Epigallocatechin-3-gallate Compositions for Cancer Therapy and

Chemoprotection Field of Invention

This invention relates to methods and compositions comprising Epigallocatechin-3-gallate (EGCG) or analogues and derivatives thereof which are useful in the inhibition of tumour cell growth.

Background of Invention

Adenosine is a potent endogenous regulator of a variety of physiological processes through specific receptors on the cell surface. Adenosine binds to four different types of G protein- coupled cell surface molecules, termed the Al, A2A, A2B, and A3 adenosine receptors . The AlAR and A3AR are coupled to Gi protein and cause inhibition of adenylate cyclase, whereas the A2AR, which consists of the A2A and A2B receptor subtypes, is coupled to Gs protein and causes an increase in adenylate cyclase activity. The binding of adenosine to cell surface receptors alters immune cell production of soluble mediators such as cytokines, free radicals, and arachidonic acid metabolites (Linden, 2001) .

However, the utilization of adenosine as a therapeutic agent is restricted, as its rapid metabolization to inosine and AMP limits its ability to exert a systemic effect . Pharmacological studies carried out by Fishman's group (Fishman et al . 2001) indicated that agonists to the A3 adenosine receptors (A3AR) act similarly to adenosine, while having the advantage of being stable, non- degradable and bioavailable molecules. The binding of adenosine to A3AR inhibits NF-KB activation through a pathway that involves a decrease on cAMP, the inhibition of PKA and downregulation of the PKB/Akt arm (Fishman et al . , 2004; Delghandi et al . , 2005).

Therefore, activation of A3AR by the natural ligand or by synthetic agonist induces an inhibitory effect on tumour cell growth. The finding that these receptors are over expressed in tumour cells indicates that a molecule targeting them could be a promising candidate for cancer chemotherapy. The results, first described by

Fishman (Madi et al . , 2004), have been confirmed in our laboratory- using normal and cancer tissues from breast and colon cancer. For example, we found that in 86-90% of the pairs analyzed (n = 30) a 2- 3 fold increase in A3AR occurred in cancer versus normal tissues. Figure 1 shows the comparative results in breast tissues.

Recently, tea polyphenol (-) -epigallocatechin-3-gallate (EGCG) has been shown to be an efficient inhibitor of human dihydrofolate reductase (DHFR) (Navarro-Peran et al . , 2005) . As with other antifolate compounds, EGCG acts by disturbing the folic acid metabolism in cells, causing the inhibition of DNA and RNA synthesis, alteration of DNA methylation and modulation of cell signalling pathways.

Summary of the Invention

The present inventors have now discovered that EGCG compounds exert a synergistic effect in combination with A3 adenosine receptor agonists and/or thymidylate synthase inhibitors in the inhibition of the growth of tumour cells.

Aspects of the present invention provide compositions of matter and methods of treatment of tumor cells comprising an effective amount of EGCG, or a derivative thereof, and an effective amount of either (a) an A3 adenosine receptor agonist or (b) thymidylate synthase inhibitor, or (c) both an A3 adenosine receptor agonist and a thymidylate synthase inhibitor.

Brief Description of the Drawings

Figure 1 shows western blot analysis of A3AR in normal (N) and tumoral (T) breast tissues.

Figure 2 shows the effect of EGCG on TNF-α mediated activation of NF- _K B. Effect of different treatments on TNF-α mediated degradation of IKBOC. Caco-2 cells were treated with vehicle only or the specified concentration of EGCG for 24 h, and at the times

specified, cells were treated with TNF-α. Treatments included ξGCG alone (E) or combined with leucovorin (EL) , APCP (EAPCP) or DMPX (EDMPX) . IκBα protein expression was assessed by immunoblot analysis in the cytosolic fraction using β-actin as an internal control. The data shown here are from a representative experiment repeated three times with similar results. The columns represent the mean of these experiments; bars, ± SD.

Figure 3 shows Akt and p-Akt levels in the cytoplasm of Caco-2 treated for 24 h with different concentrations of EGCG in the absence (E) or the presence of leucovorin (EL) , and then with and without TNF-α. The data shown here are from a representative experiment repeated three times with similar results. The columns represent the mean of three experiments; bars, ± SD.

Figure 4 shows the effect of EGCG on the expression of A3AR in the surface of Caco-2 cells. A: Fluorescence confocal microscopy of Caco-2 after 3 days of treatments with vehicle only (NT) , or 20 μM EGCG in the absence (E) or presence of leucovorin (EL) . B: Induction folds of A3AR calculated by both fluorescence confocal microscopy (three separate experiments) and western blot analysis (five separate experiments) . Bars represent the means of the eight experiments carried out by both techniques ± SD.

Figure 5 shows the time-course of the effect of individual and combined treatments on the growth of Caco-2 cells.

Figure 6 shows the individual effect of EGCG (100 μM) and 5-FU (50 μM) or in combination on Caco-2 growth.

Figure 7 shows the effect of hypoxanthine (H, 100 μM) and thymine (T, 100 μM) on the growth inhibition of Caco-2 cells by EGCG (20 μM) after 3 days of treatment. The data are expressed assuming 100% growth for the untreated control. Bars represent the average growth

for three individual experiments, and the error bars represent the SD of the data.

Figure 8 shows the effect of different treatment on Caco-2 growth. EGCG (E; 20 μM) or adenosine (Ado; 10 μM) alone or EGCG (20 μM) combinations with adenosine (10 μM) , APCP (50 μM) and ADA (10 μg/ml) . Growth was determined at 4 days of treatment and the data expressed assuming 100% growth for the untreated control. Bars represent the average growth for three independent experiments, and the error bars represent the SD of the data. *P < 0.0001 compared with corresponding values for individual treatments with EGCG or adenosine .

Figure 9A shows the structure of EGCG and figure 9B shows the structure of ECG.

Figure 10 shows examples of EGCG compounds.

Figure 11 shows alkaline phosphatase (ALP) activity induction relative to control (C) in Caco-2 cells following treatment with

EGCG 20 μM (E) alone or in combination with either leucovorin 100 μM (L) , or HT medium (HT) , or thymidine 100 μM (T) , or hypoxanthine 100 μM (H), or adenosine 10 μM (A) for 5 days.

Detailed Description

One aspect of the invention provides a composition comprising an EGCG compound and a) an A3 adenosine receptor agonist or (b) thymidylate synthase inhibitor, or (c) both an adenosine receptor agonist and a thymidylate synthase inhibitor.

An A3 adenosine receptor agonist is a compound which binds and activates an A3 adenosine receptor (A3AR or ADORA3 ; MIM: 600445 GenelD: 140, reference sequence isoform 2 NP_000668.1 GI: 4501953, reference sequence isoform 1, NP_065734.5 GI: 46559769).

Preferably, the A3 adenosine receptor agonist is specific for the A3 adenosine receptor and shows little or now binding or activation of other adenosine receptors, such as the Al, A2A or A2B receptor. For example, an A3 adenosine receptor agonist may show at least 100 fold, at least 500 fold or al least 1000 fold greater activation of the A3 adenosine receptor relative to the Al, A2A or A2B receptor.

A suitable A3AR agonist may be selected from the group consisting of if- (3-iodobenzyl) -5 ' -N-methylcarboxamidoadenosine (IB-MECA) , Cl-IB- MECA (2-ChIOrO-JV ⁶ - (3-iodobenzyl) -5 ' -W-methylcarboxamidoadenosine) , 4-thio-2-chloro-J\7 ⁶ - (3-iodobenzyl) -5 ' -.W-methylcarboxamidoadenosine, if- (4-aminobenzyl) -5 ' -iV-methylcarboxamidoadenosine (AB-MECA) , 5ι-N- ethylcarboxamidoadenosine (NECA) and 2-chloro-4 ' -thioadenosine-5 ' - methyluronamide and esters, salts and prodrugs of any of these.

Assays suitable for the identification of A3AR agonists are well- known in the art .

A thymidylate synthase inhibitor is a compound which inhibits or reduces the activity of thymidylate synthase (TYMS, MIM: 188350; GenelD: 7298; EC 2.1.1.45; reference sequence NP_001062.1 GI: 4507751) . Preferably, the thymidylate synthase inhibitor is specific for thymidylate synthase and shows little or now binding or activation of other related enzymes . Assays suitable for the identification of thymidylate synthase inhibitors are well-known in the art.

A suitable thymidylate synthase inhibitor may be selected from the group consisting of fluorouracil (5-FU) , raltitrexed (tornudex) , nolatrexed, LY231514, ZD9331, N ¹⁰ -propargyl-5, 8-dideazafolic acid (CB3717) , ZD1694, AG337 and esters, salts and prodrugs of any of these.

Prodrugs of 5-FU are compounds which are metabolised to 5-FU in vivo and include 5 ' -deoxy-5-fluorouridine (doxifluridine) ,

fluorodeoxyuridine, tegafur, l-tetrahydrofuranyl-5-fluorouracil, capecitabine (Xeloda) and S-I (BMS-247616) .

Other 5-FU pro-drugs are described, for example, in Wang et al Curr Eye Res 1991 Jan; 10 (1) : 87-97, Shibaraoto Int J Rad One 58 2 397-402 Y, Taylor et al J Pharm Sci 87 1 15-20 and Malet-Martino et al The Oncologist 2002 7 4 288-323.

EGCG compounds include both unmodified (-) -epigallocatechin gallate (EGCG) and modified forms of EGCG, for example, analogues, variants and derivatives of EGCG. Suitable analogues, variants and derivatives may comprise a gallate moiety, or a moiety with an analogous structure. A suitable gallate moiety may be ester bonded. In some embodiments, the EGCG compound may be a polyphenol, for example a flavanoid, such as a flavan-3-ol.

A modified form of EGCG may have the structure of EGCG with one or more modifications. Modifications to the EGCG structure may include removal or replacement of the non-ester trihydroxybenzene moiety, removal or methylation of one or more hydroxyl groups of the ester bonded gallate moiety, introduction of a hetero atom instead of the carbon atom between the two hydroxyl groups of the catechol ring of the EGCG structure and the addition or substitution of one or more atoms or groups in the EGCG structure with one or more of hydrogen,- an optionally substituted Ci _-7 alkyl group; a C _3-20 heterocyclyl group; a C _5-20 aryl group; an optionally substituted heterocyclic ring having from 4 to 8 ring atoms,- or one or more of the following substituent groups: Halo: -F, -Cl, -Br, and -I; Hydroxy: -OH; Ether: -OR, C _1-7 alkoxy: -OR, wherein R is a C _1-7 alkyl group; C _1-2 alkdioxylene ; Oxo (keto, -one) : =0; Imino (imine) : =NR; Formyl :

C(=O)H; Acyl (keto): -C(=O)R; Ester: -C(=O)0R; Acyloxy: -OC(=O)R; Amido: -Ct=O)NR ¹ R ² ; Acylamido: -NR ¹ Cf=O)R ² ;

Thioamido: -Cf=S)NR ¹ R ² ; Tetrazolyl; • Amino: -NR ¹ R ² ; Amidine: - C(=NR)NR ₂ ; Nitro: -NO ₂ ; Nitroso: -NO; Azido: -N ₃ ; Cyano: -CN; Isocyano: -NC; Cyanato: -OCN; Isocyanato: -NCO; Thiocyano: -SCN;

Isothiocyano (isothiocyanato) : -NCS; SuIfhydryl : -SH; Thioether: -SR; Disulfide: -SS-R; SuIfone: -S(=O) ₂ R; SuIfine: -S(=O)R; Sulfonyloxy: -OSt=O) ₂ R; Sulfinyloxy: -OS(=O)R; SuIfamino: -NR ¹ Sf=O) ₂ OH; Sulfonamino: -NR ¹ Sl=O) ₂ R; SuIfinamino: -NR ¹ Sl=O)R, SuIfamyl: -Sl=O)NR ¹ R ² ; Phosphoramidite : -OP (OR ¹ ) -NR ² ₂ ; and Phosphoramidate : -OP (=0) (OR ¹ J-NR ² _Z .

C _1-7 alkyl is a monovalent moiety obtained by removing a hydrogen atom from a C _1-7 hydrocarbon compound having from 1 to 7 carbon atoms, which may be aliphatic or alicyclic, or a combination thereof, and which may be saturated, partially unsaturated, or fully unsaturated.

C _3-20 heterocyclyl is a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a C _3-2O heterocyclic compound, said compound having one ring, or two or more rings (e.g., spiro, fused, bridged), and having from 3 to 20 ring atoms, atoms, of which from 1 to 10 are ring heteroatoms, and wherein at least one of said ring(s) is a heterocyclic ring. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms. "C _3-20 " denotes ring atoms , whether carbon atoms or heteroatoms .

C _5-20 aryl is a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of a C _5-20 aromatic compound, said compound having one ring, or two or more rings (e.g. fused), and having from 5 to 20 ring atoms, and wherein at least one of said ring(s) is an aromatic ring. Preferably, each ring has from 5 to 7 ring atoms. The ring atoms may be all carbon atoms or the ring atoms may include one or more heteroatoms, for example oxygen, nitrogen, and sulphur.

In many cases, substituents may themselves be substituted. For example a C _1-7 alkyl group, a C _3-20 heterocyclyl group, a C _5-20 aryl group, or heterocyclic ring as described above may comprise one or more substituent groups .

In some embodiments, the ester bond of EGCG may be replaced by an amide or other linkage (Figure 10B) .

Examples of EGCG compounds are shown in Figures 9B, IOA and 1OB. Other suitable EGCG compounds and substituents are described in Anderson et al Bioorg. & Med. Chem. Lett (2005) 15 2533-2635, Anderson et al Tetrahedron (2005) , Lam et al Bioorg Med Chem 2004 Nov 1; 12(21) :5587-93, Waleh et al Anticancer Res 2005 Jan-Feb; 25 (IA) :397-402 and US7109236.

A preferred EGCG compound for use in accordance with the present methods is EGCG or a salt, ester or prodrug thereof. The structure of EGCG is set out in Figure 9A.

A composition as described above may be a pharmaceutical composition which further comprises a pharmaceutically acceptable excipient. Such a composition may be produced by formulating an EGCG compound with a) an A3 adenosine receptor agonist or (b) thymidylate synthase inhibitor, or (c) both an A3 adenosine receptor agonist and a thymidylate synthase inhibitor, and a pharmaceutically acceptable excipient .

The term "pharmaceutically acceptable" as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

Another aspect of the invention provides an EGCG compound and a) an A3 adenosine receptor agonist or (b) thymidylate synthase inhibitor, or (c) both an A3 adenosine receptor agonist and a thymidylate synthase inhibitor, for example in a composition as described above, for use in method of treatment of the human or animal body, for example in the treatment of cancer .

Another aspect of the invention provides the use of an EGCG compound and a) an A3 adenosine receptor agonist or (b) thymidylate synthase inhibitor, or (c) both an A3 adenosine receptor agonist and a thymidylate synthase inhibitor in the manufacture of a medicament for use in the treatment of cancer.

Another aspect of the invention provides a method of treating cancer comprising administering an EGCG compound and a) an A3 adenosine receptor agonist or (b) thymidylate synthase inhibitor, or (c) both an A3 adenosine receptor agonist and a thymidylate synthase inhibitor to an individual in need thereof .

The EGCG compound, A3 adenosine receptor agonist and/or thymidylate synthase inhibitor may be administered simultaneously or sequentially, by the same or different routes of administration according to an appropriate treatment regimen under the direction of a medical practitioner.

Cancer suitable for treatment as described herein may be any type of solid or non-solid cancer or malignant lymphoma and especially leukaemia, sarcomas, skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, oesophageal cancer, pancreatic cancer, renal cancer, stomach cancer and cerebral cancer. Cancers may be familial or sporadic.

A individual suitable for treatment as described herein may include a eukaryote, an animal, a vertebrate animal, a mammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse) , a murine (e.g. a mouse), a canine (e.g. a dog), a feline (e.g. a cat), an equine (e.g. a horse) , a primate, such as a simian (e.g. a monkey or ape) , a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, gibbon) , or a human.

The term "treatment", as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e. prophylaxis) is also included.

While it is possible for the active compounds (e.g. an EGCG compound and/or a) an A3 adenosine receptor agonist or (b) thymidylate synthase inhibitor, or (c) both an A3 adenosine receptor agonist and a thymidylate synthase inhibitor) to be administered alone, it is preferable to present them as one or more pharmaceutical compositions (e.g., formulation) comprising at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.

Pharmaceutical compositions comprising an EGCG compound and/or a) an A3 adenosine receptor agonist or (b) thymidylate synthase inhibitor, or (c) both an A3 adenosine receptor agonist and a thymidylate synthase inhibitor, for example, an inhibitor admixed or formulated together with one or more pharmaceutically acceptable carriers,

excipients, buffers, adjuvants, stabilisers, or other materials, as described herein, may be used in the methods described herein.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a ¹ carrier which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.

The compounds or pharmaceutical composition comprising the compounds may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of ■ desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal ; by implant. of a depot, for example, subcutaneously or intramuscularly.

Formulations suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or

tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

A tablet may be made by conventional means, e.g., compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); and preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid) . Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile . Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Formulations suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal) , include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-

aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 μg/ml, for example, from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi- dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.

It will be appreciated that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side- effects.

Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment . Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

In general, a suitable dose of the active compound is in the range of about 100 μg to about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

Another aspect of the invention provides a method of screening for a compound for use in combination with an EGCG compound in the treatment of cancer comprising: contacting a test compound with an A3AR polypeptide and; determining the activity of the A3AR in the presence relative to the test compound.

An increase in activity of the A3AR polypeptide in the presence relative to the absence of test compound is indicative that the compound is useful in combination with an EGCG compound in the treatment of cancer.

A suitable A3AR polypeptide may have the amino acid sequence of NCBI database entry NP_000668.1 GI: 4501953 or NCBI database entry NP 065734.5 GI: 46559769, or may be a variant thereof.

The A3AR polypeptide is preferably expressed on the surface of a cell.

The activity of the A3AR polypeptide may be determined by any convenient technique. For example, A3AR activity may be determined by measuring the level or amount of phosphorylation of extracellular signal-regulated kinase (ERKl/2) in a cell (Schulte MoI Pharm (2002) 62, 5, 1137-1146) or by measuring the amount of forskolin-stimulated cAMP accumulation (Salvatore et al Proc Natl Acad Sci U S A. 1993 November 1; 90(21): 10365-10369).

Another aspect of the invention provides a method of screening for a compound for use in combination with an EGCG compound in the treatment of cancer comprising: contacting a test compound with thymidylate synthase, and; determining the activity of the thymidylate synthase in the presence relative to the test compound,

An increase in activity in the presence relative to the absence of test compound is indicative that the compound is useful in combination with an EGCG compound in the treatment of cancer.

A suitable thymidylate synthase may have the amino acid sequence of NCBI database entry sequence NP_001062.1 GI: 4507751 or may be a variant thereof .

Thymidylate synthase activity may be determined by any convenient technique. For example, a labelled fluorodeoxyuridylic acid (FdUMP) binding assay may be employed (Jenh et al (1985) J. Cell Physiol. 122 149-154) .

A polypeptide which is a variant of a wild-type or reference sequence may comprise an amino acid sequence which shares greater than about 60% sequence identity with the wild-type sequence, greater than about 70%, greater than about 80%, greater than about

90% or greater than about 95%. The sequence may share greater than about 60% similarity with the wild-type kinase sequence, greater than about 70% similarity, greater than about 80% similarity or greater than about 90% similarity.

Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Genetics Computer Group, Madison, WI) . GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al . (1990) J. MoI. Biol. 215: 405- 410) , FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and

Waterman (1981) J. MoI Biol. 147: 195-197), or the TBLASTN program, of Altschul et al . (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used. Sequence identity and similarity may also be determined using GenomequestTM software (Gene-IT, Worcester MA USA) .

Sequence comparisons are preferably made over the full-length of the relevant sequence described herein.

Similarity allows for "conservative variation" where one amino acid is substituted for another amino acid of similar chemical structure and may have no effect on the protein function, e.g. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.

The effect of amino acid substitution on a protein function depends on the role of the particular residue in protein activity. Using

established techniques, such as in vitro mutagenesis, it is routine to test whether particular amino acids are necessary for protein function.

The precise format for performing the methods described herein may be varied by those of skill in the art using routine skill and knowledge .

Compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms which contain several characterised or uncharacterised components may also be used.

Combinatorial library technology provides an efficient way of testing a potentially vast number of different compounds for ability to modulate an interaction. Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others. The use of peptide libraries may be preferred in certain circumstances.

The amount of test compound or compound which may be added to a method of the invention will normally be determined by serial dilution experiments. Typically, from about 0.001 nM to 1 mM or more of putative inhibitor compound may be used, for example from 0.01 nM to lOOμM, e.g. 0.1 to 50 μM, such as about 10 μM.

A method may comprise identifying the test compound as an inhibitor of thymidylate synthase or an A3AR agonist. Such a compound may, for example, be useful in inhibiting the growth or proliferation of cancer cells, for example in the treatment of cancer, as described herein.

A test compound identified using one or more initial screens as having ability to activate the A3AR receptor or inhibit thymidylate

synthase may be assessed further using one or more secondary- screens. A secondary screen may, for example, involve testing for a' biological function such as an effect on tumour growth, proliferation or metastasis in an animal model in combination with an EGCG compound.

The test compound may be isolated and/or purified or alternatively, it may be synthesised using conventional techniques of recombinant expression or chemical synthesis. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals, in combination with an EGCG compound, for the treatment of a cancer condition. Methods of the invention may thus comprise formulating the test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier for therapeutic application, as discussed above.

Following identification of a compound which activates the A3AR receptor or inhibits thymidylate synthase and may therefore be useful in increasing the sensitivity of cancer cells to EGCG compounds, a method may further comprise modifying the compound to optimise the pharmaceutical properties thereof .

A compound may be optimised by making modifications to the structure, for example, by adding molecular scaffolding, adding or varying functional groups, or connecting the molecule with other molecules (e.g. using a fragment linking approach) such that the chemical structure of the modulator molecule is changed while its original modulating functionality is maintained or enhanced. Such optimisation is regularly undertaken during drug development programmes to e.g. enhance potency, promote pharmacological acceptability, increase chemical stability etc. of lead compounds.

Modifications to a compound structure will be those conventional in the art known to the skilled medicinal chemist, and will include, for example, substitutions or removal of groups containing residues which interact with the target receptor or enzyme. For example, the replacements may include the addition or removal of groups in order to decrease or increase the charge of a group in a test compound, the replacement of a charge group with a group of the opposite charge, or the replacement of a hydrophobic group with a hydrophilic group or vice versa.

As described above, a compound identified and/or obtained using the present methods may be formulated into a pharmaceutical composition.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure .

All documents mentioned in this specification and the sequences and other contents of database entries recited in this specification are hereby incorporated herein by reference .

The invention encompasses each and every combination and sub- combination of the features that are described above.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above and tables described below.

Table 1 shows the effect of EGCG and MTX on thymidine incorporation in caco-2 cells.

Examples

Whether EGCG inhibits NF-κB activation through the release of adenosine, by disturbing folate metabolism was investigated in our

laboratory by co-treating Caco-2 cells with EGCG and a competitive inhibitor of ecto-5' nucleotidase (α, β-methylene adenosine-5' - diphosphate; APCP) or EGCG with an antagonist of the A2AAR (3,7- dimethyl-1-propargylxanthine; DMPX) . The results show that by inhibiting adenosine production or by blocking its binding to A2AAR, it was possible to reverse the EGCG-mediated suppression of NF-KB activation in the presence of TNF-α (Figure 2) .

We next investigated the link between adenosine production and EGCG- mediated suppression of NF-KB activation in the presence of TNF-α by studying the levels of Akt activation in treated cells (Figure 3) . It has been proposed that TNF-α activates the PI3K/PDK-l/Akt signalling pathway (Ozes et al . , 1999) . This pathway culminates in the phosphorylation of IKKα by Akt, which is necessary for IκBα degradation and NF-KB activation. EGCG was found to inhibit Akt phosphorylation, while co-treatment of cells with EGCG and leucovorin restored cell p-Akt . Although adenosine, at low concentrations, binds preferentially to the high affinity AlAR, it seems that in these conditions its signalling pathway is switched off (Cutolo et al . , 2001). Activation of A2AAR produces a constellation of effects that can attenuate inflammation. An increase of intracellular cAMP would have an inhibitory effect on the P13K/PDKl/Akt signalling pathway by blocking coupling between Akt and its upstream regulator, PDKl, in the plasma membrane (Kim et al., 2001) . Inactivation of the PI3K/PDK1/Akt signalling pathway would affect multiple components of the apoptotic cascade such as caspases, GSK-3β, ceramide, BAD/Bcl-2, CREB, and NF-KB (Song et al . , 2005) . Thus, EGCG may induce apoptosis by regulating multiple molecules in the Akt and NF-KB pathway. The suppression of TNF-α- induced NF-κB-mediated gene transcription may also downregulate several genes involved in inflammation, angiogenesis and metastasis, including COX-2, iNOS, MMP-9, cell surface adhesion molecules (e.g. ICAM-I, E-selectin, and VCAM-I), urokinase-type plasminogen activator, TNF-α, IL-I, IL-2, IL-6, and GM-CSF. Downregulation of

these genes has been described in several cell models treated with EGCG or MTX (Mello et al . , 2000; Hussain et al . , 2005; Robbins et al . , 1998; Lin and Lin, 1997; Kim and Moon, 2005; Gingras and Beliveau, 2004; Johnston et al.,-2005).

We investigated the constitutive activation of NF-KB in the absence of TNF-α. It has been show that NF-KB is constitutively activated in human colorectal carcinoma tissue (Yu et al . , 2004) . Here we show that EGCG differentially modulates NF-KB under these conditions in a time-dependent manner. After treatment of Caco-2 cells with 20 μM

EGCG for one day, NF-KB was activated by more than 50% compared with an untreated control . This activation was effectively reversed by co-treatment with leucovorin or APCP, but DMPX had the opposite effect, potentiating this activation. These results clearly show that the adenosine induced by EGCG treatment was responsible for such activation, and that a different signalling pathway operates in the absence of TNF-α. By binding to AlAR, adenosine activates NF-KB though a pathway that involves the decrease of cAMP, the liberation of calcium from endoplasmic reticulum and activation of the PKC pathway (Basheer et al . , 2001) . This pathway was the dominant event when the antagonist DMPX was present in the culture medium together with EGCG, demonstrating the implication of both AlAR and A2AAR in this short-term response to EGCG. However, the lack of NF-KB activation after longer treatments (i.e. three days) with the same EGCG concentration cannot be explained by the involvement of these two adenosine receptors. A third type of receptor, A3AR, has also been implicated in NF-KB modulation. Recently, it has been shown that an A3AR agonist inhibits colon carcinoma growth by modulation of GSK-3β and NF-KB (Fishman et al . , 2004) . We therefore analyzed the levels of adenosine receptors in the plasmatic membranes of

Caco-2 cells and their response to EGCG treatment by both Western blot analysis and confocal microscopy. EGCG differentially modulated expression of adenosine subtype receptors . Although AlAR and A2AR were not significantly increased after 3 days' treatment with 20 μM

EGCG, we found that A3AR did show significant increase after this time (Figure 4) . This A3AR increase was reversed by co-treatment of the cells with EGCG plus leucovorin (Figure 4) . The data clearly indicate that the A3AR signalling pathway is the predominant event in more prolonged treatments with EGCG, and its action was able to suppress even the constitutive activation of NF-KB in colon cancer cells. The binding of adenosine to A3AR inhibits NF-KB activation through a pathway that involves a decrease on cAMP, the inhibition of PKA and downregulation of the PKB/Akt arm (Fishman et al . , 2004; Delghandi et al . , 2005) . PKA and PKB/Akt utilize GSK-3β as a substrate, upon phosphorylation, GSK-3β activity is inhibited. The latter has been widely implicated in cell homeostasis, for its ability to phosphorylate a broad range of substrates, including β- catenin, a key component of the Wnt pathway. In normal cells, GSK-3β phosphorylates β-catenin, thereby inducing its ubiquitination and degradation by the proteosome system. However, in tumor cells, GSK- 3β fails to phosphorylate β-catenin, leading to its accumulation in the cytoplasm. It then translocates to the nucleus, where it acts in concert with LEF-I to induce the transcription of the cell cycle progression genes such as cyclin Dl and c-Myc (Fishman et al . ,

2004) . Downregulation of these two oncogenes by EGCG has been widely described (Huh et al . , 2004; Ju et al . , 2005) . Activation of A3AR by EGCG treatment would suppress the activation of cyclin Dl and c-Myc, leading to cell cycle arrest and induction of apoptosis (Fishman et al . , 2004). Moreover, the PKA signalling cascade is also connected with several pathways that have been shown to be affected by EGCG, such as the MAPKs including extracellular regulated kinases 1/2 (ERK1/2) or p38 (Sah et al . , 2004) . By modulating the PKA cascade, EGCG can interfere with pathways involved in glucose utilization and lipolysis (Gonzalez-Yanes and Sanchez-Margalet, 2006) , which justifies its traditional inclusion in weight loss diets.

Combinations of EGCG with A3AR agonists were tested and found to have a synergistic action. We treated Caco-2 cells with different

concentrations of EGCG in the absence or in the presence of IB-MECA. Time-course results showed that combinations of EGCG and IB-MECA inhibited the growth of Caco-2 cells to a greater extent than individual treatments or the sum of individual treatments (Figure 5) . The synergy of both compounds on the inhibition of growth of cancer (tumor) cells is evident from the results of Figure 5. Low concentrations of IB-MECA (100 nM) , which had no effect on the growth of Caco-2, inhibited the growth of these cells by more than 50% in the presence of 50 μM of EGCG. The statistical analysis of the data showed that the values differ significantly from values without IB-MECA. It is anticipated that this is a general effect that will be achieved to greater or lesser extents depending on the EGCG derivative used and the specific A3AR agonist utilized in a given treatment regiment .

Fluoropyrimidine 5-fluorouracil (5-FU) is an antimetabolite drug that is widely used for the treatment of cancer, particularly for colorectal cancer. 5-FU exerts its anticancer effects through inhibition of thymidylate synthase (TS) and incorporation of its metabolites into RNA and DNA. Modulation strategies, such as co- treatment with leucovorin and methotrexate, have been developed to increase the anticancer activity of 5-FU. The effects of combination of EGCG and 5-FU was investigated. The synergy of both drugs on Caco-2 growth is apparent in Figure 6. Combinations of EGCG and derivatives thereof with other thymidylate synthase inhibitors are therefore likely to exhibit enhanced inhibition of tumor cell growth to that observed in the combination of EGCG and 5-FU.

EGCG disturbs DNA and RNA synthesis in Caco-2 cells. To determine the effects of EGCG on DNA synthesis we have studied the degree of incorporation of [3H] -Thymidine in the DNA of cells subject to treatments with EGCG and MTX. To avoid the inhibition of DNA and RNA synthesis cold hypoxanthine was added to the cell medium. The effects of antifolates on [3H] -Thymidine incorporation on DNA is well known21. By inhibiting DHFR, these drugs block the de

novo methylation of dUMP to TMP. Thus, the addition, of MTX to cancer cells enhances the incorporation of exogenous [3H] -Thymidine into DNA (Table I) . A similar effect was observed by treating the cells with EGCG (Table I) , demonstrating that this drug disturb DNA synthesis.

Demonstration of the effect of EGCG on RNA synthesis was obtained with co-treatment of EGCG with hypoxanthine or thymine . Although single treatment with thymine showed low reversion of EGCG cell growth inhibition, hypoxanthine showed an opposite effect, resulting in enhanced EGCG cytotoxicity (Fig. 7) . These results are completely consistent with DHFR being the site of action of EGCG. By inhibiting DHFR, antifolates deplete cellular stores of reduced folates, resulting in the inhibition of DNA and RNA synthesis. Inhibition of RNA synthesis arrests cells in the Gl phase of the cell cycle, preventing such cells from entering S phase and rendering them insensitive to antifolates. It has been described that hypoxanthine potentates MTX cytotoxicity by maintaining RNA synthesis, allowing cells that might be arrested in Gl to progress into the cytotoxic S phase .

Adenosine is required for EGCG cytotoxicity

Next, we evaluated the contribution of the blockage of the purine biosynthesis pathway and the involvement of adenosine on the inhibition of Caco-2 growth by EGCG. First, we observed that adenosine had a little inhibitory effect on cell growth but this effect was significantly enhanced by co-treatment with EGCG, acting both compound in a synergistic manner (Fig. 8) . To establish that EGCG suppressed Caco-2 growing by enhancing adenosine release, we designed a series of experiments orientated to deplete extracellular adenosine. Caco-2 cells were treated with EGCG in the presence of ADA or APCP, a competitive inhibitor of ecto-5' nucleotidase, and both treatments were able to completely reverse EGCG effect (Fig. 8) . These results provide indication that the inhibitory effect of EGCG on Caco-2 growth is mediated by adenosine. However, adenosine

alone did not inhibit cell growth. The data indicate that induction of A ₃ AR in EGCG treated cells was required for adenosine toxicity.

The epithelial cells of the mammalian intestinal tract are hierarchically arranged so that cells become more differentiated as they ascend the crypt-villus axis. The process is maintained and regulated by a population of intestinal stem cells, which reside within the crypts of the small and large intestine and generate all the intestinal epithelial lineages. Intestinal differentiation is accompanied by changes in morphology, temporal and spatialspecific expression of genes, such as the brush border enzyme alkaline phosphatase (ALP) , and is regulated by a myriad of biochemical pathways. Aberrations in the biochemical signalling cascades within stem cells play an integral part in the development of gastrointestinal malignancy. Several compounds, such as the antifolate methotrexate (MTX) , can induce differentiation in colon and other cancer cells. The overall process of differentiation involves the reversion of malignant tumour cells into more benign forms, in which cellular proliferation is lowered resulting in reduced tumor growth.

Caco-2 cells were treated for 4 days with 20 μM EGCG, 5 days with 10 μM EGCG and 6 days with 5 μM EGCG. EGCG treatment was found to result in enterocyte-like differentiation.

To determine whether DHFR inhibition caused the observed EGCG induced intestinal cell differentiation, we co-administered EGCG with either leucovorin (100 mM) , or hypoxanthine-thymidine (HT) medium, or thymidine (100 μM) , or hypoxanthine (100 μM) or adenosine (10 μM) to the colon cancer cell line, Caco-2 (Fig. 11) . The cells were harvested after 5 days, lysed and the extracts assayed for brush-border enzyme ALP activity. Both leucovorin and HT medium treatments resulted in ALP activity induction reversion. Interestingly, hypoxanthine supplementation reversed more ALP induction than thymidine supplementation. In contrast, EGCG co-

administration with adenosine (Fig. 11) enhanced ALP activity. The effect of EGCG on cellular differentiation is shown by these results to be mainly due to folate cycle disruption as a consequence of DHFR inhibition. Adenosine is shown to act synergically with EGCG on cellular differentiation.

These findings together with the observed synergy between adenosine and EGCG against colon cancer cells indicated that combined therapies with EGCG and adenosine agonists, such as IB-MECA, may be a promising strategy for the treatment of cancer.

[ ³ H] -Thy [ ³ H] -Thy incorporated

Addition incorporated Cell number x P ^{er cel1}

(dpm) ^a 10 ^" ( dpm/cell )

None 7814 ± 275 9.6 ± 0.6 0.08 ± 0.01

EGCG (20 μM) 11564 ± 310 8.3 ± 0.5 0.14 + 0.01

MTX ( 1 μM) 13140 ± 500 7.3 + 0.6 0.18 ± 0.01 ^a Results are the mean ± SD of five determinations from three independent experiments .

Table 1

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