CANCER

TECHNICAL FIELD

[0001] The present invention relates to pharmaceutical compositions and methods for treatment of cancer.

BACKGROUND ART

[0002] Cystine/cysteine starvation in target cells makes these cells more vulnerable to oxidative stress, due to inhibition of the X _c ^~ cystine/glutamate antiporter, which mediates exchange of extracellular cystine, i.e., the oxidized form of cysteine, for intracellular glutamate with a stoichiometry of 1: 1. There is an increasing amount of evidence suggesting that the X _c ^~ transporter has an important role in uptake of cystine/cysteine by cancer cells that depend on supply of the amino acid from the microenvironment for growth and viability. Thus, the X _c ^~ transporter has a key role in the in vivo secretion by somatic cells, e.g., activated macrophages and dendritic cells, of cysteine, which can be readily taken up by neighboring cells via, e.g., the universally expressed alanine, serine, and cysteine (ASC) transport system. In contrast, cystine is not readily taken up by cells, and X _c ^~ , if expressed by cancer cells, can directly mediate their uptake of cystine. Intracellularly, cystine is rapidly reduced to cysteine, essential for biosynthesis of proteins and in particular as a rate-limiting substrate of glutathione (GSH), a tripeptide thiol playing an essential role in cellular defense against oxidative stress (Chung et al., 2005).

[0003] GSH, a major cellular scavenger of free radicals, is considered essential for protection of cells from oxidative stress, particularly generated in cancer cells due to their relatively high metabolism. As a rate-limiting GSH precursor, the amino acid cystine/cysteine is of critical importance for maintenance of intracellular levels of GSH and thus has a vital role in the protection of cellular growth and viability. GSH has a short half-life and intracellular cysteine depletion can readily lead to GSH depletion with subsequent growth arrest and reduced defense against oxidative stress. Inhibition of the X _c ^~ transporter leading to cystine/cysteine starvation and subsequent GSH depletion therefore represents a potential therapeutic approach for malignancies dependent on extracellular cystine/cysteine (Guan et al., 2009). [0004] Sulfasalazine (salicylazosulfapyridine, SASP), an anti-inflammatory drug used in clinical therapy of inflammatory bowel disease and rheumatoid arthritis, is a potent X _c ^~ inhibitor. SASP-induced X _c ^~ inhibition can readily lead to cystine starvation of a variety of experimental cancer cell lines, including lymphoma, prostate and breast cancer cell lines, with subsequent reduction of intracellular GSH levels and growth arrest in vitro and in vivo. In addition, it has been shown that GSH plays a role in multidrug resistance (MDR), as it can combine with anticancer drugs to form less toxic and more water-soluble GSH conjugates (a reaction catalyzed by GSH S-transferases), which can then be exported from cells by multidrug-resistant proteins (MRPs) (Narang et al., 2007). There is increasing evidence that GSH depletion in cancer cells can inhibit drug efflux capability of MRPs, leading to increased intracellular accumulation of drugs and reversal of drug resistance. It thus may be assumed that GSH depletion can enhance therapeutic efficacy of anticancer agents and could provide a new strategy urgently needed in cancer therapy. Finally, it has been suggested that sulfasalazine induces apoptosis through direct inhibition of NF-kB (nuclear factor kappa- light-chain-enhancer of activated B cells) activation, also contributing to its anti-cancer potential (Sebens et al., 2008; Hermisson and Weller, 2003).

SUMMARY OF INVENTION

[0005] It has been found, in accordance with the present invention, that a combination of sub-therapeutic doses of sulfasalazine and an additional drug, more particularly thalidomide; the non-steroidal anti-inflammatory drug (NSAID) celecoxib or etodolac; or capecitabine, i.e., a combination of said drugs each in a concentration having alone very low anti-proliferative effect or no such effect at all, has unexpected synergistic antiproliferative effects in cellular models of breast, lung and pancreatic cancers, showing prominent induction of cell death in various cell lines of these cancer types. As further been found, a combination of 40 mg/kg sulfasalazine (i.e., a dose that is 6-12 times lower than the reported effective dose in-vivo), and either 40 mg/kg thalidomide (i.e., a dose that is 5-10 times lower than the reported effective dose in-vivo) or 40 mg/kg etodolac (a dose that is 2-3 time lower than the reported effective dose in vivo), i.e., a combination consisting of sub-therapeutic doses of each one of the drugs, has unexpected synergistic effect in slowing tumor growth in mouse models of lung and pancreatic cancer types.

[0006] In one aspect, the present invention thus provides a pharmaceutical composition for treatment of cancer comprising a pharmaceutically acceptable carrier and a combination consisting of a sulfa drug and an additional drug selected from (i) thalidomide or a thalidomide-like compound; (ii) an NSAID; or (iii) capecitabine, i.e., a pharmaceutical composition consisting, as active agents, of a combination of a sulfa drug and an additional drug as defined above. Such compositions have synergistic anti-proliferative effects in cellular models of various cancers, showing prominent induction of cell death in cell lines of those cancer types. Moreover, such combinations consisting of sub-therapeutic doses of a sulfa drug, and either thalidomide or an NSAID, show synergistic effects in slowing tumor growth in various cancer mouse models. Particular such compositions comprise a drug combination comprising a sub-therapeutic dose of at least one of said drugs, or sub- therapeutic doses of both of said drugs.

[0007] In another aspect, the present invention relates to a method for treatment of cancer in an individual in need thereof, said method comprising concomitantly administering to said individual a combination consisting of a sulfa drug and an additional drug selected from (i) thalidomide or a thalidomide-like compound; (ii) NSAID; or (iii) capecitabine. In certain embodiments, the drug combination administered according this method comprises a sub-therapeutic dose of at least one of said drugs, or sub-therapeutic doses of both of said drugs. In a particular such aspect, the drug combination is administered from a sole pharmaceutical composition, i.e., the method of the invention comprises administering to said individual a pharmaceutical composition as defined above.

[0008] In a further aspect, the present invention relates to a drug combination consisting of a sulfa drug and an additional drug selected from (i) thalidomide or a thalidomide-like compound; (ii) an NSAID; or (iii) capecitabine, for use in treatment of cancer. Particular such combinations comprise a sub-therapeutic dose of at least one of said drugs, or subtherapeutic doses of both of said drugs.

[0009] In another aspect, the present invention relates to the use of a sulfa drug and an additional drug selected from (i) thalidomide or a thalidomide-like compound; (ii) an NSAID; or (iii) capecitabine, for the preparation of a pharmaceutical composition for treatment of cancer.

[0010] In yet another aspect, the present invention provides a kit for use according to the method defined above, said kit comprising (i) a first pharmaceutical composition consisting, as an active agent, of a sulfa drug; (ii) a second pharmaceutical composition consisting, as an active agent, of thalidomide or a thalidomide-like compound, an NSAID, or capecitabine; and (iii) instructions for concomitant administration of said pharmaceutical compositions for treatment of cancer. In particular embodiments, the pharmaceutical compositions constituting said kit comprise a sub-therapeutic dose of at least one of said drugs, or sub-therapeutic doses of both of said drugs.

[0011] In still another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a drug combination consisting of a sulfa drug and an additional drug selected from (i) a thalidomide-like compound; (ii) a non-steroidal anti-inflammatory drug; or (iii) capecitabine, i.e., a pharmaceutical composition consisting, as active agents, of a combination of a sulfa drug and said additional drug, but excluding a composition comprising a combination of either sulfasalazine and celecoxib, or sulfasalazine and thalidomide. Particular such compositions comprise a combination of the two drugs comprising a sub-therapeutic dose of at least one of said drugs, or sub-therapeutic doses of both of said drugs.

BRIEF DESCRIPTION OF DRAWINGS

[0012] Fig. 1 shows the inhibition effect of sulfasalazine (100 μΜ), thalidomide (100 μΜ) and the combination thereof on MCF-7, Calu-6, Capan-1, MDA-468 and MDA-231 cell growth, wherein cells treated without a drug used as a control. As shown, a synergistic effect was observed in all cases, as the inhibition induced by the combination of sulfasalazine and thalidomide was greater than the sum of the inhibitions induced by each one of the drugs alone.

[0013] Fig. 2 shows the inhibition effect of sulfasalazine (50 μΜ), celecoxib (100 μΜ) and the combination thereof on Calu-6 and Capan-1 cell growth, wherein cells treated without a drug used as a control. As shown, a synergistic effect was observed in both cell lines as the inhibition induced by the combination of sulfasalazine and celecoxib was greater than the sum of the inhibitions induced by each one of the drugs alone.

[0014] Fig. 3 shows the inhibition effect of sulfasalazine (100 μΜ), etodolac (100 μΜ) and the combination thereof on MDA-468, MDA-231, Calu-6, A549 and PANC-1 cell growth, wherein cells treated without a drug used as a control. As shown, a synergistic effect was observed in all cases as the inhibition induced by the combination of sulfasalazine and etodolac was greater than the sum of the inhibitions induced by each one of the drugs alone.

[0015] Fig. 4 shows the inhibition effect of sulfasalazine (100 μΜ), capecitabine (100 μΜ ) and the combination thereof on NCI-H23 and PANC-1 cell growth, wherein cells treated without a drug used as a control. As clearly shown, a synergistic effect was observed in both cases as the inhibition induced by the combination of sulfasalazine and capecitabine was greater than the sum of the inhibitions induced by each one of the drugs alone.

[0016] Fig. 5 shows the inhibition effect of the sulfasalazine+etodolac combination on Calu-6 tumor growth, wherein cells treated with the corresponded vehicle used as a control. As shown, inhibition of about 35% of tumor growth was observed after 27 daily IP injections with the combination where each drug was in a dose of 40 mg/kg.

[0017] Fig. 6 shows the inhibition effect of the sulfasalazine+etodolac combination on Capan-1 tumor growth, wherein cells treated with the corresponded vehicle used as a control. As shown, inhibition of about 30% of tumor growth was observed after 18 daily IP injections with the combination where each drug was in a dose of 40 mg/kg.

[0018] Fig. 7 shows the inhibition effect of the sulfasalazine+thalidomide combination on Calu-6 tumor growth, wherein cells treated with the corresponded vehicle used as a control. As shown, inhibition of about 30% of tumor growth was observed after 27 daily IP injections of sulfasalazine and thalidomide, each at 40 mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

[0019] In one aspect, the present invention provides a pharmaceutical composition for treatment of cancer comprising a pharmaceutically acceptable carrier and a drug combination consisting of a sulfa drug and an additional drug selected from (i) thalidomide or a thalidomide-like compound; (ii) an NSAID; or (iii) capecitabine. In other words, the invention provides a pharmaceutical composition for treatment of cancer consisting, as active agents, of a combination of a sulfa drug and an additional drug selected from thalidomide or a thalidomide-like compound, an NSAID, or capecitabine. As shown herein, such compositions have synergistic anti-proliferative effects on cancer cells both in vitro and in vivo.

[0020] The term "sulfa drug" as used herein refers to a synthetic organic compound closely related in chemical structure to sulfanilamide, chemically similar to 4- aminobenzoic acid, also known as para-aminobenzoic acid (PABA), and capable of inhibiting bacterial growth and activity by interfering with the bacterial metabolic processes that require PABA. Examples of sulfa drugs, without being limited to, include sulfasalazine, sulfapyridine, sulfanilamide, sulfathiazole, sulfaguanidine, sulfadiazine, sulfametizol, sulfamethazine, sulfamethoxazole, sulfasoxazole, sulfamonomethoxine, sulfadimethoxine, sulfacetamide, sulfadimidine and sulfachloropyridazine.

[0021] In certain embodiments, the sulfa drug is sulfasalazine. The safety and efficiency of sulfasalazine in treatment of recurrent or progressive high-grade gliomas, in particular anaplastic astrocytoma and glioblastoma, in adults has been studied in a phase 1/2 prospective, randomized clinical study, with daily dose of 1.5-6 grams of oral sulfasalazine; however, the study was early terminated and no clinical response was observed (ISRCTN45828668; Robe et al, 2009). In a different case, a phase 1 dose escalation study in patients with advanced gastric cancer who had received one or more standard chemotherapies was conducted to determine the optimal dose, change in CD44 splice variant expression and intra-tumor level of GSH pre- and post-sulfasalazine exposure and pharmacokinetics (clinical trial information: UMIN000010254). The drug was given orally four times daily (8-12 grams/day) with 2 weeks as one cycle, which were continued until progression of disease, unacceptable toxicity, or other discontinuation criteria were met. 12 grams/day was judged as maximum tolerated dose, and no dose limiting toxicity was observed among patients with 8 grams/day, which was considered the optimal dose (Shitara et al, 2016; clinical trial information: UMIN000010254; htt ://cancerres . aacrj ournal s . org/cort tent/74/ 19 Supplement/211.short) .

[0022] Thalidomide (Thalomid ^® ; a-(N-phthalimido)glutarimide), chemically known as 2- (2,6-dioxo-3-piperidyl)-isoindoline-l,3-dione, is an immunomodulatory agent. The drug was first introduced in 1956 as a sedative agent, but was subsequently withdrawn because of teratogenicity. However, in 1965, the unexpected activity of thalidomide in reactive lepromatous leprosy stimulated further studies, and in 1977, it was approved by the FDA for treatment of erythema nodosum leprosum. This stimulated new interest in thalidomide for the treatment of other inflammatory and autoimmune diseases (see, e.g., Prakash et al, 2011, disclosing the effect of thalidomide in combination with sulfasalazine in experimentally induced inflammatory bowel disease in rats). In addition, thalidomide was used in treating other diseases including AIDS and cancer such as multiple myeloma, glioblastoma, lymphoma, melanoma, renal carcinoma and prostate cancer (van Moos et al, 2003).

[0023] The glutarimide moiety contains a single asymmetric center and may thus exist in either of two optically active forms designated S-(-) and R-(+) with presumed differential activities. Nevertheless, Thalomid ^® is a racemic mixture, i.e., an equal mixture of the S-(-) and R-(+) forms having a net optical rotation of zero, available in 50-200 mg capsules for oral administration. Thalidomide is indicated for treatment of patients with newly diagnosed multiple myeloma, where the drug is typically administered as monotherapy in doses of 200-800 mg/day (Rajkumar et ah, 2001 ; Weber et ah, 2003) or in combination with dexamethasone or other chemotherapies, wherein the dosage recommended is 200 mg administered orally once daily (htlp://mediibrar ^rg lib/rK/meds/thalomid-1 /). The dosage used in clinical trials for treatment of patients with stage III non-small cell lung cancer, in combination with carboplatin, paclitaxel or radiation therapy, begins with 200 mg/day and increased by 100 mg every week as tolerated up to a max of 1000 mg (http://clinicaltrials.gov/ct2/8how NCT00004859); and the dosage used in clinical trials for treatment of metastatic, advanced or recurrent renal cell carcinoma, in combination with 5- fluorouracil (5-FU), interferon-a and interleukin-2 therapy, begins with 200 mg/day and gradually increases up to 1200 mg/day (http://ciinicaltrials.gov/ct2/show/NCT{ )277Q1,7). Li and Huang (2016) describes a clinical study in which the efficacy and toxicity of thalidomide (100 mg/day; given orally), in combination with chemotherapy, was tested in patients with advanced lung cancer, including non- small cell and small cell variants.

[0024] The mechanism of action of thalidomide is not entirely understood and it seems to be related to immune modulation, changes in cytokine levels and angiogenesis inhibition. The immunomodulatory effects of thalidomide are controversially discussed and greatly depend upon the conditions used for examination. These effects include a decrease in circulating CD4 positive T cells and a stimulation of CD8 positive T-cells, leading to a decreased CD4/CD8 ratio. It seems that thalidomide further induces a shift from T helper cell type 1 (Thl) to Th2 T-cell responses, i.e., a change from a cytotoxic T-cell dominated immune response to a mainly antibody mediated immune response, and inhibits T-cell proliferation of stimulated T-cells. In addition, thalidomide seems to block NF-kB-activity, which is a critical transcription factor involved in immune responses and cellular growth. NF-kB can translocate to the nucleus and regulate many genes including the TNFa and IL6 genes. Thalidomide has been described as an angiogenesis inhibitor as early as in 1994, and has a strong anti- angiogenic activity in vascular endothelial growth factor (VEGF)- and basic fibroblast growth factor (bFGF)-induced angiogenesis. These effects are particularly important in the treatment of diseases involving neoformation of blood vessels including most malignancies (Franks et ah, 2004). [0025] The term "thalidomide-like compound" as used herein refers to a thalidomide derivative or a thalidomide structural or functional analog having a thalidomide-like activity. In particular, the thalidomide-like compound may be a thalidomide derivative in which (i) the 2-isoindolinyl-l,3-dione is substituted at position 4, 5, 6 and/or 7 by an electron donating group such as amino, fluoro, chloro, bromo, iodo and hydroxyl; (ii) one of the two carbonyl groups of the 2-isoindolinyl-l ,3-dione is replaced by sulfonyl or the 2- isoindolinyl core has only one carbonyl at position 1 ; (iii) the nitrogen atom of the 2,6- dioxo-3-piperidyl is substituted by hydroxy or Ci-C ₄ (alkyl)morpholino, preferably methylmorpholino; (iv) the 3-piperidyl core has only one carbonyl at position 2; (v) thalidomide is substituted at position 4, 5, 6 or 7 of the 2-isoindolinyl-l,3-dione with glutamine or glutamic acid residue via its a-amino group, wherein said glutamine or glutamic acid residue is further substituted at position γ with additional 2-isoindolinyl-l,3- dione; or (vi) a hydrolysis metabolite of (i)-(v). The thalidomide-like compound may also be a thalidomide structural analog in which (vii) 2-isoindolinyl- l,3-dione is linked to 2,6- dioxo-4-piperidyl instead of to 2,6-dioxo-3-piperidyl, and each one of these radicals, or both, is optionally modified as described in (z)-(v) hereinabove; (viii) the aromatic ring of the 2-isoindolinyl-l,3-dione is replaced by a bicyclic ring such as norbornane; or (ix) a hydrolysis metabolite of (vii)-(viii). The thalidomide-like compound may further be (x) a thalidomide functional analog consisting of two 2-isoindolinyl- l,3-dione radicals, two 1- oxoisoindolin-2-yl radicals, or one of each of these radicals, wherein one of said radicals is linked via its nitrogen atom either to the nitrogen atom or at position 4, 5, 6 or 7 of the other radical; or (xi) a thalidomide functional analog in which l-oxoisoindolin-2-yl radical is linked to l'-methoxycarbonylmethyl 3,4-dimethoxybenzyl.

[0026] Non-limiting examples of thalidomide-like compounds include the thalidomide derivatives: pomalidomide (initially known as CC-4047 and marketed as Actimid™), in which the 2-isoindolinyl-l,3-dione is substituted at position 4 with amino; 4-hydroxy thalidomide and 5-hydroxy thalidomide, in which the 2-isoindolinyl-l,3-dione is substituted at position 4 or 5, respectively, with hydroxy; tetrafluoro-thalidomide, in which the 2-isoindolinyl- l,3-dione is substituted at positions 4, 5, 6 and 7, independently, with fluoro; EM 12, in which the 2-isoindolinyl core has only one carbonyl at position 1; lenalidomide (initially known as CC-5013 and marketed as Revlimid ^® ), in which the 2- isoindolinyl core has only one carbonyl at position 1 and is further substituted at position 4 with amino; N-hydroxy thalidomide, and CG601, in which the nitrogen atom of the 2,6- dioxo-3-piperidyl is substituted by hydroxy and methylmorpholino, respectively; supidimide, in which one of the two carbonyl groups of the 2-isoindolinyl-l,3-dione is replaced by sulfonyl and the 3-piperidyl core has only one carbonyl at position 2; N- phthalylisoglutamine, a thalidomide metabolite obtained by hydrolysis of the amide bond linking the nitrogen atom and the carbonyl at position 6 of the 2,6-dioxo-3-piperidyl; EM240, a metabolite obtained by hydrolysis of the amide bond in the l,2-benzisothiazol-3- one 1,1-dioxide moiety of supidimide; and 3-amino-phthalimidoglutarimide-N-phthalyl isoglutamine, in which the 2-isoindolinyl-l,3-dione is substituted at position 4 with glutamine or glutamic acid residue via its a-amino group, and said glutamine or glutamic acid residue is further substituted at position γ with additional 2-isoindolinyl-l,3-dione; the thalidomide structural analogs: EM 16, in which l-oxoisoindolin-2-yl is linked to 2,6- dioxo-4-piperidyl; CG603, in which the nitrogen atom of the 2,6-dioxo-4-piperidyl is substituted by methylmorpholino; and biglumide, in which the aromatic ring of the 2- isoindolinyl-l,3-dione is replaced by norbornane; and the thalidomide functional analogs: Ν,Ν'-biphthalimide, in which two 2-isoindolinyl-l,3-dione radicals are linked via their nitrogen atoms; N-phthalimidophthalimide, in which a 2-isoindolinyl-l,3-dione radical is linked via its nitrogen atom to position 4 of another 2-isoindolinyl-l,3-dione radical; and CC-3052, in which l-oxoisoindolin-2-yl radical is linked to l'-methoxycarbonylmethyl 3,4-dimethoxybenzyl.

[0027] Cyclooxygenase (COX) catalyzes the synthesis of prostaglandins from arachidonic acid, and COX-2 expression is linked to all stages of carcinogenesis with the enzyme localized to the neoplastic cells, microvascular endothelial cells and stromal fibroblast. The contributions of COX-2 to tumor angiogenesis include the increased expression of the pro-angiogenic VEGF, the production of eicosanoid products, thromboxane A2 (TXA2), prostaglandin E2 (PGE2) and prostacyclin (PGI2) that can directly stimulate endothelial cell migration and growth factor-induced angiogenesis, and potentially, the inhibition of endothelial cell apoptosis by stimulation of Bcl-2 or Akt activation. A growing body of evidence indicates that COX-2 inhibition suppresses tumor growth and neovascularization in different tumor types.

[0028] Celecoxib is a COX-2 selective inhibitor used for pain relief and for reducing inflammation, and it is recently being studied for prevention of several cancers. Combination treatment with celecoxib and epidermal growth factor receptor (EGFR)- selective tyrosine kinase inhibitors inhibited the growth of head and neck squamous cell carcinoma, suggesting that combination therapy with celecoxib and anti- angiogenic/tumoral drugs provides a potential strategy for highly vascularized brain tumor therapy. Celecoxib has a multi-potent activity including anti-proliferative activity to tumor and endothelial cells, and it also inhibits expression of VEGF from tumor cells, whereas UK1 activity is endothelial cell- selective and it elicits rare toxicity in vivo (Mutter et al., 2009; Zhang et al, 2008). Wildy and Wasko (2001) discloses a combination of sulfasalazine and celecoxib for treatment of rheumatoid arthritis.

[0029] The term "non-steroidal anti-inflammatory drug" (NSAID) as used herein refers to any non-steroidal anti-inflammatory drug/agent/analgesic/medicine, and relates to both COX-2 selective inhibitors such as celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib and lumiracoxib, as well as to COX-2 non-selective inhibitors such as etodolac, aspirin, naproxen, ibuprofen, indomethacin, piroxicam and nabumetone.

[0030] In certain embodiments, the COX-2 selective inhibitor is celecoxib. The daily dose of celecoxib recommended in treatment of familial adenomatous polyposis is 800 mg (400 mg twice daily)

(http://www.accessdatafda.gov/drugsatfda_.docs label/2005/020998s0171bl.pdf). The daily dose of celecoxib used in a phase II clinical trial for preoperative treatment of primary breast cancer is 800 mg (400 mg twice daily for 2-3 weeks) (Brandao et al, 2013); the daily dose used in a pilot study of allogeneic tumor cell vaccine with metronomic oral cyclophosphamide and celecoxib in patients undergoing resection of lung and esophageal cancers, thymic neoplasms, and malignant pleural mesotheliomas is 800 mg (400 mg twice daily) (http://clin.ical trial s .gov/ct2/show/NCT() 1143545); and the daily dose used in treating patients with stage I, II or IIIA non- small cell lung cancer is 800 mg (400 mg twice daily for 5 days) http / cU^ A Higher dose of 1200 mg (600 mg twice daily) of celecoxib in combination with erlotinib showed efficacy in phase II trial in patients with advanced NSCLC (Reckamp et al., 2015; https://www.ncbi.nlm.nih.gov/pmc/artjcles/ ⁾ MC48640i 1/). Similar or identical daily dosages of celecoxib were used in studies aimed at determining the biological effect of the drug in pancreatic cancer (Jimeno et al., 2006); verifying the efficacy and safety of the addition of celecoxib to FOLFIRI combination therapy in patients affected by advanced colorectal cancer (Maiello et al., 2006); and determining the maximum tolerated dose and dose-limiting toxicities of bortezomib in combination with celecoxib in patients with advanced solid tumors (Hayslip et al., 2007). [[00003311]] IInn cceerrttaaiinn eemmbbooddiimmeennttss,, tthhee CCOOXX--22 nnoonn--sseelleeccttiivvee iinnhhiibbiittoorr iiss eettooddoollaacc.. TThhee ddaaiillyy ddoossee ooff eettooddoollaacc uusseedd iinn ssaaffeettyy aanndd eeffffiiccaaccyy ssttuuddiieess ooff eettooddoollaacc aaddmmiinniisstteerreedd ttooggeetthheerr wwiitthh cchhlloorraammbbuucciill iinn ppaattiieennttss wwiitthh cchhrroonniicc llyymmpphhooccyyttiicc lleeuukkeemmiiaa ((CCLLLL)),, oorr wwiitthh pprroopprraannoollooll ((aa nnoonnsseelleeccttiivvee bbeettaa bblloocckkeerr)) iinn ppaattiieennttss wwiitthh cclliinniiccaallllyy pprrooggrreessssiivvee pprroossttaattee ccaanncceerr,, iiss 660000 mmgg 55 aanndd 668800 mmgg,, rreessppeeccttiivveellyy ((hhttttpp:://7/cclliinniiccaallttiriiaallss..ggoovv//cctt22 //sshhooww//rreeccoorrdd//NNCCTT0000115511773366;; hhttttpp::////cclliinniiccaallttrriiaallss..ggoovv//cctt22// sshhooww//rreeccoorrdd//NNCCTT0011885577881177));; aanndd tthhee ddaaiillyy ddoossee ooff eettooddoollaacc uusseedd iinn cclliinniiccaall ttrriiaallss ffoorr ppeerriiooppeerraattiivvee aaddmmiinniissttrraattiioonn ooff tthhee ddrruugg aanndd aa bbeettaa bblloocckkeerr ttoo wwoommeenn uunnddeerrggooiinngg bbrreeaasstt ccaanncceerr ssuurrggeerryy,, oorr ffoorr pprreevveennttiioonn ooff ccoolloorreeccttaall ccaanncceerr rreeccuurrrreennccee,, iiss 880000 mmgg ((440000 mmgg ttwwiiccee ddaaiillyy))

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[0032] Capecitabine (5'-deoxy-5-fluoro-N-[(pentyloxy) carbonyl]-cytidine) (Xeloda , Roche) is an orally-administered chemotherapeutic agent used in the treatment of metastatic breast and colorectal cancers with a recommended dose of 3 grams/day or more

15 Capecitabine is a prodrug that is selectively converted to its active metabolite 5-FU by thymidine phosphorylase in tumor cells, where it inhibits DNA synthesis and slows growth of tumor tissue. Although capecitabine and 5-FU have comparable end results, capecitabine offers the advantage of convenience as it can be administered orally.

20 [0033] Both normal and tumor cells metabolize 5-FU to 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) and 5-fluorouridine triphosphate (FUTP) in vivo. These metabolites cause cell injury by two different mechanisms: first, by binding to thymidylate synthetase to form a stable ternary complex that inhibit its activity in DNA synthesis, and second, FUTP, that mimics UTP, can be mistakably incorporated into the RNA thus

25 interfering with RNA processing and protein synthesis. The concentration of thymidine phosphorylase in tumor cells is 3-10 times higher than that in healthy tissues. This can enable selective drug activation of 5-FU at the tumor site and limit systemic toxicity (Malet-Martino and Martino, 2002; Sulkes, 2001).

[0034] The Examples section hereinafter shows that combinations of sulfasalazine with 30 thalidomide, celecoxib, etodolac or capecitabine, consisting of said drugs each in a subtherapeutic dose, i.e., a dose that is insufficient for producing an effect when given alone, are effective in inhibiting the viability of various cancer cell lines, clearly demonstrating a synergistic effect between the two drugs. In vivo experiments have shown that such combinations consisting of sulfasalazine and either thalidomide or etodolac, each in a dose that is sub-therapeutic in tumor growth inhibition according to the literature, cause significant anti-tumor effect, indicating the synergistic effect between the drugs in-vivo.

[0035] Example 1 describes a study testing the effect of sulfasalazine (100 μΜ) in combination with thalidomide (100 μΜ) on the cell viability of various cancer cell lines, in particular, MCF-7, Calu-6, Capan-1, MDA-468 and MDA-231. Fig. 1, showing the cell growth inhibition induced by each one of the drugs or their combination, clearly demonstrates a synergistic effect in all cases in which the drug combination was used, as the inhibition induced by this combination was greater than the sum of the inhibitions induced by each one of the drugs alone.

[0036] Example 2 describes a study testing the effect of sulfasalazine (50 μΜ) in combination with celecoxib (100 μΜ) on the cell viability of Calu-6 and Capan-1 cancer cells. Fig. 2, showing the cell growth inhibition induced by each one of the drugs or their combination, clearly demonstrates a synergistic effect in all cases in which the drug combination was used, as the inhibition induced by this combination was greater than the sum of the inhibitions induced by each one of the drugs alone.

[0037] Example 3 describes a study testing the effect of sulfasalazine (100 μΜ) in combination with etodolac (100 μΜ) on the cell viability of MDA-468, MDA-231, Calu-6, A549 and PANC-1 cancer cells. Fig. 3, showing the cell growth inhibition induced by each one of the drugs or their combination, clearly demonstrates a synergistic effect in all cases in which the drug combination was used, as the inhibition induced by this combination was greater than the sum of the inhibitions induced by each one of the drugs alone.

[0038] Example 4 describes a study testing the effect of sulfasalazine (100 μΜ) in combination with capecitabine (100 μΜ) on the cell viability of various cancer cell lines, in particular, NCI-H23 and PANC-1. Fig. 4, showing the inhibition of cell growth induced by each one of the drugs or their combination, clearly demonstrates a synergistic effect in all cases in which the drug combination was used, as the inhibition induced by this combination was greater than the sum of the inhibitions induced by each one of the drugs alone.

[0039] According to the literature, sulfasalazine, thalidomide and etodolac have been studied as monotherapies in-vivo, and their effective doses in animal models are known. In glioma mouse cancer models, 50 mg/kg of sulfasalazine did not affect tumor growth (Sehm et al., 2016), while doses of 400-500 mg/kg were required to cause inhibition of tumor growth in different mouse models of breast cancer (Timmerman et al, 2013; Doxsee et al, 2007), prostate cancer ( Doxsee et al, 2007) lung cancer (Guan et al, 2009) and pancreatic cancer (Lo et al, 2010). For thalidomide, 200 mg/kg once a day or every other day were required to inhibit tumor growth in animal lung (Lin et al. , 2006) and in pancreatic cancer, respectively (Qiao et al, 2015), while 400 mg/kg once a day were required to induce an inhibitory effect in ovarian cancer animal model (Kobayashi et al, 2005). For etodolac, 100 mg/kg/day inhibited tumor growth in colon cancer animal model (Chen et al, 2003). Surprisingly, Example 5 shows a significant tumor growth inhibition, induced in both lung and pancreatic animal cancer models by combinations of sulfasalazine with either thalidomide or etodolac, in which the dose of each one of said drugs was 40 mg/kg, i.e., clearly and remarkably lower than the dose reported in the literature as effective.

[0040] As described above, the mechanism of action of sulfasalazine involves the inhibition of the X _c ^~ transporter leading to cystine/cysteine starvation and subsequently to GSH depletion. X _c ^~ inhibition can readily lead to cystine starvation of a variety of experimental cancer cell lines with subsequent reduction of intracellular GSH levels and growth arrest in vitro and in vivo. It has further been shown that GSH plays a role in MDR, as it can combine with anticancer drugs to form less toxic and more water-soluble GSH conjugates, which can then be exported from cells by MRPs. There is increasing evidence that GSH depletion in cancer cells can inhibit drug efflux capability of MRPs, leading to increased intracellular accumulation of drugs and reversal of drug resistance.

[0041] As well-known from the literature, in the gastrointestinal tract about 85% of the sulfasalazine is metabolized by intestinal bacteria to sulfapyridine and 5-aminosalicylic acid (5-ASA; also known as mesalazine), and only about 15% of the drug per se, i.e., the unbroken (whole) sulfasalazine molecule, is absorbed into the circulation. Yet, the NF-kB inhibition and cysteine uptake inhibition properties of sulfasalazine are both attributed to the unbroken molecule rather than to one or both of its colonic metabolites (Lo et al, 2010; Chungand and Sontheimer, 2009; Gout et al, 2001; Liptay et al, 1999; Wahl et al, 1998).

[0042] The anti-proliferative effect of sulfasalazine in combination with any of the other active agents shown herein, i.e., thalidomide, celecoxib, etodolac and capecitabine, may have a beneficial additive effect. For instance, the MDR-related properties can enhance the effect of the drugs activity, and the GSH depletion may cause cell death that will be additive to the angiogenesis effect of thalidomide, or the apoptotic effect of the celecoxib. A growing body of evidence suggests that up-regulation of COX-2 and its product, prostaglandin E2 (PGE2), is important in the growth of cancer cells (Hida et ah, 1998; Wolff et ah, 1998), and it is also known that both COX-2 and the prostaglandins derived therefrom play a role in stimulating angiogenesis and apoptosis inhibition, as well as in immune response suppression (Choy and Milas, 2003). Thus, the growth arrest effect by sulfasalazine and the apoptotic effect of celecoxib may be additive, and a combination of the two agents will have an improved effect leading to increased anti-cancer activity. Similarly, in the case of thalidomide, either the angiogenesis effect or the NF-kB inhibition effect can be additive to sulfasalazine's mechanism of action to add to the combined anticancer effect. These evidence and observation provide a good reason to use these combinations in cancer therapy. However, we have surprisingly found that not only do these drugs have an additive effect, as may be expected, but they have a strong synergistic effect. As in fact shown herein, when we use doses of the drugs that individually have very little effect or no effect at all, the combination has an activity that is significantly greater than the expected additive effect. These observations imply that we could obtain the same or better therapeutic effect with significantly lower doses than currently used when the drugs are administered individually or in combination.

[0043] The results shown herein imply that combinations of a sulfa drug, e.g., sulfasalazine, and an additional drug as defined herein, including such combinations comprising a sub-therapeutic dose of one or both of said active agents, are highly efficient in treatment of cancer and may further be used for potentiation of current chemotherapeutic regimens. It is further expected that the in-vivo potency of combinations consisting of a sulfa drug and either thalidomide or a thalidomide-like compound will significantly increase as additional mechanisms such as anti-angiogenesis are activated.

[0044] The pharmaceutical composition of the present invention may comprise any combination of the two drugs, wherein preferred such combinations are those comprising a sub-therapeutic dose of at least one of said drugs, or sub-therapeutic doses of both of said drugs. Pharmaceutical compositions according to the invention may thus comprise, e.g., a sub-therapeutic dose of the sulfa drug and a therapeutic dose of the other drug being selected from thalidomide, a thalidomide-like compound, an NSAID, or capecitabine; or a therapeutic dose of the sulfa drug and a sub-therapeutic dose of the other drug; or subtherapeutic doses of both the sulfa drug and the other drug.

[0045] The term "therapeutic dose" as commonly used in the art with respect to a drug, refers to the amount or dose of said drug, when used alone for treating a particular indication, that is sufficient to produce, after a single administration or consecutive administrations, a therapeutic response or desired effect. A therapeutic dose of a drug thus refers to any dose falling within the therapeutic window of said drug, i.e., the range between the minimum effective dose and the maximum tolerated dose (highest possible but still tolerable dose level with respect to a pre-specified clinical limiting toxicity) of said drug, e.g., the dose of said drug recommended by regulation authorities for treatment of said indication. Examples of therapeutic doses of thalidomide and capecitabine, recommended in treatment of various cancer types, are mentioned above. However, for sulfasalazine, celecoxib and etodolac, no doses have yet been approved as effective, and only doses currently being tested in clinical trials, for which efficacy has not yet been shown, are known and depicted above. The term "sub-therapeutic dose" as used herein with respect to a drug thus refers to any amount or dose of said drug that is lower than that recommended by regulation authorities for treatment of a particular indication, or than that shown, e.g., in a clinical trial, to produce a therapeutic response or desired effect when used alone for treatment of said indication. In cases a particular drug has different therapeutic doses when used for treatment of different cancer types (e.g., a first therapeutic dose for treating a first cancer type, and a second therapeutic dose for treating a second cancer type), the term "sub-therapeutic dose" refers to an amount or dose of said drug that is lower than said first therapeutic dose when used for treating said first cancer type, or lower than said second therapeutic dose when used for treating said second cancer type.

[0046] Thus, (i) a therapeutic dose of sulfasalazine would be any amount of this drug that is equal to or higher than the minimal (lowest) dose producing a therapeutic response or desired effect when used alone for treatment of the particular cancer type to be treated with the composition of the invention, e.g., equal to or higher than 8 grams/day assuming these doses show efficacy in the clinical trials currently in progress, and a sub-therapeutic dose of sulfasalazine would be any amount of this drug that is lower than the minimal dose found to be effective in treating said cancer type, e.g., lower than 8 grams/day; (ii) a therapeutic dose of thalidomide would be any amount of this drug that is equal to or higher than 200 mg/day, and a sub-therapeutic dose of thalidomide would be any amount of this drug that is lower than 200 mg/day; (iii) a therapeutic dose of celecoxib would be any amount of this drug that is equal to or higher than the minimal dose producing a therapeutic response or desired effect when used alone for treatment of the particular cancer type to be treated with the composition of the invention, e.g., equal to or higher than 800 mg/day assuming this drug dose shows efficacy in the clinical trials currently in progress, and a sub-therapeutic dose of celecoxib would be any amount of this drug that is lower than the minimal dose found to be effective in treating said cancer type, e.g., lower than 800 mg/day; (iv) a therapeutic dose of etodolac would be any amount of this drug that is equal to or higher than the minimal dose producing a therapeutic response or desired effect when used alone for treatment of the particular cancer type to be treated with the composition of the invention, e.g., equal to or higher than 600-800 mg/day assuming these drug doses show efficacy in the clinical trials currently in progress, and a sub-therapeutic dose of etodolac would be any amount of this drug that is lower than the minimal dose found to be effective in treating said cancer type, e.g., lower than 600-800 mg/day (depending on the cancer type to be treated); and (v) a therapeutic dose of capecitabine would be any amount of this drug that is equal to or higher than 3 grams/day, and a subtherapeutic dose of this drug would be any amount of capecitabine that is lower than 3 grams/day.

[0047] In certain particular embodiments, the pharmaceutical composition of the invention comprises (i) a sub-therapeutic dose of sulfasalazine; and a therapeutic dose of thalidomide, celecoxib, etodolac or capecitabine; or (ii) a therapeutic dose of sulfasalazine; and a sub-therapeutic dose of thalidomide, celecoxib, etodolac or capecitabine; or (iii) a sub-therapeutic dose of sulfasalazine; and a sub-therapeutic dose of thalidomide, celecoxib, etodolac or capecitabine.

[0048] In other particular embodiments, the pharmaceutical composition of the invention comprises sulfasalazine in a dose ranging from 0.1 to 15 grams/day, e.g., 0.1-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.5-4.0, 4.0-4.5, 4.5-5.0, 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, 9.5-10.0, 10.0-10.5, 10.5-11.0, 11.0-11.5, 11.5-12.0, 12.0-12.5, 12.5-13.0, 13.0-13.5, 13.5-14.0, 14.0-14.5 or 14.5-15.0 grams/day; and (i) thalidomide in a dose ranging from 10 to 600 mg/day, e.g., 10 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, 450 to 500, 500 to 550 or 550 to 600 mg/day; or (ii) celecoxib in a dose ranging from 10 to 1200 mg/day, e.g., 10 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, 450 to 500, 500 to 550, 550 to 600, 600 to 650, 650 to 700, 700 to 750, 750 to 800, 800 to 850 or 850 to 900, 900 to 950, 950 to 1000, 1000 to 1050, 1050 to 1100, 1100 to 1150 or 1150 to 1200 mg/day; or (iii) etodolac in a dose ranging from 10 to 1200 mg/day, e.g., 10 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, 450 to 500, 500 to 550, 550 to 600, 600 to 650, 650 to 700, 700 to 750, 750 to 800, 800 to 850, 850 to 900, 900 to 950, 950 to 1000, 1000 to 1050, 1050 to 1100, 1100 to 1150 or 1150 to 1200 mg/day; or (iv) capecitabine in a dose ranging from 0.1 to 6 grams/day, e.g., 0.1-0.5, 0.5-1.0, 1.0-1.5, 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.5-4.0, 4.0-4.5, 4.5-5.0, 5.0-5.5 or 5.5-6.0 grams/day.

[0049] The pharmaceutical compositions of the present invention may also constitute any molar ratio of the two active agents. The phrase "molar ratio of the sulfa drug to the thalidomide or thalidomide-like compound, the non-steroidal anti-inflammatory drug, or capecitabine" used herein means the total molar amount of the sulfa drug in relation to the total molar amount of the other drug, i.e., the thalidomide or thalidomide-like compound, the NSAID or capecitabine, in the combination of these two drugs.

[0050] In certain embodiments, the pharmaceutical composition of the present invention comprises a fixed dose combination of the sulfa drug and the other drug, i.e., thalidomide, thalidomide-like compound, NSAID, or capecitabine, as defined above. In particular such compositions, the molar ratio of the sulfa drug to the other drug is in the range of 100: 1 to 1: 100. The term "fixed dose combination" as used herein refers to a formulation of the two drugs defined above combined in a single dosage form available in certain fixed doses, so as to improve medication compliance by reducing the pill burden of patients.

[0051] In certain embodiments, the pharmaceutical composition of the present invention comprises a fixed dose combination of a sulfa drug, e.g., sulfasalazine, with thalidomide. In particular such embodiments, the molar ratio of the sulfa drug to thalidomide is about 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 100: 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10, 1: 12, 1: 14, 1: 16, 1: 18, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1: 100.

[0052] In certain embodiments, the pharmaceutical composition of the present invention comprises a fixed dose combination of a sulfa drug, e.g., sulfasalazine, with celecoxib or etodolac. In particular such embodiments, the molar ratio of the sulfa drug to celecoxib or etodolac is about 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 100: 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10, 1: 12, 1: 14, 1: 16, 1: 18, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1: 100.

[0053] In certain embodiments, the pharmaceutical composition of the present invention comprises a fixed dose combination of a sulfa drug, e.g., sulfasalazine, with capecitabine. In particular such embodiments, the molar ratio of the sulfa drug to capecitabine is about 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60:1, 70: 1, 80: 1, 90: 1, 100: 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10, 1: 12, 1: 14, 1: 16, 1:18, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1: 100.

[0054] As well-known to any person skilled in the art, for a multi drug-based treatment, both the exact ratio between the drugs administered as well as the timing, dosing and pharmacokinetic aspects, play an extremely important role. In other words, in order to determine the optimal fixed dose combination, not only the combined/synergistic efficacy and potency are of importance, but also the relative pharmacokinetics of each drug and the optimal formulation. Thus, in certain embodiments, the molar ratio of the two drugs contained within the pharmaceutical composition of the present invention is precisely calibrated and the two drugs are preferably formulated in a single dosage form designed for optimal pharmacokinetic performance and efficacy and for patient's compliance. Moreover, the synergistic effects between the drugs in the combination may depend on the time they have to act together in the body, i.e., on the relative release profile of the drugs determined by the formulation of each one of the drugs in the combination.

[0055] According to the present invention, each one of the drugs comprised within the pharmaceutical composition may thus independently be formulated for immediate release, controlled, i.e., sustained, extended or prolonged, release, or both immediate and controlled release. Moreover, considering that only about 15% of the sulfasalazine administered orally is absorbed into the circulation, whereas about 85% of the drug degrades to its colonic metabolites; and that the NF-kB inhibition and cysteine uptake inhibition properties of sulfasalazine are both attributed to the unbroken molecule rather than to one or both of said metabolites, a pharmaceutical composition as defined above, comprising sulfasalazine formulated for controlled release, may be highly effective in maintaining a constant desired level of the drug in the circulation.

[0056] In certain embodiments, at least one of the drugs comprised within the pharmaceutical composition, or both of said drugs, is/are formulated for controlled release, e.g., either in a microencapsulated dosage form or controlled-release matrix. In particular such embodiments, the pharmaceutical composition of the invention comprises a combination, e.g., a fixed dose combination, consisting of (i) controlled release sulfasalazine, and controlled release thalidomide, celecoxib, etodolac or capecitabine; (ii) controlled release sulfasalazine, and immediate release thalidomide, celecoxib, etodolac or or capecitabine; (iii) immediate release sulfasalazine, and controlled release thalidomide, celecoxib, etodolac or capecitabine; (iv) immediate release sulfasalazine, and immediate release thalidomide, celecoxib, etodolac or capecitabine; or (v) controlled and immediate release sulfasalazine, and controlled and immediate release thalidomide, celecoxib, etodolac or capecitabine.

[0057] The pharmaceutical compositions of the present invention may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 19 ^th Ed., 1995. The compositions may be in solid, semisolid or liquid form and may further include pharmaceutically acceptable fillers, carriers or diluents, and other inert ingredients and excipients. The compositions can be administered by any suitable route, e.g., orally, intravenously, parenterally, rectally or transdermally, the oral route being preferred. The dosage will depend on the state of the patient, and will be determined as deemed appropriate by the practitioner.

[0058] The pharmaceutical compositions of the invention, when formulated for oral administration, may be in any suitable form, e.g., tablets such as matrix tablets, in which the release of a soluble active is controlled by having the active diffuse through a gel formed after the swelling of a hydrophilic polymer brought into contact with dissolving liquid (in vitro) or gastro-intestinal fluid (in vivo). Many polymers have been described as capable of forming such gel, e.g., derivatives of cellulose, in particular the cellulose ethers such as hydroxypropyl cellulose, hydroxymethyl cellulose, methylcellulose or methyl hydroxypropyl cellulose, and among the different commercial grades of these ethers are those showing fairly high viscosity. The pharmaceutical compositions may also be in the form of bi- or multi-layer tablets, made up of two or more distinct layers of granulation compressed together with the individual layers lying one on top of another, with each separate layer containing a different active agent. Bilayer tablets have the appearance of a sandwich since the edge of each layer or zone is exposed.

[0059] As stated above, the pharmaceutical compositions of the present invention may comprise one or both of the active agents formulated for controlled release, e.g., in a microencapsulated dosage form wherein small droplets of the active agent(s) are surrounded by a coating or a membrane to form particles in the range of a few micrometers to a few millimeters, or in a controlled-release matrix.

[0060] In certain embodiments, the present invention provides a pharmaceutical composition for oral administration, which is solid and may be in the form of granulate, granules, grains, beads or pellets, mixed and filled into capsules or sachets, or compressed to tablets by conventional methods. In certain embodiments, the pharmaceutical composition is in the form of a bi- or multilayer tablet, in which each one of the layers comprise one of the two active agents, and the layers are optionally separated by an intermediate, inactive layer, e.g., a layer comprising one or more disintegrants.

[0061] Another contemplated formulation is depot systems, based on biodegradable polymers. As the polymer degrades, the active agent is slowly released. The most common class of biodegradable polymers is the hydrolytically labile polyesters prepared from lactic acid, glycolic acid, or combinations of these two molecules. Polymers prepared from these individual monomers include poly (D,L-lactide) (PLA), poly (glycolide) (PGA), and the copolymer poly (D,L-lactide-co-glycolide) (PLG).

[0062] Useful dosage forms of the pharmaceutical compositions include orally disintegrating systems including, but not limited to, solid, semi-solid and liquid systems including disintegrating or dissolving tablets, soft or hard capsules, gels, fast dispersing dosage forms, controlled dispersing dosage forms, caplets, films, wafers, ovules, granules, buccal/mucoadhesive patches, powders, freeze dried (lyophilized) wafers, chewable tablets which disintegrate with saliva in the buccal/mouth cavity and combinations thereof. Useful films include, but are not limited to, single layer stand-alone films and dry multiple layer stand-alone films.

[0063] Another useful dosage form is a long lasting injectable system, such as a liposomal gel consisting of, e.g., poloxamer 407 and a liposomal solution containing the actives.

[0064] The two active agents of the composition may also be formulated in a pellets dosage form (capsules) with different release patterns: one agent for immediate release and the other for controlled release, or each of the agents both for immediate and controlled release where about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the dose is for controlled release and the remaining for immediate release.

[0065] The pharmaceutical composition of the invention may comprise one or more pharmaceutically acceptable excipients. For example, a tablet may comprise at least one filler, e.g., lactose, ethylcellulose, microcrystalline cellulose, silicified microcrystalline cellulose; at least one disintegrant, e.g., cross-linked polyvinylpyrrolidinone; at least one binder, e.g., polyvinylpyridone, hydroxypropylmethyl cellulose; at least one surfactant, e.g., sodium laurylsulfate; at least one glidant, e.g., colloidal silicon dioxide; and at least one lubricant, e.g., magnesium stearate. [0066] In another aspect, the present invention relates to a method for treatment of cancer in an individual in need thereof, said method comprising concomitantly administering to said individual a combination consisting of a sulfa drug and an additional drug selected from (i) thalidomide, a thalidomide-like compound; (ii) an NSAID; or (iii) capecitabine, each as defined above.

[0067] In certain embodiments, the drug combination administered according to the method of the invention comprises a sub-therapeutic dose of at least one of said drugs, or sub-therapeutic doses of both of said drugs, more particularly: (i) a sub-therapeutic dose of the sulfa drug and a therapeutic dose of the other drug being selected from thalidomide, a thalidomide-like compound, an NSAID, or capecitabine; or (ii) a therapeutic dose of the sulfa drug and a sub -therapeutic dose of the other drug; or (iii) sub-therapeutic doses of both the sulfa drug and the other drug. In other embodiments, the drug combination administered according to this method is a fixed dose combination of the sulfa drug and the other drug, i.e., thalidomide, thalidomide-like compound, NSAID, or capecitabine, e.g., wherein the molar ratio of the sulfa drug to the other drug is in the range of 100: 1 to 1: 100. In further embodiments, at least one of the drugs administered according to this method, or both of said drugs, is/are formulated for controlled release.

[0068] In a particular such aspect, the method of the invention comprises concomitantly administering to said individual a drug combination as defined above, wherein said drug combination is administered from a sole pharmaceutical composition, i.e., said method comprises administration of a pharmaceutical composition as defined in any one of the embodiments above, e.g., a pharmaceutical composition comprising, as active agents, sulfasalazine and one of thalidomide, celecoxib, etodolac, or capecitabine.

[0069] In a further aspect, the present invention relates to a drug combination consisting of a sulfa drug and an additional drug selected from (i) thalidomide or a thalidomide-like compound; (ii) an NSAID; or (iii) capecitabine, each as defined above, for use in treatment of cancer.

[0070] In certain embodiments, the drug combination of the invention comprises a subtherapeutic dose of at least one of said drugs, or sub-therapeutic doses of both of said drugs, more particularly: (i) a sub -therapeutic dose of the sulfa drug and a therapeutic dose of the other drug being selected from thalidomide, a thalidomide-like compound, an NSAID, or capecitabine; or (ii) a therapeutic dose of the sulfa drug and a sub-therapeutic dose of the other drug; or (iii) sub-therapeutic doses of both the sulfa drug and the other drug. In other embodiments, said drug combination is a fixed dose combination of the sulfa drug and the other drug, i.e., thalidomide, thalidomide-like compound, NSAID, or capecitabine, e.g., wherein the molar ratio of the sulfa drug to the other drug is in the range of 100: 1 to 1: 100. In further embodiments, at least one of the drugs in said drug combination, or both of said drugs, is/are formulated for controlled release. In particular embodiments, such drug combinations consist of sulfasalazine and one of thalidomide, celecoxib, etodolac, or capecitabine.

[0071] In another aspect, the present invention relates to the use of a sulfa drug and an additional drug selected from (i) thalidomide or a thalidomide-like compound; (ii) a NSAID; or (iii) capecitabine, each as defined above, for the preparation of a pharmaceutical composition for treatment of cancer.

[0072] In yet another aspect, the present invention provides a kit for use according to the method defined above, said kit comprising (i) a first pharmaceutical composition consisting, as an active agent, of a sulfa drug as defined above; (ii) a second pharmaceutical composition consisting, as an active agent, of thalidomide or a thalidomide-like compound, an NSAID, or capecitabine, each as defined above; and (iii) instructions for concomitant administration of said pharmaceutical compositions for treatment of cancer.

[0073] In certain embodiments, the first pharmaceutical composition comprised within the kit of the invention comprises a sub-therapeutic dose of said sulfa drug; or the second pharmaceutical composition comprised within the kit of the invention comprises a subtherapeutic dose of said thalidomide, a thalidomide-like compound, an NSAID, or capecitabine; or each one of said drugs are present in a sub-therapeutic dose. In other embodiments, the dosage of the active agent in said first pharmaceutical composition and the dosage of the active agent in said second pharmaceutical composition represent a fixed dose combination of said sulfa drug and said thalidomide, thalidomide-like compound, NSAID, or capecitabine, e.g., wherein the molar ratio of the sulfa drug to the other drug is in the range of 100: 1 to 1: 100. In further embodiments, at least one of the drugs, or both of said drugs, is/are formulated for controlled release. In particular embodiments, such kits comprise a first pharmaceutical composition consisting, as an active agent, of sulfasalazine, and a second pharmaceutical composition consisting, as an active agent, of thalidomide, celecoxib, etodolac, or capecitabine. [0074] In still another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a combination consisting of a sulfa drug and an additional drug selected from (i) a thalidomide-like compound; (ii) a non-steroidal anti-inflammatory drug; or (iii) capecitabine, each as defined above, i.e., a pharmaceutical composition consisting, as active agents, of a combination of a sulfa drug and said additional drug, but excluding a composition comprising the combination of sulfasalazine and either celecoxib or thalidomide. Particular such compositions comprise a combination of the two drugs comprising a sub-therapeutic dose of at least one of said drugs, or sub-therapeutic doses of both of said drugs.

[0075] Unless otherwise indicated, all numbers expressing, e.g., molar or quantitative ratios of the two active agents defined above, used in this specification are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification are approximations that may vary by up to plus or minus 10% depending upon the desired properties to be obtained by the present invention.

[0076] The invention will now be illustrated by the following non-limiting Examples.

EXAMPLES

Experimental

[0077] Cell and reagents. A549 (human lung adenocarcinoma), Capan 1 (human pancreatic adenocarcinoma), Calu-6 (human anaplastic carcinoma of the lung), MCF-7 (human breast adenocarcinoma), MDA-MB-468 (human breast adenocarcinoma), MDA- MB-231 (human breast adenocarcinoma, from metastatic site), NCI-H23 (non-small cell lung cancer, NSCLC, adenocarcinoma) and Panc-1 (human pancreatic epithelioid carcinoma) cells were obtained from the ATCC. A549 cells were maintained in F12K medium (Gibco) containing 10% bovine serum, glutamine and antibiotics. Capan- 1 cells were maintained in Iscove's Modified Dulbecco's Medium (IMDM, Biological industries) containing 20% bovine serum, glutamine and antibiotics. Calu-6 and MCF-7 cells were grown in Eagle s Minimum Essential Medium (EMEM, Biological industries) containing 10% bovine serum, glutamine and antibiotics. Panc-1 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Biological industries) containing 10% bovine serum, glutamine and antibiotics. NCI-H23 cells were grown in Roswell Park Memorial Institute (RPMI) medium 1640 (Biological industries). All cell lines were maintained at 37°C and 5% C0 ₂ .

[0078] Cell viability assay for testing potentiated drug combinations for cancer therapy .

For cytotoxicity experiment, A549 and NCI-H23 cells were plated 5x10 cells/well, while Capan-1, Calu-6 and MCF7 cells were plated lOxlO ³ , 7.5xl0 ³ and 15xl0 ³ cells/well, respectively, on 96 wells tissue culture plates (Costar). Twenty-four hours after plating, the medium was exchanged with fresh growth medium with or without the tested drug. Every drug was added in various concentrations alone and in combination with another drug. The drugs for combination assay were added simultaneously to the culture plate. Cell viability was determined after 72 hours of incubation with the tested drug or drug combination by staining using methylene blue method. Cells first fixed by 0.5% glutaraldehyde for 10 minutes at room temperature, followed by washes with double distilled (dd) H ₂ 0 and incubation with 0.1M borate buffer PH=8.5 before stained with 2% methylene blue solution for 2 hours. Excess methylene blue stain was washed away from the plate by extensive washing in ddH ₂ 0, and the samples were dried overnight at room temperature. Color extraction was done by addition of 50 μΐ of 0.1N HCL to each sample and optical density of the samples was quantified using Elisa reader at 620 nm. The percent inhibition was calculated as the ratio between the OD of cells treated with drugs or drug combinations to the OD of control untreated cells, wherein this ratio was subtracted from 100% to give the actual percent inhibition of each treatment.

[0079] Animals. Females, 6 weeks old, HSD: Athymic Nude mice (Envigo, Harlan Laboratories, Israel) were used to develop both Calu-6 and Capan xenograft models. Body weight at study initiation was 14.0-16.5 gram and the minimum and maximum weight within each group was not exceeding +20% of the group. Animals were allowed to acclimatize and recover from shipping-related stress for 7-13 days before used.

[0080] Tumor implantation. Cells were suspended in phosphate-buffered saline, and 5xl0 ⁶ cells in 200 μΐ were implanted subcutaneously in the right flank of each mouse using 27G needle. Once palpable tumors were established, animals were randomized so that all groups had similar starting mean tumor volumes of 50-80 mm . Example 1. A combination of sulfasalazine and thalidomide has a synergistic effect on various cancer cells viability

[0081] MCF-7, Calu-6, Capan-1, MDA-468 and MDA-231 cells were plated on 96 wells tissue culture plates as described in Experimental, and 24 hours after plating, the medium was exchanged with fresh growth medium containing 100 μΜ thalidomide, 100 μΜ sulfasalazine or the combination thereof (100 μΜ thalidomide+100 μΜ sulfasalazine). Cells treated without a drug used as a control. Cell viability was determined after 72 hours of incubation by staining using methylene blue method as described in Experimental.

[0082] Fig. 1 shows the cell growth inhibition induced by thalidomide, sulfasalazine and the combination thereof, in the five cancer cell lines tested. As clearly shown, a synergistic effect was observed in all cases as the inhibition induced by the drug combination was greater than the sum of the inhibitions induced by each one of the drugs alone.

Example 2. A combination of sulfasalazine and celecoxib has a synergistic effect on various cancer cells viability

[0083] Calu-6 and Capan-1 cells were plated on 96 wells tissue culture plates as described in Experimental, and 24 hours after plating, the medium was exchanged with fresh growth medium containing 100 μΜ celecoxib, 50 μΜ sulfasalazine or the combination thereof (100 μΜ celecoxib+50 μΜ sulfasalazine). Cells treated without a drug used as a control. Cell viability was determined after 72 hours of incubation by staining using methylene blue method as described in Experimental.

[0084] Fig. 2 shows the cell growth inhibition induced by celecoxib, sulfasalazine and the combination thereof, in the two cancer cell lines tested. As clearly shown, a synergistic effect was observed in both cases as the inhibition induced by the drug combination was greater than the sum of the inhibitions induced by each one of the drugs alone. Example 3. A combination of sulfasalazine and etodolac has a synergistic effect on various cancer cells viability

[0085] MDA-468, MDA-231, Calu-6, A549 and PANC-1 cells were plated on 96 wells tissue culture plates as described in Experimental, and 24 hours after plating, the medium was exchanged with fresh growth medium containing 100 μΜ etodolac, 100 μΜ sulfasalazine or the combination thereof (100 μΜ etodolac+100 μΜ sulfasalazine). Cells treated without a drug used as a control. Cell viability was determined after 72 hours of incubation by staining using methylene blue method as described in Experimental.

[0086] Fig. 3 shows the cell growth inhibition induced by etodolac, sulfasalazine and the combination thereof, in the five cancer cell lines tested. As clearly shown, a synergistic effect was observed in all cases as the inhibition induced by the drug combination was greater than the sum of the inhibitions induced by each one of the drugs alone.

Example 4. A combination of sulfasalazine and capecitabine has a synergistic effect on pancreatic and NSCLC cancer cells viability

[0087] NCI-H23 and PANC-1 cells were plated on 96 wells tissue culture plates as described in Experimental, and 24 hours after plating, the medium was exchanged with fresh growth medium containing 100 μΜ capecitabine, 100 μΜ sulfasalazine or the combination thereof (100 μΜ capecitabine+100 μΜ sulfasalazine). Cells treated without a drug used as a control. Cell viability was determined after 72 hours of incubation by staining using methylene blue method as described in Experimental.

[0088] Fig. 4 shows the cell growth inhibition induced by capecitabine, sulfasalazine and the combination thereof, in the two cancer cell lines tested. As clearly shown, a synergistic effect was observed in the presented cases as the inhibition induced by the drug combination was greater than the sum of the inhibitions induced by each one of the drugs alone. Example 5. A combination of sulfasalazine and either etodolac or thalidomide inhibits pancreatic cancer and NSCLC growth in subcutaneous xenograft cancer models

Test drugs and vehicles

[0089] Vehicle A contains 10% DMSO and 90% PEG ₄₀₀ pH 7.5; Vehicle B contains 0.1N NaOH/PBS pH 12; Vehicle C contains 30% ethanol and 70% propylene glycol, pH 6.8.

[0090] Sulfasalazine obtained from Tecoland Batch No: 20151129 was prepared in stock solution of 20 mg/ml in Vehicle B and kept stable at room temperature for two weeks. Thalidomide obtained from Olon s.p.a. Batch Nr.: 13001PR10A was freshly prepared every day in Vehicle A to final concentration of 10 mg/ml. Etodolac Micronized USP/EP obtained from Taro Batch No: S 156170/2 was prepared in stock solution of 20 mg/ml in Vehicle C and kept stable at room temperature for two weeks. [0091] In this study, two in vivo subcutaneous xenograft cancer models were used in order to evaluate the effect of a combination therapy on tumor growth: Capan-1 model for pancreatic cancer and Calu-6 for non-small cell lung (NSCLC) cancer. The drug combinations evaluated in each one of the models were sulfasalazine and etodolac; and sulfasalazine and thalidomide.

[0092] Drug treatment in both models was started when the tumors reached mean size of

75 mm 3 (range of 50-80 mm 3 ). The compounds (40 mg/kg of each) were daily administered (7 days a week) by IP injections. Animals from group 1 (n=10) were administered daily with a total volume of 100 μΐ sulfasalazine+etodolac mixed together in a 1: 1 ratio just before injections. Final concentration of the sulfasalazine and etodolac was 10 mg/ml. Animals from group 3 (n=10) were administered with the corresponding vehicles (C+B) which were premixed and ready for injection. Animals from group 2 (n=10) were injected daily with 100 μΐ of sulfasalazin and then with 100 μΐ thalidomide at concentration of 10 mg/ml each. Animals from group 4 (n=10) were administered with the corresponding vehicles (B+A) injected in the same manner as group 2.

[0093] Tumor measurements and mouse weights were taken three times per week. Tumor size was measured using caliper. The longest (L) and perpendicular (S) diameter were measured and the tumor volume (V) was calculated using the formula: V=[LxS ]/2. At the end of the experiment, 24 hours after the last treatment, the mice were sacrificed with C0 ₂ and the tumors were isolated, weighted and photographed.

[0094] Fig. 5 shows the inhibition effect of the sulfasalazine+etodolac combination on Calu-6 tumor growth, wherein cells treated with the corresponded vehicle used as a control (see Example 5). As shown, inhibition of about 35% of tumor growth was observed after 27 daily IP injections with the combination where each drug was in a dose of 40 mg/kg.

[0095] Fig. 6 shows the inhibition effect of the same sulfasalazine+etodolac combination on Capan-1 tumor growth, wherein cells treated with the corresponded vehicle used as a control (see Example 5). As shown, inhibition of about 30% of tumor growth was observed after 18 daily IP injections with the combination where each drug was in a dose of 40 mg/kg.

[0096] Fig. 7 shows the inhibition effect of the sulfasalazine+thalidomide combination on Calu-6 tumor growth, wherein cells treated with the corresponded vehicle used as a control (see Example 5). As shown, inhibition of about 30% of tumor growth was observed after 27 daily IP injections of sulfasalazine and thalidomide, each at 40 mg/kg. REFERENCES

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