WO2011130486A2 - Combination treatments and formulations for cancer - Google Patents

Abstract

This invention relates to methods of treating cancer in a subject afflicted therewith comprising periodically administering to the subject an amount of phospho-valproic acid, phospho- ibuprofen, phospho-sulindac, P-A2, or a pharmaceutically acceptable salt thereof, together with a bioavailability enhancer, cimetidine, QW-3. curcumin, DFMO, or P-A1 in amounts effective to treat cancer in the subject.

Description

COMBINATION TREATMENTS AND FORMULATIONS FOR CANCER This application claims priority of U.S. Provisional Application No. 61/325,203, filed April 16, 2010, the contents of which are hereby incorporated by reference.

This invention was made with government support under grant numbers CA092423 and CA139454 awarded by the National Institutes of Health. The government has certain rights in the invention.

Throughout this application, certain publications are referenced in brackets. Full citations for these publications may be found immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention relates.

Background of the Invention

Lung, breast, colon and pancreatic cancer are among the leading cancers in the US and worldwide in terms of prevalence and/or mortality. For example, pancreatic cancer, the fourth-leading cause of cancer mortality in the US with a five year survival of <5%, is one of the most lethal cancers. Because current chemotherapy is often either partially effective (e.g breast cancer) or completely inadequate (e.g., pancreatic cancer), the need for new agents against these cancers is pressing, if these lethal diseases are to be controlled. Cimetidine (CIM), a histamine type-2 (H-2) receptor antagonist used in the past to treat peptic ulcers, has been reported to improve the outcome of patients with gastrointestinal cancers, including pancreatic cancer [1-2]. Its mechanism of anticancer action, not entirely clear, may include its effect on histamine; down-regulation of the cell surface expression of E-selectin, impairing tumor cell interaction (adhesion) with the tumor vasculature; or enhancment of cell-mediated immune response against cancer [3j.

Curcumin, a diferuloylmethane derived from the Indian spice, turmeric (popularly called "curry powder") is considered to have antineoplastic activity against leukemia, lymphoma and multiple cancers, including those of the gastrointestinal and genitourinary tracts, breast cancer, ovarian and lung cancer [4-5], Curcumin interferes with multiple cell signaling pathways, including cell cycle, apoptosis, proliferation and tumor invasion, Difluoromethylornithine (DFMO) inhibits the enzyme ornithine decarboxylase, which catalyzes the rate-limiting step in polyamine synthesis. Meyskens et al recently demonstrated the remarkable efficacy of the combination of sulindac and DFMO in preventing colon cancer [6]. The rationale for this combination is simple yet quite powerful [71: DFMO inhibits the enzyme ornithine decarboxylase, while sulindac stimulates polyamine acetylation and export, the end result being profoundly reduced polyamine levels that suppress the growth of cancer cells.

Anticancer agents, such as those described herein, have molecular targets mediating their effect. Likely relevant to the action of compounds herein are molecular targets that are members of the signal transducer and activator of transcription (STAT) pathway and the NF- KB pathway. In particular, STAT, activated when ligands bind to cytokine and growth factor receptors, is involved in such cellular events as differentiation, proliferation and apoptosis 18, 9|. Of the seven STAT proteins, STAT3 likely has a causal role in oncogenesis. STATs are believed to be important therapeutic targets against cancer. Nuclear factor κΒ (NF-κΒ), involved in cancer in general, plays an essential role in pancreatic, colon, lung and breast cancer [10, 11]. For example, NF-κΒ is constitutively activated in 70% of human pancreatic cancers but not in normal pancreatic tissues [12], and it promotes proliferation and inhibits apoptosis, thus favoring malignant growth. Herein, novel combinations and/or formulations of anticancer compounds that enhance their efficacy are described.

Summary of the Invention

This invention provides a method of treating cancer in a subject afflicted therewith comprising periodically administering to the subject an amount of phospho-valproic acid, phospho-ibuprofen, phospho-sul indac, diphospho-aspirin (P-A2), or a pharmaceutically acceptable salt thereof, together with a bioavailability enhancer, cimetidine, QW-3, curcumin, DFMO, or P-Al in amounts effective to treat cancer in the subject.

This invention provides a method of treating cancer in a subject afflicted therewith comprising periodically administering to the subject an amount of phospho-valproic acid, or a pharmaceutically acceptable salt thereof, in combination with cimetidine in amounts effective to treat cancer in the subject.

This invention provides a method for preventing cancer in a subject comprismg administering to the subject an amount of phospho-valproic acid, phospho-ibuprofen, phospho-sulindac, P A2, or a pharmaceutically acceptable salt thereof, together with a bioavailability enhancer, cimetidine, QW-3, curcumin, DFMO, or P-Al in amounts effective to prevent cancer in the subject.

Brief pescripttion of the Figures

Structures of phospho-valproic acid (P-V), phnspho-ibuprofen (P-I), phospho-sulindac (P-S), phospho-aspirin (two derivatives, P-Al and P- A2) cimetidine and curcumin.

P-V prevents pancreatic cancer in the xenograft model. A: Nude mice bearing BxPC-3 pancreatic cancer cell xenografts were treated orally with P-V 50 mg/kg or valproic acid (VPA) 250 mg/kg, starting 1 wk prior to tumor implantation. B: Following 40 days of P-V treatment (ON), the drug administration was suspended for two weeks (OFF) and tumor growth resumed at a pace similar to controls. When the drug was re-administered (ON) tumor growth was again retarded. Values are mean±SEM. C: Nude mice bearing pancreatic cancer cell xenografts were treated orally with P- V 50 mg/kg, starting when the tumor was about 200 mm³ in volume.

Synergy between cimetidine and P-V in vitro and in vivo. A: In the two isobolograms for BxPC-3 and Panc- 1 pancreatic cancer cell lines, the experimental points are below the additivity line, demonstrating synergy. B: Nude mice bearing BxPC-3 pancreatic cancer cell xenografts were treated with P-V 50 mg/kg orally and/or cimetidine (CIM) 100 mg/kg ip. The combination of the two agents eliminated pancreatic tumors in all animals. C: Synergy between cimetidine and P-V in vivo. The tumor weight, determined when animals were sacrificed and expressed as percent control, is shown and corresponds to the values obtained by volume determination (B).

Figure 4. Mechanism of action of P-V in pancreatic cancer. Figure 5. P-V inhibits STAT3 expression in BxPC-3 xenografts. Tumor sections from P-V-treated and control mice were stained using an anti-STAT3 mAb (left; 40x). The luminosity of randomly selected fields (right) was determined using the Photoshop program. (n=7/group; Mean ± SEM; p<0.0001).

Partial metabolic profile of P-V. Three metabolites of P-V were identified in cultured pancreatic cells. The mass spectra of the corresponding compounds are shown.

P-V and Cimetidine in nanoparticles and their effects on cancer, (a):

Conceptual diagram of P-V encapsulated in polymer micelles, which, in turn, was loaded into a liposome that bears incorporated into its lipid bilayer CIM. (b): TEM image of lipsomes and polymer micelles, (a) Liposomes with a clear lipid bilayer structure; their average size is 200 nm. (b) Polymer micelles; they have an average size of 50 nm. (c): Liposomes (LPV)inhibit the growth of xenografts of MIA PaCa-2 human pancreatic cancer cells compared to vehicle (PBS)-treated mice(values are mean±SEM).

Synergy between cimetidine and P-I in breast cancer. A: Isobologarms establishing the synergy in two breast cancer cell lines. B: Treatment of BALB/C nude mice bearing MCF-7 breast cancer xenografts with vehicle, P-I 300 mg/kg or liposome encapsulated P-I (Lipo-PI) 300 mg/kg started when the tumor volume reached about 100 mm³. Mice were treated once a day 5 days/week for 34 days.

Treatment of pancreatic cancer with P-I in liposomes. Pancreatic cancer xenografts were treated with P-I administered intraperitoneally dissolved in PBS or incorporated into liposomes. The latter maintained the tumor volume at a baseline value, whereas control tumors and those treated with P-I in PBS grew 160% and 75% from baseline, respectively.

The inhibitory effect of P-S and SLN-P-S on A549 human lung cancer xenografts. Results are mean+SEM. *p<0.05, **p<0.01, ***p<0.005. Figure 11. Inhibitory effect of P-S and SLN-P-S on MIA PaCa-2 xenografts.

Results are mean±SEM. *p<0.05, **rx0.01, ***p<0.005. Figure 12. Synergy between SLN-P-S and curcurain. *, SLN-P-S and cur cumin each alone; *, P-S and curcumin in combination in SLN.

Figure 13. The inhibitory effect of SLN-P-S combined with the anticancer peptide QW-3 on human pancreatic cancer xenografts.

Figure 14. P-S inhibits colon cancer and synergizes with DFMO. A: P-S prevents the growth of colon cancer xenografts in nude mice. B: P-S inhibits intestinal carcinogenesis in Min mice. Results for the small intestine and colon are shown. C: DFMO and P-S nearly eliminate intestinal tumors in Min mice. Figure 15. P-Al synergizes with P-A2 to inhibit the growth of human breast cancer xenografts. Nude mice bearing MDA-MB231 human breast cancer xenografts were treated with P-Al 25 mg/kg or P-A2 110 mg/kg, or both given at the same doses. These compounds were given orally once a day 5d/wk for 1 1 days. Figure 16. Pharmacokinetics of P-S and sulindac sulfide in mice after the administration of SLN-P-S. Figure 17. Tissue distribution of P-S and its metabolite, sulindc, in nude mice bearing xenografts. Similar results were obtained for sulindac sulfone and sulindac sulfide (not shown), although the enhancement varied among tissues. Detailed Description of the Invention

This invention provides a method of treating cancer in a subject afflicted therewith comprising periodically administering to the subject an amount of phospho-valproic acid, phospho-ibuprofen, phospho-sulindac, diphospho-aspirin (P-A2), or a pharmaceutically acceptable salt thereof, together with a bioavailability enhancer, cimetidine, QW-3, curcumin, DFMO, or P-Al in amounts effective to treat cancer in the subject. in an embodiment, phospho-valproic acid, phospho-ibuprofen, phospho-sulindac, diphospho- aspirin (P-A2), or a pharmaceutically acceptable salt thereof, is administered together with a bioavailability enhancer.

In another embodiment, phospho-valproic acid, phospho-ibuprofen, phospho-sulindac, P-A2, or a phannaceutically acceptable salt thereof, is administered in combination with cimetidine, QW-3, curcumin, DFMO, or P A I . In a further embodiment, phospho-valproic acid, phospho-ibuprofen, phospho-sulindac, P-A2, or a pharmaceutically acceptable salt thereof, is administered in combination with cimetidine, QW-3, curcumin, DFMO, or P-Al, together with a bioavailability enhancer.

In an embodiment, phospho-sulindac, or a pharmaceutically acceptable salt thereof, is administered in combination with QW-3 or curcumin, together with a bioavailability enhancer.

In a further embodiment, the bioavailability enhancer is solid lipid nanonparticles.

In an embodiment, phospho-valproic acid, or a pharmaceutically acceptable salt thereof, is administered in combination with cimetidine, together with a bioavailability enhancer.

In a further embodiment, the bioavailability enhancer is liposomes.

In another embodiment, the bioavailability enhancer is polymer micelles.

In an embodiment, phospho-sulindac, or a pharmaceutically acceptable salt thereof, is administered together with a bioavailability enhancer. In a further embodiment, the bioavailability enhancer is solid lipid nanonparticles.

In an embodiment, phospho-ibuprofen, or a pharmaceutically acceptable salt thereof, is administered together with a bioavailability enhancer.

In a further embodiment, the bioavailability enhancer is liposomes.

In an embodiment, phospho-valproic acid, or a pharmaceutically acceptable salt thereof, is administered in combination with cimetidine.

In another embodiment, phospho-ibuprofen, or a pharmaceutically acceptable salt thereof, is administered in combination with cimetidine. In an embodiment, phospho-sulindac, or a pharmaceutically acceptable salt thereof, is administered in combination with DMFO.

In another embodiment, P-A2, or a pharmaceutically acceptable salt thereof, is administered in combination with P-Al.

In a further embodiment, the cancer is pancreatic cancer. In other embodiments, the cancer is colon cancer. In yet other embodiments, the cancer is breast cancer. In other embodiments, the cancer is lung cancer.

This invention provides a method of treating cancer in a subject afflicted therewith comprising periodically administering to the subject an amount of phospho-valproic acid, or a pharmaceutically acceptable salt thereof, in combination with cimetidine in amounts effective to treat cancer in the subject. In an embodiment, the treatment of cancer is inhibition of proliferation of human tumor celts. to another embodiment, the human tumors cells are pancreatic tumor cells, colon tumor cells, or breast tumor cells.

In a further embodiment, the human tumors cells are pancreatic tumor cells, to an embodiment, the treatment of cancer is elimination of tumor cells.

This invention provides a method for preventing cancer in a subject comprising administering to the subject an amount of phospho-valproic acid, phospho-ibuprofen, phospho-sulindac, P- A2, or a pharmaceutically acceptable salt thereof, together with a bioavailability enhancer, cimetidine, QW-3, curcuinin, DFMO, or P-A 1 in amounts effective to prevent cancer in the subject.

In an embodiment, phospho-sulindac, or a pharmaceutically acceptable salt thereof, is administered together with DMFO.

In another embodiment, the cancer is colon cancer.

In an embodiment, phospho-valproic acid, or a pharmaceutically acceptable salt thereof, is administered together with cimetidine.

In a further embodiment, the cancer is pancreatic cancer.

In one embodiment, the compounds and compositions of the present invention are used for preventing cancer in a subject. In another embodiment, the cancer is lung cancer, breast cancer, or colon cancer. In yet another embodiment, these compounds are used to treat pancreatic cancer, lung cancer, breast cancer, or colon cancer.

The term "phospho" when used in conjunction with the name of a compound means that the compound comprises one or more -OP(=0)(OR_x)₂ functional groups, wherein R_x can be hydrogen or any substituent set forth herein. It is understood that the one or more - OP(=0)(OR,)2 functional groups can be incorporated into a compound by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. The one or more -OP(=0)(OR_s)2 functional groups may be substituent(s) on the compound or substituent(s) of substituent(s) on the compound.

A substituent can be a halogen (i.e., F, CI, Br, and I); an alkyl group, such as methyl, ethyl, n- propyl, isopropryl, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; an alkoxy group, such as methoxy, ethoxy, n-propoxy, and isopropoxy; an aryloxy group, such as phenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) and p-trifluoromethylbenzyloxy (4- trifluoromethylphenylmethoxy); a heteroaryloxy group; a sulfonyl group, such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; a sulfanyl group, such as methylsulfanyl, ethylsulfanyl and propylsulfanyl; cyano; an amino group, such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; carboxyl, phosphate or phosphate ester (-OP(=0)(OR_x)2). Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally. By independently substituted, it is meant that the (two or more) substituents can be the same or different. A "bioavailability enhancer" as used herein is an agent or combination of agents that enhance the rate and/or extent of absorption of a compound, such as a drug, that reaches the systemic circulation and is available at the site of action. A bioavailability enhancer may also improve tissue distribution and targeting of the compound or drug. Examples of bioavailability enhancers include, but are not limited to, liposomes, vitamin E TPGS (d-a-tocopheryl polyethylene glycol 1000 succinate); acetylated monoglycerides; mono-, di-, and triglyceride esters of medium-chain (6- 12 carbon atoms in length) and long-chain (more than 12 carbon atoms in length) fatty acids; esters of fatty acids and glycols; esters of mixed fatty acids and glycols; diesters of propylene glycol having from about 7 to about 55 carbon atoms; propylene glycol esters of capric and caprylic acids; citric acid, malic acid, ascorbic acid, fumarie acid, caproic acid, caprylic acid, cholic acid, glycocholic acid, sodium cholate, sodium lauryl sulfate, palmitoyl carnitin, cyclosporin A, polyoxyethylene/polyoxypropylene copolymers and other soluble polymers, solid lipid nanoparticles, and mixtures thereof. Soluble bioavailability-enhancing polymers to which compounds may be coupled to as targetable drug carriers or as prodrugs include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenoi, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.

Liposomes include, but are not limited to, small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. Solid lipid nanoparticles are ultrafine particles made from melted or solid lipid for use in enhancing bioavailability of a compound, such as a drug. Methods of producing and derivatizing solid lipid nanoparticles are described in Oner and Yener, "Importance of solid lipid nanoparticles (SLN in various administration routes and future perspectives", Int. J. Nanomedicine (2007), 2(3), pp. 289-300, and references cited therein, which is hereby incorporated by reference in its entirety.

The compounds of the instant invention may be in a salt form. As used herein, a "salt" is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. In the case of compounds used for treatment of cancer, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium. The term "pharmaceutically acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, paimitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like, (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", /. Pharm. Sci. 66: 1-19).

As used herein, the term "control of cancer" shall mean either treatment or prevention of cancer.

As used herein, "cancer prevention" is the administration of a pharmaceutical agent alone or in combination with other pharmaceutical or natural agents for the prevention of the development of cancer or of its recurrence in subjects at risk for the development of a given cancer or precancerous condition.

As used herein, "treating" means slowing, stopping or reversing the progression of a disease. An embodiment of "treating cancer" is inhibition of proliferation of tumor cells. As used herein, "administering" an agent may be performed using any of the various methods or delivery systems well known to those skilled in the art. The administering can be performed, for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery, subcutaneously, intraadiposally, intraarticularly, intrathecally, into a cerebral ventricle, intraventicularly, intratumorally, into cerebral parenchyma or mtraparenchchymally.

The compositions of this invention may be administered in various forms, including those detailed herein. The treatment with the compound may be a component of a combination therapy or adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds. Combination therapy is treatment by at least two different compounds or methods. Combination therapy can be accomplished by sequential administration where the patient is treated first with one compound and then another compound. Combination therapy can also be accomplished by simultaneous or substantially simultaneous administration. The compounds can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.

Sequential combination therapy means that the patient is administered first one compound and then another compound at a later time. The time between administrations can be 3, 6, 12, 24, 48, or 72 hours, or it can be weekly. The administration of compound A may precede administration of different compound B. Alternatively, for example, administration of compound B may precede administration of different compound A.

When combination therapy is simultaneous, two compounds are administered at the same time, and optionally as one dosage unit. When combination thereapy is substantially simultaneous, two compounds are each administered as separate dosage units, which may be the same or different, with a short time period in between administrations. For example, administration of a tablet of one compound followed by a short time period, for example, less than 1 hour, and then subsequent injection of another compound would be substantially simultaneous.

As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind.

The dosage of the compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with the compounds; and the desired therapeutic effect. A dosage unit of the compounds may comprise a single compound or mixtures thereof with anti-cancer compounds, or tumor growth inhibiting compounds, or with other compounds also used to treat cancer-related diseases. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection or other methods, into the cancer, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. The compounds can be administered in admixture with suitable phannaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone but are generally mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. In one embodiment the carrier can be a monoclonal antibody. The active agent can be co-administered in the form of a tablet or capsule, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen. Specific examples of pharmaceutical acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U, S. Pat, No. 3,903,297 to Robert, issued Sept. 2, 1975. Techniques and compositions for making dosage forms useful in the present invention are described-in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition ( 1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Dmg Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein. Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. It can also be administered parentally, in sterile liquid dosage forms. Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

For oral administration in liquid dosage form, the oral drug components are combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.

Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. The compounds of the instant invention may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.

Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

The compounds and compositions of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.

The compounds of the present invention include hydrates, solvates, and complexes of the compounds used by this invention. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein. Compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. The compounds described in the present invention are in racemic form or as individual enantiomers. The enantiomers can be separated using known techniques, such as those described in Pure and Applied Chemistry 69. 1469-1474, (1997) IUPAC. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within tills invention whether existing in equilibrium or predominantly in one form.

It will be noted that the structure of the compounds of this invention may include an asymmetric carbon atom and thus the compounds occur as racemates, racemic mixtures, and isolated single enantiomers. All such isomeric forms of these compounds are expressly included in this invention. Each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereoniers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis, such as those described in "Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.

The subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

It will be noted that any notation of a carbon in structures throughout this application, when used without further notation, are intended to represent all isotopes of carbon, such as ^l2C, ⁱ³C, or ^l4C. Furthermore, any compounds containing ^{l 3}C or ^l4C may specifically have the structure of any of the compounds disclosed herein.

It will also be noted that any notation of a hydrogen in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as Ή, ²H, or ³H. Furthermore, any compounds containing ²H or ³H may specifically have the structure of any of the compounds disclosed herein.

Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non- labeled reagents employed.

The compounds, compositions, and methods of the present invention are useful in the inhibition of tumor proliferation and treatment and/or prevention of a variety of cancers, including, but not limited to, breast colon, and pancreatic cancers.

All combinations of the various elements described herein are within the scope of the invention. This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

Experimerital Details

Reagents: Phospho-compounds were synthesized for us by Chem-Master International, Inc. (Setauket, NY) following the methodology of Penning et al [13]. All other reagents were obtained from commercial sources. The compounds reported herein are: phospho-ibupofen (P-I); phospho-valproic acid (P-V); phospho-aspirin, including monophospho-aspirin (P-Al) and diphospho- aspirin (P-A2); phospho-sulindac (P-S), cimetidine (CIM) and curcumin. Their structures are shown in Fig. 1. They have been herein abbreviated as shown in parentheses.

Cell culture: Human pancreatic (BxPC-3, Panc-1 and MIA PaCa-2), breast (MCF-7 and MDA-MB 231), lung (A549) and colon (HT-29, and SW-480) cancer cell lines obtained from the American Type Culture Collection, (ATCC, Manassas, VA) were grown as monolayers in the specific medium suggested by ATCC, following standard protocols.

Cell viability assay: We used an assay based on the reduction of 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide dye (MTT), and followed the manufacture's protocol (Promega, Madison, WI, USA). Tumor xenografts in nude mice: Typically, female immune deficient nude mice at 5 weeks of age, maintained in pathogen-free conditions and fed irradiated chow, were implanted with lx10⁶ cultured human cancer cells/site in 0.1 ml sterile PBS, often bilaterally subcutaneously (sc) in their back. For chemotherapy studies, when palpable tumors formed, mice were divided randomly into groups receiving vehicle (control) or the test drug(s), according to specific study protocols. For chemoprevention studies, administration of the test agent started one week prior to the implantation of the cancer cells; the remaining of the process was the same as for the chemoprevention studies. Body weight was measured once a week and the size of the tumors was determined twice weekly. Tumor size was calculated using the formula: [length x width x (length + width/2) x 0.56] in millimeters. At the end of the experiments, animals were sacrificed by C(¼ asphyxiation and after their careful resection, tumors were weighed. Statistical Analyses: Statistical evaluation of the data was performed by one-factor analysis of variance followed by Tukey test for multiple comparisons or by t-test. P<0.05 was regarded statistically significant. The data, obtained from at least three independent experiments, were expressed as the mean ± SEM.

Example 1. Evaluation of P-V used alone and in combination with CIM for the control of pancreatic cancer

The effect of P-V, a derivative of valproic acid (VPA) on the growth of seven human cancer cell lines was evaluated by determining the 24-h IC50 of both P-V and VPA (Table 1). P-V showed enhanced potency in inhibiting cell growth compared to conventional VPA, with the potency enhancement ranging between 35 and 245-fold,

P-V prevents pancreatic cancer in a xenograft model

The chemopreventive potential of P-V in vivo was tested using BxPC-3 pancreatic cancer xenograft model in athymic nude mice. Starting 1 wk prior to the implantation of BxPC-3 cells, we gave orally P-V 50 mg/kg and VPA 250 mg/kg (these and all subsequent doses are per kg body weight; the reference to body weight is omitted for simplicity); these doses represent 25% of their respective maximum tolerated dose. As shown in Fig. 2A, P-V was consistently more effective than VPA in inhibiting tumor growth. At sacrifice, P-V decreased tumor volume by 68% compared to controls (p<0.01), whereas VPA only decrease tumor volume by 34% (p<0.05). P-V achieved this effect a) by reducing cancer cell proliferation by 49.1% compared to control, (p<0.001) and b) by inducing apoptosis briskly: 94.6% increase of the apoptosis index (p<0.003) compared to control. In another study (Fig. 2B), P-V administration for 40 days ("ON" in the figure) inhibited tumor growth; when P-V was discontinued for 2 wks ("OFF"), tumor growth increased; re- administration of P-V ("ON") retarded tumor growth again.

P- V inhibits pancreatic cancer

Using the same xenograft model, the therapeutic potential of P-V was assessed in vivo (Fig. 2C). Once the BxPC-3 xenografts reached -200 mm³, mice were treated with P-V 25 mg/kg or vehicle. By day 17, P-V reduced the tumor volume by 64% compared to control (p<0.01). P-V was well tolerated, with no weight loss or other side effects. Xenografts of MIA PaCa-2 cells gave similar results.

Synergy between cimetidine and P-V

Using isobolograms and fractional analyses, various drug combinations were evaluated in cultured pancreatic cancer cells. As shown in Fig. 3 A, there is a clear-cut pharmacological synergy between P-V and CIM.

The pharmacological synergy between P-V and CIM was then evaluated in animals. As shown in Fig. 3B,C, P-V combined with CIM had a dramatic effect: by day 58, all tumors in all animals were eliminated; each agent alone showed only a partial effect. Mechanism of action of P-V

Key parts of the mechanism of action of P-V have been unraveled (Fig. 4). The central mechanistic event is the inhibition of the activation of STAT3 (activation by phosphorylation), which a) suppresses the transcription of apoptosis-related genes; b) has mitochondrial effects, including e- transfer and Ras-dependent transformation; and c) induces p21. P-V also induces MAPKs and suppresses PI3K/AKT. All these effects culminate in the cytokinetic changes manifested as the dramatic inhibition of pancreatic carcinogenesis that was observed in the animal studies. Contrary to expectation, the effect of P-V on histone acetylation is so modest as to appear non-contributory to its action. P-V markedly reduced the levels of STAT3 in xenografts of BxPC3 cells in nude mice. The expression of STAT3 was determined in tumor tissue sections from P-V-treated and control mice by immunohistochemistry, using a mAb recognizing STAT3, both phosphorylated and non-phosphorylated. STAT3 levels were determined by evaluating 10 randomly selected fields and recording the mean luminosity per sample, P-V reduced the expression of STAT3 by 81%, compared to control (rxO.Ol; Fig, 5). Changes in molecular targets of P-V, such as those exemplified by the response of STAT3 in the cancer tissue of treated mice, represent markers of response to treatment with P-V alone or in combination and can be clinically useful.

Metabolism, pharmacokinetics and pharmacodynamics of P- V

The metabolism of P-V was evaluated (Fig. 6). BxPC-3 cells were incubated for 2 h with P- V at its IC₅₀ concentration. Organic extracts of the cells were subjected to structure analysis using LC-MS-MS and ¹ H MR. We documented: a) ester hydrolysis of P-V generating valproic acid and 4-(diethoxy-phosphorylomethyl)-phenolate; the latter was identified as both an ion and a sodium salt; and b) a conjugate between valproic acid and glutathione, which may have significant implications for redox homeostasis [14], Ongoing work has revealed additional metabolites of P-V, whose structure has not been finalized.

Example 2: The combination of P-V with CIM formulated in liposomes and polymer micelles is effective in the treatment of pancreatic cancer

To maximize the delivery of P-V combined with CIM to the target tissues and to ensure that a given cancer cell receives both compounds (thus increasing the efficacy of the drug combination), the following drug formulation was designed. P-V was encapsulated in polymer micelles, which, in turn, were loaded into a liposome that bears CIM incorporated into its lipid bilayer (Fig. 7A). Preparation of micelles and loading with P-V

The block copolymer micelles are made of an amphiphilic polymer, and, in this case, of poly(ethylene oxide)-poly(ε-caprolactone) (PEO-b-PCL). The PEO-b-PCL block copolymer and P-V were dissolved in an unselective solvent, acetone, and an excess of deionized water was subsequently added dropwise with stirring. The remaining acetone was removed by vacuum.

Formation of micelle encapsulated liposomes

Egg yolk phosphatidylcholine (EPC) and CIM were dissolved in chloroform and dried as a thin lipid film. Hydrating this thin film with micelle solution produced a suspension of micelles encapsulated into liposomes. This suspension was then extruded three times through double polycarbonate membranes and the liposomes that were obtained were further purified on a Sephadex G-50 column to remove the free (unincorporated) drugs.

Drug concentrations were determined by HPLC. A Waters alliance 2695 Separations Module equipped with a Waters 2998 photodiode array detector and a thermo Hypersil BDS C18 column ( 150 x 4.6 mm, particle size 3 μm ) was used. Nuclear magnetic resonance (NMR, Varian INOVA 500-MHz spectrometer) was also used to confirm the composition and stability of the components. Zeta Potential Analyzer (Zetaplus, Brookhaven Instrument, Holtsville, NY) and Transmission Electron Microscope (TEM, FEI BioTwinG2) were used to determine the size and morphology of each nanoparticle (liposome and micelle). The liposome vesicle structure was visualized by TEM with negative staining (1% uranium acetate). The hydrodynamic radius (/¾) and polydispersity index (PDI) of the liposomes were measured by the dynamic light scattering (Zetaplus, Brookhaven Instrument, NY).

Fig. 7B shows the morphology of the liposomes and the polymer micelles. Self assembled polymer micelles are a monodispersed nano-sized structure (R_h =50 nm). P-V and CIM in this formulation inhibit the growth of human pancreatic cancer xenografts

We evaluated the effect of this combination on xenografts of MIA PaCa-2 human pancreatic cancer cells in nude mice. Each animal had two xenografts, one on each side. When the xenografts reached a volume of about 75-100 mm³, we started administering liposomes ip 5 d/wk for 51 days. Each liposome dose provided P-V 50 mg/kg and CIM 10 mg/kg. As shown in Fig. 7C, the liposomes inhibited the growth of the xenografts at all time points. At the time of animal sacrifice, on day 51 , the liposomes inhibited tumor growth by 88% compared to the vehicle -treated control group (p<0.01). Example 3: The combination of P-I with CIM inhibits the growth of breast cancer We evaluated the effect of the combination of P-I with CIM on the growth of breast cancer.

Breast cancer cell lines

We evaluated the inhibitory effect of P-I combined with CIM on the growth of two human breast cancer cell lines, MCF - 7. which is estrogen receptor positive |ER(+)], and MDA-MB- 231, which is triple negative [ER(-), HER2 (-) and progesterone receptor (-)]. The respective ICJOS were as follows: For P-I: 120 μΜ in MCF-7 cells and 30 μM in MDA-MB-231 cells. For CIM: 13 mM in MCF-7 cells and 45 niM in MDA-MB-231 cells.

We then evaluated their potential synergy. MCF-7 cells were treated with either P-I alone (15, 30, 60, 120, or 240 μΜ), or P-I (same concentrations) and 3 mM CIM (about ¼ of its ICso). MDA-MB-231 cells were treated with either P-I alone (3.25, 7.5, 15, 30, or 60 μΜ), or P-I (same concentrations) and CIM 6 mM (about 1/7 of its ICso). The IC₅₀s of P-1 plus CIM was 84 μ.Μ in MCF-7 cells and 17 μΜ in MDA-MB-231 cells. Thus the effect of the combination was synergistic in MDA-MB-231 cells and additive in MCF-7 cells. (Fig. 8).

Example 4: Formulation of P-I and liposomes enhances the anticancer effect of P-I on breast and pancreatic cancer

P-I liposomes

PEGylated liposomes were used as a delivery system foro P-I, following standard protocols. Briefly, 50 mg of PC, 19 mg of PEG-DSPE and 30 mg of P-I were dissolved in 2 mJL chloroform. The chloroform was evaperated and the resultant components were dried as a thin lipid film. Hydrating this thin film with 1 mL PBS solution (pH 7.4) produced a suspension of liposomes. This suspension was then extruded three times through double polycarbonate membranes (0.2 μΜ). The free drug was removed by thoroughly dialyzing the liposome solution againt a PBS solution. The resultant liposome solution contains 50 mg/ml of L-a-phosphatidylcholine (PC), 19 mg/ml 1,2-dioleoyl-.s7i-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol)-2000] (PEG-DSPE) and 28.2 mg/ml P-I.

Liposome encapsulated P-I regresses MCF-7 xenografts Three days before implanting MCF-7 breast cancer cells in Balb/C nude mice, a p-estradiol pellet (Innovative Research of America, Sarasota, FL) was inserted subcutaneous! y in the interscapular area of the mice using the device recommended by the manufacturer, lintraperitoneal treatment with vehicle or P-I 300 mg/kg or liposome encapsulated P-I (Lipo- PI) 300 mg/kg started when the tumor volume reached about 100 mm³. Mice were treated once a day 5 d/wk for 34 days.

As shown in Figure 8B, P-I inhibited the growth of the xenografts compared to control- treated animals. However, liposome-encapsulated P-I was much more efficacious, regressing theses xenografts. On day 34, the % increase of tumor volume from day 1 was 64% in the control group, 17% in the P-I group, and -14% in the Lipo-P-I group. Compared to controls, P-I suppressed tumor growth by 47% (p=0.046), and lipo-P-I suppressed tumor growth by 78% (p<0.01). Liposome encapsulated P-I arrests the growth of BxPC-3 pancreatic xenografts

BxPC-3 human pancreatic cancer xenografts were treated ip with vehicle, or P-I 300 mg/kg ip or liposome encapsulated P-I (liposome-PI) providing P-I 300 mg/kg. All groups were treated daily. 5 d/wk for 21 days, starting when the tumor volume reached about 100 mm³. As shown in Figure 9, P-I inhibited the growth of the xenografts compared to control-treated animals. However, liposome-P-I was much more efficacious, arresting the growth of xenografts. On day 21, the % increase of tumor volume from day 1 was 160% in the control group, 76% in the P-I group, and -0.3% in the Liposome-P-I group. Compared to controls, P-I suppressed tumor growth by 84% (p=0.01 ), and liposome-P-I suppressed it by 160.3% (p<0.001).

Example 5: Formulation of P-S in solid lipid nanoparticles enhances its efficacy against lung cancer xenografts

P-S in solid lipid nanoparticles (SLN) was formulated to take advantage of their desirable properties as drug delivery vehicles (27-29).

Preparation ofSLN-P-S

200 mg stearic acid, 100 mg lecithin and l lOmg drug P-S were dissolved in 10 mL chloroform in a glass flask (organic phase). 250 mg sucrose stearate was resolved in 30 mL distilled water in a three-neck flask and heated up to 75 °C using a water bath (aqueous phase). We added the organic phase to the aqueous phase, removed the flask from the water bath and added 10 mL distilled water pre-cooled on ice; stirring of the mixture at 1000 rpm continued for 2 h. The resulting suspension was washed twice with distilled water by centrifuging at 40,000x# for 4 h, removing the supernatant. Thus the free drug (unreacted P S) was removed. The pellet was resuspended in distilled water, and stored at -20 ° C for 2 h, followed by freeze-drying that converted it to powder form.

Evaluation of SLN-P-S in vitro

We determined the effect of SLN-P-S on the growth of A549 human lung cancer cells. The IC50 of SLN-P-S was 16 μΜ while that of P S 81 μΜ.

SLN-P-S inhibits the growth of human lung cancer xenografts

Nude mice bearing A549 xenografts were treated daily, 5 d/wk, with PBS (control) ip, or SLN-P-S 300 mg/kg ip, or P-S 150 mg/kg orally starting when the average tumor volume was about 180 mm³. (P-S was administered at 150 mg/kg because this is its highest safe dose in these mice.) As shown in Fig. 10, P-S failed to inhibit significantly the growth of the xenografts. In contrast, SLN-P-S had a profound inhibitory effect on the growth of these xenografts, regressing them between days 4 and 9 and severely retarding their growth between days 9 and 16. On day 16, compared to controls, SLN-P-S reduced tumor volume by 78% (p<0.005).

Example 6: P-S formulated in SLN inhibits the growth of pancreatic cancer xenografts Nude mice bearing MIA PaCa-2 human pancreatic cancer xenografts were treated with PBS or SLN-P-S 200 mg/kg ip daily, 5d/wk starting when the average tumor volume was about 100 mm³. As shown in Fig. 11, SLN-P-S had a profound inhibitory effect on the growth of these xenografts, maintaining the tumor volume stable (tumor stasis) between days 4 and 9, and severely retarding their growth between days 9 and 23. On day 23, compared to controls, SLN-P-S reduced tumor volume by 78% (p<0.005).

Example 7: SLN-P-S synergizes with curcumin to inhibit the growth of human lung cancer cells Curcumin, an agent with pleiotropic effects on various human cancers, is generally believed to have anticancer properties (2). Therefore, we evaluated its effect against lung cancer in combination with P-S.

Incorporation of P-S and curcumin into SLN

P-S and curcumin were incorporate into SLN as in Example 5, with the exception that we also added 55 rng curcumin at the first step (preparation of the organic phase). Synergy between SLN-P-S and curcumin in vitro

We assessed the potential synergy between SLN-P-S and curcumin by treating A-549 human lung cancer cells with these two agents alone and in combination. As demonstrated by the isobolograms in Fig. 12, SLN-P-S and curcumin display true pharmacological synergy in inhibiting the growth of A-549 human lung cancer cells.

Example 8: SLN-P-S and the anticancer peptide QW-3 synergize against pancreatic cancer

We evaluated for the treatment of pancreatic cancer the combination of P-S with a recently described anticancer peptide [21 ]. Nude mouse xenografts of the human pancreatic cancer cell line MIA PaCa-2 were treated with SLN-P-S providing P-S 100 mg/kg ip and QW-3 80 pg/mouse ip. Both were given once a day, 5d/wk for 21 d. Treatment started when tumors were around 100 mm^'1. As shown in Fig. 13, QW-3 had no effect on the growth of the xenografts and SLN-P-S had a minimal and statistically not significant effect. In contrast, on day 18, the combination of the two agents reduced the growth of the xenografts by 65% compared to control (p<0.05).

Example 9: P-S alone and in combination with DFMO inhibits colon carcinogenesis in vivo

We evaluated the chemotherapeutic potential of P-S in a xenograft model of HT-29 human colon cancer cells. At sacrifice, P-S 100 mg/kg po reduced tumor growth by 63%, compared to controls; the tumor volume (mean±SEM) for control and P-S groups was 920±148 and 337±60 mm⁵, respectively (p<0.05; Fig. 14A). In Min mice (predisposed to the development of tumors in the small intestine and colon [ 15]), P-S 100 mg/kg po once daily for 4 weeks decreased the number of tumors in the small intestine by 57.2% compared to controls (p<0.002); in the colon such reduction was 61.8 % (p<0.02; Fig. 14B). In both studies P-S was well tolerated with no weight loss during treatment. Of note, the dose of 100 mg/kg is significantly below its maximum tolerated dose.

In another study, P-S 100 mg/kg po and DFMO 2% in drinking water were administered alone and combined to Min mice between 6 and 13 weeks of age. DFMO and P-S alone reduced the number of all intestinal tumors by 50.8% and 52.1 %, respectively, but their combination reduced it by 90.2% (p<0.0001 ; Fig. 14C).

Example 10: P-Al synergizes with P-A2 to inhibit the growth of human breast cancer xenografts

Nude mice bearing MDA-MB231 human breast cancer xenografts were treated with P-Al 25 mg/kg or P-A-2 1 10 mg/kg, or both given at the same doses. These compounds were given orally once a day 5d/wk for 1 1 days. As seen in Fig. 15, by day 1 1, P-A l decreased the growth rate of the xenografts by 13%, P-A2 by 5%, but the combination of the two by 38%, which is more than twice the sum of the effects of P-Al and P-A2 given alone. These data establish pharmacological synergy between the two compounds.

Example 11: Formulation of P-S in SLN: Pharmacokinetics and tissue distribution We evaluated the effect of formulating P-S in SLN on the pharmacokinetics and tissue distribution of P-S in mice. SLN-P-S was prepared as described hereinabove. Six-wk old BALB/c mice were given ip or po 50 mg/kg of P-S or the equivalent amount of SLN-P-S in terms of P-S content and levels of P-S and its main metabolites were determined in plasma and select organs by HPLC over a 24-hr period post-dosing.

As shown in Fig. 16, the levels of P-S and those of sulindac sulfide, an active metabolite of conventional sulindac, are higher when P-S is given incorporated in SLN than when not incorporated in SLN. Table 3 provides the numerical results. The highest difference in metabolite levels between P-S and SLN-PS administration is noted in the case of sulindac sulfide administered ip, with the ratio of the corresponding AUC (area under the curve) values being 48. Longer periods of observation are expected to show even more pronounced differences.

The tissue distribution of P-S in nude mice bearing MIA PaCa-2 pancreatic cancer xenografts given SLN-P-S or P-S po or ip was also evaluated. One hour post-dosing mice were sacrificed and their heart, lung, liver, intestine, kidney and tumor xenograft were removed and drug levels in each one of them were determined by HPLC. As shown in Fig. 17, formulation of P-S in SLN invariably led to higher tissue levels, coampred to plain P-S. Pronounced chages are noted in the lung, intestine and the xenograft.

Example 12: Formulation of similar compounds (e.g, H₂S and NO donating compounds) in liposomes and SLN enhances their pharmacological activity.

In recent years a series of compounds have appeared that represent modifications of existing molecules. Such compounds include the so-called nitric oxide-donating compounds, with nitric oxide-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs) being their best- characterized representatives 116]. An additional group of compounds are the sulfhydril releasing compounds [17-19]. The pharmacological action of such compounds is believed to be based on the biological moiety they release (or "donate"), e.g., NO orなS, although other parts of the molecule may be pharmacologically relevant [20]. Similar to compounds described in the present invention (e.g., P-V), NO-donating, lなS-releasing and other similar compounds are susceptible to hydrolytic cleavage resulting in loss or reduction of their efficacy. Our results indicate that formulating the NO-donating, H₂S-releasing and other similar compounds in the same manner as described hereinabove enhances their pharmacological efficacy. These formulations, especially the formulation in SLN, which is particularly suitable for oral administration, enhance the efficacy of our compounds and those mentioned herein for other applications, such as treatment of inflammatory conditions, including but not limited to arthritis, of pain and of fever.

Example 13. Evaluation of P-V in combination with curcumin, DFMO, QVV-3, or P-A2

P-V is evaluated in combination with curcumin, DFMO, QW-3, and Ρ-Λ2 according to the procedure set forth in Example 1. The cancer tumor inhibitory effect is analogous to the result observed hereinabove in Example 1.

Example 14. Evaluation of P-V together with solid lipid nanoparticles or polymer micelles

P-V is evaluated together with solid lipid nanoparticles or polymer micelles according to the procedure set forth in Example 4. The cancer tumor inhibitory effect is analogous to the result observed hereinabove in Example 4. Example 15. Evaluation of P-I in combination with curcumin, DFMO, QW-3, or P-A2

P-I is evaluated in combination with curcumin, DFMO, QW-3, or P-A2 according to the procedure set forth in Example 3. The cancer tumor inhibitory effect is analogous to the result observed hereinabove in Example 3.

Example 16. Evaluation of P-I together with solid lipid nanoparticles or polymer micelles

P-I is evaluated together with solid lipid nanoparticles or polymer micelles according to the procedure set forth in Example 4. The cancer tumor inhibitory effect is analogous to the result observed hereinabove in Example 4.

Example 17. Evaluation of P-S in combination with cimetidine or P-A2 P-S in combination with cimetidine or P-A2 is evaluated according to the procedure set forth in Example 9. The cancer tumor inhibitory effect is analogous to the result observed hereinabove in Example 9,

Example 18. Evaluation of P-S together with liposomes or polymer micelles

P-S is evaluated together with liposomes or polymer micelles according to the procedures set forth in Examples 5 and 6. The cancer tumor inhibitory effect is analogous to the results observed hereinabove in Examples 5 and 6.

Example 19. Evaluation of P-Al in combination with cimetidine, curcumin, QW-3, or DFMO P-Al in combination with cimetidine, curcumin, QW-3, or DFMO is evaluated according to the procedure set forth in Example 10. The cancer tumor inhibitory effect is analogous to the result observed hereinabove in Example 10.

Example 20. Evaluation of P-Al together with liposomes, polymer micelles, or SLNs

P-Al together with liposomes, polymer micelles, or SLNs are evaluated according to the procedure set forth in Example 4, wherein P-I is replaced with P-Al. Liposomes are replaced by polymer micelles or SLNs. The cancer tumor inhibitory effect is analogous to the result observed hereinabove in Example 4.

Discussion

Of importance to cancer control are: a) the need, at times, to combine therapeutic and/or cancer preventive agents to achieve maximal results; and b) the need to formulate anticancer agents in ways that enhance their efficacy in the control of cancer. Drug combinations offer two advantages: a) they enhance the therapeutic efficacy of the combined agents; such enhanced efficacy can be additive or synergistic, as conventionally understood in pharmacology; and b) it may reduce side effects, either through the inherent properties of the combined agents or by reducing their respective doses, in which case dose-dependent side effects are reduced or even eliminated. On the other hand, the manner in which a drug or drug combinations are formulated can at times be a critical determinant of drug efficacy.

As exemplary applications of drug combinations, the following drug combinations: a) P-V with CIM for the control of pancreatic cancer; b) P-S with difluoromethylorni thine (DFMO) for the control of colon cancer; c) P-I with CIM for the control of breast cancer; d) P S with curcumin for the control of lung cancer; and e) P-S with the peptide QW-3 have been described. As exemplary applications of drug formulations, the following drug formulations: a) P-V with CIM incorporated into liposomes; b) P-I in liposomes for the control of breast cancer; c) P-S in solid lipid nanoparticles (SLN) for the control of pancreatic cancer; and d) P-S incorporated in SLN alone or combined with curcumin for the control of lung cancer have been described.

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Claims

Claims What is claimed is:

1. A method of treating cancer in a subject afflicted therewith comprising periodically administering to the subject an amount of phospho-valproic acid, phospho- ibuprof en, phospho-sulindac, diphospho-aspirin (P-A2), or a pharmaceutically acceptable salt thereof, together with a bioavailability enhancer, cimetidine, QW-3, curcumin, DFMO, or P-Al in amounts effective to treat cancer in the subject.

2. The method of claim 1, wherein phospho-valproic acid, phospho-ibuprofen, phospho- sulindac, diphospho-aspirin (P-A2), or a pharmaceutically acceptable salt thereof, is administered together with a bioavailability enhancer,

3. The method of claim 1, wherein phospho-valproic acid, phospho-ibuprofen, phospho- sulindac, P-A2, or a pharmaceutically acceptable salt thereof, is administered in combination with cimetidine. QW-3, curcumin, DFMO, or P-Al.

4. The method of claim 1, wherein phospho-valproic acid, phospho-ibuprofen, phospho- sulindac, P-A2, or a pharmaceutically acceptable salt thereof, is administered in combination with cimetidine, QW-3, curcumin, DFMO, or P-Al, together with a bioavailability enhancer.

5. The method of claim 4, wherein phospho-sulindac, or a pharmaceutically acceptable salt thereof, is administered in combination with QW-3 or curcumin, together with a bioavailability enhancer.

6. The method of claim 5, wherein the bioavailability enhancer is solid lipid nanonparticles.

7. The method of claim 4, wherein phospho-valproic acid, or a pharmaceutically acceptable salt thereof, is administered in combination with cimetidine, together with a bioavailability enhancer.

8. The method of claim 7, wherein the bioavailability enhancer is liposomes.

9. The method of claim 7, wherein the bioavailability enhancer is polymer micelles.

10. The method of claim 2, wherein phospho-sulmdac, or a pharmaceutically acceptable salt thereof, is administered together with a bioavailability enhancer.

11. The method of claim 10, wherein the bioavailability enhancer is solid lipid nanonparticles.

12. The method of claim 2, wherein phospho-ibuprofen, or a pharmaceutically acceptable salt thereof, is administered together with a bioavailability enhancer.

13. The method of claim 12, wherein the bioavailability enhancer is liposomes.

14. The method of claim 3, wherein phospho-valproic acid, or a pharmaceutically acceptable salt thereof, is administered in combination with cimetidine.

15. The method of claim 3, wherein phospho-ibuprofen, or a pharmaceutically acceptable salt thereof, is administered in combination with cimetidine.

16. The method of claim 3, wherein phospho-sulindac, or a pharmaceutically acceptable salt thereof, is administered in combination with DMFO.

17. The method of claim 3, wherein P-A2, or a pharmaceutically acceptable salt thereof, is administered in combination with P-Al .

18. The method of any one of claims 1-17, wherein the cancer is pancreatic cancer.

19. The method of any one of claims 1-17, wherein the cancer is colon cancer.

20. The method of any one of claims 1-17, wherein the cancer is breast cancer.

21. The method of any one of claims 1-17, wherein the cancer is lung cancer.

22. A method of treating cancer in a subject afflicted therewith comprising periodically administering to the subject an amount of phospho-valproic acid, or a pharmaceutically acceptable salt thereof, in combination with cimetidine in amounts effective to treat cancer in the subject.

23. The method of claim 22, wherein the treatment of cancer is inhibition of proliferation of human tumor cells.

24. The method of claim 23, wherein the human tumors cells are pancreatic tumor cells, colon tumor cells, or breast tumor cells.

25. The method of claim 23, wherein the human tumors cells are pancreatic tumor cells.

26. The method of claim 25, wherein the treatment of cancer is elimination of tumor cells.

27. A method for preventing cancer in a subject comprising administering to the subject an amount of phospho-valproic acid, phospho-ibuprofen, phospho-sulindac, P-A2, or a pharmaceutically acceptable salt thereof, together with a bioavailability enhancer, cimetidine, QW-3, curcumin, DFMO, or P-Al in amounts effective to prevent cancer in the subject.

28. The method of claim 27, wherein phospho-sulindac, or a pharmaceutically acceptable salt thereof, is administered together with DMFO.

29. The method of claim 28, wherein the cancer is colon cancer.

30. The method of claim 27, wherein phospho-valproic acid, or a pharmaceutically acceptable salt thereof, is administered together with cimetidine.

31. The method of claim 30, wherein the cancer is pancreatic cancer.