WO2005082407A1

WO2005082407A1 - Use of quercetin and resveratrol to treat and prevent oral cancer

Info

Publication number: WO2005082407A1
Application number: PCT/US2004/036682
Authority: WO
Inventors: Thomas Walle
Original assignee: Musc Foundation For Research Development
Priority date: 2003-11-03
Filing date: 2004-11-03
Publication date: 2005-09-09

Abstract

A pharmaceutical composition comprising a topically effective amount of quercetin and a topically effective amount of resveratrol in a pharmaceutically acceptable carrier is provided. A method of treating oral cancer in a subject is provided, comprising topically administering to the mouth of the subject a topically effective amount of quercetin and a topically effective amount of resveratrol in a pharmaceutically acceptable carrier. A method of preventing oral cancer in a subject is provided, comprising topically administering to the mouth of the subject a topically effective amount of quercetin and a topically effective amount of resveratrol in a pharmaceutically acceptable carrier.

Description

USE OF QUERCETIN AND RESVERATROL TO TREAT AND PREVENT ORAL CANCER

STATEMENT OF GOVERNMENT RIGHTS This invention was made with government support under Grants GM55561 and

DOD Contract # N6311602MD200, awarded by The National Institutes of Health and the Department of Defense, respectively. The government has certain rights in the invention.

BACKGROUND

Oral cancer and its morbidity and mortality are serious and costly problems. Early detection has been a major difficulty, and once detected, this cancer is difficult to treat and the prognosis is poor. Smoking and alcohol consumption are major risk factors in oral cancer [1, 2]. In fact, about 90 percent of people with oral cavity cancers use tobacco. This type of cancer responds poorly to treatment. Although dietary interventions have not received much attention in this cancer, epidemiological studies suggest that this could be a critically important protective factor [2-6]. For example, epidemiological studies suggest that a diet rich in fruits and vegetables could be an important protective factor. Many dietary polyphenols have been shown to have antiproliferative effects on cancer cells [8] with only limited reports on oral cancer cells [57, 58, 59]. However, most of these polyphenols are occurring naturally as glycosides or other conjugates. As presumably only the aglycones are biologically active, the glycosides first have to be hydrolyzed by the bacterial flora of the intestine before they can be absorbed [9, 10]. Thus, dietary polyphenols have not been viewed as useful for oral application.

SUMMARY OF THE INVENTION

Provided herein is a pharmaceutical composition comprising a topically effective amount of quercetin and a topically effective amount of resveratrol in a pharmaceutically acceptable carrier. Also provided is a pharmaceutical composition comprising a topically effective amount of quercetin and a topically effective amount of genistein in a pharmaceutically acceptable carrier. A method of treating oral cancer in a subject is provided, comprising topically administering to the mouth of the subject a topically effective amount of quercetin and a topically effective amount of resveratrol in a pharmaceutically acceptable carrier. Also provided is a method of treating oral cancer in a subject, comprising topically administering to the mouth of the subject a topically effective amount of quercetin and a topically effective amount of genistein in a pharmaceutically acceptable carrier. A method of preventing oral cancer in a subject is provided, comprising topically administering to the mouth of the subject a topically effective amount of quercetin and a topically effective amount of resveratrol in a pharmaceutically acceptable carrier. Also provided is a method of preventing oral cancer in a subject, comprising topically administering to the mouth of the subject a topically effective amount of quercetin and a topically effective amount of genistein in a pharmaceutically acceptable carrier. Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the hydrolysis of the 7-O-glucoside of genistein present in high concentrations in soy food to the genistein aglycone when incubated with saliva from a normal healthy volunteer. Figure 2 shows that quercetin produced a dramatic inhibition of the number of metabolically competent oral squamous cell carcinoma SCC-9 cells with a significant reduction occurring at the very low concentration of 5-20 μM. Figure 3 shows the effects of various concentrations of quercetin and quercetin-4- glucoside on SCC-9 cells. Figure 4 shows an autoradiogram of lysate from Caco-2 cells treated with

[14c]quercetin. Figure 5 shows the rate of uptake of quercetin by oral epithelial cells. Figure 6 illustrates the method used to culture and test the oral tissue model at the Air Liquid Interface (ALI). Figure 7 shows the light micrographs of oral tissue and a native human buccal tissue control. Figure 8 shows immunohistochemistry results for cytokeratin K13. Figure 9 shows immunohistochemistry results for the human beta defensins. Figure 10 shows the effects of SLS in the concentration range (0-3%) normally found in common dentifrices. Figure 11 shows the effects on tissue viability of two toothpastes containing SLS. Figure 12 shows the that genistein produced a dramatic inhibition of the number of metabolically competent oral squamous cell carcinoma SCC-9 cells with a significant reduction occurring at low concentration, e.g., minimum effective concentration in this study of 10 μM. Figure 13 shows the effect of treatment for 24 hours with 25 μM quercetin or resveratrol or a combination of 12.5 μM of both polyphenols on the incorporation of [3H]thymidine in newly synthesized DNA in human oral squamous carcinoma SCC-9 cells. Mean values ± SE of triplicate samples from three independent experiments are shown. Statistical analysis showed that the combination of quercetin and resveratrol was more effective than either compound alone. Figures 14A and 14B show the transport of quercetin and resveratrol through the EpiOral tissue (expressed in pmol) over time. The polyphenols were applied to the mucosal side of the tissue mounted in permeable inserts. The transport was measured on the opposite (serosal) side of the tissue.

DETAILED DESCRIPTION

As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "flavonoid" includes mixtures of flavonoids, reference to "a pharmaceutically acceptable carrier" includes mixtures of two or more such carriers, and the like. Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed, the "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout the application data are provided in a number of different formats and that these data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. ^/ Provided is a pharmaceutical composition comprising a topically effective amount of quercetin and a topically effective amount of resveratrol in a pharmaceutically acceptable carrier. Also provided is a pharmaceutical composition comprising a topically effective amount of quercetin and a topically effective amount of genistein in a pharmaceutically acceptable carrier. As used herein, a "topically effective amount" refers to an amount that is applied topically to (i.e., to the surface of) a lesion that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side affects. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. A "pharmaceutically acceptable carrier" refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled A topically effective amount of quercetin is from at least about 5μM to 100 μM.

The effective concentration of quercetin can also be from about 25 μM to about 50μM. The IC50 of quercetin is from about lOμM to about 50μM. The term quercetin is used herein to refer to the biologically active form of the compound, rather than to any of its forms that require further processing, e.g., hydrolysis. A topically effective amount of resveratrol can be from at least about 25μM to about

100 μM. The effective concentration of resveratrol can also be from about 40 μM to about 80 μM. The IC50 of resveratrol is from about 25 μM to about 75 μM. The term resveratrol is used herein to refer to the biologically active form of the compound, rather than to any of its forms that require further processing, e.g., hydrolysis. A topically effective amount of genistein can be from at least about 5μM to about 100 μM. The effective concentration of genistein can also be from about 50μM to about 100 μM. The IC50 of genistein is about 70 μM. The term genistein is used herein to refer to the biologically active form of the compound, rather than to any of its forms that require further processing, e.g., hydrolysis. A topically effective amount of quercetin and a topically effective amount of resveratrol can penetrate the stratified layers of the oral epithelium and reach the basal cell layer of the human oral mucosal tissue where abnormal cellular proliferation begins and develops into dysplasia or cancer. There are two pathways by which quercetin and resveratrol, after being applied to the surface cell layer of the oral mucosa, can reach the basal cell layer. One route is a paracellular route, whereby the compositions permeate the tissue and reach the basal cell layer by going between the cells in the various layers of the oral mucosal tissue, as shown in Figures 14A and 14B. Another route is a transcellular route, whereby the compositions enter mucosal surface cells, pass through and out of the surface cells, and are taken up and pass through contiguous cells that are progressively closer to the basal cell layer. A method of treating oral cancer in a subject is provided. The method comprises topically administering to the mouth of the subject a topically effective amount of quercetin in a pharmaceutically acceptable carrier and a topically effective amount of resveratrol in a pharmaceutically acceptable carrier. These two flavonoids can be delivered in separate carriers, or they can be delivered together in a single carrier formulation. Also provided is a method of treating oral cancer in a subject, comprising topically administering to the mouth of the subject a topically effective amount of quercetin in a phannaceutically acceptable carrier and a topically effective amount of genistein in a pharmaceutically acceptable carrier. These two flavonoids can be delivered in separate carriers, or they can be delivered together in a single carrier formulation. As used herein, "subject" includes, but is not limited to, a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent), a fish, a bird or a reptile or an amphibian. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. The present methods can include administering quercetin and resveratrol together. This can mean they are delivered at the exact same time (e.g., via a single pharmaceutical formulation containing both), or it can mean they are present in the mouth at overlapping times, i.e., they are present together. As shown in the Examples below, administering quercetin and resveratrol together produces a synergistic effect. For example, the IC₅₀ for quercetin and resveratrol combined at equal concentrations is from about 2 μM to about 5 μM for each. In contrast, the IC₅₀ for quercetin administered alone is from about 10 μM to about 50 μM, and the IC₅o for resveratrol administered alone is from about 25 μM to about 75 μM. Because a lower than expected concentration of quercetin can be used in combination with a lower than expected concentration of resveratrol to kill approximately 50% of test cells compared to the required concentrations of each drug, if each drug were administered separately, there is a synergistic effect when quercetin and resveratrol are administered in combination. The present methods also can include administering quercetin and genistein together. This can mean they are delivered at the exact same time (e.g., via a single pharmaceutical formulation containing both), or it can mean they are present in the mouth at overlapping times, i.e., they are present together. A method of preventing oral cancer in a subject is provided, comprising topically administering to the mouth of the subject a topically effective amount of quercetin and a topically effective amount of resveratrol in a pharmaceutically acceptable carrier. Also provided is a method of preventing oral cancer in a subject, comprising topically administering to the mouth of the subject a topically effective amount of quercetin and a topically effective amount of genistein in a pharmaceutically acceptable carrier. In the methods of preventing oral cancer, the subject has been identified as having a pre-cancerous condition. The pre-cancerous condition can be a dysplasia, for example, an abnormality in size, shape and organization of oral epithelial cells. Topical administration to the mouth comprises administering the composition comprising quercetin and resveratrol or the composition comprising quercetin and genistein in a formulation selected from the group consisting of mouth rinses, mouth washes, pastes, oral bands, powders, gels, chewing gum, mouth sprays, dissolvable lozenges, chewable lozenges, dissolvable tablets, chewable tablets, and dissolvable polymers with the composition contained therein. These and other carriers are described below. It is also recognized that other pharmaceutically acceptable carriers for administration of quercetin, resveratrol, and genistein that are known or later developed can be used in the methods and compositions for treating and/or preventing oral cancer. Sites of Oral Cancer The oral cavity (mouth) and the oropharynx (the part of the throat at the back of the mouth) includes many parts: the lips; the lining inside the lips and cheeks, called the buccal mucosa; the teeth; the bottom (floor) of the mouth under the tongue; the front two-thirds of the tongue; the bony top of the mouth (hard palate); the gums (gingiva); and the small area behind the wisdom teeth (retromolar trigone). The oropharynx includes the back one-third of the tongue, the soft palate, uvula, the tonsils, and the part of the throat behind the mouth. Salivary glands throughout the oral cavity make saliva, which keeps the mouth moist and helps digest food. The most common subsites of oral/pharyngeal cancer are the tonsil, base of tongue, other and unspecified parts of tongue, floor of mouth, lip and parotid gland. The methods and compositions provided herein can also be used to treat or prevent cancer in any part of the oral cavity, oropharynx, pharynx and hypopharynx. Administration of the Compositions The compositions are useful in therapeutic and prophylactic methods for the treatment, prevention or reduction of oral cancer in humans. Such compositions are suitable for use in a variety of drug delivery systems. Suitable formulations are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). A brief review of methods for drug delivery is provided by Langer, Science 249:1 527 1533 (1990). The compositions are suitable for single administrations or a series of administrations. The pharmaceutical compositions are intended for parenteral, topical, oral or local administration. Preferably, the pharmaceutical compositions are administered topically to the oral cavity. Thus, pharmaceutical formulations are used that deliver effective amounts of quercetin, resveratrol, and genistein to the tonsils, the base of tongue, other parts of tongue, the floor of mouth, the insides of the cheeks, the lips, the oropharynx, the pharynx, the hypopharynx and the parotid glands. The compositions for administration comprise a solution of the agents described above dissolved dispersed or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers are used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions are sterilized by conventional, well known sterilization techniques, or are sterile filtered. The compositions contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. For solid compositions, conventional nontoxic solid carriers are used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10 - 95% of active ingredient and more preferably at a concentration of 25% - 75%. The composition comprising quercetin and resveratrol and the composition comprising quercetin and genistein can be administered topically to the mouth by the means applicable to oral delivery. For example, they can be delivered in the form of mouth rinses or washes, pastes (e.g., tooth pastes), oral bands, powders, gels, chewing gum, mouth sprays, dissolvable or chewable lozenges (including breath mints), dissolvable or chewable tablets, dissolvable polymers and rapidly dissolving adhesive membranes, having the effective amount of quercetin and genistein contained therein. An example of an adhesive membrane is a mucosal adhesive film, a three-layer hydroxypropyl cellulose film comprising a nonadhesive layer, an intermediate layer containing the compositions of the invention, and an adhesive layer. The mucosal adhesive film can remain on the wet mucosal surface for about 5 to 6 hours despite chewing or tongue movement [60]. United States Patent No. 4,454,110 issued June 12, 1984 to Caslavsky, et al. describes a self-gelling liquid composition for topical application in the oral cavity. A self- gelling composition for topical application, particularly in the oral cavity, which composition comprises: an aqueous solution of ethyl orthosilicate monomer or prepolymer; an active ingredient, such as a provided therapeutic or preventive anticancer composition for sustained release in the area of topical application; and a gelation agent, to provide, on mixing of the composition, the in situ gelling of the composition within a predetermined time period after topical application, to provide the penetration properties of a low-viscosity solution with the sustained release of the active ingredient from the gel. "Therapeutic or preventive composition" as used herein includes compositions that are both therapeutic and preventive as well as those that are primarily or exclusively either therapeutic or preventive, but not both. United States Patent No. 6,319,510 issued November 20, 2001 to Yates, et al. describes a gum pad for delivery of medication to mucosal tissues. The gum pad is a laminate composed of: (a) a synthetic base or backing layer which is configured to be held in place on the gingiva (gums) in the mouth; (b) an intermediate, reservoir layer for containing medication therein; and (c) a semi-permeable outer layer facing outwardly toward oral mucosal tissues in the mouth which will allow saliva to enter and dissolve the medication in the reservoir layer into solution and pass the diffused saliva-medication solution outwardly to the oral mucosal tissues. The backing layer is placed on the gum so that the semi-permeable outer layer faces outwardly toward the buccal mucosa. Saliva enters the semi-permeable layer and dissolves the therapeutic or preventive anticancer composition in the reservoir layer, then diffuses outwardly through the semi-permeable layer to the mucosal tissues in the mouth where it is readily absorbed into the circulatory system. United States Patent No. 6,017,516 issued January 25, 2000 to Mody et al. describes a pharmaceutical oral formulation for topical application of anticancer compositions. The formulation includes a gelled hydrophilic and water-dispersible polymer having free carboxylic groups, an aqueous base, a penetration enhancer and a chelating agent. The formulation is for topical application in the form of an aqueous gel in the treatment of oral cancer and other oral diseases (e.g., periodontal diseases including gingivitis, stomatitis, aphthous ulcers and post-extraction infection). United States Patent No. 6,136,297 issued to Sagel et al. describes a delivery system for an oral cancer therapeutic or preventive composition using a strip of material having low flexural stiffness. Disclosed is a system for delivering a therapeutic or preventive composition to the oral surface, the delivery system comprising a strip of flexible material having a sufficient flexibility to form to the contours of the oral surface. The strip of material is readily conformable to oral surfaces without permanent deformation when the delivery system is placed there against. The therapeutic or preventive composition is applied to the strip of material such that when the delivery system is placed on the oral surface, the active composition contacts the surface. The delivery system also provides adhesive attachment between the strip of material and the oral surface so as to hold the delivery system in place for a sufficient amount of time to allow the anticancer therapeutic or preventive composition to act upon the oral surface. Methods of delivering the therapeutic or preventive composition to the oral surface include pre-coating the strip of material, having the wearer apply therapeutic or preventive composition to the strip of material, or having the wearer apply the therapeutic or preventive composition directly to the oral surface and immediately applying the strip of material over the applied therapeutic or preventive composition. United States Patent No. 6,514,484 issued February 4, 2003 to Rajaiah et al. describes a system for delivering a therapeutic or preventive composition to oral surfaces using an integral carrier. Systems for delivering cosmetic and therapeutic or preventive compositions to the oral cavity employ a strip comprising a first layer of material, a second layer comprising polybutene with a molecular weight of about 300 to about 3000, and a therapeutic or preventive composition included within the second layer. Therapeutic or preventive or cosmetic compositions in delivery systems comprising polybutene prevent, inhibit or treat oral cancer and other oral diseases. A further option is to deliver the compositions with tobacco products (smoking or chewing), themselves, to provide a protective effect against the carcinogenic effect of tobacco. The tobacco product can be treated with the composition so that the composition is absorbed by the tobacco product, and is released into the mouth when the tobacco product is consumed (combusted or chewed). The technology for putting additives into tobacco products is well know and routinely applicable in the present context.

Therapeutic concentrations for quercetin, resveratrol, and genistein: The IC50, the concentration that leads to a 50% inhibition of oral cancer cell growth, for quercetin is from about 10 μM to about 50 μM. This would correspond to a saliva concentration of about 15 μg/ml (50μM at mol. wt. 308), which would be present, if at all, for a very short time period after dietary intake. For genistein with an IC50 of about 70 μM, the amount required to be present in saliva would be even higher and, thus, even less likely to occur from dietary sources. In contrast, topical application via a rapidly dissolving adhesive membrane reaches this concentration faster and lasts longer. In identifiable circumstances, a once per day application of about 0.25 mg to about 2 mg, for example about 1 mg, of each of quercetin and resveratrol or of quercetin and genistein is sufficient for maintenance of an effective concentration. It is reasonable to expect that multiple administrations can be used, over a period of weeks or months, providing daily effective doses in the amounts described herein or those later identified through optimization. Combination Therapy In the present compositions and methods, other known flavonoids and plant polyphenols can be included in the present composition comprising quercetin and resveratrol and in the present composition comprising quercetin and genistein. These compositions and methods treat and prevent oral cancer and any additional cancers treated by the component polyphenols, e.g., tannin-polyphenols, saponins, mimosine, flavonoids, terpenoids, phytates, catechins (epigallocatechin-3-gallate, epigallocatechin and epicatechin), theaflavin (TF), thearubigin (TR), trans-resveratrol, chrysin (5,7- dihydroxyflavone), apigenin (5,7,4'-trihydroxyflavone), morin and flavone. The compositions or methods do not contain the flavonoid morin alone. However, morin can also be present in the compositions and methods along with other known flavonoids and polyphenols. In the present compositions and methods, other anticancer compounds can be included in the present composition comprising quercetin and resveratrol and in the present composition comprising quercetin and genistein. The combination therapy can include anti- neoplastic agents, such as the following: Acivicin; Aclarubicin; Acodazole Hydrochloride; AcrQnine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefmgol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflomithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil 1 131 ; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl;

Interferon Alfa-n3; Interferon Beta- 1 a; Interferon Gamma- 1 b; Iproplatin; Irinotecan

Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole

Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;

Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine;

Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin;

Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole;

Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;

Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine

Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine;

Rogletimide; Safmgol; Safmgol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium;

Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid;

Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide;

Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine;

Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate;

Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate;

Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine

Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole;

Zeniplatin; Zinostatin; Zorubicin Hydrochloride. Other anti-neoplastic compounds include: 20-epi-l,25 dihydroxyvitamin D3; 5- ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin;

ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; atrsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-

CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1 ; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocannycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; fmasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MLF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A +myobacterium cell wall sk; mopidamol; multiple drug resistance genie inhibitor; multiple tumor suppressor 1 -based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N- acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone +pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenyl acetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safmgol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1 ; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1 ; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfmosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfm; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatin stimalamer. The combination therapy can further include anti-cancer supplementary potentiating agents, such as the following: Tricyclic anti-depressant drugs (e.g., imipramine, desipramine, amitryptyline, clomiprainine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline); non-tricyclic anti-depressant drugs (e.g., sertraline, trazodone and citalopram); Ca⁺⁺ antagonists (e.g., verapamil, nifedipine, nitrendipine and caroverine); Calmodulin inhibitors (e.g., prenylamine, trifluoroperazine and clomipramine); Amphotericin B; Triparanol analogues (e.g., tamoxifen); anti arrhythmic drugs (e.g., quinidine); antihypertensive drugs (e.g., reserpine); Thiol depleters (e.g., buthionine and sulfoximine) and Multiple Drug Resistance reducing agents such as

Cremaphor EL. The compounds of the invention also can be administered with cytokines such as granulocyte colony stimulating factor. Additionally, the anticancer therapeutic or preventive compositions comprising quercetin and resveratrol and the anticancer therapeutic or preventive compositions comprising quercetin and genistein can be used with any of the known oral compositions that prevent dental caries or prevent or treat gingivitis or periodontal disease. Any of the known fluoride-containing dentifrices can be used in combination with the anticancer therapeutic or preventive composition. An example of such a composition comprises 0.04 to 10% by weight of stannous fluoride and not less than 0.1% by weight of a phytic acid compound, the molar ratio of the phytic acid compound to stannous fluoride being in the range of from 0.01:1 to 4:1 and the composition being acidic, is effective in inhibiting dental caries and shows a prolonged effect after application. The oral combination therapy can be delivered by the means described herein and the like. The methods of treating and preventing oral cancer can use combination therapies with additional active compounds as described above. The polyphenols and/or flavonoids can be delivered to the site of the oral cancer in the same or different phannaceutically acceptable carriers as described above. The quercetin and resveratrol components of the methods are present at overlapping times at the site of the oral cancer, although they and the other components may also be present before or after each other in some cases. Likewise, the quercetin and genistein components of the methods are present at overlapping times at the site of the oral cancer, although they and the other components may also be present before or after each other in some cases. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some ereors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in C or is at ambient temperature, and pressure is at or near atmospheric.

EXAMPLES

Example 1 :

Conversion of polyphenol glucosides to their aglycones by saliva and their effects on proliferation and apoptosis of oral cancer cells. This example involves studies of the enzymatic hydrolysis of quercetin-4'-O-glucoside, genistin and ellagitannins to their aglycone forms. Product identification is done by

LC/MS. Kinetic studies are done by determining K_m and V_max values and in particular the total hydrolytic capacity. The effect of chlorhexidine is evaluated in order to determine the contribution of bacterial enzymes to the hydrolysis rate [17]. Hydrolysis rates in situ directly in the oral cavity are determined. This example also focuses on the effects of the intact glycosides and tannins as well as their hydrolysis products (aglycones) on proliferation and apoptosis of the human oral cancer cell line SCC-9. A wide range of concentrations (0.01-200 μM) as in Fig. 2 is tested using the well established cell proliferation and viability assay MTT [18, which is incorporated herein by reference for its teaching of the cell proliferation and viability assay MTT] and the annexin V assay for apoptosis [19, which is incorporated herein by reference for its teaching of the annexin V assay for apoptosis]. Thus, the minimum effective concentration of these agents is established. Negative controls are solvent blanks. Polyphenols to be Studied: The compounds selected for study are four of the major anticancer dietary polyphenols, shown to be inhibitors of cancer cell proliferation and inducers of apoptosis in various cancer cells, although not oral cancer cells. Three of these must, presumably, first be hydrolyzed to produce their effect (two glucosides, one tannin). All of them have been shown to accumulate in epithelial cells of the digestive tract [20-22]. Each of the four compounds appears to have a different mechanism of action. Quercetin-4'-O-glucoside and its aglycone: The glucoside is isolated from the red onion, as described in previous studies [23, which is incorporated herein by reference for its teaching of the isolation of quercetin]. Quercetin is a selective inhibitor of both PI 3-kinase and protein kinase C [12]. Genistin (genistein-7-O-glucoside) and its aglycone genistein: Genistein is a specific inhibitor of tyrosine kinase (e.g., IGF and EGF receptor intrinsic tyrosine kinase) [12]. Ellagitannins (composite of ellagic acid conjugates) and their hydrolysis product ellagic acid: Ellagitannins are obtained as described [24, which is incorporated herein by reference for its teaching of the isolation ellagitannins]. Ellagic acid is a potent inhibitor of esophageal cancer cell proliferation and inducer of apoptosis with unknown mechanism of action [25, which is incorporated herein by reference for its teaching of the activity of ellagic acid]. Epigallocatechin gallate (EGCG) is not a conjugate that needs hydrolysis to produce its effect. EGCG is the major anticancer flavonoid present in tea. It inhibits cancer proliferation via the tumor necrosis factor-α and multiple other pathways [26]. Methods: Genistin (genistein 7-glucoside), quercetin 4'-glucoside and ellagitannins (0.1-200 μM) are incubated at 37° with human saliva for various times. The samples are purified by solid phase extraction, reconstituted in mobile phase and subjected to reversed-phase HPLC analysis for glucosides and aglycones, using photodiode array detection at 270 nm for genistin and genistein and at 370 nm for quercetin and its glucoside [23, 35, which are incorporated herein by reference for their teaching of purification and HPLC analysis for glucosides and aglycones]. Saliva is obtained by asking normal volunteers to spit into a test tube after rinsing the oral cavity with tap water. The saliva samples are diluted with an equal volume of water. The effects of the polyphenols, glucosides and aglycones are investigated in the human oral cell line SCC-9, derived from a tongue squamous cell carcinoma. The cells are cultured in Ham's F12/DMEM medium with 10% fetal bovine serum and hydrocortisone and are subcultured in 96-wells for cell viability and cell proliferation assays. With the MTT assay, the effect of 48-72 hr treatment with 0-200 μM of the polyphenols on the number of viable cells is used as a measure of inhibition of cell survival and proliferation. Fresh polyphenol-containing medium is added every 24 hr. This spectrophotometric assay measures the cellular cleavage of the MTT substrate (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyl tetrazolium bromide) to colored formazan by mitochondrial dehydrogenase in living cells [18]. The results are expressed as percent inhibition compared to control treatment. Cytotoxicity of the polyphenols (0-200 μM) after treatment of the cells for 24 hr is measured by trypan blue exclusion, where only dead cells are stained. Translocation of phosphatidylserine from the inner (cytoplasmic) leaflet of the plasma membrane to the outer (cell surface) leaflet, an early event in the apoptotic process [19], is quantified by the annexin V assay. This protein, with a fluorescent tag, binds specifically to phosphatidylserine with high affinity. SCC-9 cells in 24-well plates are exposed for 24 hr to 0-200 μM concentrations of the polyphenols. The cells are then stained with annexin-EGFP and propidium iodide (Clontech kit) and visualized by confocal microscopy with dual filters for EGFP and propidium iodide. The percentage of cells with green membrane staining is used as an index of apoptosis. Results: When genistin, the 7-O-glucoside of genistein present in high concentrations in soy food, was incubated with saliva from a normal healthy volunteer, hydrolysis to the genistein aglycone occurred (Fig.l). Quercetin-4'-O-glucoside, present in high concentrations in apples and onions, is hydrolyzed by saliva to quercetin. This represents a novel finding for glycosidic flavonoids and would put their putative biologically active forms directly in contact with the oral epithelium. The present results (e.g., Fig 1) establish the capacity of human saliva to hydrolyze dietary polyphenolic conjugates, including flavonoid glucosides, to their potentially biologically active aglycones. Oral squamous cell carcinoma SCC-9 cells were treated with quercetin and its effect on cell proliferation was measured, using the MTT assay [18]. As shown in Fig. 2, quercetin produced a dramatic inhibition of the number of metabolically competent cells with a significant reduction occurring at the very low concentration of 5-20 μM. Interestingly, quercetin has been reported to inhibit the cell proliferation signaling enzyme PI 3-kinase at the same low concentration [11, 12]. These experiments are repeated to establish minimum effective concentrations. Once quercetin is formed, it is rapidly absorbed by the epithelial oral cancer cells, as previously shown to occur with colonic and hepatic cancer cells [15, 16], to produce its biological effects. The present results (e.g., Fig. 2, Fig. 3 and Fig. 12) also establish the inhibitory action of polyphenolic aglycones on cell proliferation and induction of apoptosis in oral cancer cells.

Example 2:

Nature of interactions between polyphenol aglycones and the cell proliferation signaling systems of oral cancer cells. This example involves the establishment of the time course, extent and kinetics of [¹⁴C] quercetin accumulation by the SCC-9 cells. The example further assesses the covalent binding of [^I4C]quercetin to SCC-9 cell proteins, using the quantitative solvent extraction approach [29, 30]. Further, the labeled proteins are separated both by ID SDS-PAGE as in Fig. 4 and by 2D gel electrophoresis, with a focus on the p85 and p55 bands. Although it appears clear that the mechanism by which quercetin blocks cancer cell proliferation is via the PI 3-kinase pathway [11, 12, 27], it is not clearly understood how. In previous preliminary studies with the human colonic adenocarcinoma cell line Caco-2, a novel set of steps for this and other effects have been recognized. First, quercetin appears to be oxidized within the cells by reactive oxygen species (ROS), the levels of which are markedly elevated in cancer cells. Second, the product of this oxidation, a semiquinone or quinone methide [28], is highly reactive and combines covalently with proteins with high affinity for quercetin [30]. This is shown in Fig. 4 with [¹⁴C]quercetin, demonstrating protein bands of molecular weights of 30-85 kDa (reduced) as well as bands at 12-17 kDa (non-reduced). When compared to the Coomassie blue staining, it is apparent that the radiolabeled proteins shown are highly specific protein targets, which are proposed to be linked to the antiproliferative and apoptotic mechanisms of action of quercetin. This is the first report of its kind for any dietary anticancer polyphenol. The two bands at ~ 85 and ~ 55 kDa represent the two known regulatory subunits of PI 3-kinase, i.e. p85 and p55 (splice variant). Surprisingly, the catalytic subunit pi 10a, with which quercetin has been assumed to interact [27], was not labeled. This raises an alternative hypothesis for how quercetin blocks cancer cell proliferation via the PI 3-kinase pathway. Methods: The uptake of genistein, quercetin, ellagic acid and EGCG by the SCC-9 cells are measured after exposing the cells (in 6-well plates) to 50 μM flavonoid in buffer for 1 hr. After repeated rapid washing of the cell surface, the cell monolayers are extracted twice with methanol. The combined methanol extracts are analyzed by HPLC as above. The effect of exposure time and flavonoid concentration on the cellular uptake is also investigated. [¹⁴C]-Labeled quercetin, obtained from NCI, is also used. Total radioactivity in the cells is measured after digestion of the cells. The covalent binding of [¹⁴C] quercetin to SCC-9 cellular protein is first quantified after exposure of the cells to < 50 μM flavonoid for 10 min up to 6 hr. TCA precipitation of cell homogenate proteins is followed by exhaustive solvent extraction of non-covalently bound radioactivity [29, which is incorporated herein by reference for its teaching of precipitation and extraction techniques]. The protein pellet is then digested with NaOH and subjected to liquid scintillation spectrometry. The covalent binding of [¹⁴C]quercetin to specific SCC-9 cellular proteins is explored by SDS-PAGE analysis with Coomassie blue staining and autoradiography. The cells are treated with [¹⁴C]quercetin as above and lysed with buffer with or without β- mercaptoethanol (i.e. under reducing or non-reducing conditions). Cell lysates or subcellular fractions (cytosolic and nuclear fractions) are loaded on 4-20% gradient polyacrylamide gels together with appropriate molecular weight markers and the stained and dried gels are exposed to autoradiography films for up to a month before development. 2-Dimentional gel electrophoresis of SCC-9 cell lysates after the optimum exposure time and [¹⁴C]quercetin concentration use isoelectric focusing with pH 5-8 strips as the first dimension and 10% SDS-PAGE as the second dimension. The gels are silver-stained and subjected to autoradiography. Western blot analysis of the SCC-9 lysates with PI 3-kinase antibodies after various flavonoid treatments is done with both SDS-Page gels and 2-D gels (IEF + SDS-Page). Immunoprecipitation is performed using agarose-conjugated p85 antibodies (Upstate Technologies). Mass spectrometric identification of proteins and flavonoid binding sites mainly use trypsin digests from 2D gel stabs or immunoaffinity-purified protein fractions with MALDI-TOF analysis (molecular weight information) or electrospray LC/MS for detailed structural information. A shift in the migration of p85 and p55 bands in quercetin-treated vs. control cell lysates shows that p85 and p55 are the major targets of covalent binding of quercetin. This indicates that quercetin's actions are via the regulatory rather than the catalytic subunits of PI 3-kinase, thus a major deviation from accepted but unproven dogma. To confirm that quercetin binds covalently to both the p85 and p55 subunits, the focus is on p85α, for which an agarose-conjugated antibody is available. After incubation of the SCC-9 cells with [¹⁴C] quercetin, p85 is immunoprecipitated from the lysates with or without the addition of purified PI 3-kinase and subjected to 2D-gel electrophoresis. The gels are silver-stained and subjected to autoradiography. Purified PI 3-kinase as mobility standard is prepared from bovine brain by several chromatographic steps [36, incorporated herein by reference for its teaching of the preparation of purified PI 3-kinase] or by immunoprecipitation from human platelets [37, incorporated herein by reference for its teaching of immunoprecipitation of Pl-kinase from human platelets] and tested for activity with phosphatidylinositol and labeled ATP [37, incorporated herein by reference for its teaching of testing PI 3-kinase for activity with phosphatidylinositol and labeled ATP]. Then MS of the quercetin-labeled immunoprecipitate after trypsin digestion is performed using standard methods. A further focus is on the site(s) of quercetin binding to p85α. This involves MS studies, initially of adducts between quercetin and a suitable model protein, then with purified PI 3-kinase (see above). Quercetin is activated for binding by H₂O₂ HRP [22, which is incorporated herein by reference for its teaching activation of quercetin for binding]. Analogous studies of the nature of the low molecular weight quercetin adducts (12- 17 kDa) and their potential role in quercetin's biological actions are facilitated by their smaller size (for MS analysis) and the strong suggestion that they represent binding of quercetin to -SH residues. The importance of the present studies to other oral cancer cells is confirmed simply be repeating the present observations with the SCC-9 cells with other oral cancer cells.

Example 3: Oral Epithelial Uptake of Using protocols similar to those described in example 1 and a protocol similar to a published study on uptake into colonic cells (Walle et al., Biochem Pharmacol 65:1603- 1610, 2003) , the uptake of flavonoids by oral epithelium was studied. The oral (tongue) epithelial cells show a very rapid and high uptake of both quercetin and genistein. The uptake of quercetin by the SCC-9 oral epithelial cells is extensive and very rapid during the first 2 minutes, reaching 75% of maximum concentrations within this time period ( Fig. 5).

Example 4: Buccal Epithelial Tissue Model Oral epithelia function as microbial barriers against bacteria and yeast that are resident in the oral cavity. These microbes can be categorized as non-pathogenic (commensal) and pathogenic (resulting in cavities, gingivitis, and periodontal disease, etc.). The epithelium is actively involved in recognition and response to bacteria, setting into motion the innate immune responses that are an important part of periodontal health and disease. An example of a response of natural epithelium is the expression of antimicrobial peptides of the human beta-defensin (hBD) family. A three-dimensional model of the human buccal epithelium, based on normal human oral epithelial cells cultured in serum free medium, has been developed. Histologically, the tissue is 20-30 cell layers thick with cells becoming increasingly squamous toward the apical surface. No evidence of cornification is present and immuno-staining shows the expression of cytokeratin K13 in the suprabasal layers. Cytokeratin K4, the expression partner of K13, has also been detected by immuno-b lotting. These features are characteristic of buccal epithelium. Additional immuno-staining revealed the presence of hBD-1 and the recently discovered hBD-3 in the suprabasal layers; hBD2 was not expressed. The hBD-1 and hBD-2 results parallel those of un-inflamed epithelia, however the hBD-3 result has not been previously reported. In this current study, the constitutive expression of human beta defensins and other markers of intact oral epithelium in a human buccal tissue model, EpiOral™ (MatTek Corporation), was investigated. Culture Preparation The tissue model described herein is an organotypic culture of normal human oral keratinocytes cultured in serum free medium to form a three-dimensional differentiated tissue which histologically is similar to buccal mucosa. Cell culture inserts are coated with an extracellular matrix preparation upon which the gingival keratinocytes are seeded. After several days of submerged culture, the culture inserts containing the developing tissues are elevated to the air liquid interface (Fig. 6), which induces stratification and differentiation. Additional background information on the oral tissue model (commercially available under the trade name EpiOral) can be obtained from MatTek Corporation (information@mattek.com or www.mattek.com). Histology Tissue samples were fixed in 10% formalin and cut from cell culture inserts using an 8-mm diameter dermal punch and cuticle scissors. Next, tissues were dehydrated in a graded series of ethanol, embedded in paraffin, and 5-7 micron cross sections were cut. The sectioned tissues were then stained using hematoxylin and eosin (H&E) and photographed using a Nikon Diaphot microscope outfitted with a CoolPix 990 digital camera. Fig. 7 compares the histology of the tissue model with that of native human buccal tissue. Immunohistochemistry Procedures for immunohistochemistry followed those of Dale et al. (43). In brief, tissues were fixed in Carnoy's fixative (acetic acid: ethanol: chloroform, 10:60:30) overnight at RT, embedded in paraffin, and sectioned for routine histology. Sections were deparaffinized and rehydrated: endogenous peroxide was blocked using 1% H₂0₂/Tris buffered saline (TBS). After blocking with 3% normal serum of the source of secondary biotinylated antibody, the sections were incubated with primary antibody. A monoclonal antibody to cytokeratin K13 and polyclonal antibodies to beta-defensins hBD-1, hBD-2, and hBD-3 were used as per (6). Detection was via the avidin-biotin-peroxidase complex method using diaminobenzidene as substrate. MTT Tissue Viability Assay The MTT (7, which is incorporated herein by reference for its teaching of the MTT assay) tissue viability assay has proven to be an effective, quantitative means of predicting irritation using MatTek's other reconstructed tissue models, e.g., EpiDerm™, the epidermal skin tissue (8, 9, which are incorporated herein by reference for their teaching regarding epidermal skin tissue models), and EpiOcularTM, the non-cornified cornea-like epithelial tissue (10, which is incorporated herein by reference for its teaching of cornea-like tissue models). Thus, the correlation of in vivo irritation from dentifrice, anticancer drugs, and other oral care products with the EpiOral tissue results using the MTT assay is warranted. The MTT assay protocol was performed as follows: the test compounds were diluted 1 : 1 in ultrapure water and 40 μL of the resulting solution were applied to the apical surface of the tissue. Tissues were then incubated in a 6- well plate containing 0.9 ml of medium at 37°C and 5% CO2 for 60 minutes. After this time period, the tissues were rinsed with PBS to remove the applied dose. Cultures were then loaded with a 1 mg/ml solution of MTT in culture medium for 3 hours in the incubator, after which tissues were blotted, and extracted in 2.0 ml of isopropyl alcohol (LPA) overnight at room temperature in the dark. Next, 200 μL of this extractant solution were pipetted into a 96-well assay plate and the optical density (OD) was determined at 570 nm using a plate reader (Molecular Devices). Percent viability was calculated from the ratio of the ODs for the treated and negative control tissues. A time of exposure which reduced the viability to 50% (ET-50) was then calculated mathematically by interpolating between 2 time points for which the tissue viability was above and below 50%. Complete assay details can be obtained in the protocol entitled "MTT EFFECTIVE TLME-50 (ET-50) PROTOCOL" available from MatTek Corporation. Testing of compounds and actives in the model Sodium lauryl sulfate (SLS) is one of the most widely used synthetic detergents in dentifrices (48), (49). Nonetheless, it has been implicated in irritation of the oral mucosal caused by toothpastes (49). Therefore, an initial study was undertaken to determine if the oral tissue model here described could differentiate between differing levels of SLS. Towards this end, solutions of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% SLS in ultrapure water were applied to the tissue model. Solutions were diluted 1 : 1 and 40 μL were applied for 1 hour (potentially an exaggerated exposure time). Afterl hour, the tissues were rinsed and the viability of the tissue was determined using the MTT assay, as described above. Two commercial toothpastes produced by the same manufacturer were purchased at a local drug store. One toothpaste was labeled "Sensitive, Maximum Strength" and the other "Whitening with Tartar Control." Both contain an unspecified amount of SLS. 40 mg of each toothpaste was applied to the flat end of a dosing device which was then contacted with the apical surface of the oral tissue for 1 hour. After 1 hour, the tissues were rinsed, and the viability of the tissues was determined using the MTT assay, as described above. Results Fig. 6 illustrates the method used to culture and test the oral tissue model at the Air Liquid Interface (ALI). Growth or assay medium is placed beneath the cultures (5.0 or 0.9 ml, respectively). Nourishment to the tissue is supplied by medium which permeates through the microporous membrane on the bottom of the cell culture insert. In contrast to the use of monolayer cultures submerged in culture medium, the ALI method improves tissue development and differentiation and allows topical application of dentifrice samples, as would occur in vivo. Fig. 7 shows the light micrographs of oral tissue and a native human buccal tissue control. Both micrographs show examples of typical non-keratinized epithelia. Neither tissue contains a stratum corneum or granular layer. Rather, the tissues consist of 3 strata (basal, filamentosum, and distendum) and terminally differentiated cells of the stratum distendum (SD) retain their nuclei, similar to characteristics described in the literature (50). Fig. 8 shows immunohistochemistry results for cytokeratin K13. As expected, K13 was expressed in the suprabasal layers of the tissue. Cytokeratin K13 (and K4) is uniquely expressed in the suprabasal layers of non-cornified oral epithelia (51). Furthermore, K13 and K4 are not expressed in skin or in the non-cornified corneal epithelium (52). Thus, K13 and K4 expression is a specific marker for buccal/soft palate tissue. Interestingly, the K13 appears to be expressed paracellularly, i.e. only the cell membranes stain. Fig. 9 shows immunohistochemistry results for the human beta defensins. The human beta defensins (hBD) are antimicrobial peptides produced by oral epithelial cells to control the many commensal and pathogenic bacteria in the oral cavity (53). It has been shown that hBDl is expressed in the suprabasal layers of gingival and buccal epithelia whereas hBD2 is only expressed following bacterial challenge (17). In fact, in the NHBE tissue, hBDl was expressed subrabasally and hBD2 was not expressed (Figures 9A & 9B). hBD3, a newly discovered defensin (55) which is the most potent antimicrobial peptide of the hBD-1 , hBD-2, hBD-3 group (56), has been detected using reverse transcriptase polymerase chain reaction (RT-PCR) in both oral epithelial and fibroblast cells from inflamed and non-inflamed tissue (55). The immunolocalization of hBD3 as shown in Figure 9C was reported by Kubilus et al. (J. Invest. Dermatol., 121, (1), Abstract #0045, 2003). Fig. 9 shows the effects of SLS in the concentration range (0-3%) normally found in common dentifrices. As shown therein, there is close to a linear relationship between tissue viability and the SLS concentration between 0-2.5 %. Fig. 11 shows the effects on tissue viability of two toothpastes, one of which is formulated for "Sensitive teeth" and the other for teeth "Whitening." As would be expected, the effects on the tissue are reduced in the Sensitive teeth toothpaste versus the teeth Whitening sample.

Conclusion A serum free, reconstructed oral tissue model containing normal human oral epithelial cells is described and used. Histologically this tissue most closely resembles the non-cornified buccal tissue of the oral cavity. The reconstructed tissue expresses cytokeratin K13 in the suprabasal tissue layers. This matches the expected expression pattern for human buccal tissue reported in the literature. Human β-defensin 1 (hBD-1) and β-defensin 3 are constitutively expressed in the reconstructed tissue in the apical cell layers of the tissue; human α-defensin 2 was not expressed. For hBD-1 and hBD-2 these findings corroborate literature reports; however, localization of hBD-3 in buccal tissue has not been previously reported. Based on the results presented, the oral tissue model appears sensitive enough to discriminate between concentrations of a compound. In addition, since the model is representative of oral epithelium, it is expected to validate the delivery of compounds (e.g., quercetin and genistein) to the basal membrane or the oral epithelium. These findings support the effectiveness of the oral delivery approach in an environment very similar to that in the human study.

Example 5:

Inhibitory effects of quercetin and other polyphenols on human oral cancer cell proliferation - involvement of the PI3K/AKT pathway and its downstream targets. The effects of 25 dietary polyphenols on the proliferation of the oral squamous carcinoma SCC-9 cells and the signaling mechanisms involved were studied. The MTT cell survival assay showed that all unglycosylated polyphenols tested were active with minimum effective concentrations ranging from 5 to 100 μM, with some of the most potent polyphenols being apigenin, chrysin, luteolin, quercetin and genistein. Interestingly, two polyphenol glycosides, i.e. diosmin, which is in clinical use, and genistin, were quite active, although most other glycosides, e.g. rutin and quercitrin, were not. When the effect of some of these polyphenols was also examined with the thymidine incorporation assay, all produced effects similar to the MTT assay. Interestingly, when combining treatment such as quercetin with resveratrol, there was a synergistic effect on the inhibition of cell proliferation. When cellular uptake of some of these polyphenols was examined in the SCC-9 cells, high accumulation of most polyphenols occurred. Because quercetin has previously been shown to interact with phosphoinositol 3-kinase (PI3K), initial cell signaling studies were performed on this pathway. LY294002 and wortmannin, two prototype PI3K inhibitors, were potent inhibitors of SCC-9 cell proliferation, reflective of their PI3K binding affinity. The effect of quercetin on PI3K-inducible AKT phosphorylation was examined next. Quercetin exposure in serum-stimulated SCC-9 cells produced a concentration-dependent inhibition over 24-72 hr of treatment, with complete inhibition at 50 μM. In addition, a concentration-dependent inhibition of cyclin Dl protein expression, a well-established risk factor in oral cancer, was observed. Examination of a limited number of additional polyphenols on cell signaling showed that only resveratrol and, to a minor extent, ellagic acid inhibited AKT phosphorylation. These polyphenols were the only polyphenols that also inhibited cyclin Dl expression. In contrast, silymarin and silibinin resulted in a 3-fold increase in cyclin Dl expression. The protective effects of quercetin and resveratrol (and maybe ellagic acid) appear to involve the PI3K/cyclin Dl pathway; other polyphenols have different mechanisms of action.

Example 6:

Quercetin and other polyphenols inhibit human oral cancer cell proliferation synergistically The effects of selected dietary polyphenols on the proliferation of the SCC-9 oral cancer (tongue) cells, using the thymidine incorporation assay, were studied. After the cells were treated with polyphenols at 0, 10, 25 and 50 μM in serum-free medium overnight, [3H]thymidine was added for 3 hr. The cells were harvested, washed and treated with trichloroactic acid. After centrifugation and digestion of the cell pellet, the amount of incorporated [3H]thymidine was measured by liquid scintillation spectrometry. Quercetin, one of the major polyphenols in the human diet, particularly abundant in apples and onions, showed a dose-dependent inhibition of the thymidine incorporation with about 85% inhibition at 25 μM and >95% inhibition at 50 μM. Identical results were obtained with SCC-25 cells, in which poor responses had been obtained in a previous study. In contrast to quercetin, rutin (quercetin rutinoside), a flavonoid glycoside present in many vegetables, had no effect. This was consistent with the notion that rutin does not enter cells. Resveratrol, an active polyphenol in red grapes and wine, was the second most potent of the compounds examined, also producing a dose-dependent inhibition with about 65% inhibition at 25 μM and 80% inhibition at 50 μM. Several other polyphenols showed potencies similar to that of resveratrol. Based on previous studies, quercetin and resveratrol may affect cell proliferation by different mechanisms. A combination of these two polyphenols was therefore examined. A clear synergistic effect was seen with 75% inhibition at 5 μM of each compound and >90% inhibition at the higher concentrations.

Example 7:

Chemoprevention by polyphenols in oral cancer cells: antiproliferative effect and interaction with signaling pathways. Chemoprevention refers to the administration of naturally occurring or synthetic agents to prevent the initiation and the promotion of carcinogenesis. In this regard, several naturally occurring agents, such as polyphenols, which are widely distributed in fruit and vegetables, present significant potential. The effects of different polyphenols, for example, quercetin (Q), resveratrol (RN), and acacetin, were evaluated on cell growth, DΝA synthesis and cell cycle progression, with regard to the human oral squamous carcinoma cell line SCC-9. Cell growth was determined by the MTT assay; DΝA synthesis was measured by the incorporation of [3-H]- thymidine in nuclear DΝA; and cell cycle progression was observed by flow cytometry. All three compounds induced significant dose-dependent (10, 25, 50 μM) inhibition in cell growth as well as in DΝA synthesis. Because increased expression of epidermal growth factor receptor, a characteristic of oral cancer, is correlated with activation of the phosphoinositide-3-kinase PI3K Akt pathway, the effect of these polyphenols on Akt phosphorylation was next determined. In this study, Q inhibited Akt activation in a time and dose-dependent manner, while RV at 50 μM also inhibited Akt phosphorylation. Glycogen synthase kinase-3 beta (GSK-3 beta), an important proapoptotic signaling enzyme, is a key enzyme in the PI3K/Akt pathway: Akt phosphorylates and deactivates GSK-3 beta. In this study, 50 μM Q inhibited pGSK-3 beta after 1, 3 and 6 hr incubations, while after long-term incubations (72h) Q stimulated GSK-3 beta phosphorylation in a concentration-dependent manner. Also, Cyclin Dl expression has been shown to be upregulated in SCC cells as compared with normal oral mucosa. In this study, Q inhibited cyclin Dl expression in a time and dose-dependent manner. Preliminary studies of the cell cycle distribution show that all the acacetin doses (5- 50 μM) used cause an increase in the S-phase population in a dose-dependent manner. Example 8:

Covalent binding of the flavonoid quercetin to human serum albumin. Quercetin is oxidized in cells to products capable of covalently binding to cellular proteins, a process that may be important for its biological activities. In the present study, proteins to which oxidized quercetin is binding irreversibly were identified, using radiolabeled drug, and quantifying the products after electrophoretic separation of the proteins. The binding of quercetin to human serum albumin (HSA) in fresh human blood and the effect of stimulation of neutrophilic myeloperoxidase on this binding were also measured. The in vitro binding of quercetin to 8 different proteins in the presence of catalytic amounts of horseradish peroxidase and hydrogen peroxide was highly selective for HSA. For all proteins the binding was dramatically decreased by GSH. In the blood samples, the stimulation of myeloperoxidase by nanomolar concentrations of a phorbol ester caused a 3-fold increase in the binding of quercetin to HSA. In summary, quercetin oxidized by peroxidase/hydrogen peroxide covalently links to proteins with a particularly high affinity for HSA. This also may occur in vivo after exposure to quercetin. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application, for the teaching referred to in the sentence for which the reference is made, in order to more fully describe the state of the art to which this invention pertains.

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It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

What is claimed is:

1. A pharmaceutical composition comprising a topically effective amount of quercetin and a topically effective amount of resveratrol in a pharmaceutically acceptable carrier.

2. The composition of claim 1, wherein the topically effective amount of quercetin is from about 2 μM to about 75 μM.

3. The composition of claim 2, wherein the topically effective amount of quercetin is about 50 μM.

4. The composition of claim 2, wherein the topically effective amount of quercetin is about 5 μM.

5. The composition of claim 2, wherein the topically effective amount of quercetin is about 2 μM.

6. The composition of claim 1, wherein the topically effective amount of resveratrol is from about 2 μM to about 75 μM.

7. The composition of claim 6, wherein the topically effective amount of resveratrol is about 50 μM

8. The composition of claim 6, wherein the topically effective amount of resveratrol is about 5 μM.

9. The composition of claim 6, wherein the topically effective amount of resveratrol is about 2 μM.

10. A method of treating oral cancer in a subject, comprising topically administering to the mouth of the subject a topically effective amount of quercetin and a topically effective amount of resveratrol in a pharmaceutically acceptable carrier.

1 1. A method of preventing oral cancer in a subject, comprising topically administering to the mouth of the subject a topically effective amount of quercetin and a topically effective amount of resveratrol in a pharmaceutically acceptable carrier.

12. The method of claim 11, wherein the subject has been identified as having a precancerous condition.

13. The method of claim 12, wherein the pre-cancerous condition is a dysplasia.

14. The method of any of claims 10 to 13, wherein the topical administration to the mouth comprises administering a formulation selected from the group consisting of mouth rinses, mouth washes, pastes, oral bands, powders, gels, chewing gum, mouth sprays, dissolvable lozenges, chewable lozenges, dissolvable tablets, chewable tablets, mucosal adhesive films, and dissolvable polymers with the composition contained therein.