EP2403497A1 - Compounds, compositions and use for anticancer therapy - Google Patents
Compounds, compositions and use for anticancer therapyInfo
- Publication number
- EP2403497A1 EP2403497A1 EP10748321A EP10748321A EP2403497A1 EP 2403497 A1 EP2403497 A1 EP 2403497A1 EP 10748321 A EP10748321 A EP 10748321A EP 10748321 A EP10748321 A EP 10748321A EP 2403497 A1 EP2403497 A1 EP 2403497A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cell
- neoplasms
- wst
- apigenin
- ikk
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
- C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D257/04—Five-membered rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
- A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- 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.
- 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.
- 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.
- the success rate of such new generation of cancer treatment is limited. Majority of such developments are used as adjunct therapy to conventional chemotherapies.
- cancer cells 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).
- tPMET trans-plasma membrane electron transport
- 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.
- NOX NADH Oxidases
- 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.
- tPMET 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.
- 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.
- tNOX tumor specific NADH Oxidase
- ECTO-NOX a tumor specific NADH Oxidase
- doxorubicinjhydrdrochloride Adria-Mycin
- phenoxodiol for cancer display capability of inhibiting tNOX as one of their underlying mechanisms.
- 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.
- 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.
- HIF hypoxia inducible factor
- 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- ⁇ B 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.
- 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.
- 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.
- 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- ⁇ B 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.
- 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.
- 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.
- tPMET trans plasma membrane electron transport
- 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.
- 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.
- 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.
- WST-3 2-(4-Iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H- tetrazolium, sodium salt
- IVA is the symbol of "Intermediate Electron Acceptor”.
- mPMS l-methoxy-5-methyl-phenazinium methyl sulfate
- coenzyme Ql (2,3-Dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-l,4-benzoquinone) is a chemical compound act as an IEA.
- IKK represents Inhibitory kappaB Kinase, which phosphorylate IKB that leads to NF- KAPPAB activation.
- IKK Inhibitory kappaB Kinase
- Two IKK isoforms have been identified. They are IKKl (IKKo ⁇ and IKK2 (IKK ⁇ ).
- 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.
- the IKB 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.
- 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.
- 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.
- 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-
- 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.
- 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.
- flavonoids also called bioflavonoids also collectively know as Vitamin P and citrin, are a class of plant secondary metabolites.
- 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,
- 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- l ⁇ and HIF- l ⁇ ), HIF-2 (a dimmer composed of HIF-2oc and HIF-2 ⁇ ), HIF-3 (a dimmer composed of HIF-3oc and HIF-3 ⁇ ).
- 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.
- tNOX' represents a tumor specific cell surface NADH oxidase. It is also called ECTO2.
- tNOX inhibitors represents the compounds that are capable of inhibiting the tNOX activity.
- the tNOX inhibits 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
- 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.
- 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
- TTFB 4,5,6,7-tetrachloro-2-(trifluoromethyl)-lH-benzimidazole
- 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, benzimidazo
- 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.
- 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.
- 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.
- terapéuticaally 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.
- 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.
- 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.
- 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.
- 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.
- Concurrently means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.
- 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.
- one oxygen atom will accept two electrons and two H + to form one molecule of water.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- DNP 2,4-Dinitrophenol
- 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.
- the cell plasma impermeable group 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.
- 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-
- 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
- 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.
- WSTs water soluble tetrazoliums
- 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.
- 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.
- 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.
- hypoxia inducible factor HIF
- 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.
- 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).
- IAA intermediate electron acceptor
- 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.
- 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.
- 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 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- ⁇ B, IKK and induction of generation of reactive oxygen species (ROS) and more.
- ROS reactive oxygen species
- 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, rut
- 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,
- 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
- 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
- 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.
- 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.
- 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.
- the third compound 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.
- 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, Para
- 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.
- WST-3 Water-soluble tetrazolium salts 3
- Fig IA 2-(4-Iodophenyl)-3-(2,4- dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, salt, Fig IA
- 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.
- 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.
- mPMS l-methoxy-5-methyl-phenazinium methyl sulfate
- 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.
- WST-3 contains a 2,4-dinitrophenol (DNP) group directly linked to the tetrazolium ring (Fig 1).
- DNP 2,4-dinitrophenol
- 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.
- 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).
- WST-3 represents classes of cell plasma membrane impermeable compounds that are capable of blocking cell surface respiration by uncoupling cell surface oxidative- phosphorylation.
- 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-(phenylhydra
- WST-3 the uncoupling oxidative-phosphorylation
- WST-3 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 (-)-epigallocate
- 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- ⁇ B, induction of p53 activation, suspend cell cycle progression to maintain genomic stability, induction of generation of ROS and more.
- HIF-I hypoxia induced factor- 1
- CK2 casein kinase II
- NF- ⁇ B 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.
- 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 Flavonoids
- 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,
- 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 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
- 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),
- 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).
- IAA intermediate electron acceptor
- 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.
- 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.
- 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.
- 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 0.001-500 ⁇ M or lower, but can be higher as well.
- the in vitro effective dose of apigenin may range between 0.001-500 ⁇ M.
- the in vitro effective dose of mPMS, the third compound may range between 0.001- lOO ⁇ M.
- 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.
- 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.
- 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.
- 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
- 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,
- 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.
- 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 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.
- 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,
- 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- ⁇ B, 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- ⁇ B 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- ⁇ B 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.
- 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.
- 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 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 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 0.001-500 ⁇ M in vitro.
- 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 0.001-500 ⁇ M or lower in a pharmaceutical acceptable medium.
- 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.
- 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.
- 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.
- 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.
- 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.
- this treatment has shown the advantages in treating cancer with high efficiency, less cytotoxic and broad spectrum of cancer types.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the WST-3 and the apigenin each represents classes of chemical compounds with similar function.
- 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.
- 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.
- pharmaceutical compositions of the present specification comprise, among other things, pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
- 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.
- 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.
- 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.
- 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).
- polymeric materials may be employed (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, FIa.
- 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).
- 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).
- 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 ⁇ M 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 lO ⁇ M Apigenin
- AP 30 30 ⁇ M Apigenin
- AP 100 lOO ⁇ M Apigenin.
- 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 ⁇ M and mPMS+WST-1 from HT1080 cells was 5 ⁇ M verses 60 ⁇ M from untreated control cells.
- mPMS IC50 of combination treatment of apigenin 100 ⁇ M and mPMS+WST-1 from UM-SCC6 cells was 30 ⁇ M verses 80 ⁇ M from untreated control cells.
- 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 50 ⁇ M 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 50 ⁇ M, while the IC50 from Cal27, UM-SCC6 and HT1080 cells were 20, and 30 ⁇ M respectively.
- UM-SCC6, HT1080, Cal27, SK-Mel-5, and HEKa cells were treated with 50 or lOO ⁇ M WST-3 or 10 or 30 ⁇ M 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 50 ⁇ M WST-3 (WST-3) or 30 ⁇ M 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).
- the SK-Mel-5 cells showed synergistic cell death response to combination treatment of 50 ⁇ M WST-3 and 30 ⁇ M apigenin.
- the WST-3 IC50 from this combination treatment of the SK-Mel-5 cells was 20 ⁇ M, 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.
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