FIELD OF THE INVENTION

The present invention relates to novel metal complexes. The invention also relates to methods of treating inflammatory conditions in humans and animals.

BACKGROUND

Non-steroidal anti-inflammatory drugs (NSAIDs) are used to treat a variety of inflammatory conditions in humans and animals. 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 effects in humans and animals, particularly adverse gastrointestinal effects.

Indomethacin (IndoH) is a NSAID and is effective in treating inflammatory conditions in humans and animals. However, indomethacin can cause severe adverse effects in humans and animals, particularly when administered orally. In humans, oral administration of indomethacin can cause ulcerations in the esophagus, stomach, duodenum and intestines, and some fatalities have been reported. In dogs, oral administration of indomethacin causes fatal gastrointestinal hemorrhaging. Other adverse efϊects associated with oral administration of indomethacin, include: (a) inhibition of platelet aggregation, (b) cardiovascular effects (fluid retention and peripheral oedema), (c) adverse ocular effects (corneal deposits and retinal disturbances), (d) adverse central nervous system effects (headaches and dizziness), (c) masking of infections due to antipyretic properties, and (f) adverse renal effects. It has also been reported that the topical administration of indomethacin causes severe adverse effects ("Anti-inflammatory activity of Indomethacin following topical application", Amico-Roxas, M., Mater, M., Caruso, A,, Puglisi, G., Bernadini, R., Rinaldo, G., European Review for Medical & Pharmacological Sciences, 1982, IV,

1999, 204). Adverse gastrointestinal effects have also been reported for administration of indomethacin by suppository.

It has been reported that dinuclear metal complexes of indomethacin (containing two metal coordination centres) cause less adverse gastrointestinal effects, and result in increased uptake of the drug, compared to free indomethacin. United States No. 5,466,824, for example, describes various dinuclear metal complexes of indomethacin. Compositions for oral administration containing the dinuclear Cu(II) complex of indomethacin, bis (N,N-dimethylformamide)telrakis-μ- (O,O -indomethacin)dicopper(II) complex ([Cu ₂ (Indo) ₄ (DMF) ₂ ]), sold under the name Cu-Algesic, have been used in veterinary practice in Australia, New Zealand,

South Africa and other countries.

The mechanism of the reduced gastrointestinal toxicity of dinuclear metal complexes of indomethacin has not been elucidated. However, it is believed that the main reason is the reduced interaction of the indomethacin with the COX-I enzyme in the gastrointestinal tract.

Acemetacin, (1-(4-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid carboxymethyl ester), is a glycolic acid ester of indomethacin. Acemetacin was first described in 1974 by Troponwerke Dinklage and Co. and was developed with the intention of reducing the side effects of NSAIDs and increasing the tissue distribution of NSAIDs (Boltze, K. H.; Brendler, O.; Dell. H. D.; Jacobi, H Patent: Antiinflammatory

[[1-(4-Chlorobenzoyl)-5-methoxy-2-methylindol-3-yl]acetox y]acetic acid In Ger. Offen ; Troponwerke Dinklage und Co. DR Patent 2234651. 1974; 23 pp; Heiter, A,; Tausch, G.; Eberl, R. Arzneim-Forsch, 1980, 30, 1460-1463; Lonauer, G.; Wirth, W. Arzneim- Forsch. 1980, 30, 1440-1444; Brackertz, D.; Muller, M. Therapiewoche 1982, 32, 2493- 2498).

In this specification, the abbreviation "ACMH" is used to refer to the uncharged form of acemetacin, and the abbreviation "ACM" is used to refer to the deprotonated anionic form. Similarly, the abbreviation "lndoH" is used to refer to the uncharged form of indomethacin, and the abbreviation "Indo" is used to refer to the deprotonated anionic form.

The structures of ACMH and IndoH are:

The pharmacological activity of ACMH is the consequence of the activity of both ACMH and its major metabolite Indo. ACMH, like its congener IndoH, possesses potent anti-inflanimatory, analgesic, and antipyretic activity. Clinical studies have shown that the clinical efficacy of ACMH is equal to that of IndoH in various forms of arthritis.

It is considered that the reduced gastrointestinal toxicity of ACMH compared to IndoH is due to the reduced ability of ACM, compared to Indo, to interact with the COX-I enzyme in the gastrointestinal tract. Indo binds tightly in the pockets of the COX enzymes and down-regulates the production of prostaglandins, which otherwise protect the gastric mucosa. When the IndoH is converted to the ester ACMII, the resultant ACM is too large to fit easily into the pocket of the COX-I enzyme and, therefore, this COX enzyme is only weakly inhibited (Tavares. I. A.; Bennett, A. J. Gastroenterol Hepatol. 1998, 13, S190-S192; Tavares, I. A.; Bennett,

A. Int. J. Tissue Reactions 1993. 15, 49-53). While ACMH has reduced gastrointestinal toxicity compared with IndoH, some adverse gastrointestinal effects associated with the administration of ACMH have been reported, including ulceration in the small intestines. The occurrence of intestinal damage by ACM has been attributed to the formation of the metabolite Indo and its subsequent effect on the small intestine from the enterohepatic circulation of Indo (Yamda, T.; Detch, E.; Specian, R. D.; Perry, M. A.; Sartor, R. B.; Grisham, M.

B, Med. Cent. 1993, 17, 641-662; Hucker, H. D.; Zacchei, A. G.; Cox, S, V.; Brodie, D. A.; Cantwell, N. H. R..J Pharmacol Exper. Therap. 1966, 153, 237*249; Yesair, D. W.; Callahan, M.; Remington, T,.; Kensler, C, J. Biochem. Pharmacol. 1970, 19,

1579-1592; Arita, T.; Miyazaki, K.; Kohri, N.; Saitoh, H. Yakugaku Zasshi 1982, 102, 477-483; Beck, W, S.; Schneider, H. T.; Dietzel, K.; Nuernberg, B.; Brune, K. Arch. Toxicol. 1990, 64, 210-217).

SUMMARY OF THE INVENTION

The present inventors have surprisingly foυnd that metal complexes of ACM, and metal complexes of other ligands of the formula L ² (where L ³ is as defined below), have reduced gastrointestinal toxicity compared Io the compound L ² H. As the reduced gastrointestinal toxicity of ACMH compared to indomethacin is considered to be due to the weak interactions of ACM with the COX-I enzyme in the gastrointestinal tract, and the residual small intestine toxicity of ACM has been attributed to the secondary circulation of the lndo metabolite, the formation of a metal complex of ACM would not be expected to reduce the gastrointestinal effects of ACMH. Similarly, the ester group in other anions of the formula L ² would be expected to block the anion from interacting to any significant degree with the COX-I enzyme in the gastrointestinal tract and, therefore, the formation of a metal complex of the anion would not be expected to reduce the gastrointestinal effects of L . However, the present inventors have surprisingly found that metal complexes of ACM and other ligands of the formula L ² cause less adverse gastrointestinal effects than the compound L ² H, irrespective of whether they are administered orally or topically.

In a first aspect, the present invention provides a metal complex containing a Iigand of the formula L ² :

wherein:

R ¹ is H or halo (i.e., Cl, F, Br or I);

R ² is H; a C ₁ to C ₆ alkyl, an alkenyl or an alkynyl, where the C ₁ to C ₆ alkyl, alkenyl or alkynyl may be optionally substituted; or

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

C ₁ to C ₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C ₁ to C ₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted; .

R ³ is H or halo; each R ⁵ is independently selected from the group consisting of halo, -CH ₃ , -

CN, -OCH ₃ , -SCH ₃ and -CH ₂ CH ₃ , where the -CU ₃ , -OCH ₃ -SCH ₃ or -CH ₃ CH ₃ may be optionally substituted; and n is 1 , 2, 3, 4 or 5.

When R ² is a C ₁ to C ₆ alkyl, an alkenyl or an alkynyl, the C ₁ to C ₆ alkyl, alkenyl or alkynyl may be substituted with one or more substitucnts. The one or more substituents may, for example, be independently selected from the group consisting of halo, -OHU -COOH and -NH ₂ .

When R ^2A is a C ₁ to C ₆ alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or art arylalkyl, the C ₁ to C ₆ 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 -NH ₂ .

When R ⁵ is CH ₃ , -OCH ₃ -SCH ₃ or --CH ₂ CH ₃ , the -CH ₃ , -OCH ₃ , -SCH ₃ or - CH ₂ CH ₃ 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 -NH ₂ .

Typically the complex is a complex of formula (1), (2) or (3):

where M is a divalent or trivalent metal ion, L ² is a ligand of the Formula:

wherein:

R ¹ is H or halo (i.e., Cl, F, Br or 1);

R ² is H; a C ₁ to C ₆ alkyl, an alkenyl or an alkynyl, where the C ₁ to C ₆ alkyl, alkenyl or alkynyl may be optionally substituted; or

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

C ₁ to C ₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C ₁ to C ₆ , alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted; R ³ is H or halo; each R ⁵ is independently selected from the: group consisting of halo, -CH ₃ , - CN, -OCH ₃ , -SCH ₃ and -CH ₂ CH ₃ , where the -CH ₃ , -OCH ₃ , -SCH ₃ or -CH ₂ CH ₃ 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 1 or 2; and p is the charge of the complex;

where each M is independently selected and is a divalent or ttivalent metal ion, L ² is a ligand of the formula:

wherein:

R ¹ is H or halo (i.e., Cl, F, Br or I);

R ² is H; a C ₁ to C ₆ alkyl, an alkenyl or an alkynyl, where the C ₁ to C ₆ alkyl, alkenyl or alkynyl may be optionally substituted; or

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

C ₁ to C ₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C ₁ to C ₆ alkyl, alkcnyl, alkynyl, aryl, cycloalkyl or arylalkyl maybe optionally substituted; R ³ is H or halo; each R ⁵ is independently selected from the group consisting of halo, -CH ₃ , - CN, -OCH ₃ , -SCH ₃ and -CH ₂ CH ₃ , where the -CH ₃ , -OCH ₃ , -SCH ₃ or -CH ₂ CH ₃ 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 0, 1 or 2, and p is the charge of the complex;

where each M' is independently selected and is a trivalent or tetravalent metal ion, L ² is a ligand of the formula:

wherein:

R ¹ is H or halo (i.e., Cl, F, Br or I);

R ² is H; a C ₁ to C ₆ alkyl, an alkenyl or an alkynyl, where the C ₁ to C ₆ alkyl, alkenyl oτ alkynyl may be optionally substituted; or

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

C ₁ to C ₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the C ₁ to C ₆ alkyl, alkenyl, alkynyl, aryl, cycloalkyl or arylalkyl may be optionally substituted; R ³ is H or halo; each R ⁵ is independently selected from the group consisting of halo, -CH ₃ , - CN, -OCH ₃ , -SCH ₃ and -CH ₂ CH ₃ , where the -CH ₃ , -OCH ₃ , -SCH ₃ or -CH ₂ CH ₃ may be optionally substituted; and n is 1, 2, 3, 4 or 5; each L is independently selected and is a monodentatc ligand; and p is the charge of the complex. In formulas (1), (2) and (3) when R ² is a C ₁ to C ₆ alkyl, an. alkenyl or an alkynyl, the C ₁ to C ₆ 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 -NH ₂ .

In formulas ( 1 ), (2) and (3), when R ^2A is a C ₁ to C ₆ alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or an arylalkyl, the C ₁ to C ₆ 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 -NH ₂ .

In formulas (1), (2), and (3), when R ⁵ is-CH ₃ , -OCH ₃ , -SCH ₃ or -CH ₂ CH ₃ , the -CH ₃ , -OCH ₃ , -SCH ₃ or- CH ₂ CH ₃ 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 -NH ₂ . R ¹ is typically H.

R ³ is typically H.

R ² is typically CH ₃ .

In some embodiments, L ² is ACM.

M may be any divalent metal or a trivalent metal ion. M is preferably copper, zinc, cobalt, nickel, chromium, molybdenum, tungsten or ruthenium, more preferably copper.

M' may be any trivalent or tetravalent metal ion. M' is preferably iron, vanadium, manganese, chromium or ruthenium, and more preferably iron or ruthenium. The ligand L may be any monodentate ligand. L may be charged or uncharged. L may for example, be water, an alcohol, N,N-dimethylformaamide (DMF), N-methylpyrrolidone, dimethylsulfoxide or N,N-dimethylacetamide (DMA). L is preferably a pharmaceutically acceptable ligand. By a "pharmaceutically acceptable ligand" it is meant a ligand that does not cause any or a substantial adverse reaction when the complex is administered to a human or animal patient. Such ligands include water, alcohols, N-methylpyrrolidone , dimethylsulfoxide or N,N- dimethylacetamide. However, complexes of formula (1), (2) or (3) where one or more L is not a pharmaceutically acceptable ligand fall within the scope of the invention. Such complexes may be used, for example, as an intermediate in the preparation of complexes in the formula (1), (2) or (3 ) where each L is a pharmaceutically acceptable ligand.

The complexes of formula (1), (2) or (3) may be dissolved in a solvent, or

may be in the form of a solid . Crystal s of a complex of formula (1), (2 ) or (3 ) may include solvents of crystallisation, and crystals of a complex of formula (1), (2) or (3) including solvents of cyrstallisation fall within the scope of the present invention, Crystals of a complex of formula (1), (2) of (3) may also include waters of crystallisation, and crystals of a complex of formula (1), (2) or (3) including waters of crystallisation fall within the scope of the present invention.

The complex of formula (1), (2) or (3) may be charged or neutral in charge. For complexes of formula (1), when M is a divalent metal, and each L is a neutral ligand, the complex will be uncharged (i.e. p is 0). However, in some embodiments, p may be 1- or 2-. For complexes of formula (2), when each M is a divalent metal and each L is a neutral ligand, the complex will be uncharged (i.e., p is 0). However, in some embodiments, p may be 1- or 2-. For complexes of formula (3), when each M' is a trivalent metal and each L is neutral, the complex will have a charge of 1 + (i.e, p is 1+)- However, in some embodiments, p may be 2-, 1-, 0 or 2+. When the complex of formula (1), (2 ) or (3) is charged, a counter ion will be present in crystals of the complex.

In a second aspect, the present invention provides a pharmaceutical composition comprising a complex according to the first aspect of the present invention and a pharmaceutically acceptable carrier. The composition may be suitable for administration by oral administration, topical application, or by some other route. Typically the complex is a complex of formula (1), (2) or (3) as defined above.

In a third aspect, the present invention provides a method of treating an inflammatory condition in a human or animal, the method comprising administrating to the human or animal a therapeutically effective amount of a complex according to the first aspect of the present invention. The animal may, for example, be a dog, a cat, a cow, a horse, a camel, etc. The complex may be administered orally, topically, by injection, by suppository, by inhalation or by some other route. Typically the complex is a complex of formula (1), (2) or (3) as defined above, In a fourth aspect, the present invention provides the use of a complex according to the first aspect of the present invention in the manufacture of a medicament for the treatment of an inflammatory condition.

AIl publications mentioned in this specification are herein incorporated by reference. Any discussion or documents, acts, materials, device, 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, hut 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 DRAWINGS

Figure 1 is the solid-state IR spectra of: (a) [Cu ₂ (CH ₃ COO) ₄ (OH ₂ ) ₂ ]; (b) Product 1 prepared as described in Example 1 (containing [Cu ₂ (ACM) ₄ (DMF) ₂ ] and [Cu(ACM) ₂ (DMF) ₂ ]); and (c) ACMH in a KBr matrix,

Figure 2 is the UV-Vis solution spectra of: (a) [Cu ₂ (lndo) ₄ (DMA) ₂ ] (0.113 and 1.133 mg/ml in DMA); and (b) Product 1 prepared as described in Example 1 (containing [Cu ₂ (ACM) ₄ (DMF) ₂ ] and [Cu(ACM) ₂ (DMF) ₂ ]) (0.1064 and 1.064 mg/ml in DMF). Figure 3 is the X-band powder EPR spectra of Product 2 prepared as described in Example 1 ([Cu(ACM) ₂ (DMF) ₂ ]): at 135 K and at RT.

Figure 4 is the X-band powder EPR spectra of Product 1 prepared as described in Example 1 (containing [Cu ₂ (ACM) ₄ (DMF) ₂ ] and [Cu(ACM) ₂ (DMF) ₂ ]): at 135 K and at RT. Figure 5 is the solid slate X-band EPR spectra of [Cu(ACM) ₂ (OH ₂ ) ₂ ] at 77 and 295 K.

Figure 6 consists of graphs of the % inhibitions versus treatment ((1) mg/kg or (2) log[mg/kg]) for carrageenan-induced paw oedema in rats 4 hr after oral administration of: (a) Product 1 prepared as described in Example 1 (containing a mixture of [Cu ₂ (ACM) ₄ (DMF) ₂ ] and [Cu(ACM) ₂ (DMF) ₂ ]); (b) [Cu ₂ (Indo) ₄ (DMF) ₂ ]; and (c) IndoH in CMC solution. Error bars represents the mean ± SEM for 3 ..10 rats. The lines represent the best fit estimated using PRISM 3 program. Nonlinear regression: (a) R ² =0.9878; (b) R ² = 0.9933; and (c) R ² = 0.9947 for (1) ; (a) R ² = 0.9883; (b) R ² = 0.9948; (c) R ² = 0.9970 for (2).

Figure 7 is a graph of the change in paw volume (% of baseline) in rats 4 h after oral administration of: (a) 2% (w/v) CMC solution (control); (b) lndoH (10 mg/kg); and equimolar Indo or Cu doses of: (c) Cu-acetate; (d) a physical mixture of Cu-acetate and IndoH; (c) [Cu ₂ (Indo) ₄ (DMF) ₂ ]; (h) ACMH; and (i) Product 1 prepared as described in Example 1 (containing a mixture of [Cu ₂ (ACM) ₄ (DMF) ₂ ] and [Cu(ACM) ₂ (DMF) ₂ ]) in 2% (w/v) CMC solution. Each bar represents the mean ± SEM from 3-10 rats, control 26 rats. * P < 0.01 from control.

Figure 8 contains graphs of the macroscopic gastrointestinal ulcerations observed in rats following oral administration with: (a) 2% (w/v) CMC solution (control); (b) IndoH (10 mg/kg) and equimolar Indo doses in 2% (w/v) CMC solution of: (C) [Cu ₂ (Indo) ₄ (DMF) ₂ ]; (d) ACMH; and (e) Product 1 prepared as described in Example 1 (containing a mixture of [Cu2(ACM) ₄ (PMF) ₂ ] and [Cu(ACM) ₂ (DMF) ₂ ]) in (1) the stomach and (2) the small intestine. Each bar represents the mean ± SEM from 4-18 rats. Significant difference at P < 0.01 : *, from control; #, from TndoH. Figure 9 is a graph of the % edema inhibition and gastric ulcers (mm) 4 h after oral administration of Product 1 prepared as described in Example 1 (containing a mixture of [Cu ₂ (ACM) ₄ (DMF) ₂ ] and [Cu(ACM) ₂ (DMF) ₂ ]) in CMC solution versus dose (doses listed in Tables 1 and 2). Error bars represents the mean ± SEM tor 4-18 rats.

Figure 10 is a graph of the percent paw change (% Δmm ³ ) against time determined over 3 h following edema formation induced in rat paws by carrageenan, in rats previously orally administered MCT OrganoGel (Control); [Zn(ACM) ₂ (OH ₂ ) ₂ ] (2.56 mg kg ^-1 bw) (Sample A); [Cu(ACM) ₂ (OH ₂ ) ₂ ] in MCT OrganoGel (2.6 mg kg ^{- 1}

bw) (Sample B); and ACMH in MCT OrganoGel (2.3 mg kg ^{- 1} bw). Data are presented as the means ± sem ( m ^m2 ) between four rats per treatment group, The control cohort represents nil treatment. The greater the value of the percent paw change for a treatment, the smaller is the treatment anti-inflammatory response. A significant difference is found between the treatment group and control ((P < 0.05(*) and P < 0.01(**)).

Figure 11 is a graph of the percent paw change (% Δmm ³ ) against time determined over 3 h following edema formation induced in rat paws by carrigeenan, in rats previously orally administered MCT organogel (Control); ACMH in MCT organogel ( 1 1.6 mg kg ^{- 1} bw); [Cu(ACM) ₂ (OHz) ₂ ] in MCT organogel ( 12-8 mg kg ^-1 bw); [Zn(ACM) ₂ (OH ₂ ) ₂ ] in MCT organogel (12.7 mg kg ^-1 bw). The control cohort represents nil treatment. The greater the value of the percent paw change for a treatment, the smaller is the treatment anti-inflammatory response. A significant difference is found between the control and treatment groups P < 0.01 (**). Figure 12 is a graph of the gastric ulcerogenic effects in rats after oral administration of MCT organogel Control; ACMH in MCT organogel (1 1.6 mg kg ^{- 1} bw); [Cu (ACM) ₂ (OH ₂ )2] in MCl organogel (12.9 mg kg ^-1 bw); [7.n(ACM) ₂ (OH ₂ )2| in MCT organogel (12.8 mg kg ^-1 bw), A significant difference is found between the control and treatment group (P < 0.05(*) and P < 0.01 (**)). Figure 13 is a graph of the smalI intestine ulcerogenic effects in rats after oral administration of MCT organogel Control; ACMH in MCT organogel (11.6 mg kg ^{- 1} bw); [Cu(ACM) ₂ (H ₂ O) ₂ ] in MCT organogel (12.9 mg kg ^-1 bw); [Zn(ACM) ₂ (OH ₂ ) ₂ ] in MCT organogel (12.8 mg kg ^-1 bw). A significant difference in found between the control and treatment group (P < 0.05(*)).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

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 poly-cyclic

alkyl. Examples of straight chain and branched alkyl include methyl, ethyl, propyl, isopropyl, butyl, isbutyl, sec-butyl, tert-butyl, amyl, isoamyl, 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, and 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 poly-cyclic alkyl having 3 to 12 carbons.

In this specification, the term "alkenyl" refers to a straight chain, branched or cyclic alkenyl having one or more double bonds. Preferably the alkenyl is a C ₂ to C ₂₀ alkenyl, and more preferably a C ₂ to C ₆ alkenyl. Examples of alkenyl include vinyl, allyl, 1 -methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 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-cyclohcxadienyl, 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 having one or more triple bonds, preferably a C ₂ to C ₂₀ alkynyl, more preferably a C ₂ to C ₆ alkynyl.

In this specification, the term "aryl " used either alone or in compound words such as "arylalkyl", refers to u 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 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. The present invention relates to metal complexes containing the ligand L ² as defined above, including metal complexes of Formula (1), (2) and (3) as defined above. The present inventors have surprisingly found that metal complexes

containing the ligand L ² cause less adverse gastrointestinal effects than an equimolar dose of L ² in the form of the compound L ² H.

Complexes of formula (1) or (2) can be prepared by mixing a stoichiometric amount of a compound of the formula L ² H wherein L ² 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). The mixture is then heated until precipitation occurs and it is then cooled and the solid in 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 L ² H, a divalent or trivalent metal salt and the ligand L arc added to a solvent more weakly coordinating than I . 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 oreomplcxcs of formula (1) or (2) using a trivalent or tetravalent metal salt. Such complexes are obtained by mixing L ² H 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.

Examples of complexes of formula (1) include [Cu(ACM) ₂ (DMF ₂ )] and [Cu(ACM) ₂ (OH ₂ ) ₂ ].

In some embodiments, the complex of formula (2) is [Ru ₂ (ACM) ₄ ] or [Ru ₂ (ACM) ₄ L ₂ ] ^p wherein Ru is Ru(II), L is as defined above for formula (2). and p is the charge of the complex (typically, 0, 1- or 2-), or [Ru ₂ ( ACM) ₄ L] ^p wherein one Ru is Ru(Il) and the other is Ru(III), L is as defined above ibr formula (2) and p is the charge of the complex (typically 0 or 1+).

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

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

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

The composition of the present invention comprises a complex according to the first aspect of the present invention together with a pharmaceutically acceptable carrier. Typically the complex is a complex of formula (1), (2) or (3) as defined above. The composition may comprise two or more complexes according to the first aspect of the present invention, 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 may be liquid or solid and is selected with the planned manner of administration in mind. The carrier is "pharmaceutically acceptable" in the sense of being not biologically of otherwise undesirable, i.e., the carrier may be administered to a human or animal along with the complex without the carrier causing any or a substantial adverse reaction.

As used herein, the term "therapeutically effective amount" means an amount effective to yield a desired therapeutic response, for example, to treat an inflammatory condition. 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 may be determined in the same way, that is, by continuously monitoring the response of the subject and modifying the interval between dosages.

The metal complex is typically administered to the human or animal by administering a composition containing the complex. The complex may be administered by any route or mode suitable for the disease or condition being treated. The metal complex may also be administered alone or co-administered in combination with one or more active agents conventionally used in the treatment of inflammation. By "co-administered" is meant simultaneous administration in the same composition or different compositions by the same of different routes, or sequential or nonsequential administration by the same or different routes. By "sequential" administration is meant one is administered one after the other. By "non-sequential" administration is meant that the one administration may not be coupled in time with another type of administration .

As described in the applicant's co-pending International Patent Application Filed 24 March 2006 entitled "Methods for the prophylaxis or treatmenl of carcinoma" claiming priority from Australian Provisional Application No. 2005901463 , one or more embodiments of metal complexes of the present invention and compositions incorporating them may also be used in the prophylaxis or treatment of carcinomas such as one or more carcinomas selected from the group consisting of basal cell carcinomas, squamous cell carcinomas, melanoma, colon cancer, colorectal cancer, breast cancer, lυng cancer and other cancers of the epithelium, and the contents of the

International Patent Application is hereby incorporated by cross-reference in its entirety.

As will be understood, the use of one or more embodiments of a metal complex of the present invention in combination with other anti-inflammatory or anticancer drug may enhance the effectiveness of the other drug. In the prophylaxis or treatment of carcinoma, this may include both carcinomas that are responsive to treatment by the other drug and carcinomas that are otherwise resistant to the other drug.

In particularly preferred embodiments a composition embodied by the invention may be formulated as described in International Application No. PCT/AU2005/000442 filed 30 March 2005, 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 may 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 limited 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. 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 in administered are able to interact with the complex and which cause the complex to dissociate before it is absorbed. For such 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 of metal complexes of the invention 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, palmic and stearic acids, and include sorbitan trioleate (Span 85), sorbitan monooleate (Span 80), sorbitan tristcarate (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 polyoxy ethylene group of surfactants, such as Termul 1284 and caster oil cthoxylate. Further surfactants in this class include the polyoxyethylene sorbitan fatty acid esters group of surfactants, including polyoxyethylene (20) sorbitan monolaurate (Tween 20), polyoxyethylene (4) sorbitan monolaurate (Tween 21), and polyoxyethylene (20) sorbitan monooleate (Tween 80),

Other suitable surfactants 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 L101 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, lauriec palmic, oleic, and stearic acids, including but not limited to PEG 200 monolaurate, PEG 300 dilaurate, ethylene glycol distcarate, PEG 300 monooleate, PEG 400 monooleate, PEG 350 monostearate, PEG 300 monostearate, PEG 400 Monostearate, PEG 600 Monostearate, PEG 1000 monostearate, PEG 1.800 monostearate, PEG 6500 monostearate, PEG 400 mono-iso stearate, PEG 600 mono-iso-slearate, PEG 200 dilaurate, PEG 600 distearate, PEG 6000 distearate, PEG 200 distearate, PEG 300 distearate, and PEG 400 distearate.

A composition as described herein may also optionally further comprise one or more solvents, co-solvents or solubilising components for increasing the solubility of the metal carboxylate complex in the composition. The solvent or co-solvent may, for example, be tetraglycol (IUPAC name: 2-[2-[(tetrahydro-2- furanyl)methoxy]ethoxy]ethanol; other names:

2-[2-(tetrahydrofurfuryloxy)ethoxy]ethanol; tetrahydrofurfuryldiethyleneglycol ether) or other glycofurols (also known as tetrahydrofurfurylpolyethyleneglycol ethers),

polyethylene glycols, glycerol, propylene glycol, butyl glycol or other pharmaceutically acceptable glycol. Further suitable co-solvents include ethoxylated alcohols and aromatic alcohols including cetyl alcohol, stearyl alcohol, lauryl alcohol, benzyl alcohol, and ethoxydiglycol. An example of a solubitising component is a polyvinylacohol/povidone mixture. The composition may also further comprise a thickener such as Aerosil 200, clay or another inorganic filler.

Suitable viscosity imparting or suspending agents include sorbitol, povidone, soya bean lecithin, cholesterol and egg yolk phospholipid.

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. Preferably, the composition is also not strongly acidic or basic as strong acids and bases can cause metal carboxylate complexes to dissociate.

Preferably, in one or more embodiments of compositions of the invention, more than 80%, preferably more than 90%, and more preferably more than 95%, of the total amount of the copper atom is present in the composition as part of the metal complex, and less than 10% of metal complex dissociates when the composition is stored for 12 months in the absence of light at room temperature ( 18°C to 25°C). The degree of dissociation of the metal complex in the composition can be readily determined by a person skilled in the art using known methods such as EPR. spectroscopy.

More generally, the metal complex may be dissolved in the composition or may be present in the composition as a solid. The solid complex may 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.

Compositions useful for administering metal complexes embodied by the invention include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), transdermal, ophthalmological, vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, and for instance, administration by inhalation.

The composition may 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 consists of two or more components. 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 ingredients making up the carrier may be mixed in any order. However, it will be appreciated that the components are mixed in a manner that minimises dissociation of the metal complex during the preparation of the composition.

A composition for oral administration of a metal complex in accordance with an embodiment of the invention may be in the form of a viscous paste, a digestible tablet, a capsule, a chewable composition, or any other form suitable for oral administration. If desired, the composition may be encapsulated in a soft or hard capsule by techniques known in the art. Moreover, the metal complex may 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. Compositions for oral administration include, for example, a composition containing 2% (w/v) of a complex of formula (1), (2) or (3) in CMC solution. Another example of a composition for oral administration is a paste formulation comprising 2% (w/v) of a complex of formula (1), (2) or (3), one or more glycofurols (e.g. tetraglycol), one or more surfactants, one or more thickeners and a medium chain triglyceride.

A composition for oral use may 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 of alginic acid, lubricants such as magnesium stearate, flavouring agents, colouring agents and preserving agents eg, such as sorbic acid, in order to produce pharmaceutically elegant and palatable preparations.

A chewable composition may for example comprise the complex of formula

(1), (2) or (3), 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. For example, a chewable composition for horses may comprise the complex of formula (1), (2) or (3), flavour, the base (comprising pre-gel starch, gelatine, flour and water), and 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 dogs may comprise the complex of formula (1), (2) or (3), meat emulsion, an acidulate (e.g. phosphoric acid), one or more antifungal agents (eg, benzoic acid and sorbic acid), sugar or sugar alcohol, and salt. A composition of the present invention for topical application may comprise the complex of formula (1), (2) or (3) 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 in the form of an emulsion or are immiscible with water.

An example of a composition for topical application is a composition comprising 0.5-2% w/w of the complex of formula (1), (2) or (3) in an emulsifying cream comprising chlorocresol (4-chloro-3-methylphenol) as a preservative as follows: cetomacrogol emulsifying wax 15 g liquid paraffin 10 g white soil paraffin 10 g chlorocresol 0.1 g propylene glycol 5 ml purified and cooled water to 100 g.

Another example of a topical composition is a composition consisting of 0,5- 2% w/w of the complex of formula (1), (2) or (3) in wool fat. This composition is immiscible with water.

Another example of a topical formulation for a metal complex embodied by the invention is as follows.

Ingredient Amount (% by weight of the composition)

Oil Phase

Myrj 45 10.00 isopropyl Lanolate 3.00

Lantrol 1.00

Modulan 0.50

Isopropyl myristate 5.00

Mineral oil 4.75

Emulan 3.00

CetyI alcohol 0.10

Complex 0.25

Water Phase

Veegum 1.00

Water 66.80

Propylene, glycol 3.00

Methyl paraben 0.20

100.00 This computation is an emollient oil-in-water cream. The composition may bo prepared by separately preparing the oil phase and water phase by mixing the components of each phase, and then adding the water phase to the oil phase at 65 °C alter blending the Veegum into the water. The complex is dissolved in the oil phase prior to emulsification. For a less viscous lotion, the % w/w of Veegum may be reduced to 0.50% to 0.75%.

A yet further 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 as follows:

Ingredient Amount

cetomacrogol emulsifying wax 15 g liquid paraffin 10 g white soft paraffin 10 g chiorocresol 0.1 g propylene glycol 5 mL purified and cooled water to 100 g

An example of a composition for rectal administration of a metal complex as described herein such as to infants or for paediatric use may be prepared as follows. Amounts shown are % w/w of the composition.

Ingredient Amount

One or more metal complexes having anti-inflamniatory activity 10%

Macrogol 400 20%

Macrogol 4000 70% This composition is an un-reactive non-greasy, water miscible suppository base which does not ionise in the presence of water. The proportions of the macrogols (ethylene glycol polymers) are determined to provide a melting point of the suppositories which is not higher than 37°C. Allowances are made for volume occupied by the metal complex in each suppository, i.e based on densities of the complex relative to the base.

Typically, the metal complex will constitute about 0.025% to about 20% by weight of a composition embodied by the present invention, 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 prophylaxis of skin carcinoma, a composition of the invention for topical application to the skin will typically comprise the metal complex in an amount of about 1% by weight of the composition or less.

In some embodiments, a composition of the invention does not comprise any therapeutically active ingredients in addition to the complex of formula (1), (2) or (3). In other embodiments, a composition embodied by the invention may include one or more therapeutically active agent(s) in addition to the complex of formula (1), (2) or (3). The active agent(s) may for instance be selected from drugs conventionally used for the prophylaxis or treatment of inflammation or other conditions.

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 by reference in its entirety.

The human or animal may be any human or animal having a disease or condition in need of treatment by a method embodied by the present invention. The animal is typically a mammal, and may be a non-human primate or non-primate. The mammal may 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 mammals include members of the Orders Primates, Rodentia, Lagomorpha, Cetacea, Carnivora, Perissodactyla and Artiodactyla. Typically, the subject will be a dog, primate, or a human being. The inflammatory condition may for example be rheumatoid arthritis, osteoarthritis, acute musculoskeletal disorders (such as tendonitis, sprains and strains), or lower back pain (commonly referred to as lumbago). The inflammatory condition may also be inflammation, pain or edema following surgical or non- surgical procedures, or any other inflammatory disease or condition responsive to treatment as described herein.

The invention is described further below by reference to a number υf non- limiting examples.

The invention is described further below by reference to a number of non- limiting examples. In the following examples, the abbreviation "Pyrro" refers to pyrrolidine.

EXAMPLE 1 Synthesis of bis(N,N-dimethytformamide)tetrakis-μ-(O,O'-

ACM)dicopper(II) tetrahydrate ([Cu ₂ (ACM) ₄ (DMF) ₂ ].4H ₂ O) and bis( N, N-dimethylformamide)bis-(O,O'-ACM)copper(II) dihydrate ([Cu(ACM) ₂ (DMF) ₂ ].2H ₂ O)

Chemicals

ACMH was of pharmaceutical grade and obtained from Sigma Aldrich. All other chemicals were used as supplied. [Cu ₂ (Indo) ₂ (DMA) ₂ ] was prepared as reported in 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.

Synthesis

A warm solution of Cu(II) acetate monohydrate (0.24 g, 1.20 mmol) dissolved in DMF (2 ml) was added to a stirred solution of ACMH ( 1.00 g, 2.41 mmol) in DMP (1.5 ml) heated at 35 °C. The reaction was maintained at 35 °C for a further 2 hr, then stirred at room temperature overnight, during which time a pale green precipitate formed. The mixture was filtered and the green product was washed with ice-cold DMF 3 ml/3 times and dried under the vacuum. Yield: 0.89 g (74%). Anal, (Cu ₂ C ₉₀ H _9O CI ₄ N ₆ O ₃₀ ) C, H ₅ N ₁ Cu: calcd, 53.92, 4.53, 4.19, 6.34; found: 53.68, 4.0S, 4.08, 6,61%. This synthesis resulted in a mixture of a dimer and a monomer as shown by EPR spectroscopy with ∼30-40% of the copper present as the dimer and the rest as a monomer ("Product 1"). Similar reactions performed with somewhat more dilute solutions resulted in the precipitation of the monomer only as shown by EPR spectroscopy ("Product 2").

Elemental microanalyses

Copper analyses were performed with a varian AA-800 air-acetylene flame atomic absorption spectrophotometer. The C, H, N microanalyses were performed by the Department of Chemistry, University of Otago.

Infrared spectroscopy

Fourier transform IR spectra were acquired from samples within pressed disks of KBr matrix on a Bio-Rad Win-IR FTS-40 infrared spectrometer (400-4000 cm ^{- 1} ).

UV-Vis spectroscopy

Diffuse-reflectance solid-state UV-Vis spectra were recorded using a Varian Cary IE spectrophotometer. UV-Vis spectra of solutions were obtained in 1-cm quartz cells in a Hewlett-Packard 8452A diode-array (190-820 nm) or a Varian Cary 5E UV-VIS-NIR spectrophotometer. Each complex was dissolved in the same solvent as its solvent ligand.

X-Band electron paramagnetic resonance spectroscopy

X-band (∼9.5 GHz) EPR spectra of powdered and solution samples of the complexes were acquired using a Bruker EMX EPR spectrometer equipped with a standard ER4120 X-band cavity, EMX 03SM NMR gaussmeter, EMX 032 field controller, EMX 08I magnet power supply, Bruker EMMX 048T microwave bridge control, and BVT2000 variable temperature unit.

Results

Figure 1 compares the infrared spectra of the reactants, copper(ll) acetate (a) and ACMH (c), and Product 1 (b). It is clear from the spectrum for Product 1 that the ACM ligand is bound to the metal, since many of the ACM bands that are not in copper acetate are in Product 1 and additionally, some bands change on the ACMH ligand due to its deprotoriation and coordination to the Cu(II) (notably the loss of the carboxylic acid O-H stretch above 2,000 cm ^{- 1} and the large changes in the peaks due to the carbonyl group of carboxylic acid/carboxylate). The UV/Vis absorption spectra (Figure 2) of Product 1 in DMF (b) and [Cu ₂ (Indo) ₄ (DMA) ₂ ] in DMA (a), shows the intensity of the band in the UV/Vis for Product 1 is about half that for the Indo complex, whereas the ligand-centred bands are of comparable intensities. These results show that while the Indo complex is almost completely dimeric in DMA, Product 1 is almost completely monomeric in solution in DMF. The same result is

obtained if the dissolved complex is Product 1 or Product 2 showing a rapid interconversion in solution. The spectrum also demonstrates that the structure of the monomer is a tetragonally distorted octahedron. The structure of the Cu-monomer complex, was established from the lack of Cu-dimer signals in the EPR spectrum of Product 2 (Figure 3) compared with the spectrum of Product 1 (Figure 4), which shows dimer peaks, which are inherently weaker than that of the monomer, and even though the monomer signal appears to dominate the spectrum, it contains substantial amounts of dimer. By comparison of the peak ratios of the monomer and dimer with those reported for Cu-lndo dimers and related species, the percentage of Cu as the dimer in Product 1 is estimated to be ∼30-40% with the rest present as the monomer.

(A) EXAMPLE 2: Monomeric Cu(II) complexes

Bis(O,O'-acemetacin)diaquacopper(lI)-3-water ([CU(ACM) ₂ (OH ₂ ) ₂ ].3H ₂ O)

Experimental

The [Cυ(ACM) ₂ (OH ₂ ) ₂ ] monomer complex can be prepared as described for the Indo aqua dimer in Anti-Inflammatory Dinuclear Copper(II) Complexes with lndomethacin. Synthesis, Magnetism and EPR Spectroscopy; Crystal Structure of the N,N-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, or more preferably, as follows. Cu(II) acetate monohydrate (1 g, 0.005 mmol) in ethanol (50 ml) was added drop wise to acemetacin (4.158 g, 0.01 mmol) dissolved in ethanol (50ml) at room temperature. Warming the ethanol mildly (~40 °C) helped solubilise the acemetacin before adding the copper acetate solution. On addition of the Cu(II) acetate monohydrate in ethanol, a bright green complex started to form immediately. To this,150 ml of ethanol was added and left stirring overnight, during which time it converts to a blue complex. Following this, the precipitate was filtered, washed with ethanol and dried. Anal. Found: C, 54.35; H, 3.88; Ν, 3.06; Cu, 6.82; Cl, 7.60%. CaIc. for CuC ₄₂ H ₃₈ CI ₂ N ₂ O ₁₄ : C, 54.3; H, 4.12; N, 3.01; Cu, 6.84, Cl 7.60 %.

Bis(O,O '-acemetacin)bis(imidazole)copper(Il) ([Cu(ACM) ₂ (Im) ₂ ])

[Cu(ACM) ₂ (Im) ₂ ] was prepared by dissolving Cu acetate monohydrate (0.559 mmol, 0.1116 g) in MeOH (8 mL) with 12 drops of water and sonication of the mixture for 5 min. Accmetacin (1.118 mmol, 0.465 g) and imidazole (1.118 mmol, 0.076 g) were dissolved in MeOH (12 rnL), the copper solution was added dropwise and the mixture was stirred for 20 min. A colour change from green to blue to purple was followed by a copious precipitate within minutes. This was filtered, washed once with MeOH (3 mL) and the solid was dried at RT under N ₂ . Anal.

Found: C, 56.01; H, 4.11; N, 8.16; CaIc. for CuC ₄₈ H ₄₂ Cl ₂ N ₆ O ₁₂ : C, 56.01 ; H, 4,11; N, 8.17.

The experimental procedures used to obtain the spectra were the same as for the DMF complexes described in Example 1. Low temperature X-band EPR spectra of [Cu ₂ (ACM) ₄ (OH) ₂ ] were measured at X-band frequencies of ~9.5 GHz using a Bruker EMX EPR spectrometer with a standard ER4120 X-band cavity, EMX 035 gaussmeter, EMX 032T field controller, EMX 081 magnet power supply, Bruker EMX 048T microwave bridge control and BVT 2000 variable temperature unit. The following parameters were used to obtain the spectra: microwave power (2.0 mW), modulation frequency (100 kHz), modulation amplitude (10 G), centre field (3500 G) sweep width (7000 G), and number of scans (10). Powdered solids were placed in quartz EPR tubes (2-mm o.d.) for low-temperature data collection.

Results and discussion

While the colour of the initially formed complex in solution is green, which is typical of a dimer, the precipitate turns to blue in the reaction flask. It is likely that the initial green dimer is the alcohol complex, since the blue monomer also turns green when dissolved in a variety of alcohols, but it turns blue as water displaces the alcohol from the coordination sphere for form the monomer. The EPR spectrum of the complex only showed the presence of the monomer (Figure 5), since the dimer

peaks, in Figure 4 were absent even at room temperature. The EPR spectrum is consistent with a tetragonally distorted octahedron structure with unsymmetric carboxylate bidcntate ligands forming one short and one long bond to Cu(II). The distinctive resonances for the Cu(II) complexes in the X-band EPR spectra exhibited a broad resonance at 3300 G indicative of monomeric species, and a very weak feature at -5980 G, The patterns for DMF solution state spectra at 77 K and 295 K were similar, and the spectra displayed . X-band EPR spectra of formulations prepared all showed a predominance of monomeric species with broad resonance at 3300 G. Likewise, the solid-state RT EPR for [Cu(ACM) ₂ (Im) ₂ ] displayed a distinctive monomer signal at 3300 G .

EXAMPLE 3: Diaquabis(O,O'- acemetacin)zinc(ll)-6-water ([Zn(ACM) ₂ (OH ₂ ) ₂ ]-6H ₂ O)

The [Zn(ACM) ₄ (OH ₂ ) ₂ ] complex was prepared in a way similar to that used in preparation of the [Cu ₂ (ACM) ₄ (OH ₂ ) ₂ ] complex. Acemetacin (2.079 g, 0.01 mol) was dissolved in ethanol (25 mL) with gradual heating to ~60°C. Zn acetate dihydrate (0.504 g, 0.005 mol) was similarly dissolved in ethanol (25 ml) with continuous stirring and the solution was added to the acemetacin solution, followed by an additional 75 ml of ethanol. The resultant precipitate was filtered, washed with ethanol and dried. Anal Found: C, 52.41, ; H, 4.38; N, 2.81 ; CaIc. for [ZnC ₄₂ H ₃₈ Cl ₂ N ₂ O _14. 6H ₂ O: C, 52.16; H, 4.38; N, 2,9.

EXAMPLE 4: Pentaammine(acemetacin)cobalt(Ill) trifluoromethanesuIfonate-2-dimethylsulfoxide ([Co(NH ₃ ) ₅ (ACM)](SO ₃ CF ₃ ) ₂ .2(CH ₃ ) ₂ SO)

The [Co(NH ₃ ) ₅ (ACM)](SO ₃ CF ₃ ) ₂ complex using the general method described in Ion Association und the Reactions of Cobalt(III)-Acido complexes. 4. Origin of the Products in the Base Hydrolysis of [Co(NH ₃ ) ₅ X] ^(3-n) Complexes. Brasch, N E.; Buckingham, D.A.; Clark, C.R.; Finnie, K.S. Inorg. Chem. 1989, 238, 4567- 4574._[Co(NH ₃ ) ₅ (OSO ₂ CF ₃ )](CF ₃ SO ₃ ) ₂ , (0.84 ol, m 0m,5 g) was slowly added to excess acemetacin (2.8 mmol, 1.16 g) in triethylamine (2.8 mmol, 0.38 ml) and

DMSO (5 ml), and the mixture was stirred over an oil bath for 3 hr at 50 °C. The solution was then heated to 80 °C for 2 hr. It was cooled and slowly added to diethyl ether (800 ml) with brisk stirring. The oily residue was stirred for an hour, the cloudy diethyl ether was replaced with fresh diethyl ether and the mixture was refrigerated overnight. It was sonicated for 10 minutes resulting in formation of a red solid, which was filtered and dried under vacuum. Anal. Found: C, 31.9; H, 4.16; N, 8.26; CaIc. for CoC ₂₇ H ₄₄ ClN ₆ O ₁₄ S ₄ F ₆ : C, 32,00; H, 4.38; N, 8.30.

EXAMPLE 5 Efficacy and toxicity studies

Experimental

Animals

Sprague-Dawley rats weighing 200-250 g were housed in metabolic cages four days before study and were allowed free access to standard laboratory rat chow (Purina Rat Chow, Ralston Purina, St Louis MO, USA) and tap water. Animals were supplied by the laboratory animal services at the University of Sydney and housed in the Bosch animal house facility of the University of Sydney 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.

Chemicals

IndoH, ACMH, copper(II) acetate, carboxymethylcellulose (CMC) and carrageenan Type 1 were purchased from Sigma Aldrich. Medium chain triglyceride (MCT) paste (200 mg/5 g) was obtained from Nature Vet Pty Ltd. Technical grade formaldehyde was purchased from Ajax Chemicals (Auburn, Australia),

Dosing forms and administration

Rats were orally dosed via a curved feeding needle (Harvard Apparatus) attached to a 1 -mL syringe. The rats were orally dosed with IndoH, ACMH, an equimolar Indo or ACM dose of [Cu ₂ (Indo) ₄ (DMF) ₂ ] or Product 1 (prepared as described in Example 1) respectively, an equimolar Indo dose of Cu(Il) acetate and

Indo, or Cu(II) acetate suspended in 0.5 mL of 2% (w/v) CMC solution. The dose of each compound used with CMC as a vehicle is listed in Tabic 1. Product 1 prepared as described in Example 1 is referred to below as "CuACM".

Table 1 : Dose of each compound in the animal tests

In Vivo anti-inflammatory activity

The carrageenan-induced acute paw edema model in rats (CARR) was used. Rats were deprived of food but not water 24 hours prior to dose and 4 hours post dose. Each group of rats (n = 3--6 per group) was orally dosed with IndoH, ACMH, or an equimolar Indo or ACM dose (except for the dose-response curve experiments) of test compound listed Table 1 (ie [Cu ₂ (Indo) ₄ (DMF) ₂ ], CuACM, or lndoH together with [Cu ₂ (CH ₃ COO) ₄ (OH ₂ ) ₂ ]) or vehicle. Acute paw edema was induced one hour after dosing with the test compounds or vehicle, by injecting with carrageenan (0.1 mL, 1 % w/v in 0.9% saline isotonic with body fluid) through a 26- gauge needle into the plantar region of the right hind paw. Paw volume was measured prior to dosing and at 3 and 5 h after carrageenan injection by submerging the right hind paw in water up to an ink mark on the skin over the lateral malleus. The vessel containing the water was tared to zero on a top pan balance and the volume of fluid displaced was measured directly as a positive force (in grams). As the density of water is 1 g mL ^{- 1} , a measurement of 1 g corresponds to a volume of 1 mL. The mean percent edema or

percent inhibition of edema was determined as:

Acute macroscopic gastric damage

Method 1

Rats were fasted with access to water for 24 h prior to dose and 3 hours post dose. Each group of rats (n = 3-6 per group) was orally dosed with indoH, ACMH, or an equimolar lndo or ACMH dose of test compound listed in Table 1, or the vehicle. Three hours after administration of the test compound, the rats were euthanased and the stomach was excised and opened by incision along the greater curvature. The stomach was rinsed, submerged in 10% formaldehyde for 1 h and the extent of macroscopic gastric toxicity was examined. The gastric ulceration is expressed as the summation of the area of macroscopic ulcerations (mm ² ).

Method 2

After the aforementioned anti-inflammatory activity experiments, the rats were immediately euthanased. Similarly, the stomach was excised and opened by incision along the greater curvature for the examination of the macroscopic ulcerations (mm ² ).

As there were no significant differences in the results obtained by the two methods, the results shown in Figure 8 (1) are those of the two methods combined.

Small intestinal macroscopic damage

Rats were allowed free access to food and water throughout and prior to the assay period. Each group of rats (n = 3-6 per group) was orally dosed with IndoH, ACMH, or an equimolar lndo or ACM dose of test compound listed in Table 1 , or the vehicle. At 24 h after dosing, rats were euthanased and the entire small intestine was

excised and flushed with water to expel the intestinal contents and opened along the anti-mesenteric side. The intestine was examined from 10 cm distal to the ligament of Treitz to the ileocecal junction for macroscopic ulcerations. The degree of ulcerations is expressed as the summation of the area of macroscopic ulcerations (mm ² ).

Statistical Analyses

All inhibition of carrageenan-induced paw edema and gastrointestinal ulceration data are expressed as the standard error of the mean (±sem). Comparisons among the control and treatment groups were made using one-way analysis of variance followed by a Student-Newman-Keuls t-test using the GraphPad Instat statistical program. With all analyses, an associated probability (P value) of less than 5% (P < 0.05) was considered significant. The calculation of the power of the experiment to compare two treatment groups with a P-value threshold of 0.05 was determined using the GraphPad StatMate program (GraphPad lnstat; version 3,01 for WIN95/NT, GraphPad Software Inc., 1998).

Results

Efficacy and safety of ACM /DMF complexes

The efficacy curve for CuACM (containing a mixture of [Cu ₂ (ACM) ₄ (DMF) ₂ ] and [Cu(ACM) ₂ (DMF) ₂ ]) versus [Cu ₂ (Indo) ₄ (DMF) ₂ ] ("Culndo") in CMC shows that CuACM has a higher potency than Culndo at low doses (Figure 6). It should be noted that similar results are obtained when pure monomer ([Cu(ACM) ₂ (DMF) ₂ ]) is used, which is as expected since both CuACM

(containing a mixture of dimer and monomer) and the pure monomer give exclusively the monomer in CMC.

A comparison of hyperbolic or sigmoidal dosc-efficacy curves (Figure 6) with respect to inhibition of carrageenan-induced acute inflammation in paw oedema further confirmed that CuACM, Culndo and IndoH have similar efficacies but different potencies. The potencies of the drugs as shown in Figure 6 is: CuACM > [Cu ₂ (Indo) ₄ (DMF) ₂ ] > IndoH. In other words, the drugs CuACM, Culndo, and

IndoH have similar maximum efficacy (E _max ) but a higher contentration of Culndo or IndoH was required to produce an effect comparable to that of CuACM. As shown in Figure 6, the dose-efficacy curves reached plateau values at oral doses of CuACM of 5,0 to 13.5 mg/kg with an E _max value of 57.4 ± 8.0% and an ED ₅₀ value of 0.9 ± 2.2 mg/kg; Culndo of 6.5 lo 13.0 mg/kg with an E _max value of 53.0 ± 4.8% and an ED ₅₀ value of 1.5 ± 1.3 mg/kg; and IndoH started to reach a plateau value at 10 mg/kg, with an E _max value of 54.6 ± 5.2% and an ED ₅₀ value of 7.8 ± 1.7 mg/kg.

At high equivalent doses, Figure 7 clearly shows that CuACM and Culndo, have similar anti-inflammatory effects as the parent drugs IndoH, ACMH, and a physical mixture of IndoH and Cu-acetate. All of these compounds significantly reduced the carrageenan-induced paw oedema as compared to the control (P < 0.01). Cu-acetate does not show significant anti-inflammatory effects compared with those of the control (P > 0.05).

The results of the macroscopic gastric ulcerations at doses approximately an order of magnitude above an effective dose were similar using either method one or two. Culndo and CuACM and ACMH showed considerable gastric protection with significant reductions (P < 0.01 ) in gastric ulcerations as compared to IndoH (Fig 8(1 )). Both ACM and CuACM were superior to Culndo with regard to gastric protection. Figure 8(2) shows that small intestinal ulcerations induced in rats treated with high equivalent doses of CuACM, and Culndo in 0.5 mL of 2% (w/v) CMC solution were significantly lower (P < 0.01) than those induced by ACMH or IndoH.

Figure 9 and Table 2 depict the further highly desirable properties of the pharmacodynamic (PD) relationship between the drug dose and drug effect of CuACM and the highly protective effect of this complex against gastric ulceration.

In particular, even at doses that are approximately twice the minimum required to reach maximum efficacy, there is no significant gastric ulceration. Even at approximately three times the minimum dose required to reach the maximum efficacy, the gastric ulceration is very small. This is particularly the caae given that the rats were deprived of food for 24 hr prior to dosing in order to maximise the senstivity of the rats to gastric ulceration.

Table 2 Comparison of dose-response data for gastric ulceration of CuACM at various doses.

The results reported in Example 3 used CMC as the vehicle, which results in the conversion of the [Cu2( ACM) ₄ (DMF) ₂ ] dimer to the monomer. However, the [Cu ₂ (ACM) ₄ (DMF) ₂ ) dimer is retained in MCT paste formulations, and the results of further studies conducted by the inventors using such formations show similar efficacy to the monomer.

EXAMPLE 6 Efficacy and safety of [Cu(ACM) ₂ (O H ₂ ) ₂ ] formulations

Oral Dosing

For efficacy and safety experiments used for oral administration in rats, these were performed as outlined in Example 3, except that the compound was administered in an MCT paste formulation, The MCT paste formulation was prepared using the test agent ([Cu(ACM) ₂ (OH ₂ ) ₂ ] or ACMH) as the active ingredient.

The MCT paste formulation comprised the following ingredients;

Ingredient Amount

Test agent 55.0 mg Tetraglycol 300.0 mg Termul 1284 100.0 mg

Aerosil 200 50.0 mg

Delios V MCT oil qs 1.0 g

Delios V MCT oil is a medium chain triglyceride oil, Aerosil 200 is a silica based flow enhancing agent. The composition was prepared as follows:

1. Add tetraglycol to mixer and heat to 75°C while stirring.

2. Add and dissolve CuACM. Stir until dissolved, then remove heat/

3. Add Delios V MCT oil, while stirring. 4. Add Termυl 1284, while stirring.

5. Add Aerosil 200 slowly, taking care to add it Io the mixing vortex, while bulk is still hot. Stir for 15 minutes until homogenous, then allow to cool.

The composition was a single phase dark green paste.

Subcutaneous and topical dosing

All doses were calculated as equivalent concentrations of IndoH. Rats were allowed free access to food and water except for gastric toxicity studies, when they were fasted for 24 h but still with free access to water. For subcutaneous administration, rats were injected with 125-200-μL volumes of the test agents ([Cu(ACM) ₂ (OH ₂ ) ₂ ], [Cu ₂ (Indo) ₄ (OH2) ₂ ] and IndoH) diluted in medium chain triglyceride oil (MCT) (the formulations were prepared as described above for the MCT paste formulation for oral dosing, except that termul was excluded). The control cohort was injected with equivalent volumes of neat MCT. Subcutaneous injections were made in the lower dorsal surface. For topical administration, rats were treated with the test agents ([Cu(ACM) ₂ (OH ₂ ) ₂ ], IndoH, [Cu ₂ (lndo) ₄ (OH ₂ ) ₂ ] and ACMH) thoroughly mixed in an emulsifying cream consisting of: cetomacrogol emulsifying wax, 15 g; liquid paraffin, 10 g; white soft paraffin, 10 g; chlorocresol, 0.1 g; propylene glycol, 5 mL; purified and cooled water to 100 g. An amount of 0.2 g was applied to the right hind paw of each animal and gently massaged in for 1 min at three hourly intervals. Inflammation was induced (1 h after dosing by injection

administrations or at the final topical application) with 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 for subcutaneous injection or 4 h for topical administration after carrageenan injection by procedures outlined in Example 3. All other procedures for determining efficacy and safety are as outlined in Example 3.

Results

Oral administration

Preliminary studies with [CU(ACM) ₂ (OH ₂ ) ₂ ] delivered otally in an MCT paste formulation indicate similarly desirable properties in terms of efficacy and safety at the high doses to those discussed above in Example 3 for CuACM (containing a mixture of [Cu ₂ (ACM) ₄ (DMF) ₂ ] and [Cu(ACM) ₂ (DMF) ₂ ]). At an equivalent dose of 10 mg/kg IndoH, [Cu(ACM) ₂ (OH ₂ ) ₂ ] produced only 0, 0, 5, 12 and 30 mm ² of small intestine ulcers in 5 rats, while ACMH produced 100, 80, 20, 5, and 105 mm ² ulcers.

Subcutaneous injection While [Cu(ACM) ₂ (OH ₂ ) ₂ ], [Cu ₂ (Indo) ₄ (OH ₂ ) ₂ ] and IndoH when delivered by subcutaneous injection all resulted in anti-inflammatory effects, at the minimum dose where a small amount of ulceration was apparent with IndoH (4,5 mg/kg Indo; 2, 2, 0, and 1 mm ² in 4 rats), an equivalent dose of [Cu(ACM) ₂ (OH ₂ ) ₂ ] (5.58 mg/kg) or the Culndo complex resulted in zero ulceration in four rats.

Topical formulation

Preliminary studies on the topical formulations using the emulsifying cream with a ~1% equivalent amount of active ingredients again showed the efficacy of [Cu(ACM) ₂ (OH ₂ ) ₂ ] as an anti-inflammatory drug (Table 3). In Table 3, "CuACM" refers to [Cu(ACM) ₂ (OH ₂ ) ₂ ]. It is clear that [Cu(ACM) ₂ (OH ₂ ) ₂ ] is superior to IndoH,

Cυ-lndo and somewhat better than ACMH in terms of efficacy. Small intestine

toxicity was seen following treatment with all compounds, but generally was minimal except for IndoH and one rat with ACMH,

Table 3 Comparison of efficacy and safety of equivalent doses (∼1 %) of topical formulations of anti-inflammatories to the paws of rats (using emulsifying cream us the vehicle)

^a [Cu(ACM) ₂ (OH ₂ ) ₂ ]. ^b One animal had ulceration amounting to 112 mm ² and blood in its faeces. ^c One animal had an extreme ulcerogenic response, probably due to biological diversity. It was deemed to be a statistical outlier and eliminated from this calculation. ^d Value including rat with severe ulceration.

Discussion

Oral administration

As summarised in Figures 8 to 9 and Table 4, the CuACM complex (a mixture of the monomer and dimer) and the pure [Cu(ACM) ₂ (DMF) ₂ ] monomer are superior to [Cu ₂ (Indo) ₄ (DMF) ₂ ] (CuIndo) in that they have higher efficacy at low therapeutic doses and are also safer in terms of gastric and small intestine ulceration. Unexpectedly, the ACM complex is much safer than ACMH in the small intestine. ACMH was designed to be a safe drug for oral administration since the ester group on the indomethacin minimises the interaction of the drug with the COX-I enzyme in the gastrointestinal tract. However, the results reported above show that the administration of ACMH induces extensive small intestine ulceration at high doses. In contrast, the ACM complex exhibited low ulceration in the small intestine even at doses that arc an order of magnitude higher than the therapeutic dose, providing an excellent therapeutic window.

Table 4: Comparison of small intestinal and gastric ulceration induced by different drugs in rats (given in CMC as the vehicle)

The better gastrointestinal toleration of ACMH to gastric ulceration on oral administration in CMC solution than that of IndoH, and the fact that ACM induced intestinal damage, which still exceeded that of control in rats, is consistent with the literature data. It has been reported that ACM is eliminated by both hepatic and renal routes. ³ In particular, it has been reported that ACM is eliminated partly in the bile, mainly as free molecule and as its glucuronide, but additionally as the metabolites Indo and Indo-glucuronide in small amounts. ⁴ Several suggested reasons have been given in the literature for the better gastrointestinal tolerance of ACMH than IndoH including: (1) the amount of free IndoH and IndoH glucuronide in the bile is much higher after administration of IndoH compared with ACMH; ⁴ (2) ACMH does not decrease the blood circulation in the gastrointestinal tract nor does it inhibit the smooth muscle as much as does IndoH; ⁵ (3) ACM only weakly inhibits COX-I; ⁶ ' ⁷ (4) renal excretion of metabolites of ACM after administration of ACMH in the rat was three-fold greater that of IndoH; ⁸ and (5) IndoH resulted in significantly increased intestinal permeability (IP) but ACMH did not. The mechanism of this increased IP is due to lack of mucosal prostaglandins in the small intestine. ⁹ It appears the occurrence of intestinal damage by ACMH, which is a troublesome side- effect, is attributed to the formation of a metabolite Indo followed by the enterohepatic circulation of Indo. ¹ ' ^10-13 The complexation of ACM surprisingly resulted in a large decrease in intestinal ulceration compared with equivalent amounts of ACMH and IndoH. ACMH is known to be absorbed quickly from the gastric and

intestinal loops ¹² and to be metabolized through ether splitting, amide splitting, estcrolysis, and conjugation to glycine and sulfuric acid. ^4,10,14,15 Accordingly, the formation of a metal complex of ACM may be expected to enhance esterolytic cleavage of ACM to lndo, compared to ACMH, which would have the consequence of an increase in Indo enterohepatic circulation that would result in intestinal damage. However, the results discussed above indicate that this is not the case and that other protective effects are in operation, which only occur on complexation.

While Culndo and CuACM exhibited similar maximum anti-inflammatory efficacy to the parent drugs IndoH and ACMH, CuACM was more potent than IndoH, ACMH or Culndo, which means that a lower therapeutic dose is required to give the same anti-inflammatory effect, which, together with the inherent safety of CuACM, shows the excellent properties of complexes of ACM in terms of its therapeutic window. The results for oral administration of [Cu(ACM) ₂ (OH ₂ ) ₂ ] in MCT paste indicate that this complex also exhibits the same desirable properties as those observed for CuACM in CMC.

Other modes of delivery

The preliminary data on both subcutaneous injections and topical applications of the [Cu(ACM) ₂ (OH ₂ ) ₂ ] monomer indicate that the superior efficacy and safety of ACM complexes compared to IndoH, [Cu ₂ (lndo) ₄ (DMF) ₂ ] and ACMH are generalised for different modes of administration and point to their wide potential applications as veterinary and human pharmaceuticals. This is a surprising but important result as the small intestinal toxicity is reported to arise from secondary circulation of the lndo metabolite of ACMH and it is difficult to understand how complexation of ACM influences this process. The reduced toxicity is unlikely be due to slower release of ACM into the bloodstream when delivered as the metal complex, as this would result in a decrease is efficacy and not the observed increase.

The results of Examples 3 and 4, therefore, demonstrate that metal complexes of ACM cause less adverse gastrointestinal effects than ACMH and, in some cases, have higher efficacy. This is surprising as ACMH (containing an ester group) was designed to minimise interaction with the COX-I enzyme in the gastrointestinal tract, as the effect of NSAIDs on the COX-I enzyme is considered to

be the reason why NSAIDs cause adverse gastrointestinal effects, such as gastric ulceration. However, while ACMH is safe in the stomach as expected, it does cause significant ulceration in the small intestine as reported elsewhere. The present inventors have found that metal complexes of ACM cause much less ulceration in the small intestines than ACMH whether administered orally or topically. This would not be expected if the mechanism of such ulceration is the interaction of ACM with the COX-I enzymes, since ACMH is designed to minimise such interactions. Similarly, the complexation of ACM would not be expected to prevent the secondary circulation of the Indo metabolite from causing small intestinal ulceration. Moreover, if the reason for the small intestinal toxicity of ACMH is a combination of the hydrolysis of ACM to tndo in the small intestine and secondary circulation of Indo, the metal complex of the present invention would not be expected to inhibit such a hydrolysis, since metal ions are well known to catalyse ester hydrolysis. Indeed, in light of this, it would be expected that metal complexes of ACM would have a greater gastrointestinal toxicity than ACMH.

EXAMPLE 7 Oral Efficacy and Safety of ACMH, [Cu(ACM) ₂ (OH ₂ ) ₂ ] and [Zn(ACM) ₃ (OH ₂ ) ₂ ] in organogel MCT pastes

Examples of the efficacy and safety of the MCT organogel is described below in a series of in vivo studies for the assessment of the test composition as anti- inflammatory agents and for their ability to induce acute intestinal and or gastrointestinal ulceration.

7.1 Experimental

Test compositions:

Freshly prepared compositions containing the parent NSAID or the copper or zinc complex (active ingredient) in the MCT organogel was prepared by formulating as described below.

The MCT organogel composition comprised the following ingredients:

Ingredient Amount

Tetraglycol 400.0 mg

Span 80 200.0 mg Aerosil 200 70.0 mg

Delios V MCT oil qs 1.0 g

Delios V MCT oil is a medium chain triglyceride oil. Aerosil 200 is a silica based flow-enhancing agent. The composition was prepared as follows:

1 Add tetraglycol to mixer and heat to 65 °C while stirring.

2. Add and dissolve the NSAID or NSAlD complex. Stir until dissolved, then remove heat.

3, Add Delios V MCT oil heated to 65°C, while stirring. 4. Add Span 80 heated to 65°C, while stirring.

6. Add Aerosil 200 slowly, taking care to add it to the mixing vortex while bulk is still hot. Stir for 15 minutes until homogenous, then allow to cool,

7. De-gas the resulting composition by means of a vacuum system until the formulation to translucent.

Methods

Samples

The composition containing the metal complexes in MCT organogel paste were freshly prepared for each experiment.

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 in Building A27, University of Sydney, at ambient temperature and humidity with a I1-h light-dark cycle. The experimental animal protocols were approved by the Animal Ethics Committee of the University of Sydney.

In vivo anti-inflammatory activity and gastric toxicity

These experiments were conducted as described in Example 5.

In vivo small intestinal toxicity These experiments were conducted as described in Example 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. For groups greater than five, the Tukey-Kramer test was used in place of the Bonferroni test (Copyright 1992- 1998 GraphPad Software Inc. www.graphpad.com). For comparison of test treatments to control but not to each other, the Dunnett's test was used. All graphs show the outcome of the Dunnett's test (Copyright 1992-1998 GraphPad Software Inc. www.graphpad.com).

Data are expressed as the mean ± SEM. All reported P values are two-sided, and P<0.05 was considered statistically significant.

7.2 Results and discussion

Acute GI ulceration and inhibition of carrageenan-induced paw edema

Oral administration at an Indomethacin Equivalence (EI) treat dose of 2 mg kg ^{- 1} bw of [Zn(ACM) ₂ (OH ₂ ) ₂ ], [Cu(ACM) ₂ (H ₂ O) ₂ ], and acemetacin (ACMH) in MCT organogel compositions all resulted in a significant reduction in paw oedema at

P < 0.01 (**), P < 0.05(**) and P < 0.01(**), respectively, compared to the control cohort. All three treatment groups (Sample A, B and C) resulted in no significant (P>0.05) gastric ulceration compared to the control cohort. In addition, no significant differences (P>0.05) were found between the treatment groups with respect to anti- inflammatory effect or gastropathy. Anti-inflammatory effects of the compositions are shown in Figure 10.

Oral administration at an lndomethacin Equivalence (EI) treat dose of 10 mg kg ^{- 1} bw of ACM, [Cu(ACM) ₂ (OH ₂ ) ₂ ] and [Zn(ACM) ₂ (OH ₂ ) ₂ ], gave a significant anti-inflammatory response at P< 0.01(**) compared to the control cohort (Figure 1 1 ). The gastric and intestinal ulcerations are shown in Figures 12 and 13. Whilst there was no statistically significant difference in intestinal ulceration between the treatments of ACMH and [Zn(ACM) ₂ (OH ₂ ) ₂ ] compared to control cohort (P < 0.05), there was a significant small intestine ulceration (P< 0,05) compared to control with respect to the [Cu(ACM) ₂ (H ₂ O) ₂ ] treatment. However, as the standard error was so large for [Cu(ACM) ₂ (H ₂ O) ₂ ], all treatments were found to be comparable with respect to potential small intestine ulceration (P > 0.05), There was no significant gastric ulceration (P < 0.05) compared to control with respect to [Zn(ACM) ₂ (OH ₂ ) ₂ ]. There was, however, significant gastric ulceration with respect to ACMH (P< 0.05 (*)) and [Cu(ACM) ₂ (H ₂ O) ₂ ] (P< 0.01 (**)) compared to the control cohort. Between the treatments, [Zn(ACM) ₂ (OH ₂ ) ₂ ] resulted in significantly less gastric ulcerations (P<0.05) than [Cu(ACM) ₂ (H ₂ O) ₂ ].

Discussion

At IE treat dose of 2 mg kg ^-1 bw, the MCT organogel compositions of the Cu and Zn complexes of ACM were equi-effective and gastro-protective as the MCT composition of the parent NSAID, ACM, in inhibiting carageenan-induced paw oedema. The results indicate the gastro-protective effects of the MCT organogel composition with respect to the NSAID ACM. At an IE treat dose of 10 mg kg ^-1 bw, ACMH and ACM complexes were efficacious, but only the Zn complex of ACM resulted in significantly less gastric ulceration - comparable to the control cohort. These results suggest that the Zn ACM complex is safer than the ACMH and Cu complex when dissolved in MCT organogel paste. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

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