Abstract:
Tumor cells exhibit consistent abnormalities in calcium regulation. The present disclosure teaches methods by which such differences are exploited to induce Apoptosis selectively in tumor/cancer cells while sparing normal cells. These methods are based upon employing drugs that, acting in synergistic combinations, trigger selective killing of malignant cells. Since the invention is based upon fundamental cell cycle requirements, to the extent that calcium handling abnormalities are a general characteristic of the malignant state, the methods presented here are widely applicable regardless of tissue of origin and degree of cellular de-differentiation.

Description:
RELATED APPLICATIONS 
       [0001]    This application is a continuation in part of U.S. patent application Ser. No. 12/911,723, filed Oct. 25, 2010, which is a continuation in part of U.S. patent application Ser. No. 10/588,079, filed Nov. 22, 2005, entitled “Methods For the Selective Treatment of Tumors by Calcium-Mediated Induction of Apoptosis,” which claims priority to U.S. provisional application Ser. No. 60/475,063 entitled “Methods For the Selective Treatment of Tumors by Calcium-Mediated Induction of Apoptosis,” filed May 30, 2003; the entire disclosures of which are hereby incorporated by reference. Any disclaimers that may have occurred during the prosecution of the above-referenced applications are hereby expressly rescinded, and reconsideration of all relevant art is respectfully requested. 
     
    
     TECHNICAL FIELD 
       [0002]    This present disclosure is in the field of medical therapeutics, more particularly in the field of clinical treatment of malignancy and cancer therapy. The methods allow a broad range of human tumors or cancer types to be treated by selectively inducing apoptosis. Apoptosis is induced in tumors by disrupting intracellular calcium distribution in a manner that leaves normal growing or non-growing cells unharmed. 
       BACKGROUND 
       [0003]    Warburg described a metabolic “defect” in energy utilization exhibited by most cancer cells. This “defect” is now known to result from a change in mitochondrial function. Many different mutations in initial growth factor dependent pathways function to produce a state in which cells are made capable of continuously passing the Pardee Restriction Point (RP) or point of no return towards the end of the G1 phase of the cell cycle. It is demonstrated that traverse through G1 prior to this point is dependent on the continuous availability of EC (extra celluler) Ca 2+ . Any growth factor requirement for passing the RP is bypassed completely by Ca++-specific ionophores as long as there is a ready supply of EC Ca 2+  Carcinogenic Phorbol analogs, which act to stimulate certain forms of Ca 2+  dependent PKC, can replace the growth factor requirement for crossing the RP, as long as there is sufficient EC Ca 2+  present in the growth medium. The present disclosure teaches these steps can be short-circuited and effectively bypassed by providing a ready supply of EC Ca 2+  consistent with the known requirement for IC (intra cellular) but not EC Ca 2+  upon passing the RP. Effectively, malignant transformation mimics the effect of Ca 2+  ionophores and Phorbol compounds and the initiating event in cancer is any mutation which produces an increased new steady state of continuous Ca 2+  influx. In order for such cells to escape Ca 2+ -induced apoptosis, several adaptations in IC Ca 2+ -handling must occur if such a potentially cancerous cell is to survive to a detectable disease state. This does not exclude the influence of known mutations in tumor suppressor or tumor promoter genes either prior to or selected for once the initiating stimulus for malignancy occurs in exacerbating the malignant state, but these mutations must be secondary to satisfying the Ca 2+  requirement for passing the RP. 
         [0004]    The present disclosure teaches the use of calcium manipulation for the treatment of cancer. 
       SUMMARY OF THE EMBODIMENTS 
       [0005]    The disclosure teaches a method for treating a cancer in a patient comprising administering to said patient effective amounts of two or more drugs at concentrations which interact synergistically, that stimulate an increase in the Ca 2+  burden of smooth endoplasmic reticulum and mitochondria. The term cancer can mean a tumor in a patient. In one embodiment, the drug concentrations are submaximal. In one embodiment, at least one of said drugs stimulates Smooth-Endoplasmic-Reticulum Ca 2+ -ATPase (SERCA) and wherein at least one of said drugs is an antagonist of SER Ca 2+  gates. 
         [0006]    The disclosure teaches a method for treating a tumor in a patient comprising administering to said patient effective amounts of two or more drugs at concentrations which interact synergistically, that stimulate an increase in the Ca 2+  burden of smooth endoplasmic reticulum and mitochondria. 
         [0007]    In one embodiment at least one of said drugs stimulates Smooth-Endoplasmic-Reticulum Ca-ATPase (SERCA) and wherein at least one of said drugs is an antagonist of SER Ca 2+  gates. 
         [0008]    In one embodiment at least one of said drugs is selected from the group consisting, inhibitors of SER IP3-sensitive Ca 2+  gates and SERCA agonists, and one of said drugs are selected from the group of drugs which are stimulators of particulate guanylate cyclase. In one embodiment at least one of said drugs is selected from the group consisting of inhibitors of SER IP3-sensitive Ca 2+  gates and agonists of SERCA and wherein at least one of said drugs is an effective elevator of cGMP levels including activators of particulate guanylate cyclases and inhibitors of cGMP phosphodiesterases. 
         [0009]    In one embodiment at least one of said drugs is a calmodulin (CAM) antagonist, including antagonists of the CAM targets calcineurin/protein phosphatase 2B (e.g. members of the class but not limited to cyclosporine A or the cell permeable calcineurin autoinhibitory domain poly-arginine-based polypeptide) and CAM-dependent protein kinase II (for example, members of the class but not limited to KN-62) and wherein at least one of said drugs is a Protein Kinase C (PKC) agonist (e.g. members of the class but not limited to ceramide C6). 
         [0010]    In one embodiment at least one of said drugs is a protein kinase C agonist and wherein at least one of said drugs is an inhibitor of cGMP phosphodiesterases. 
         [0011]    In one embodiment, at least one of said drugs is a protein kinase C agonist and wherein two additional drugs of the classes CAM-dependent protein kinase II antagonists and calcineurin/protein phosphatase 2B antagonists are combined, each at submaximal effective drug concentrations. 
         [0012]    In one embodiment at least one of said drugs is a CAM-dependent protein kinase II antagonist and wherein at least one of said drugs is a calcineurin/protein phosphatase 2B antagonist. In one embodiment at least one of the drugs is a submaximal concentration. In one embodiment, all of the drugs are at submaximal concentration. 
         [0013]    In one embodiment at least one of said drugs is a DNA damaging agent. In one embodiment at least one of said drugs is an anti-mitotic drug. 
         [0014]    The disclosure teaches a method of treating a tumor in a patient comprising administering to said patient effective amounts of two or more drugs that stimulate mitochondrial Ca 2+  loading. In one embodiment further comprising administering to said patient an effective amount of a DNA damaging agent. In one embodiment further comprising administering to said patient an effective amount of an anti-mitotic drug. 
         [0015]    The disclosure teaches a method for treating a cancer in a patient comprising administering to said patient effective amounts of two or more drugs at concentrations which interact synergistically, that stimulate an increase in the Ca 2+  burden of smooth endoplasmic reticulum and mitochondria, wherein the drugs comprise W-7 and C 6 C. In one embodiment wherein the drugs comprise PMA and W-7. In one embodiment the drugs comprise SKi and W-7. In one embodiment the drugs comprise a PP2B Antagonist and C 6 C. In one embodiment the drugs comprise (AIP) PP2B Antagonist and C 6 C. In one embodiment the drugs comprise Cyclosporin and C 6 C. In one embodiment wherein the drugs comprise an Akt/Protein Kinase B Antagonist and C 6 C. In one embodiment wherein the drugs comprise calcium, vitamin D and IP 6 . 
         [0016]    The disclosure teaches any of the methods listed above further comprising the drug DCA. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0017]      FIG. 1 . Regulatory enzymatic tetrad controlling calcium targets and calcium distribution required for transitions between two sequential cell cycle phases. 
           [0018]      FIG. 2 . Time Course of Effect of the Calmodulin Antagonist W-7 on Induction of Apoptosis in MEL-STR Cells. 
           [0019]      FIG. 3 . Dose Response Comparison of the Effect of W-7 on Induction of Apoptosis in Malignant MEL-STR and Non-Malignant MEL-STVP Cells. 
           [0020]      FIG. 4 . Dose Response Comparison of the Effect of the PP2A and PKC Agonist C 6 C on Induction of Apoptosis in Malignant MEL-STR and Non-Malignant MEL-STVP Cells. 
           [0021]      FIG. 5 . Potentiation Between C 6 C and W-7 on Induction of Apoptosis in Malignant MEL-STR Cells. 
           [0022]      FIG. 6 . Potentiation between the PKC Agonist PMA and W-7 on Induction of Apoptosis, Growth Inhibition, and Microscopic or FACS Morphology in Malignant MEL-STR Cells. 
           [0023]      FIG. 7 . Potentiation between a Sphingosine Kinase Antagonist, SKi (4-[[4-(4-Chlorophenyl)-1,3-thiazol-2-yl]amino]phenol), and W-7 on Induction of Apoptosis in Malignant MEL-STR Cells. 
           [0024]      FIG. 8 . Selective Potentiation of Apoptosis between the Cell Permeable Auto-Inhibitory Peptide (AIP) PP2B Antagonist and C 6 C in Malignant MEL-STR but Not Non-Malignant MEL-STVP Cells. 
           [0025]      FIG. 9 . Potentiation of Apoptosis by the PP2B Antagonist Cyclosporin by C 6 C in Malignant MEL-STR Cells as Measured by Inhibition of Population Doubling Time. 
           [0026]      FIG. 10 . Potentiation of Apoptosis and Inhibition of Growth Rate using an Akt/Protein Kinase B Antagonist (Triciribine) in Combination with C 6 C in Malignant MEL-STR Cells. 
           [0027]      FIG. 11 . Prophetic Example in a Patient Diagnosed with Prostate Cancer and Subjected to a Treatment Regimen Designed to Produce Endoplasmic Reticulum Calcium Overload Using an Over-The-Counter 3 Component Mixture of Agents. 
           [0028]      FIG. 12 . Prophetic Example in a Patient Diagnosed with Inoperable Metastasized Pancreatic Cancer with a 6 Month Survival Estimate and Subjected to a Treatment Regimen Designed to Produce Endoplasmic Reticulum Calcium Overload Using an Over-The-Counter 3 Component Mixture of Agents. 
       
    
    
       [0029]    In the following description, for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the described embodiments. It will be apparent to one skilled in the art, however, that other embodiments of the present invention may be practiced without some of these specific details. Several embodiments are described herein, and while various features are ascribed to different embodiments, it should be appreciated that the features described with respect to one embodiment may be incorporated with other embodiments as well. By the same token, however, no single feature or features of any described embodiment should be considered essential to every embodiment of the invention, as other embodiments of the invention may omit such features. 
       DETAILED DESCRIPTION 
       [0030]    Unless otherwise indicated, all numbers used herein to express quantities, dimensions, and so forth used should be understood as being modified in all instances by the term “about.” In this application, the use of the singular includes the plural unless specifically stated otherwise, and use of the terms “and” and “or” means “and/or” unless otherwise indicated. Moreover, the use of the term “including,” as well as other forms, such as “includes” and “included,” should be considered non-exclusive. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one unit, unless specifically stated otherwise. 
         [0031]    Submaximal concentration is defined as a concentration of a drug that is at least 50% lower than the concentration given for the drugs maximal effect when given alone. The concentration may be 10 fold lower than its maximal effect when given alone. 
         [0032]    Drugs that are SERCA agonists (stimulate) include but are not limited to: Ceramide, C2-Ceramide, C6-Ceramide, HK654, PMA, and functional equivalents thereof (see Table 1, Protein Kinase C Agonists). 
         [0033]    Drugs that are inhibitors/antagonists of SER IP3-sensitive Ca 2+  gates include but are not limited to: IP6, IP5, and functional equivalents thereof (see Table 3, Endoplasmic Reticulum Ca 2+  Overload—IP3-Receptor Antagonists). 
         [0034]    Drugs that are agonists (activators/stimulators) of particulate guanylate cyclases include but are not limited to: Ceramide, C2-Ceramide, C6-Ceramide, HK654, PMA, and functional equivalents thereof (see Table 1, Protein Kinase C Agonists) 
         [0035]    Drugs that are effective elevators of cGMP levels include but are not limited to: Ceramide, C2-Ceramide, HK654, PMA, and functional equivalents thereof (see Table 1, Protein Kinase C Agonists). 
         [0036]    Drugs that are inhibitors of cGMP phosphodiesterases include but are not limited to: Viagra, Clalis, Levitra, Sulindac (and derivatives), and functional equivalents thereof (See Table 2, Endoplasmic Reticulum Ca2+Overload—cGMP PDE Antagonists). 
         [0037]    Drugs that are calmodulin (CAM) antagonists include but are not limited to: W-7 and functional equivalents thereof (See Table 1, Calmodulin Antagonists). 
         [0038]    Drugs that are Protein Kinase C (PKC) agonists include but are not limited to: Ceramide, C2-Ceramide, C6-Ceramide, HK654, PMA and functional equivalents thereof (see Table 1, Protein Kinase C Agonists). 
         [0039]    Drugs that are Protein Phosphatase 2A agonists include but are not limited to: Ceramide, C2-Ceramide, C6-Ceramide, and functional equivalents thereof (see Table 1, Protein Phosphatase 2A Agonists). 
         [0040]    Drugs that are CAM-dependent protein kinase II antagonists include but are not limited to: CK59, KN-93, KN-62, and functional equivalents thereof (see Table 1, Calmodulin-dep. Protein Kinase—II Antagonists). 
         [0041]    Drugs that are Calcineurin/CAM-dependent protein phosphatase 2B antagonists include but are not limited to: CN585, Cell Permeable Calcineurin Autoinhibitory Peptide, Cyclosporin A, FK-506, and functional equivalents thereof (see Table 1, Calmodulin-dep. Protein Phosphatase 2B Antagonists). 
         [0042]    Drugs that are Warburg Metabolic Antagonists include but are not limited to: Various salts of DCA, and functional equivalents thereof (see Table 3, Warburg Metabolic Antagonists). 
         [0043]    Drugs that are DNA damaging agents include but are not limited to: Ara-C I[Cytosine β-D-arabinofuranoside] and functional equivalents thereof. 
         [0044]    Drugs that are anti-mitotic drugs include but are not limited to: Vinblastine. [dimethyl (2β,3β,4β,5α,12β,19α)-15-[(5S,9S)-5-ethyl-5-hydroxy-9-(methoxycarbonyl)-1,4,5,6,7,8,9,10-octahydro-2H-3,7-methanoazacycloundecino[5,4-b]indol-9-yl]-3-hydroxy-[6-methoxy-1-methyl-6,7-didehydroaspidospermidine-3,4-dicarboxylate] and functional equivalents thereof. 
         [0045]    The disclosure teaches regulation of cell cycle traverse involved a series of alternating switches consisting of elevated cGMP, Ca 2+  uptake and sequestration within the ER, and reduced cytosolic [Ca 2+ ]. These phases are followed by periods of elevated cAMP, release of ER Ca2+ increased cytosolic [Ca 2+ ], and net Ca2+ efflux from the cell. Some of these switches correlate with known cell cycle transitions. The correlated cell cycle phenomena include the relationships between the Cyclin Kinase and calcium regulatory systems. Cytosolic [Ca 2+ ] is measured in synchronized cells and is in agreement, quantitatively and temporally. The relationships between calcium, cyclic nucleotides, Cyclin Kinases, and checkpoint control systems, are used for the treatment of cancer. 
         [0046]    The disclosure teaches uses for predicting new avenues for treating malignancy and it has been tested experimentally with positive results. The disclosure teaches an approach that is generalizable in many cancers, as it is based on one fundamental cell cycle aberration common to most if not every form of cancer. Cancers include but are not limited to melanoma, prostate, pancreatic, breast, lymphoma, lung, colon, etc. 
         [0047]    The Warburg effect is a metabolic “defect” in energy utilization exhibited by most cancer cells. This so-called “defect” results from a change in mitochondrial function. This disclosure teaches that this “defect” is not really a defect at all but rather is a normal process that is shared by other very rapidly growing cell such as early embryonic cells. This disclosure teaches that malignant cells merely co-opt an existing system which somehow is consistent with or enables rapid proliferation. 
         [0048]    Many different mutations in initial growth factor dependent pathways function to produce a state in which cells are made capable of continuously passing the so-called Pardee Restriction Point or point of no return towards the end of the G1 phase of the cell cycle. Traversal through G1 prior to this point is dependent on the continuous availability of EC Ca 2+ . Any growth factor requirement for passing the RP is bypassed completely by Ca 2+  specific ionophores as long as there is a ready supply of EC Ca 2+  Carcinogenic Phorbol analogs, which act to stimulate certain forms of Ca 2+  dependent PKC, can replace the growth factor requirement for crossing the RP, as long as there is sufficient EC Ca 2+  present in the growth medium. This disclosure teaches that for a normal cell to become irreversibly committed to pass through the cell cycle, these steps are effectively bypassed by providing a ready supply of EC Ca 2+  consistent with the known requirement for IC but not EC Ca 2+  upon passing the RP. Malignant transformation mimics the effect of Ca 2+  ionophores and Phorbol compounds and the initiating event in cancer is any mutation which produces an increased new steady state of continuous Ca 2+  influx. In order for such cells to escape Ca 2+  induced apoptosis, several adaptations in IC Ca 2+ -handling occur if such a potentially cancerous cell is to survive to a detectable disease state. This does not exclude the influence of known mutations in tumor suppressor or tumor promoter genes either prior to or selected for once the initiating stimulus for malignancy occurs in exacerbating the malignant state. However, all of such mutations must be secondary to satisfying the Ca 2+  requirement for passing the RP. 
         [0049]    This disclosure teaches the anticancer mechanism of Vit D3 is through short term elevation of Ca 2+  availability through intestinal absorption and short increase in Ca 2+  uptake by cancer cells. Suppression of and lower incidence of cancer occurrence requires only a slight increase in Ca 2+  overload in malignant cells. The efficacy of Vitamin D plus Ca 2+  supplements are potentiated by drugs designed to reduce release of Ca2+ from the ER. In one embodiment, the drug would be an antagonist of the ER IP3 receptor. 
         [0050]    Cell cycle checkpoints occur during periods of Ca 2+  sequestration and elevated cGMP levels. Cells can be prevented from passing out of these phases either directly or indirectly. Prolonged exposure to Ca 2+  influx triggers apoptosis significantly more easily in cancer cells compared to normal cells. Once normal cells pass the Pardee RP, they can complete one pass through the cell cycle in the absence of external growth factors. Only the intrinsic apoptotic pathway is used to trigger apoptosis in the event of uncorrectable genetic and chromosomal errors, as governed by cell cycle checkpoints. This pathway converges on the mitochondrion and involves Ca 2+  The mitochondrial Ca 2+  uptake pathway normally requires facilitated transfer of Ca 2+  directly from the ER as opposed to some cell-wide increase in Ca 2+  This disclosure teaches the use of drugs which shift the equilibrium from ER Ca 2+  release to ER Ca 2+  uptake. This disclosure teaches 2 (or more)-drug combinations directed against a tetrad of specific enzymes to achieve synergistic interactions and lower the possibility of unwanted side effects. Non limiting examples of drugs are found in Table 1, 2 and 3. This tetrad and the mediators of Ca 2+  distribution into and out of various compartments is illustrated in  FIG. 1 . 
         [0051]    Three main cell cycle checkpoints coincide with Ca 2+  storage phases. The Warburg phenomenon is related to changes in mitochondrial Ca 2+  content. Preventing cells from passing out of the Ca 2+  storage phases leads to mitochondrial Ca 2+  overload and subsequent apoptosis. The Ca 2+  regulatory enzyme tetrad is a means of not only controlling exit from Ca 2+  storage phases but also towards a method for converting cells residing in the Ca 2+  release phases to a state of continuous Ca 2+  storage and ultimate apoptosis. This predicts how cancer cells can be forced to undergo apoptosis by pharmaceutical intervention of Calmodulin- and PKC/PP2A-dependent processes. 
         [0052]    Three major “Checkpoints” have been identified which, in the face of uncorrectable errors in DNA integrity (including proper chromosomal separation at anaphase), arrest cell cycle progression and lead to apoptosis. The timing of these three Checkpoints coincides with cell cycle phases during which EC Ca 2+  is sequestered within the ER. A fourth checkpoint is known to occur during G2 but only leads to a slowing of cell cycle traverse rather than apoptosis and does not coincide with Ca 2+  sequestration. 
         [0053]    The intrinsic apoptosis pathway which operates during the cell cycle depends on the transference of Ca 2+  into the ER and ultimately into the mitochondria. 
         [0054]    Progression of cells through the cell cycle is dependent on the ordered synthesis of specific Cyclins and activation of their partnering kinases. Likewise, cell cycle progression is also obligatorily dependent on activation of specific Ca 2+ -sensitive intracellular receptors such as Calmodulin and Ca 2+ -sensitive forms of Protein Kinase C. Errors in the operation of either of these two regulatory systems have the power to arrest cells at specific transition points in the cell cycle. These two systems function in an obligatorily inter-related manner. 
         [0055]    Cancer cells differ from normal cells in their Ca 2+  handling. If cells could be pharmacologically arrested in Ca 2+  sequestering phases by interfering with Ca 2+  dependent mechanisms necessary to transition out of these phases, it triggers apoptosis. The extra burden of sequestered Ca 2+  in cancer cells allows for the selective induction of apoptosis in cancer cells before harming non-malignant cells. The present disclosure teaches the selective induction of apoptosis of cancer cells with reduction of toxic side-effects using novel 2 (or more)-drug combinations which are mutually synergistic. 
         [0056]      FIG. 1 . shows the Regulatory Enzymatic Tetrad Controlling Calcium Targets and Calcium Distribution in Two Different, Contiguous Cell Cycle Phases. Abbreviations used: CAM-PP2B, Calmodulin-Dependent Protein Phosphatase 2B; CAM-PKII, Calmodulin-Dependent Protein Kinase Type II; PKC, Protein Kinase C (Ca 2+ -stimulated subtypes); PP2A, Protein Phosphatase 2A; cAMP, Cyclic Adenosine Monophosphate; PKA, Cyclic AMP-Dependent Protein Kinase; cGMP, Cyclic Guanosine Monophosphate; PKG, Cyclic GMP-Dependent Protein Kinase; SOCE, Store Operated Calcium Entry; STIM 1, Stromal Interaction Molecule 1; PMCA, Plasma Membrane Calcium ATPase; PM Ca2+ Gates (also known as CRAC or ORAI), Ca 2+  specific plasma membrane influx channels; CICR, Calcium-Induced Calcium Release; IP 3 —R, Inositol Triphosphate Receptor; RY-R, Ryanodone Receptor; SERCA-A/B, Smooth Endoplasmic Reticulum Calcium ATPase. 
         [0057]    This illustration summarizes the cellular targets which regulate Ca 2+  distribution between various compartments as cells pass from one phase or regulatory switch-point to the next during the cell cycle. Each of the Tetrad enzymes acting directly, or secondarily through cyclic nucleotide dependent protein kinases, exert highly coordinated regulation of the functional activity of targets that control movement of Ca 2+  between cellular compartments and in and out of the cell. Of the various targets regulating Ca 2+  movements, some are activated and some are inactivated by phosphorylation. In each case, cells proceed from one switch point to the next. These phosphorylation events are reversed by opposing phosphatases. Thus, CAM-PKII is opposed by PP2A and PKC is opposed by PP2B. Steady state levels of cytosolic Ca 2+  vary between high and low levels for the entire length of each particular phase. These switch-points obligatorily control whether a cell will successfully transition from one phase to the next and successfully proceed through that phase. Pairs of contiguous phases are characterized by net Ca 2+  uptake, sequestration of said Ca 2+  into the SER compartment, and concomitant lowering of cytosolic Ca 2+  below the CAM activation threshold ([Ca 2+ ]&lt;0.1 M). The following phase is characterized by release of sequestered Ca 2+  into the cytosol in coordination with activation of the PMCA efflux pump exactly balanced to elevate cytosolic [Ca 2+ ] above the CAM activation threshold and below the PKC activation range (&gt;0.1 uM&lt;1.0 uM) and to gradually reduce SER-sequestered and total cellular Ca 2+  over time. 
         [0058]    By pharmacologically manipulating the activity of the Tetrad enzymes by appropriate stimulation or inhibition, progression through the cell cycle is arrested and all cells in the population are forced into a state of continuous Ca 2+  accumulation. Ultimately this leads to SER and mitochondrial Ca 2+  overload and triggering of apoptosis. Pharmacological manipulation of any pair of the Tetrad enzymes will interact synergistically to trigger an apoptotic response and thus can be used to reduce drug concentrations and toxicity clinically as well as shortening treatment duration. Apoptotic sensitivity of malignant cells to such treatments will be significantly greater than normal cells as a result of a greater burden of sequestered SER and mitochondrial Ca 2+  in cancer cells. 
         [0059]    In each of the treatment methods provided, there is a therapeutic window for selectively initiating an Apoptotic cascade in tumor cells without simultaneously inducing undesirable side effects in normal Ca 2+ -dependent physiological processes of normal cells. This treatment window can easily be determined by the routine experimentation of one skilled in the art. While inhibitors of plasma membrane efflux pumps may provide some clinical efficacy, employing submaximal combinations of drugs that interact synergistically to increase cellular Ca 2+  loading provides an unexpected means to reduce undesirable side effects and to increase therapeutic indices. 
         [0060]    The duration of treatment required to initiate an Apoptotic response in patients is relatively brief, on the order of 8 to 16 hours. In one embodiment, on the order of 3 to 6 hours. In one embodiment, 2 to 20 hours. In one embodiment, 4 to 6 hours. In one embodiment, 5 to 7 hours. Individual drugs or drug combinations are administered by standard means according to the absorptive and pharmacokinetic requirements of efficacious drug candidates. The therapeutic agents are administered orally or intravenously in amounts calculated to achieve measured blood concentrations approximating those determined to be effective from tissue culture studies. Each drug is used at the lowest dosage shown to produce mutual potentiation of apoptosis. In one embodiment, submaximal concentrations are used. 
         [0061]    The dosage of each drug is calculated to provide clinically effective blood levels for a period of 3 to 5 hours based on animal and Phase I trials. This short duration of treatment is based upon the minimum time required to force tumor cells into irreversible commitment to apoptosis. Resorption of a patient&#39;s tumor can be followed at appropriate intervals thereafter using ultra-sensitive techniques such as PET or SPECT molecular imaging. This regimen can be repeated daily if required based upon the severity, if any, of side-effects and by the rate of tumor shrinkage. Given the thresholds of sensitivity to calcium-induced apoptosis between normal and cancerous cells, such side-effects are likely to be fairly innocuous. 
         [0062]    Blood levels of given therapeutic agents are monitored by suitable assay methods specifically developed for this purpose in order to maximize therapeutic ratios. Depending on the severity of any side effects, this treatment regimen is repeated at regular intervals as often as necessary to maximize tumor regression. In one embodiment, drug responsiveness and treatment efficacy are monitored during the course of drug administration by assay of blood levels apoptotic markers, namely any of several caspases released by cells undergoing Apoptosis specifically developed for this purpose. In this way, patients are spared unnecessarily prolonged drug exposure and the clinician is furnished with immediate evidence of treatment efficacy. 
         [0063]    Tables 1, 2 and 3 list drugs for the synergistic effects as described above. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 PRIMARY APOPTOTIC TARGETS 
               
               
                 TABLE 1 - PRIMARY APOPTOTIC TARGETS 
               
             
          
           
               
                 PRIMARY ENZYME TETRAD 
                 DRUG/CHEMICAL 
                 DRUG/CHEMICAL 
               
               
                 TARGETS 
                 COMMON NAME 
                 CHEMICAL NAME 
               
               
                   
               
               
                 Calmodulin-dep. Protein Kinase - 
                 CK59 
                 2-(2-Hydroxyethylamino)-6-aminohexylcarbamic acid tert- 
               
               
                 II Antagonists 
                   
                 butyl ester-9-isopropylpurine 
               
               
                   
                 KN-93 
                 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino- 
               
               
                   
                   
                 N-(4-chlorocinnamyl)-N-methylbenzylamine) 
               
               
                   
                 KN-62 
                 1-[N,O-bis-(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- 
               
               
                   
                   
                 phenylpiperazine 
               
               
                 Calmodulin-dep, Protein 
                 CN585 
                 6-(3,4-dichloro-phenyl)-4-(N,N-dimethylaminoethylthio)-2- 
               
               
                 Phosphatase 2B Antagonists 
                   
                 phenyl-pyrimidine 
               
               
                   
                 Calcineurin Autoinhibitory 
                 11R-CaN-AlD, Ac- 
               
               
                   
                 Peptide, Cell-permeable 
                 RRRRRRRRRRRGGGRMAPPRRDAMPSDA-NH 2   
               
               
                   
                 Cyclosporin A, 
                 {R—[R*,R*—(E)]}-cyclic-(L-alanyl-D-alanyl-N-methyl-L-leucyl- 
               
               
                   
                 
                   Tolypocladium inflatum 
                 
                 N-methyl-L-leucyl-Nmethyl-L-valyl-3-hydroxy-N,4-dimethyl-L- 
               
               
                   
                   
                 2-amino-6-octenoyl-L-α-amino-butyric-N-methyl-glycinyl- 
               
               
                   
                   
                 Nmethyl-L-leucyl-L-valyl-N-methyl-leucyl) 
               
               
                   
                 FK-506,  Streptomyces   
                 (3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)- 
               
               
                   
                 sp. 
                 5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a 
               
               
                   
                   
                 Hexadecahydro-5,19-dihydroxy-3-[(1E)-2--[(1R,3R,4R)-4- 
               
               
                   
                   
                 hydroxy-3-methoxycyclohexyl]-1-methylethenyl]-14,16- 
               
               
                   
                   
                 dimethoxy-4,10,12,18-tetramethyl-8-(2-propen-1-yl)-15,19- 
               
               
                   
                   
                 epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclotricosine- 
               
               
                   
                   
                 1,7,20,21(4H,23H) tetrone 
               
               
                 Calmodulin Antagonists 
                 W-7 
                 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide 
               
               
                   
                   
                 hydrochloride 
               
               
                 Protein Phosphatase 2A 
                 Ceramide 
                 D-erythro-Sphingosine 
               
               
                 Agonists 
                 C2-Ceramide 
                 N-Acetyl-D-sphingosine 
               
               
                   
                 C6-Ceramide 
                 N-Hexanoyl-D-erythro-Sphingosine 
               
               
                 Protein Kinase C Agonists 
                 Ceramide 
                 D-erythro-Sphingosine 
               
               
                   
                 C2-Ceramide 
                 N-Acetyl-D-sphingosine 
               
               
                   
                 C6-Ceramide 
                 N-Hexanoyl-D-erythro-Sphingosine 
               
               
                   
                 HK654 
                 Diacylglycerol-lactone analog (cell permeable) 
               
               
                   
                 PMA 
                 Phorbol-12-Myristate-13-Acetate 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 SECONDARY APOPTOTIC TARGETS 
               
               
                 TABLE 2 - SECONDARY APOPTOTIC TARGETS 
               
             
          
           
               
                 SECONDARY APOPTOTIC 
                 DRUG/CHEMICAL 
                 DRUG/CHEMICAL 
               
               
                 TARGET 
                 COMMON NAME 
                 CHEMICAL NAME 
               
               
                   
               
               
                 Endoplasmic Reticulum 
                 Ski, Ski-2 
                 4-[[4-(4-Chlorophenyl)-1,3-thiazol-2-yl]amino]phenol 
               
               
                 Ca2 + Overload - 
               
               
                 Sphingosine Kinase Antagonist 
               
               
                 Endoplasmic Reticulum 
                 Triciribine 
                 6-amino-4-methyl-8-(beta.-D-ribofuranosyl) pyrrolo 
               
               
                 Ca2 + Overload - 
                   
                 [4,3,2-de]pyrimido[4,5-c]pyridazine 
               
               
                 Akt/Protein Kinase B Antagonist 
               
               
                 Endoplasmic Reticulum 
                 Viagra 
                 1-[4-ethoxy-3-(6,7-dihydro-1-methyl- 
               
               
                 Ca2 + Overload - 
                   
                 7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl) 
               
               
                 cGMP PDE Antagonists 
                   
                 phenylsulfonyl]-4-methylpiperazine 
               
               
                   
                 Cialis 
                 (6R-trans)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a- 
               
               
                   
                   
                 hexahydro-2-methyl-pyrazino [1′,2′:1,6] pyrido[3,4-b] 
               
               
                   
                   
                 indole-1,4-dione 
               
               
                   
                 Levitra 
                 4-[2-Ethoxy-5-(4-ethylpiperazin-1-yl)sulfonyl-phenyl]-9- 
               
               
                   
                   
                 methyl-7-propyl-3,5,6,8-tetrazabicyclo[4.3.0]nona-3,7,9- 
               
               
                   
                   
                 trien-2-one 
               
               
                   
                 Sulindac and 
                 {(1Z)-5-fluoro-2-methyl-1-[4-(methylsulfinyl) 
               
               
                   
                 Derivatives 
                 benzylidene]-1H-indene-3-yl}acetic acid 
               
               
                   
               
             
          
         
       
     
         [0000]                                  TABLE 3                   SECONDARY APOPTOTIC TARGETS - Over-the-Counter Supplements       TABLE 3 - SECONDARY APOPTOTIC TARGETS - Over-the-Counter Supplements            SECONDARY APOPTOTIC   DRUG/CHEMICAL   DRUG/CHEMICAL       TARGET   COMMON NAME   CHEMICAL NAME               Endoplasmic Reticulum   IP6, IP5   Inositol-1,2,3,4,5,6-hexakisphosphate (Inositol       Cal ++  Overload -       Hexaphosphate), myo-Inositol 1,3,4,5,6-       IP 3 -Receptor Antagonists       pentakisphosphate, (Inositol Pentaphosphate)       Endoplasmic Reticulum   Ca ++     Calcium Citrate       Ca ++  Overload -   Vitamin D3   Cholecalciferol       Plasma Membrane Ca ++  Channel       Agonists       Warburg Metabolic Antagonists   DCA   Sodium di-chloro-acetate                    
In as much as DCA reverses the Warburg effect and thus changes the sensitivity threshold for Ca 2+  dependent release of mitochondrial cytochrome C into the cytoplasm and consequent activation of caspase apoptotic mediators, this compound is claimed to be usable to potentiate the actions of either IP6 or Ca 2+  plus Vitamin D3 either alone or in various combinations. This allows the use of DCA clinically at sub-toxic levels as well as shortening treatment duration for effective induction of apoptosis in malignant cells.
 
       EXAMPLES 
       [0064]    The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. 
       Example 1 
       [0065]      FIG. 2 . Shows the Time Course of Effect of the Calmodulin Antagonist W-7 on Induction of Apoptosis in MEL-STR Cells. Malignant (MEL-STR) and non-malignant (MEL-STVP) cells used in these experiments were derived originally from normal human foreskin melanocytes obtained from the Weisberg lab. These original surgical samples were genetically modified to grow continuously in vitro and are non-tumor-forming in a nude mouse model. These same cells were genetically modified further to generate the tumor-forming cell line and were propagated. Thus, this represents an ideal pair of highly similar cells by which drug specificity between normal and malignant cells can be assessed. 
         [0066]    Transformed MEL-STR cells were incubated over a period of 24 hrs in the presence of a previously determined ineffective concentration (10 μm) of the CAM antagonist W-7 or the drug vehicle DMSO (1%) as controls and a concentration of 60 μm W-7 as illustrated. Apoptotic+dead cells were assayed in this experiment and those that follow below on a Becton-Dickenson flow cytometer using an Annexin V-FITC Apoptosis Detection Kit as described by the manufacturer. 
         [0067]    The results in this experiment show the time course for induction of apoptosis in the malignant cell line (measured by the Annexin Assay) by a highly-specific antagonist of the primary intracellular Ca 2+  receptor, Calmodulin. Calmodulin is known to be required for traverse of late G1, G2, and specific periods during mitosis and coincides with periods of elevated cAMP levels. Surprisingly, induction of apoptosis can be seen as soon as 3 hours of drug exposure. Morphological rounding of cells can be observed microscopically of by changes in FACS light scatter as early as 1 hrs. This is to be compared with typical studies on drug-induced apoptosis which require 48-72 hrs. of exposure. This is especially important because patient exposure and unwanted side-effects can be minimized in vivo. Essentially all of the population (at least in excess of 90%) scores positively for apoptosis. Given the ubiquitous function of Calmodulin in every cell of the body, use of the drug (or more potent congeners) has not been previously used for development by the pharmaceutical industry as far too toxic for clinical use. 
       Example 2 
       [0068]      FIG. 3  show a Dose Response Comparison of the Effect of W-7 on Induction of Apoptosis in Malignant MEL-STR and Non-Malignant MEL-STVP Cells. These results are critically important and highly unusual. Transformed malignant cells are more sensitive than non-transformed cells to a completely specific drug which acts only to antagonize a single target, that is, the main IC calcium receptor Calmodulin. The degree of this difference in sensitivity is approximately one order of magnitude. This is large enough that allows the use W-7 clinically. However, Calmodulin regulates many processes throughout the body and despite this sensitivity advantage may still trigger unwanted side effects. 
       Example 3 
       [0069]      FIG. 4  shows a Dose Response Comparison of the Effect of the PP2A and PKC Agonist C 6 C on Induction of Apoptosis in Malignant MEL-STR and Non-Malignant MEL-STVP Cells. The sensitivity difference observed in  FIG. 3  is not unique to the Calmodulin antagonist W-7 and a similar order of magnitude in EC50&#39;s is observed using a drug originally thought to stimulate specific PKC and PP2A targets at the time these experiments were carried out. This drug is now thought to act specifically on PP2A alone. These results show that malignant cells exhibit a very significant difference from normal cells in triggering Apoptosis and teach that cancer cells survive by reaching a stable condition of Ca 2+  overload higher than non-malignant cells. 
       Example 4 
       [0070]      FIG. 7  shows Potentiation Between C 6 C and W-7 on Induction of Apoptosis in Malignant MEL-STR Cells. In this experiment, it is shown that when combined with the Calmodulin antagonist (W-7) the effects of this drug on induction of Apoptosis are potentiated. Given the accepted specificity of W-7, this experiment provides evidence that both drugs are affecting processes that share Ca 2+  in common. 
       Example 5 
       [0071]      FIG. 9  shows Potentiation between the PKC Agonist PMA and W-7 on Induction of Apoptosis, Growth Inhibition, and Microscopic or FACS Morphology in Malignant MEL-STR Cells. The involvement of a form of PKC in the enzyme tetrad that is involved in Ca 2+ -dependent, cell cycle phase transitions is given in  FIG. 6 . Here the classic PKC activator, Phorbol Myristate Acetate, is found to be potentiated by W-7. Four different means for assaying the effect of these two chemicals are shown. The standard Annexin assay for detecting Apoptosis shows potentiation (Panel A). However, the more sensitive cell density assay as measured by Coulter Counter shows an even greater potentiation (Panel B). Potentiation is also confirmed by two different measures of cell shape, Light scatter by FACS (Panel C) and by direct microscopic examination (Panel D; results shown are the average of 3 microscope fields selected at random). Throughout all of the results reported here, the morphological effect can be observed as early as 1 hr. drug exposure and has been a sensitive indicator of the subsequent apoptotic fate of the MEL-STR cells under study. It is significant that such morphological shape changes are not observed in MEL-STVP cells. It is also important to realize that the only element in common with PMA and W-7 is Ca 2+  and these results provide additional evidence supporting the enzymatic tetrad regulatory system in triggering apoptosis. 
       Example 6 
       [0072]      FIG. 8  shows Potentiation between a Sphingosine Kinase Antagonist, SKi (4-[[4-(4-Chlorophenyl)-1,3-thiazol-2-yl]amino]phenol), and W-7 on Induction of Apoptosis in Malignant MEL-STR Cells. This result is especially interesting because it provides evidence that Ca 2+  must be involved in the action of Sphingosine Kinase. Given the widespread distribution of this enzyme in normal cells, and given the 10-fold potency advantage over non-malignant MEL-STVP cells enjoyed by W-7 (see  FIG. 1 ), these results represent another method for toxicity reduction during cancer therapy and a way of using a drug like W-7 that normally would be expected to be too toxic for use clinically. The synergy between W-7 and SKi provides evidence that both are converging upon a common element, namely Ca 2+ . 
       Example 7 
       [0073]      FIG. 5  is Selective Potentiation of Apoptosis between the Cell Permeable Auto-Inhibitory Peptide (AIP) PP2B Antagonist and C 6 C in Malignant MEL-STR but Not Non-Malignant MEL-STVP Cells. These results illustrate potentiation between C6C and a completely specific activator of the Calmodulin plus Ca 2+  requiring enzyme PP2B which is a cell-permeable auto-inhibitory polypeptide (abbr. PP2B-AIP) which acts to block the catalytic site of PP2B. Four observations can be drawn from these results. The first and foremost is that this result was predicted as a consequence of the Calcium Storage/Release Model in 2002. The synergistic interaction with C6C provides yet another example of convergence of two highly dissimilar targets which share Ca 2+  in common. This potentiation is seen only in the transformed MEL-STR cell line, not in untransformed MEL-STVP cells, thus providing a third example of differential sensitivity between malignant and non-malignant cells. Lastly, over the concentration range tested, AIP exerted no visible induction of apoptosis in either cell line. 
         [0074]    In this and other experiments using this protocol, it has never been possible to kill more than 50% of the MEL-STR cells over a 5 hr. exposure. This is in marked contrast to the potent effect of W-7 ( FIG. 2 ). This implicates at least one other target involved in the actions of W-7. This target is likely to be Calmodulin-dependent Protein Kinase II. This enzyme completes the 4 th  element of the regulatory enzymatic tetrad as illustrated in  FIG. 1 . Thus, pharmacological (antagonists of Calmodulin effectors, PP2B and PCAM-PK II; agonists of PKC and PP2A) manipulation any pair of tetrad enzymes is expected to interact synergistically and be usable in clinical practice. 
       Example 8 
       [0075]      FIG. 6  shows Potentiation of Apoptosis by the PP2B Antagonist Cyclosporin by C 6 C in Malignant MEL-STR Cells as Measured by Inhibition of Population Doubling Time. As a test of the specificity of AIP, the effect of Cyclosporin (a known inhibitor of PP2B) was tested for pro-apoptotic potential. At quite high concentrations, this compound displayed only slight stimulation of apoptosis or growth inhibition measured in this experiment by a change in doubling time (an indirect assay of cell death). This effect was dramatically potentiated by C6C in MEL-STP cells in the same manner as the previous experiment with AIP ( FIG. 7 ). In MEL-STVP cells, no inhibition of cell growth was observed with Cyclosporin at this or lower concentrations nor was there any potentiation observed between these two compounds, thus providing a fourth example of differential sensitivity between malignant and non-malignant cells. 
       Example 9 
       [0076]      FIG. 10  shows Potentiation of Apoptosis and Inhibition of Growth Rate using an Akt/Protein Kinase B Antagonist (Triciribine) in Combination with C 6 C in Malignant MEL-STR Cells. Because PKB has pro-survival/anti-apoptotic properties and, when activated, can overcome checkpoint arrests in both G1 and G2 (periods in which cAMP is normally elevated during traverse of these phases), because cAMP has been implicated in many cells types as an anti-apoptotic agent, and because cAMP is known to stimulate the release of ER Ca 2+  the question of whether these observations shared a common mechanism involving ER Ca 2+  reduction was tested experimentally. The effect of low dose C6C on cells treated over a wide concentration range of the PKB inhibitor Triciribine was examined. The results of the highest dose of Triciribine tested are shown in  FIG. 8 . Clear potentiation was observed consistent with the hypothesis that ER Ca 2+  overload can promote Apoptosis in cancer cells and that PKB antagonists could be used synergistically with other drugs which modulate cellular Ca 2+  distribution and as a means of reducing off-target side-effects. 
         [0077]    There are other ways of effecting clinical treatment of any and all cancer cell types. For example, any treatment which delivers excess Ca 2+  to the right location within cells, even on a short term basis, could be combined with an agent that inhibits release of Ca 2+  from the ER, the obligatory organelle that transfers Ca 2+  to the mitochondria and induces an apoptotic response. Calcitriol (the active form of Vitamin D) reduces the incidence of certain cancers to a small but significant degree (ca. 17-20%). This cannot be demonstrated when only 400 IU of Vitamin D is taken as a supplement, nor can it be shown when only 1000 mg of Calcium is taken. Only when the two are combined is any effect observed, albeit quite modest. If this regimen is combined with an inhibitor of ER Ca 2+  release, such as IP6 at doses up to 1000-1600 mg/day, or in another embodiment, at 500-800 mg; taken twice daily, then together this 3-component combination synergistically interacts to produce a much larger reduction of cancer incidence as well as reducing or even eliminating established cancers. Below are two prophetic examples illustrating different forms of cancer and the responses that can be expected as measured by antigen markers. 
       Example 10 
       [0078]    Since this 3-part regimen, at the levels shown, should have no detectable side effects, it may be used in conjunction with either male or female hormone replacement therapies in order to nullify any chance of elevated cancer risk associated with testosterone or estrogen supplementation. 
         [0079]      FIG. 11  shows a Prophetic Example in a Patient Diagnosed with Prostate Cancer and Subjected to a Treatment Regimen Designed to Produce Endoplasmic Reticulum Calcium Overload Using an Over-The-Counter 3 Component Mixture of Agents. A patient is prescribed 1000 mg Calcium Citrate (or mixed salts of citrate, malate, and carbonate), 2000 IU of Vitamin D3, and 500 mg IP6 to be taken twice daily 12 hrs. apart. This regimen is continued for at least 6 months. The dose of Calcium salt and IP6 (but not Vitamin D3) can be increased to thrice daily without side effects. This treatment should be combined with adequate exposure to sunlight. Relief of symptoms can be expected within the first 2-3 weeks of treatment and the effects of this regimen can be followed objectively by standard PSA measurements as illustrated in this figure or more specific prostate cancer-specific antigens in development. 
       Example 10 
       [0080]      FIG. 12  shows a Prophetic Example in a Patient Diagnosed with Inoperable Metastasized Pancreatic Cancer with a 6 Month Survival Estimate and Subjected to a Treatment Regimen Designed to Produce Endoplasmic Reticulum Calcium Overload Using an Over-The-Counter 3 Component Mixture of Agents. The same regimen as described in  FIG. 11  is provided. Enlargement of metastasized tumors should be arrested and some tumors may be eliminated entirely. The effect of the treatment regime is illustrated in this figure by radioimmunoassay of the pancreatic cancer antigen CA-19-9 over time. 
         [0081]    The description of the various embodiments has been presented for purposes of illustration and description, but is not intended to be exhaustive or limiting of the invention to the form disclosed. The scope of the present invention is limited only by the scope of the following claims. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments described and shown in the figures were chosen and described in order to explain the principles of the invention, the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated. All references cited herein are incorporated in their entirety by reference.