu(Indo-Gly)]⁺ (475.9) and [Cu(Indo- Gly)(Imidazole)]⁺ (544.0). From the IR spectra typical peaks of ligands of Indomethacin-glycine and imidazole are observed.

Indomethacin-glycyl-glycyl-glycine

EtsN, H₂O

Scheme 10

To a mixture of Indomethacin (75 mg, 0.21 mmol) and HOBt (31 mg, 0.23 mmol) was added acetonitrile (5 ml) and the resulting yellow suspension was stirred at room temperature. To this slurry was added N,N'-dicyclohexylcarbodiimide (DCC) (47 mg, 0.23 mmol) and the mixture was left to stir at room temperature. After 1 hour, a solution of glycyl-glycyl-glycine (52 mg, 0.27 mmol) in water (1 ml) and triethylamine (0.04 ml, 27 mg, 0.27 mmol) was added dropwise. The batch was stirred overnight at room temperature.

After 16 hrs, water (50 ml) was added and the mixture was extracted twice with EtOAc (50 ml, then 15 ml). The combined organic extracts were washed with brine (20 ml) and then concentrated in vacuo to give a pale yellow solid: 0.36 g obtained. The crude product was recrystallised from MeCN to give a white solid, 0.17 g. This is > 100% yield and NMR confirmed that the product was contaminated with DCC-urea.

Indomethacin-Gly-Gly-His

Et3N, H2O

Scheme 11 To a stirred suspension of Indomethacin (93 mg, 0.26 mmol) and HOBt (39 mg, 0.29 mmol, 1.1 eq) in acetonitrile (5 ml) at room temperature was added DCC (60 mg, 0.29 mmol, 1.1 eq). The DCC was washed in with additional acetonitrile (2 ml) and all the solid dissolved. The solution was stirred at room temperature for 2 hrs and a precipitate formed during this time.

A solution of Gly-Gly-His (84 mg, 0.31 mmol, 1.2 eq) in water (1 ml) and triethylamine (0.04 ml, 31 mg, 0.31 mmol, 1.2 eq) was added dropwise to the batch. The reaction was stirred at room temperature for 16 hrs. The batch was filtered to remove by-product DCC-urea, and the filter-cake was washed with EtOAc (2 x 4 ml). However, the desired product Indo-Gly-Gly-His was out of solution at the end of reaction and was also isolated on the filter-cake. After drying, this solid weighed 164 mg; the expected amount of DCC-urea was 65 mg. NMR confirmed that the solid was a combination of Indo-Gly-Gly-His and DCC-urea. By NMR, the purity was 88% Indo- Gly-Gly-His (by weight). dH (300MHz, DMSO-d6) 8.33-8.29 (IH, m, one of CONH), 8.21-8.14 (2H, m, two of CONH), 7.70-7.62 (4H, m, C₆H₄Cl), 7.57 (IH, s, one of Histidine-CH), 7.14 (IH, d, J2.4 Hz, one OfC₆H₃OCH₃), 6.93 (IH, d, J9.0 Hz, one OfC₆H₃OCH₃), 6.69 (IH, dd, J9.0, 2.5 Hz, one Of C₆H₃OCH₃), 3.79-3.69 (8H, m, CH₃O, 2 x CH₂NH, and NHC^*HCH₂), 3.59 (2H, s, indole-CH₂), 2.96-2.80 (2H, m, C^*HCH₂), and 2.23 (3H, s, indole-CH₃).

[peaks for DCC-urea were also observed: 5.57 (d, J8.0 Hz), and 1.80-0.97 (m)]

Indomethacin-Ala-Ala-Ala

Et₃N HjO, Et₃N

Scheme 12 To a stirred suspension of Indomethacin (200 mg, 0.56 mmol) in acetonitrile (10 ml) at room temperature was added triethylamine (0.09 ml, 62 mg, 0.62 mmol, 1.1 eq) and the solid all dissolved to give a yellow solution. HBTU (O-(Benzotriazol-l-yl)- ΛζN,N',iV-tetramethyluronium hexafluorophosphate) (235 mg, 0.62 mmol, 1.1 eq) was added portionwise. This solid all dissolved initially but a precipitate formed during the next five minutes.

After 1.5 hrs, a solution of Ala-Ala-Ala (143 mg, 0.62 mmol, 1.1 eq) in water (4 ml) and triethylamine (0.09 ml, 62 mg, 0.62 mmol, 1.1 eq) was added dropwise, and the batch was stirred overnight at room temperature. After 16 hrs, TLC confirmed that all the Indomethacin had been consumed

(silica, DCM : MeOH, 90 : 10). Water (50 ml) and EtOAc (50 ml) were added and the aqueous phase was adjusted to pH 4-5 using dil. HCl (aq). Separation was poor, but the two phases did eventually separate. The aqueous was extracted with further EtOAc (25 ml). The combined EtOAc phases were washed with sat. brine (50 ml) and the brine was then re-extracted with EtOAc (25 ml). The combined EtOAc phases were then concentrated in vacuo to give a pale yellow solid (0.74 g obtained). The crude product was slurried in a mixture of IPA (60 ml) and water (5 ml) and heated to reflux. The material did not dissolve so the hot mixture was filtered to isolate the off-white solid, 0.25 g obtained after drying (78% yield). NMR confirmed the correct structure and indicated that the material was of high purity. dH (300MHz, DMSO-d6) 12.75-12.35 (0.8H, br s, CO₂H), 8.34 (IH, d, J7.5 Hz, one of NH), 8.05 (IH, d, J7.2 Hz, one of NH), 7.99 (IH, d, J8.1 Hz, one of NH), 7.69-7.62 (4H, m, C₆H₄Cl), 7.15 (IH, d, J2.4 Hz, one Of C₆H₃OCH₃), 6.94 (IH, d, J9.0 Hz, one OfC₆H₃OCH₃), 6.69 (IH, dd, J9.0, 2.5 Hz, one OfC₆H₃OCH₃), 4.33-4.14 (3H, m, 3 x NHC^*IΪCH₃), 3.76 (3H, s, CH₃O), 3.56 (2H, br s, indole-CH₂), 2.22 (3H, s, indole-CH₃), and 1.25-1.19 (9H, m, 3 x NHC^*HCH₃). Indomethacin-Gly-Tyr-Ala

Et₃N H₂O, Et₃N

Scheme 13

To a stirred suspension of Indomethacin (226 mg, 0.63 mmol) in acetonitrile (6 ml) at room temperature was added triethylamine (0.1 ml, 70 mg, 0.70 mmol, 1.1 eq) and the solid all dissolved to give a bright yellow solution. HBTU (O-(Benzotriazol-l- yl)-N,N,iV,iV-tetramethyluronium hexafluorophosphate) (268 mg, 0.70 mmol, 1.1 eq) was added portionwise. This solid all dissolved. Traces of HBTU were washed in using acetonitrile (2 ml).

After 2.5 hrs, a white precipitate had formed. A solution of Gly-Tyr-Ala (215 mg, 0.70 mmol, 1.1 eq) in water (6 ml) and triethylamine (0.1 ml, 70 mg, 0.70 mmol, 1.1 eq) was added dropwise and washed in with more water (1 ml). Most of the solid dissolved and the batch was stirred overnight at room temperature.

After 16 hrs, all solid had dissolved to give a pale yellow solution. TLC confirmed that all the Indomethacin had been consumed (silica, DCM : MeOH, 90 : 10). Water (25 ml) and EtOAc (40 ml) were added and the aqueous phase was adjusted to pH 4 using dil. HCl (aq). Separation was poor, but the two phases did eventually separate with gentle heating. The aqueous had to be readjusted to pH 4 using dil. HCl (aq). This aqueous was then extracted with further EtOAc (25 ml). The aqueous was again adjusted to pH 3-4 using dil. HCl (aq) and extracted with EtOAc (25 ml). The combined EtOAc phases were washed with water (25 ml) then sat. brine (25 ml), then concentrated in vacuo to give a pale yellow foam (0.67 g obtained). Recrystallisation 1: EtOAc (15 ml) was added and the mixture was heated to reflux. However, the solid did not dissolve. The batch was allowed to cool to room temperature and then filtered to afford a yellow solid which was dried under vacuum at 4O⁰C to give 0.36 g. This was shown by NMR to be the correct product but 92% purity. Yield 0.36 g @ 92% purity = 0.33 g @ 100% purity, i.e. 80% yield. dH (300MHz, DMSO-d6) 12.75-12.40 (0.8H, br s, CO₂H), 9.23 (IH, s, phenolic OH), 8.34 (IH, d, J7.3 Hz, one of NH)₅ 8.28-8.24 (IH, m (pseudo t), one of NH), 8.00 (IH, d, J8.5 Hz, one of NH), 7.69-7.63 (4H, m, C₆H₄Cl), 7.15 (IH, d, J2.4 Hz, one Of C₆H₃OCH₃), 7.01 (2H, d, J8.4 Hz₅ two of C₆H₄OH), 6.93 (IH, d, J9.0 Hz₅ one Of C₆H₃OCH₃), 6.68 (IH, dd, J9.0, 2.5 Hz₅ one Of C₆H₃OCH₃), 6.62 (2H₅ d, J8.4 Hz₅ two OfC₆H₄OH)₅ 4.49-4.41 (IH₅ m, NHC^*HCH₃), 4.23-4.14 (IH, m, NHC^*IΪCH₂Ar), 3.75 (3H, s, CH₃O), 3.79-3.71 and 3.58-3.51 (2H, m, NHCH₂), 3.55 (2H, s, indole-CH₂), 2.94-2.85 and 2.64-2.55 (2H₅ m, CfHCH₂Ar), 2.21 (3H, s, indole- CH₃), and 1.27 (3H, d, J7.3 Hz, C^*HCH₃).

1.1.6 Sugar derivatives as ligands

These ligands can be prepared by the following reaction schemes and can coordinate to metal ions via the diol functions.

THF

Scheme 14 1.1.7 Copper Ketorolac complexes

[Cu(Ketorolac)₂(OH₂)₂].2H₂0

Ketorolac tris salt (0.452 g, 1.20 mmol) was dissolved in water (5 mL). CuCl₂ solution prepared from CuCl₂-2H₂O (0.204 g) in H₂O (2.5 mL) was added to ketorolac solution with stirring. Initially bright green precipitate were formed immediately after the addition, and changed to light blue after addition completion. The light blue precipitate was filtered off and washed with water, dried in vacuum for 2 h at room temperature. Yield 0.245 g. EPR spectroscopy confirmed it is the monomer. Cu(Ketorolac)₂(H₂O)₄: Elemental Analysis, CaId for C₃₀H₃₀CuN₂O₁₀: C, 56.11; H, 4.71; N, 4.36; Cu, 9.90; Found: C, 56.15; H, 4.91, N, 4.55, Cu, 9.46.

[Cu₂(KCtOrOIaC)₄(CH₃CH₂OH)₂]

Light blue [Cu(Ketorolac)₂(OH₂)₂].2H₂O was dissolved in a minimum amount of ethanol. The green solution was filtered through the filter paper into a round-bottom flask, evaporated to dryness on a rotary evaporator. [Cu₂(Ketorolac)₄(CH₃CH₂θH)₂]: CaId for C₆₄H₅₆Cu₂N₄O₁₄; C, 62.38; H, 4.58; N, 4.55; Cu, 10.31; Found: C, 61.04; H, 4.45, N, 4.51, Cu, 10.34. The compound was confirmed to be a dimer by EPR spectroscopy.

1.2.1 Results and discussion

The above examples demonstrate a diversity of complex types and metals that can be used to prepare drugs for prophylaxis or treatment of inflammation. Complexes such as the Co(III) complex described in Example 1.1.3 offer the potential of systemic delivery of even higher concentrations of NSAIDs through oral, injectable, and topical delivery and incorporation into slow release patches. As demonstrated by this example, the complex exhibits significant anti-inflammatory.

EXAMPLE 2 In vivo toxicity of [Cu₂(IiIdO)₄(I)MF)₂] compared to indomethacin 2.1 Summary

A study was conducted to evaluate gastrointestinal permeability, mitochondrial oxidative damage and renal toxicity of [CU₂(LKIO)₄(DMF)₂] compared to indomethacin. Rats were dosed with indomethacin and [Cu₂(Indo)₄(DMF)₂] for 28 days. Acute gastrointestinal toxicity was measured using gastrointestinal permeability markers, gastrointestinal ulceration and bleeding, and measurement of an acute phase protein haptoglobin. The effects of acute and chronic administration of indomethacin and [Cu₂(Indo)₄(DMF)₂] on urinary electrolyte concentrations were examined.

It was found that [Cu₂(Indo)₄(DMF)₂] was considerably safer in all measures of gastrointestinal toxicity than indomethacin. Indomethacin reduced urinary electrolytes at an ulcerogenic dose of 10 mg/kg acutely and chronically at 3 mg/kg for 28 days. [Cu₂(Indo)₄(DMF)₂] at equimolar Indo doses affected urine electrolytes after acute dosing but did not after chronic dosing for 28 days. It is concluded that

[Cu₂(Indo)₄(DMF)₂] has both gastrointestinal and renal sparing properties.

2.2 Methods and materials

Indomethacin, carboxymethylcellulose (CMC), phosphate buffered saline, foetal bovine serum, trypsin-EDTA, halothane, HEPES, penicillin-streptomycin, sodium bicarbonate sucrose, McCoy's 5 A medium and methylene blue were obtained from Sigma Aldrich (St. Louis MO, USA). [Cu₂(Indo)₄(DMF)₂] was used as supplied by Biochemical and Veterinary Research Limited (Mittagong, NSW, Australia) and Vetafarm (Wagga Wagga, Australia). Sprague-Dawley rats weighing 200-250 g were used throughout this study.

Animals were housed in polypropylene cages and allowed free access to standard laboratory rat chow (Purina Rat Chow, Ralston Purina, St Louis MO, USA) and tap water. Animals were housed in an animal care facility at ambient temperature and humidity with a 12-h light-dark cycle. The experimental animal protocols were approved by animal ethics committees at The University of Sydney, Australia and Washington State University, USA.

2.2 Plasma haptoglobin

Rats (n = 4 for each treatment) were deprived of food, but not water, for 18 h and fasted overnight. The rats received either oral indomethacin at doses of 10 mg/kg or

[Cu₂(Indo)₄(DMF)₂] at doses of 13.3 mg/kg via gavage. Plasma was obtained by cardiac puncture using a 23 gauge (G) needle, attached to a 10 mL syringe, under halothane anaesthesia. Haptoglobin concentration (milligrams per millilitre) was measured using a commercially available kit Dade Behring (Mannheim, Germany) by radial immunodiffusion using 5-μL samples in each well. Normal range values are a diffusion zone of approximately 6 mm of diameter (1.22 g/L). The diameter of the precipitin zone is directly proportional to the concentration of the relevant protein in the sample

(Nabumetone, an effective anti-inflammatory agent, lacks gastrointestinal irritancy in the rat when dosed orally for one month: comparison with tiaprofenic acid and etodolac. Melarange, R.; Gentry, C; Blower, P.R.; Toseland, CD.; Spangler, R. Eur. J. Rheumatol. Inflamm. 1994, 14, 15-22) (see Figure 4).

2.3 Gastric mucosal damage

Rats were divided into two groups (n = 4 each). The rats received either oral indomethacin at doses of 10 mg/kg or [Cu₂(Indo)₄(DMF)₂] at doses of 13.3 mg/kg via gavage. Since [CU₂(LKIO)₄(DMF)₂] consists of a copper moiety and an indomethacin moiety, a higher dose of this compound was given so that an equivalent amount of the NSAID moiety was being delivered as in the indomethacin-treated rats. The compounds were suspended in 2% carboxymethylcellulose. Rats were deprived of food, but not water, for 18 h and were administered indomethacin, [Cu₂(Indo)₄(DMF)₂] or vehicle. Animals were anesthetized with halothane 3 h after the dose and the stomach was excised and opened by an incision along the greater curvature for assessment of macroscopic visible damage by an observer unaware of the treatment the rats had received using a method described previously (Aspirin causes rapid up-regulation of cyclooxygenase-2 expression in the stomach of rats. Davies, N.M.; Sharkey, K. A.; Asfaha, S.; Macnaughton, W.K.; Wallace, J.L. Aliment. Pharmacol. Ther. 1997, 11, 1101-18).

The length of lesions was measured (in millimetres) using digital callipers and then the lengths of all lesions observed in each stomach were added together (see Figure 2). After scoring, the gastric tissue was fixed in neutral buffered formalin and processed by routine techniques prior to embedding in paraffin, sectioning and staining with hematoxylin and eosin. 2.3.1 Gastric permeability

Sucrose permeability changes were measured using a previously reported method (Sucrose urinary excretion in the rat using a simple assay: a model of gastroduodenal permeability. Davies, N.M.; Coπigan, B.W.; Jamali, F. Pharm. Res. 1995, 12, 1733-6).

Rats (n = 4 for each treatment) were deprived of food, but not water, for 18 h and fasted overnight. The rats received either oral indomethacin at doses of 10 mg/kg or [Cu₂(Indo)₄(DMF)₂] at doses of 13.3 mg/kg via gavage. Two hours post-dosing, an aqueous solution (0.5 mL) containing 0.5 g/mL of sucrose was administered orally to each rat. Urine was collected 0-24 h following the administration of the sucrose solution. Relative permeability was determined by calculating the sucrose present in each urine sample as a percent of the administered dose (see Figure 3).

2.3.2 Small intestinal ulceration

Rats (» = 4 for each treatment) received either oral indomethacin at doses of 10 mg/kg or [Cu₂(Indo)₄(DMF)₂] at doses of 13.3 mg/kg via gavage. To assess small intestinal damage, the animals were sacrificed 24 h after dosing and the intestines removed. A vertical mid-line abdominal incision was made, and the entire length of the small intestine was isolated, excised, and examined extending 10 cm distal to the ligament of Treitz to the ileocecal junction. A 26 G needle attached to 5-mL syringe was used to flush the intestine in order to avoid distension. After expelling the intestinal contents, the entire intestine was submerged in 10% technical grade formaldehyde 37/7 for 1 h. The intestine was then opened along the anti-mesenteric side and pinned to a thin piece of cardboard. Intestinal ulceration was determined by measuring the length of lesions in millimetres using digital callipers and the lengths of all lesions observed in each intestine summed (NO-naproxen vs. naproxen: ulcerogenic, analgesic and antiinflammatory effects. Davies, N.M.; Roseth, A.G.; Appleyard, CB. ; et al. Aliment. Pharmacol. Ther. 1997, 11, 69-79) (see Figure 5). In order to ensure that the damage being assessed were indeed ulcers, sections of tissue were obtained from damaged areas for histology. Tissue sections were embedded in plastic using a commercially available kit (JB-4 embedding kit, Polysciences. Inc. Warrington, PA). Embedded tissues were cut to thin sections (1-1.5 μm) and the sections were stained with Lee's methylene blue- basic fuchsin for 30 seconds. Sections were then examined by light microscopy.

2.3.3 Small intestinal permeability

Rats (n = 4 for each treatment) received either oral indomethacin at doses of 10 mg/kg or [Cu₂(Indo)₄(DMF)₂] at doses of 13.3 mg/kg via gavage. To test intestinal permeability, 0.5 rnL of a solution containing 10 μCi/mL Of⁵¹Cr-EDTA was administered orally following the dose of placebo or NSAID. Rats were housed in special metabolic cages where urine and faeces were collected separately. Urine was collected 0-24 h following the administration Of ⁵¹Cr-EDTA. The urine was collected in cups and transferred to scintillation vials. Urine samples were counted by a gamma counter Beckman Gamma 8000 (Irvine, California) for 1 min in a counting window scanning within a range of 0-2 MeV. At least two standards of 100 μL of the administered ⁵¹Cr-EDTA solution were counted with every set of urine samples. Relative permeability was determined by calculating the activity present in each urine sample as a percent of the administered dose after correcting for background radiation (Anti-inflammatory drug-induced small intestinal permeability: the rat is a suitable model. Davies, N.M.; Wright, M.R.; Jamali, F. Pharm. Res. 1994, U(Il), 1652-6) (see Figure 6).

2.3.4 Caecal haemoglobin

The caecum {n = 4) was excised from rats dosed with indomethacin (10 mg/kg), [Cu₂(Indo)₄(DMF)₂] (13.3 mg/kg) or controls (no treatment). The contents were removed and diluted with distilled water up to 5 mL and vortex-mixed to a homogeneous consistency. The contents of each test tube were divided into two tubes and centrifuged for 10 min at 3000 g and the supernatants were stored at -20°C. Aliquots (0.5 mL) were assessed for the quantitative, colorimetric determination of haemoglobin concentration measured spectrophotometrically in accordance to Sigma Diagnostics Total Hemoglobin^® (Sigma Chemical Co., St. Louis, MO, USA) (Nabumetone, an effective anti-inflammatory agent, lacks gastrointestinal irritancy in the rat when dosed orally for one month: comparison with tiaprofenic acid and etodolac. Melarange, R.; Gentry, C; Blower, P.R.; Toseland, CD.; Spangler, R. Eur. J. Rheumatol. Inflamm. 1994, 14, 15-22) (see Figure 7).

2.3.5 Enteric bacterial numbers Rats (n = 4 for each treatment) received either oral indomethacin at doses of

10 mg/kg or [Cu₂(Indo)₄(DMF)₂] at doses of 13.3 mg/kg via gavage. In order to assess enteric bacterial translocation, the animals were sacrificed 24 h after dosing and the intestines were removed. A vertical mid-line abdominal incision was made, and the entire length of the small intestine was isolated, excised, and examined extending 10 cm distal to the ligament of Treitz to the ileocecal junction. Intestinal tissue samples (~1 g) were obtained from the distal ileum, placed into Petri dishes containing sterile phosphate-buffered saline (pH 7.4), and weighed. The tissues were homogenized and plated onto MacConkey agar No. 2 (Oxoid, NSW, Australia) and incubated at 37°C for 24 h under aerobic conditions. Plates containing between 20 and 200 colony-forming units (CFU) were analysed to determine total enteric bacterial numbers per gram of tissue (Nonsteroidal anti-inflammatory drug enteropathy in rats: role of permeability, bacteria, and enterohepatic circulation. Reuter, B. K.; Davies, N.M.; Wallace, J.L. Gastroenterology 1997, 112, 109-17) (see Figure 8).

2.3.6 Mitochondrial DNA

The small intestine (n = 5) was excised from rats dosed with indomethacin (10 mg/kg), [Cu₂(Indo)₄(DMF)₂] (13.3 mg/kg) or controls (no treatment). Quantitative polymerase chain reaction (QPCR) was used as previously described (Chemotherapy induced gastrointestinal toxicity in rats: involvement of mitochondrial DNA, gastrointestinal permeability and cyclooxygenase-2. Yanez, J.A.; Teng, X.W.; Roupe,

K.A.; Fariss, M.W.; Davies, N.M. J. Pharm. Pharm. Sd. 2003, 6, 308-314).

DNA was isolated from afflicted intestinal tissue using Qiagen® genomic tip and genomic DNA buffer set kit for mammalian DNA extractions (Valencia, CA, USA). DNA quantitation utilized the PicoGreen® dsDNA Quantitation Kit (Molecular Probes, Eugene, OR, USA). Picogreen® was used to quantify dsDNA fragment. QPCR involved the use of GeneAmp XL PCR kit (Applied Biosystems, Branchburg, NJ, USA) and dNTPs (Pharmacia, Peapack, NJ, USA). Primers were based on sequences already optimized by Van Houten (Analysis of gene-specific DNA damage and repair using quantitative polymerase chain reaction. Torres, S.A.; Chen, Y.; Svoboda, T.; Rosenblatt, J.; Van Houten, B. Methods 2000, 22, 135-147; and Measuring oxidative mtDNA damage and repair using quantitative PCR. Santos, J.H.; Mandavilli, B. S.; Van Houten, B. Methods MoI Biol 2002, 197, 159-176).

Fluorescent readings for all products were taken using a CytoFluor fluorescence multiwell plate reader Series 4000 (Applied Biosystems, Framingham, MMA, USA) and subtracted from the no-template controls and relative amplification was calculated (see Figure 9).

2.4 NAG (N-acetyl-β-D-glucosaminidase)

Rats (n - 4 for each treatment) received either oral indomethacin at doses of 10 mg/kg or [Cu₂(Indo)₄(DMF)₂] at doses of 13.3 mg/kg via gavage. Rats were housed in special metabolic cages where urine and faeces were collected separately 0-24 h after administration. The colorimetric assay kit was obtained from Boehringer Mannheim. The instructions and procedures were followed as documented by the manufacturer. S-Cresolsulfonphthalenyl-N-acetyl-β-D-glucosaminide, sodium salt was hydrolysed by NAG with the release of 3-cresolsulfonphthalein, sodium salt (3-cresol purple), which was measured photometrically at 580 nm (see Figure 10).

2.5 Urine electrolytes

Rats (n = 3-5 for each treatment) received either oral indomethacin at doses of 3 or 10 mg/kg or [Cu₂(LIdO)₄(DMF)₂] at doses of 3.8-13.3 mg/kg. Rats were housed in special metabolic cages where urine and faeces were collected separately 0-24 h after administration. Sodium, potassium, chloride and phosphate were assayed at the

Veterinary Pathology laboratory (Faculty of Veterinary Science, The University of Sydney), using commercially prepared reagents obtained from Beckman Coulter Inc. a Beckman, Synchrome, and EI-ISE, Electrolite system (Beckman Coulter Inc. Brea, CA USA) (See Table 2). Table 2: Effect of Acute and Chronic IndoH and [Cu₂(Indo)₄(DMF)₂] Dosing on Urine Electrolytes Composition

2.6 Results

The results were expressed as the mean + standard error of the mean (SEM). Student's t test, 2-tailed ANOVA and Bonferroni's multiple-comparison test were used to determine significance levels. P < 0.05 was considered significant.

Administration of indomethacin induced statistically significant gastric and intestinal damage in the rat at 10 mg/kg. However, [Cu₂(Indo)₄(DMF)₂] administration, at an equivalent dose of Indo, significantly attenuated the ulcerogenic properties and renal toxicity of the parent compound (see Figures 1-9). Acute administration of ulcerogenic doses of indomethacin also resulted in decreased urinary excretion of Na , K⁺, and Cl and increased urinary excretion of NAG and phosphate (see Table 2, Figure 10). Acute administration of [CU₂(LKIO)₄(DMF)₂] also decreased Na⁺, K⁺, and Cl but did not result in an increase in urinary NAG or phosphate excretion. The chronic administration of indomethacin at a non-ulcerogenic dose resulted in a decrease in Na⁺, K⁺, Cl, but not phosphate, whereas chronic administration of [Cu₂(Indo)₄(DMF)₂] did not significantly change the urinary electrolytes concentrations studied.

Two animals m the indomethacin group died in the first week of treatment with indomethacin. Together with the measures of GI and renal toxicity, it was demonstrated that IndoH at a dose of 10 mg/kg was too toxic, but the rats tolerated Culndo at an equivalently high dose surprisingly well in terms of GI and renal toxicity.

2.7 Discussion [CU₂(LKIO)₄(DMF)₂] has been shown to be less ulcerogenic to the gastrointestinal tract of rats than indomethacin (Copper-indomethacinate associated with zwitterionic phospholipids prevents enteropathy in rats: effect on inducible NO synthase. Bertrand, V.; Guessous, F.; Le Roy, A.L.; et al. Dig. Dis. Sd. 1999, 44, 991-9; Gastrointestinal Toxicity, Antiinflammatory Activity, and Superoxide Dismutase Activity of Copper and Zinc Complexes of the Antiinflammatory Drug Indomethacin. Dillon, C. T.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; Zhou, Q.; Davies, N. M.; Biffin, J. R.; Regtop, H. L. Chem. Res. Toxicol. 2003, 16, 28-37). It appears that formulating indomethacin in a coordination complex with copper also modulates the gastrointestinal permeability. This, therefore, limits bacteria translocating across the intestinal mucosa through tight junctions of enterocytes and consequently, there is less ulceration and the sequelae of tissue damage in terms of up-regulated acute phase proteins such as haptoglobin and caecal haemoglobin and oxidative damage to the enterocytes. Although it has been established that [CU₂(LKIO)₄(DMF)₂] is less gastrointestinally (GI) toxic than Indo, an unexpected result in terms of long-term chemotherapy is that chronic administration of [Cu2(Indo)₄(DMF)₂] is much safer in terms of renal toxicity than IndoH. Since renal toxicity is a serious and limiting side- effect, this observation is of high significance in terms of Culndo being used for prophylaxis or treatment of cardiovascular inflammation, particularly long term treatment. The remarkable renal and GI safety of Culndo may be further enhanced by the use of formulations for stabilising the complex. Suitable such formulations as described in International application No. PCT/AU2005/000442 filed 30 March 2005, the contents of which are incorporated herein in its entirety by reference. The use of such formulations may enable higher tolerated doses of the metal complexes.

It is also noted that a physical mixture of IndoH and a Cu salt is even more GI toxic than IndoH alone. Hence, the Culndo complex should be appropriately formulated in order to maintain its integrity in the GI tract. This will maximise its therapeutic window which is desirable for the high safety required for long-term treatment of cardiovascular inflammation.

2.8 Conclusion [Cu₂(Indo)₄(DMF)₂] is much safer in the gastrointestinal tract and indices of kidney toxicity in rats than is IndoH, which is also highlighted by the death of two rats during chronic dosing of IndoH, whereas none of the control rats or Culndo treated rats died or showed any outward signs of toxic effects. These data show that Culndo has a much wider therapeutic window than IndoH. This is important in the treatment of cardiovascular diseases or conditions where high doses are required for acute conditions, the patient has pre-existing GI and/or renal conditions that would normally prevent the use of NSAIDs, or the patient is a premature baby. This is also important in the long term prophylactic use of lower concentrations of the drug to slow the progression of cardiovascular conditions.

EXAMPLE 3 Treatment of inflammation by intramuscular and subcutaneous injections

3.1 Methodology 3.1.1 Test compositions.

A composition comprising the [Cu₂(Indo)₄(OH₂)₂] complex in MCT oil was prepared for subcutaneous and intramuscular injections. The composition comprised the following ingredients: Ingredient: Amount:

[Cu₂(Indo)₄(OH₂)₂] complex 40 mg

Tetraglycol 300.0 mg

Delios V MCT oil qs l.O g

Tetraglycol is the solvent; Delios V MCT oil is a medium chain triglyceride oil. The composition was prepared as follows:

1 Add tetraglycol to mixer and heat to 75°C while stirring. 2. Add and dissolve Copper Indomethacin complex. Stir until dissolved, then remove heat.

3. Add Delios V MCT oil, while stirring.

4. Stir for 15 minutes until homogenous, then allow to cool.

The composition was a single-phase dark green oil immiscible in water. The composition contained >95% of Indo in the composition as part of the dimer ([Cu₂(Indo)₄(OH₂)2] as shown by EPR spectroscopy. For comparison, a similar composition containing IndoH in MCT oil was prepared by the same process using IndoH instead of ([Cu₂(Indo)₄(OH₂)₂].

3.1.2 Animals

Sprague-Dawley rats (weighing 200-250 g) used for these studies were supplied by the laboratory animal services at The University of Sydney. Animals were housed in polypropylene cages and allowed free access to standard laboratory rat chow (Purina Rat Chow, Ralston Purina, St Louis MO) and tap water. Animals were housed in the animal care facility of the Faculty of Pharmacy at ambient temperature and humidity with a 12-h light-dark cycle. The experimental animal protocols were approved by the Animal Ethics Committee of the University of Sydney.

3.1.3 In Vivo anti-inflammatory activity and gastric toxicity

Groups of four rats were used for all studies. All doses were calculated as equivalent concentrations of Indo. Rats were allowed free access to food and water except for gastric toxicity studies, when they were fasted for 24 h but with free access to water. For subcutaneous and intramuscular administration, rats were injected with 125-200 μL volumes of the test compositions (IndoH or [Cu₂(Indo)₄(OH₂)₂] in MCT oil). The control cohort was injected with equivalent volumes of neat MCT. Subcutaneous injections were made in the lower dorsal surface and intramuscular injections were made in the right hind thigh muscle. Inflammation was induced 1 h after dosing by injection of the formulation by an injection of carrageenan (0.1 mL, 1% w/v in isotonic saline) into the plantar region of the hind paw.

Paw volume was measured prior to dosing and at 3 h after carrageenan injection by immersing the left hind paw (to the lateral malleus) into a vessel filled with water and measuring the volume of water displaced as decribed in International Patent Application No. PCT/AU2005/000442 filed 30 March 2005, the contents of which is incorporated herein by cross-reference in its entirety. Immediately after paw volume measurements, 24 h-fasted animals were euthanased and the stomach was excised and opened by incision along the greater curvature. The stomach was rinsed and examined to determine the extent of macroscopic gastric toxicity, which is reported as the summation of the area of macroscopic ulcerations (mm²).

3.1.4 In Vivo small intestinal toxicity Groups of four rats were used for all studies and were treated similarly as described in Example 4.1.3, except that they were allowed free access to food and water during the assay. At 24 h after dosing, the entire small intestine was excised and flushed with water to expel the intestinal contents. The intestine was examined from 10 cm distal to the ligament of Treitz to the ileocecal junction, and the toxicity is reported as the summation of the area of macroscopic ulcerations (mm²).

3.1.5 Statistical analysis

The Student t test was used to compare mean values between two groups and repeated measures ANOVA followed by Bonferroni correction for comparisons was used to compare mean values between more than two groups. Data are expressed as the mean ± SEM. All reported P values are two-sided, and P<0.05 was considered statistically significant. 3.2 Results

3.2.1 Subcutaneous injection

The results of dose-response data for the composition containing [Cu₂(Indo)₄(OH₂)₂] in MCT oil are listed in Table 3 together with the data for the composition containing IndoH in MCT at the highest dose at which no small intestinal toxicity was observed for the MCT oil composition containing [Cu₂(Indo)₄(OH₂)₂] in MCT oil. None of the rats exhibited soreness, swelling, redness or any other adverse effect at the site of the injection at all doses.

Table 3. Efficacy and Safety of Subcutaneous Treatments of Inflammation (Rat Paw Oedema) using [Cu₂(Indo)₄(OH₂)₂] or IndoH in MCT oil.

All doses are given as the amount of Indo delivered in the form of [Cu₂(Indo)₄(OH₂)₂], except for the one example indicated in Table 3, where Indo was delivered in the form of IndoH. Although the treatment regimes were not optimized for maximum efficacy, it is clear that this mode of administration has a strong anti-inflammatory effect that plateaus around 5-7.5 mg kg¹ of Indo (the amount of Indo in the complex). At these concentrations, there is no gastrointestinal toxicity induced by the composition containing [Cu₂(Indo)₄(OH₂)₂], either as acute gastric ulcers in fasted rats, or intestinal ulcers due to secondary circulation. By contrast, the composition containing IndoH alone resulted in small intestinal ulceration in all four rats at 7.5 mg/kg and greater ulceration than that observed at 10 mg/kg of Indo for the composition containing [Cu₂(Indo)₄(OH₂)₂]. AU gastric side-effects could be easily prevented, even at the very high dose of 20 mg/kg of Indo (administered using the composition containing [Cu₂(Indo)₄(OH₂)₂]) if the rats were not fasted, but at these high concentrations small intestinal ulceration was substantial.

3.2.2 Intramuscular injection

The results of intramuscular injection into the right hind thigh muscle on rat paw oedema are given in Table 4. Table 4. Efficacy and Safety of Intramuscular Injection Treatments of Inflammation (Rat Paw Oedema) in Fasting Rats using [Cu₂(Indo)₄(OH₂)₂] or IndoH in MCT oil.

Equivalent dose of Indo

While only preliminary data have been obtained, the composition containing [Cu₂(Indo)₄(OH₂)₂] in MCT oil has a similar efficacy and safety profile in rats as those observed following subcutaneous injections, although the efficacy for treatment of inflammation is higher in the plateau region of the dose-response curve. Again, while the compositions containing [Cu₂(Indo)₄(OH₂)₂] and IndoH have similar efficacy, the composition containing [Cu2(Indo)₄(OH₂)2] in MCT oil resulted hi less GI toxicity. At both 5 and 10 nig kg^"1 of IndoH, the GI toxicity of the composition containing IndoH in MCT oil was comparable to that observed for twice the dose of Indo when it is delivered as the composition containing [Cu₂(Indo)₄(OH₂)₂] in MCT oil.

3.3 Discussion

Both subcutaneous and intramuscular administration of the composition containing the complex [Cu₂(Indo)₄(OH₂)₂] in MCT oil have considerable efficacy, with the latter mode of administration being more efficacious. If the composition was delivered as a physical mixture of a Cu salt and IndoH or the composition caused the complex to dissociate with the release of free Indo, then toxicity effects similar to those of IndoH are expected.

EXAMPLE 4 Sub-cutaneous injection study

2mL of [Cu₂(Indomethacin)₄(H₂O)2] 50mg/mL in benzyl alcohol (the concentrate) was freshly prepared. The concentrate was filtered by means of a 0.22- micron filter prior to administration. Benzyl alcohol has local anesthetic effects. 200μL of the Culndo preparation was then administered subcutaneously (sc) (n = 4 rats) into the shaved lower dorsal area just above the base of the tail. Single sc injection of lOmg of Culndo in 200μL of benzyl alcohol equates to a dose of 40mg/kg bw. Following injection, the rats were observed for 24 hours. The observations are summarised in Table 5.

Table 5: Gastro-intestinal toxicity following subcutaneous injection of [Cu₂(Indomethacin)₄(H₂O)₂] in rats

⁺ Euthanased without further examination. Whilst a 40mg/kg bw dose of [Cu₂(Indomethacin)₄(H₂O)2] by means of subcutaneous injection is toxic to the gastro-intestinal tissue of the rat, no overt irritant or necrotic effects were observed in the sub-cutaneous tissue at the site of injection.

EXAMPLE 5 Rabbit model for treatment of cardiovascular disease

To study the role of inflammation and novel anti-inflammatory drugs in the protection against cardiovascular disease a rabbit model of inflammation in the artery wall was developed. This model involves placing a plastic collar around the neck artery of a rabbit leading to irritation and inflammation in the artery wall not unlike the inflammation that occurs when blockages build up in human arteries prior to myocardial infarction.

5.1 Methodology Male New Zealand White (NZW) rabbits, aged 3 months, underwent implantation of silastic, non-occlusive collars around the carotid arteries under general anaesthesia. This was achieved as follows: a midline neck incision was made; the muscle and connective tissue layers were separated to expose the carotid arteries; the collars were placed around the arteries, taking care not to damage the blood vessels and then filled with sterile saline; and the muscle, connective tissue and skin layers were then sutured and covered with antibiotic powder. A sham operated group {n = 2) acted as a surgical control. The maximum inflammatory response was also assessed with animals receiving the collar only and no treatment (n = 8). The remaining treatment groups were treated with the following test compounds using oral gavage for the indomethacin and ACM drugs and the anti-inflammatory effects were monitored, with two animals tested for each dose of these compounds. Lipid Free Al (ApoA-1) (8 mg/kg) was infused into animals intravenously to act as a positive control. Group Metal complex

A. Indomethacin (3 mg/kg)

B. [Cu₂(Indo)₄(OH₂)₂] (1 or 3 mg/kg of Indo) C. [Cu(ACM)₂(OH₂)₂] (3 mg/kg equivalent of Indo)

The animals were pre-medicated with a sedative, administered in the form of subcutaneous acetylpromazine (2 mg/kg) and atropine sulfate (0.2 mg/kg). This occurred prior to venepuncture, surgery and sacrifice. For general anaesthesia, animals received an intramuscular injection of ketamine (7.5 mg/kg) and xylazine (3 mg/kg) and were then maintained with isofluorane (4-5% for induction and 1.5-2% for maintenance). Analgesia was supplied by administration of buprenorphine IM shortly after induction of GA, and repeated 8 hourly until sacrifice if the animal appeared to have wound discomfort. Local anaesthetic was applied at the wound site during the procedure and after wound closure topical antiseptic wound coverage was applied.

During recovery (usually 1-2 hr) each animal was placed on soft bedding in a clean recovery cage with a heating lamp. Analgesia was supplied by administration of buprenorphine IM shortly after induction of GA, and repeated 8 hourly until sacrifice if the animal appeared to have wound discomfort. Local anaesthetic was applied at the wound site during the procedure and after wound closure topical antiseptic wound coverage was applied. Animals were given normal rabbit chow throughout the study. Food was withheld one hour prior to general anaesthesia and animals were individually housed in floor pens.

All animals were sacrificed 24 hr following application of the collars by lethal infusion of barbiturate administered via a cannula placed in the marginal ear vein.

Heparin (100 U/kg) was administered prior to sacrifice. An aliquot of 3 mL of blood was sampled from the marginal ear vein prior to entry in to the study (ie. before the commencement of drug treatment), at the time of collar implantation and immediately prior to sacrifice. Blood pressure was also recorded at these times for each animal. The carotid arteries were removed from all animals following sacrifice. A proximal non-collared segment of artery was also removed from each to serve as a control. The arteries were placed in physiological buffer and the collar and fatty tissue surrounding the arteries was removed. The segments were divided into four rings. The first ring was assessed fresh for the presence of oxidative stress. The second ring was frozen in fixative solution for later analysis of inflammatory activity by immunohistochemistry. The third and fourth rings were snap frozen in liquid nitrogen for analyses at a later date for levels of mRNA and by vibrational and X-ray microprobe techniques for biochemical changes. Blood was assessed for drug concentrations, platelet aggregation and markers of renal toxicity.

The stomach and small intestine were also thoroughly examined postmortem for evidence of gastro-intestinal ulceration. The stomach was excised and opened by incision along the greater curvature. The stomach was then rinsed, submerged in 10% formaldehyde for 1 h and examined to determine the extent of macroscopic gastric damage. The damage was reported as the summation of the area of macroscopic ulcerations (mm²). Similarly, the small intestine was examined at 24 h after dosing, with the entire small intestine excised and flushed with water to expel the intestinal contents. The small intestine was examined from 10 cm distal to the ligament of Treitz to the ileocecal junction and damage was likewise reported as the summation of the area of macroscopic ulcerations (mm ).

5.2 Results

All preparations were well tolerated by the rabbits and there were no gastrointestinal lesions (stomach or small intestine) in any of the treatment groups. The level of neutrophils in the artery were quantified as a measure of the inflammation induced by the collar. These are shown as dark patches in the crossections of the artery (see Figure 12). Quantification of the results for different treatments are shown in

Figure 11. The dinuclear Culndo complex (81% reduction in neutrophils) was at least as anti-inflammatory as the standard IndoH (67% reduction in neutrophils) at the same dose of Indo and there was a dose-dependent effect for Culndo. There was some heterogeneity in the rabbit response, especially for the lower dose of Culndo. The CuACM complex (94% reduction in neutrophils) was the same as the control and the

ApoA-1 positive control (89% reduction in neutrophils), even though the Cu complex was given at a lower dose and orally, as opposed to the intravenous infusion of ApoA-1. 5.3 Discussion

The results show that some of the complexes are at least as effective as the current best treatment for arterial wall inflammation similar to that which occurs when blockages build up in human arteries prior to myocardial infarction. Moreover, the metal NSAID drugs were given orally under conditions that minimize the GI and other toxic effects normally associated with oral ingestion of NSAIDs. These results demonstrate the outstanding potential for metal complexes to be used in the treatment and prevention of both acute and chronic cardiovascular disease and conditions, especially given that they are effective at lower doses than apoA-1, which also needs to be given by intraveneous infusion.

EXAMPLE 6 Effect of metal-NSAID complexes on VCAM-I and ICAM-I expression in rabbit model of cardiovascular disease

A further study was undertaken to evaluate the anti-inflammatory properties of metal-NSAID complexes in vivo using the New Zealand White (NZW) rabbit model of acute vascular inflammation described in Example 5. The metal-NSAIDs [Cu₂(Indo)₄(OH₂)₂] (Cu-Indo), [Cu(ACM)₂(OH₂)₂] (Cu-ACM), [Zn(Indo)₂(OH₂)₂] (Zn-Indo), [Zn(ACM)₂(OH₂)₂] (Zn-ACM) were used in this study.

6.1 Methods

6.1.1 Animals

Male New Zealand White (NZW) Rabbits (Merungora Stud Farm, Wauchope, NSW, Australia) weighing approximately 2.5 kg were maintained on a normal chow diet throughout the studies.

6.1.2 Surgical Procedure

As in Example 5, prior to carotid surgery the animals were sedated with subcutaneous acetylpromazine (0.5 mg/kg), then anesthetized with inhaled isofluorane (4-5% for induction and 1.5-2% for maintenance). In this study, the left common carotid artery was exposed surgically and cleared of connective tissue along a 30-mm length. The hollow, non-occlusive, silastic, peri-arterial collar (length, 20 mm; internal diameter along bore, 4mm; internal diameter at ends, lmm) was positioned around the artery and held in place with a nylon sleeve. The space inside the collar was again filled with sterile saline (0.9%, wiv). After collar insertion, muscle, fat and skin layers were sutured. The collars were left in place until the animals were sacrificed. The non-collared right carotid artery was used as a control for each collared artery. The collar was placed for 24h. The NSAID drugs were administered via laparotomy under general anaesthesia. The rabbits (n=4 in each arm) received either no treatment, unmodified indomemthacin (Indo) (3mg/kg), Zn-ACM (3mg/kg), Cu-ACM (3mg/kg), Cu-Indo (2mg/kg) or Cu-Indo (3mg/kg). The drug was administered at the collar insertion.

6.1.3 Tissue harvesting

All the animals were scarified 24 h after collar insertion. Animals were euthanized with an i.v overdose of sodium pentobarbital (100 mg/kg). The collared segment of the left carotid artery and a 30 mm segment of the contra-lateral carotid artery were excised and placed in ice-cold phosphate buffered saline (PBS) (pH 7.4). The collars were subsequently removed and the arteries were cleaned of fat and connective tissue. For immunohistochemical analysis, ring sections were cut from the arteries, fixed in cold ethanol and wax embedded using known protocols.

6.1.4 Immunohistochemistry

Sections (5 mm) of the vessels were cut, dewaxed, and rehydrated. Mouse anti- rabbit VCAM-I and ICAM-I antibodies (gifts from Dr M. Cybulsky, University of Toronto) were used to assess endothelial expression of VCAM-I and ICAM-L. The antibodies were diluted in TBS (0.01 M Tris, 0.15 M NaCl, 0.006% (w/v) NaN₃ and 0.005% (w/v) EDTA-Na₂, pH 7.4) containing 10% (IA) heat-inactivated horse serum. The diluted antibody was applied to the sections, which were then incubated for 1 hr at room temperature. The sections were then washed twice with TBS, incubated with 300 μl of the Dalcocytometim Envision HRP system for 30 min at room temperature, then developed as instructed by the manufacturer for 3 min with DAB solution (DAKO Australia Pty, Ltd). The sections were lightly counterstained with haematoxylin. The specificity for anti-rabbit ICAM-I or VCAM-I antibody was determined by the use of an isotype control (irrelevant Ig).

6.1.5 Quantification Endothelial expression of the VCAM-I and ICAM-I adhesion molecules was determined by quantitative immunohistochernistry using ImagePro Plus 4.5 software (Media Cybernetics, Silver Spring, MD). The threshold for positive staining was defined by a pathologist who was blinded to the treatment. This threshold was used to analyse all subsequent samples. The results are shown in Figs 13 and 14, are expressed as image units and represent the average positive staining above the threshold for individual arterial sections. Systematic sampling of each artery quadrant generated a single mean image unit value for staining for each artery. The mean of these values represents the amount of staining per treatment group used for subsequent statistical comparison. AU results are expressed as mean ± SEM. Statistical comparisons were made by two-tailed Student's t-tests using GraphPad Prism Version 4.0 (San Diego, CA). A value of p < 0.05 was considered statistically significant.

6.2 Results

There was marked up-regulation in endothelial expression of VCAM-I in response to the carotid collar. This was substantially decreased by the treatments with unmodified Indo (75+27%, p<0.05), Cu-ACM (88+22%, pO.Ol), and Cu-Indo 3mg/kg (67+21%, p<0.05). Decreases in endothelial VCAM-I expression observed for the Zn- ACM (39+27%) and Cu-Indo 2 mg/kg (51 ±31%) treatments did not achieve achieve statistical significance. (Fig. 13). Endothelial expression of ICAM-I was also markedly increased in response to the carotid collar. This up-regulation was substantially decreased by the unmodified Indo (79±9%, ρθ.0001), Zn-ACM (84+8%, pO.OOOl) and Cu-Indo 2 mg/kg (64±16%, p<0.01) treatments. The decreases in endothelial ICAM-I expression obtained by the Cu-ACM (43±19%) and Cu-Indo 3 mg/kg (28+33%) treatments did not achieve statistical significance (Fig. 14). 6.3 Conclusions

This study confirms that metal-NSAID complexes can have marked antiinflammatory actions in rabbit carotid arteries in vivo. In particular, significant down regulation of VCAM-I, a key player in early artherogenesis, was obtained by the Cu- ACM and Cu-Indo treatments. Moreover, endothelial ICAM-I expression was decreased with Zn-ACM and Cu-Indo. The magnitude of the inhibition of the CAMs was at least comparable to that achieved with unmodified Indo.

These data provide supporting evidence for the use of the metal-NSAID complexes in the treatment of states of acute vascular inflammation in vivo. There were no adverse effects noted with any of the treatments and indeed, no macroscopic or microscopic evidence of gastrointestinal or renal effects was found. Moreover, the dosing of Cu complexes at the therapeutic levels used herein may enhance the treatment of a range of acute cardiovascular conditions, eg., hypertrophic cardiomyopathy, (Jiang, Y, et al. J. Exp Med. 2006, 2007 204: 657-666). Chronic treatment with the drugs is also likely to be beneficial in the prophylaxis of chronic cardiovascular conditions, since there is evidence that Cu supplementation at the levels used in these studies is advantageous in reducing cardiovascular and other degenerative diseases as many people have insufficient Cu hi their diet, which is a risk factor in such diseases. (Saari, J. T. Copper deficiency and cardiovascular disease: role of peroxidation, glycation, and nitration. Canad. J. Physiol. Pharmacol. (2000), 78, 848-855; Marginal copper deficiency and atherosclerosis. Hamilton, I. M. J.; Gilmore, W. S.; Strain, J. J. Biol. Trace Elem. Res. (2000), 78, 179-189; Effect of copper supplementation on copper status and risk markers of cardiovascular disease in young healthy women. Bugel, S.; Harper, A.; Rock, E.; O'Connor, J. M.; Bonham, M. P.; Strain, J. J. Brit. J. Nutrit. (2005), 94, 231-236).

In addition, the indobufen analogs of these complexes, [Cu₂(Indob)₄L₂] and [Cu(Indob)₂L₂], would have a three-fold benefical effect in the prophylaxis and treatment of cardiovascular conditions. These include: the beneficial effects of Cu, as described above; the anti-inflammatory effect of indobufen; and anti-thrombotic effect of indobufen, while simultaneously reducing the GI toxicity (Endoscopic evaluation of the effects of indobufen and aspirin in healthy volunteers. Marzo A; Crestani S; Fumagalli I; Giusti A; Lowenthal D T, Am. J. Therapeutics (2004), 11, 98-102. Since indobufen is comparable to warfarin in its anti-thrombotic effect, and a significant problem with its use is the potential for ulceration of the GI tract, these complexes are expected to be extremely useful in the prophylaxis and treatment of cardiovascular disease. Although the invention has been described with reference to particular examples, it will be appreciated by those skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. All such variations and/or modifications are to be considered within the scope of the present invention the nature of which is to be determined from the foregoing description. It will be appreciated by those skilled in the art that the invention may be embodied in many forms. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

1. A method for the prophylaxis or treatment of cardiovascular inflammation in a mammalian subject, comprising administering to the subject a therapeutically effective amount of a complex of a metal and a carboxylate, or a derivative of a carboxylate, having anti-inflammatory activity, wherein the carboxylate or derivative is other than salicylate or a derivative of salicylate.

2. A method according to claim 1 wherein the derivative is selected from the group consisting of a hydroxamate, a hydroximate, an amide, and an ester.

3. A method according to claim 2 wherein the derivative is a hydroxamate or amide.

4. A method according to claim 1 , wherein the metal complex is a complex of a metal and the deprotonated form of a carboxylic acid is selected from the group consisting of:

Suprofen ((+)-α-methyl-4-(2-thienyl-carbonyl)phenylacetic acid (SupH));

Tolmentin (l-methyl-5-(p-toluoyl)-lH-pyrrole-2 -acetic acid (ToIH)); Naproxen (6-methoxy-α-methyl-2-naphthaleneacetic acid (NapH)); Ibuprofen ((+)-α-methyl-4-(isopropylmethyl)benzeneacetic acid (IbuH)); Flufenamic Acid ((iV-trifluoromethylρhenyl)anthranilic acid) (FlufenH)); Niflumic Acid (2-((3-trifluoromethyl)phenylamino)-3-pyridinecarboxylic acid

(NifH));

Diclofenac (2-[(2,6-dichlorophenyl)amino]phenylacetic acid (DicH));

Indomethacin (1 -(4-chlorobenzoyl)-5-methoxy-2 -methyl- 1 H-indole-3 -acetic acid (IndoH)); and Acemetacin (1 -(4-chlorobenzoyl)-5-methoxy-2-methylindole-3 -acetic acid carboxymethyl ester (ACMH)).

5. A method according to claim 4, wherein the carboxylate having anti- inflammatory activity is selected from the indomethacin anion and the acemetacin anion.

6. A method according to any one of claims 1 to 3, wherein the complex is selected from the group consisting of:

Where ^a Suprofen = (+)-α-methyl-4-(2-thienyl-carbonyl)phenylacetic acid (SupH); ^b Tolmentin = l-metiiyl-5-(p-toluoyl)-lH-pyrrole-2-acetic acid (ToIH); ^c DMSO = dimethylsulfoxide; ^d Naproxen = ό-methoxy-α-methyl^-naphthaleneacetic acid (NapH); ^e Ibuprofen = (+)-α-methyl-4-(isopropylmethyl)benzeneacetic acid (IbuH);

^Metronidazole = 2-methyl-5-nitrobenzimidazole ^g Flufenamic Acid = (iV-trifluoromethylphenyl)anthranilic acid (FlufenH); ^ Niflumic Acid = 2-((3-trifluoromethyl)phenylamino)-3-pyridinecarboxylic acid

(NifH);

¹ Indomethacin = l-(4-chlorobenzoyl)-5-methoxy-2-methyl-lH-indole-3 -acetic acid (IndoH);

^J Diclofenac = 2-[(2,6-dichlorophenyl)amino]phenylacetic acid (DicH); and M^a is a metal ion.

7. A method according to claim 6 wherein the metal ion is a copper or zinc ion.

8. A method according to claim 1, wherein the complex is a mononuclear, dinuclear, trinuclear or polynuclear complex of a metal and a ligand of the formula L :

wherein: R¹ is H or halo;

wherein each R^2A is independently selected from the group consisting of H, C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted; R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN, -OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH_3, -SCH₃ Or-CH₂CH₃ maybe optionally substituted; and n is i, 2, 3, 4 or 5.

9. A method according to claim 8, wherein the complex of a metal and a carboxylate, hydroxamate or amide having anti-inflammatory activity is selected from the group consisting of complexes of the following formulas (1), (2) and (3):

[M(L²)₂L_m]^p (1)

where M is a divalent or trivalent metal ion; L² is a ligand of the formula:

wherein:

R¹ is H or halo;

wherein each R^2A is independently selected from the group consisting of H, C₁ to Ce alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted; R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN, -OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH_3, -SCH₃ or -CH₂CH₃ may be optionally substituted; n is 1, 2, 3, 4 or 5; each L is independently selected and is a monodentate ligand; m is 1 or 2; and p is the charge of the complex;

[M₂(L²)₄L_m]^p (2)

where each M is independently selected and is a divalent or trivalent metal ion; L ^■ 2 is a ligand of the formula:

wherein: R¹ is H or halo;

wherein each R^2A is independently selected from the group consisting of H, C₁ to

C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted; R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN, -OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH_3, -SCH₃ or -CH₂CH₃ may be optionally substituted; n is 1, 2, 3, 4 or 5; each L is independently selected and is a monodentate ligand, m is 0, 1 or 2; and p is the charge of the complex;

[M'₃O(L²)₆L₃]^P (3) where each M' is independently selected and is a trivalent or tetravalent metal ion;

L² is a ligand of the formula:

wherein: R¹ is H or halo;

wherein each R^2A is independently selected from the group consisting of H, C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted; R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN, -OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH₃, -SCH₃ or -CH₂CH₃ may be optionally substituted; n is 1, 2, 3, 4 or 5, each L is independently selected and is a monodentate ligand, and p is the charge of the complex.

10. A method according to claim 9, wherein L is ACM.

11. A method according to claim 9 or 10, wherein M is selected from the group consisting of copper, zinc, cobalt, nickel, chromium, molybdenum, tungsten and ruthenium.

12. A method according to claim 11 , wherein M is ruthenium, copper or zinc.

13. A method according to claim 9 or 10, wherein M' is selected from the group consisting of iron, vanadium, manganese, chromium and ruthenium.

14. A method according to claim 13, wherein M' is iron or ruthenium.

15. A method according to any one of claims 9 to 14, wherein L is selected from the group consisting of water, an alcohol, iV,N-dimethylformamide (DMF), N-methylpyrrolidone (ΝMP), dimethylsulfoxide (DMSO) and iV^V-dimethylacetamide (DMA).

16. A method according to any one of claims 9 to 15, wherein the complex is [Cu(ACM)₂(DMF)₂], [Cu(ACM)₂(OH₂)₂] [Cu₂(ACM)₄(DMF)₂], [Zn(ACM)₂(DMF)₂], [Zn(ACM)₂(OH₂)₂], [Ru₂(ACM)₄L]^p or [Ru₂(ACM)₄L₂f, where L and p are as defined in claim 6 for formula (2).

17. A method according to claim 1, wherein the complex is a complex of the following formula (4A):

where M is a divalent or trivalent metal ion;

"η²-L^l5' is a bidentate ligand of the formula L¹:

wherein: R¹ is H or halo;

R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN, -OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH₃, -SCH₃ or -CH₂CH₃ may be optionally substituted; n is i, 2, 3, 4 or 5; each L is independently selected and is a monodentate ligand; and p is the charge of the complex.

18. A method according to claim 17, wherein L¹ is Indo.

19. A method according to claim 17 or 18, wherein each R⁵ is selected from the group consisting of fluoro, chloro, bromo and iodo.

20. A method according to any one of claims 17 to 19, wherein the complex is selected from the group consisting of [Cu(Indo)₂(Pyrro)₂], [Cu(Indo)₂(Im)₂], [Zn(Indo)₂(L)₂], where L=OH₂ or an alcohol, [Co(Indo)₂(OH₂)₂], [Co(Indo)₂(EtOH)₂] and [Ni(Indo)₂(OH₂)].

21. A method according to claim 1 , wherein the complex is a complex of the following formula (5):

[M₂(μ-L¹)₄L_m]^p (5)

wherein: R¹ is H or halo;

wherein each R^2A is independently selected from the group consisting of H, C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted; R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN, -OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH₃, -SCH₃ or -CH₂CH₃ may be optionally substituted; n is 1, 2, 3, 4 or 5; each L is independently selected and is a monodentate ligand; m is 0, 1 or 2; and p is the charge of the complex.

22. A method according to claim 21 , wherein L¹ is Indo.

23. A method according to claim 21 or 22, wherein M is selected from the group consisting of copper, zinc, cobalt, nickel, chromium, molybdenum, tungsten and ruthenium.

24. A method according to claim 23, wherein M is selected from copper, zinc or ruthenium.

25. A method according to any one of claims 21 to 24, wherein L is selected from the group consisting of water, an alcohol, ΛζTV-dimethylformamide (DMF), iV-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO), AζiV-dimethylacetamide (DMA), pyridine (Py), acetonitrile (AN) and tetrahydrofuran (THF).

26. A method according to any one of claims 18 to 25, wherein the complex is [Cu₂(IMo)₄(DMF)₂], [Cu₂(Indo)₄(DMA)₂], [Cu₂(Indo)₄(NMP)₂], [Cu₂(Indo)₄(DMSO)₂], [Cu₂(Indo)₄(THF)₂], [Cu₂(Indo)₄(Py)₂], [Cu₂(IMo)₄(AN)₂], [Cu₂(Indo)₄(OH₂)₂], [Cu₂(Indo)₄(OH₂)₂].nH₂O, [Zn₂(IMo)₄(DMA)₂], [Zn₂(IMo)₄(NMP)₂] or [Zn₂(IMo)₄(Py)₂] , wherein n is the number of waters of crystallisation.

27. A method according to claim 1 , wherein the complex of a metal and a carboxylate, hydroxamate or amide having anti-inflammatory activity is a complex of the folio wing formula (6):

[M'₃O(μ-L¹)₆L₃]^p (6) where each M' is independently selected and is a trivalent or tetravalent metal ion; and μ-L¹, L and p are as defined in claim 21.

28. A method according to claim 27, wherein M' is selected from the group consisting of copper, zinc, cobalt, nickel, chromium, molybdenum, tungsten and ruthenium.

29. A method according to claim 28, wherein M' is ruthenium.

30. A method according to any one of claims 27 to 29, wherein L is selected from the group consisting of water, an alcohol, iV,N-dimethylformamide (DMF), iV-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO), iV^V-dimethylacetamide (DMA), pyridine (Py), acetonitrile (AN) and tetrahydrofuran (THF).

31. A method according to claim 1 wherein the complex is a complex of the following formula (7):

[Zn₂M(L³)₆(L⁴)₂] (7) where:

M is a divalent metal ion;

32. A method according to claim 31 wherein L³ is a monodentate, bidentate or bridging ligand of formula L¹ or L² as follows:

wherein: R¹ is H or halo;

C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C₁ to C₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted; R³ is H or halo; each R⁵ is independently selected from the group consisting of halo, -CH₃, -CN, -OCH₃, -SCH₃ and -CH₂CH₃, where the -CH₃, -OCH₃, -SCH₃ or -CH₂CH₃ may be optionally substituted; and n is 1, 2, 3, 4 or 5.

33. A method according to claim 31 or 32 wherein M is selected from the group consisting of zinc, cobalt, nickel, magnesium, copper and calcium.

34. A method according to any one of claims 31 to 33 wherein the heterocyclic base comprises one or more N atoms.

35. A method according to claim 34 wherein the heterocyclic base is optionally substituted and is selected from the group consisting of isoquinolyl, quinolyl, piperidinyl, pyridinyl, 2-methylpyridinyl, imadazoyl, pyranyl, pyrrolyl, pyrimidinyl, indolyl, purinyl and quinolizinyl.

36. A method according to claim 35 wherein the heterocyclic base is quinolyl.

37. A method according to claim 1 wherein the metal complex is a complex of the following formula (8):

[M(L⁵)_m(L⁶)_n]^p (8) wherein

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁵ is independently selected and is a monodentate carboxylate or amide ligand (O or N bound), having anti-inflammatory activity; each L⁶ is independently selected and is NH₃, a monodentate ligand, a polydentate ligand, or a macrocyclic ligand; m is 1 , 2, 3 or 4 n is O, 1, 2, 3, 4 or 5; and p is the charge of the complex.

38. A method according to claim 1 wherein the metal complex is a complex of the following formula (8 a):

[M(L⁵ULV (8a) where

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁵ is independently selected and is NH₃, a monodentate ligand, a polydentate ligand, or a macrocyclic ligand; each L⁶ is independently selected and is a chelating derivative of a carboxylate such as a hydroximate, hydroxamate, hydrazine, ester, amino acid, peptide or sugar, or a amide chelating ligand (O or N bound), having anti-inflammatory activity; m is 0, 1, 2, 3, 4, or 5; n is 1, 2, 3 or 4; and p is the charge of the complex.

39. A method according to claim 1 wherein the metal is a complex of the following formula (9): [M(L⁷)_m(L⁸)_n]^p (9) wherein

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁷ is independently selected and is NH₃, a monodentate ligand, a polydentate ligand, or a macrocyclic ligand; each L⁸ is independently selected and is a chelating derivative of a carboxylate having anti-inflammatory activity; m is 1, 2, 3 or 4; n is 1, 2, 3 or 4; and p is the charge of the complex.

40. A method according to claim 1 wherein the metal complex is a complexe of the following formula (10):

[M_qO^UI^ML³)^ (10) wherein

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L¹ is independently selected and is NH₃, another monodentate ligand, a polydentate ligand, or a macrocyclic ligand; each L² is independently selected and is a chelating derivative of a carboxylate having anti-inflammatory activity; each L³ is independently selected and is a bridging ligand; m is an integer from 0 to 5q; n is an integer from 1 to 2q; p is the charge of the complex; q is an integer between 2 and 20 inclusive; and r is an integer from 1 to 60.

41. A method according to claim 1 wherein the metal is a complex of the following formula (11): [M(L⁵)₀(L⁷)_m(L⁸)_n]^p (11) wherein

M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁵ is independently selected and is a monodentate carboxylate or amide ligand (O or N bound) having anti-inflammatory activity; each L⁷ is independently selected and is a monodentate or a polydentate ligand; each L⁸ is independently selected and is a chelating derivative of a carboxylate or amide ligand (O or N bound), having anti-inflammatory activity; o is 1, 2, 3, 4 or 5 m is 1, 2, 3 or 4; n is 1, 2, or 3; and p is the charge of the complex.

42. A method according to claim 1 wherein the metal complex is a complex of the following formula (12):

[M_q(L⁵)_m(L⁷)_n(L⁸)_r]^p (12) wherein M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L⁵ is independently selected and is a monodentate or bidentate carboxylate, or monodentate amide ligand (O or N bound), having anti-inflammatory activity; each L⁷ is independently selected and is a monodentate or a polydentate ligand; each L⁸ is independently selected and is a bridging ligand; m is an integer from 0 to 5q; n is an integer from 1 to 5q; p is the charge of the complex; q is an integer between 2 and 20 inclusive; and r is an integer from 1 to 60.

43.. A method according to any one of claims 1 to 42 for prophylaxis or treatment of a cardiovascular disease or condition selected from the group consisting of acute and chronic cardiovascular inflammation including as a result of surgery or other trauma, cardiovascular disease, angina pectoris, atheroma, atherosclerosis, arteriosclerosis, congestive heart failure, coronary heart disease, cardiomyopathy, myocardial infarction, stroke, ischeamic conditions, ischaemic cardiomyopathy, patent ductus arteriosus, high blood pressure, pulmonary hypertension peripheral artery disease, coronary artery disease, coronary artery spasm and pericarditis.

44. A method according to any one of claims 1 to 43 wherein the complex is administered systemically.

45. A method according to any one of claims 1 to 44 wherein the mammalian subject is a human.