WO2006099685A1 - Method for the prophylaxis or treatment of carcinomas - Google Patents

Method for the prophylaxis or treatment of carcinomas Download PDF

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Publication number
WO2006099685A1
WO2006099685A1 PCT/AU2006/000403 AU2006000403W WO2006099685A1 WO 2006099685 A1 WO2006099685 A1 WO 2006099685A1 AU 2006000403 W AU2006000403 W AU 2006000403W WO 2006099685 A1 WO2006099685 A1 WO 2006099685A1
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Prior art keywords
complex
alkenyl
alkynyl
independently selected
alkyl
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PCT/AU2006/000403
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French (fr)
Inventor
Peter Lay
Trevor Hambley
Brenden Kennedy
Neal Davies
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Medical Therapies Limited
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Priority claimed from AU2005901463A external-priority patent/AU2005901463A0/en
Application filed by Medical Therapies Limited filed Critical Medical Therapies Limited
Priority to PCT/IB2006/002423 priority Critical patent/WO2007026240A1/en
Publication of WO2006099685A1 publication Critical patent/WO2006099685A1/en
Priority to PCT/IB2007/000766 priority patent/WO2007110745A1/en
Priority to PCT/AU2007/000375 priority patent/WO2007109843A1/en
Priority to PCT/AU2007/000376 priority patent/WO2007109844A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/295—Iron group metal compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/30—Copper compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/32—Tin compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the prophylaxis and treatment of carcinomas in humans and animals.
  • One or more embodiments of the invention may find application in the prophylaxis or treatment of one or more of basal cell carcinomas, squamous cell carcinomas, melanomas, colorectal cancer, breast cancer, lung cancer and other cancers of the epithelial tissues.
  • Non-steroidal anti-inflammatory drugs are used in the treatment of 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 oedema following surgical or non-surgical procedures.
  • GI adverse gastrointestinal
  • Indomethacin is a NSAID and is effective in treating inflammatory conditions in humans and animals.
  • the structure of indomethacin is as follows:
  • indomethacin can cause severe adverse gastrointestinal effects in humans and animals, particularly when administered orally.
  • oral administration of indomethacin can cause ulcerations in the oesophagus, stomach, duodenum and intestines, and some fatalities have been reported.
  • oral administration of indoniethacin causes fatal gastrointestinal haemorrhaging.
  • indomethacin effects associated with oral administration of indomethacin include: (a) inhibition of platelet aggregation, (b) 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, (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). Studies have also shown that administration of indomethacin by other routes, e.g.
  • indomethacin indomethacin
  • dinuclear metal complexes of indomethacin i.e. complexes containing two metal coordination centres
  • indomethacin cause less adverse gastrointestinal effects, and result in improved uptake of the drug, compared to the free indomethacin.
  • the oral administration of the dinuclear copper(II) complex of indomethacin, bis(N,N-dimethylformamide)tetrakis- ⁇ -(O, O -Indo)dicopper(II) [Cu2(Indo) 4 (DMF)2], Indo is the deprotonated form of indomethacin
  • DMF dimethylformamide
  • Indo is the deprotonated form of indomethacin
  • the mechanism of the reduced gastrointestinal toxicity has not been elucidated, but is believed to be due to reduced interaction of the indomethacin with the COX-I enzyme in the gastrointestinal tract.
  • Compositions containing this complex sold under the name Cu- Algesic have been used in veterinary practice in Australia, New Zealand, South Africa and other countries. These compositions are in the form of a tablet or a paste.
  • NSAIDs including indomethacin and related NSAIDs, have been reported to produce a chemoprotective effect against colorectal cancers (Turchanowa, L.;
  • Cu-salicylate complexes [Cu 2 (3,5-di-rso-propylsalicylate) 4 L 2 ], have been shown to have a limited effect (no-statistically significant difference) in tumorogenisis in female C3H/HeNCR mice models of mammary cancer (Crispins, Jr., C. G.; Sorenson, J. R. J. Anti-Cancer Res. 1992, 12, 1271-1273). These complexes had anti-cancer activity against reticulum cell sarcoma in SJL/J mice if injected subcutaneously but were toxic if injected via the i.p route (Crispins, Jr., C. G.; Sorenson, J. R. J.
  • a method for the prophylaxis or treatment of a carcinoma 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, the carboxylate or derivative being other than salicylate or a derivative of salicylate.
  • carcinoma refers to a malignant neoplasm of the internal or external lining (epithelium) of the human or animal body.
  • Carcinoma includes basal cell carcinomas, squamous cell carcinoma, melanoma, colorectal cancer, breast cancer and lung cancer. Carcinomas account for 80-90% of all cancer cases in humans.
  • the carboxylate or the derivative of the carboxylate having anti-inflammatory activity will generally have such activity when administered to a human or animal.
  • the derivative of the carboxylate may be hydrolysed in vivo, and the hydrolysed compound may have anti-inflammatory activity.
  • the derivative of the carboxylate maybe 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.
  • a method for the prophylaxis or treatment of a carcinoma 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 may 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:
  • Suprofen ((+)- ⁇ -methyl-4-(2-thienylcarbonyl)phenylacetic acid (“SupH”)); Tolmetin (l-methyl-5-(p-toluoyl)-lH- ⁇ yrrole-2-acetic acid (“ToIH”)); Naproxen (6-methoxy- ⁇ -methyl-2-naphthaleneacetic acid (“NapH”)); Ibuprofen ((+)- ⁇ -methyl-4-(isopropylmethyl)benzeneacetic acid (“IbuH”)); Flufenamic Acid ((N-trifluororaethylphenyl)anthranilic acid (“FlufenH”));
  • Niflumic Acid ((2-(3-trifluoromethyl)phenylamino)-3-pyridinecarboxylic acid (“NifH”)
  • Diclofenac (2-[(2,6-dichlorophenyl)amino] ⁇ henylacetic acid (“DicH”)
  • Acemetacin (l-(4-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid carboxyniethyl ester (“ACMH”).
  • the inclusion of the "H" at the end of an abbreviation for a carboxylate (e.g., any one of the carboxylic acid listed above) or 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.
  • IndoH refers to the uncharged form of indomethacin
  • Indo is used to refer to the deprotonated anionic form of indomethacin
  • ACMH refers to the uncharged form of acemetacin
  • ACM refers to the deprotonated anionic form.
  • carboxylate, or hydroxamate, hydroximate, ester or amide derivative having anti-inflammatory activity is a non-steroidal anti-inflammatory drug (NSAID).
  • NSAID non-steroidal anti-inflammatory drug
  • the NSAID may be indomethacin (IndoH), or an ester derivative of indomethacin such as acemetacin, or a hydroxamate or amide derivative of indomethacin or acemetacin.
  • IndoH indomethacin
  • Acemetacin, 1 -(4-chlorobenzoyl)-5-methoxy-2-methylindole-3 -acetic acid carboxymethyl ester is a glycolic acid ester of indomethacin.
  • the complex of the metal and a carboxylate, or hydroximate, hydroxamate, ester or amide derivative may be any complex comprising at least one metal ion and at least one carboxylate, or hydroxamate, hydroximate, ester or amide derivative having antiinflammatory activity.
  • the carboxylate or hydroximate, hydroxamate, ester or amide derivative is coordinated with the metal ion via the carboxylate, hydroximate, hydroxamate, amide and other groups attached to the amide or ester linkages.
  • 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 carboxylate, or hydroxamate, hydroximate, ester, or amide ligands having anti-inflammatory activity.
  • the complex includes other ligands in addition to the carboxylate, or hydroximate, hydroxamate, ester or amide ligands having anti-inflammatory activity, and these other ligands may also have anti-inflammatory and/or anti-cancer activities.
  • the complex is one of the following complexes (eg see Copper Complexes of Non-steroidal Anti-inflammatory Drugs: An Opportunity yet to be
  • each L is independently selected and Dimer is water, ethanol, dimethylsulfoxide or methanol
  • a Suprofen (+)- ⁇ -methyl-4-(2-thienyl-carbonyl)phenylacetic acid (SupH);
  • Tolmentin l-methyl-5-(p-toluoyl)-l/7-pyrrole-2-acetic acid (ToIH);
  • c DMSO dimethylsulfoxide;
  • Naproxen ⁇ -methoxy- ⁇ -methyl ⁇ -naphthaleneacetic acid (NapH);
  • Ibuprofen (+)- ⁇ -methyl-4-(isopropylmethyl)benzeneacetic acid (IbuH);
  • Indomethacin l-(4-chlorobenzoyl)-5-methoxy-2-methyl-lH-indole-3- acetic acid (IndoH);
  • M a is a metal ion and preferably, a transition metal ion.
  • M a will be copper ion.
  • the complex is any one of the complexes referred to in the table above where the metal ion is a transition metal ion other than copper (eg zinc, nickel, or cobalt ions, and preferably zinc, 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.
  • the metal ion is a transition metal ion other than copper (eg zinc, nickel, or cobalt ions, and preferably zinc, as described in Copper and Zinc Complexes as Anti-Inflammatory Drugs. Dillon, C. T.; Hambley, T. W.; Kennedy,
  • 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 L 2 :
  • R 1 is H or halo (i.e. Cl, F, Br or I);
  • R 2 is H; a Cj 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
  • each R 2A is independently selected from the group consisting of H, C 1 to
  • 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.
  • R 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 .
  • R 2A is a C 1 to C 6 alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or an arylalkyl
  • the Cj 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 .
  • 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 .
  • the complex may be a complex of formula (1), (2) or (3):
  • L is a ligand of the formula:
  • 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 2 ⁇ 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; n is 1, 2, 3, 4
  • each M is independently selected and is a divalent or trivalent metal ion
  • L is a ligand of the formula:
  • 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 Ci to C 6 alkyl, alkenyl or alkynyl may be optionally substituted; or
  • each R 2 ⁇ 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; 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;
  • each M' is independently selected and is a trivalent or tetravalent metal ion
  • L 2 is a ligand of the formula:
  • R 1 is H or halo (i.e. Cl, F 5 Br or I);
  • R 2 is H; a Ci to C 6 alkyl, an alkenyl or an alkynyl, where the Ci to C 6 alkyl, alkenyl or alkynyl may be optionally substituted; or
  • each R 2 ⁇ is independently selected from the group consisting of H, Cj 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,
  • R 2 is a C 1 to C 6 alkyl, an alkenyl or an alkynyl
  • the Ci 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 .
  • R 2A is a C 1 to C 6 alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or an arylalkyl
  • the Cj 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 .
  • 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 .
  • L 2 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, ⁇ TV-dimethylformamide (DMF), N-methylpyrrolidone, dimethylsulfoxide or ⁇ ,N-dimethylacetamide (DMA).
  • the complexes of formula (1), (2) or (3) maybe 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.
  • the complex is a mononuclear complex of the following formula (4):
  • M is a divalent or trivalent metal ion
  • L 1 is a ligand of the formula:
  • 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
  • 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, 0 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 5 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;
  • the mononuclear complex of formula (4) is a complex of O formula (4A):
  • M is a divalent or trivalent metal ion
  • ⁇ 2 -L 1 is a bidentate ligand of the formula L 1 :
  • 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 Cj to C 6 alkyl, alkenyl or alkynyl may be optionally substituted; or
  • 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 Ci 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,
  • R 2 is a Ci to C 6 alkyl, an alkenyl or an alkynyl
  • the Cj 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 .
  • R 2A is a C 1 to C 6 alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or an arylalkyl
  • the Ci 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 .
  • R 5 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 .
  • each R 5 is typically halo (i.e. F, Cl, Br or I), and n is typically 1, 2 or 3.
  • L 1 may for example be Indo.
  • 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.
  • the complex is a dinuclear complex of the formula (5):
  • each M is independently selected and is a divalent or trivalent metal ion
  • ⁇ -L 1 is a ligand of the formula L :
  • 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
  • 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,
  • R 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 .
  • 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 .
  • R 5 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 .
  • L 1 may for example be Indo.
  • 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.
  • M is copper ion.
  • the ligand L may be any monodentate ligand.
  • L may, for example, be water (OH 2 ), an alcohol, dimethylsulfoxide (DMSO), tetrahydrofuran (THF), or L may be a ligand containing a tertiary amide or cyclic tertiary amide.
  • the complex is a trinuclear complex of the following formula (6):
  • each M' is independently selected and is a trivalent or tetravalent metal ion; and ⁇ -L 1 , L and p are as defined above for formula (5).
  • the complex is a trinuclear complex of the following formula (7):
  • M is a divalent metal ion
  • L 3 is a carboxylate having anti-inflammatory activity
  • L 4 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 L 3 is independently selected; and each L 4 is independently selected.
  • L 3 may be a monodentate, bidentate or bridging ligand of formula L 1 or L 2 as follows:
  • 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
  • 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.
  • M may, for example, be selected from the group consisting of zinc ion, cobalt ion, nickel ion, magnesium ion, copper ion and calcium ion.
  • the heterocyclic base comprises one or more N atoms.
  • 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.
  • the heterocyclic base is quinolyl.
  • the metal complex may be a complex of the following formula (8):
  • L 6 is NH 3 or a monodentate, polydentate, or macrocyclic amine ligand, such as 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), Ru(III) or Pt(IV); or [M(O 2 R 1 ) m (NR 2 R 3 R 5 ) (4-m) ] p where M is Pd(II), Pt(II) or Au(III) and more preferably Pt(II); or [M(O 2 R 1 )(NR 2 R 3 R 5 ) 3 ] + or c ⁇ 5 orJr ⁇ M-[M(O 2 R 1 ) 2 (NR 2 R 3 R 5 ) 2 ] + , and R 1 , R 2 R 3 and R 5 are as defined in any one of formulae (1) to (7) above.
  • M is selected from Co(III), Cr(III), Ir(III), Os(III),
  • the metal complex may be a complex of the following formula (9):
  • L 7 is NH 3 or a monodentate, polydentate, or macrocyclic amine ligand.
  • L 7 is [M(NR 2 R 3 R 5 ) (6-2n )(L 8 ) n ] p when L 8 is a bidentate ligand, [M(NR 2 R 3 R 5 ) 3 (L 8 )] P when L 8 is a tridentate ligand, or [M(NR 2 R 3 R 5 ) 2 (L 8 )] P when L 8 is a tetradentate ligand, where M is selected from Co(III), Cr(III), Ir(III), Os(III), Rh(III), Ru(III), Ru(II) or Pt(IV) and more preferably, Co(III), Ru(III), Ru(II) or Pt(IV), and Rl, R2, R3 and R5 are as defined in any one of formulae (1) to (7) above.
  • L 7 is [M(NR 2 R 3 R 5 ) 2 (L 8 )] P when L 8 is a bidentate ligand or [M(NR 2 R 3 R 5 )(L 8 )f when L 8 is a tridentate ligand, where M is Cu(II), Ni(II), Pd(II), Pt(II) or Au(III) and more preferably Cu(II) or Pt(II), and Rl, R2, R3 and R5 are as defined in any one of formulae (1) to (7) above .
  • the complex is [M(L 8 ) n ] p .
  • L 8 when L 8 is a bidentate ligand n is 3, or when L 8 is a tridentate ligand n is 2, where M is preferably Co(III), Cu(II), Ga(III), Pt(IV), Ru(III), or Ru(II); or when L 8 is a bidentate ligand n is 2, or when L 8 is a tetradentate ligand n is 1, where M is preferably Cu(II), Ni(II), Pd(II), Pt(II) or Au(III) and more preferably Cu(II) or Pt(II).
  • the metal complex may be a complex of the following formula (10):
  • One or more of the ligands L 5 and L 8 in any combination may also form dimeric, trimeric, tetrameric, oligomeric or polumeric complexes with one or more metal ions.
  • the ancilliary ligands may be chosen from ligands that exert a separate anti-cancer activity, such as dithiocarbamates.
  • the carcinoma may be a lesion or tumour of the epithelium.
  • the lesion may, for example, be a skin lesion such as basal cell carcinoma, squamous cell carcinoma or melanoma.
  • the carcinoma may also be other cancers of the epithelium, such as lung cancer and cancer of the oesophagus.
  • the complex may be administered to the mammalian subject systemically or may be locally applied to the site of the carcinoma.
  • the complex may also be administered to the mammalian subject by both methods concurrently.
  • the complex may be topically applied to a skin cancer. If the carcinoma is a lung cancer, the complex may be administered to the cancer site by inhalation of the complex. If the carcinoma is a cancer of the nose, the complex may be administered by nasal spray.
  • the complex may be administered to the cancer site by oral administration (including buccal or sublingual administration for mouth cancers) or by suppository for colorectal cancers.
  • the complex may be administered by direct injection into the lesion or organ containing the lesion.
  • the complex may also be administered to the mammalian subject in combination with other anti-cancer drugs or cancer treatments. Such administration may be given to improve the response to chemotherapy, or to reduce the dose of a chemotherapeutic agent to reduce toxic side-effects while maintaining a comparable efficacy.
  • the present invention also provides the use of a complex of a metal and a carboxylate, or derivative of a carboxylate, having anti-inflammatory activity in the manufacture of a medicament for the prophylaxis or treatment of a carcinoma in a mammalian subject.
  • the present invention further provides the use of a complex of a metal and a carboxylate, or derivative of a carboxylate, having anti-inflammatory activity for the prophylaxis or treatment of a carcinoma in a mammalian subject.
  • Figure 1 shows the chemical structure of [Cu 2 (Indo) 4 (DMF) 2 ]
  • Figure 2 is a graph showing the extent of gastric ulceration measured in mm 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;
  • 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 [Cu 2 (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 11 shows photographs of a control rat colon (left panel) and azoxymethane-induced aberrant crypt focci (ACF) (right panel) in the colon of a rat treated with azoxymethane (AOM) at doses of 15 mg/kg for four weeks then sacrificed 6 weeks after the final injection of AOM;
  • ACF azoxymethane-induced aberrant crypt focci
  • Figure 12 shows graphs indicating the effects observed in rats following treatment with equimolar Indo doses of IndoH or [Cu 2 (Indo) 4 (DMF)2] in 2% w/w CMC, compared with control rats treated with CMC only, on AOM-induced ACF in rats
  • the concentration scale is the equivalent dose of Indo;
  • the log concentration scale is the equivalent dose of Indo;
  • Figure 15 is a graph showing the effect of IndoH suspended in medium chain triglyceride (MCT) oil on the viability of A549 cells;
  • Figure 16 is a graph showing the effect of IndoH suspended in MCT oil on the 0 viability of A2780 cells
  • Figure 17 is a graph showing the effect of Cu-Indo suspended in MCT oil on viability of A549 cells, where the concentration range refers to the equivalent concentration of Indo and not the copper complex;
  • Figure 18 is a graph showing the effect of Cu-Indo suspended in MCT oil on the 5 viability of A2780 cells, where the concentration range refers to the equivalent concentration of Indo and not the copper complex;
  • Figure 19 is a graph showing the effect of ACM, CuACM and ZnACM suspended in MCT oil on the viability of A549 cells, where the concentration range refers to the equivalent concentration of Indo and not the zinc or copper complexes;
  • O Figure 20 is a graph showing the effect of aspirin and [Cu(asp) 2 (3-pic) 2 ] suspended in MCT oil on the viability of A549 cells, where the concentration range refers to the equivalent concentration of aspirin and not the copper complex;
  • Figure 21 is a graph showing the effect of ibuprofen and [Cu(ibup) 2 (2-Meim) 2 ] suspended in MCT oil on the viability of A549 cells, where the concentration range refers to the equivalent concentration of ibuprofen and not the copper complex;
  • Figure 22 is a graph showing the effect of [V v O(IndoHAH)(IndoHA)] and IndoHAH 2 suspended in MCT oil on the viability of A549 cells, where the concentration range refers to the equivalent concentration of IndoHA and not the copper complex;
  • Figure 23 is a graph showing the effect of [Zn(Indo) 2 (OH 2 ) 2 ] suspended in MCT oil on the viability of A549 and Hep-2 cells, where the concentration range refers to the equivalent concentration of Indo and not the zinc complex;
  • the log concentration scale is the equivalent dose of Indo;
  • Figure 29 is a graph showing mean increase in rat paw volume 3 hours post treatment with 1% [Cu 2 (Indo)4(OH 2 ) 2 ]; the labels are the same as in Figure 26; and Figure 30 is a graph showing mean increase in rat paw volume 4 hours post treatment with [Cu2(Indo) 4 (OH 2 )2]; the labels are the same as in Figure 26.
  • a reference to “copper indomethacin” or “Culndo” is a reference to [Cu 2 (Indo) 4 (DMF) 2 ].
  • 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-rnethylimidazole;
  • a reference to “Asp” is a reference to aspirin (acetylsalicyclic acid),
  • 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;
  • halo refers to fluoro, chloro, bromo or iodo.
  • alkyl used either alone or in a compound word such as “arylalkyl”, refers to a straight chain, branched or mono- or polycyclic alkyl.
  • straight chain and branched alkyl examples include methyl, ethyl, propyl, iso-propyl, butyl, iso-bv ⁇ yl, sec-butyl, tert-butyl, amyl, w ⁇ -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,
  • cyclic alkyl examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • cycloalkyl refers to a saturated monocyclic or polycyclic alkyl having 3 to 12 carbons.
  • alkenyl refers to a straight chain, branched or cyclic alkenyl with one or more double bonds.
  • the alkenyl is a C 2 to C 2 o alkenyl, more preferably a C 2 to C 6 alkenyl.
  • alkenyl examples include vinyl, allyl, 1-methylvinyl, butenyl, wo-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-cycloheptatrien
  • alkynyl refers to a straight chain, branched or cyclic alkynyl with one or more triple bonds, preferably a C 2 to C 20 alkynyl, more preferably a C 2 to C 6 alkynyl.
  • 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.
  • 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.
  • arylalkyl refers to an alkyl substituted with an aryl group.
  • An example of arylalkyl is benzyl.
  • the term “bidentate ligand” refers to a ligand having two co-ordination bonds to a metal atom. Bidentate ligands include unsymmetric bidentate ligands with one weaker and one relatively stronger bond to the metal atom, hi this specification, the term “monodentate ligand” refers to a ligand having a single co-ordination bond with a metal atom.
  • the present invention in one or more embodiments provides a method for the prophylaxis or treatment of carcinomas in a mammalian subject, the method comprising the administration to the subject of a therapeutically effective amount of a complex of a metal and a carboxylate, or a hydroximate, hydroxamate, ester or amide derivative having anti-inflammatory activity.
  • complexes of a metal and a carboxylate, or derivative of a carboxylate, having anti-inflammatory activity have application in preventing or treating carcinomas including skin cancers and colorectal cancers, and for skin cancers may be more effective in preventing or treating the carcinomas in terms of efficacy and/or safety than the carboxylate or the derivative of the carboxylate itself.
  • the present inventors have found that complexes of a metal and indomethacin are more effective in preventing or treating carcinomas, in terms of efficacy and/or safety, than indomethacin itself, even in cases when IndoH is completely inactive.
  • copper complexes formed in vivo from the chelation of copper by the copper chelators may themselves have a strong anti-cancer effect rather than the anti-cancer effect being due solely to the removal of copper in addition to the reduction in angiogenesis when Cu is sequestered. More particularly, the efficacy of the complexes is considered to be due to a combination of both the ligand (i.e., carboxylate, or hydroximate, hydroxamate ester or amide derivative) and the metal atom.
  • the ligand i.e., carboxylate, or hydroximate, hydroxamate ester or amide derivative
  • Complexes of a metal and a carboxylate, or hydroximate, hydroxamate, ester or amide derivatives thereof having anti-inflammatory activity have not previously been shown to be useful in the general prophylaxis or treatment of carcinomas.
  • 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.
  • Suprofen (+)- ⁇ -methyl-4-(2-thienyl-carbonyl)phenylacetic acid (SupH);
  • Tolmentin l-methyl-5-(p-toluoyl)-lH-pyrrole-2-acetic acid (ToIH);
  • c DMSO dimethylsulfoxide;
  • Naproxen 6-methoxy- ⁇ -methyl-2-naphthaleneacetic acid (NapH);
  • Ibuprofen (+)- ⁇ -methyl-4-(isopropylmethyl)benzeneacetic acid (IbuH);
  • Indomethacin l-(4-chlorobenzoyl)-5-methoxy-2-methyl-lH-indole-3-acetic acid (IndoH);
  • J Diclofenac 2-[(2,6-dichlorophenyl)amino]phenylacetic acid (DicH).
  • the complexes listed above may be prepared by methods known in the art, or prepared by methods described below. Methods known in the art are described in, for example, US Patent No. 5,466,824 or the paper: Anti-inflammatory Dimiclear Copper(II) Complexes with Indomethacin. Synthesis, Magnetism and EPR Spectroscopy; Crystal Structure of the N,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.
  • the complex may be a complex comprising at least one metal ion and at least one hydroximate, hydroxamate, ester or amide derivative of a carboxylate having anti- inflammatory activity.
  • the hydroxamate or amide having anti-inflammatory activity may be the hydroximate, hydroxamate, ester or amide derivative of a carboxylic acid NSAID.
  • 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 form chelates of deprotonated amides or amide monodentate complexes with a metal ion in the complex.
  • 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 with anti-inflammatory activity such as Indo and esters of Indo (e.g. ACM), are particularly preferred.
  • RCO 2 H is the parent carboxylic acid with anti-inflammatory activity.
  • R Alk or Ar hydroxamato hydroximato complex complex
  • 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).
  • 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.
  • 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 stagentate 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.
  • 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).
  • 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.
  • 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- 1 ,2-benzothiazine-3-carboxamide-l ,1 -dioxide), tenoxicam (4-hydroxy-2-methyl-N-2- pyridinyl-2H-thieno(2,3-e)-l,2-thiazine-3-carboxamide-l,l-dioxide) and meloxicam.
  • the complex is a complex of the formula (A):
  • M is a metal ion
  • L 4 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.
  • M is a divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion.
  • the complex used in the method of the present invention may be a mononuclear, dinuclear, trinuclear, oligomeric or polymeric complex containing the ligand L 2 as defined above.
  • the complex maybe 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 2 H (where 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, 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.
  • a compound L 2 H where L 2 is as defined above
  • a divalent or trivalent metal salt preferably a basic salt such as M(OAc) 2
  • the product may need to be recrystallised until elemental, spectroscopic and/or diffraction methods demonstrate that the complex is of the required purity.
  • 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 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.
  • 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 a neutral ligand.
  • a complex of formula (1) or (2) may have a charge, for example, p may be 1 ⁇ or T.
  • Complexes of formula (3) have a charge of I + 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 " , Y, 0, or 2 + .
  • Examples of complexes of formula (1) include [Cu(ACM) 2 (DMF) 2 ], [Cu(ACM) 2 (OH 2 ) 2 ], [Zn(ACM) 2 (DMF) 2 ] and [Zn(ACM) 2 (OH 2 ) 2 ] .
  • Examples of complexes of formula (2) include [Ru 2 (ACM) 4 L] p and [Ru 2 (ACM) 4 L 2 ] 13 (where Ru is Ru(II)), and [Ru 2 (ACM) 4 Lf (a Ru(II)/Ru(III) mixed- valence complex), where p is the charge of the complex.
  • the complex of formula (2) is a complex of the formula (2A):
  • the complex is a mononuclear complex of the following formula (4):
  • the mononuclear complex of formula (4) is a complex of formula (4A):
  • the complex of formula (4) maybe charged or neutral.
  • Preferred complexes include [Cu(Indo) 2 (Im) 2 ] and [Zn(Indo) 2 (OH 2 ) 2 ].nH 2 O.
  • 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 T) 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:
  • Y is a counter ion having a 2 + charge and Y is a counter ion having a I + charge.
  • Cu(II) complexes of formula (4) are formed when copper(II) indomethacin complexes are formed using strong donor ligands, as described in a co-pending International Patent Application in the name of Medical Therapies Limited entitled “Copper complexes” filed 24 March 2006 and claiming priority from Australian Provisional Patent Application No. 2005901464, the contents of which is 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).
  • L is a ligand containing an JV-heterocyclic group.
  • Ligands containing an N-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.
  • L is an amine, e.g. NH 3 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 may 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 L 1 H where L 1 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 L 1 in a weaker donor solvent.
  • complexes of formula (4) can be prepared by re-crystallisation of a dinuclear complex, such as [Cu 2 (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).
  • a dinuclear complex such as [Cu 2 (Indo) 4 (DMF) 2
  • a solvent having a solvent donor number of about 30 or greater such as pyrrolidine
  • 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 L 1 H 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 complex is a dinuclear complex of the formula (5):
  • the ligand L may be any monodentate ligand.
  • the ligand L may be charged or uncharged.
  • L may, for example, be water (OH 2 ), an alcohol, dimethylsulfoxide (DMSO), pyridine (Py), acetonitrile (AN), tetrahydrofuran (THF), or L may be a ligand containing a tertiary amide or cyclic tertiary amide.
  • L may be a molecule of a tertiary amide of the formula:
  • R 1 is an alkyl having from 1 to 4 carbon atoms, and each R 1 may be the same or different, and R 2 is a cycloalkyl having from 2 to 7 carbon atoms.
  • the tertiary amide or cyclic tertiary amide may be, for example, N,N-dimethylformamide, N.N-dirnethylacetamide or iV-methylpyrrolidone.
  • ⁇ -L 1 is a ligand of the formula L 1 :
  • M is Cu or Zn; each L is independently selected and is a monodentate ligand; and p is the charge of the complex.
  • L 1 may for example be Indo.
  • the complex is a dinuclear metal/indomethacin complex of the formula (5B):
  • M is Cu or Zn
  • Y is water (OH 2 ) 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 0 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 cancer cell (i.e. there is greater absorption of the complex than IndoH). The present inventors further believe that both M and indomethacin are effective in inducing cancer cell apoptosis via different mechanisms, 5 leading to significantly enhanced anti-cancer activity than due to either M alone or indomethacin alone.
  • the present inventors believe that the role of M in inducing angiogenesis may be important in delivering the bioactive components of the complex to the core of the tumour, which assists in the more effective treatment of solid tumours than the anti-inflammatory drug alone.
  • the greatly reduced adverse O effects of the complex compared with the free indomethacin enables higher doses and, therefore, even higher efficacy than is possible with indomethacin alone.
  • Complexes of formula (5B) include, for example, [Cu 2 (Indo) 4 (DMF) 2 ], 5 [Cu 2 (Indo) 4 (DMA) 2 ], [Cu 2 (Indo) 4 (NMP) 2 ], [Cu 2 (Indo) 4 (DMSO) 2 ], [Cu 2 (Indo) 4 (THF) 2 ], [Cu 2 (Indo) 4 (Py) 2 ], [Cu 2 (Indo) 4 ( AN) 2 ], [Cu 2 (Indo) 4 (OH 2 ) 2 ], [Zn 2 (Indo) 4 (DMA) 2 ], [Zn 2 (Indo) 4 (NMP) 2 ] and [Zn 2 (Indo) 4 (Py) 2 ], wherein NMP is N-methylpyrrolidone.
  • a preferred complex is [Cu 2 (Indo) 4 (OH 2 ) 2 ].nH 2 O, wherein n is the number of waters of crystallisation. The number of waters of crystallisation will vary depending on 0 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 N,N-Dimethylformamide Adduct. Weder, J.
  • Trinuclear metal complexes with the ligand L 1 Trinuclear metal complexes with the ligand L 1
  • the complex is a trinuclear complex of the following formula (6):
  • each M' is independently selected and is a trivalent or tetravalent metal ion, and ⁇ -L 1 , L and p are as defined above for formula (5).
  • Trinuclear metal complexes with the ligand L 3 and L 4 Trinuclear metal complexes with the ligand L 3 and L 4
  • the complex is a trinuclear complex of the following formula (7):
  • M is a divalent metal ion
  • L 3 is a carboxylate having anti-inflammatory activity
  • L 4 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 L 3 is independently selected; and each L 4 is independently selected.
  • L 3 may be a monodentate, bidentate or bridging ligand of formula L 1 or L 2 as follows:
  • R 1 is H or halo (i.e. Cl, F, Br or I);
  • R 2 is H; a Cj 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
  • each R 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.
  • 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 0 of halo, -OH, -COOH and -NH 2 .
  • 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, 5 -COOH and -NH 2 .
  • R 5 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 .
  • 0 R 1 is typically H.
  • R 3 is typically H.
  • R 2 is typically CH 3 .
  • L is ACM.
  • M may be selected from the group consisting of zinc, cobalt, 5 nickel, magnesium, copper and calcium.
  • the heterocyclic base comprises one or more N atoms.
  • the heterocyclic base is optionally substituted.
  • the heterocyclic base may be selected from the group consisting of isoquinolyl, quinolyl, piperidinyl, pyridinyl, 2-methylpyridinyl, imadazoyl, pyranyl, pyrrolyl, pyrimidinyl, O indolyl, purinyl and quinolizinyl.
  • the heterocyclic base is quinolyl.
  • the term "therapeutically effective amount” means an amount effective to yield a desired therapeutic response, for example, to prevent or treat a carcinoma or to provide a chemopreventative effect to reduce the incidence or reoccurrence of cancer caused by carcinomas.
  • the metal may be administered alone or co-administered in combination with one or more chemotherapeutic or anti-cancer agents conventionally used in the treatment of carcinoma.
  • co-administered is meant simultaneous administration in the same formulation or a plurality of formulations by the same or different routes, or sequential administration by the same or different routes.
  • sequential administration is meant one is administered one after the other.
  • agents include metal and non-metal based drugs.
  • the conventional metal based drugs may be organic, inorganic, mixed ligand co-ordination compounds or chelates, including complexes of platinum and palladium.
  • platinum based chemotherapeutic drugs include cisplatin (cw-diamminedichloroplatinum(II), oxaliplatin ([Pt((lR),(2R)- cyclohexane-l,2-diamine)(oxalato)] complex, and carboplatin (cis- diammine(cyclobutane- 1 , 1 -dicarboxylato)platinum(II) .
  • non-metal chemotherapeutic drugs include paclitaxel, gleevac, docetaxel, taxol, 5-fluorouracil, doxorubicin, cyclophosphamide, vincristine (Oncovin), vinblastine, vindesin, camplothecin, gemcitabine, adriamycin, and topoisomerase inhibitors such as irinotecan (CPT-11).
  • a metal complex in one or more methods embodied by the invention in combination with another anti-cancer agent may enhance the effectiveness of the other anti-cancer drug. This may include the treatment of both carcinomas that are susceptible to the other drug and carcinomas that are otherwise resistant to the other drug. Accordingly, the present invention expressly extends to such combination treatments.
  • 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.
  • a low dosage may initially be administered which is subsequently increased at each administration following evaluation of the response of the subject.
  • 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 complex may be administered to the mammalian subject systemically or may be locally applied to the site of the carcinoma.
  • the complex may also be administered to the mammalian subject by both methods concurrently.
  • the complex may be topically applied to a skin cancer. If the carcinoma is a lung cancer, the complex may be administered to the cancer site by inhalation of the complex. If the carcinoma is a cancer of the nose, the complex may be administered by nasal spray.
  • the complex may be administered to the cancer site by oral administration (including buccal or sublingual administration for mouth cancers) or by suppository for colorectal cancers.
  • the complex may be administered by direct injection into the lesion or organ containing the lesion.
  • the complex will generally be administered in the form of a composition comprising the complex together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier 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.
  • a formulation having a colloidal structure or which forms a colloidal structure post administration is particularly desirable for administration of metal complexes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition is immiscible with water, and is thus immiscible with aqueous biological fluids whereby a colloidal system is thereby formed.
  • 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.
  • the composition 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, palmic 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).
  • 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.
  • 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).
  • a composition as described herein may also optionally further comprise one or more solvents or solubilising components for increasing the solubility of the metal carboxylate complex in the composition.
  • the 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, or other pharmaceutically acceptable glycol.
  • IUPAC name 2-[2-[(tetrahydro-2-furanyl)methoxy]ethoxy]ethanol
  • other names 2-[2- (tetrahydrofurfuryloxy)ethoxy]ethanol
  • compositions may also further comprise a thickener such as Aerosil 200, clay or another inorganic filler.
  • a thickener such as Aerosil 200, clay or another inorganic filler.
  • such compositions 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 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 (18 0 C to 25 0 C).
  • 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 Antiinflammatory Dinuclear and Mononuclear Zn(II) Complexes of Indomethacin. Zhou, Q.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A. Inorg. Chem.
  • the compositions preferably do not comprise, or are substantially tree of, peptides, carboxylate donors, reductants and thiolate groups, apart from those in the derivatives of the NSAID utilised.
  • the composition is also not strongly acidic or basic as strong acids and bases can cause metal carboxylate complexes to dissociate.
  • 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.
  • 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 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.
  • the carrier may 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.
  • composition for use in the method of the invention may be suitable for oral, rectal, nasal, topical (including buccal and sublingual), ophthalmological, vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, or for administration respiratoraly, intratrachaely, nasopharanyngealy, intraoccularly, intrathecally, intranasally, by infusion, or via IV group patch and by implant.
  • suitable routes are via injection into blood vessels which supply a tumour or particular organs to be treated. Agents may also be delivered into cavities such as for example the pleural or peritoneal cavity, or be injected directly into tumour tissue.
  • compositions may 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.
  • the carrier comprises two or more ingredients.
  • 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 may be in the form of a viscous paste, a tablet, a capsule, a chewable composition, or any other form suitable for oral administration.
  • the composition may be encapsulated in a hard or soft capsule (e.g. gelatine) by techniques known in the art.
  • the metal complex maybe provided in the form of ingestible tablets, 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 skilled addressee.
  • 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 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.
  • 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 such as stearate
  • colouring agents e.g. such as sorbic acid
  • a chewable composition may, 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.
  • 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 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.
  • 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:
  • compositions for application to skin are 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 may include one or more pharmaceutically active components in addition to the complex that have anti-cancer activity or other therapeutic activity.
  • active components include conventionally used anti-inflammatory drugs, and conventionally used metal and non- metal based chemotherapeutic and anti-cancer agents such as those identified above.
  • the metal complex constitutes 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.
  • a composition of the invention for topical application to the skin will typically comprise the metal complex in an amount of about 0.1% 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 may 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 Artiodactyla.
  • the subject will be a primate and more usually, a human being.
  • the method of the present invention may for instance be used to prevent or treat basal cell carcinomas, squamous cell carcinoma, melanoma, colon, colorectal cancer, breast cancer, lung cancer and other cancers of the epithelial tissues such as epithelial cancers of the tongue, salivary glands, gums and other areas of the mouth, oropharynx, nasopharynx, hypopharynx, oesophagus, pancreas, stomach, small intestine, duodenum, gall bladder, pancreas, larynx, trachea, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid, and mestastic cancers thereof.
  • basal cell carcinomas squamous cell carcinoma, melanoma, colon, colorectal cancer, breast cancer, lung cancer and other cancers of the epithelial tissues
  • epithelial cancers of the tongue salivary glands,
  • 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.
  • Acetylsalicylhydroxamic Acid (AcSHA) and its copper complex
  • Salicylhydroxamic acid (SHAH 2 ) (7.65 g, 50 mmol) was mixed with acetic anhydride (9.5 ml, 100 mmol). The solution was acidified with H 3 PO 4 (1 ml) and was stirred in a water bath at 60 °C for 30 min. Distilled water (5 ml) was added to the solution in order to decompose the unreacted acetic anhydride, and the resulting solution were stirred at room temperature until the vapor from the solution gave no acid reaction 0 towards litmus paper. Finally, the reaction mixture was mixed with distilled water (50 ml) and AcSHAH 2 precipitated as a white powder solid (68.2%). Anal. Calcd.
  • VOSO 4 -5H 2 O 50.6 mg, 0.200 mmol
  • IndoHAH 2 149 mg, 0.400 mmol
  • methanol MeOH, HPLC grade, 5.0 mL
  • the colour of the solution immediately turned dark-red.
  • This solution was added to ice-cold H 2 O (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.
  • 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 the high (10 mg/kg Indo molar equivalent and dissolved into an MCT paste).
  • Vanadium and chromium complexes of this ligand are prepared by similar methods as those described for IndoHA, but electrospray mass spectrometry indicates that the complexes are polymeric rather than monomers.
  • Example 1.1.3 offer the potential of systemic delivery of even higher concentrations of NSAIDs through oral, injectable, and topical delivery and to incorporate them into slow release patches.
  • the complex still exhibits significant anti-inflammatory, and hence is expected to have systemic anti-cancer activity, but completely eliminates gastric side-effects by being absorbed in the GI tract before release of the drug.
  • ACF Aberrant crypt focci
  • Rats were dosed with indomethacin and [Cu 2 (IiIdO) 4 (DMF) 2 ] in a rat model of adenocarcinoma for 28 days and aberrant crypt focci were evaluated.
  • 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 [Cu 2 (LtIdO) 4 (DMF) 2 ] on urinary electrolyte concentrations were examined.
  • HCT- 116 colorectal cancer cells were exposed to indomethacin and [Cu 2 (Indo) 4 (DMF) 2 ] (0-250 ⁇ g/mL) and cell viability was measured.
  • indomethacin and [Cu 2 (Indo) 4 (DMF) 2 ] resulted in a significant reduction in aberrant crypt focci in azoxymethane-treated rats.
  • Indomethacin reduced urinary electrolytes at an ulcerogenic dose of 10 mg/kg acutely and chronically at 3 mg/kg for 28 days.
  • 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- ⁇ L 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.
  • Rats were deprived of food, but not water, for 18 h and were administered indomethacin, [Cu 2 (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.
  • 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.; Corrigan, B.W.; Jamali, F. Pharm. Res.
  • 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
  • Relative permeability was determined by calculating the sucrose present in each urine sample as a percent of the administered dose (see Figure 3).
  • 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. 1991, 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 ⁇ m) and the sections were stained with Lee's methylene blue-basic fuchsin for 30 seconds. Sections were then examined by light microscopy.
  • Rats (n 4 for each treatment) received either oral indomethacin at doses of 10 mg/kg or [Cu 2 (IiIdO) 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. Urine was collected 0-24 h following the administration Of 51 Cr-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 ⁇ L of the administered 51 Cr-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, 11(11), 1652-6) (see Figure 6).
  • Rats (n 4 for each treatment) received either oral indomethacin at doses of 10 mg/kg or [Cu 2 (IiIdO) 4 (DMF) 2 ] at doses of 13.3 mg/kg via gavage.
  • 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).
  • 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.
  • Rats (n 4 for each treatment) received either oral indomethacin at doses of
  • the rats were given weekly intraperitoneal injections of azoxymethane (AOM) at doses of 15 mg/kg for four weeks and then sacrificed 6 weeks after the final injection of AOM for assessment of numbers of aberrant crypt focci (ACF).
  • AOM azoxymethane
  • the rats were randomized into treatment groups at the beginning of the period of treatment with AOM.
  • Groups of five rats were given 2% carboxymethylcellulose (CMC) vehicle, indomethacin (3.00 mg/kg), or an equimolar dose Of [Cu 2 (In(Io) 4 (DMF) 2 ] (3.81 mg/kg) orally each day for 28 consecutive days during the AOM dosing.
  • CMC carboxymethylcellulose
  • DMF equimolar dose Of [Cu 2 (In(Io) 4 (DMF) 2 ] (3.81 mg/kg) orally each day for 28 consecutive days during the AOM dosing.
  • indomethacin The dose of indomethacin was selected on the basis of a previous study of intestinal toxicity of indomethacin and colorectal carcinogenesis in an animal model.
  • Non-steroidal anti-inflammatory drugs with activity against either cyclooxygenase 1 or cyclooxygenase 2 inhibit colorectal cancer in a DMH rodent model by inducing apoptosis and inhibiting cell proliferation.
  • the tissues were then stained with 0.2% methylene blue in 0.9% saline and then coded before scoring blindly.
  • the colon from each animal was cut into convenient 2-3 cm pieces and each piece was placed mucosal side up between two slides as a wet mount, and the mounted sample was examined microscopically at 10Ox magnification.
  • Aberrant crypts were distinguished from the surrounding normal crypts by their increased size, bright blue staining, significantly increased distance from lamina to basal surface of cells, and the easily discernible pericryptal zone (see Figure 11).
  • the left panel of Figure 11 shows a control rat colon, and the right panel of Figure 11 shows azoxymethane-induced aberrant crypt foci in the colon of a rat treated with azoxymethane.
  • the parameters used to assess the aberrant crypts were occurrence and multiplicity. Crypt multiplicity was determined as the number of crypts in each focus and categorized as those containing up to three, or four or more aberrant crypts/focus
  • 2.7 CeU Culture HCT-116 cells were cultured in McCoy's 5 A medium containing sodium bicarbonate, penicillin-streptomycin, 25 mM HEPES and 10% heat-inactivated FBS in a 5% CO 2 atmosphere at 37°C. The cells were passed twice a week on reaching confluence. The cells were trypsinized, washed with PBS and plated with fresh medium in a 96-well plate at a density of 3 x 10 4 cells/well and cultured overnight prior to discarding the culture medium. The treatment was started by adding fresh medium with or without cytotoxins (indomethacin and [Cu 2 (Indo) 4 (DMF) 2 ]) of different concentrations and the cells were further incubated for 72 h.
  • cytotoxins indomethacin and [Cu 2 (Indo) 4 (DMF) 2 ]
  • Acute administration of ulcerogenic doses of indomethacin also resulted in decreased urinary excretion OfNa + , K , and Cl and increased urinary excretion of NAG and phosphate (see Table 2, Figure 10).
  • Acute administration of [Cu 2 (Indo) 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 [Cu 2 (Indo) 4 (DMF) 2 ] did not significantly change the urinary electrolytes concentrations studied.
  • indomethacin 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.
  • Hepatomas are primary carcinomas of the liver. As is the case for the human colorectal and melanoma cancer cell lines, there is a significantly greater increase in cytotoxicity of Culndo compared to IndoH suggesting that topical administration of Culndo will increase the efficacy of treatment of a large range of carcinomas.
  • Trypsin-EDTA, trypan blue, phosphate-buffered saline (PBS), resazurin, sodium bicarbonate, McCoy's 5 A medium, penicillin-streptomycin, and insulin were purchased from Sigma (St. Louis, MO, USA).
  • HPLC grade reagent alcohol was purchased from J. T. Baker (Phillipsburg, NJ, USA).
  • Dulbecco's Modified Eagle Medium (D-MEM) and RMPI 1640 medium were purchased from Gibco Industries Inc. (Langley, OK, USA).
  • Foetal bovine serum (FBS) was purchased form Equitech-Bio Inc. (Kerrville, TX, USA).
  • the Hep-G2 (human hepatoma) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD). The Hep-G2 cells were maintained in Dulbecco's Modified Eagle Medium (D-MEM). The cell line was supplemented with 10% heat-inactivated foetal bovine serum (FBS), penicillin-streptomycin (10 mg/1 L) and were incubated at 37 0 C in a 5% CO 2 atmosphere. The Hep-G2 cell line was additionally supplemented with insulin (4 mg/mL).
  • D-MEM Dulbecco's Modified Eagle Medium
  • FBS heat-inactivated foetal bovine serum
  • penicillin-streptomycin 10 mg/1 L
  • the optimal cell seeding numbers were determined by preliminary cell seeding number experiments. Cells were seeded in numbers 1 x 10 4 , 2 x 10 4 , 3 x 10 4 and so on until the final cell seeding number 1 x 10 5 per well in a 96 well plate (Costar 3595). Cell plates were incubated at 37 0 C in a 5% CO 2 atmosphere for 72 hours. Following incubation, the medium was aspirated and alamar blue (resazurin) fluorescent dye solution was diluted in fresh medium to make a 10% resazurin solution. The 10% solution was added directly to cells. The cell plates were incubated at 37°C in a 5% CO 2 atmosphere for 3 hours.
  • the cell plates were subsequently removed from the incubator and placed at room temperature in a darkened drawer to protect from light for 30 min.
  • the cell plates were subsequently placed into the Cytoflour®4000 fluorescence multi-well plate reader (Applied Biosystems, USA). Fluorescence was read at an excitation of 485 run and an emission of 530 run. Standard curves of cell seeding number against fluorescence were generated. From these studies Hep-G2 cells were seeded at a density of 5000 cells/well.
  • Alamar blue (resazurin) fluorescent dye is an easy and accurate assay that has recently gained popularity in determining the cytotoxicity of many cell lines
  • resazurin Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. O'Brien, J.; Wilson, L; Orton, T.; Pognan, F. Eur. J. Biochem. 2000, 267, 5421-5426).
  • the resazurin non-fluorescent compound is metabolized into the fluorescent compound resorufin by intact and viable cells. This emission of fluorescence can be quantified using a cell plate reader and the number of viable cells following treatment can be determined.
  • the cytotoxicity of metal complexes on cancer cell lines A549, A2780 and Hep- 2 were assessed using MTT and crystal violet blue assays.
  • Advanced DMEM medium was used in all the cell culture work.
  • the medium did not contain certain components needed to facilitate cell growth. Therefore, for the A549 cells, antibiotics-actimycotic (0.5 mL), (100 U m "1 penicillin, 100 ⁇ g mL “1 streptomycin and 0.25 ⁇ g mL “1 amphotericin B), 200 mM glutamine solution (0.5 mL) and fetal calf serum (2 %, 0.8 mL) were added to the medium (40 mL) before proceeding with any cell work.
  • antibiotics-actimycotic 0.5 mL
  • penicillin 100 ⁇ g mL "1 streptomycin and 0.25 ⁇ g mL “1 amphotericin B
  • 200 mM glutamine solution 0.5 mL
  • fetal calf serum 2 %, 0.8 mL
  • Advanced DMEM medium 40 mL was supplemented with antibiotics-actimycotic (0.5 mL), (100 U m "1 penicilin, 100 ⁇ g mL “1 streptomycin and 0.25 ⁇ g mL “1 amphotericin B), 200 mM glutamine solution (1.0 mL) and fetal calf serum (5 %, 4 mL).
  • antibiotics-actimycotic 0.5 mL
  • 200 mM glutamine solution 1.0 mL
  • fetal calf serum 5 %, 4 mL
  • AU of the above components were obtained from Gibco Industries Inc. (Langley, OK, USA). All other reagents used in the cell work were obtained from Sigma (St. Louis, MO, USA).
  • Frozen cells were stored in liquid nitrogen. The cells were rapidly warmed in a 37 0 C water bath for approximately 5 min. The cell suspension was then transferred to a 10- mL centrifuge tube with 9 mL of medium and centrifuged for 3 min at 2000 rpm. The medium was removed from the resultant pellet and fresh medium (1 mL) was added to resuspend the cells, then transfer the cells to a 10-cm plate with fresh medium (10 mL) added to it. Cells were incubated at 37 0 C in a humidified atmosphere containing 5 % CO 2 for 3 days. 4.1.3 Subculturing of A549, A2780 and Hep-2 cells
  • the medium was removed from the cells and the cell layer was washed with phosphate buffer solution (PBS, 10 mL) prior to trypsination with 0.25 % trypsin EDTA solution (4 mL). Cells were then incubated for 6 min at 37 0 C, after which medium with serum (5 mL) was added to inactivate the trypsin. The cell suspension was then collected into a centrifuge tube and the mixture was centrifuged at 2000 rpm for 3 min. The medium was subsequently removed from the cell pellet and fresh medium (1 mL, A549, and 1 mL Hep-2) was added to resuspend the cells.
  • PBS phosphate buffer solution
  • the cell suspension (0.58 mL, A549, 0.24 mL, A2780 and 1.6 mL, Hep-2) was transferred from the total cell suspension to a centrifuge tube. Further, medium (3 mL, for A549, A2780 and Hep-2) was added to the centrifuge tube and the cells were counted using a haemocytometer.
  • the cell suspension (100 ⁇ L per well) was transferred to four sets of ninety six- well plates with each well having approximately the same amount of cells (1x10 4 cells/well/100 ⁇ L for A549 and Hep-2 and IxIO 5 cells/well/ 100 ⁇ L for A2780). The plates were incubated overnight at 37 0 C prior to the addition of the test compound.
  • Indomethacin and [Cu 2 (Indo) 4 (OH 2 ) 2 ] were tested over a range of concentrations (10-300 ⁇ M) while ACMH, [Cu(ACM) 2 (OH 2 ) 2 ], [Zn(ACM) 2 (OH 2 ) 2 ], aspirin, IndoHAH 2 (oxamethacin), [V v O(IndoHAH) 2 ], ibuprofen, [Cu(asp) 2 (3-pic) 2 ] and [Cu(ibup) 2 (2- Mim) 2 ] were tested over a range of concentrations (20-400 ⁇ M).
  • the medium was removed from all the wells via a vacuum pump. A number of wells were left without addition of the test compound and were used as control wells. Appropriate concentrations of the test compound (10-300 ⁇ M) in complete medium were added to the remainder of the wells. After treatment, the plates were incubated at 37 0 C for 3 days.
  • the medium was removed from all the wells via a vacuum pump. The first and the last row of wells were left without addition of the test compound and were used as control wells. To the remainder of the wells appropriate concentrations of the test compound (20-400 ⁇ M) in complete medium were added into the wells. After treatment, the plates were incubated at 37 0 C for 3 days.
  • MTT MTT (1 mg/mL) was added to all the wells and the cells were further incubated for approximately 4 h at 37 0 C to allow sufficient time for it to interact with the cells. The medium was then carefully discarded and the cellular contents were extracted using DMSO (100 ⁇ L per well).
  • crystal violet blue assay the medium was removed from the plates and the cells were washed twice with saline (0.9% NaCl). To all the wells, crystal violet blue dye (1 mL in 5% PBS) was added and the mixture was left for ⁇ 1 min to stain the cells. The dye was then removed and the cells were extracted using a solution of propan-2-ol (1 mL).
  • Absorption at 595 nm was determined using an ELISA plate reader.
  • the percent survival was determined by the intensity of the absorbance obtained, which correlated to the amount of cells present in each well.
  • the negative control wells were arbitrarily assigned as 100% survival.
  • the MTT assay provides a measure only for viable cells.
  • Statistical analysis of data of all the cell work after quantification with the plate reader was achieved using Origin 6.1 software (Microcal Inc., 1999). Comparison of test compound cytotoxicities was analyzed using a two-tailed t test.
  • the Zn Indo complex was also compared against two cell lines Hep-2 and A549 and it was shown to have an equivalent effect on cell viability across the two cell lines (Figure 23).
  • the A-375 (human melanoma) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD).
  • the optimised seeding densities used in the studies on A-375 cells were at a density of 1500 cells/well.
  • the B 16 melanoma cell line was grown in Dulbecco's minimal essential medium supplemented with 10% foetal calf serum, glutamine and penicillin/streptomycin. Groups of 10 female C57B1 mice were shaved of dorsal hair and pretreated with a 0.1% ethanolic solution of either indomethacin or Culndo (50 ⁇ L) applied directly to the dorsal skin daily for 10 days.
  • the B 16 cells (5 x 10 4 suspended in 0.1 mL of serum- free medium) were injected intradermally into the shaved ventral skin, and the daily application of the drugs was continued while tumour appearance and growth was monitored. Tumour incidence indicates the percentage of mice in the group displaying palpable tumour growth at the inoculation site. The area of the tumours was calculated as the product of two perpendicular diameters, and the group average area was calculated. 5.3 Results
  • tumour areas were quite widely dispersed within each treatment group, but showed consistently that tumour growth was fastest in the control mice, and that Culndo appeared to have slowed tumour growth more effectively than IndoH (Figure 26).
  • squamous cell carcinoma may have some important clinical applications, especially when the tumour occurs on regions of the face where it is difficult to operate or where an operation could cause disfigurement. Studies against this type of tumour are also important since it is related to similar cancers of the epithelium, such as lung and breast cancers, that are difficult to treat with chemotherapy. In the results of this experiment, Culndo caused 100% tumour regression in a mouse model of the human disease. 6.2 Experimental
  • the T79 cell line was established from a squamous cell carcinoma induced with chronic solar simulated UV irradiation in the inbred Skh:hr-1 hairless mouse.
  • Groups of 10 female Skh:hr- 1 mice were pretreated on the dorsal skin with a 0.1 % ethanolic solution of either IndoH or Culndo (50 ⁇ L), which was applied directly by pipette to the dorsal skin daily for 10 days.
  • the T79 cells (10 5 cells in 0.1 mL of serum-free medium) were injected intradermally into the dorsal skin, and the daily application of the drugs was continued while tumour growth was monitored.
  • the T79 cell line is known as a "regressor" cell line that requires UV radiation- induced immunosuppression of the recipient mouse for continuing growth.
  • This study demonstrates the regression of the established tumour with time in the absence of immunosuppression in the control mice, and shows that Culndo, but not IndoH, facilitates this regression.
  • Culndo, but not IndoH-treated mice plateaus after 15 days, there are no longer any tumours remaining in the Culndo-treated mice. This shows the strong anti-cancer effect of Culndo on squamous cell carcinomas that are also closely related to lung cancers.
  • the early tumour growth enhancement by Culndo may be relevant to the efficacy of Culndo as an anti-cancer drug.
  • the inventors postulate the effectiveness of the drug is associated with the property of Cu in promoting angiogenesis. While this is generally considered to be undesirable in that it promotes tumour growth, one of the biggest issues associated with the treatment of solid tumours is the difficulty in delivering drugs to the interior of the tumour because of poor blood flow. For effective treatment of cancer, this is essential because the cancer cells that lead to metastases are those between the outer edge of the tumour and the necrotic core, and this is why most chemotherapies have limited success, i.e.
  • the angiogenic nature of the Cu in the compound promotes the growth of blood vessels into solid tumours and thereby provides a highly effective method of delivering the compound into the interior of the tumours.
  • Rats were orally administered (non-anaesthetized) 1% [Cu 2 (Indo) 4 (OH2)2] at 7 mg/kg body weight via oral gavage.
  • the control cohort was dosed solely with CMC
  • AU topical non-ionic formulations (10 g) were prepared in a water bath by heating (50-55 0 C), mixing Phase A consisting of the active ingredient, i.e., [Cu 2 (Indo) 4 (OH2)2] in propylene glycol or glycerol and mixing in the relevant oils.
  • Phase B (water) was heated (50-55 0 C) in a water bath and added to phase A mixing vigorously until cooled.
  • Treatment A Control group (i.e, nil treatment)
  • Treatments B, C, D and E were all cetomacrogol formulations differing only in the amount of propylene glycol or glycerol.
  • the lotion (D) is the most aqueous.
  • Treatment F White Soft Paraffin is the most hydrophobic base of the five formulations.
  • the % mean inhibition of rat paw oedema for the formulations is shown in Table 5.
  • the ANOVA statistical analysis showed all formulations were significantly and comparably anti-inflammatory compared to nil treatment.
  • the propylene glycol cream resulted in significantly less inflammation compared to controls when measured against either cetomacrogol lotion or white soft paraffin.
  • the propylene glycol cream resulted in the greatest inhibition of inflammation and white soft paraffin resulted in the least inhibition of inflammation. While cetomacrogol lotion and white soft paraffin were the least efficacious of the five formulations, both were still significantly anti-inflammatory compared to nil treatment. The results are shown graphically in Figure 28. The asterisk symbol denotes a significant anti-inflammatory response compared to nil treatment. Of all the formulations, the propylene glycol cream was the only formulation at two hours that resulted in significantly less inflammation compared to either cetomacrogol lotion or white soft paraffin.
  • cetomacrogol-based creams i.e., the cetomacrogol cream, glycerol cream and propylene glycol cream
  • the cetomacrogol cream resulted in the least gastric irritation, albeit all formulations afforded minimal gastric irritation ( ⁇ 20 mm 2 ).
  • the cetomacrogol lotion and the white soft paraffin were the least efficacious of the formulations and were not significantly antiinflammatory at three hours post treatment.
  • results further show activity of the test compound in terms of both maximum efficacy and time profiles can be controlled by the formulation used.
  • This is of relevance in different anti-cancer and chemopreventative applications where it may be desirable to achieve maximum residency time in the skin or for the metal complex to diffuse more rapidly through tissues, depending on the size and type of carcinoma and whether systemic, as well as topical, treatment was desirable.
  • the low GI effects which would be negligible when a larger animal (e.g. a dog or a horse) or a human were treated for skin lesions since the dose (mg/kg body weight) received would be below that needed to induce toxic side effects.
  • this concentrate was used to develop formulations that were suitable for injection into lesions, using an oil base to minimize diffusion, where the concentrate was diluted with various amounts of benzyl alcohol, before it was mixed with the oil.
  • the choice of formulation can result in the metal complex either having a localized effect where it is desirable to localize the drug into a lesion that has been injected with the metal complex, or a systemic effect that may be used to help control metastases. Again the extent to which treatment is localized or systemic can be optimised by changing the amount of oil and alcohol in the formulation.

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Abstract

There are described methods for the prophylaxis or treatment of carcinomas in a mammalian subject comprising administering a metal complex having anti-inflammatory activity to the mammal. Typically, the complex is a complex of at least one metal and at least one non-steroidal anti-inflammatory drug (NSAID). Suitable complexes include a complex of a metal and a carboxylate, or a derivative of a carboxylate, having anti-inflammatory activity.

Description

METHOD FOR THE PROPHYLAXIS OR TREATMENT OF CARCINOMAS
FIELD OF THE INVENTION
The invention relates to the prophylaxis and treatment of carcinomas in humans and animals. One or more embodiments of the invention may find application in the prophylaxis or treatment of one or more of basal cell carcinomas, squamous cell carcinomas, melanomas, colorectal cancer, breast cancer, lung cancer and other cancers of the epithelial tissues.
BACKGROUND
Non-steroidal anti-inflammatory drugs (NSAIDs) are used in the treatment of 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 oedema following surgical or non-surgical procedures. However, many NSAIDs cause adverse effects in humans and animals, particularly adverse gastrointestinal (GI) effects.
Indomethacin (IndoH) is a NSAID and is effective in treating inflammatory conditions in humans and animals. The structure of indomethacin is as follows:
Figure imgf000002_0001
However, indomethacin can cause severe adverse gastrointestinal effects in humans and animals, particularly when administered orally. In humans, oral administration of indomethacin can cause ulcerations in the oesophagus, stomach, duodenum and intestines, and some fatalities have been reported. In dogs, oral administration of indoniethacin causes fatal gastrointestinal haemorrhaging. Other effects associated with oral administration of indomethacin include: (a) inhibition of platelet aggregation, (b) 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, (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). Studies have also shown that administration of indomethacin by other routes, e.g. as a suppository or by topical application (e.g. Amico-Roxas, M.; Matera, M.; Caruso, A.; Puglisi, G.; Bemardi, R.; Rinaldo, G. Rivista Europeaper Ie Scienze Mediche e Farmacologiche 1982, 4, 199-204), also results in adverse effects.
These 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. For example, the oral administration of the dinuclear copper(II) complex of indomethacin, bis(N,N-dimethylformamide)tetrakis-μ-(O, O -Indo)dicopper(II) ([Cu2(Indo)4(DMF)2], Indo is the deprotonated form of indomethacin), has been found to cause less gastrointestinal toxicity than indomethacin. The mechanism of the reduced gastrointestinal toxicity has not been elucidated, but is believed to be due to reduced interaction of the indomethacin with the COX-I enzyme in the gastrointestinal tract. Compositions containing this complex sold under the name Cu- Algesic have been used in veterinary practice in Australia, New Zealand, South Africa and other countries. These compositions are in the form of a tablet or a paste.
Complexes of copper and dithiocarbamate have previously been studied for the treatment of melanoma. The mechanism proposed for the anti-melanoma effect of such complexes is that both the copper and the ligand affect the intracellular redox status, which makes melanoma cells more susceptible to these drugs because of the susceptibility of melanoma cells to apoptosis induced by reactive oxygen species. However, it appears that copper is not essential for the anti-melanoma effect as complexes of the redox-inactive Zn(II) and dithiocarbamate were also found to be effective (Cen, D.; Gonzalez, R. L; Buckmeier, J. A.; Kahlon, R. S.; Tohidian, N. B.; Meyskens, F. L., Jr. MoI. Cancer Ther. 2002, 1, 197-204). It therefore seems that the presence of the dithiocarbamate Hgand is necessary for the complex to exhibit anti- melanoma effects. In mouse models of melanoma no response to indomethacin was observed (Indomethacin and telomerase activity in tumor growth retardation. Lonnroth, C; Andersson, M.; Lundholm, K. Int. J. Oncology 2001, 18, 929-937). A minimal effect of indomethacin on human melanoma lung metastases in nude mice models has been reported, although a combination of interleukin-2 (IL-2) with indomethacin led to a cure of the metastases in some cases (Cure of human melanoma lung metastases in nude mice with chronic indomethacin therapy combined with multiple rounds of IL-2: characteristics of killer cells generated in situ. LaIa, P. K.; Elkashab, M.; Kerbel, R. S.; Parhar, R. S. Int. Immunol. 1990, 2, 1149-1158).
A range of human clinical trials have been conducted on the effects of indomethacin in combination therapy for the treatment of cancer (IL- 1 /indomethacin, IL-2/indomethacin, or IL-2/ranitidine/indomethacin). While there is some conflict as to whether there is any benefit of indomethacin in these clinical trials, the majority of the trials conclude that the benefit is marginal and that it contributes to adverse side effects such as renal toxicity (Phase II trial of interleukin-1 alpha and indomethacin in treatment of metastatic melanoma. Janik, J. E.; Miller, L. L.; Longo, D. L.; Powers, G. C; Urba, W.
J.; Kopp, W. C; Gause, B. L.; Curti, B. D.; Fenton, R. G.; Oppenheim, J. J.; Conlon, K. C; Holmlund, J. T.; Sznol, M.; Sharfman, W. H.; Steis, R. G.; Creekmore, S. P.; Alvord, W. G.; Beauchamp, A. E.; Smith, J. W., 2nd. J. Natl. Cancer Inst. 1996, 88, 44-49; Sustained indomethacin and ranitidine with intermittent continuous infusion of interleukin-2 in advanced malignant melanoma: a phase II study. Mertens, W. C; Bramwell, V. H.; Banerjee, D.; Gwadry-Sridhar, F.; LaIa, P. K. Clin. Oncol. (Roy. College Radiol. (Gt Brit.)) 1993, 5, 107-113; Indomethacin, ranitidine, and interleukin-2 in melanoma. Hamblin, T. J. Lancet 1992, 340, 8826; Marginal effect and interfered with treatment Randomized trial of recombinant alpha 2b-interferon with or without indomethacin in patients with metastatic malignant melanoma. Miller, R. L.; Steis, R. G.;
Clark, J. W.; Smith, J. W. 2nd; Crum, E.; McKnight, J. E.; Hawkins, M. J.; Jones, M. J.; Longo, D. L.; Urba, W. J. Cancer Res. 1989, 49, 1871-1876; Repetitive weekly cycles of interleukin-2. II. Clinical and immunologic effects of dose, schedule, and addition of indomethacin. Sosman, J. A.; Kohler, P. C; Hank, J. A.; Moore, K. H.; Bechhofer, R.; Storer, B.; Sondel, P. M. J. Natl. Cancer Inst. 1988, 80, 1451-1461).
NSAIDs, including indomethacin and related NSAIDs, have been reported to produce a chemoprotective effect against colorectal cancers (Turchanowa, L.;
Dauletbaev, N.; Milovic, V.; Stein, J. Eur. J. Clin. Invest. 2001, 31, 887-893) and to enhance the anti-cancer activities of known anti-cancer drugs (Touhey, S.; O'Connor, R.; Plunkett, S.; Maguire, A.; Clynes, M. Eur. J. Cancer 2002, 38, 1661-1670). However, in some models of colorectal cancer, indomethacin actually increased mortality and increased metastases compared to control animals (Danzi, M.; Ferulano, G. P.; Abate, S.; Califano, G. Carcinogenesis 1984, J, 287-289). Although other studies have reported anti-cancer activity in chemically induced colorectal cancers in rats, no such effect was found when a cultured cell line was injected into rats (Olsson, N. O.; Caignard, A.; Martin, M. S.; Martin, F. Int. J. Immunopharmac. 1984, 6, 329-334). The evidence from epidemiological studies about the effectiveness of NSAIDs that are non-selective COX inhibitors is variable, although there is evidence that indomethacin has a chemopreventative effect (Collet, J.-P.; Sharpe, C; Belzile, E.; Boivin, J.-F.; Hanley, J.; Abenhaim, L. Brit. J. Cancer 1999, 81, 62-68). More recent research shows that advanced solid tumour patients treated with indomethacin survive twice as long as do such patients who receive supportive care alone (Blanke, C. D. Oncology (Williston Park,
N. Y.) 2002, 16 (4 Suppl 3)).
Cu-salicylate complexes, [Cu2(3,5-di-rso-propylsalicylate)4L2], have been shown to have a limited effect (no-statistically significant difference) in tumorogenisis in female C3H/HeNCR mice models of mammary cancer (Crispins, Jr., C. G.; Sorenson, J. R. J. Anti-Cancer Res. 1992, 12, 1271-1273). These complexes had anti-cancer activity against reticulum cell sarcoma in SJL/J mice if injected subcutaneously but were toxic if injected via the i.p route (Crispins, Jr., C. G.; Sorenson, J. R. J. Anti-Cancer Res. 1988, 8, 77-79). While the literature indicates Cu-salicylate complexes may have anti-cancer activity, such complexes are unstable when exposed to biological fluids and so have the normal toxicity of the parent organic drug. More efficacious complexes and improved delivery modes are needed for the treatment of cancer. SUMMARY OF THE INVENTION
In a first aspect of the present invention there is provided a method for the prophylaxis or treatment of a carcinoma in a mammalian subject, the method 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, the carboxylate or derivative being other than salicylate or a derivative of salicylate.
As used herein, the term "carcinoma" refers to a malignant neoplasm of the internal or external lining (epithelium) of the human or animal body. "Carcinoma" includes basal cell carcinomas, squamous cell carcinoma, melanoma, colorectal cancer, breast cancer and lung cancer. Carcinomas account for 80-90% of all cancer cases in humans.
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 may be hydrolysed in vivo, and the hydrolysed compound may have anti-inflammatory activity. The derivative of the carboxylate maybe 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 a carcinoma in a mammalian subject, the method 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 may 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:
Suprofen ((+)-α-methyl-4-(2-thienylcarbonyl)phenylacetic acid ("SupH")); Tolmetin (l-methyl-5-(p-toluoyl)-lH-ρyrrole-2-acetic acid ("ToIH")); Naproxen (6-methoxy-α-methyl-2-naphthaleneacetic acid ("NapH")); Ibuprofen ((+)-α-methyl-4-(isopropylmethyl)benzeneacetic acid ("IbuH")); Flufenamic Acid ((N-trifluororaethylphenyl)anthranilic acid ("FlufenH"));
Niflumic Acid ((2-(3-trifluoromethyl)phenylamino)-3-pyridinecarboxylic acid ("NifH")); Diclofenac (2-[(2,6-dichlorophenyl)amino]ρhenylacetic acid ("DicH"));
Indomethacin (l-(4-chlorobenzoyl)-5-methoxy-2-methyl-lH-indole-3-acetic acid ("IndoH")); and
Acemetacin (l-(4-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid carboxyniethyl ester ("ACMH"))..
In this specification, the inclusion of the "H" at the end of an abbreviation for a carboxylate (e.g., any one of the carboxylic acid listed above) or 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. Preferably, the carboxylate, or hydroxamate, hydroximate, ester or amide derivative having anti-inflammatory activity is a non-steroidal anti-inflammatory drug (NSAID). For example, the NSAID may be indomethacin (IndoH), or an ester derivative of indomethacin such as acemetacin, or a hydroxamate or amide derivative of indomethacin or acemetacin. Acemetacin, 1 -(4-chlorobenzoyl)-5-methoxy-2-methylindole-3 -acetic acid carboxymethyl ester, is a glycolic acid ester of indomethacin.
Figure imgf000008_0001
Acemetacin Indomethacin
The complex of the metal and a carboxylate, or hydroximate, hydroxamate, ester or amide derivative may be any complex comprising at least one metal ion and at least one carboxylate, or hydroxamate, hydroximate, ester or amide derivative having antiinflammatory activity. As will be apparent to a person skilled in the art, the carboxylate or hydroximate, hydroxamate, ester or amide derivative is coordinated with the metal ion via the carboxylate, hydroximate, hydroxamate, amide and other groups attached to the amide or ester linkages.
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 carboxylate, or hydroxamate, hydroximate, ester, or amide ligands having anti-inflammatory activity.
Typically, the complex includes other ligands in addition to the carboxylate, or hydroximate, hydroxamate, ester or amide ligands having anti-inflammatory activity, and these other ligands may 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). Metal/carboxylate complex Structure
[Ma 2(Sup)4(CH3CN)2] ° Dimer
[Ma 2(Sup)4(OH2)2]β Dimer
[Ma(Tol)2(pyridine)2] b Monomer [Ma 2(Tol)4(DMSO)2]δ'c Dimer
[Ma(Nap)2(pyridine)2] d Monomer [Ma 2(Nap)4(DMSO)2] c'd Dimer
[Ma(Ibu)2(pyridine)2] e Monomer [Ma 2(Ibu)4(DMSO)4] c'e Dimer
[Ma(Ibu)2(imidazole)2] e Monomer
[Ma(Ibu)2(2-methylimidazole)2] e Monomer
[Ma 2(Ibu)4(caffeine)2] e Dimer
[Ma 2(Ibu)4(metronidazole)2] e/ Dimer
[Ma 2(Flufen)4L2]g where each L is independently selected and Dimer is caffeine or papaverine.
[Ma(Flufen)2L2],s where each L is independently selected and Monomer is nicotine, nicotinamide or N,N-diethylnicotinamide.
[Ma(Νif)2L2] '' where each L is independently selected and Monomer, Polymer is 3-ρyridylmethanol, or water.
[Ma 2(Nif)4(DMSO)2] c'" Dimer
[Ma 2(Indo)4L2],' where each L is independently selected and Dimer is water, N^N-dimethylacetamide, N-methyl-2-pyrrolidone, tetrahydrofuran, acetonitrile, acetone, or dimethylsulfoxide
Figure imgf000009_0001
where each L is independently selected and Dimer is water, ethanol, dimethylsulfoxide or methanol Where a Suprofen = (+)-α-methyl-4-(2-thienyl-carbonyl)phenylacetic acid (SupH); b Tolmentin = l-methyl-5-(p-toluoyl)-l/7-pyrrole-2-acetic acid (ToIH); c DMSO = dimethylsulfoxide; d Naproxen = β-methoxy-α-methyl^-naphthaleneacetic acid (NapH); e Ibuprofen = (+)-α-methyl-4-(isopropylmethyl)benzeneacetic acid (IbuH);
•^Metronidazole = 2-methyl-5-nitrobenziniidazole s Flufenamic Acid = (iV-trifluoromethylphenyl)anthranilic acid (FlufenH); h Niflumic Acid = 2-((3-trifluoromethyl)phenylamino)-3-pyridinecarboxylic acid (NifH);
' 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
Ma is a metal ion and preferably, a transition metal ion.
Preferably, Ma will be copper ion. In other embodiments, the complex is any one of the complexes referred to in the table above where the metal ion is a transition metal ion other than copper (eg zinc, nickel, or cobalt ions, and preferably zinc, 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 = N1N- 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, 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:
Figure imgf000011_0001
wherein:
R1 is H or halo (i.e. Cl, F, Br or I);
R2 is H; a Cj to C6 alkyl, an alkenyl or an alkynyl, where the C1 to C6 alkyl, alkenyl or alkynyl may be optionally substituted; or
Figure imgf000011_0002
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 Ci 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 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.
When R2Ais a C1 to C6 alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or an arylalkyl, the Cj 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, -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.
The complex may be a complex of formula (1), (2) or (3):
[M(L2)2Lm]p (1)
where M is a divalent or trivalent metal ion, L is a ligand of the formula:
Figure imgf000012_0001
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
Figure imgf000013_0001
wherein each R2Λ 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 1 or 2; and p is the charge of the complex;
[M2(L^4L1n]P (2)
where each M is independently selected and is a divalent or trivalent metal ion;
L is a ligand of the formula:
Figure imgf000013_0002
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 Ci to C6 alkyl, alkenyl or alkynyl may be optionally substituted; or
Figure imgf000014_0001
wherein each R2Λ 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;
[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:
Figure imgf000014_0002
wherein:
R1 is H or halo (i.e. Cl, F5 Br or I);
R2 is H; a Ci to C6 alkyl, an alkenyl or an alkynyl, where the Ci to C6 alkyl, alkenyl or alkynyl may be optionally substituted; or
Figure imgf000015_0001
wherein each R2Λ is independently selected from the group consisting of H, Cj 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.
In formulas (1), (2) and (3), when R2 is a C1 to C6 alkyl, an alkenyl or an alkynyl, the Ci 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 R2A is a C1 to C6 alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or an arylalkyl, the Cj 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, -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.
R1 is typically H. R3 is typically H.
R2 is typically CH3.
In 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, ΛζTV-dimethylformamide (DMF), N-methylpyrrolidone, dimethylsulfoxide or Λζ,N-dimethylacetamide (DMA).
The complexes of formula (1), (2) or (3) maybe 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 formula (4):
[M(L1^L1n]13 (4)
where M is a divalent or trivalent metal ion,
L1 is a ligand of the formula:
Figure imgf000017_0001
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
Figure imgf000017_0002
wherein each R2A 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, 0 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 5 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;
In some embodiments, the mononuclear complex of formula (4) is a complex of O formula (4A):
[M(^-U)2Uf (4A)
where M is a divalent or trivalent metal ion; "η2-L1" is a bidentate ligand of the formula L1:
Figure imgf000018_0001
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 Cj to C6 alkyl, alkenyl or alkynyl may be optionally substituted; or
Figure imgf000018_0002
wherein each R2A is independently selected from the group consisting of H, C1 to C6 alkyl, alkenyl, alkynyl, aryl, cycloalkyl and arylalkyl, where the Ci 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.
In formulas (4) and (4A), when R2 is a Ci to C6 alkyl, an alkenyl or an alkynyl, the Cj 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 R2Ais a C1 to C6 alkyl, an alkenyl, an alkynyl, an aryl, a cycloalkyl or an arylalkyl, the Ci 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, -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 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):
[M2(μ-Lι)4Lm]p (5)
wherein each M is independently selected and is a divalent or trivalent metal ion, μ-L1 is a ligand of the formula L :
Figure imgf000019_0001
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
Figure imgf000020_0001
wherein each R2A 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 0, 1 or 2; and p is the charge of the complex.
In formula (5), 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 formula (5), when R2Ais 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):
[W3O(μ-Lι)6UT (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; 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:
Figure imgf000022_0001
Figure imgf000022_0002
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
Figure imgf000022_0003
wherein each R2A 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. In the complex of formula (7), M may, 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 may be 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 a monodentate or a polydentate ligand; m is 1, 2, 3 or 4 n is 0, 1, 2, 3, 4 or 5; and p is the charge of the complex.
In some embodiments of the complex of formula (8), L6 is NH3 or a monodentate, polydentate, or macrocyclic amine ligand, such as
Figure imgf000023_0001
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), Ru(III) or Pt(IV); or [M(O2R1)m(NR2R3R5)(4-m)]p where M is Pd(II), Pt(II) or Au(III) and more preferably Pt(II); or [M(O2R1)(NR2R3R5)3]+ or cϊ5 orJrαM-[M(O2R1)2(NR2R3R5)2]+, and R1, R2 R3 and R5 are as defined in any one of formulae (1) to (7) above.
In still other embodiments, the metal complex may be a complex of the following formula (9):
Figure imgf000023_0002
wherein M is a monovalent, divalent, trivalent, tetravalent, pentavalent or hexavalent metal ion; each L7 is independently selected and is a monodentate or a polydentate ligand; each L is independently selected and is a chelating derivative of a carboxylate having anti-inflammatory activity, such as a hydroximate, hydroxamate, amine acid, peptide or sugar or amide NSAID chelating ligand (O or N bound) with antiinflammatory 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 complex of formula (9), L7 is NH3 or a monodentate, polydentate, or macrocyclic amine ligand.. Preferably, L7 is [M(NR2R3R5)(6-2n)(L8)n]p when L8 is a bidentate ligand, [M(NR2R3R5)3(L8)]P when L8 is a tridentate ligand, or [M(NR2R3R5)2(L8)]P when L8 is a tetradentate ligand, where M is selected from Co(III), Cr(III), Ir(III), Os(III), Rh(III), Ru(III), Ru(II) or Pt(IV) and more preferably, Co(III), Ru(III), Ru(II) or Pt(IV), and Rl, R2, R3 and R5 are as defined in any one of formulae (1) to (7) above. Alternatively, L7 is [M(NR2R3R5)2(L8)]P when L8 is a bidentate ligand or [M(NR2R3R5)(L8)f when L8 is a tridentate ligand, where M is Cu(II), Ni(II), Pd(II), Pt(II) or Au(III) and more preferably Cu(II) or Pt(II), and Rl, R2, R3 and R5 are as defined in any one of formulae (1) to (7) above . In other embodiments of formula (9) the complex is [M(L8)n]p. In this instance, when L8 is a bidentate ligand n is 3, or when L8 is a tridentate ligand n is 2, where M is preferably Co(III), Cu(II), Ga(III), Pt(IV), 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, where M is preferably Cu(II), Ni(II), Pd(II), Pt(II) or Au(III) and more preferably Cu(II) or Pt(II).
In still other embodiments, the metal complex may be a complex of the following formula (10):
[M(L5)0(L7)ra(L8)nf (10) 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 having anti-inflammatory activity or amide ligand (O or N bound) with 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.
One or more of the ligands L5 and L8 in any combination may also form dimeric, trimeric, tetrameric, oligomeric or polumeric complexes with one or more metal ions.
In all of the formula (I)-(IO) or other complexes containing NSAID ligands and/or derivatives thereof, the ancilliary ligands may be chosen from ligands that exert a separate anti-cancer activity, such as dithiocarbamates.
The carcinoma may be a lesion or tumour of the epithelium. The lesion may, for example, be a skin lesion such as basal cell carcinoma, squamous cell carcinoma or melanoma. The carcinoma may also be other cancers of the epithelium, such as lung cancer and cancer of the oesophagus. The complex may be administered to the mammalian subject systemically or may be locally applied to the site of the carcinoma. The complex may also be administered to the mammalian subject by both methods concurrently. The complex may be topically applied to a skin cancer. If the carcinoma is a lung cancer, the complex may be administered to the cancer site by inhalation of the complex. If the carcinoma is a cancer of the nose, the complex may be administered by nasal spray. If the carcinoma is a cancer of the gastrointestinal tract, including those of the mouth, oesophagus, stomach, small intestine and colorectal regions, the complex may be administered to the cancer site by oral administration (including buccal or sublingual administration for mouth cancers) or by suppository for colorectal cancers. For any carcinoma, the complex may be administered by direct injection into the lesion or organ containing the lesion.
The complex may also be administered to the mammalian subject in combination with other anti-cancer drugs or cancer treatments. Such administration may be given to improve the response to chemotherapy, or to reduce the dose of a chemotherapeutic agent to reduce toxic side-effects while maintaining a comparable efficacy.
The present invention also provides the use of a complex of a metal and a carboxylate, or derivative of a carboxylate, having anti-inflammatory activity in the manufacture of a medicament for the prophylaxis or treatment of a carcinoma in a mammalian subject.
The present invention further provides the use of a complex of a metal and a carboxylate, or derivative of a carboxylate, having anti-inflammatory activity for the prophylaxis or treatment of a carcinoma in a mammalian subject.
All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like that 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 mm 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(Indo)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(ItIdO)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(MdO)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(bido)4(DMF)2] {n = 5, Mean±SEM);
Figure 10 is a graph showing TV-acetyl-β-D-glucosaminidase (NAG) concentration in urine collected for 0-24 h after dosing of rats with equimolar Lido doses of IndoH or [Cu2(Indo)4(DMF)2] in 2% w/w CMC compared with control rats (n = 4; Mean±SEM);
Figure 11 shows photographs of a control rat colon (left panel) and azoxymethane-induced aberrant crypt focci (ACF) (right panel) in the colon of a rat treated with azoxymethane (AOM) at doses of 15 mg/kg for four weeks then sacrificed 6 weeks after the final injection of AOM;
Figure 12 shows graphs indicating the effects observed in rats following treatment 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, on AOM-induced ACF in rats
(n = 3-5; Mean±SEM). Upper panel: control; middle panel: IndoH 3.00 mg/kg per day for 28 consecutive days; lower panel: [Cu2(Indo)4(DMF)2] 3.81 mg/kg per day for 28 consecutive days. The rats received 15 mg/kg of AOM for four weeks while the rats were being dosed with the drug or the CMC control and the rats were sacrificed 6 weeks after the final dose of AOM;
Figure 13 is a graph of viable cell number (% of control) in in vitro cytotoxicity assays of IndoH and [Cu2(Indo)4(DMF)2] against cultured HCT-116 cells (n = 5; Mean±SEM). The concentration scale is the equivalent dose of Indo;
Figure 14 is a graph of viable cell number (% of control) in in vitro cytotoxicity 5 assays of IndoH and [Cu2(IrIdO)4(DMF)2] against cultured Hep G2 (human hepatoma) cells (n = 6; Mean±SEM). The log concentration scale is the equivalent dose of Indo;
Figure 15 is a graph showing the effect of IndoH suspended in medium chain triglyceride (MCT) oil on the viability of A549 cells;
Figure 16 is a graph showing the effect of IndoH suspended in MCT oil on the 0 viability of A2780 cells;
Figure 17 is a graph showing the effect of Cu-Indo suspended in MCT oil on viability of A549 cells, where the concentration range refers to the equivalent concentration of Indo and not the copper complex;
Figure 18 is a graph showing the effect of Cu-Indo suspended in MCT oil on the 5 viability of A2780 cells, where the concentration range refers to the equivalent concentration of Indo and not the copper complex;
Figure 19 is a graph showing the effect of ACM, CuACM and ZnACM suspended in MCT oil on the viability of A549 cells, where the concentration range refers to the equivalent concentration of Indo and not the zinc or copper complexes; O Figure 20 is a graph showing the effect of aspirin and [Cu(asp)2(3-pic)2] suspended in MCT oil on the viability of A549 cells, where the concentration range refers to the equivalent concentration of aspirin and not the copper complex; Figure 21 is a graph showing the effect of ibuprofen and [Cu(ibup)2(2-Meim)2] suspended in MCT oil on the viability of A549 cells, where the concentration range refers to the equivalent concentration of ibuprofen and not the copper complex;
Figure 22 is a graph showing the effect of [VvO(IndoHAH)(IndoHA)] and IndoHAH2 suspended in MCT oil on the viability of A549 cells, where the concentration range refers to the equivalent concentration of IndoHA and not the copper complex;
Figure 23 is a graph showing the effect of [Zn(Indo)2(OH2)2] suspended in MCT oil on the viability of A549 and Hep-2 cells, where the concentration range refers to the equivalent concentration of Indo and not the zinc complex; Figure 24 is a graph of viable cell number (% of control) in in vitro cytotoxicity assays of IndoH and [Cu2(Indo)4(DMF)2] against cultured A375 human melanoma cells (n = 6; Mean±SEM). The log concentration scale is the equivalent dose of Indo;
Figure 25 is a graph of the incidence of tumours (%) in C57BI mice (« = 10) administered with equivalent doses of IndoH and [Cu2(Indo)4(DMF)2] (daily doses of 50 μL of 0.1% Indo concentrations in ethanolic solutions 10 days prior to and daily during the monitoring period after the injection of cultured B16 melanoma cells);
Figure 26 is a graph showing the effects of equivalent doses of IndoH and [Cu2(Indo)4(DMF)2] (daily doses of 50 μL of 0.1% Indo concentrations in ethanolic solutions 10 days prior to and daily during the monitoring period after the injection of cultured B 16 melanoma cells) on the tumour area in female C57BI mice (n = 10);
Figure 27 is a graph showing the effects of equivalent doses of IndoH and [Cu2(Indo)4(DMF)2] (daily doses of 50 μL of 0.1% Indo concentrations in ethanolic solutions 10 days prior to and daily during the monitoring period after the injection of cultured T79 UV-induced squamous cell carcinoma cells) on the tumour area in female Skh:hr-l mice (« = 10);
Figure 28 is a graph showing the mean increase in rat paw volume at 2 hours post treatment with 1% [Cu2(mdo)4(OH2)2] topical non-ionic formulations: A = nil treatment; B = Cetomacrogol Cream; C = Glycerol Cream; D = Propylene Glycol Cream, E = Cetomacrogol Lotion, F = White Soft Paraffin, compared to control (Treatment A = nil treatment);
Figure 29 is a graph showing mean increase in rat paw volume 3 hours post treatment with 1% [Cu2(Indo)4(OH2)2]; the labels are the same as in Figure 26; and Figure 30 is a graph showing mean increase in rat paw volume 4 hours post treatment with [Cu2(Indo)4(OH2)2]; the labels are the same as in Figure 26.
DETAILED DESCRIPTION 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-rnethylimidazole; a reference to "Asp" is a reference to aspirin (acetylsalicyclic acid), 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 "InCIoHAH2" 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 ΛyV-dimethylacetamide; "DMSO" refers to dimethylsulfoxide; "NMP" refers to iV-methylpyrrolidone; and "DMF" refers to iV,N-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, iso-propyl, butyl, iso-bvΛyl, sec-butyl, tert-butyl, amyl, wø-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. hi 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 C2o alkenyl, more preferably a C2 to C6 alkenyl. Examples of alkenyl include vinyl, allyl, 1-methylvinyl, butenyl, wo-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 C20 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 unsymmetric bidentate ligands with one weaker and one relatively stronger bond to the metal atom, hi this specification, the term "monodentate ligand" refers to a ligand having a single co-ordination bond with a metal atom.
The present invention in one or more embodiments provides a method for the prophylaxis or treatment of carcinomas in a mammalian subject, the method comprising the administration to the subject of a therapeutically effective amount of a complex of a metal and a carboxylate, or a hydroximate, hydroxamate, ester or amide derivative having anti-inflammatory activity.
The present inventors have surprisingly found that in one or more embodiments of the invention, complexes of a metal and a carboxylate, or derivative of a carboxylate, having anti-inflammatory activity have application in preventing or treating carcinomas including skin cancers and colorectal cancers, and for skin cancers may be more effective in preventing or treating the carcinomas in terms of efficacy and/or safety than the carboxylate or the derivative of the carboxylate itself. For example, the present inventors have found that complexes of a metal and indomethacin are more effective in preventing or treating carcinomas, in terms of efficacy and/or safety, than indomethacin itself, even in cases when IndoH is completely inactive. This enhanced activity is substantially consistent across a range of cancer cell lines including melanoma, colorectal, gastric, hepatic, ovarian and non-small lung cancer lines. While studies have indicated that IndoH itself has some anti-cancer activity in carcinomas (especially colorectal cancers), which is believed to be due to a range of effects including inhibition of the COX enzymes that is upregulated in cancer cells (Vane, J. R.; Bakhle, Y. S.; Botting, R. M. Annu. Rev. Pharmacol. Toxicol. 1998, 38, 97-120) and reduction of angiogenesis, it is surprising that a complex of Indo and a metal such as copper(II) is more effective in preventing or treating carcinomas than indomethacin. This is surprising since it has been shown that the reduction of availability of copper by treatment with copper chelators has an anti-cancer effect due to reduction of angiogenesis
(Brewer, G. J. Curr. Opinion Chem. Biol. 2003, 7, 207-212), which would counteract the anti-cancer effect of IndoH that results from a reduction in angiogenesis. The discovery by the inventors that copper complexes of indomethacin exhibit a strong anti-cancer effect, that is often much stronger than indomethacin alone, is counterintuitive. This suggests that there might be a completely different reason to explain the anti-cancer activity of copper chelators.
' Without wishing to be bound by theory, it is the hypothesis of the inventors that copper complexes formed in vivo from the chelation of copper by the copper chelators may themselves have a strong anti-cancer effect rather than the anti-cancer effect being due solely to the removal of copper in addition to the reduction in angiogenesis when Cu is sequestered. More particularly, the efficacy of the complexes is considered to be due to a combination of both the ligand (i.e., carboxylate, or hydroximate, hydroxamate ester or amide derivative) and the metal atom.
Complexes of a metal and a carboxylate, or hydroximate, hydroxamate, ester or amide derivatives thereof having anti-inflammatory activity, such as copper/indomethacin and zinc/indomethacin complexes, have not previously been shown to be useful in the general prophylaxis or treatment of carcinomas. 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(L!)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).
Figure imgf000033_0001
Figure imgf000034_0001
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 = 6-methoxy-α-methyl-2-naphthaleneacetic acid (NapH); e Ibuprofen = (+)-α-methyl-4-(isopropylmethyl)benzeneacetic acid (IbuH);
■^Metronidazole = 2-methyl-5-nitrobenzimidazole
8 Fhifenamic Acid = (iV-trifluoromethylplienyl)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);
J Diclofenac = 2-[(2,6-dichlorophenyl)amino]phenylacetic acid (DicH).
The complexes listed above may be prepared by methods known in the art, or prepared by methods described below. Methods known in the art are described in, for example, US Patent No. 5,466,824 or the paper: Anti-inflammatory Dimiclear Copper(II) Complexes with Indomethacin. Synthesis, Magnetism and EPR Spectroscopy; Crystal Structure of the N,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.
The complex may be a complex comprising at least one metal ion and at least one hydroximate, hydroxamate, ester or amide derivative of a carboxylate having anti- inflammatory activity.
The hydroxamate or amide having anti-inflammatory activity may be the hydroximate, hydroxamate, ester or amide derivative of a carboxylic acid NSAID.
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 form chelates of deprotonated amides or amide monodentate complexes with a metal ion in the complex.
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 with 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 hydroximate and hydroximato in the context of the present invention are interchangeable. In scheme 1 , RCO2H is the parent carboxylic acid with anti-inflammatory activity.
Figure imgf000036_0001
R = Alk 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.
Figure imgf000037_0001
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.
ROH + coupling agent
Figure imgf000037_0003
Figure imgf000037_0002
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- 1 ,2-benzothiazine-3-carboxamide-l ,1 -dioxide), tenoxicam (4-hydroxy-2-methyl-N-2- pyridinyl-2H-thieno(2,3-e)-l,2-thiazine-3-carboxamide-l,l-dioxide) and meloxicam. In some embodiments, the complex is a complex of the formula (A):
[M(L4)nLm]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 NS AIDs 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 maybe 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 L2H (where L2 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. 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 a neutral ligand. However, in some embodiments, a complex of formula (1) or (2) may have a charge, for example, p may be 1~ or T.
Complexes of formula (3) have a charge of I+ 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", Y, 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]13 (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]:
Figure imgf000040_0001
Mononuclear metal complexes with the ligand L1
In some embodiments, the complex is a mononuclear complex of the following formula (4):
Figure imgf000040_0002
as defined above.
In further embodiments, the mononuclear complex of formula (4) is a complex of formula (4A):
[M(^-V)2L2? (4A) as defined above.
The complex of formula (4) maybe charged or neutral. Preferred complexes include [Cu(Indo)2(Im)2] and [Zn(Indo)2(OH2)2].nH2O. 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 T) 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 Y'^M^-L1)^] (4C)
wherein M, η -L 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 I+ 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 a co-pending International Patent Application in the name of Medical Therapies Limited entitled "Copper complexes" filed 24 March 2006 and claiming priority from Australian Provisional Patent Application No. 2005901464, the contents of which is 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 JV-heterocyclic group. Ligands containing an N-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 may 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 L1H when administered topically.
Dinuclear metal complexes with the ligand L1
In other embodiments, the complex is a dinuclear complex of the formula (5):
[M2(μ-L1)4Lm]p (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:
Figure imgf000043_0001
or a molecule of a cyclic tertiary amide (iV-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, N,N-dimethylformamide, N.N-dirnethylacetamide or iV-methylpyrrolidone.
An example of a complex of formula (5) is a dinuclear complex of the formula (5A):
[M^-L1)^ (5A)
wherein μ-L1 is a ligand of the formula L1:
Figure imgf000043_0002
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 0 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 cancer cell (i.e. there is greater absorption of the complex than IndoH). The present inventors further believe that both M and indomethacin are effective in inducing cancer cell apoptosis via different mechanisms, 5 leading to significantly enhanced anti-cancer activity than due to either M alone or indomethacin alone. In addition, the present inventors believe that the role of M in inducing angiogenesis may be important in delivering the bioactive components of the complex to the core of the tumour, which assists in the more effective treatment of solid tumours than the anti-inflammatory drug alone. In addition, the greatly reduced adverse O effects of the complex compared with the free indomethacin (such as gastrointestinal and particularly, the newly discovered reduction in renal effects on oral administration) enables 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], 5 [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(Indo)4( AN)2], [Cu2(Indo)4(OH2)2], [Zn2(Indo)4(DMA)2], [Zn2(Indo)4(NMP)2] and [Zn2(Indo)4(Py)2], wherein NMP is N-methylpyrrolidone.
A preferred complex is [Cu2(Indo)4(OH2)2].nH2O, wherein n is the number of waters of crystallisation. The number of waters of crystallisation will vary depending on 0 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 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; 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(Indomethacm)4(L2)] (L = iV,N-dimethylacetamide, Pyridine, 1-Methyl- 2-pyrrolidinone) and [Zn(Indomethacin)2(L!)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
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).
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:
Figure imgf000046_0001
Figure imgf000046_0002
wherein:
R1 is H or halo (i.e. Cl, F, Br or I);
R2 is H; a Cj to C6 alkyl, an alkenyl or an alkynyl, where the C1 to C6 alkyl, alkenyl or alkynyl may be optionally substituted; or
Figure imgf000046_0003
wherein each R 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.
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 0 of halo, -OH, -COOH and -NH2.
In formula (7), when R2A 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, 5 -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 consisting of halo, -OH, -COOH and -NH2. 0 R1 is typically H.
R3 is typically H.
R2 is typically CH3.
In some embodiments, L is ACM.
In formula (7), M may be selected from the group consisting of zinc, cobalt, 5 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, quinolyl, piperidinyl, pyridinyl, 2-methylpyridinyl, imadazoyl, pyranyl, pyrrolyl, pyrimidinyl, O indolyl, purinyl and quinolizinyl. Preferably, the heterocyclic base is quinolyl.
As used herein, the term "therapeutically effective amount" means an amount effective to yield a desired therapeutic response, for example, to prevent or treat a carcinoma or to provide a chemopreventative effect to reduce the incidence or reoccurrence of cancer caused by carcinomas. The metal may be administered alone or co-administered in combination with one or more chemotherapeutic or anti-cancer agents conventionally used in the treatment of carcinoma. By "co-administered" is meant simultaneous administration in the same formulation or a plurality of formulations by the same or different routes, or sequential administration by the same or different routes. By "sequential" administration is meant one is administered one after the other. Such agents include metal and non-metal based drugs. The conventional metal based drugs may be organic, inorganic, mixed ligand co-ordination compounds or chelates, including complexes of platinum and palladium. Examples of platinum based chemotherapeutic drugs include cisplatin (cw-diamminedichloroplatinum(II), oxaliplatin ([Pt((lR),(2R)- cyclohexane-l,2-diamine)(oxalato)] complex, and carboplatin (cis- diammine(cyclobutane- 1 , 1 -dicarboxylato)platinum(II) . Examples of non-metal chemotherapeutic drugs include paclitaxel, gleevac, docetaxel, taxol, 5-fluorouracil, doxorubicin, cyclophosphamide, vincristine (Oncovin), vinblastine, vindesin, camplothecin, gemcitabine, adriamycin, and topoisomerase inhibitors such as irinotecan (CPT-11). As will be understood, the use of a metal complex in one or more methods embodied by the invention in combination with another anti-cancer agent may enhance the effectiveness of the other anti-cancer drug. This may include the treatment of both carcinomas that are susceptible to the other drug and carcinomas that are otherwise resistant to the other drug. Accordingly, the present invention expressly extends to such combination treatments.
Further examples of metal complexes and methods for their preparation are described in the present applicant's co-pending International Patent Application filed 24 March 2006 claiming priority from Australian Provisional Patent Application
No. 2005901464, and the applicant's further co-pending International Patent Application filed 24 March 2006 claiming priority from Australian Provisional Patent Application No. 2005901462. The contents of both of these International Patent Applications are incorporated herein by cross-reference in their entirety. 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 complex may be administered to the mammalian subject systemically or may be locally applied to the site of the carcinoma. The complex may also be administered to the mammalian subject by both methods concurrently. The complex may be topically applied to a skin cancer. If the carcinoma is a lung cancer, the complex may be administered to the cancer site by inhalation of the complex. If the carcinoma is a cancer of the nose, the complex may be administered by nasal spray. If the carcinoma is a cancer of the gastrointestinal tract, including those of the mouth, oesophagus, stomach, small intestine and colorectal regions, the complex may be administered to the cancer site by oral administration (including buccal or sublingual administration for mouth cancers) or by suppository for colorectal cancers. For any carcinoma, the complex may be administered by direct injection into the lesion or organ containing the lesion.
The complex will generally be administered in 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 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 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, palmic 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.
Other suitable 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).
A composition as described herein may also optionally further comprise one or more solvents or solubilising components for increasing the solubility of the metal carboxylate complex in the composition. The 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, or other pharmaceutically acceptable glycol. An example of a solubilising component is a polyvinylacohol/povidone mixture. The composition may also further comprise a thickener such as Aerosil 200, clay or another inorganic filler. Preferably, such compositions 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 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 Antiinflammatory 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 tree 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 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.
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 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 may 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), ophthalmological, vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, or for administration respiratoraly, intratrachaely, nasopharanyngealy, intraoccularly, intrathecally, intranasally, by infusion, or via IV group patch and by implant. With respect to the intravenous route, particularly suitable routes are via injection into blood vessels which supply a tumour or particular organs to be treated. Agents may also be delivered into cavities such as for example the pleural or peritoneal cavity, or be injected directly into tumour tissue. The composition may 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 may be in the form of a viscous paste, a tablet, a capsule, a chewable composition, or any other form suitable for oral administration. If desired, the composition may be encapsulated in a hard or soft capsule (e.g. gelatine) by techniques known in the art. Moreover, the metal complex maybe provided in the form of ingestible tablets, 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 skilled addressee.
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 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 may, 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 may include one or more pharmaceutically active components in addition to the complex that have anti-cancer activity or other therapeutic activity. Such active components include conventionally used anti-inflammatory drugs, and conventionally used metal and non- metal based chemotherapeutic and anti-cancer agents such as those identified above.
Typically, the metal complex constitutes 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 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 0.1% 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 may 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 Artiodactyla. Typically, the subject will be a primate and more usually, a human being. The method of the present invention may for instance be used to prevent or treat basal cell carcinomas, squamous cell carcinoma, melanoma, colon, colorectal cancer, breast cancer, lung cancer and other cancers of the epithelial tissues such as epithelial cancers of the tongue, salivary glands, gums and other areas of the mouth, oropharynx, nasopharynx, hypopharynx, oesophagus, pancreas, stomach, small intestine, duodenum, gall bladder, pancreas, larynx, trachea, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid, and mestastic cancers thereof.
A number of embodiments of the present invention will now be described below by reference to the following non-limiting examples.
EXAMPLE l
1.1 Preparation of compounds
1.1.1 Metal carboxylate complexes
[Cu2(Indo)4(OH2)2].nH20
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].nH2O 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(Indo)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(mdo)2(Pyrro)2]-2PyrroΗ2O complex.
[Cu(Indo)2(Im)2] complex
Cu(OAc)2-H2O (0.108 g, 0.5408 mmol) in methanol (8 mL) and water (1 mL) 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 dropwise to the solution of indomethacin and imidazole at -30 °C 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; Cl, 7.76, Cu, 6.96%. Found: C5 58.18; H, 4.49; N, 8.99; Cu, 6.64%.
1.1.2 Metal hydroxymate complexes
Acetylsalicylhydroxamic Acid (AcSHA) and its copper complex
Figure imgf000058_0001
Scheme 4: Synthetic route for AcSHAH2
Salicylhydroxamic acid (SHAH2) (7.65 g, 50 mmol) was mixed with acetic anhydride (9.5 ml, 100 mmol). The solution was acidified with H3PO4 (1 ml) and was stirred in a water bath at 60 °C for 30 min. Distilled water (5 ml) was added to the solution in order to decompose the unreacted acetic anhydride, and the resulting solution were stirred at room temperature until the vapor from the solution gave no acid reaction 0 towards litmus paper. Finally, the reaction mixture was mixed with distilled water (50 ml) and AcSHAH2 precipitated as a white powder solid (68.2%). Anal. Calcd. for CgH9NO4: C, 55.39; H, 4.65; N, 7.18. Found: C, 55.23; H, 4.61; N, 7.06%. NMR (acetone-d6): δ 7.82 (dd, IH, Ph-H), 8.82 (bm, IH, -NHOH), 7.67 (m, 4H, -C6H4Cl), 7.15-6.66 (m, 3Η, - C6H3OCH3), 3.77 (s, 3H, -OCH3), 3.37 (s, 2H, O=CCH2), 2.50 (s, 3H, CCH3). 5 The ligand compound was characterized by elemental analysis and infrared spectrometry. The IR spectrum of AcSHAH2 gave two absorption bands centred at 3322 and 3272 cm"1, ascribed to the (OH) and (NH) stretching vibrations, respectively. In the ^-n.m.r spectrum of AcSHAH2, the phenolic OH resonance of SHA at δ 12.44 ppm disappeared and a new singlet appeared at d 2.25 ppm for CH3. Acetylation of SHA O influenced the resonances of the hydroxamic OH and NH which are shifted from 11.52 to 10.74 ppm and 9.35 to 10.41 ppm, respectively. In the IR spectrum, extensive coupling occurs for several vibrations making qualitative deductions difficult. However, AcSHA shows two bands centered at 3322 and 3272 cm"1, ascribed to the m(0H) and m(NH) stretching vibrations, respectively. The appearance of a sharp and strong peak located at 5 ca. 1787 cm"1 in the spectrum of AcSHA clearly indicates the presence of the acetyl carbonyl, in addition to a peak due to the hydroxamic carbonyl at ca. 1640 cm"1. The broadening of the hydroxamic carbonyl band is an indication of its partition to the intramolecular H-bonding. Copper complex Of AcSHA-[Cu(AcSHAH)(OH)]
Cu(OAc)2-H2O (1.0 g, 5 mmol) was added to a solution OfAcSHAH2 (1.77 g, 10 mmol) in EtOAc (20 mL). The resulting green complex was filtrated, washed with EtOAc and dried in vacuo. Anal. Calcd. for C9H9CuNO5: C, 39.35; H, 3.30; Cu, 23.13; N, 5.10; Found: C, 41.45; H, 3.13; Cu, 23.17; N, 5.08%.
IndoHA and its Copper Complex
Figure imgf000059_0001
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 O0C. The indomethacin acid chloride remaining was then reconstituted in anhydrous distilled tetrahydrofuran (THF). (See Meyer, G.A., Lostritto, R.T., and Johnson, J.F., Journal of Applied Polymer Science 1991, 42, 2247-2253 and Davaran, S. and Entezami, A. A., Journal of 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 Ci9H17ClN2O4: 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): δ 10.65 (s, IH, NOH), 8.82 (bm, IH, -NHOH), 7.67 (m, 4H, -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(IHdOHAH)(OH)]
IndoHA (0.932 g, 2.5 mmol) in ethanol (15 niL) 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
VOSO4-5H2O (50.6 mg, 0.200 mmol) and IndoHAH2 (149 mg, 0.400 mmol) were dissolved in methanol (MeOH, HPLC grade, 5.0 mL). The colour of 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 [VvO(IndoHAH)(IndoHA)]-2MeOH-1.5H2O.. The most intense signal in the electrospray mass spectrum (solution in MeOH,
~1 mg mL"1): m/z -807.1 (corresponds to [VVO(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 [VvO(IndoHAH)(IndoH A)] -2MeOH- 1.5H2O (C40H42Cl2N4O1Z5V): 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:
Figure imgf000061_0001
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)2](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)2]"1"- 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+-LH""); -855.0 (100%; [Cr(LH)2(OMe)2]"); -917.9 (62%; [Cr(L)(LH)J-NO3 ""); -792.2 (55%; [CrL2J"); and -1649.8 (30%;
[Cr(LH)2(OMe)2J--[Cr(L)(LH)J). AU 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): C5 49.95%; H, 4.41%; N, 7.66%. Found: C, 50.86%; H, 4.29%; N, 7.21%.
1.1.3 Metal amine complexes with mononuclear Iigands
[Co(NH3)5(Indo)](CF3Sθ3)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 °C, 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: C, 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-^): δ 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-^6): 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 the high (10 mg/kg Indo molar equivalent and dissolved into an MCT paste).
1.1.4 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 3SC1), 100%, and 415 (L" for 37Cl), 40%.
Figure imgf000063_0001
Scheme 6
[Cu(IiIdO-GIy)(Im)2]
Cu(OAc)2-H2O (0.108 g, 0.5408 mmol) in methanol (8 mL) and water (1 mL) 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. The solution of Cu2+ was added dropwise to the solution of indomethacin-glycine and imidazole at ~30 °C with stirring, 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-Gly)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.
Vanadium and chromium complexes of this ligand are prepared by similar methods as those described for IndoHA, but electrospray mass spectrometry indicates that the complexes are polymeric rather than monomers.
1.2.1 Results and Discussion
The examples above demonstrate a great diversity of complex types and metals that can be used to prepare such anti-cancer drugs. While all of the prior art has concentrated on labile metals such as those of Cu(II), inert metals, such as Co(III)
(Example 1.1.3) offer the potential of systemic delivery of even higher concentrations of NSAIDs through oral, injectable, and topical delivery and to incorporate them into slow release patches. As demonstrated by this example, the complex still exhibits significant anti-inflammatory, and hence is expected to have systemic anti-cancer activity, but completely eliminates gastric side-effects by being absorbed in the GI tract before release of the drug.
EXAMPLE 2 Inhibition of experimental colorectal cancer (adenomas and carcinomas) 2.1 Summary
Aberrant crypt focci (ACF) are early pre-neoplastic microscopic lesions that have consistently been observed in a number of experimental models of colorectal carcinogenesis, and are also present in the mucosa in human colorectal cancer, where they have been suggested to be precursor lesions from which adenomas and carcinomas develop.
A study was conducted to evaluate the efficacy of a dinuclear copper/indomethacin complex, [Cu2(Indo)4(DMF)2], to inhibit aberrant crypt focci (ACF) formation in azoxymethane-induced adenocarcinoma model and examine cell viability in HCT-116 colorectal cancer cell line and gastrointestinal permeability, mitochondrial oxidative damage and renal toxicity.
Rats were dosed with indomethacin and [Cu2(IiIdO)4(DMF)2] in a rat model of adenocarcinoma for 28 days and aberrant crypt focci were evaluated. 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(LtIdO)4(DMF)2] on urinary electrolyte concentrations were examined. In an in vitro assay of anti-cancer activity, HCT- 116 colorectal cancer cells were exposed to indomethacin and [Cu2(Indo)4(DMF)2] (0-250 μg/mL) and cell viability was measured.
It was found that both indomethacin and [Cu2(Indo)4(DMF)2] resulted in a significant reduction in aberrant crypt focci in azoxymethane-treated rats. However, [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. In parallel, indomethacin and [Cu2(Indo)4(DMF)2] also reduced cell viability in in vitro assays with cultured HCT-116 colorectal cancer cells. It is concluded that [Cu2(Indo)4(DMF)2] has both gastrointestinal and renal sparing properties while maintaining efficacy in the adenocarcinoma model.
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). HCT-116 cells were obtained from American Type Culture Collection (Manassas, Virginia, USA), and Alamar Blue from Trek Diagnostic
Systems, Cleveland, Ohio, USA. 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.; Corrigan, 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(IMO)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 (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 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. 1991, 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(IiIdO)4(DMF)2] at doses of 13.3 mg/kg via gavage. To test intestinal permeability, 0.5 mL 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 Of51Cr-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, 11(11), 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 -2O0C. 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 antiinflammatory 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(IiIdO)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(LIdO)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-iV-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(Indo)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(ItIdO)4(DMF)2] Dosing on Urine Electrolytes Composition
Figure imgf000071_0001
2.6 Colonic Aberrant Crypt Focci
Briefly, at the commencement of the experiment, the rats were given weekly intraperitoneal injections of azoxymethane (AOM) at doses of 15 mg/kg for four weeks and then sacrificed 6 weeks after the final injection of AOM for assessment of numbers of aberrant crypt focci (ACF). The rats were randomized into treatment groups at the beginning of the period of treatment with AOM. Groups of five rats were given 2% carboxymethylcellulose (CMC) vehicle, indomethacin (3.00 mg/kg), or an equimolar dose Of [Cu2(In(Io)4(DMF)2] (3.81 mg/kg) orally each day for 28 consecutive days during the AOM dosing. The dose of indomethacin was selected on the basis of a previous study of intestinal toxicity of indomethacin and colorectal carcinogenesis in an animal model. (Non-steroidal anti-inflammatory drugs with activity against either cyclooxygenase 1 or cyclooxygenase 2 inhibit colorectal cancer in a DMH rodent model by inducing apoptosis and inhibiting cell proliferation. Brown, W. A.; Skinner, S. A.; Malcontenti-Wilson, C; Vogiagis, D.; O'Brien, P.E. Gut 2001, 48, 660-6; and Toxicokinetics of indomethacin- induced intestinal permeability in the rat. Wright, M.R.; Davies, N.M.; Jamali, F. Pharmacol. Res. 1997, 35, 499-504). Six weeks after the final AOM injection, the rats were sacrificed by an overdose of halothane, a laparotomy was performed and the entire colon was excised. After gentle flushing with 0.9% saline, the colon was tied at both ends with silk sutures and insufflated with 10% phosphate-buffered formalin (pH 7.4). After 2 h, the colons were opened along the mesenteric border, pinned flat, mucosal side up, and then submerged in formalin for a further 24 h. The tissues were then stained with 0.2% methylene blue in 0.9% saline and then coded before scoring blindly. The colon from each animal was cut into convenient 2-3 cm pieces and each piece was placed mucosal side up between two slides as a wet mount, and the mounted sample was examined microscopically at 10Ox magnification. Aberrant crypts were distinguished from the surrounding normal crypts by their increased size, bright blue staining, significantly increased distance from lamina to basal surface of cells, and the easily discernible pericryptal zone (see Figure 11). The left panel of Figure 11 shows a control rat colon, and the right panel of Figure 11 shows azoxymethane-induced aberrant crypt foci in the colon of a rat treated with azoxymethane. The parameters used to assess the aberrant crypts were occurrence and multiplicity. Crypt multiplicity was determined as the number of crypts in each focus and categorized as those containing up to three, or four or more aberrant crypts/focus (see Figure 12).
2.7 CeU Culture HCT-116 cells were cultured in McCoy's 5 A medium containing sodium bicarbonate, penicillin-streptomycin, 25 mM HEPES and 10% heat-inactivated FBS in a 5% CO2 atmosphere at 37°C. The cells were passed twice a week on reaching confluence. The cells were trypsinized, washed with PBS and plated with fresh medium in a 96-well plate at a density of 3 x 104 cells/well and cultured overnight prior to discarding the culture medium. The treatment was started by adding fresh medium with or without cytotoxins (indomethacin and [Cu2(Indo)4(DMF)2]) of different concentrations and the cells were further incubated for 72 h. After incubation, the medium was discarded and fresh medium containing 10% Alamar Blue was added to each well as well as to blank wells, and the cells were incubated for another 3 h. The plate was placed at room temperature in the dark for 15 min and the fluorescence was read with a CytoFluor (PerSeptive Biosystems, MA) at Ex. 532/20, Em 590/50 gain 40 (see Figure 13).
2.7.1 Statistical Analysis
The results set out below are 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 OfNa+, K , and Cl and increased urinary excretion of NAG and phosphate (see Table 2, Figure 10). Acute administration of [Cu2(Indo)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.
Administration of azoxymethane resulted in the development of widespread ACF, predominantly in the distal colon (see Figure 11). Figure 12 shows that both indomethacin and [Cu2(Indo)4(DMF)2] significantly decreased the number of induced ACF compared with controls. Indomethacin was more effective than [Cu2(Indo)4(DMF)2], however, two animals in 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. Interestingly, there were proportionally more ACF with two crypts in both the control and indomethacin groups, whereas with the [Cu2(UIdO)4(DMF)2] group, ACF with one and two crypts were approximately equal (see Figure 12). Both indomethacin and [Cu2(Indo)4(DMF)2] demonstrated pharmacological activity in the colorectal cancer cell line HCT-116 in a concentration-dependent manner (see Figure 13). At the highest concentrations tested, [Cu2(Indo)4(DMF)2] demonstrated significantly greater activity than indomethacin.
2.8 Discussion
In cell culture, with colorectal cancer cell line HCT-116, [Cu2(Indo)4(DMF)2] was shown to be more efficacious than IndoH, which is consistent with the data obtained for other cancer cell lines (see Examples 3 to 5 below).
[Cu2(Indo)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. ScL 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(hido)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 has been a serious and limited side-effect in clinical trials in which indomethacin has been used as a chemotherapeutic agent, this observation is of high significance in terms of Culndo being used as both a chemopreventative agent and in chemotherapy.
This study demonstrates for the first time that dinuclear copper/indomethacin complexes are effective in experimental models of colorectal cancer. The remarkable renal and GI safety of Culndo will be further enhanced by more appropriate formulations that are designed to stabilise the complex, 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, which will enable much higher tolerated doses for chemotherapy than have previously been contemplated with NSAIDs. Such formulations may also be designed to improve targeting of the colorectal region of the GI tract through suppositories or other suitable oral formulations.
It is also noted that a physical mixture of IndoH and a Cu salt, such as Cu acetate, cannot be used to treat colorectal cancer, since such a physical mixture 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 colorectal cancers.
2.9 Conclusion [Cu2(IiIdO)4(DMF)2] is effective in significantly reducing the number of ACF in the rat colorectal cancer model and is more cytotoxic than IndoH in the HCT-116 colorectal cancer cell line. This compound 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.
EXAMPLE 3 Efficacy of Culndo complex in in vitro cytotoxicity assays with cultured Hep-G2 (human hepatoma)
3.1 Summary Hepatomas are primary carcinomas of the liver. As is the case for the human colorectal and melanoma cancer cell lines, there is a significantly greater increase in cytotoxicity of Culndo compared to IndoH suggesting that topical administration of Culndo will increase the efficacy of treatment of a large range of carcinomas.
3.2 Chemicals and reagents
Trypsin-EDTA, trypan blue, phosphate-buffered saline (PBS), resazurin, sodium bicarbonate, McCoy's 5 A medium, penicillin-streptomycin, and insulin were purchased from Sigma (St. Louis, MO, USA). HPLC grade reagent alcohol was purchased from J. T. Baker (Phillipsburg, NJ, USA). Dulbecco's Modified Eagle Medium (D-MEM) and RMPI 1640 medium were purchased from Gibco Industries Inc. (Langley, OK, USA). Foetal bovine serum (FBS) was purchased form Equitech-Bio Inc. (Kerrville, TX, USA).
3.3 Cell Culture
The Hep-G2 (human hepatoma) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD). The Hep-G2 cells were maintained in Dulbecco's Modified Eagle Medium (D-MEM). The cell line was supplemented with 10% heat-inactivated foetal bovine serum (FBS), penicillin-streptomycin (10 mg/1 L) and were incubated at 370C in a 5% CO2 atmosphere. The Hep-G2 cell line was additionally supplemented with insulin (4 mg/mL).
3.4 Cell Number
The optimal cell seeding numbers were determined by preliminary cell seeding number experiments. Cells were seeded in numbers 1 x 104, 2 x 104, 3 x 104 and so on until the final cell seeding number 1 x 105 per well in a 96 well plate (Costar 3595). Cell plates were incubated at 370C in a 5% CO2 atmosphere for 72 hours. Following incubation, the medium was aspirated and alamar blue (resazurin) fluorescent dye solution was diluted in fresh medium to make a 10% resazurin solution. The 10% solution was added directly to cells. The cell plates were incubated at 37°C in a 5% CO2 atmosphere for 3 hours. The cell plates were subsequently removed from the incubator and placed at room temperature in a darkened drawer to protect from light for 30 min. The cell plates were subsequently placed into the Cytoflour®4000 fluorescence multi-well plate reader (Applied Biosystems, USA). Fluorescence was read at an excitation of 485 run and an emission of 530 run. Standard curves of cell seeding number against fluorescence were generated. From these studies Hep-G2 cells were seeded at a density of 5000 cells/well.
3.5 Alamar Blue Assay
Alamar blue (resazurin) fluorescent dye is an easy and accurate assay that has recently gained popularity in determining the cytotoxicity of many cell lines
(Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. O'Brien, J.; Wilson, L; Orton, T.; Pognan, F. Eur. J. Biochem. 2000, 267, 5421-5426). The resazurin non-fluorescent compound is metabolized into the fluorescent compound resorufin by intact and viable cells. This emission of fluorescence can be quantified using a cell plate reader and the number of viable cells following treatment can be determined.
Cells were counted and seeded on 96 well plates. The seeded cells were incubated at 37°C in a 5% CO2 atmosphere for 24 hours. Piceatannol was dissolved in methanol on the day of the experiment and was diluted in medium to yield concentrations ofθ.1, 1, 10, 50 and 100 μg/mL. Following aspiration of the medium, the cells were treated with the piceatannol solutions. Additional cells were treated with either methanol diluted in medium or medium only. Treated and control cells were incubated at 37°C in a 5% CO2 atmosphere for 72 hours. After the cell plates were removed from the incubator, the medium was aspirated and replaced with 10% alamar blue (resazurin) fluorescent dye diluted in fresh medium. Cell plates were subsequently treated as described in Example
3.4, and fluorescence read at an excitation wavelength of 485mm and an emission of 530mm. The viable cell number (as a percent of control) in each cell line exposed to varying concentrations of piceatannol was measured and the /C50 for each cell line was determined.
3.6 Results
There is enhanced activity of Culndo (/C50 = ~70 μg/mL) whereas the effect of IndoH plateaued at only a 20% cytotoxic effect and the /C50 value could not be reached even at concentrations as high as 250 μg/mL. The results demonstrate that Culndo is very significantly more active against this cancer cell line than is IndoH which shows little activity. EXAMPLE 4 Cytotoxicity Studies of the NSAIDs on the A549 (non-small lung), A2780 (Pt-resitant ovarian) and Hep-2 cancer cells
4.1 Experimental
The cytotoxicity of metal complexes on cancer cell lines A549, A2780 and Hep- 2 were assessed using MTT and crystal violet blue assays.
4.1.1 Medium Preparation for A549, A2780 and Hep-2 cell lines
Advanced DMEM medium was used in all the cell culture work. The medium did not contain certain components needed to facilitate cell growth. Therefore, for the A549 cells, antibiotics-actimycotic (0.5 mL), (100 U m"1 penicillin, 100 μg mL"1 streptomycin and 0.25 μg mL"1 amphotericin B), 200 mM glutamine solution (0.5 mL) and fetal calf serum (2 %, 0.8 mL) were added to the medium (40 mL) before proceeding with any cell work. For the Hep-2 and A2780 cells, Advanced DMEM medium (40 mL) was supplemented with antibiotics-actimycotic (0.5 mL), (100 U m"1 penicilin, 100 μg mL"1 streptomycin and 0.25 μg mL"1 amphotericin B), 200 mM glutamine solution (1.0 mL) and fetal calf serum (5 %, 4 mL). AU of the above components were obtained from Gibco Industries Inc. (Langley, OK, USA). All other reagents used in the cell work were obtained from Sigma (St. Louis, MO, USA).
4.1.2 Thawing of frozen A549, A2780 and Hep-2 cancer cells
Frozen cells were stored in liquid nitrogen. The cells were rapidly warmed in a 37 0C water bath for approximately 5 min. The cell suspension was then transferred to a 10- mL centrifuge tube with 9 mL of medium and centrifuged for 3 min at 2000 rpm. The medium was removed from the resultant pellet and fresh medium (1 mL) was added to resuspend the cells, then transfer the cells to a 10-cm plate with fresh medium (10 mL) added to it. Cells were incubated at 37 0C in a humidified atmosphere containing 5 % CO2 for 3 days. 4.1.3 Subculturing of A549, A2780 and Hep-2 cells
The medium was removed from the cells and the cell layer was washed with phosphate buffer solution (PBS, 10 mL) prior to trypsination with 0.25 % trypsin EDTA solution (4 mL). Cells were then incubated for 6 min at 37 0C, after which medium with serum (5 mL) was added to inactivate the trypsin. The cell suspension was then collected into a centrifuge tube and the mixture was centrifuged at 2000 rpm for 3 min. The medium was subsequently removed from the cell pellet and fresh medium (1 mL, A549, and 1 mL Hep-2) was added to resuspend the cells. The cell suspension (0.58 mL, A549, 0.24 mL, A2780 and 1.6 mL, Hep-2) was transferred from the total cell suspension to a centrifuge tube. Further, medium (3 mL, for A549, A2780 and Hep-2) was added to the centrifuge tube and the cells were counted using a haemocytometer.
4.1.4 Seeding of A549, A2780 and Hep-2 cells for cytotoxicity experiments
The cell suspension (100 μL per well) was transferred to four sets of ninety six- well plates with each well having approximately the same amount of cells (1x104 cells/well/100 μL for A549 and Hep-2 and IxIO5 cells/well/ 100 μL for A2780). The plates were incubated overnight at 37 0C prior to the addition of the test compound.
4.2 Sample preparation for indomethacin and lCu2(Indo)4(OH2)2], [Zn(Indo)(OH2)2], ACMH, [Cu(ACM)2(OH2)2], [Zn(ACM)2(OH2)2], aspirin,
[Cu(asp)2(3-pic)2], IndoHAH2 (oxamethacin), [VvO(IndoH AH)2], ibuprofen and [Cu(ibup)2(2-Meim)2]
Indomethacin and [Cu2(Indo)4(OH2)2] were tested over a range of concentrations (10-300 μM) while ACMH, [Cu(ACM)2(OH2)2], [Zn(ACM)2(OH2)2], aspirin, IndoHAH2 (oxamethacin), [VvO(IndoHAH)2], ibuprofen, [Cu(asp)2(3-pic)2] and [Cu(ibup)2(2- Mim)2] were tested over a range of concentrations (20-400 μM). After all the samples were finely dispersed in medium chain triglycerides (MCT) oil with sonication, the sample suspension (100 μL) was added to plastic vials with subsequent addition of medium (100 μL). The vials were vigorously vortexed to obtain a homogenous suspension. The whole procedure was kept sterile by rinsing every single pasteur pipettes with ethanol (70%) and flicking it dry. Blank assays with the MCT oil only had no effects on the cells. 4.2.1 Treatment of A549 and A2780 cells with indomethacin and [Cu2(Indo)4(OH2)2]
The medium was removed from all the wells via a vacuum pump. A number of wells were left without addition of the test compound and were used as control wells. Appropriate concentrations of the test compound (10-300 μM) in complete medium were added to the remainder of the wells. After treatment, the plates were incubated at 37 0C for 3 days.
4.2.2 Treatment of A549 and Hep-2 cells with [Zn(Indo)2(OH2)2]
The medium was removed from all the wells via a vacuum pump. The first and the last row of wells were left without addition of the test compound and were used as control wells. To the remainder of the wells appropriate concentrations of the test compound (20-400 μM) in complete medium were added into the wells. After treatment, the plates were incubated at 37 0C for 3 days.
4.2.3 Treatment of A549 cells with ACMH, [Cu(ACM)2(OHz)2], [Zn(ACM)2(OH2)2], aspirin, [Cu(asp)2(3-pic)2], IndoHAH2 (oxamethacin), [VvO(IndoHAH)2], ibuprofen and [Cu(ibup)2(2-Mim)2] The medium was removed from all the wells via a vacuum pump. The first and the last row of wells were left without addition of the test compound and were used as control wells. To the rest of the wells, appropriate concentrations of the test compound (20-400 μM) in complete medium were added into the wells. After treatment, the plates were incubated at 37 °C for 3 days.
4.2.4 Quantification of A549, A2780 and Hep-2 cancer cells
For the MTT assay, the medium was removed from the plates, MTT (1 mg/mL) was added to all the wells and the cells were further incubated for approximately 4 h at 37 0C to allow sufficient time for it to interact with the cells. The medium was then carefully discarded and the cellular contents were extracted using DMSO (100 μL per well). For the crystal violet blue assay, the medium was removed from the plates and the cells were washed twice with saline (0.9% NaCl). To all the wells, crystal violet blue dye (1 mL in 5% PBS) was added and the mixture was left for ~ 1 min to stain the cells. The dye was then removed and the cells were extracted using a solution of propan-2-ol (1 mL).
Absorption at 595 nm was determined using an ELISA plate reader. The percent survival was determined by the intensity of the absorbance obtained, which correlated to the amount of cells present in each well. The negative control wells were arbitrarily assigned as 100% survival. The MTT assay provides a measure only for viable cells. Statistical analysis of data of all the cell work after quantification with the plate reader was achieved using Origin 6.1 software (Microcal Inc., 1999). Comparison of test compound cytotoxicities was analyzed using a two-tailed t test.
4.3 Cytotoxicity results The cytotoxicity of the drugs and cell lines tested according to the above protocol is presented in the following table and discussion. Data are expressed as the mean ± SEM. AU reported P values are two-sided, and P<0.05 was considered statistically significant. Although not statistically significant, the results are consistent with the other cell lines described in Examples 2, 3 and 5 where the dinuclear complex is more active than IndoH against both the A549 and A2780 cell lines (see Figs 15 to 18 and Table 3).
Complexation of ACM to either Cu(II) or Zn(II) decreased the LC50 values in the A549 cell lines (Figure 19). Unlike IndoH and ACMH, none of the parent NSAIDs, aspirin, ibuprofen, AcSHAH2, or IndoHAH2 had significant effects on the cell viability for A549 even up to 400 μM. While the Cu complexes of aspirin and ibuprofen did effect cell viability, the LC50 values were the same (~300 μM) in both cases and probably reflected normal Cu toxicity, without any specific effect of the complex (Figures 20 and 21). No significant effect on cell viability of the Cu complex of AcSHAH was observed. Table 3: LC50 values for IndoH and [Cu2(Indo)4(OH2)2] in A549 and A2780 cells
Figure imgf000082_0001
By contrast, complexation of IndoH A with a metal ion had a dramatic effect on the cell viability and the V(V) complex had the greatest effect on cell viability of all of the metal complexes (Figure 22).
The Zn Indo complex was also compared against two cell lines Hep-2 and A549 and it was shown to have an equivalent effect on cell viability across the two cell lines (Figure 23).
Discussion
These results indicate that Culndo is more effective than Indo against a broad range of carcinomas if applied topically or injected directly into the lesion or tissue containing a lesion (a practice which has been shown to be more effective in chemotherapy for a variety of external and internal lesions and has been increasingly accepted as a preferred mode of treatment by some oncologists). Similarly, the Zn and Cu ACM complexes appear to be more effective than ACM, which has also been demonstrated to have an anti-cancer effect and enhances the efficacy of cytotoxins. Of particular interest is the relative high activity of the V-IndoHA complex. It is considered that this lipophilic complex will transfer both the ligand and the metal inside the cells, where they are likely to exert their activities separately: the ligand by Cox inhibition and the V by phosphatase activity. EXAMPLE 5: Growth-inhibitory effect of IndoH and Culndo against melanoma
5.1 Summary
Consistent with reports in the literature as discussed above, IndoH is not effective in treating melanoma in animal models. The present study found that Culndo is more active against the human melanoma A-375 cell line than Indo in in vitro cytotoxicity assays. The efficacy of Culndo is also highlighted by observations indicating Culndo was able to cause complete regression of melanoma tumours.
5.2 Experimental 5.2.1 In vitro cytotoxicity assays in the human melanoma A-375 cell line
These experiments were performed in the same manner as described in Examples 3 and 4, with the following changes. The A-375 (human melanoma) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD). The optimised seeding densities used in the studies on A-375 cells were at a density of 1500 cells/well.
5.2.2 Growth-inhibitory effect of IndoH and Culndo against the transplantable murine B16 melanoma in C57BI mice
The B 16 melanoma cell line was grown in Dulbecco's minimal essential medium supplemented with 10% foetal calf serum, glutamine and penicillin/streptomycin. Groups of 10 female C57B1 mice were shaved of dorsal hair and pretreated with a 0.1% ethanolic solution of either indomethacin or Culndo (50 μL) applied directly to the dorsal skin daily for 10 days. The B 16 cells (5 x 104 suspended in 0.1 mL of serum- free medium) were injected intradermally into the shaved ventral skin, and the daily application of the drugs was continued while tumour appearance and growth was monitored. Tumour incidence indicates the percentage of mice in the group displaying palpable tumour growth at the inoculation site. The area of the tumours was calculated as the product of two perpendicular diameters, and the group average area was calculated. 5.3 Results
5.3.1 In vitro cytotoxicity assays
Enhanced activity of Culndo (/C50 = ~20 μg/mL) compared to IndoH (ICso = ~80 μg/mL) was observed in the in vitro cytotoxicity experiments. IndoH appeared to promote cell growth somewhat at low doses (<80 μg/mL) (Figure 24). The results demonstrate that Culndo is more active against this cancer cell line in vitro than IndoH.
5.3.2 Animal studies The progressive tumour incidence showed clearly that the appearance of the tumours was delayed in the Culndo-treated mice (Figure 25), such that after 10 days, no tumours were present in any of the mice treated with Culndo, whereas 10 and 20% of the IndoH-treated and control rats, respectively, exhibited tumours. Even after 15 days, 40% of the Culndo treated rats remained free of tumours, whereas only 5% of the combined control and IndoH-treated rats were free of tumours.
The average tumour areas were quite widely dispersed within each treatment group, but showed consistently that tumour growth was fastest in the control mice, and that Culndo appeared to have slowed tumour growth more effectively than IndoH (Figure 26).
5.4 Discussion
The present results are consistent with reports in the literature that IndoH is not active against animal models of human melanomas. This is shown by the modest activity of IndoH in in vitro cytotoxicity assays compared to Culndo found in the present study. Surprisingly, while IndoH has been shown to be inactive against animal models of melanoma in the studies reported here and those reported in the literature, Culndo has been found by the present inventors to have a significant chemopreventative effect on preventing the growth of melanoma tumours, with 40% of the mice remaining free of this aggressive phenotype of melanoma tumour 15 days after application to the cells. Culndo also reduced the growth of tumours once they had formed. This behaviour differs from the dithiocarbamate-based drugs where the ligand pro-drug (disulfuram) is active as well as the Cu and Zn complexes of disulfuram. The results on animal studies of human models of the disease were found to be consistent with the minimal effects observed in clinical trials involving the co-administration of IndoH with another chemotherapeutic agent.
While the reason for the marked difference in anti-cancer activities of IndoH and Culndo is not entirely clear, it is proposed that the increased lipophilicity of the drug when complexed to Cu(II) and the effect of Cu(II) itself on the cells are both likely to contribute to the observed anti-cancer activity of Culndo.
In a separate preliminary study, a similar application of the Culndo drug to melanomas on white ponies resulted in the complete disappearance of the tumours, whereas IndoH had no effect on these tumours.
The present results show that IndoH, while being a powerful COX inhibitor, is ineffective (Figure 25) in the prevention and treatment of melanomas, whereas Culndo has a very significant effect on both the prevention and treatment of human melanomas in an animal model. The results reported herein also demonstrate that Culndo is active against animal models of human melanomas and animal melanomas, while IndoH is not. The results further demonstrate that Culndo is considerably safer in terms of the dose- limiting toxicities of gastric and renal toxicity, and this offers a much wider therapeutic window for human therapeutics.
It is anticipated that even stronger effects and higher safety may be achieved with other more lipophilic complexes, including complexes of a metal and ACM.
EXAMPLE 6 Assay of the effect of IndoH and Culndo on the growth of transplantable murine UV radiation-induced squamous cell carcinoma T79 in Skh:hr hairless mice
6.1 Summary The effective topical treatment of squamous cell carcinoma may have some important clinical applications, especially when the tumour occurs on regions of the face where it is difficult to operate or where an operation could cause disfigurement. Studies against this type of tumour are also important since it is related to similar cancers of the epithelium, such as lung and breast cancers, that are difficult to treat with chemotherapy. In the results of this experiment, Culndo caused 100% tumour regression in a mouse model of the human disease. 6.2 Experimental
The T79 cell line was established from a squamous cell carcinoma induced with chronic solar simulated UV irradiation in the inbred Skh:hr-1 hairless mouse. Groups of 10 female Skh:hr- 1 mice were pretreated on the dorsal skin with a 0.1 % ethanolic solution of either IndoH or Culndo (50 μL), which was applied directly by pipette to the dorsal skin daily for 10 days. The T79 cells (105 cells in 0.1 mL of serum-free medium) were injected intradermally into the dorsal skin, and the daily application of the drugs was continued while tumour growth was monitored.
6.3 Results
The results are shown in Figure 27. All recipients produced tumour growth that was maximal at 8 days post-inoculation. At day 6 and day 8, the Culndo-treated mice had tumours that were significantly larger than in either the control or the IndoH-treated mice. After day 8 the tumours began to regress. By day 10, the tumour area in Culndo-treated mice was significantly (P < 0.01) less than in the control or the IndoH-treated mice. By day 15, there was no further reduction in tumour area in the control and IndoH treated mice, but there were no longer any visible tumours in any of the Culndo-treated mice.
6.4 Discussion
The T79 cell line is known as a "regressor" cell line that requires UV radiation- induced immunosuppression of the recipient mouse for continuing growth. This study demonstrates the regression of the established tumour with time in the absence of immunosuppression in the control mice, and shows that Culndo, but not IndoH, facilitates this regression. In addition, while the regression in the control and IndoH-treated mice plateaus after 15 days, there are no longer any tumours remaining in the Culndo-treated mice. This shows the strong anti-cancer effect of Culndo on squamous cell carcinomas that are also closely related to lung cancers.
The early tumour growth enhancement by Culndo may be relevant to the efficacy of Culndo as an anti-cancer drug. The inventors postulate the effectiveness of the drug is associated with the property of Cu in promoting angiogenesis. While this is generally considered to be undesirable in that it promotes tumour growth, one of the biggest issues associated with the treatment of solid tumours is the difficulty in delivering drugs to the interior of the tumour because of poor blood flow. For effective treatment of cancer, this is essential because the cancer cells that lead to metastases are those between the outer edge of the tumour and the necrotic core, and this is why most chemotherapies have limited success, i.e. most chemotherapies succeed in killing the outer cancer cells causing the tumour to shrink, but do not kill the crucial cells in the interior of the tumour that ultimately lead to fatal metastases. Thus, the angiogenic nature of the Cu in the compound promotes the growth of blood vessels into solid tumours and thereby provides a highly effective method of delivering the compound into the interior of the tumours.
6.5 Conclusions
The broad spectrum activity of Culndo against such a diverse range of cancers of the epithelium (melanomas, colorectal cancers, squamous cell carcinomas and hepatomas) in both animal models and in vitro cell assays, combined with the unusually high safety for a NSAID or anti-cancer drug in general, provide enormous opportunity for its application in the clinic either as a chemotherapeutic agent alone or in combination therapy with other anti-cancer drugs. In addition, the powerful analgesic and antiinflammatory properties of the drug provide additional benefits for both the treatment and palliative care in late stage cancers for the relief of pain and the treatment of arthritis in patients who cannot tolerate the side effects of NSAIDs when they are undergoing chemotherapy.
EXAMPLE 7 Comparison of topical formulations: Rat paw oedema and gastric ulceration
7.1 Experimental
Rats were orally administered (non-anaesthetized) 1% [Cu2(Indo)4(OH2)2] at 7 mg/kg body weight via oral gavage. The control cohort was dosed solely with CMC
(2%) solution. Inflammation was induced one hour after dosing with the metal complex (or vehicle), by injecting with carrageenan (0.1 mL, 2% w/v in isotonic saline) into the plantar region of the hind paw in = 3) (Winter, C. A.; Flataker, L., Pharmacol. Exp. Ther. 1965, 150, 165-171). The paw oedema assay was performed by measuring the increase in paw volume, as described in International Application No. PCT/AU2005/000442, the contents of which are incorporated herein in its entirety by reference
All inhibition of carrageenan-induced paw oedema data are expressed as the standard error of the mean (± SEM). Comparisons with the control were made using one-way analysis of variance (ANOVAR) using the conservative Bonferroni approach. With all analyses, an associated probability (P-value) of less than 5% (P- value < 0.05) was considered significant.
7.2 Formulations
AU topical non-ionic formulations (10 g) were prepared in a water bath by heating (50-55 0C), mixing Phase A consisting of the active ingredient, i.e., [Cu2(Indo)4(OH2)2] in propylene glycol or glycerol and mixing in the relevant oils. Phase B (water) was heated (50-55 0C) in a water bath and added to phase A mixing vigorously until cooled.
For the purposes of quality control, all creams and the white soft paraffin ointment were rubbed down with a spatula onto a glass slab. The lotion was mixed until cooled with a hand held electric whisk.
Table 4: Formulations
Figure imgf000088_0001
+ APF = Australian Pharmaceutical Formulary. Treatment A = Control group (i.e, nil treatment) Treatments B, C, D and E were all cetomacrogol formulations differing only in the amount of propylene glycol or glycerol. Of the four cetomacrogol-based formulations (B, C, D and E), the lotion (D) is the most aqueous. Treatment F (White Soft Paraffin) is the most hydrophobic base of the five formulations.
7.3 Results
7.3.1 Anti-inflammatory response
The % mean inhibition of rat paw oedema for the formulations is shown in Table 5.
Table 5: % mean inhibition of rat paw oedema
Figure imgf000089_0001
At two hours post treatment, the ANOVA statistical analysis showed all formulations were significantly and comparably anti-inflammatory compared to nil treatment. In addition, the propylene glycol cream resulted in significantly less inflammation compared to controls when measured against either cetomacrogol lotion or white soft paraffin.
Of the five formulations tested, the propylene glycol cream resulted in the greatest inhibition of inflammation and white soft paraffin resulted in the least inhibition of inflammation. While cetomacrogol lotion and white soft paraffin were the least efficacious of the five formulations, both were still significantly anti-inflammatory compared to nil treatment. The results are shown graphically in Figure 28. The asterisk symbol denotes a significant anti-inflammatory response compared to nil treatment. Of all the formulations, the propylene glycol cream was the only formulation at two hours that resulted in significantly less inflammation compared to either cetomacrogol lotion or white soft paraffin.
7.3.2 Anti-inflammatory effect three hours post treatment
At three hours post treatment the ANOVA statistical analysis showed the cetomacrogol cream, glycerol cream and propylene glycol cream were significantly and comparably anti-inflammatory compared to nil treatment. A graph of the mean increase in paw volume at three hours post treatment compared to control (Treatment A = nil treatment) is shown in Figure 29.
7.3.3 Anti-inflammatory effect four hours post treatment
At four hours post treatment the ANOVA statistical analysis showed the cetomacrogol cream and propylene glycol cream were significantly and comparably antiinflammatory compared to nil treatment. A graph of the mean increase in paw volume at four hours post treatment compared to control (Treatment A = nil treatment) is shown in Figure 30.
7.4 Gastric ulceration
Whilst gastric irritation was mild (with erosions rather than necrosis), the least gastric irritation observed by means of magnification was with the cetomacrogol cream. Of note were a number of necrotic patches in one of four rats treated with white soft paraffin.
Table 6: Gastric ulceration of rats
Figure imgf000090_0001
+ Not examined under magnification 7.5 Discussion
While all five formulations initially afforded a significant anti-inflammatory effect compared to controls, only the propylene glycol cream and the cetomacrogol cream were significantly anti-inflammatory over the entire four-hour observation period. Of all the formulations, the propylene glycol cream also initially afforded the greatest antiinflammatory effect and as such was significantly better than either the cetomacrogol lotion or white soft paraffin.
At three hours post treatment, (the typical testing time), all the cetomacrogol- based creams (i.e., the cetomacrogol cream, glycerol cream and propylene glycol cream), prepared by the melt-and-mix method afforded statistically significant and comparable anti-inflammatory effects with approximately a 40% inhibition of inflammation. The cetomacrogol cream resulted in the least gastric irritation, albeit all formulations afforded minimal gastric irritation (≤ 20 mm2). The cetomacrogol lotion and the white soft paraffin were the least efficacious of the formulations and were not significantly antiinflammatory at three hours post treatment.
Importantly, the results further show activity of the test compound in terms of both maximum efficacy and time profiles can be controlled by the formulation used. This is of relevance in different anti-cancer and chemopreventative applications where it may be desirable to achieve maximum residency time in the skin or for the metal complex to diffuse more rapidly through tissues, depending on the size and type of carcinoma and whether systemic, as well as topical, treatment was desirable. Of particular interest are the low GI effects, which would be negligible when a larger animal (e.g. a dog or a horse) or a human were treated for skin lesions since the dose (mg/kg body weight) received would be below that needed to induce toxic side effects.
8 Sub-cutaneous injection study
8.1 Experimental [Cu2(Indo)4(OH2)2] (2 mL, 50 mg/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. The Culndo preparation (200 μL) 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 10 mg of Culndo in 200 μL of benzyl alcohol equates to a dose of 40 mg/kg bw, which was expected to be strongly GI toxic in rats. Analysis of GI toxicity was performed using assays described in the above examples.
In further experiments, this concentrate was used to develop formulations that were suitable for injection into lesions, using an oil base to minimize diffusion, where the concentrate was diluted with various amounts of benzyl alcohol, before it was mixed with the oil.
8.2 Results
Following injection, the rats were observed for 24 hours. The observations are summarised in Table 7, Whilst a 40 mg/kg bw dose of [Cu2(Indo)4(OH2)2] by means of sub-cutaneous 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.
When the formulation was diluted with MCT oil (MCT:benzyl alcohol 9:1 v/v) to give a 1 mg/mL solution of [Cu2(UIdO)4(OHa)2], no efficacy and no side effects were observed (including not overt irritation or necrotic effects at the site of injection). Since this is a therapeutic dose when given orally, this shows that the drug remains localized in the oil.
Table 7: Gastro-intestinal toxicity following subcutaneous injection of [Cu2(Indo)4(OH2)2] in rats
Figure imgf000093_0001
+ Euthanased without further examination.
8.3 Discussion
The choice of formulation can result in the metal complex either having a localized effect where it is desirable to localize the drug into a lesion that has been injected with the metal complex, or a systemic effect that may be used to help control metastases. Again the extent to which treatment is localized or systemic can be optimised by changing the amount of oil and alcohol in the formulation.
The advantage of including benzyl alcohol in the formulation is that it also acts as local anesthetic. It is particularly noteworthy that none of the formulations cause any adverse side effects at the site of the injection and that while the high dose is GI toxic in rats, this is not a serious problem in humans or animals that would be treated with such a formulation as the mg/kg dose is below that which induces such side-effects. 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. 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 a carcinoma in a mammalian subject, the method 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, the carboxylate or derivative being other than salicylate or a derivative of salicylate.
2. A method according to claim 1 wherein the derivative is selcted 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 selected from the group consisting of:
Suprofen ((+)-α-methyl-4-(2-thienyl-carbonyl)phenylacetic acid (SupH));
Tolmentin (l-methyl-5-0?-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-trifluoromethylphenyl)anthranilic acid) (FlufenH)); Niflumic Acid (2-((3-trifiuoromethyl)phenylamino)-3-pyridinecarboxylic acid
(MfH));
Diclofenac (2-[(2,6-dichlorophenyl)amino]phenylacetic acid (DicH));
Indomethacin (l-(4-chlorobenzoyl)-5-methoxy-2-methyl-lH-indole-3-acetic acid (IndoH)); Acemetacin ( 1 -(4-chlorobenzoyl)-5-methoxy-2-methylindole-3 -acetic acid carboxymethyl ester (ACMH));
Acemetacin (l-(4-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid carboxymethyl ester ("ACMH"));
5. A method according to claim 4, wherein the carboxylate having antiinflammatory activity is selected from the indomethacin anion and the acemetacin anion.
6. A method according to claim 4, wherein the complex is selected from the group consisting of:
Figure imgf000096_0001
Figure imgf000097_0001
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 = 6-methoxy-α-methyl-2-naphthaleneacetic acid (NapH); e Ibuprofen = (+)-α-methyl-4-(isopropylmethyl)benzeneacetic acid (IbuH);
■^Metronidazole = 2-methyl-5-nitrobenzimidazole
8 Flufenamic Acid = (iV-trifluoromethylphenyl)anthranilic acid (FlufenH); Λ 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 Ma 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 L2:
Figure imgf000098_0001
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
Figure imgf000098_0002
wherein each R2A 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.
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):
Figure imgf000098_0003
where M is a divalent or trivalent metal ion; L2 is a ligand of the formula:
Figure imgf000099_0001
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
Figure imgf000099_0002
wherein each R2A 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 maybe optionally substituted; 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;
Figure imgf000099_0003
where each M is independently selected and is a divalent or trivalent metal ion; L2 is a ligand of the formula:
Figure imgf000100_0001
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
Figure imgf000100_0002
wherein each R2A 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;
[M'3O(L2)6L3]P (3) where each M' is independently selected and is a trivalent or tetravalent metal ion;
L is a ligand of the formula:
Figure imgf000101_0001
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
Figure imgf000101_0002
wherein each R2A 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 maybe 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 L2 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, ΛζJV-dimethylformamide (DMF), iV-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO) and N,N-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(OHz)2] [Cu2(ACM)4(DMF)2], [Zn(ACM)2(DMF)2], [Zn(ACM)2(OH2)2], [Ru2(ACM)4Lf or [RU2(ACM)4L2]11, 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):
Figure imgf000102_0001
where M is a divalent or trivalent metal ion;
"r^-L1" is a bidentate ligand of the formula L1:
Figure imgf000103_0001
wherein:
R1 is H or halo (i.e. Cl, F5 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
Figure imgf000103_0002
wherein each R2A 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 maybe 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.
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 fiuoro, 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(In(Io)2(L)2], where L=OH2 or an alcohol, [Co(Indo)2(OH2)2], [Co(Indo)2(EtOH)2] and
Figure imgf000104_0001
21. A method according to claim 1, wherein the complex is a complex of the following formula (5):
[M2(μ-Lι)4Lm]p (5)
wherein each M is independently selected and is a divalent or trivalent metal ion, μ-L1 is a ligand of the formula L1:
Figure imgf000104_0002
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
Figure imgf000104_0003
wherein each R2A 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 maybe optionally substituted; n is i, 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, N,N-dimethylformamide (DMF), N-methylpyrrolidone (ΝMP), dimethylsulfoxide (DMSO), N^-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(ϊndo)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(Indo)4(AN)2], [Cu2dndo)4(OH2)2], [Cu2(Indo)4(OH2)2].nH2O, [Zn2(Indo)4(DMA)2], [Zn2(Mo)4(NMP)2] or [Zn2(Indo)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 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 in claim 18.
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, i\ζiV-dimethylformamide (DMF), N-methylpyrrolidone (ΝMP), dimethylsulfoxide (DMSO), ΛζiV-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;
L 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.
32. A method according to claim 31 wherein L3 is a monodentate, bidentate or bridging ligand of formula L1 or L2 as follows:
Figure imgf000107_0001
Figure imgf000107_0002
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
Figure imgf000107_0003
wherein each R2A 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.
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 a monodentate or a polydentate ligand; m is 1, 2, 3 or 4 n is 1, 2, 3, 4 or 5; and p is the charge of the complex.
38. 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 a monodentate or a polydentate 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.
39. A method according to any one of claims 1 to 38, wherein the carcinoma is a lesion or tumour of the epithelium.
40. A method according to any one of claims 1 to 38, wherein the carcinoma is melanoma.
41. A method according to any one of claims 1 to 40, wherein the complex is administered systemically.
42. A method according to any one of claims 1 to 40, wherein the complex is administered to the site of the carcinoma.
43. A method according to any one of the claims 1 to 40, wherein the complex is coadministered at both the site of the carcinoma and systemically.
44. A method according to any one of the claims 1 to 43, wherein the complex is administered in combination with other chemotherapeutic agents or cancer treatments.
45. A method according to any one of claims 1 to 44, wherein the mammalian subject is a human being.
PCT/AU2006/000403 2005-03-24 2006-03-24 Method for the prophylaxis or treatment of carcinomas WO2006099685A1 (en)

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Cited By (10)

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WO2007110745A1 (en) * 2006-03-24 2007-10-04 Medical Therapies Limited Combination therapy for treatment of cancer
CN107501303A (en) * 2017-08-04 2017-12-22 广西师范大学 Copper (II) complex and its synthetic method and application that a kind of brufen is constructed with the formaldehyde schiff bases of quinoline 8
WO2020023191A1 (en) * 2018-07-09 2020-01-30 Tyme, Inc Tumor reduction formulations and methods of use thereof
CN112584841A (en) * 2018-07-09 2021-03-30 迪美公司 Tumor reduction formulations and methods of use thereof
US11534420B2 (en) 2019-05-14 2022-12-27 Tyme, Inc. Compositions and methods for treating cancer
CN110642875A (en) * 2019-09-05 2020-01-03 广西师范大学 Copper complex constructed based on 8-methoxyquinoline and preparation method and application thereof
CN110642875B (en) * 2019-09-05 2021-02-09 广西师范大学 Copper complex constructed based on 8-methoxyquinoline and preparation method and application thereof
CN110903307A (en) * 2019-10-28 2020-03-24 广西师范大学 Dinuclear metal complex with 9-aldehyde-10-mianthracene hydrazone as ligand and synthetic method and application thereof
CN110903307B (en) * 2019-10-28 2021-05-14 广西师范大学 Dinuclear metal complex with 9-aldehyde-10-mianthracene hydrazone as ligand and synthetic method and application thereof
US11607418B2 (en) 2020-05-14 2023-03-21 Tyme, Inc. Methods of treating SARS-CoV-2 infections

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