WO2010099731A1

WO2010099731A1 - Compounds, compositions and use for anticancer therapy

Info

Publication number: WO2010099731A1
Application number: PCT/CN2010/070813
Authority: WO
Inventors: Ming Yu
Original assignee: Ming Yu
Priority date: 2009-03-01
Filing date: 2010-03-01
Publication date: 2010-09-10
Also published as: AU2010220755A1; EP2403497A1; EP2403497A4; CN102438620A; AU2010220755B2

Abstract

Cell plasma membrane impermeable compounds and compositions thereof used for anticancer treatment are provided. The compounds contain functional groups capable of blocking and/or inhibiting trans-plasma membrane electron transport (tPMET), cell surface respiration and uncoupling cell surface oxidative-phosphorylation. The compositions comprise at least one of the cell plasma membrane impermeable compounds in combination with compounds of multi-function inhibitor capable of inhibiting cancer cell hypoxia responses.

Description

TITLE: COMPOUNDS, COMPOSITIONS AND USE FOR ANTICANCER THERAPY DESCRIPTION OF THE INVENTION

RELATED APPLICATION

This application is a continuation in part claims the benefit of U.S. Provisional Application No. 61/156,507, filed Mar. 1, 2009, which are herein incorporated by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

This invention relates to the fields of oncology and chemotherapy. Specifically, the invention provides novel methods, pharmaceutical composition and targets for more efficient and less or non cytotoxic treatments of cancer.

BACKGROUND ART OF THE INVENTION

Cancer is a deadly disease and has been the leading cause of death worldwide, claiming 7.4 million lives (about 13% of all deaths) in 2004. This number has been projected to continue rising, with an estimated 12 million deaths worldwide in 2030. Even though the cancer death in US has been stabilized during the past decade, cancer incidence, prevalence and mortality from the developing countries are still in the rising phase. An effective and safe cure for cancer has been badly sought.

Up to date, chemotherapy and radiation therapy are still the mainstream for cancer treatment. These treatments were based on targeting proliferating cells rather than cancer cells only, which is also the intrinsic cause of their lethal side effects that, in turn, limit the treatment doses and, hence, the efficacy.

Targeted therapy, a new generation of cancer treatment, is aimed to target cancer specific changes of molecules and signaling pathways to induce cancer cell death, with the assumption of specifically targeting cancer cells while limiting the effects on normal cells. Enormous efforts have been made and are currently making for finding the targets and the ways of targeting them in the cells as a treatment. However, due to the complexity and overlapping regulation in biological systems, up to date, the success rate of such new generation of cancer treatment is limited. Majority of such developments are used as adjunct therapy to conventional chemotherapies.

Unlike normal cells that oxidize pyruvate and synthesize ATP in mitochondria, cancer cells predominantly metabolize glucose and produce energy by glycolysis in the cytosol. Nobel laureate Otto Heinrich Warburg pointed out that it is the aerobic fermentation lead to the formation of cancer cells (Warburg O., Science 123:309, 1956) over half century ago. This Aerobic glycolysis is a unique feature of cancer cell energy metabolism. More recently, PKM2 has been identified to be responsible for this energy metabolic switch. However, using PKM2 inhibitors to inhibit glycolysis as an anticancer treatment caused intolerable neurotoxicity. Associated with the aerobic glycoslysis, these cancer cells consume oxygen through trans-plasma membrane electron transport (tPMET, Heart, PM, Curr MoI Med, 2006, 6:895).

The tPMET is mediated by NADH Oxidases (NOX) located on cell plasma membrane. This process oxidizes intracellular NADH to form NAD⁺ that recycles the NAD+ to maintain the NADH/NAD+ ratio and to support glycolytic. Several across cell plasma membrane electron transfer systems have been identified. These tPMET systems include the NADH:Ferricyanide Oxidoreductase PMET system, the NADH: Ubiquinone: WST-I Reductase system and cell surface NADH-Oxidase activities.

As normal cells consume oxygen at mitochondrial, the tPMET is also unique for cancer cells. In general, the higher degree aggressive tumors are associated with increased glycolysis rate and tPMET activity. Compounds that affect tPMET also affect cancer cell survival. As ATP production contributes substantially to fulfilling the energy requirements of rapidly dividing cells, such as cancer cells, and that tPMET is the major source for cancer cell energy production, targeting tPMET could be a strategy for cancer specific treatment.

This concept was initially proposed by Herst PM, and Berridge MV based on the available data reporting the compounds that affecting tPMET resulted in cancer cell death and the link between the plasma membrane redox mechanism and the activation of acid sphingomyelinase and the formation of ceramide-enriched membrane islands with the resulted apoptosis (Herst PM, Berridge MV, Curr MoI Med 6:895, 2006).

Baesd on this hypothesis, Herst PM, and Berridge MV proposed that to target tPMET it will need to discover or make compounds that specifically locate to the plasma memebrane without entering the cell (Herst PM, Berridge MV, Curr MoI Med 6:895, 2006). However, no further development has been reported.

On the other hand, a novel tumor specific NADH Oxidase (tNOX), an ECTO-NOX, has been identified and has been used as a target for anti-cancer drug development (Davies SL, Bozzo J, Drug News Perspect 2006 19:223). Several products either currently utilized, such as non-steroidal anti-inflammatory drugs, (-)-epigallocatecin gallate, and doxorubicinjhydrdrochloride (Adria-Mycin) or under development, phenoxodiol, for cancer display capability of inhibiting tNOX as one of their underlying mechanisms. These developments are designed to inhibit the NOX enzymes, but not the direct targeting the tPMET.

Phenoxoldiol is a modified isoflevene capable of interact with cell membrane tNOX as well as inhibiting multiple regulatory processes. Phenoxodiol has demonstrated its effect in sensitizing cancer cells to chemotherapy drugs. It is currently under clinical evaluation as adjunct treatment in combination with conventional chemotherapy drugs. Phenoxodiol has also been used as a single treatment for selected tumors.

In addition, cancer cells also exhibit increased hypoxia responses, including increased expression levels and activities of hypoxia inducible factor (HIF) activities, which also contribute to the aerobic glycolysis. Inhibiting HIF has shown promising efficacy effects.

Apigenin is one of the naturally exited flavonoids contained in a wide range of fruits and vegetables such as apple and celery. Apigenin has been identified as multi-function inhibitor. It induced p53 activation, suspends cell cycle progression, maintaining genomic stability, and inhibits CK2, NF-κB HIF, Her2 etc activities and their corresponding pathways. Apigenin also induces reactive oxygen species generation. Over 100 patent applications have been filed regarding apigenin. Among those, several of them claimed apigenin as a drug for treating inflammatory diseases and autoimmune diseases. Apigenin has also been claimed for cancer prevention drug and for combination therapy with chemotherapy drugs for cancer treatment. Other isoforms of apigenin, other flavonoids, isoflavonoids including, naturally existed, modified or synthetic including phenoxodiol a synthetic isoflevene, have also been found with similar function of apigenin. All of those need to be combined with chemotherapy drugs for cancer treatment.

Previously, the inventor described a combination treatment composition for the use as anticancer therapy. This composition comprised of a WST-I reagent, the mixture of a water soluble tetrazolium salt WST-I and an intermediate electron acceptor mPMS, in combination with apigenin, a flavonoid. According to the description, the WST-I reagent is capable of interfere with tPMET and could be substituted by either combination of other tetrazolium salts and intermediate electron acceptors, or individual tetrazolium salts or intermediate electron acceptors, including WST-3 and WST-3 plus mPMS. Also according to the description, apigenin can be substituted by IKK inhibitors, CK2 inhibitors, inhibition of NF- KB, other flavonoids, as well as pUC19 DNA vector and corresponding siRNA sequences and all other means against the listed target genes. The proposed mechanism included inhibition of NF-κB activity, induction of reactive oxygen species, prolonged activation of JNK and inhibition of HIF. However, it did not touch blocking the ATP syntheses, and the combination of inhibition of cell surface respiration and energy production and inhibition of HIF or cell hypoxia response. Therefore, not the corresponding substitutes for the same.

As cancer cells rely on the aerobic glycolysis and the tPMET for their repiration and ATP production, which may represent the features that are unique for cancer cells, therefore, direct targeting the tPMET processes, including the process of coupling of electron transfer and oxidative phosphorylation will be useful for developing cancer specific anti-cancer treatment. However, most of the research efforts on tPMET have been focused on developing inhibitors against tumor specific NOX (tNOX). Little attention has been putting on direct targeting the tPMET process and the process of coupling the electron transfer and the oxidative phosphorylation involved in tPMET.

SUMMERY OF THE INVENTION

The present invention describes compounds that are cell plasma membrane impermeable and containing functional groups capable of blocking and/or inhibiting trans plasma membrane electron transport (tPMET), cell surface respiration and uncoupling cell surface oxidative- phosphorylation on cell surface, compositions of combining blocking cancer cell tPMET and cell surface respiration with inhibiting cancer cell hypoxia responses, and use these compounds and compositions for cancer specific, high efficient, less toxic and broad spectrum anticancer therapy. One embodiment of this invention described cell plasma membrane impermeable compound containing functional groups capable of blocking and/or inhibiting trans plasma membrane electron transport (tPMET), cell surface respiration and uncoupling cell surface oxidative-phosphorylation for treating cancer in patient.

Another embodiment of this invention describes composition comprising at least one cell plasma membrane impermeable compounds capable of blocking and/or inhibiting trans plasma membrane electron transport (tPMET), cell surface respiration and uncoupling cell surface oxidative-phosphorylation in combination with a the second compound of multi function inhibitor capable of inhibiting cancer cell hypoxia responses for anticancer therapy.

Yet another embodiment of this invention described synergistic induction of cancer cell death by combination of WST-3 with apigenin for an effective and cancer specific treatment of cancer.

Yet other embodiments of this invention described the means to design the compounds, means to block tPMET, cell surface respiration and uncoupling the cell surface oxidative- phosphorylation and means to inhibit cancer cell hypoxia responses as well as the use of the said compounds and compositions for synergista induction of cancer cell specific cell death as anticancer therapy.

BRIEF DESCRIPTION OF DRAWINGS

Fig 1. Chemical Structure of DNP and WST-3

Fig 2 Chart of Dose-Response of Apigenin and mPMS Combination Treatment

Fig 3 Chart of Differential cellular responses to mPMS treatment

Fig 4 Chart of Effect of Combination WST-3 with Apigenin On Cell Death

DETAILED DESCRIPTION OF THE INVENTION

I. General Description

Cancer cells can be treated by selectively blocking cancer cell respiration in combination with blocking their hypoxia response. Detailed descriptions for designing the compounds that can specifically block cell surface tPMET, respiration and oxidative-phosphorylation and compositions of such said compound in combination with inhibition of cell responses to hypoxia are provided for the treatment of cancer in the patients and in a mmamal.

II. Definitions

The term "WST" represents the collection of a class of compounds of water soluble tetrazolium salts including, but not limited to WST-3, WST-4, WST-5, WST-8, WST-9, WST-IO, WST-Il, XTT, and MSN. These compounds are also impermeable to cell plasma membrane.

The term "WST-3" 2-(4-Iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H- tetrazolium, sodium salt The term "IEA" is the symbol of "Intermediate Electron Acceptor".

The term "mPMS" (l-methoxy-5-methyl-phenazinium methyl sulfate) is a chemical compound acts as an "electron coupling agent/IEA" when combined with tetrazolium salts.

The term "coenzyme Ql", (2,3-Dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-l,4-benzoquinone) is a chemical compound act as an IEA.

The term "IKK" represents Inhibitory kappaB Kinase, which phosphorylate IKB that leads to NF- KAPPAB activation. Two IKK isoforms have been identified. They are IKKl (IKKoø and IKK2 (IKKβ). The term "NF-kappaB" Nuclear factor kappaB is a family of rel proteins that act as transcription factors regulating gene expression. Normally NF-KAPPAB proteins forms a dimmer which also complex with an inhibitory kappa B (IKB) molecule stay in inactive form in the cytoplasm. Upon signal activation, the IKB is phosphorylated by IKK and dissociate from the NF-kappaB dimmer, which release the NF- KAPPAB to entering the nuclear for activating transcription of a special set of genes that are regulated by NF-KAPPAB. The dissociated IKB will be degraded by protesomes. Activation of NF-kappaB favors cell proliferation and survival. NF-kappaB activity has been found to associate with and contribute to carcinogenesis process, tumor progression and resistance of cancer cells to chemo and radiation therapies.

The term "IKK inhibitor" refers to an agent capable of inhibiting the activity of Inhibitor kappaB kinase (IKK) and thereby inhibiting the kinase activity of IKK and its function of activating NF-kB. Therefore, inhibits NF-KB activity. An IKK inhibitor may be a competitive, noncompetitive, or irreversible IKK inhibitor. "A competitive IKK inhibitor" is a compound or a peptide that reversibly inhibits IKK enzyme activity at the catalytic site; "a noncompetitive IKK Inhibitor" is a compound that reversibly inhibits IKK enzyme activity at a non-catalytic site; and "an irreversible IKK inhibitor" is a compound that irreversibly destroys IKK enzyme activity by forming a covalent bond with the enzyme. The term "IKK inhibitors" include, without limitation, i) compounds previously established to exhibit IKK inhibitory properties including, but not limited to: SPC839 (Signal Pharmaceutical Inc.), Anilino- Pyrimidine Derivative(Signal Pharmaceutical Inc.), PS1145(Millennium Pharmaceutical Inc.), BMS- 34554 l*(Bristol-Myers Squibb Pharmaceutical Research Institute, IKK inhibitor III), SC- 514*(Smithkilne Beecham Corp.), Amino-imidazolecarboxamide derivative(Smithkilne Beecham Corp.), Ureudo-thiophenecarboxamide derivatives(AstraZeneca ), Diarylpybidine derivative(Bayer ), Pyridooxazinone derivative(Bayer ), Indolecarboxamide derivative(Aventis Pharma), Benzoimidazole carboxamide derivative(Aventis Pharma), Pyrazolo[4,3-c]quinoline derivative(Pharmacia Corporation), Imidazolylquinoline-carbxaldehyde semicarbazide derivative(Tulark Inc.), Pyridyl Cyanoguanidine derivate(Leo Pharma), IkB Kinase Inhibitor Peptide(CalBiochem), IKK-2 Inhibitor IV [5-(p- Fluorophenyl)-2-ureido]thiophene-3-carboxamide(CalBiochem), IKK Inhibitor II, Wedelolactone(CalBiochem), IKK Inhibitor VII (CaIB iochem), IKK-2 Inhibitor V N-(3,5-Bis- trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide IMD-0354(CalBiochem), IKK-2 Inhibitor VI (5- Phenyl-2-ureido)thiophene-3-carboxamide(CalBiochem), IKK-2 Inhibitor VIII ACHP 2-Amino-6-(2- (cyclopropylmethoxy)-6-hydroxyphenyl)-4-(4-piperidinyl)-3-pyridinecarbonitrile(CalB iochem). ii) In a certain embodiment, the group of IKK inhibitors may additionally include compounds discovered to have IKK inhibitory activity, in accordance with the present specification, and previously identified to have anti-tumor activity, including, but not limited to PS 1145(Millennium Pharmaceutical Inc.), BMS- 34554 l*(Bristol-Myers Squibb Pharmaceutical Research Institute). The term "CK2 inhibitor" represents all protein kinase casein kinase2 inhibitors. The preferred CK2 inhibitors is, but not limited to Apigenin.

The term "Apigenin" CAS Registry Number: 520-36-5, Chemical Abstracts Service Name: 4H- 1- benzopyran-4-one,5,7-dihydroxy-2-(4-hydroxy-phenyl)- (9CI). It is also named as Apigenine; Chamomile; Apigenol; Spigenin; and Versulin and is a member of Flavones, a subclass of flavonoids. Apigenin is a multi function signal transductor modulator that reduces DNA oxidative damage; inhibit the growth of human leukemia cells and induced these cells to differentiate; inhibit cancer cell signal transduction and induce apoptosis; act as an anti-inflammatory; and as an anti-spasmodic or spasmolytic. Apigenin inhibits activity of NF-KB, IKK-I and IKK-2, protein kinase 2 (CK2), mape kinase (MPK), hypoxia inducing factor 1 (HIF), vescular epithelium growth factor (VEGF) and some other molecules and regulatory pathways such as cell cycle and angiogenesis, induce p53 activity, maintaining genomic stability by holding cell cycle for mismatch repair or arrest cell cycle and induce apoptosis etc. Apigenin is know to have the effects of anti-UV radiation caused oxidation, and chemoprevention for cancer. The apigenin, herein, is also described as a representative of the subclasses of flavonoids, the flavones including, but not limited to: tricin, luteolin, tangeritin, ,6-hydroxyflavone, Baicalein, Scutellarein, Wogonin, Diosmin, Flavoxate, Chrysin, the glycosided forms of these flavones, and other subclasses of the flavonoids with similar biological activities include, but not limited to Isoflavones, Flavonols, Flavanones, 3-Hydroxyflavanones, Flavan-3-ols, Anthocyanidins, 3-deoxyanthocyanidin, Anthocyanins, Acetylated and glycosides, and Tannins, as well as isoflavonoids and neoflavonoids.

The term "Flavonoids" also called bioflavonoids also collectively know as Vitamin P and citrin, are a class of plant secondary metabolites. Herein flavonoids represent all of the three ketone-containing compounds (flavonoid and flavonols) according to IUPAC nomenclature classifications: i) the flavonoids derived from 2-phenylchromen-4-one (2-phenyl-l,4-benzopyrone) structure; ii) isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-l,4-benzopyrone) structure; and iii) neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-l,2-benzopyrone) structure; as well as the non -ketone polyhydroxy polyphenol compounds including: flavanoids, flavan-3-ols and catechins. Sample compounds include, but not limited to Isoflavone:Biochanin A, Daidzein, Daidzin, Formononetin, Genistein, Coumestrol, Puerarin; flavan-3-ols: catechins (catechin, epicatechin (EG), epicatechin, gallate (EGC), and epigallocatechin gallate (EGCG)); flavonol: myricetin, quercetin, and Kaempferol; Isoflavenes: phenoxodiol; Anthocyanins: Antirrhinin, Chrysanthenin, Malvin, Myrtillin, Oenin Primulin, Protocyanin, Tulipanin; 3-deoxyanthocyanidin: Apigeninidin, Columnidin, Diosmetinidin, Luteolinidin, Tricetinidin; Anthocyanidins: Aurantinidin, Cyanidin, Delphinidin, Europinidin, Luteolinidin, Malvidin, Pelargonidin, Peonidin, Petunidin, Rosinidin; 3- Hydroxyflavanones: Dihydrokaempferol, Dihydroquercetin; Flavanones: Eriodictyol, Hesperetin, Homoeriodictyol, Naringenin; Flavonols: Fisetin, Isorhamnetin, Kaempferol, Myricetin, Pachypodol, Quercetin, Rhamnazin, Morin; and their glycoside forms.

The term "HIF" hypoxia inducible factor represents a family of transcription factors that response to decrease of available oxygen or hypoxia in the cellular environment. Three family members have been identified. They are HIF-I (a dimmer composed of HIF- lα and HIF- lβ), HIF-2 (a dimmer composed of HIF-2oc and HIF-2β), HIF-3 (a dimmer composed of HIF-3oc and HIF-3β).

The term "HIF inhibitors" are the biological and non-biological compounds that inhibit HIFs and/or cellular responses to hypoxia, including, but not limited to: 2,2-dimethybenzopyran compounds, chetomin, 2-methoxyestradiol (2ME2), PX-478, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), EZN-2968, camptothecins, NSC 644221 , 3-(5'-hydroxymethyl-2'-furyl)-l-benzylindazole (YC-1 ), rapamycin, and decoy oligonucleotides against HIF-I RX-0047.

The term "tNOX' represents a tumor specific cell surface NADH oxidase. It is also called ECTO2.

The term "tNOX inhibitors" represents the compounds that are capable of inhibiting the tNOX activity. The tNOX inhibits, herein, includes, but not limited, catechins: catechin, (-)- epicatechin (EC), (-)-epicatechin (EG), (-)-epicatechin gallate (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epigallocatechin gallate (EGCG); Capsaicin, Capsicum, Capsicum vanilloids, Capsibiol-T, and a isoflavenes analogue derivative, the phenoxodiol; BTS derivative, the 6-[N-(2- phenylethyl)]benzo[b] thiophenesulphonamide 1,1 -dioxide; Non-steroidal anti-inflammatory drugs (NSAIDS), piroxicam, aspirin, ibuprofen, naproxen and celecoxib; and doxorubicin hydrochloride (Adria- mycin).

The term "Oxidative Phosphorylation" is a process that coupling the oxidation of the protons with the synthesis of ATP, which transfer and store the energy derived from glucose metabolism to the ATP as cellular energy source.

The term "Uncoupler" means to uncouple the cellular oxidative phosphrylation process that blocks the ATP synthesis, the energy metabolism in the cell. The known unucouplers including, but not limited to: dinitrophenol (DNP),

D5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13), sodium 2,3,4,5,6- pentachlorophenolate (PCP), 4,5,6,7-tetrachloro-2-(trifluoromethyl)-lH-benzimidazole (TTFB), Flufenamic acid (2-[3-(trifluoromethyl)anilino]benzoic acid), 3,5-di-tert-butyl-4-hydroxy- benzylidenemalononitrile (SF6847), carbonyl cyanide m-chloro phenyl hydrazone (CCCP), Carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP), and alpha- (phenylhydrazono)phenylacetonitrile derivatives.phenylacetonitrile derivatives; and weak acids comprising: Weakly Acidic Phenols, benzimidazoles, N-phenylanthranilates, salicylanilides, phenylhydrazones, salicylic acids, acyldi-thiocarbazates, cumarines, and aromatic amines.

The term "Cancer Cells" represents the cells in culture that were derived from human cancer or tumors, which have malignant features, such as lost of contact inhibition.

The term "Cancer" describes a diseased state in which a carcinogenic agent or agents causes the transformation of a normal cell into an abnormal cell, the invasion of adjacent tissues by these abnormal cells, and lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites, i.e., metastasis. The term "Effective dose" As used herein, the term "effective dose" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a cell, tissue, system, animal or human that is being sought, for instance, by a researcher or clinician.

The term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.

The term "Treatment of cancer" describes the drug or reagents administrated to the cells or to a mammal, the duration of the treatment, the method used to administrate these drugs, or reagents and the order and intervals of between these treatments.

The term "Synergistic effect/Synergize" refers to a combination of two or more treatments, which is more effective to produce advantageous results than the additive effects of these agents.

The term a "therapeutically effective amount" of a compound or a pharmaceutical composition refers to an amount sufficient to modulate cancer cell proliferation in culture, tumor growth or metastasis in an animal, especially a human, including without limitation decreasing tumor growth or size or preventing formation of tumor growth in an animal. This term may also mean the effective amount(s) needed to cause cancer cell death or selective cancer cell death while not causing side effects in normal cells.

The term "Pharmaceutically acceptable" indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

The term a "carrier" refers to, for example, a diluent, adjuvant, excipient, auxilliary agent or vehicle with which an active agent of the present specification is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. It also include the transfection reagents as used for deliver of DNA and/or RNA into cells either in vitro or in vivo.

The term "Concurrently" means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

The term "Sequentially" refers to the administration of one active agent used in the method followed by administration of another active agent. After administration of one active agent, the next active agent can be administered substantially immediately after the first, or the next active agent can be administered after an effective time period after the first active agent; the effective time period is the amount of time given for realization of maximum benefit from the administration of the first active agent.

III. Cancer Specific Target for Targeting tPMET Processes Normal cells obtain energy from metabolizing glucose mainly through the Tri-Citric- Acid (TCA) cycle located in mitochondria matrix. The energy that released through this process is carried in the form of NADH, which, then, releases to the H⁺ that are further pumped out of the inner mitochondrial along with the electrons translfer by the electron transfer system located in the inner mitochondrial membrane. This electron transfer process build an H⁺ gradient across the inner mitochonfrial memebrane. Driven by the H⁺ concentration gradient, the H⁺ will, then, enter into the mitochondria through a proton selective ional pore, the ATP syntheses, to phosphorylate the ADP to form ATP. At the same time, one oxygen atom will accept two electrons and two H⁺ to form one molecule of water. This is the Oxidative-Phosphorylation coupling process, through which the energy that released from the H+ gradient will be stored into the ATP (Fig IB), the major energy source for the living cells. This process consumes oxygen to form water and is also called cell respiration.

In the case of glycolysis, cells obtain energy through glucose fermentation in the cytoplasm. No oxygen is consumed during this process. Accompanying this process, the NADH that generated through glycolysis will be oxidized to NAD⁺ by reducing pyruvate, the end product of glycolysis, to form lactate in order to recycle the NAD⁺ in cytoplasm to maintain the NADH/ND⁺ ratio.

As mentioned above, cancer cells metabolize glucose by glycolysis even in the presence of sufficient oxygen and that this switch to aerobic glycolysis process is also accompanying with tPMET. The NADH that generated through glycolysis in the cytoplasm is further oxidized to NAD⁺ through tPMET to maintain the NADH/NAD⁺ ratio. This process consumes oxygen on cell surface through cell plasma membrane electron transport (tPMET). Potential oxidative- phosphorylation on the cell plasma membrane has been predicted.

Therefore, one significant difference between normal cells and cancer cells is the different sub cellular location of cell respiration and energy production. Normal cells respiration to consume oxygen and produce ATP in mitochondria, while cancer cells do so at cell surface. As cell respiration and energy metabolism are crucial to cell survival, this differential sub cellular location of cell respiration and energy production makes the cell surface respiration and ATP syntheses a special cancer selective target for anticancer therapy.

One embodiment of this present invention provides a cancer specific drug target on cancer cell surface for anticancer therapy. This target is located on/in cell plasma membrane. Its function is involved in trans-plasma electron transport systems, cell surface respiration and oxidative-phosphorylation coupling processes. They can be selectively targeted by compounds that are impermeable to cell plasma membrane.

A more specific embodiment is that the target molecules include, but not limited to a cell surface proton specific ional pore with ATP syntheses activity located in the cell plasma membrane. The inhibition of this process can be direct inhibition of the function of the molecule and/or indirect interfere the proton gradient across the plasma membrane.

Yet another embodiment is that each of the molecule of the tPMET systemes and their corresponding function, including, but not limited to the molecules that form the NADH:Ferricyanide Oxidoreductase system, the NADH: Ubiquinone: WST-I Reductase system and cell surface NADH-Oxidases are also the target to inhibit and/or block cell surface respiration. These molecules can be targeted directly or indirectly for anticancer therapy. The function of these systems can be targeted directly and indirectly for anticancer therapy.

VI. Compounds targeting Cell Surface Respiration for Anticancer Therapy

One embodiment of the present invention provides a principle for designing and making compounds that can be used for targeting cell surface respiration. The said compounds are composed of two functional chemical groups: 1) at least one of the first functional group that is capable of binding to and/or interfering and/or blocking the electron transport process of the tPMET systems, blocking the coupling of oxidative phosphorylation, and/or inhibiting the tNOX, therefore, to block cell surface respiration and oxygen consumption; and 2) at least one of the second chemical group or a combination of chemical groups that make(s) the entire compound impermeable to cell plasma membrane and capable of blocking the said compound penetrating the cell plasma membrane and entering the cell. By integrating these functional groups into single molecule, the said compound is capable of interfering, inhibiting and/or blocking the tPMET, or the oxidative phosphorylation process or the coupling of the oxidative phosphorylation and cell surface respiration specifically on cell surface, but not affecting the mitochondrial respiration in normal cells.

One embodiment of the present invention provides such a compound, WST-3, Chemical name: 2-(4-Iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, sodium salt (WST-3, Japanese patent JP,2592436,B, 1995), that can be used for targeting cell surface respiration, the oxidating-phosphorylation process.

Fig.l diagrams the chemical structure of WST-3, which is composed of two types of functional chemical groups: a 2,4-Dinitrophenol (DNP), chemical structure linked to the tetrazolium ring as the said functional chemical group for uncoupling the said oxidative- phosphorylation and the [1,3-benzenedilsulfonate] and a [4-lodophenyl] to enhance its hydrophilic feature that makes the compound impermeable to cell plasma membrane.

The 2,4-dinitrophenol (DNP), a cellular metabolic poison, represents a class of six manufactured chemical compounds that can dissolve in the inner mitochondria membrane, acts as a proton specific ionophore, an agent that can shuttle protons (hydrogen ions) across biological membranes driven by the proton concentration gradient built by the electron transport process to enter into the mitochondria that bypass the ATP syntheses, the oxidative- phosphorylation process, where it uncouples oxidative phosphorylation by carrying protons across the mitochondrial membrane, leading to a rapid consumption of energy without generating ATP. DNP defeats the proton gradient across mitochondria and chloroplast membranes, collapsing the proton motive force that the cell uses to produce most of its ATP chemical energy. Instead of producing ATP, the energy of the proton gradient is lost as heat. Cells counteract the lowered yields of ATP by oxidizing more stored reserves such as carbohydrates and fat. For this reason, DNP has been used as weight loss treatment for burning extra fats. However, it is toxic to the cells by exoughsting cell energy sources and leads to cell death.

By integrating the said DNP with the said second group, the cell plasma impermeable group, it keeps the DNP from entering the cell, but can only function on the surface of cell plasma membrane. As cancer cells respiration mainly rely on tPMET and cell surface oxygen consumption, the WST-3 will only blocks the cell surface respiration of cancer cells, but, will not affect the oxidative phosphorylation in mitochondrial of normal cells, hence, the treatment will be cancer specific.

The DNP as the said first functional chemical group represents an uncoupler of oxidative phosphorylatoin and may also implicate a principle of other ways of blocking tPMET and cell surface respiration. Accordingly, the said DNP can be substituted by 1) the compounds of oxidative-phsphorylation decoupling agents comprising: 5-chloro-3-tert-butyl-2'-chloro-4'- nitrosalicylanilide (S-13), sodium 2,3,4,5,6-pentachlorophenolate (PCP), 4,5,6,7-tetrachloro-2- (trifluoromethyl)-lH-benzimidazole (TTFB), Flufenamic acid (2- [3-

(trifluoromethyl)anilino]benzoic acid), 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF6847), carbonyl cyanide m-chloro phenyl hydrazone (CCCP) and Carbonyl cyanide p- [trifluoromethoxy]-phenyl-hydrazone (FCCP), and alpha-(phenylhydrazono)phenylacetonitrile derivatives.phenylacetonitrile derivatives; and T) general structure feature of uncouplers, weak acids, the chemical groups of which comprising: Weakly Acidic Phenols, benzimidazoles, N- phenylanthranilates, salicylanilides, phenylhydrazones, salicylic acids, acyldi-thiocarbazates, cumarines, and aromatic amines.

Additionally, the cell surface respiration can also be inhibited and/or blocked by the compound with the functional groups that can directly and/or indirectly interfere, inhibit or block the function of the tPMET and/or cell surface respiration selected from the groups comprising 1) intermediate electron acceptor that direct interact with tPMET, including with no limitation: mPMS and coenzyme Ql; T) tPMET substrates, such as NADH; 3) the respiration inhibitors such as cyanic group (C≡N), ferricyanide and arsenic and arsenide groups; 4) tNOX inhibitors include, but not limited to catechins: catechin, (-)-epicatechin (EC), (-)-epicatechin (EG), (-)- epicatechin gallate (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epigallocatechin gallate (EGCG); Capsaicin, Capsicum, Capsicum vanilloids, Capsibiol-T, and a isoflavenes analogue derivative, the phenoxodiol; BTS derivative, the 6-[N-(2-phenylethyl)]benzo[b] thiophenesulphonamide 1,1-dioxide; Non-steroidal anti-inflammatory drugs (NSAIDS), piroxicam, aspirin, ibuprofen, naproxen and celecoxib; and doxorubicin hydrochloride (Adria- mycin). All of these functional groups can be used as substitute of DNP to form the cell plasma membrane impermeable compound.

The said second chemical group or combinations of groups are the chemical groups that are capable of keeping the compound impermeable to cell plasma membrane. Their chemical structures can be designed and/or produced by a skilled person in the field. Examples include, but not limited to the chemical groups that were used for modifying the tetrazolium to form the water soluble tetrazoliums (WSTs), such as the chemical structures of the substituted phenol rings in the WST-I, WST-3, WST-4, WST-5, WST-8, WST-9, WST-10, WXST-I l, XTT, MSN that keep the compound impermeable to the cell plasma membrane.

The function of the said WST-3 can be substituted by the compounds of any combination of at least one of the first functional groups that are capable of interfere, inhibit, block tPMET, cell surface respiration or oxidative-phoasphorylation and at least one of the second functional group that can keep the molecule impermeable to cell plasma membrane. In another word, the function of the said WST-3 can be substituted by the compounds of the cell plasma membrane impermeable oxidative-phoasphorylation uncoupler, the tPMET inhibitor, and the cell surface respiration blocker.

V. Combinational Composition For Anticancer Therapy

1. A Cancer Treatment Strategy by Combination of Inhibition of HIF and Cell surface Respiration

A living cell relies on energy. Unlike normal cells that consume oxygen and generate ATP in mitochondrial, cancer cells consume oxygen on cell surface through tPMET. This cellular geographic difference between cancer cells and normal cells makes the PMET a unique site for cancer specific targeting. In addition, cancer cells are resistant to hypoxia due to increased levels and activities of hypoxia inducible factor (HIF). Therefore, blocking the PMET while inhibiting the HIF induces synergistic and cancer specific cell death for treating cancer in a cancer patient.

Accordingly, one embodiment of the present invention provides pharmaceutical compositions comprising (1) at least one of the first compound that is impermeable to cell plasma membrane and is capable of interfering, and/or blocking tMPET and/or cell surface respiration, such as WST-3 or their valid substitute, in combination with (2) at least one of the second compound that is a multifunction inhibitor with the capability of inhibiting HIF, or inhibition of cellular response to hypoxia or their valid substitutes. The at least one of the second compound that is capable of suppressing cellular survival signaling, such as NF- KB activities, and/or cellular responses to hypoxia, such as apigenin or its valid substitute, HIF inhibitors, IKK inhibitors, flavonoids and valid substitutes. Such a pharmaceutical composition may be administered, in a therapeutically effective amount, in optimized concentrations in pharmaceutical acceptable medium, to a patient in need for the treatment of cancer.

The therapeutic effect of the said combination treatment can be further enhanced by administering an additional third compound of and an intermediate electron acceptor (IEA).

The first compound comprises all the compounds as listed above in the "Compounds targeting Cell Surface Respiration for Anticancer Therapy" section comprising the compounds that are impermeable to cell plasma membrane and also contain at least one of the functional chemical groups comprising: A) at least one functional groups that is capable of interfering and/or blocking the trans plasma membrane electron transport process of the tPMET systems, B) at least one functional groups that is capable of blocking the cell surface respiration, and/or C) at least one functional groups that is capable of uncoupling oxidative phosphorylation, and/or D) at least one functional groups that is capable of blocking or inhibiting the tNOX, therefore, to block tPMET, cell surface respiration and oxygen consumption. By integrating these functional groups into a single molecule that is impermeable to cell plasma membrane, these functional groups will can only act on cell surface to block or inhibit cell surface respiration by cancer cells, but shall not affect the mitochondrial respiration from normal cells.

As listed above in the "Compounds targeting Cell Surface Respiration for Anticancer Therapy" section, the first compounds include, but not limited to 1) intermediate electron acceptor that direct interact with tPMET, including with no limitation: mPMS and coenzyme Ql; T) tPMET substrates, such as NADH; 3) the respiration inhibitors such as cyanic group (C≡N), ferricyanide and arsenic and arsenide groups; 4) tNOX inhibitors include, but not limited to catechins: catechin, (-)-epicatechin (EC), (-)-epicatechin (EG), (-)-epicatechin gallate (EGC), (-)- epicatechin-3-gallate (ECG), and (-)-epigallocatechin gallate (EGCG); Capsaicin, Capsicum, Capsicum pepper vanilloids, Capsibiol-T, and a isoflavenes analogue derivative, the phenoxodiol; BTS derivative, the 6-[N-(2-phenylethyl)]benzo[b] thiophenesulphonamide 1,1- dioxide; Non-steroidal anti-inflammatory drugs (NSAIDS), piroxicam, aspirin, ibuprofen, naproxen and celecoxib; and doxorubicin hydrochloride (Adria- mycin); 5) cell plasma membrane impermeable molecules containing oxidative-phosphorylation uncouplers including, but not limited to DNP, 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13), sodium 2,3,4,5,6-pentachlorophenolate (PCP), 4,5,6,7-tetrachloro-2-(trifluoromethyl)- lH-benzimidazole (TTFB), Flufenamic acid (2-[3-(trifluoromethyl)anilino]benzoic acid), 3,5-di-tert-butyl-4- hydroxy-benzylidenemalononitrile (SF6847), carbonyl cyanide m-chloro phenyl hydrazone (CCCP), Carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP), and alpha- (phenylhydrazono)phenylacetonitrile derivatives.phenylacetonitrile derivatives; and weak acids comprising: Weakly Acidic Phenols, benzimidazoles, N-phenylanthranilates, salicylanilides, phenylhydrazones, salicylic acids, acyldi-thiocarbazates, cumarines, and aromatic amines.

The said at least one of the second compound that is a multifunction inhibitor with the capability of inhibiting HIF, or inhibition of cellular response to hypoxia or their valid substitutes are selected from the groups comprising 1) the flavonoids and its subclasses such as flavorones, T) HIF inhibitors, 3) inhibitors that inhibit NF- KB, IKK, CK2 activities, such including, but no limited to IKK inhibitors.

Apigenin is a representative of the second compound. It is a flavone, a subclass of flavonoids, and it is also a multi-function signal transduction modulator and/or inhibitor to cells. Apigenin is known as a HIF inhibitor that inhibits lHIFoc expression levels and activities of hypoxia induced factor- 1. Apigenin can also activate p53, suspend cell cycle progression for maintaining genomic stability), inhibit the activities of casein kinase II, NF-κB, IKK and induction of generation of reactive oxygen species (ROS) and more.

The second compound and the valid substitutes of apigenin are selected from the groups comprising (1) At least one flavones, include, but not limited to nature existed flavones, such as: tricin, Luteolin, Tangeritin, Chrysin, 6-hydroxyflavone, Baicalein, Scutellarein, Wogonin and synthetic flavones, such as: Diosmin, Flavoxate, additional subgroups of flavones, flavonols, flavannones, flacanonols, catechins, isoflavones; or (2) at least one from other subgroups of Flavonoid (Bioflavonoids) and their isoforms, with or without acetylated or glycosided, comprising naturally existed, artificial modified ketone isoforms and synthetic compounds including, but not limited to flavonoids, derived from 2- phenylchromen-4-one (2-phenyl-l,4-benzopyrone) structure (examples: quercetin, rutin); isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-l,4-benzopyrone) structure; neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-l,2-benzopyrone) structure, and flavanoids as a non-ketonepolyhydroxy polyphenol compounds, including: flavanoids, flavan-3-ols and catechins. Sample compounds include, but not limited to Isoflavone:Biochanin A, Daidzein, Daidzin, Formononetin, Genistein, Coumestrol, Puerarin; flavan-3-ols: catechins (catechin, epicatechin (EG), epicatechin, gallate (EGC), and epigallocatechin gallate (EGCG)); flavonol: myricetin, quercetin, and Kaempferol; Isoflavenes: phenoxodiol; Anthocyanins: Antirrhinin, Chrysanthenin, Malvin, Myrtillin, Oenin Primulin, Protocyanin, Tulipanin; 3- deoxyanthocyanidin: Apigeninidin, Columnidin, Diosmetinidin, Luteolinidin, Tricetinidin; Anthocyanidins: Aurantinidin, Cyanidin, Delphinidin, Europinidin, Luteolinidin, Malvidin, Pelargonidin, Peonidin, Petunidin, Rosinidin; 3-Hydroxyflavanones: Dihydrokaempferol, Dihydroquercetin; Flavanones: Eriodictyol, Hesperetin, Homoeriodictyol, Naringenin; Flavonols: Fisetin, Isorhamnetin, Kaempferol, Myricetin, Pachypodol, Quercetin, Rhamnazin, Morin; and their glycoside forms; or

(3) At least one HIF inhibitors that can inhibit cellular responses to hypoxia including, but not limited to: 2,2-dimethybenzopyran compounds, chetomin, 2-methoxyestradiol (2ME2), PX-478, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), EZN-2968, camptothecins, NSC 644221 , 3-(5'- hydroxymethyl-2'-furyl)-l-benzylindazole (YC-1 ), rapamycin, and decoy oligonucleotides against HIF- 1 RX-0047; or

(4) At least one IKK inhibitors as listed above and following embodiments include compounds which exhibits IKK inhibitory activity in pharmaceutically acceptable medium. The at least one IKK inhibitor may be selected from compounds of the group consisting of, without limitation, i) compounds previously established to exhibit IKK inhibitory properties including, but not limited to: SPC839 (Signal Pharmaceutical Inc.), Anilino-Pyrimidine Derivative(Signal Pharmaceutical Inc.), PS1145(Millennium Pharmaceutical Inc.), BMS -345541*( IKK inhibitor III, Bristol-Myers Squibb Pharmaceutical Research Institute), SC-514*(Smithkilne Beecham Corp.), Amino-imidazolecarboxamide derivative(Smithkilne Beecham Corp.), Ureudo-thiophenecarboxamide derivatives(AstraZeneca ), Diarylpybidine derivative(Bayer ), Pyridooxazinone derivative(Bayer ), Indolecarboxamide derivative(Aventis Pharma), Benzoimidazole carboxamide derivative(Aventis Pharma), Pyrazolo[4,3-c]quinoline derivative(Pharmacia Corporation), Imidazolylquinoline-carbxaldehyde semicarbazide derivative(Tulark Inc.), Pyridyl Cyanoguanidine derivate(Leo Pharma), IKB Kinase Inhibitor Peptide(CalBiochem), IKK-2 Inhibitor IV [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide(CalBiochem), IKK Inhibitor II (Wedelolactone(CalBiochem), IKK Inhibitor VII (CalBiochem), IKK-2 Inhibitor V (N-(3,5-Bis- trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide IMD-0354, CalBiochem), IKK-2 Inhibitor VI (5- Phenyl-2-ureido)thiophene-3 -carboxamide, CalBiochem), IKK-2 Inhibitor VIII (ACHP 2-Amino-6-(2- (cyclopropylmethoxy)-6-hydroxyphenyl)-4-(4-piperidinyl)-3-pyridinecarbonitrile, CalBiochem). ii) In a certain embodiment, the group of IKK inhibitors may additionally include compounds discovered to have IKK inhibitory activity, in accordance, and previously identified as anti-tumor agents, including, but not limited to PS1145(Millennium Pharmaceutical Inc.), BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb Pharmaceutical Research Institute).

One embodiment provides a treatment protocol for the use of treating cancer in a patient by inducing cancer cell death and tumor regression. In accordance, by combination of the said the first compound that inhibit cancer cell surface respiration and the said second compound that inhibits cancer cell hypoxia response this treatment uses combination treatment to inhibit cancer cell surface respiration with the compositions as described above and in effective doses, this treatment can be used to reach synergistic induction of cancer cell death for anticancer therapy.

It is yet another embodiment to treat cancer cells with the said first compound and the said the second compound as described above simultaneously and/or sequentially in any order for each of the above and following embodiments, forming a more preferred embodiment.

Each of the treatment agents may be administrated via oral, intra peritonea injection, intra muscular injection, intra venous injection, intra venous infusion, intra artery infusion, intra artery injection, as well as via dermal penetration.

When the third compound is used, it can be administered in any order with the treatment the other two compounds.

The treatment doses and schedule can be varied by skilled person in the field.

This treatment strategy targets the fundamental change of energy metabolism in cancer cells. Therefore, this treatment can be used for a broad spectrum of tumor types. Cancers that may be treated using the combinatorial protocol with WST-3 or its valid substitutes in combination with apigenin or its valid substitutes are: Abdominal Neoplasms, Peritoneal Neoplasms, Retroperitoneal Neoplasms, Anal Gland Neoplasms, Bone Neoplasms, Adamantinoma, Femoral Neoplasms, Skull Neoplasms, Spinal Neoplasms, Breast Neoplasms, MaleBreast Neoplasms, Breast DuctalCarcinoma, Phyllodes Tumor, Digestive System Neoplasms, Biliary Tract Neoplasms, Gastrointestinal Neoplasms, Liver Neoplasms, Pancreatic Neoplasms, Peritoneal Neoplasms, Endocrine Gland Neoplasms, Adrenal Gland Neoplasms, Multiple Endocrine Neoplasia, Ovarian Neoplasms, Pancreatic Neoplasms, Paraneoplastic Endocrine Syndromes, Parathyroid Neoplasms, Pituitary Neoplasms, Testicular Neoplasms, Thyroid Neoplasms, Eye Neoplasms, Conjunctival Neoplasms, Orbital Neoplasms, Retinal Neoplasms, Uveal Neoplasms, Head and Neck Neoplasms, Esophageal Neoplasms, Facial Neoplasms, Mouth Neoplasms, Otorhinolaryngologic Neoplasms, Parathyroid Neoplasms, Thyroid Neoplasms, Tracheal Neoplasms, Hematologic Neoplasms, Bone Marrow Neoplasms, AnimalMammary Neoplasms, ExperimentalMammary Neoplasms, Nervous System Neoplasms, Central Nervous System Neoplasms, Cranial Nerve Neoplasms, Acoustic Neuroma, Nervous System Paraneoplastic Syndromes, Peripheral Nervous System Neoplasms, Pelvic Neoplasms, Skin Neoplasms, Acanthoma, Sebaceous Gland Neoplasms, Sweat Gland Neoplasms, Soft Tissue Neoplasms, Muscle Neoplasms, Vascular Neoplasms, Splenic Neoplasms, Thoracic Neoplasms, Heart Neoplasms, Mediastinal Neoplasms, Respiratory Tract Neoplasms, Thymus Neoplasms, Urogenital Neoplasms, Female Genital Neoplasms, Male Genital Neoplasms, Urologic Neoplasms, Veterinary Venereal Tumors; Malignant including Histiocytic Disorders: Follicular Dendritic Cell Sarcoma, Interdigitating Dendritic Cell Sarcoma, Histiocytic Sarcoma, Langerhans Cell Sarcoma; Leukemia: Enzootic Bovine Leukosis, Feline Leukemia, Hairy Cell Leukemia, Lymphoid Leukemia, Mast-CellLeukemia, Myeloid Leukemia, Plasma CellLeukemia, Radiation- InducedLeukemia; Lymphatic Vessel Tumors: Lymphangioma, Lymphangiomyoma, Lymphangiosarcoma; Lymphoma: Hodgkin Disease, Immunoproliferative Small Intestinal Disease, Non-Hodgkin Lymphoma; Complex and MixedNeoplasms: Adenolymphoma, PleomorphicAdenoma, Adenomyoepithelioma, Adenomyoma, Adenosarcoma, AdenosquamousCarcinoma, Carcinosarcoma, Hepatoblastoma, Mesenchymoma, MalignantMixed Tumor, MesodermalMixed Tumor, MullerianMixed Tumor, Myoepithelioma, MesoblasticNephroma, Pulmonary Blastoma, Rhabdoid Tumor, Endometrial StromalSarcoma, Thymoma, Wilms Tumor; Connective and Soft Tissue Neoplasms: Adipose Tissue Neoplasms, Connective Tissue Neoplasms, Muscle Tissue Neoplasms, Perivascular Epithelioid Cell Neoplasms, Sarcoma; Germ Cell and EmbryonalNeoplasms: EmbryonalCarcinoma, Chordoma, Germinoma, Gonadoblastoma, Mesonephroma, Neuroectodermal Tumors, Teratocarcinoma, Teratoma, Trophoblastic Neoplasms; Glandular and Epithelial Neoplasms: Adenoma, Carcinoma, Adnexal and Skin Appendage Neoplasms, Basal Cell Neoplasms, Mucinous and Serous Cystic Neoplasms, Lobular and Medullary Ductal Neoplasms, Fibroepithelial Neoplasms, Mesothelial Neoplasms, Neuroepithelial Neoplasms, Squamous Cell Neoplasms; Gonadal TissueNeoplasms: Gonadoblastoma, Sex Cord-Gonadal Stromal Tumors; Nerve Tissue Neoplasms: Meningioma, Nerve Sheath Neoplasms, Neuroectodermal Tumors; Plasma Cell Neoplasms: Multiple Myeloma, Plasmacytoma, Waldenstrom Macroglobulinemia; Vascular TissueNeoplasms: Angiofibroma, Angiokeratoma, Glomus Tumor, Hemangioma, Hemangiopericytoma, Hemangiosarcoma, Meningioma, KaposiSarcoma; Nevi and Melanomas: Melanoma, Nevus; Odontogenic Tumors: Ameloblastoma, Cementoma, CalcifyingOdontogenic Cyst, SquamousOdontogenic Tumor, Odontoma.

2. Pharmaceutical Composition of WST-3 And Apigenin And Use of the Combination Treatment For Cancer Therapy

One specific embodiment of the present invention provides pharmaceutical compositions comprising (1) at least one Water-soluble tetrazolium salts 3 (WST-3, 2-(4-Iodophenyl)-3-(2,4- dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, salt, Fig IA) or its valid substitutes in combination with (2) at least one apigenin or its valid substitutes. Such a pharmaceutical composition may be administered, in a therapeutically effective amount, in optimized concentration in pharmaceutical acceptable medium, to a patient in need for the treatment of cancer.

The combination of WST-3 and apigenin resulted in synergistic cancer specific death. This effect can be further enhanced by adding a third compound, l-methoxy-5-methyl-phenazinium methyl sulfate (mPMS), and an intermediate electron acceptor.

As described above, WST-3 is a water soluble tetrazoliums (WST) that were developed by Dojindo Inc., whose WSTs have sulfate groups added directly or indirectly to the phenyl ring to improve water-solubility that also makes the compound impermeable to cell plasma membrane. Different from all other WSTs, WST-3 contains a 2,4-dinitrophenol (DNP) group directly linked to the tetrazolium ring (Fig 1).

The DNP is an oxidative-phosphorylation uncoupler by dissolving in the inner mitochondria membrane and forming proton specific ional pores, which caused the protons enter the mitochondrial driven by the proton concentration gradient that bypass the ATP syntheses, the oxidative-phosphorylation process. This process leads to a rapid consumption of energy without generating ATP. Therefore, DNP is a mitochondria poison. It exhausts energy and ATP in the cell that leads to cell death.

However, as WST-3 is impermeable to the cell plasma membrane, the incorporating DNP into the water soluble tetrozolium salts WST-3 that keeps the DNP impermeable to cell plasma membrane, hence, makes WST3 capable of mimicking the DNP effect to act on the surface of cell plasma membrane for uncoupling oxidative phosphorylation that interrupts tPMET and cell surface respiration, but does not affect mitochondria in normal cells (Fig 1).

Thus, WST-3 represents classes of cell plasma membrane impermeable compounds that are capable of blocking cell surface respiration by uncoupling cell surface oxidative- phosphorylation. In this way such a compound shall be able to specifically block the tPMET electron transport, cell surface respiration and/or oxidative-phosphorylation on cell surface, hence, specifically inhibit cell surface respiration and/or ATP production in cancer cells, while not affect mitochondrial respiration in normal cells.

The valid substitutes of WST-3 include, but not limited to the compounds that are impermeable to cell plasma membrane and also carry at least one of the oxidative phosphorylation uncouplers comprising 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S- 13), sodium 2,3,4,5, 6-pentachlorophenolate (PCP), 4,5,6,7-tetrachloro-2-(trifluoromethyl)-lH- benzimidazole (TTFB), Flufenamic acid (2-[3-(trifluoromethyl)anilino]benzoic acid), 3,5-di-tert- butyl-4-hydroxy-benzylidenemalononitrile (SF6847), carbonyl cyanide m-chloro phenyl hydrazone (CCCP) and Carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP), and alpha-(phenylhydrazono)phenylacetonitrile derivatives as described above in the "Compounds targeting Cell Surface Respiration for Anticancer Therapy" section.

Additionally, the function of WST-3, the uncoupling oxidative-phosphorylation, can also be substituted by compounds that are impermeable to cell plasma membrane and also with means to direct or indirect inhibit and/or block the function of tPMET and cell surface respiration, inhibition of tNOX as described above in the "Compounds targeting Cell Surface Respiration for Anticancer Therapy" section comprising 1) intermediate electron acceptor that direct interact with tPMET, including with no limitation: mPMS and coenzyme Ql; T) tPMET substrates, such as NADH; 3) the respiration inhibitors such as cyanic group (C≡N), ferricyanide and arsenic and arsenide groups; 4) tNOX inhibitors include, but not limited to catechins: catechin, (-)-epicatechin (EC), (-)-epicatechin (EG), (-)-epicatechin gallate (EGC), (-)- epicatechin-3-gallate (ECG), and (-)-epigallocatechin gallate (EGCG); Capsaicin, Capsicum, Capsicum vanilloids, Capsibiol-T, and a isoflavenes analogue derivative, the phenoxodiol; BTS derivative, the 6-[N-(2-phenylethyl)]benzo[b] thiophenesulphonamide 1,1-dioxide; Non-steroidal anti-inflammatory drugs (NSAIDS), piroxicam, aspirin, ibuprofen, naproxen and celecoxib; and doxorubicin hydrochloride (Adria- my tin)..

The said Apigenin is a flavonoid and is a multi-function inhibitor to cells. Its function includes, but not limited to inhibiting expression and /or activities of hypoxia induced factor- 1 (HIF-I), casein kinase II (CK2), NF-κB, induction of p53 activation, suspend cell cycle progression to maintain genomic stability, induction of generation of ROS and more.

The valid substitutes of apigenin are selected from the groups comprising: at least one flavones, include, but not limited to nature existed flavones, such as: Tricin, Luteolin, Tangeritin, Chrysin, 6-hydroxyflavone, Baicalein, Scutellarein, Wogonin and synthetic flavones, such as: Diosmin, Flavoxate.

The function of apigenin can also be substituted by at least one of the flavonoids selected from additional subgroups of flavones: flavonols, flavannones, flacanonols, catechins, isoflavones; at least one from other subgroups of Flavonoid or Bioflavonoids and their isoforms including naturally existed, artificial modified isoforms and synthetic compounds including, but not limited to* flavonoids, derived from 2-phenylchromen-4-one (2-phenyl-l,4-benzopyrone) structure (examples: quercetin, rutin); isoflavonoids, derived from 3-phenylchromen-4-one (3- phenyl-l,4-benzopyrone) structure; neoflavonoids, derived from 4-phenylcoumarine (4-phenyl- 1 ,2-benzopyrone) structure, and flavanoids as a non-ketonepolyhydroxy polyphenol compounds comprising at least one from other subgroups of Flavonoid (Bioflavonoids) and their isoforms including naturally existed, artificial modified ketone isoforms and synthetic compounds, with or without acetylated or glycosides including, but not limited to flavonoids, derived from 2- phenylchromen-4-one (2-phenyl-l,4-benzopyrone) structure (examples: quercetin, rutin); isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-l,4-benzopyrone) structure; neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-l,2-benzopyrone) structure, and flavanoids as a non-ketonepolyhydroxy polyphenol compounds, including: flavanoids, flavan-3-ols and catechins. Sample compounds include, but not limited to Isoflavone:Biochanin A, Daidzein, Daidzin, Formononetin, Genistein, Coumestrol, Puerarin; flavan-3-ols: catechins (catechin, epicatechin (EG), epicatechin, gallate (EGC), and epigallocatechin gallate (EGCG)); flavonol: myricetin, quercetin, and Kaempferol; Isoflavenes: phenoxodiol; Anthocyanins: Antirrhinin, Chrysanthenin, Malvin, Myrtillin, Oenin Primulin, Protocyanin, Tulipanin; 3- deoxyanthocyanidin: Apigeninidin, Columnidin, Diosmetinidin, Luteolinidin, Tricetinidin; Anthocyanidins: Aurantinidin, Cyanidin, Delphinidin, Europinidin, Luteolinidin, Malvidin, Pelargonidin, Peonidin, Petunidin, Rosinidin; 3-Hydroxyflavanones: Dihydrokaempferol, Dihydroquercetin; Flavanones: Eriodictyol, Hesperetin, Homoeriodictyol, Naringenin; Flavonols: Fisetin, Isorhamnetin, Kaempferol, Myricetin, Pachypodol, Quercetin, Rhamnazin, Morin; and their glycoside forms; or

Additionaly, the function of apigenin can also be substituted by at least one of the HIF inhibitors and/or inhibition of cellular responses to hypoxia including, but not limited to: 2,2- dimethybenzopyran compounds, chetomin, 2-methoxyestradiol (2ME2), PX-478, 17-N-allylamino-17- demethoxygeldanamycin (17-AAG), EZN-2968, camptothecins, NSC 644221 , 3-(5'-hydroxymethyl-2'- furyl)-l-benzylindazole (YC-1 ), rapamycin, and decoy oligonucleotides against HIF-1 RX-0047. The function of apigenin can also be substituted by at least one of IKK inhibitors comprising compounds that exhibits IKK inhibitory activity in pharmaceutically acceptable medium. The at least one IKK inhibitor may be selected from the compounds of the group consisting of, without limitation, i) compounds previously established to exhibit IKK inhibitory properties including, but not limited to: SPC839 (Signal Pharmaceutical Inc.), Anilino-Pyrimidine Derivative( Signal Pharmaceutical Inc.), PS 1145(Millennium Pharmaceutical Inc.), BMS -345541*( IKK inhibitor III, Bristol-Myers Squibb Pharmaceutical Research Institute), SC-514*(Smithkilne Beecham Corp.), Amino-imidazolecarboxamide derivative(Smithkilne Beecham Corp.), Ureudo-thiophenecarboxamide derivatives(AstraZeneca ), Diarylpybidine derivative(Bayer ), Pyridooxazinone derivative(Bayer ), Indolecarboxamide derivative(Aventis Pharma), Benzoimidazole carboxamide derivative(Aventis Pharma), Pyrazolo[4,3- c]quinoline derivative(Pharmacia Corporation), Imidazolylquinoline-carbxaldehyde semicarbazide derivative(Tulark Inc.), Pyridyl Cyanoguanidine derivate(Leo Pharma), IKB Kinase Inhibitor Peptide(CalBiochem), IKK-2 Inhibitor IV [5-(p-Fluorophenyl)-2-ureido]thiophene-3- carboxamide(CalBiochem), IKK Inhibitor II (Wedelolactone(CalBiochem), IKK Inhibitor VII (CalBiochem), IKK-2 Inhibitor V (N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide IMD-0354, CalBiochem), IKK-2 Inhibitor VI (5-Phenyl-2-ureido)thiophene-3-carboxamide, CalBiochem), IKK-2 Inhibitor VIII (ACHP 2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxyphenyl)-4-(4- piperidinyl)-3-pyridinecarbonitrile, CalBiochem). ii) In a certain embodiment, the group of IKK inhibitors may additionally include compounds discovered to have IKK inhibitory activity, in accordance, and previously identified as anti-tumor agents, including, but not limited to PS1145(Millennium Pharmaceutical Inc.), BMS -345541*(IKK inhibitor III, Bristol-Myers Squibb Pharmaceutical Research Institute).

The said the third compound, mPMS, as mentioned above is and can be substituted by an intermediate electron acceptor (IEA). The valid substitutes of mPMS include, but not limited to coenzyme Ql.

One embodiment provides use and treatment protocol for inducing cancer cell death and tumor suppression to treat cancer in a patient. In accordance with this protocol, it has been discovered that the combination of WST-3 or its valid substitutes with an apigenin or its valid substitutes for synergistic induction of cancer cell death and suppression of tumor growth.

Accordingly, cancer cells are treated with effective dose(s) of WST-3 or at least one of its valid substitutes in combination with apigenin or at least one of its valid substitutes with effective doses in pharmaceutical acceptable medium for effective time period.

It is yet another embodiment to treat cancer cells with WST-3 with at least one apigenin or any of its valid substitutes for both WST-3 and apigenin simultaneously and/or sequentially in any order for each of the above and following embodiments, forming a more preferred embodiment.

It is yet another embodiment to treat cancer cells with WST-3 with at least one IKK inhibitor or all other valid substitutes for both WST-3and IKK inhibitor simultaneously and sequentially in any order for each of the above and following embodiments, forming a more preferred embodiment.

In the case when the third compound is administered, the treatment can be in any order. That means that the third compound can be treated simultaneously or sequentially in any order. The in vitro effective dose of WST-3 may range between 0.001-500μM or lower, but can be higher as well.

The in vitro effective dose of apigenin may range between 0.001-500μM.

The in vitro effective dose of mPMS, the third compound, may range between 0.001- lOOμM.

The WST-3 or at least one of its valid substitutes and apigenin or at least one of its valid substitutes may be administered to cancer cells or to cancer patients concurrently, separately and/or sequentially in any order.

The treatment time of WST-3 or at least one of its valid substitutes may be between 15 minutes to 8 hours of initial treatment or continuously for a longer time.

The treatment time of apigenin or at least one of its valid substitutes may be last for 15 min to 24 hours consecutively or longer.

In other words, the WST-3 or at least one of its valid substitutes may be treated first with effective dose for 15 minutes to 8 hours in the absence of apigenin, then, administer the apigenin or at least one of its valid substitutes to the cancer cells for another 1 to 24 hours; or

Alternatively, administering the apigenin or at least one of its valid substitutes to the cancer cells for another 1 to 24 hours, and then, administering the WST-3 or its valid substitutes to cancer cells for 15 minutes to 8 hours; or

Alternatively, administering the apigenin or at least one of its valid substitutes and the WST-3 or its valid substitutes to the cancer cells for 15 minutes to 8 hours, then remove the treatments and administering the apigenin or at least one of its valid substitutes for another 1-24 hours, or

Alternatively, administering the apigenin or at least one of its valid substitutes for 24 hours, then, administering the WST-3 or at least one of its valid substitutes to the treatment of cancer cells for 15 minutes to 8 hours,

Alternatively, administering of apigenin or at least one of its valid substitutes and the WST-3 or its valid substitutes can be concurrently to the cancer cells for 15 minutes to 8 hours.

Alternatively, administering of apigenin or at least one of its valid substitutes and the WST-3 or its valid substitutes can be concurrently to the cancer cells continuously.

The actual treatment doses of WST-3 and apigenin and the treatment time of these compounds can be adjusted by a physician or a skilled person.

The preferred embodiment for the treatment is to administer the apigenin or at least one of its valid substitutes and the WST-3 or its valid substitutes to the cancer cells for 4 hours, then remove the treatments and administering the apigenin or at least one of its valid substitutes for another 24 hours. This is because we have the most date for.

As described above, this combination composition of WST-3 and apigenin and treatment protocol targets the fundamental switches of cancer cells energy metabolism that makes this treatment effective over broad spectrum of tumor types. Cancers that may be treated using the combinatorial protocol with WST-3 or its valid substitutes in combination with apigenin or its valid substitutes are carcinomas and sarcomas include, but are not limited to those carcinomas and sarcomas that may be treated using the present protocol include, but are not limited to: Abdominal Neoplasms, Peritoneal Neoplasms, Retroperitoneal Neoplasms, Anal Gland Neoplasms, Bone Neoplasms, Adamantinoma, Femoral Neoplasms, Skull Neoplasms, Spinal Neoplasms, Breast Neoplasms, MaleBreast Neoplasms, Breast DuctalCarcinoma, Phyllodes Tumor, Digestive System Neoplasms, Biliary Tract Neoplasms, Gastrointestinal Neoplasms,

Liver Neoplasms, Pancreatic Neoplasms, Peritoneal Neoplasms, Endocrine Gland Neoplasms, Adrenal Gland Neoplasms, Multiple Endocrine Neoplasia, Ovarian Neoplasms, Pancreatic Neoplasms, Paraneoplastic Endocrine Syndromes, Parathyroid Neoplasms, Pituitary Neoplasms, Testicular Neoplasms, Thyroid Neoplasms, Eye Neoplasms, Conjunctival Neoplasms, Orbital Neoplasms, Retinal Neoplasms, Uveal Neoplasms, Head and Neck Neoplasms, Esophageal Neoplasms, Facial Neoplasms, Mouth Neoplasms, Otorhinolaryngologic Neoplasms, Parathyroid Neoplasms, Thyroid Neoplasms, Tracheal Neoplasms, Hematologic Neoplasms, Bone Marrow Neoplasms, AnimalMammary Neoplasms, ExperimentalMammary Neoplasms, Nervous System Neoplasms, Central Nervous System Neoplasms, Cranial Nerve Neoplasms, Acoustic Neuroma, Nervous System Paraneoplastic Syndromes, Peripheral Nervous System Neoplasms, Pelvic Neoplasms, Skin Neoplasms, Acanthoma, Sebaceous Gland Neoplasms, Sweat Gland Neoplasms, Soft Tissue Neoplasms, Muscle Neoplasms, Vascular Neoplasms, Splenic Neoplasms, Thoracic Neoplasms, Heart Neoplasms, Mediastinal Neoplasms, Respiratory Tract Neoplasms, Thymus Neoplasms, Urogenital Neoplasms, Female Genital Neoplasms, Male Genital Neoplasms, Urologic Neoplasms, Veterinary Venereal Tumors; Malignant including Histiocytic Disorders: Follicular Dendritic Cell Sarcoma, Interdigitating Dendritic Cell Sarcoma, Histiocytic Sarcoma, Langerhans Cell Sarcoma; Leukemia: Enzootic Bovine Leukosis, Feline Leukemia, Hairy Cell Leukemia, Lymphoid Leukemia, Mast-CellLeukemia, Myeloid Leukemia, Plasma CellLeukemia, Radiation-InducedLeukemia; Lymphatic Vessel Tumors: Lymphangioma, Lymphangiomyoma, Lymphangiosarcoma; Lymphoma: Hodgkin Disease, Immunoproliferative Small Intestinal Disease, Non-Hodgkin Lymphoma; Complex and MixedNeoplasms: Adenolymphoma, Pleomorphic Adenoma, Adenomyoepithelioma, Adenomyoma, Adenosarcoma, AdenosquamousCarcinoma, Carcinosarcoma, Hepatoblastoma, Mesenchymoma, MalignantMixed Tumor, MesodermalMixed Tumor, MullerianMixed Tumor, Myoepithelioma, MesoblasticNephroma, Pulmonary Blastoma, Rhabdoid Tumor, Endometrial StromalSarcoma, Thymoma, Wilms Tumor; Connective and Soft Tissue Neoplasms: Adipose Tissue Neoplasms, Connective Tissue Neoplasms, Muscle Tissue Neoplasms, Perivascular Epithelioid Cell Neoplasms, Sarcoma; Germ Cell and EmbryonalNeoplasms: EmbryonalCarcinoma, Chordoma, Germinoma, Gonadoblastoma, Mesonephroma, Neuroectodermal Tumors, Teratocarcinoma, Teratoma, Trophoblastic Neoplasms; Glandular and Epithelial Neoplasms: Adenoma, Carcinoma, Adnexal and Skin Appendage Neoplasms, Basal Cell Neoplasms, Mucinous and Serous Cystic Neoplasms, Lobular and Medullary Ductal Neoplasms, Fibroepithelial Neoplasms, Mesothelial Neoplasms, Neuroepithelial Neoplasms, Squamous Cell Neoplasms; Gonadal TissueNeoplasms: Gonadoblastoma, Sex Cord-Gonadal Stromal Tumors; Nerve Tissue Neoplasms: Meningioma, Nerve Sheath Neoplasms, Neuroectodermal Tumors; Plasma Cell Neoplasms: Multiple Myeloma, Plasmacytoma, Waldenstrom Macroglobulinemia; Vascular TissueNeoplasms: Angiofibroma, Angiokeratoma, Glomus Tumor, Hemangioma, Hemangiopericytoma, Hemangiosarcoma, Meningioma, KaposiSarcoma; Nevi and Melanomas: Melanoma, Nevus; Odontogenic Tumors: Ameloblastoma, Cementoma, CalcifyingOdontogenic Cyst, SquamousOdontogenic Tumor, Odontoma.

Accordingly, one of the embodiments of this invention provides a method for the use of treating cancer in a patient by combination of (1) means of blocking tPMET, cellsurface respiration, and/or uncoupling the oxidative-phosporylation on the cell plasma membrane, and tNOX inhibitors, in combination with (2) means of inhibiting cellular responses to hypoxia, HIF, NF-κB, IKK activities or mimic one or more of predetermined apigenin effects on cancer cells.

The means to block tPMET and/or uncouple oxidative phosporylation on the cell plasma membrane include, but not limited to: the cell plasma membrane impermeable tMPET inhibitor, cell surface oxidative phosphorylation uncoupler or their valid substitutes, and tNOX inhibitors as listed above are the compounds that can inhibit the trans plasma membrane electron transfer process, or the oxidative phosphorylation process or the coupling of electron transport and the oxidative phosphorylation and impermeable to cell plasma membrane as listed above.

The means of inhibiting cellular responses to hypoxia, HIF, NF-κB activity, or mimic one or more of predetermined apigenin effects on cancer cells including, but not limited to treatment with apigenin, or its valid substitutes, Flavonoids, HIF inhibitors, IKK inhibitors as listed above.

The order of the treatment to cancer cells or cancer patients of the means of blocking tPMET and/or oxidative phosporylation on the cell plasma membrane or tNOX inhibitors, in combination with the means of inhibiting cellular responses to hypoxia, HIF, NF-κB activity, or mimic one or more of predetermined apigenin effects on cancer cells can be concurrently or sequentially in any order at effective doses and effective time period for the treatment.

The therapeutic effect of the said combination treatment as described immediate above can be further enhanced by administering a third compound, an IEA. Suitable IEA that can be used for this treatment inculds mPMS and coenzyme Ql in effective dose and in pharmaceutical acceptable medium.

The third compound can be administered in any order in addition to the said treatment.

The present invention also provides additional methods for the use of inducing cancer cell death and suppressing tumor growth in cancer patients. In accordance with the present invention, it has been discovered that the combination of a flavonoid, apigenin, or its valid substitutes, with the WST-3 or the valid substitutes at effective concentration for synergistic induction of cancer cell death. Accordingly, the present invention provides a pharmaceutical composition and protocol for the treatment of cancer in a patient in need with effective dose comprising of at least one flavonoid, specifically, apigenin, or its valid substitutes as described above, with WST-3 or at least one of the valid substitutes of the WST-3 in a pharmaceutical acceptable medium.

Suitable flavonoids include, but not limited to, apigenin and valid substitutes of apigenin as described above in this description in pharmaceutically acceptable medium.

The valid substitutes of apigenin include the compounds that exhibit inhibitory activity as at least one of the effects of that Apigenin does in pharmaceutically acceptable medium.

The suitable at least one of the valid substitutes for the WST-3, as noted herein above in this description, include, but are not limited to the individual components that are comprises the active group as represented by DNP and the valid substitutes for tetrazolium salts that make the compound impermeable to cell plasma membrane at optimized concentrations in pharmaceutically acceptable medium.

The effective concentration of apigenin that were used may vary depending on cell type. The preferred dose is at the range of 0.001-500μM in vitro.

For all the above and following embodiments, the effective concentration of WST-3 and the valid substitutes may vary depending on the individual composition and the effective concentration of each of the composition may or may not be the same concentration as that in the WST-3 and may vary from each of the compositions and their valid substitutes and between in vitro and in vivo usage. The preferred in vitro concentration range for in vitro treatment of WST-3 is 0.001-500μM or lower in a pharmaceutical acceptable medium.

In a specific embodiment of the present invention, the administration of the WST-3 or at least one valid substitutes of WST-3, the apigenin or at least one of the valid substitutes of apigenin can be in any type of order. Specifically, the WST-3 or at least one valid substitutes of WST-3, and the apigenin or at least one of the valid substitutes of apigenin may be administered to the cells or patient concurrently or sequentially. In other words, the apigenin or at least one of the valid substitutes of apigenin or the WST-3 or the at least one substitute of WST-3 may be administered first, or the WST-3 or at least one valid substitutes of WST-3, and the apigenin or at least one of the valid substitutes of apigenin may be administered at the same time. The preferred order of the treatment in this invention is to administer the WST-3 or the valid substitutes of WST-3 and the apigenin or the valid substitutes of apigenin simultaneously and then, after removal of the WST-3, add apigenin again and keep in contact with cells for another period of time.

In a particular embodiment, the treatment of WST-3 is in contact with cells for 15 minutes to 8 hours. The preferred time is between 30 min to 4 hours. The more preferred time is between 2-4 hours. A removal of the WST-3 or its valid substitute's from treatment is required for all the above and following embodiments to induce cancer cell death of the treated cells by this method thereof. Moreover, the present invention provides a method for the treatment of cancer by administering to a patient, in need thereof, a therapeutically effective dose of at least one of the WST-3 or its valid substitutes and apigenin or at least one of its valid substitutes mentioned above in pharmaceutical acceptable medium.

A broad spectrum of cancers may be treated using this combinatorial protocol. Cancers that may be treated using the present protocol are as listed previously in this description.

ADVANTAGES

According to the above description, this treatment has shown the advantages in treating cancer with high efficiency, less cytotoxic and broad spectrum of cancer types.

2. The chemical structure of WST-3 represents a model of a class of cell plasma membrane impermeable compounds that is capable of interfere the tPMET and cell surface respiration that restricts its activity on cell surface without affecting the mitochondrial respiration in the normal cells. Cell respiration is crucial to cell survival, and as cancer cells rely on cell surface oxygen consumption, this model provides a mean of cancer specific targeting.

3. This combination treatment targets the tPMET, cell surface respiration to block cell energy metabolism, and at the same time inhibit the HIF or cell responses to hypoxia. This combination strategy further enhances cancer cell sensitivity to hypoxia that lead to synergistic induction of cancer cell specific death. It is a more effective anticancer treatment.

4. As the aerobic glyclysis and tPMET are cancer specific changes and are common in almost all types of cancer cells, this treatment strategy targets the fundamental changes of cancer cells in common. This treatment can be applied to a broad spectrum of tumor type for therapy.

5. This combination treatment utilizes two non cytotoxic compounds to reach a synergistic and cancer specific induction of cancer cell death. This combination treatment is safe or at least less toxic comparing to the conventional chemotherapy.

In summary this present invention provides a new concept of combinational treatment strategy for anticancer drug development. This combination treatment will selectively block the cell surface respiration of cancer cell while inhibiting their capability to response to hypoxia, therefore, to inhibit cancer cell respiration and hence the energy metabolism from two direction to obtain synergistic inducible cancer cell death. In addition, these treatments utilize non cytotoxic compounds result in synergistic cancer specific cell death, which provides a new strategy for anti-cancer drug development to reach a highly efficient, safe and broad spectrum and cancer specific anticancer therapy.

Although the description above contains much specificity, these should not be construed as limiting the scope of the embodiments but as merely providing illustrations of some of the presently preferred embodiments. For example, the WST-3 and the apigenin each represents classes of chemical compounds with similar function. Also the combination of WST-3 and apigenin represents a new strategy and a new avenue of cancer drug development by targeting tPMET in combination with inhibition of cellular responses to hypoxia and some other related process.

Thus the scope of the embodiments should be determined by the appended claims and their legal equivalents, rather than by the examples given.

V. ADMINISTRATION OF PHARMACEUTICAL COMPOSITIONS AND COMPOUNDS

The pharmaceutical compositions can be administered by any suitable route, for example, by injection, by intra vaneus infusion, by intra artery infusion, by oral, pulmonary, nasal, transdermal or other methods of administration. In general, pharmaceutical compositions of the present specification comprise, among other things, pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions can include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). The compositions can be incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of pharmaceutical compositions. Please see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference. The pharmaceutical compositions can be prepared, for example, in liquid form, or can be in dried powder form (e.g., lyophilized). Particular methods of administering pharmaceutical compositions are described hereinabove.

In yet another embodiment, the pharmaceutical compositions can be delivered in a controlled release system, such as using an intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In a particular embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. (1987) 14:201; Buchwald et al., Surgery (1980) 88:507; Saudek et al., N. Engl. J. Med. (1989) 321:574). In another embodiment, polymeric materials may be employed (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sd. Rev. Macromol. Chem. (1983) 23:61; see also Levy et al., Science (1985) 228: 190; During et al., Ann. Neurol. (1989) 25:351; Howard et al., J. Neurosurg. (1989) 71: 105). In yet another embodiment, a controlled release system can be placed in proximity of the target tissues of the animal, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, (1984) vol. 2, pp. 115-138). In particular, a controlled release device can be introduced into an animal in proximity of the site of inappropriate immune activation or a tumor. Other controlled release systems are discussed in the review by Langer (Science (1990) 249: 1527-1533).

The conclusion that this is cancer specific programmed cell death is formed by the observation that normal cells had no cytotoxic reaction and further, that a over 90% kill rate is more than substantial evidence of a significant finding. Therein, because this specification touches a programmed cancer cell death pathway that was prior untouched, or in the alternative, that this invention may activates a known pathway or a novel unknown pathway in a manner not able to be duplicated by other inventions, the very sequence of events defined in this specification activates programmed cell death in the cancer cells and as such, presents a valid model for further study. In other words, through processes known to those of skill, the very core molecular event leading to the over 90% kill rate, can be explored because we have the working model to induce such events. Therein, the invention is also claimed as an important model for further research, study and pathway illumination/elucidation.

Although this description above and below have shown specific experimentation and data, One of skill in the art of cancer preclinical and clinical protocol structure, execution and analysis will recognize upon reading this document, through variation of the dosages of the named components, the order in which they are applied and the time frames between applications, valid substitutions of the named components there are a myriad of variable applications which may result in the same or similar outcome. To the extent that these variables can be applied to any cancer in any mammal, the inventor notes than nothing contained within this document or any subsequent documentation provided by the inventor is intended to be limiting. The inventor also notes that this specification is intended to work alone, and reduce cytotoxic effects of traditional cancer therapy, such as chemotherapy and radiation, however, nothing herein is intended to limit the use of this specification to the extent that chemotherapeutic and radiation combination therapies can be utilized in combination with this specification. Further, that the use of chemotherapy and radiation therapy combinations, in conjunction with this specification, may reduce the cytotoxicity of the chemotherapy or radiation therapy because the dosages of the chemotherapy and radiation therapy can be reduced when used in combination with this specification. And finally, that the named sprcification may further sensitize cancer cells selectively over normal cells such that subsequent application of chemotherapy and radiation, as well as combination chemo/radiation therapies, will work more efficiently again, allowing for the reduction of chemotherapy and radiation and combination chemo/radiation dosages.

The foregoing description of the present specification provides illustration and description, but is not intended to be exhaustive or to limit the specifications to the precise one disclosed. Modifications and variations consistent with the above teachings may be acquired from practice of the specification. Thus, it is noted that the scope of the invention is defined by the claims and their equivalents.

The present specification will now be illustrated in more detail in the following examples. It is to be understood that these examples serve only to describe the specific embodiments of the present specification, but do not in any way intend to limit the scope of the claims. It is of further note to one of skill that unique sequence data has been provided in this application. To the extent that each of these new sequence data represents novel targets for the development of cancer therapeutics, nothing contained herein is intended to be limiting. Said targets are noted as potential targets for further development under this application using the above methods and other methods known to those of skilled person. Although not mentioned in this specification elsewhere, use of radiation as a distinct step, or other small molecule drugs, DNA, RNA, siRNA and all other methods for cancer therapy known to those of skill are noted as possible adjuvant to these protocols. Examples

Example 1 mPMS Dose-Response

Method: A & B: HT1080 (Fig 2A) and UM-SCC6 (Fig2B) cells were treated with variable concentrations of mPMS as indicated in combination with ImM WST-Ic and 10, 30 or 100 μM apigenin for 4 hours and, then, changed to normal growth medium for another 24 hours. Cell viabilities were measured by CCK8 Kit. 1 mM WST-I only, 0.12mM mPMS only and 10% WST-Ir were used as parallel control. AP 0: Untreated Control, AP 10: lOμM Apigenin, AP 30: 30μM Apigenin, AP 100: lOOμM Apigenin.

Result: Data showed mPMS and apigenin dose dependent cell death of both HT 1080 and UM- scc6 cells (Fig 2A &B). mPMS IC50 of combination treatment of apigenin 100 μM and mPMS+WST-1 from HT1080 cells was 5μM verses 60μM from untreated control cells. mPMS IC50 of combination treatment of apigenin 100 μM and mPMS+WST-1 from UM-SCC6 cells was 30μM verses 80μM from untreated control cells.

Example 2

Differential Cellular Responses to mPNS Treatment

Non cancer human keratinocyte (HEKa), SK-Mel-5 human malonoma cell line (SK5), human head and neck cancer cell Cal27 line (Cal27) and UM-SCC6 line (SCC6) cells and human soft tissue sarcoma cell line HT1080 (HT1080) were treated with 30, 40 or 50μM mPMS for 4 hours and then cultured in normal growth medium for another 24 hours. Cell viabilities were measured with CCK8 kit. Data showed mPMS dose dependent cell death and differential sensitivities to mPMS treatment from each of the cell lines (Fig3). Among those, the non cancer primary cultured HEKa cells showed the least sensitivity to mPMS treatment with IC50 50μM, while the IC50 from Cal27, UM-SCC6 and HT1080 cells were 20, and 30μM respectively.

Example 3

Effect of combination treatment of WST- 3 with apigenin on induction of cancer cell death

Method: UM-SCC6, HT1080, Cal27, SK-Mel-5, and HEKa cells were treated with 50 or lOOμM WST-3 or 10 or 30μM apigenin alone, or combination of WST-3 and apigenin at different concentrations for 4 hours with untreated cells as control, then, changed to normal growth medium and remained culture in this medium for another 24 hours. After the 24 hours culture, cell viabilities were measured with CCK8 Kit. Data were normalized to % of untreated control cells.

Result: A: Summary of differential cell responses to WST-3, apigenin and combination treatments. Comparing to untreated cells (Ctrl) treatment of 50μM WST-3 (WST-3) or 30μM apigenin (Apigenin) alone showed no or limited effect of cell death to all tested cell line. Combination of WST-3 and apigenin (Apigenin+ WST-3) resulted in synergistic cell death of SK-Mel-5, Cal27, UM-SCC6 and HT 1080 all four tested human cancer cell lines, but limited cell death from non cancer human keratnocytes (Fig4A). B & C: Comparison of Dose-Response of WST-3 and apigenin between non cancer HEKa and human melanoma cell line SK-Mel-5 cells. Data showed both WST-3 and apigenin induced and dose dependent but limited cell death from both HEKa cells (Fig4B) and SK-Mel-5 (Fig5C) cells. HEKa cells showed limited cell death in response to apigenin or WST-3 alone treatment. WST-3 IC50 of combination of 30μM apigenin and WST-3 was 40μM. Further increase WST-3 concentration showed no more cell death from HEKa cells (Fig4B). However, the SK-Mel-5 cells showed synergistic cell death response to combination treatment of 50μM WST-3 and 30μM apigenin. The WST-3 IC50 from this combination treatment of the SK-Mel-5 cells was 20μM, one fold less than that from HEKa cells (Fig4C). The HEKa cells were much more resistant to this combination treatment. Similar results were also observed from other cancer cells.

Claims

WHAT IS CLAIMED

1. A compound comprising

(a) at least one functional group capable of inhibiting cell trans-plasma membrane electron transport (tPMET) or cell surface respiration or uncoupling cell surface oxidative phosphorylation, or inhibiting tNOX activity and

(b) at least one functional group that keeps the compound impermeable to cell plasma membrane,

Therein the compound are a cell plasma membrane impermeable inhibitors that inhibits tPMET, cell surface respiration or/and cell surface oxidatiove-phosphorylation.

2. The compound of claim 1, wherein said the at least one functional group of uncoupling oxidative-phosphorylation is selected from the group of oxidative uncouplers consisting: DNP, CCCP, FCCP, SF6847, S-13, PCP, TTFB, and alpha-(phenylhydrazono) phenylacetonitrile derivatives.

3. The compound of claim 1, wherein said the at least one functional group of tPMET inhibitor or cell surface respiration inhibitor are selected from the group of IEA, consisting mPMS and Coemzyme Ql; tNOX inhibitors consisting capsaicin, green tea catechin, epigallocatechin-3- gallate (EGCG), dicoumarol and phenoxodiol;

4 A composition comprising:

(a) a first agent that blocks tPMET, or/and cell plasma membrane respiration, and

(b) a second agent that blocks at least one of HIF, cell hypoxia responses, NF- B, CK2, IKK activities and, and, optionally,

(c) an optional third agent comprising an IEA,

(d) a pharmaceutically acceptable carrier.

5. The composition of claim 4, wherein the first agent comprises the compound of claim 1.

6. The compound of claim 1 and the first agent of the composition of claim 4, is WST-3.

7. The composition of claim 4, wherein the second agent comprise at least one flavonoid. selected from the groups consisting of at least one flavones of apigenin, tricin, Luteolin, Tangeritin, Chrysin, 6-hydroxyflavone, Baicalein, Scutellarein, Wogonin, Diosmin, and Flavoxate; at least one IKK inhibitors selected from the group consisting of BMS-345541, SC-514. IKK-2 Inhibitor IV [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide, IKK Inhibitor VII, IKK Inhibitor II, Wedelolactone, IKK-2 Inhibitor V N-(3,5-Bis- trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide IMD-0354, and IKK-2 Inhibitor VI (5-Phenyl-2-ureido)thiophene-3-carboxamide, and at least one of the HIF inhibitor selected from the group consisting of 2,2-dimethybenzopyran compounds, chetomin, 2- methoxyestradiol (2ME2), PX-478, 17-N-allylamino-17-demethoxygeldanamycin (17- AAG), EZN-2968, camptothecins, NSC 644221, 3-(5'-hydroxymethyl-2'-furyl)-l- benzylindazole (YC-I), rapamycin, and decoy oligonucleotides against HIF-I RX-0047.

8. The composition of claim 4, wherein the second agent comprises apigenin.

9. The composition of claim 4, wherein the third agent comprises mPMS and coenzyme Ql.

10. A composition comprising WST-3 and apigenin or at least one IKK inhibitors.

11. A composition comprising at least one of WST-3, mPMS or an IEA, and apigenin or at least one IKK inhibitors.

12. The use for selectively killing cancer cells comprising contacting a population of cells with a composition of claim 4 in an amount effective to block the tPMET and/or cell surface respiration and/or uncoupling cell surface oxidative-phosphorylation process in combination with inhibiting cancer cell hypoxia responses.