Abstract:
Disclosed are neuroblastoma tumor-initiating cell inhibiting compositions comprising chemical entities capable of affecting neuroblastoma tumor-initiating cells. Pharmaceutical preparations that include these chemical entities are also provided for the treatment of neuroblastoma. These pharmaceutical preparations are suitable for the treatment of humans, and are particularly suited for the treatment of children of 12 years of age or younger having neuroblastoma. The compositions and pharmaceutical preparations posses reduced normal cell cytotoxicity. The compositions and pharmaceutical preparations may be used alone or together with other conventional neuroblastoma preparations as part of a clinical regimen in the treatment and management of neuroblastoma.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application makes reference to the following co-pending U.S. patent application. The application is U.S. App. No. 60/739,337, entitled “Cancer Stem Cells and Uses Thereof,” filed Nov. 23, 2005. The entire disclosure and contents of the above application is hereby incorporated by reference. 
     
    
     BACKGROUND  
       [0002]     1. Field of the Invention  
         [0003]     The present invention relates to the field of pharmacologically active chemical compositions useful in affecting neuroblastoma tumor-initiating cells, and the use of such compositions in the treatment of neuroblastoma and related conditions.  
         [0004]     2. Related Art  
         [0005]     Neuroblastoma (NB) is the most common extracranial solid tumor in children, with poor survival rates in children with metastatic disease. NB is estimated to be responsible for about 15% of cancer-related deaths in children (1,2). The survival rate for metastatic NB is estimated to be less than 30%. In the majority of these cases, conventional cancer therapies have been ineffective.  
         [0006]     Little is reported concerning the precise molecular alterations that give rise to NB, its cell of origin, or why NB cells metastasize and become resistant to chemotherapeutic agents. Unfortunately, genetic mutations that contribute to the origin and progression of 98% of NB cases have not been identified.  
         [0007]     One identifiable hallmark of NB is the appearance of proliferating cells with characteristics of neural crest-derived sympathetic neuronal precursors (neuroblasts). NB tumors also frequently contain other neural crest cell types, including neuroendocrine and Schwann cells. Moreover, NB appears in tissues that developmentally derive from the neural crest including sympathoadrenal precursors which differentiate into both sympathetic neurons and adrenal chromaffin cells, the paravertebral and preaortic sympathetic ganglia, and the adrenal gland.  
         [0008]     The clinical behavior of NB is unique. Tumors that arise in children under one year of age may spontaneously regress by differentiation or apoptosis, even after arising in or metastasizing to liver and skin. In contrast, NB tumors in children over one-year-old often grow aggressively, disseminate to the bone and bone marrow, and are fatal in the vast majority of cases.  
         [0009]     Mass screening of infants showed that NB is much more frequent than previously thought. Many of these tumors regress without clinical diagnosis. Regressing or favorable-prognosis tumors have been reported to express high levels of the TrkA/NGF receptor and display phenotypes of differentiated peripheral neural cells, while malignant or unfavorable-prognosis tumors resemble proliferating sympathoadrenal precursors, often expressing TrkB, amplified N-myc, and many genes involved in neural crest development.  
         [0010]     The only reported germline NB predisposition gene is Phox2b, which is mutated in many familial cases of NB, and is required for proper differentiation of sympathetic neurons from neural crest precursors (NCPs) (3,4). In the regressive form of the disease, the transformed precursors ultimately differentiate or die, while in older children, these molecular transformations instead result in a population of persistently proliferating and highly migratory transformed neuroblasts.  
         [0011]     The concept of tumor-initiating cells (TIC) (also called tumor or cancer stem cells) postulates that only rare cells in tumors are endowed with tumorigenic potential, and was initially developed to explain why (i) most tumors are comprised of both undifferentiated proliferating progenitors and post mitotic differentiated cells, (ii) only a very small fraction of tumor cells form colonies after plating in vitro, and (iii) large numbers of tumor cells are required to seed the growth of a new tumor in mice (4-10).  
         [0012]     Dick et al. and others reported that clonally-derived tumor cells of acute myelogenous leukemia (AML) patients could be physically separated into tumorigenic and non-tumorigenic fractions (11,12). Brain and breast tumors have also been reported to contain a subpopulation of TICs (13,14). Thus, in solid tumors, a rare tumor cell population may fuel tumor growth and seed metastasis. This hypothesis has major implications for treating cancer patients. For example, many current therapies kill the bulk of proliferating tumor cells, but these cells may not be intrinsically tumorigenic, and in many cases the TICs may escape the effects of the therapeutic agents, leading to tumor relapse. Thus, it is essential to identify and characterize TICs from various tumors in order to develop and target therapies against this critical cell type.  
         [0013]     TICs have also been shown to share phenotypic characteristics with stem cells derived from their tissue of origin. For example, for a given tissue, the tissue stem cells and TICs both (i) self-renew, (ii) express common phenotypic markers, (iii) grow in a similar fashion in response to mitogens, and (iv) yield tissue-appropriate progeny (13,14). However, whereas tissue stem cells generate mature differentiated cell types, differentiation of TICs is generally arrested at the level of one or more tissue progenitor cells resulting in tumors comprising a hierarchy of progenitors and some differentiated progeny (4).  
         [0014]     Many pediatric and adult tissues contain resident stem cells (4). It is currently unknown if TICs originate by transformation of tissue stem cells. Observations have been made that oncogenic mutations commonly affect genes required for normal stem cell renewal and differentiation (4). This may be particularly relevant for children&#39;s tumors, since developing tissues contain a higher proportion of tissue stem cells than do adult tissues.  
         [0015]     Tumor initiating cells from some solid tissue tumors, such as breast and brain tumors, have been described. However, a tumor initiating cell population from tumor tissue in a patient with neuroblastoma has not been isolated. One reported observation in some infantile forms of NB (called stage 4S) is that large tumors are frequently found in skin (15). It was previously assumed that skin was a preferred metastatic target for NB. However, a population of tumor initiating cells from such solid tumor tissue has not yet been reported.  
         [0016]     The above and other observations in the field reveal a continuing medical need continues to exist in the art to determine why and in which cell type NB arises, and why some neuroblastoma tumors spontaneously regress and others are fatal. In addition, new effective drug targets and therapeutics tailored to identifying and treating specific forms and stages of neuroblastoma are needed.  
       SUMMARY  
       [0017]     The above and other long-felt needs in the art are met in the present invention.  
         [0000]     Compositions/Pharmaceutical Preparations:  
         [0018]     In one aspect, the invention provides compositions comprising novel chemical entities that are capable of affecting neuroblastoma. In some embodiments, these chemical entities may be described as compounds that specifically kill neuroblastoma tumor-initiating cells, or that arrest the growth of neuroblastoma tumor-initiating cells. In other aspects, these chemical entities and compositions containing one or more of them may be described as having specifically cytostatic or cytotoxic activity toward neuroblastoma tumor-initiating cells.  
         [0019]     In some embodiments, the anti-neuroblastoma composition may be described as comprising one or more active ingredients comprising:  
         [0020]     2.3-Dimethoxy-1.4-naphthoquinone,  
         [0021]     Aklavine Hydrochloride,  
         [0022]     Amodiaquin dihydrochloride dehydrate;  
         [0023]     Amsacrine Hydrochloride;  
         [0024]     Azaguanine-8;  
         [0025]     beta-peltatin;  
         [0026]     Camptothecine (S.+);  
         [0027]     CGP-74514A hydrochloride;  
         [0028]     Chelerythrine chloride;  
         [0029]     Cholestan-3beta.5alpha.6beta-Triol;  
         [0030]     Ciclopirox Olamine;  
         [0031]     Clofazimine;  
         [0032]     Colchicine;  
         [0033]     Convallatoxin;  
         [0034]     Crassin Acetate;  
         [0035]     Crinamine;  
         [0036]     Dequalinium analog. C-14 linker;  
         [0037]     Dequalinium dichloride;  
         [0038]     Digitoxin;  
         [0039]     Digoxigenin;  
         [0040]     Dihydrogambogic acid;  
         [0041]     Dihydroouabain;  
         [0042]     Erysolin;  
         [0043]     Gambogic acid;  
         [0044]     Mechlorethamine;  
         [0045]     Meclizine hydrochloride;  
         [0046]     MG 624;  
         [0047]     Mitoxanthrone Hydrochloride;  
         [0048]     Ouabain;  
         [0049]     Oxybendazole;  
         [0050]     Oxybendazole;  
         [0051]     Paclitaxel;  
         [0052]     Parthenolide;  
         [0053]     Patulin;  
         [0054]     Periplocymarin;  
         [0055]     Peru voside;  
         [0056]     Primaquine diphosphate;  
         [0057]     Quinacrine dihydrochloride;  
         [0058]     Sanguinarine chloride; or  
         [0059]     Tomatine,  
         [0060]     In some embodiments, the chemical entities of the invention may be described as compounds that possess specific cytostatic or cytotoxic activity toward neuroblastoma tumor-initiating cells. In other embodiments, the neuroblastoma-inhibiting composition further comprises ancitabine hydrochloride, doxorubicin hydrochloride, etoposide, vincristine sulfate, or a combination thereof. In yet other embodiments, the neuroblastoma inhibiting composition may be further described as having reduced non-neuroblastoma tumor-initiating cell cytotoxicity.  
         [0061]     In some embodiments, the chemical entities may be described as possessing potent anti-neuroblastoma tumor-initiating cell activity, and a reduced cytotoxicity to normal, non-neuroblastoma tumor-initiating cells, relative to conventional neuroblastoma treatment preparations. In some embodiments, the compositions are described as essentially free of non-neuroblastoma tumor cell inhibiting activity.  
         [0000]     Methods of Inhibiting Neuroblastoma Tumor Initiating Cells/Methods of Treating and/or Inhibiting Neuroblastoma in an Animal  
         [0062]     In yet another aspect, the invention provides methods for inhibiting neuroblastoma tumor-initiating cells. In some embodiments, the method comprises administering an effective amount of a composition comprising a neuroblastoma tumor-initiating cell inhibiting ingredient. In some embodiments, the neuroblastoma tumor-initiating cell inhibiting ingredient comprises one or more active ingredients comprising:  
         [0063]     2.3-Dimethoxy-1.4-naphthoquinone,  
         [0064]     Aklavine Hydrochloride,  
         [0065]     Amodiaquin dihydrochloride dehydrate;  
         [0066]     Amsacrine Hydrochloride;  
         [0067]     Azaguanine-8;  
         [0068]     beta-peltatin;  
         [0069]     Camptothecine (S.+);  
         [0070]     CGP-74514A hydrochloride;  
         [0071]     Chelerythrine chloride;  
         [0072]     Cholestan-3beta.5alpha.6beta-Triol;  
         [0073]     Ciclopirox Olamine;  
         [0074]     Clofazimine;  
         [0075]     Colchicine;  
         [0076]     Convallatoxin;  
         [0077]     Crassin Acetate;  
         [0078]     Crinamine;  
         [0079]     Dequalinium analog. C-14 linker;  
         [0080]     Dequalinium dichloride;  
         [0081]     Digitoxin;  
         [0082]     Digoxigenin;  
         [0083]     Dihydrogambogic acid;  
         [0084]     Dihydroouabain;  
         [0085]     Erysolin;  
         [0086]     Gambogic acid;  
         [0087]     Mechlorethamine;  
         [0088]     Meclizine hydrochloride;  
         [0089]     MG 624;  
         [0090]     Mitoxanthrone Hydrochloride;  
         [0091]     Ouabain;  
         [0092]     Oxybendazole;  
         [0093]     Oxybendazole;  
         [0094]     Paclitaxel;  
         [0095]     Parthenolide;  
         [0096]     Patulin;  
         [0097]     Periplocymarin;  
         [0098]     Peruvoside;  
         [0099]     Primaquine diphosphate;  
         [0100]     Quinacrine dihydrochloride;  
         [0101]     Sanguinarine chloride; or  
         [0102]     Tomatine.  
         [0103]     In some embodiments, the effective amount of the neuroblastoma tumor initiating cell inhibiting ingredient is an amount effective to arrest the growth of and/or kill neuroblastoma tumor-initiating cells, or effective to induce differentiation of said cells to cell types that no longer proliferate. In other embodiments, the method may further comprise administering a composition further comprising ancitabine hydrochloride, doxorubicin hydrochloride, etoposide, vincristine sulfate, or a combination thereof.  
         [0104]     In some embodiments, the composition may further include a pharmaceutically acceptable carrier solution.  
         [0105]     In yet other embodiments, the neuroblastoma tumor-inhibiting cells are in an animal having neuroblastoma. In some embodiments, the animal is a human. In some embodiments, the human is 12 years of age or younger. That is, it is anticipated that the invention is particularly useful in the treatment of children afflicted with neuroblastoma, and will have a profound effect on reducing the high rate of mortality in this population of neuroblastoma patients.  
         [0106]     The method may be further described as administering a composition that has a reduced non-neuroblastoma tumor-initiating cell cytotoxicity. It is expected that the methods and compositions of the present invention will provide fewer and/or less pronounce undesirable side affect in the treatment of a patient as a result. In some embodiments, the composition employed in the method is essentially free of non-neuroblastoma tumor cell inhibiting activity.  
         [0107]     The compositions may be described as comprising a mixture of any or all of the compounds listed below:  
                       TABLE 1                                       2.3-Dimethoxy-1.4-naphthoquinone           AKLAVINE HYDROCHLORIDE           Amodiaquin dihydrochloride dihydrate           AMSACRINE HYDROCHLORIDE           ANCITABINE HYDROCHLORIDE           Azaguanine-8           beta-PELTATIN           Camptothecine (S.+)           CGP-74514A hydrochloride           Chelerythrine chloride           CHOLESTAN-3beta.5alpha.6beta-TRIOL           CICLOPIROX OLAMINE           Clofazimine           Colchicine           CONVALLATOXIN           CRASSIN ACETATE           CRINAMINE           Dequalinium analog. C-14 linker           Dequalinium dichloride           Digitoxin           Digoxigenin           Digoxin           DIHYDROGAMBOGIC ACID           Dihydroouabain           ERYSOLIN           Etoposide           GAMBOGIC ACID           Idarubicin           MECHLORETHAMINE           MECLIZINE HYDROCHLORIDE           MG 624           MITOXANTHRONE HYDROCHLORIDE           OUABAIN           OXYBENDAZOLE           Paclitaxel           Parthenolide           PATULIN           PERIPLOCYMARIN           PERUVOSIDE           Podophyllotoxin           Primaquine diphosphate           Quinacrine dihydrochloride           Sanguinarine chloride           TENIPOSIDE           TOMATINE           Vinblastine sulfate salt           Vincristine sulfate                      
 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0108]      FIG. 1 , according to one embodiment of the invention, presents a diagrammatic flow chart demonstrating the design of the high throughput, dual-cell (Normal or Tumor cells) screening assay employed in the selection of candidate test compounds that target neuroblastoma tumor-initiating cells. Normal or tumor-initiating cell spheres are dissociated; 3,000 single cells/well are plated in 96 well plates; candidate test compound is added; cell proliferation assayed by Alamar Blue signal. Blue/nonfluorescent compound is converted to a red/fluorescent compound under reducing conditions such as those produced by live cells. The magnitude of the fluorescent signal is proportional to the metabolic activity of the cell sample.  
         [0109]      FIG. 2 , according to one embodiment of the invention, presents the results from a study wherein FS90 (normal human SKPs, passage 3) cells were treated with the LOPAC library of chemical compounds. Alamar Blue was added after 30 hours and fluorescence intensity read after an additional 24 hours. The hit cutoff is indicated in the graph by the thick line across the graph at the Y axis value of about 69.00% Control Alamar Blue Signal (which corresponds to 3 standard deviations from the mean of all test samples). Nine compounds whose Alamar Blue signals fall below this line were identified as primary hits in this study. (X axis presents the Compound ID number (n=80×8 plates); Y axis presents the % Control Alamar Blue Signal).  
         [0110]      FIG. 3A-3C , according to one embodiment of the invention, presents the study results from primary screens of the chemical libraries examined. 3A presents the results of the primary screen in Venn diagram form. The Venn diagrams depict the primary hits from each library. Compounds in the gray-bordered circles (left circle) affected the tumor-initiating cells, while compounds in the black-bordered circles (right circle) affected normal cells. Compounds that affected both cell types lie in the overlap region. Note that there is some compound redundancy between the libraries. 3B presents the confirmed primary hits in Venn diagram form. Primary hits were retested against NB12, FS90 and FS105 (normal human SKPs). 87% of the primary hits were confirmed in this step, yielding 54 unique compounds that target tumor-initiating cells, 4 unique compounds that target normal cells, and 46 compounds that have activity against both normal and tumor cells (overlap region). 3C presents in a pie-format the classification of primary hits by mechanism of action. (Solid light gray area=DNA damaging agents/cell cycle inhibitors; Solid dark gray area=Na+/K+ ATPase inhibitors; Diagonal striped area=Neuronal receptor effectors; Vertical striped area=Other; Solid white area=Metabolic inhibitor; Checkerboard area=Neuronal channel effectors; Dotted area=Specific protein effectors).  
         [0111]      FIG. 4A-4E , according to one embodiment of the invention, presents the IC50 values that were determined for the 64 selected candidate compounds. Compounds were chosen for further testing based on differential cell type selectivity, mechanism of action, and pharmacological interest. Tumor-initiating cells and normal cells were treated with 10 serial dilutions of compounds (5 μM to 9 nM). Representative graphs are shown in  FIG. 4A  (Complete Response), 4B (Partial Response), and 4C (Threshold Effect). Compounds that affected the tumor-initiating cells at a much lower dose than normal cells (4D graph, left) or compounds that had a greater effect on the tumor-initiating cells than normal cells (4E, right graph), were selected for secondary in vitro screens in addition to those compounds that only affected tumor-initiating cells. (FS90=normal cells; NB12=tumor-initiating cells).  
         [0112]      FIG. 5A-5C , according to one embodiment of the invention, presents the results from secondary screens of the candidate compounds. Compounds of interest are being tested against additional normal primary cultures (FS89, FS105), a stage 1V neuroblastoma primary culture (NB25), and a neuroblastoma cell line (KCNR) using a sphere formation assay. 5A presents a flow diagram of the secondary in vitro screen. The candidate compound is added at 0 days and at 3 days. Spheres are counted at 7 days.  FIG. 5B  presents a dose response curve of various cell lines (FS89, FS90, FS105, NB12, NB25 and KCNR) to amsacrine.  FIG. 5C  presents a dose response curve of various cell lines (FS89, FS90, FS105, NB12, NB25 and KCNR) to MG624.  
         [0113]      FIG. 6A - 6 FF, according to one embodiment of the invention, presents IC 50  values for 32 selected compounds from the LOPAC and Prestwick collections. Tumor-initiating cells (NB12) and normal cells (FS90) were treated with 10 serial dilutions of compounds ranging from 5 μM to 9 nM. Cell survival/growth was assayed using Alamar Blue and the percentage of control Alamar Blue signal was plotted versus log [compound] nM. IC50 values for NB12 are given above each plot.  
         [0114]      FIG. 7A - 7 FF, according to one embodiment of the invention, represents IC50 values determined for 32 selected compounds from the LOPAC, Prestwick, and Spectrum collections. Tumor-initiating cells (NB12) and normal cells (FS90) were treated with 10 serial dilutions of compounds ranging from 5 μM to 9 nM. Cell survival/growth was assayed using Alamar Blue and the percentage of control Alamar Blue signal was plotted versus log [compound] nM (FS90 in dashed line, NB12 in bolded line). IC50 values for NB12 and FS90 are given beside each plot.  
     
    
     DETAILED DESCRIPTION  
       [0000]     Definitions  
         [0115]     Where the definition of terms departs from the commonly used meaning of the term, applicant intends to utilize the definitions provided below, unless specifically indicated.  
         [0116]     For the purposes of the present invention, “a”, “an” and “the” include reference to the plural unless the context as herein presented clearly indicates other wise.  
         [0117]     For purposes of the present invention, the term “active agent” is defined as a chemical entity, group of chemical entities or compound that is capable of providing an affect on neuroblastoma tumor initiating cells or neuroblastoma cells in vitro or in vivo. The affect of the active agent may be a reduction in cytotoxicity relative to the level of cytotoxicity demonstrated in the absence of the active agent under similar conditions, or a cytostatic affect on neuroblastoma or on neuroblastoma tumor initiating cells that results in a reduced rate of neuroblastoma or neuroblastoma tumor-initiating cell proliferation and/or growth, or a reduction of the rate or occurrence of differentiation into neuroblastoma cell types, precursors, or any other cell type that is related to the progression of a neuroblastoma pathology, or to an increase in the inducement of the differentiation of neuroblastoma tumor-initiating cells into cell types (for example, neurons) that no longer proliferate (for example, retinoic acid is a common differentiation therapy for neuroblastoma that is used as an adjunct therapy after removal of a tumor, differentiation therapy).  
         [0118]     For purposes of the present invention, the term “enriched” is defined as containing at least 50% of the identified biological moiety, such as a cancer stem cell.  
         [0119]     For purposes of the present invention, the term “neuroblastoma tumor initiating cell” (NB TIC) is defined as a cell that is capable of giving rise to neuroblastoma or a tumor cell that is identifiable with a condition of neuroblastoma, such as a tumor cell that may be identified to have particular identifiable cell surface markers associated with neuroblastoma (such as NB84, CD44, TrkA, GD2, CD34, p75NTR, and/or versican) and/or is without cell surface markers that are characteristic of tumor cells that are not from neuroblastoma (such as CD133, TrkB, and/or CD31).  
         [0120]     For purposes of the present invention, the term “neuroblastoma tumor-initiating cell inhibiting activity” is defined as an activity for affecting neuroblastoma tumor-initiating cell survival, proliferation, or that promotes cell differentiation into benign cell types.  
         [0121]     For purposes of the present invention, the term, “effective amount” is defined as an amount of a compound that will inhibit and/or reduce neuroblastoma tumor initiating cell survival, proliferation, or that will promote the differentiation of neuroblastoma tumor-initiating cells into benign cell types.  
         [0000]     Description  
       EXAMPLES  
     Example I Materials and Methods  
       [0122]     The present example provides a description of the screening method used to identify the chemical entities capable of affecting neuroblastoma cells reported in the present series of studies.  
         [0123]     Malignant neuroblastoma (NB) is the most common extra-cranial solid tumor in children. Survival of patients older than 1 year remains less than 30% with conventional therapies. Candidate NB tumor-initiating cells (TICs) were isolated, and it was hypothesized that TICs are related to SKPs (SKin-derived Precursors). Both SKPs and TICs originate from the neural crest, express similar neural crest markers, and differentiate in vitro into similar cell types. The availability of two neural crest stem cell sources, one from the NB tumor and the other from the skin of the same patient, affords us a unique opportunity for therapeutic target discovery.  
         [0000]     Study 1 Screen:  
         [0000]     Materials and Methods:  
         [0124]     To identify compounds that suppress the growth and survival of neuroblastoma (NB) tumor-initiating cells and not nontransformed normal cells (SKPs), a cell-based assay was established and used in which NB tumor-initiating cells from a multiple relapse NB patient (NB12, passage 6-17) and normal SKPs (FS90, passage 2-5) were tested in parallel to detect specific alterations of cell viability/proliferation. For each cell type, cells were passaged 5 days prior to screening. Three thousand (3000) cells in 100 □L SKPs growth media (B27, bFGF, EGF, P/S, fungizone in DMEM:F12 with 50% hFS conditioned media) were robotically plated in uncoated 96 well plates and treated with test compound for 30 hours, prior to a 24 hour incubation in the presence of Alamar Blue and subsequent fluorometric reading. Under these conditions, the Alamar Blue signal displayed a linear response with time, background was minimal, and the dynamic range satisfactory (i.e. the Alamar Blue reading at 0 hours vs. 24 hours was &gt;10 fold different).  
         [0125]     The robustness of the screen was initially evaluated by using a collection of 1280 bioactive compounds (LOPAC library, Sigma). For both normal SKPs and NB tumor-initiating cells, variability of signals was low, with CV values ranging between 3.5-4.5% across the plates, and the dimensionless, statistical parameters Z′ and Z factors were &gt;0.5, suggesting an excellent assay quality. “Hits” were defined as the compounds whose signals were shifted away by at least 3× standard deviations (99.73% confidence interval) from the mean of the general sample population.  
         [0000]     Results:  
         [0126]     The screen of the LOPAC library at 5 μM yielded 13 “hits” which were found to affect both normal and NB cells. We also identified 18 compounds that selectively target NB cells. Four compounds selectively targeted normal cells.  
                         TABLE 2                       13 compounds that affect both normal and NB cells:                                    Ancitabine hydrochloride           Brefeldin A from  Penicillium brefeldianum             Calmidazolium chloride           CGP-74514A hydrochloride           Dihydroouabain           Diphenyleneiodonium chloride           Emetine dihydrochloride hydrate           Idarubicin           Mitoxantrone           Ouabain           Quinacrine dihydrochloride           Ammonium pyrrolidinedithiocarbamate           Sanguinarine chloride                      
 
         [0127]    
       
         
               
             
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                   
               
               
                 18 compounds that selectively target NB cells. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Loratadine 
               
               
                   
                 MG 624 
               
               
                   
                 Melphalan 
               
               
                   
                 Podophyllotoxin 
               
               
                   
                 Ro 25-6981 hydrochloride 
               
               
                   
                 Rotenone 
               
               
                   
                 DL-Stearoylcarnitine chloride 
               
               
                   
                 Taxol 
               
               
                   
                 Vincristine sulfate 
               
               
                   
                 Vinblastine sulfate salt 
               
               
                   
                 Chelerythrine chloride 
               
               
                   
                 Colchicine 
               
               
                   
                 Cytosine-1-beta-D-arabinofuranoside hydrochloride 
               
               
                   
                 Dequalinium dichloride 
               
               
                   
                 (S)-(+)-Camptothecin 
               
               
                   
                 Dequalinium analog, C-14 linker 
               
               
                   
                 2,3-Dimethoxy-1,4-naphthoquinone 
               
               
                   
                 Etoposide 
               
               
                   
                   
               
             
          
         
       
     
         [0128]                              TABLE 4                       4 compounds selectively target normal cells:                                    8-Methoxymethyl-3-isobutyl-1-methylxanthine           Oligomycin A           Sphingosine           Thapsigargin                        
 Study 2 Screen: 
 
 Materials and Methods 
 
         [0129]     The Prestwick library was screened at 5 μM using FS90 and NB12 and at 1 μM using NB12 only due to the high number of “hits” at 5 μM. This screen identified 9 compounds that selectively target NB12 and 15 compounds that affect both NB12 and FS90.  
         [0130]     Results:  
                         TABLE 5                       9 compounds that selectively target NB12:                                    Azaguanine-8           Paclitaxel           Camptothecine (S.+)           Colchicine           Etoposide           Doxorubicin hydrochloride           Lanatoside C           Podophyllotoxin           Proscillaridin A                      
 
         [0131]                              TABLE 6                       15 compounds that affect both NB12 and FS90:                                    Disulfiram           Mitoxantrone dihydrochloride           Anisomycin           Cephaeline dihydrochloride heptahydrate           Digitoxigenin           Digoxin           Strophantine octahydrate           Puromycin dihydrochloride           Daunorubicin hydrochloride           Emetine dihydrochloride           Methyl benzethonium chloride           Strophanthidin           Cycloheximide           Thonzonium bromide           Sanguinarine                        
 Study 3 Screen: 
 
 Methods: 
 
         [0132]     The results from the LOPAC and Prestwick screens were confirmed using FS90, FS105, and NB12. Thirty-six (36) compounds were confirmed that specifically affect NB12 and 29 compounds that affect FS90/105 and NB12. Thirty-two (32) compounds were selected for IC 50  determinations using FS90, FS105, and NB12. IC 50  for the remaining compounds of interest will be tested at a later date (in combination with hits from additional libraries).  
         [0133]     Results:  
                         TABLE 7                       36 compounds that specifically affect NB12                                    (S)-(+)-Camptothecin           2.3-Dimethoxy-1.4-naphthoquinone           Ancitabine hydrochloride           Antimycin A           Azaguanine-8           Benzethonium chloride           Camptothecine (S.+)           Chelerythrine chloride           Ciclopirox ethanolamine           Clofazimine           Colchicine           Colchicine           Cycloheximide           Cytosine-1-beta-D-arabinofuranoside hydrochloride           Dequalinium analog. C-14 linker           Dequalinium dichloride           Dequalinium dichloride           Digoxigenin           Diphenyleneiodonium chloride           DL-Stearoylcarnitine chloride           Doxorubicin hydrochloride           Etoposide           Etoposide           MG 624           Mycophenolic acid           Paclitaxel           Parthenolide           Podophyllotoxin           Podophyllotoxin           Primaquine diphosphate           Quinacrine dihydrochloride           Quinacrine dihydrochloride dihydrate           Scoulerine           Taxol           Vinblastine sulfate salt           Vincristine sulfate                      
 
         [0134]    
       
         
               
             
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                   
               
               
                 29 compounds that affect FS90/105 and NB12 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Alexidine dihydrochloride 
               
               
                   
                 Ammonium pyrrolidinedithiocarbamate 
               
               
                   
                 Amodiaquin dihydrochloride dihydrate 
               
               
                   
                 Anisomycin 
               
               
                   
                 Brefeldin A from  Penicillium brefeldianum   
               
               
                   
                 Calmidazolium chloride 
               
               
                   
                 Cephaeline dihydrochloride heptahydrate 
               
               
                   
                 CGP-74514A hydrochloride 
               
               
                   
                 Daunorubicin hydrochloride 
               
               
                   
                 Digitoxigenin 
               
               
                   
                 Digoxin 
               
               
                   
                 Dihydroouabain 
               
               
                   
                 Disulfiram 
               
               
                   
                 Emetine dihydrochloride 
               
               
                   
                 Emetine dihydrochloride hydrate 
               
               
                   
                 Idarubicin 
               
               
                   
                 Lanatoside C 
               
               
                   
                 Methyl benzethonium chloride 
               
               
                   
                 Mitoxantrone 
               
               
                   
                 Mitoxantrone dihydrochloride 
               
               
                   
                 Ouabain 
               
               
                   
                 Proscillaridin A 
               
               
                   
                 Puromycin dihydrochloride 
               
               
                   
                 Sanguinarine 
               
               
                   
                 Sanguinarine chloride 
               
               
                   
                 Strophanthidin 
               
               
                   
                 Strophantine octahydrate 
               
               
                   
                 Terfenadine 
               
               
                   
                 Thonzonium bromide 
               
               
                   
                   
               
             
          
         
       
     
         [0135]                              TABLE 9                       32 compounds selected for IC50 determinations       using FS90, FS105, and NB12:                                    (S)-(+)-Camptothecin           Ammonium pyrrolidinedithiocarbamate           Amodiaquin dihydrochloride dihydrate           Antimycin A           Avermectin B1           Azaguanine-8           Chelerythrine chloride           Clofazimine           Colchicine           Dequalinium analog, C-14 linker           Dequalinium dichloride (LOPAC compound)           Dequalinium dichloride (Prestwick compound)           Digoxin           Dihydroouabain           Diphenyleneiodonium chloride           DL-Stearoylcarnitine chloride           Etoposide           Idarubicin           Loratadine           MG 624           Myophenolic Acid           Paclitaxel           Parthenolide           Podophyllotoxin           Primaquine diphosphate           Quinacrine dihydrochloride           Sanguinarine chloride           Scoulerine           Strophanthidin           Terfenadine           Vinblastine sulfate salt           Vincristine sulfate                        
 Study 4—Screen Results at 5 μM: 
 
         [0136]     The Spectrum collection was screened using the same protocol. At 5 μM, the initial screen identified 35 hits that affect NB12 and FS90, no hits that specifically target FS90, and 41 hits that specifically target NB12. The screen was repeated at 5 μM and 1 μM using NB12 and FS90 to confirm these hits and identified 34 hits that affect NB12 and FS90, no hits that specifically target FS90, and 33 hits that specifically target NB12. Following the Spectrum confirmatory screen, IC 50  determinations for an additional 32 compounds were performed.  
                         TABLE 10                       34 hits that affect NB12 and FS90:                                    3-METHYLORSELLINIC ACID           5alpha-CHOLESTAN-3beta-OL-6-ONE           5-AZACYTIDINE           AKLAVINE HYDROCHLORIDE           CETRMONIUM BROMIDE           CHELIDONINE (+)           COLCHICEINE           COLCHICINE           CYTARABINE           DACTINOMYCIN           DEOXYSAPPANONE B 7.3′-DIMETHYL ETHER           DIGITOXIN           DIHYDROGAMBOGIC ACID           DISULFIRAM           EMETINE           GENTIAN VIOLET           JUGLONE           LANATOSIDE C           LYCORINE           MITOMYCIN C           OXYPHENBUTAZONE           PATULIN           PERIPLOCYMARIN           PERUVOSIDE           PHENYLMERCURIC ACETATE           PUROMYCIN HYDROCHLORIDE           PYRITHIONE ZINC           PYRROMYCIN           RETUSOQUINONE           SANGUINARINE SULFATE           SARMENTOGENIN           STROPHANTHIDIN           THIMEROSAL           TOMATINE                      
 
         [0137]    
       
         
               
             
               
               
             
           
               
                 TABLE 11 
               
               
                   
               
               
                   
               
               
                 33 hits that specifically target NB12: 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 10-HYDROXYCAMTOTHECIN 
               
               
                   
                 4′-DEMETHYLEPIPODOPHYLLOTOXIN 
               
               
                   
                 ANDROGRAPHOLIDE 
               
               
                   
                 AMODIAQUINE DIHYDROCHLORIDE 
               
               
                   
                 AMSACRINE HYDROCHLORIDE 
               
               
                   
                 ANCITABINE HYDROCHLORIDE 
               
               
                   
                 BENZALKONIUM CHLORIDE 
               
               
                   
                 BENZETHONIUM CHLORIDE 
               
               
                   
                 BEPRIDIL HYDROCHLORIDE 
               
               
                   
                 beta-PELTATIN 
               
               
                   
                 CAMPTOTHECIN 
               
               
                   
                 CETYLPYRIDINIUM CHLORIDE 
               
               
                   
                 CHOLESTAN-3beta.5alpha.6beta-TRIOL 
               
               
                   
                 CICLOPIROX OLAMINE 
               
               
                   
                 CONVALLATOXIN 
               
               
                   
                 CRASSIN ACETATE 
               
               
                   
                 CRINAMINE 
               
               
                   
                 DIGOXIN 
               
               
                   
                 ERYSOLIN 
               
               
                   
                 GAMBOGIC ACID 
               
               
                   
                 IMIDACLOPRIDE 
               
               
                   
                 LIMONIN 
               
               
                   
                 MECHLORETHAMINE 
               
               
                   
                 MECLIZINE HYDROCHLORIDE 
               
               
                   
                 OUABAIN 
               
               
                   
                 OXYBENDAZOLE 
               
               
                   
                 PACLITAXEL 
               
               
                   
                 PARAROSANILINE PAMOATE 
               
               
                   
                 PARTHENOLIDE 
               
               
                   
                 PODOPHYLLOTOXIN ACETATE 
               
               
                   
                 STROPHANTHIDINIC ACID LACTONE ACETATE 
               
               
                   
                 TENIPOSIDE 
               
               
                   
                 VINBLASTINE SULFATE 
               
               
                   
                   
               
             
          
         
       
     
         [0138]    
       
         
               
             
               
               
             
           
               
                 TABLE 12 
               
               
                   
               
               
                   
               
               
                 32 Compounds selected for IC50 Determinations: 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Aklavine hydrochloride 
               
               
                   
                 AMSACRINE HYDROCHLORIDE 
               
               
                   
                 ANCITABINE HYDROCHLORIDE 
               
               
                   
                 ANDROGRAPHOLIDE 
               
               
                   
                 BEPRIDIL HYDROCHLORIDE 
               
               
                   
                 beta-PELTATIN 
               
               
                   
                 CGP-74514A hydrochloride 
               
               
                   
                 CHOLESTAN-3beta.5alpha.6beta-TRIOL 
               
               
                   
                 CICLOPIROX OLAMINE 
               
               
                   
                 CONVALLATOXIN 
               
               
                   
                 CRASSIN ACETATE 
               
               
                   
                 CRINAMINE 
               
               
                   
                 DIHYDROGAMBOGIC ACID 
               
               
                   
                 ERYSOLIN 
               
               
                   
                 Gambogic Acid 
               
               
                   
                 IMIDACLOPRIDE 
               
               
                   
                 JUGLONE 
               
               
                   
                 LIMONIN 
               
               
                   
                 MECHLORETHAMINE 
               
               
                   
                 MECLIZINE HYDROCHLORIDE 
               
               
                   
                 Mitomycin C 
               
               
                   
                 Mitoxantrone hydrochloride 
               
               
                   
                 OUABAIN 
               
               
                   
                 OXYBENDAZOLE 
               
               
                   
                 PARAROSANILINE PAMOATE 
               
               
                   
                 PERIPLOCYMARIN 
               
               
                   
                 PERUVOSIDE 
               
               
                   
                 Prenyletin 
               
               
                   
                 PYRITHIONE ZINC 
               
               
                   
                 TENIPOSIDE 
               
               
                   
                 Tomatidine hydrochloride 
               
               
                   
                 TOMATINE 
               
               
                   
                   
               
             
          
         
       
     
         [0139]     These results suggest that patient-specific therapeutics, as well as the molecular and biochemical alterations that lead to neuroblastoma, can be identified using this assay.  
       Example 2—Identified Compounds that Affect Normal, NB, or NB and Normal Cells  
       [0140]     The present example provides a description of the screening method used to identify and select chemical entities capable of affecting (i.e., reducing and/or inhibiting) neuroblastoma cells. The screening method is used here with the LOPAC compound collection. (LOPAC library, Sigma).  
         [0141]     Candidate NB tumor-initiating cells (TICs) were isolated. These TICs were used in the screening assay for the identification of these kinds of compounds because they are related to SKPs (SKin-derived Precursors). For example, both SKPs and TICs originate from the neural crest, express similar neural crest markers, and differentiate in vitro into similar cell types. The availability of two neural crest stem cell sources, one from the NB tumor and the other from the skin of the same patient, affords an approach for the therapeutic target discovery provided here.  
         [0000]     Materials and Methods:  
         [0000]     Methods:  
         [0142]     To identify compounds that specifically target neuroblastoma TICs, a cell-based assay in which TICs from a NB patient and normal SKPs were tested in parallel. Cells were treated with test compound prior to incubation with a cell viability dye. For both cell sources, signal variability was low and the Z′ and Z factors were &gt;0.5, suggesting excellent assay quality. Hits were defined as compounds whose signals were shifted at least 3 standard deviations from the mean.  
         [0000]     Results:  
         [0000]     Compounds that Affect NB Cells and Normal Cells  
         [0143]     From 3 libraries of compounds, the LOPAC collection, the Prestwick Collection and the Spectrum Collection, 46 compounds were found to affect both normal and NB cells. These 46 compounds are listed in Table 13.  
                         TABLE 13                       Normal and Neuroblastoma Hits                                    3-METHYLORSELLINIC ACID           5alpha-CHOLESTAN-3beta-OL-6-ONE           5-AZACYTIDINE           AKLAVINE HYDROCHLORIDE           Alexidine dihydrochloride           Ammonium pyrrolidinedithiocarbamate           Anisomycin           Brefeldin A from  Penicillium brefeldianum             Calmidazolium chloride           Cephaeline dihydrochloride heptahydrate           CETRMONIUM BROMIDE           CHELIDONINE (+)           COLCHICEINE           DACTINOMYCIN           Daunorubicin hydrochloride           DEOXYSAPPANONE B 7.3′-DMETHYL ETHER           Digitoxigenin           Digoxin           DIHYDROGAMBOGIC ACID           Dihydroouabain           Disulfiram           EMETINE           GENTIAN VIOLET           JUGLONE           LANATOSIDE C           LYCORINE           Methyl benzethonium chloride           MITOMYCIN C           Mitoxantrone           OXYPHENBUTAZONE           PATULIN           PERIPLOCYMARIN           PERUVOSIDE           PHENYLMERCURIC ACETATE           Proscillaridin A           Puromycin dihydrochloride           PYRITHIONE ZINC           PYRROMYCIN           RETUSOQUINONE           Sanguinarine           SARMENTOGENIN           Strophanthidin           Terfenadine           THIMEROSAL           Thonzonium bromide           TOMATINE                      
 
 Table 13: 54 Identified Compounds that Affect NB Cells 
 
         [0144]     Fifty-four (54) compounds selected from the LOPAC collection, Prestwick Collection and the Spectrum Collection, were found to selectively target NB cells. These 56 compounds appear in Table 14.  
                         TABLE 14                       Neuroblastoma Specific Hits                                    10-HYDROXYCAMTOTHECIN           2.3-Dimethoxy-1.4-naphthoquinone           4′-DEMETHYLEPIPODOPHYLLOTOXIN           Amodiaquin dihydrochloride dihydrate           AMSACRINE HYDROCHLORIDE           Ancitabine hydrochloride           ANDROGRAPHOLIDE           Antimycin A           Azaguanine-8           BENZALKONIUM CHLORIDE           Benzethonium chloride           BEPRIDIL HYDROCHLORIDE           beta-PELTATIN           Camptothecin (S.+)           CETYLPYRIDINIUM CHLORIDE           CGP-74514A hydrochloride           Chelerythrine chloride           CHOLESTAN-3beta.5alpha.6beta-TRIOL           Ciclopirox ethanolamine           Clofazimine           CONVALLATOXIN           CRASSIN ACETATE           CRINAMINE           Cycloheximide           Cytosine-1-beta-D-arabinofuranoside hydrochloride           Dequalinium analog. C-14 linker           Dequalinium dichloride           Diphenyleneiodonium chloride           DL-Stearoylcarnitine chloride           Doxorubicin hydrochloride           ERYSOLIN           Etoposide           GAMBOGIC ACID           Idarubicin           IMIDACLOPRIDE           LIMONIN           Loratadine           MECHLORETHAMINE           MECLIZINE HYDROCHLORIDE           MG 624           Mycophenolic acid           Ouabain           OXYBENDAZOLE           Paclitaxel           PARAROSANILINE PAMOATE           Parthenolide           Podophyllotoxin           Primaquine diphosphate           Quinacrine dihydrochloride           Scoulerine           Taxol           TENIPOSIDE           Vinblastine sulfate salt           Vincristine sulfate                      
 
         [0145]     Four (4) compounds selected from the LOPAC collection, Prestwick Collection and the Spectrum Collection, were found to successfully treat a NB patient and were selected as NB specific hits according to the assay criteria provided herein. These compounds serve as positive controls in the selection and screening methods. These results emphasize the validity of the assay in identifying active agents for treating neuroblastoma. These 4 compounds are listed in Table 15.  
         [0000]     Table 15: 4 Identified Compounds that are Used to Treat the NB Patient  
         [0000]     Patient Hits (i.e. drugs that were used to treat patient AND were selected as NB specific hits)  
         [0000]     Ancitabine hydrochloride (aka cyclocytidine)  
         [0000]     Doxorubicin hydrochloride (aka adriamycin)  
         [0000]     Etoposide  
         [0000]     Vincristine sulfate  
         [0146]     These results suggest that patient-specific therapeutics as well as novel molecular effectors of neuroblastoma can be identified using this assay.  
       Example 3—Cumulative Screening Assay Selection Results  
       [0147]     The present example presents the tabulated data obtained with the various chemical library screens conducted.  
                                       TABLE 16                                   NB                               hit   NB + FS   IC50       Library   Name   Repeated   only   hit   test?   target/mechanism                   S     10-HYDROXYCAMTOTHECIN     X   X           modified camptothecin       L   2.3-Dimethoxy-1.4-naphthoquinone   X   X           ROS modulator/Redox                               cycling agent used to                               study role of ROS       S   3-METHYLORSELLINIC ACID   X       X         Aspergillus terreus                                 fungal metabolite;                               possible antioxidant       S   4′-   X   X           DEMETHYLEPIPODOPHYLLOTOXIN       S   5alpha-CHOLESTAN-3beta-OL-6-ONE   X   X           Cholesterol oxidation                               product; cytotoxic due to                               oxidative stress or                               cytoskeleton disruption       S   5-AZACYTIDINE   X       X       S     ACRIFLAVINIUM                     intercalating agent that             HYDROCHLORIDE                     interferes with DNA                               replic/transcription;                               antitumor,                               antiproliferative       S   ACRISORCIN                   topical anti-infective                               from 1960s       S   AKLAVINE HYDROCHLORIDE   X       X   X   natural product; anti-                               infective; related                               structures have broad                               activity against NIH                               tumor lines       P     Alexidine dihydrochloride     X       X       phospholipase inh; oral                               gingivitis rinse       S   ALEXIDINE HYDROCHLORIDE       P   Alprostadil                   vasodilator; erectile                               dysfunction, pallative                               care for neonatal                               congenital heart defects       L     Ammonium pyrrolidinedithiocarbamate     X       X   X   blocks NOS mRNA                               translation       P   !Amodiaquin dihydrochloride dihydrate   X   X       X   antimalarial; treatment of                               CNS degeneration                               (Alzheimer, MS)       S   !AMODIAQUINE   X   X           antimalarial; 4-           DIHYDROCHLORIDE                   aminoquinoline family;                               narrow therapeutic/toxic                               window in children       S   AMSACRINE HYDROCHLORIDE   X   X       X   topo II inh; used in                               AML; may also be active                               vs malaria       L     Ancitabine hydrochloride     X   X           cyclocytidine HCl;                               DNA-synthesis inhibitor                               (cytosine analog);                               antileukemic       S     ANCITABINE HYDROCHLORIDE     X   X       X       S     ANDROGRAPHOLIDE     X   X       X   Chinese herbal medicine;                               anti-inflamm; immune                               boosting?; anti-cancer vs                               HL60, MCF7, others                               through G0/G1 block                               and apoptosis induction       P     Anisomycin     X       X       protein synthesis inh thru                               peptidyl transferase of                               80S ribosome; treatment                               activates p54, MAPK,                               SAPK       P   #Antimycin A   X   X       X   antifungal, antimicrobial;                               blocks e- transport                               between cytochrome B                               and cytochrome C; bind                               the BH3 domain of Bcl-                               xL and induce apoptosis                               in cells overexpressing                               Bcl-2 and Bcl-xL       P   !Avermectin B1               X   antiworm/insecticide       P     Azaguanine-8     X   X       X   purine analog       S   BENZALKONIUM CHLORIDE   X   X           cationic detergent; v                               common antiseptic and                               preservative       P   +Benzethonium chloride   X   X           topical antimicrobial                               used in cosmetics as                               preservative       S   +BENZALKONIUM CHLORIDE   X   X       S   !BEPRIDIL HYDROCHLORIDE   X   X       X   nonselective Ca channel                               blocker used for                               treatment of chronic                               angina pectoris; alters                               potential dep and                               receptor-operated Ca                               channels and inhibits fast                               Na inward currents       S   beta-PELTATIN   X   X       X   extracted from Mayapple                               rhizome (like                               podophyllotoxin); some                               evidence of in vitro anti-                               tumor f/x but vague       L   Brefeldin A from  Penicillium     X       X       fungal metabolite that             brefeldianum                     disrupts Golgi structure                               and function       L     Calmidazolium chloride     X       X       Potent inhibitor of                               calmodulin activation of                               phosphodiesterase;                               strongly inhibits                               calmodulin-dependent                               Ca2+-ATPase       S   CAMPTOTHECIN   X   X       L     Camptothecin (S.+)     X   X           topo 1 inh       P     Camptothecine (S.+)     X   X       X       P   Cephaeline dihydrochloride heptahydrate   X       X       ipecac alkaloid       S   CETRIMONIUM BROMIDE   X       X       cationic detergent;                               quaternary ammonium                               compound used in hair                               conditioner and as a                               antimicrobial; tested as a                               lavage during colon                               resections . . . no benefit                               and potentially toxic       S   CETYLPYRIDINIUM CHLORIDE   X   X           active ingredient in                               Scope; antiseptic used in                               oral rinses       L     CGP-74514A hydrochloride     X   X       X   Cdk1 inh       L     Chelerythrine chloride     X   X       X   PKC inhibitor; affects                               translocation of PKC                               from cytosol to plasma                               membrane       S     CHELIDONINE (+)     X   X           G2/M arrest associated                               with increased cycB1                               levels, cdc2 activity and                               SAPK/JNK activity;                               weak tubulin interaction;                               induced apoptosis at                               1 uM in Jurkat cells       S   CHOLESTAN-3beta.5alpha.6beta-   X   X       X   Cholesterol oxidation           TRIOL                   product; cytotoxic due to                               oxidative stress or                               cytoskeleton disruption       P   Ciclopirox ethanolamine   X   X           topical antifungal, anti-                               inflammatory       S   CICLOPIROX OLAMINE   X   X       X       P   Clofazimine   X   X       X   leprosy treatment; anti-                               inflammatory f/x;                               disrupts cc by binding                               DNA, may bind K+                               transporters       S     COLCHICEINE     X       X       metabolite of colchicine;                               less toxic to hepatocytes;                               less binding to tubulin                               but presumably has                               similar modeof action       L   Colchicine   X   X           binds tubulin/blocks                               mitosis by preventing                               spindle formation;                               bioactive doses would be                               toxic       P   Colchicine   X   X       X       S     COLCHICINE         S   #CONVALLATOXIN   X   X       X   derived from lily of the                               valley; digitalis-like                               action       S   CRASSIN ACETATE   X   X       X   antineoplastic vs P388                               leukemia and HT29                               colon cancer cells in                               vitro; extracted from                               marine invertebrates       S   CRINAMINE   X   X       X       P     Cycloheximide     X   X           protein synthesis inh       S     CYCLOHEXIMIDE         S   CYMARIN           X       S     CYTARABINE     X               Ara-C; DNA damage, S-                               phase block; inh DNA/                               RNA pol       L     Cytosine-1-beta-D-arabinofuranoside     X   X           Ara-C; selective inh of             hydrochloride                     DNA synthesis       S   DACTINOMYCIN   X       X       P     Daunorubicin hydrochloride     X       X       DNA intercalator;                               neuroblastoma treatment       S   DEOXYSAPPANONE B 7.3′-   X       X       flavanoid derived from           DIMETHYL ETHER                     Caesalpinia sappan  tree;                               Chinese med treatment                               for tumor, diarrhea;                               aldose reductase                               inhibitor?; one study                               suggesting activity vs                               head and neck cancer                               cell line       L     Dequalinium analog. C-14 linker     X   X       X   Protein kinase C-alpha                               (PKC-alpha) inhibitor       P   !Dequalinium dichloride   X   X       X   Selective blocker of                               apamin-sensitive K+                               channels       L   !Dequalinium dichloride   X   X       X   Member of delocalized                               lipophilic cations                               (DLCs), a family of                               compounds that                               accumulate in                               mitochondria driven by                               the negative                               transmembrane potential;                               inhibitor of NADH-                               ubiquinone reductase; A                               novel mitochondria                               delivery system is based                               on dequalinium. This                               DLC forms liposome-                               like aggregates termed                               ‘DQAsomes’. DQAsomes                               are being tested as                               mitochondria drug                               delivery systems for                               small molecules such as                               paclitaxel       P   #Digitoxigenin   X       X       Digitalis derivative;                               blocks Na+/K+ pump       S   #DIGITOXIN       P   #Digoxigenin   X   X           Digitalis derivative;                               blocks Na+/K+ pump       P   #Digoxin   X       X   X   Digitalis derivative;                               blocks Na+/K+ pump       S   #DIGOXIN   X       X       S   DIHYDROGAMBOGIC ACID   X       X   X       L   !Dihydroouabain   X       X   X   Na+/K+ pump inhibitor       L     Diphenyleneiodonium chloride     X   X       X   eNOS inh (endothelial                               NOS)       S   DISULFIRAM   X   X       P   Disulfiram   X       X       antabuse, rxn with                               alcohol use       L     DL-Stearoylcarnitine chloride     X   X       X   PKC inh       P     Doxorubicin hydrochloride     X   X           DNA synthesis inh;                               stabilizes topo II                               complex after strand                               cleavage       S     EMETINE     X       X       P     Emetine dihydrochloride     X       X       ipecac alkaloid; inh                               protein synthesis by                               blocking Rb movement                               on mRNA; inhibit DNA                               replication in S phase       L     Emetine dihydrochloride hydrate     X       X       Apoptosis inducer;                               RNA-Protein translation                               inhibitor       S   ERYSOLIN   X   X       X   organic isothiocyanate                               found in cruciferous                               veggies; increases                               accumulation of chemo                               drugs in PANC-1, MCF-                               7, NCI-H460 cell lines       P     Etoposide     X   X       X   topo II inh       L     Etoposide     X   X       P   Fosfosal                   salicylic acid derivative/                               anti-inflammatory       S     GAMBOGIC ACID     X   X       X   principle pigment of                               gambage resin (bright                               orange); caspase                               activator (not well                               characterized); growth/                               tumor inhibitory vs                               HeLa, HEL, gastic                               cancer, lung carcinoma                               cell lines       S   GENTIAN VIOLET   X       X       L     Idarubicin     X   X       X   antineoplastic, DNA                               metabolism       S   !IMIDACLOPRIDE   X   X       X   a4b2 nAChR agonist;                               activates ERK pathway;                               insecticide       S   JUGLONE   X       X   X   Pin1 inh; alkylates                               thioredoxin reductase;                               PI3K inh?; inhibits                               growth of HCT-15,                               HeLa, HL60 cell lines       P   Kaempferol                   antioxidant/flavenoid       P   #Lanatoside C   X       X       Digitalis derivative;                               blocks Na+/K+ pump       S   #LANATOSIDE C   X       X       S   LIMONIN   X   X       X   isolated from citrus fruit                               seeds; inhibits HIV1                               protease activity;                               antinociceptive, inhibits                               MCF7 growth but not                               other cancer cell lines       L   *Loratadine   X   X       X   H1 Histamine R                               antagonist       S   LYCORINE   X       X       P   +Mebendazole                   anthelmintic; blocks                               glucose/nutrient uptake                               in adult worm intestine;                               reported to be a mitotic                               spindle poison (resulting                               in chromosomal                               nondisjunction)       S     MECHLORETHAMINE     X   X       X   mustard gas derivative;                               polyfunctional alkylating                               agent = DNA breaks and                               crosslinks; non cc phase                               specific       S   *MECLIZINE HYDROCHLORIDE   X   X       X   antivert/bonine; motion                               sickness/vertigo                               treatment; piperazine                               class of antihistamines       L     Melphalan                     Antineoplastic; forms                               DNA intrastrand                               crosslinks by                               bifunctional alkylation in                               5′-GGC sequences; used                               in NB megatherapy       P   Menadione                   vitamin K3 (vitK2                               precursor); reacts with -                               SH/soaks up GSH = high                               ROS = altered Ca2+ = Ca-                               dep DNA fragmentation;                               toxic at high doses so                               vitK2 currently being                               used in cancer trials       P   +Methiazole                   anthelmintic       P   +Methyl benzethonium   X       X       topical antimicrobial       S   +METHYLBENZETHONIUM   X       X           CHLORIDE       L   !MG 624   X   X       X   Nicotinic acetylcholine                               receptor antagonist;                               selectively inhibits                               alpha-bungarotoxin                               sensitive receptors that                               contain the alpha7                               subunit       S   MITOMYCIN C   X       X   X       S     MITOXANTHRONE                 X             HYDROCHLORIDE         L     Mitoxantrone     X       X       topo II inh; used in ALL,                               breast cancer, non-                               hodgkin&#39;s lymphoma       P     Mitoxantrone dihydrochloride     X       X       P     Mycophenolic acid     X   X       X   immunosuppressant;                               blocks de novo purine                               biosynthesis       S   NERIIFOLIN       L   #Ouabain   X       X       Blocks movement of the                               H5 and H6                               transmembrane domains                               of Na+-K+ ATPases       S   #OUABAIN   X       X   X       S   +OXYBENDAZOLE   X   X       X   benzimidazole                               anthelmintic used in                               horses and other                               ruminants       S   OXYPHENBUTAZONE   X       X       Anti-inflammatory                               (Tandearil); binds                               phospholipase A2,                               human neutrophil                               elastase       P     Paclitaxel     X   X       X   taxol       S     PACLITAXEL     X   X           taxol       S   PARAROSANILINE PAMOATE   X       X   X       P     Parthenolide     X   X       X   feverfew extract; NFkB                               inh, p53 activ, increased                               ROS, JNK activ (indep                               of NFkB and ROS), inh                               of MAPK/ERK pathway       S     PARTHENOLIDE     X   X           seems to work best as a                               chemosensitizer . . . studies                               in breast, skin,                               pancreatic, thoracic cell                               lines       S   PATULIN   X       X       S   #PERIPLOCYMARIN   X       X   X   digoxin relative       S   #PERUVOSIDE   X       X   X   inhibitor of Na+K+-                               ATPase; cardiac                               glycoside class       S   PHENYLMERCURIC ACETATE   X       X       P     Podophyllotoxin     X   X       X   etoposide precursor/                               Antineoplastic glucoside;                               inhibitor of microtubule                               assembly; G2/M cc                               arrest       L     Podophyllotoxin     X   X       S     PODOPHYLLOTOXIN ACETATE     X       X       P   !Primaquine diphosphate   X   X       X   antimalarial/inh of                               DNA, RNA, protein                               synthesis/muscarinic                               AChR inh       P   #Proscillaridin A   X       X       Na+/K+ ATPase inh;                               digitalis related       P     Puromycin dihydrochloride     X       X       protein synthesis inh,                               premature strand                               termination       S     PUROMYCIN HYDROCHLORIDE     X       X       S   PYRITHIONE ZINC   X       X   X       S   PYRROMYCIN   X       X       anthracycline derivative;                               monosaccharide; induces                               erythroid diff in K562       P   +Pyrvinium pamoate                   pinworm treatment;                               prevents gluccose                               uptake; antitumor                               activity vs pancreatic cell                               line in SCID model, see                               decrease Akt phos       L   !Quinacrine dihydrochloride   X   X       X   Monoamine oxidase                               (MAO) inhibitor;                               antimalarial       P   !Quinacrine dihydrochloride dihydrate   X   X           Antimalarial, causes                               female sterility       S   RETUSOQUINONE   X       X       ?       P   !Sanguinarine   X       X       Inhibitor of Mg2+ and                               Na+/K+-ATPase;                               isolated from the leaves                               and stems of  Macleaya                                   cordata  and  microcarpa         L   !Sanguinarine chloride   X       X   X       S   !SANGUINARINE SULFATE   X   X       S   SARMENTOGENIN   X       X       P   !Scoulerine   X   X       X   opium intermediate/                               alkaloid; a1-                               adrenoreceptor inh (G-                               protein coupled R found                               on PNS sympathetic                               nerve terminals, CNS                               postsynaptically; target                               of catecholamines)       P   !Strophanthidin   X       X   X   blocks Na+/K+ ATPase                               at high conc; opposite f/x                               at low dose (Quabain)       S   !STROPHANTHIDIN   X       X       S   !STROPHANTHIDINIC ACID   X   X           !LACTONE ACETATE       P   !Strophantine octahydrate   X       X       L     Taxol     X   X           Antitumor agent;                               promotes assembly of                               microtubules and inhibits                               tubulin disassembly                               process       S     TENIPOSIDE     X   X       X   common NB treatment;                               semisynthetic                               podophyllotoxin                               derivative related to                               etoposide; topo II inh;                               induced single strand                               DNA breaks; activity in                               late S and G2 phases       P   *Terfenadine   X       X   X   nonsedating                               antihistimineoff market                               due to cardiac f/x       S   THIMEROSAL   X       X       S   THIRAM       P   Thonzonium bromide   X       X       cationic detergent       S   TOMATINE   X       X   X       P   Verteporfin                   photoreactive dye used                               in treatment of macular                               generation; anti-                               angiogenic       S     VINBLASTINE SULFATE     X   X       L     Vinblastine sulfate salt     X   X       X   Inhibitor of microtubule                               assembly       L     Vincristine sulfate     X   X       X   Inhibitor of microtubule                               assembly               132/151               repeated               (87%)                 BOLD: DNA damage/protein synthesis inhibitor/cell cycle block            italics: protein inhibitor/activator            *antihistamine            #digoxin derivative            +metabolic f/x            !ion channel inhibitor/neuro R inhibitor             
 
       Example 5—Selected Compounds of Interest  
       [0148]     The present example demonstrates the utility of the present invention for providing a composition suitable for the inhibition of neuroblastoma, and for the treatment of neuroblastoma.  
         [0149]     Forty-seven (47) compounds were selected based on differential cell toxicity and compound mechanism of action. Forty are novel compounds for the treatment of neuroblastoma. None of these 40 compounds have been used clinically in neuroblastoma therapy nor have they been examined in clinical trials. Seven compounds have been previously used for neuroblastoma treatment (marked with asterisk), and serve as positive controls in the selection and screening process of new chemical entities that may be used in the treatment of neuroblastoma according the present invention.  
                       TABLE 17                           NB12               IC50       Compounds of Interest:   (nM)   Notes:                   2.3-Dimethoxy-1.4-   nd   ROS modulator/Redox cycling agent used       naphthoquinone       to study role of ROS       AKLAVINE   778.5   natural product; anti-infective; related       HYDROCHLORIDE       structures have broad activity against NIH               tumor lines       Amodiaquin dihydrochloride   790   antimalarial; treatment of CNS degeneration       dihydrate       (Alzheimer, MS); 4-aminoquinoline family;               narrow therapeutic/toxic window in               children; 4-Aminoquinolines depress               cardiac muscle, impair cardiac conductivity,               and produce vasodilatation with resultant               hypotension       AMSACRINE   1214   topo II inh; used in AML; may also be active       HYDROCHLORIDE       vs malaria       *ANCITABINE   519.7   cyclocytidine HCl; DNA-synthesis inhibitor       HYDROCHLORIDE       (cytosine analog); antileukemic       Azaguanine-8   331   purine analog       beta-PELTATIN   1949   extracted from Mayapple rhizome (like               podophyllotoxin); some evidence of in vitro               anti-tumor f/x but vague       Camptothecine (S.+)   183.3   topoisomerase 1 inh       CGP-74514A hydrochloride       Cdk 1 inh       Chelerythrine chloride   2553   PKC inhibitor; affects translocation of PKC               from cytosol to plasma membrane       CHOLESTAN-   2410   Cholesterol oxidation product; cytotoxic due       3beta.5alpha.6beta-TRIOL       to oxidative stress or cytoskeleton disruption       CICLOPIROX OLAMINE   2048   topical antifungal, anti-inflammatory via               inhibition of 5-lipoxygenase and cyclo-               oxygenase; hydroxypyridone family; Loprox       Clofazimine   1417   leprosy treatment; anti-inflammatory f/x;               disrupts cc by binding DNA, may bind K+               transporters       Colchicine   29.3   binds tubulin/blocks mitosis by preventing               spindle formation; bioactive doses would               probably be toxic       CONVALLATOXIN   73.17   derived from lily of the valley; digitalis-like               action       CRASSIN ACETATE   1947   antineoplastic vs P388 leukemia and HT29               colon cancer cells in vitro; cembranolides               (14-member ring diterpenoid lactones)               derived from Caribbean gorgonians (marine               invertebrates)       CRINAMINE   1735   HIF-1alpha inhibitor; affinity to the               serotonin reuptake transport protein       Dequalinium analog. C-14 linker   1112   Protein kinase C-alpha (PKC-alpha)               inhibitor       Dequalinium dichloride   3617   Selective blocker of apamin-sensitive K+               channels; mitochondria toxicity       Digitoxin   nd   Na+/K+ pump inhibitor       Digoxigenin   nd   Na+/K+ pump inhibitor       Digoxin   542.2   Digitalis derivative; blocks Na+/K+ pump       DIHYDROGAMBOGIC ACID   1687       Dihydroouabain   1540   Na+/K+ pump inhibitor       ERYSOLIN   3276   organic isothiocyanate found in cruciferous               veggies; increases accumulation of chemo               drugs in PANC-1, MCF-7, NCI-H460 cell               lines       *Etoposide   693.7   topoisomerase II inh       GAMBOGIC ACID   1695   principle pigment of gambage resin (bright               orange); caspase activator (not well               characterized); growth/tumor inhibitory vs               HeLa, HEL, gastic cancer, lung carcinoma               cell lines       *Idarubicin   203.7   antineoplastic, DNA metabolism       MECHLORETHAMINE   438.2   mustard gas derivative; polyfunctional               alkylating agent = DNA breaks and               crosslinks; non cell cycle phase specific       MECLIZINE   2537   “antivert/bonine”; motion sickness/vertigo       HYDROCHLORIDE       treatment; piperazine class of antihistamines       MG 624   848   Nicotinic acetylcholine receptor antagonist;               selectively inhibits alpha-bungarotoxin               sensitive receptors that contain the alpha7               subunit       MITOXANTHRONE   60.46   topo II inh; used in ALL, breast cancer, non-       HYDROCHLORIDE       hodgkin&#39;s lymphoma       OUABAIN   122.6   Blocks movement of the H5 and H6               transmembrane domains of Na+-K+               ATPases       OXYBENDAZOLE   nd   benzimidazole anthelmintic used in horses               and other ruminants       Paclitaxel   nd   aka taxol; Antitumor agent; promotes               assembly of microtubules and inhibits               tubulin disassembly process       Parthenolide   2261   feverfew extract; NFkB inh, p53 activ,               increased ROS, JNK activ (indep of NFkB               and ROS), inh of MAPK/ERK pathway;               seems to work best as a               chemosensitizer . . . studies in breast, skin,               pancreatic, thoracic cell lines       PATULIN   nd   polyketide lactone, produced by certain               fungal species of  Penicillium ,  Aspergillus                 and  Byssochlamys  growing on fruit,               including apples, pears, grapes; crosslinks               DNA, causes p38 and JNK phosphorylation               in HEK cells       PERIPLOCYMARIN   2703   digoxin relative       PERUVOSIDE   222.5   inhibitor of Na+K+-ATPase; cardiac               glycoside class       *Podophyllotoxin   135   etoposide precursor/Antineoplastic               glucoside; inhibitor of microtubule               assembly; G2/M cc arrest       Primaquine diphosphate   nd   antimalarial/inh of DNA, RNA, protein               synthesis/muscarinic AChR inh       Quinacrine dihydrochloride   2556   Monoamine oxidase (MAO) inhibitor;               antimalarial       Sanguinarine chloride   1795   Inhibitor of Mg2+ and Na+/K+-ATPase;               isolated from the leaves and stems of                 Macleaya cordata  and  microcarpa         *TENIPOSIDE   705.5   common NB treatment; semisynthetic               podophyllotoxin derivative related to               etoposide; topo II inh; induced single strand               DNA breaks; activity in late S and G2               phases       TOMATINE   nd   alkaloid found in leaves of tomato and               unripe fruit; tetrasaccharide tomato               glycoalkaloid alpha-tomatine, trisaccharide               beta(1)-tomatine, disaccharide gamma-               tomatine, monosaccharide delta-tomatine,               and their common aglycon tomatidine;               inhibit the growth of human colon (HT29)               and liver (HepG2) cancer cells       *Vinblastine sulfate salt   113   Inhibitor of microtubule assembly       *Vincristine sulfate   61.95   Inhibitor of microtubule assembly                  
 
       Example 6—Reduced Cytotoxicity to Non-Neuroblastoma Cells  
       [0150]     The standard of care for poor prognosis neuroblastoma tumors is intensive induction chemotherapy with cisplatin, etoposide, cyclophosphamide, and doxorubicin, high-dose myeloablative therapy with bone marrow transplant, surgery, radiation therapy, and biologic or maintenance therapy to eradicate minimal residual disease. The chemotherapy regimen is designed to induce massive genomic damage and subsequent cell death in proliferating cells. This strategy results in the death of both tumor and normal cells and is extremely debilitating to young patients. Additionally, this strategy does not target the tumor-initiating cells (TICs). The survival of NB TICs may contribute to tumor relapse.  
         [0151]     The compounds identified in the present invention target cellular pathways specific to neuroblastoma tumor-initiating cells while having little or no effect on normal cells ( FIG. 5 ). Therefore, the compositions and pharmaceutical preparations of the present invention will provide a treatment method for neuroblastoma having fewer and/or less intense or pronounced toxic side effects in patients.  
         [0152]     All documents, patents, journal articles and other materials cited in the present application are hereby incorporated by reference.  
         [0153]     Although the present invention has been fully described in conjunction with several embodiments thereof with reference to the accompanying drawings, it is to be understood that various changes and modifications may be apparent to those skilled in the art. Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims, unless they depart therefrom.  
       BIBLIOGRAPHY  
       [0154]     The references listed below as well as the references cited throughout the specification are incorporated herein by reference to the extent that they supplement, explain, provide a background for or teach methodology, techniques and/or compositions employed herein. 
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