Novel chemical entities affecting neuroblastoma tumor-initiating cells

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

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

1. Field of the Invention

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.

2. Related Art

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.

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.

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.

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.

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.

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.

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).

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.

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).

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's tumors, since developing tissues contain a higher proportion of tissue stem cells than do adult tissues.

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.

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

The above and other long-felt needs in the art are met in the present invention.

Compositions/Pharmaceutical Preparations:

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.

In some embodiments, the anti-neuroblastoma composition may be described as comprising one or more active ingredients comprising:

2.3-Dimethoxy-1.4-naphthoquinone,

Aklavine Hydrochloride,

Amodiaquin dihydrochloride dehydrate;

Amsacrine 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;

Dihydrogambogic acid;

Dihydroouabain;

Erysolin;

Gambogic acid;

Mechlorethamine;

Meclizine hydrochloride;

MG 624;

Mitoxanthrone Hydrochloride;

Ouabain;

Oxybendazole;

Oxybendazole;

Paclitaxel;

Parthenolide;

Patulin;

Periplocymarin;

Peru voside;

Primaquine diphosphate;

Quinacrine dihydrochloride;

Sanguinarine chloride; or

Tomatine,

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.

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.

Methods of Inhibiting Neuroblastoma Tumor Initiating Cells/Methods of Treating and/or Inhibiting Neuroblastoma in an Animal

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:

2.3-Dimethoxy-1.4-naphthoquinone,

Aklavine Hydrochloride,

Amodiaquin dihydrochloride dehydrate;

Amsacrine 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;

Dihydrogambogic acid;

Dihydroouabain;

Erysolin;

Gambogic acid;

Mechlorethamine;

Meclizine hydrochloride;

MG 624;

Mitoxanthrone Hydrochloride;

Ouabain;

Oxybendazole;

Oxybendazole;

Paclitaxel;

Parthenolide;

Patulin;

Periplocymarin;

Peruvoside;

Primaquine diphosphate;

Quinacrine dihydrochloride;

Sanguinarine chloride; or

Tomatine.

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.

In some embodiments, the composition may further include a pharmaceutically acceptable carrier solution.

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.

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.

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

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.

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).

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).

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).

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.

FIG. 6A-6FF, according to one embodiment of the invention, presents IC₅₀ 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.

FIG. 7A-7FF, 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

Definitions

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.

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.

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).

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.

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).

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.

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.

Description

EXAMPLES

Example I Materials and Methods

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.

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.

Study 1 Screen:

Materials and Methods:

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 >10 fold different).

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 >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.

Results:

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

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

TABLE 4
4 compounds selectively target normal cells:
8-Methoxymethyl-3-isobutyl-1-methylxanthine
Oligomycin A
Sphingosine
Thapsigargin

Study 2 Screen:
Materials and Methods

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.

Results:

TABLE 5
9 compounds that selectively target NB12:
Azaguanine-8
Paclitaxel
Camptothecine (S.+)
Colchicine
Etoposide
Doxorubicin hydrochloride
Lanatoside C
Podophyllotoxin
Proscillaridin A

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:

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 IC₅₀ determinations using FS90, FS105, and NB12. IC₅₀ for the remaining compounds of interest will be tested at a later date (in combination with hits from additional libraries).

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

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

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:

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, IC₅₀ 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

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

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

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

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).

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.

Materials and Methods:

Methods:

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 >0.5, suggesting excellent assay quality. Hits were defined as compounds whose signals were shifted at least 3 standard deviations from the mean.

Results:

Compounds that Affect NB Cells and Normal Cells

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

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

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.

Table 15: 4 Identified Compounds that are Used to Treat the NB Patient

Patient Hits (i.e. drugs that were used to treat patient AND were selected as NB specific hits)

Ancitabine hydrochloride (aka cyclocytidine)

Doxorubicin hydrochloride (aka adriamycin)

Etoposide

Vincristine sulfate

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

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'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

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.

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'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

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.

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.

All documents, patents, journal articles and other materials cited in the present application are hereby incorporated by reference.

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

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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Claims

1. A neuroblastoma inhibiting composition comprising a chemical entity that selectively affects neuroblastoma tumor-initiating cells, said composition comprising one or more active ingredients comprising:

2.3-Dimethoxy-1.4-naphthoquinone,

Aklavine Hydrochloride,

Amodiaquin dihydrochloride dehydrate;

Amsacrine 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;

Dihydrogambogic acid;

Dihydroouabain;

Erysolin;

Gambogic acid;

Mechlorethamine;

Meclizine hydrochloride;

MG 624;

Mitoxanthrone Hydrochloride;

Ouabain;

Oxybendazole;

Oxybendazole;

Paclitaxel;

Parthenolide;

Patulin;

Periplocymarin;

Peruvoside;

Primaquine diphosphate;

Quinacrine dihydrochloride;

Sanguinarine chloride; or

Tomatine.

2. The neuroblastoma-inhibiting composition of claim 1 further comprising ancitabine hydrochloride, doxorubicin hydrochloride, etoposide, vincristine sulfate, or a combination thereof.

3. The neuroblastoma inhibiting composition of claim 1 further defined as having reduced non-neuroblastoma tumor-initiating cell cytotoxicity.

4. The neuroblastoma inhibiting composition of claim 1 further defined as essentially free of non-neuroblastoma tumor cell inhibiting activity.

5. A pharmaceutical formulation for the inhibition of neuroblastoma comprising an effective amount of a neuroblastoma tumor-initiating cell inhibiting composition, said composition comprising one or more active ingredients comprising:

2.3-Dimethoxy-1.4-naphthoquinone,

Aklavine Hydrochloride,

Amodiaquin dihydrochloride dehydrate;

Amsacrine 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;

Dihydrogambogic acid;

Dihydroouabain;

Erysolin;

Gambogic acid;

Mechlorethamine;

Meclizine hydrochloride;

MG 624;

Mitoxanthrone Hydrochloride;

Ouabain;

Oxybendazole;

Oxybendazole;

Paclitaxel;

Parthenolide;

Patulin;

Periplocymarin;

Peruvoside;

Primaquine diphosphate;

Quinacrine dihydrochloride;

Sanguinarine chloride; or

Tomatine.

6. The pharmaceutical formulation of claim 5 further comprising ancitabine hydrochloride, doxorubicin hydrochloride, etoposide, vincristine sulfate, or a combination thereof.

7. The pharmaceutical preparation of claim 6 further comprising a pharmaceutically acceptable carrier solution.

8. A method for inhibiting neuroblastoma tumor-initiating cells comprising administering an effective amount of a composition comprising a neuroblastoma tumor-initiating cell inhibiting ingredient.

9. The method of claim 8 wherein said neuroblastoma tumor-initiating cell inhibiting ingredient comprises one or more active ingredients comprising:

2.3-Dimethoxy-1.4-naphthoquinone,

Aklavine Hydrochloride,

Amodiaquin dihydrochloride dehydrate;

Amsacrine 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;

Dihydrogambogic acid;

Dihydroouabain;

Erysolin;

Gambogic acid;

Mechlorethamine;

Meclizine hydrochloride;

MG 624;

Mitoxanthrone Hydrochloride;

Ouabain;

Oxybendazole;

Oxybendazole;

Paclitaxel;

Parthenolide;

Patulin;

Periplocymarin;

Peruvoside;

Primaquine diphosphate;

Quinacrine dihydrochloride;

Sanguinarine chloride; or

Tomatine.

10. The method of claim 8 wherein the effective amount of the neuroblastoma tumor initiating cell inhibiting ingredient is an amount effective to arrest growth of neuroblastoma tumor-initiating cells.

11. The method of claim 8 wherein the composition further comprises ancitabine hydrochloride, doxorubicin hydrochloride, etoposide, vincristine sulfate, or a combination thereof.

12. The method of claim 8 wherein the neuroblastoma tumor-initiating cells are in an animal having neuroblastoma.

13. The method of claim 8 wherein the composition has a reduced non-neuroblastoma tumor-initiating cell cytotoxicity.

14. The method of claim 8 wherein the composition is essentially free of non-neuroblastoma tumor cell inhibiting activity.

15. A method for inhibiting neuroblastoma in an animal comprising administering an effective amount of a composition comprising a neuroblastoma tumor-initiating cell inhibiting ingredient.

16. The method of claim 15 wherein said neuroblastoma tumor-initiating cell inhibiting ingredient comprises one or active ingredients comprising:

2.3-Dimethoxy-1.4-naphthoquinone,

Aklavine Hydrochloride,

Amodiaquin dihydrochloride dehydrate;

Amsacrine 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;

Dihydrogambogic acid;

Dihydroouabain;

Erysolin;

Gambogic acid;

Mechlorethamine;

Meclizine hydrochloride;

MG 624;

Mitoxanthrone Hydrochloride;

Ouabain;

Oxybendazole;

Oxybendazole;

Paclitaxel;

Parthenolide;

Patulin;

Periplocymarin;

Peruvoside;

Primaquine diphosphate;

Quinacrine dihydrochloride;

Sanguinarine chloride; or

Tomatine.

17. The method of claim 15 wherein the composition further comprises ancitabine hydrochloride, doxorubicin hydrochloride, etoposide, vincristine sulfate, or a combination thereof.

18. The method of claim 15 wherein the animal is a human of 12 years of age or younger.

19. The method of claim 15 wherein the composition is essentially free of non-neuroblastoma tumor-initiating cell inhibiting activity.

20. The method of claim 15 wherein the effective amount of the composition is further described as an amount effective to arrest growth of neuroblastoma tumor-initiating cells.