US20070123448A1 - Novel chemical entities affecting neuroblastoma tumor-initiating cells - Google Patents

Novel chemical entities affecting neuroblastoma tumor-initiating cells Download PDF

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US20070123448A1
US20070123448A1 US11/598,034 US59803406A US2007123448A1 US 20070123448 A1 US20070123448 A1 US 20070123448A1 US 59803406 A US59803406 A US 59803406A US 2007123448 A1 US2007123448 A1 US 2007123448A1
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hydrochloride
neuroblastoma
tumor
composition
cells
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David Kaplan
Kristen Smith
Alessandro Datti
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Hospital for Sick Children HSC
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Assigned to THE HOSPITAL FOR SICK CHILDREN reassignment THE HOSPITAL FOR SICK CHILDREN ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SMITH, KRISTEN M., DATTI, ALESSANDRO, KAPLAN, DAVID R.
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Priority to US12/206,963 priority patent/US20090036387A1/en
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
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    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
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    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
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    • A61P35/00—Antineoplastic agents
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • 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.
  • NB 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.
  • NB neural crest-derived sympathetic neuronal precursors
  • nerveroblasts neural crest-derived sympathetic neuronal precursors
  • NB tumors also frequently contain other neural crest cell types, including neuroendocrine and Schwann cells.
  • 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.
  • NB predisposition gene 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).
  • NCPs neural crest precursors
  • 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.
  • TIC tumor-initiating cells
  • tumor or cancer stem cells also called tumor or cancer stem cells
  • 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).
  • Tumor initiating cells from some solid tissue tumors have been described.
  • a tumor initiating cell population from tumor tissue in a patient with neuroblastoma has not been isolated.
  • stage 4S 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.
  • a population of tumor initiating cells from such solid tumor tissue has not yet been reported.
  • compositions/Pharmaceutical Preparations are Compositions/Pharmaceutical Preparations:
  • the invention provides compositions comprising novel chemical entities that are capable of affecting neuroblastoma.
  • 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.
  • 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.
  • the anti-neuroblastoma composition may be described as comprising one or more active ingredients comprising:
  • the chemical entities of the invention may be described as compounds that possess specific cytostatic or cytotoxic activity toward neuroblastoma tumor-initiating cells.
  • the neuroblastoma-inhibiting composition further comprises ancitabine hydrochloride, doxorubicin hydrochloride, etoposide, vincristine sulfate, or a combination thereof.
  • the neuroblastoma inhibiting composition may be further described as having reduced non-neuroblastoma tumor-initiating cell cytotoxicity.
  • 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.
  • the compositions are described as essentially free of non-neuroblastoma tumor cell inhibiting activity.
  • the invention provides methods for inhibiting neuroblastoma tumor-initiating cells.
  • the method comprises administering an effective amount of a composition comprising a neuroblastoma tumor-initiating cell inhibiting ingredient.
  • the neuroblastoma tumor-initiating cell inhibiting ingredient comprises one or more active ingredients comprising:
  • 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.
  • the method may further comprise administering a composition further comprising ancitabine hydrochloride, doxorubicin hydrochloride, etoposide, vincristine sulfate, or a combination thereof.
  • the composition may further include a pharmaceutically acceptable carrier solution.
  • the neuroblastoma tumor-inhibiting cells are in an animal having neuroblastoma.
  • the animal is a human.
  • 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.
  • the composition employed in the method is essentially free of non-neuroblastoma tumor cell inhibiting activity.
  • 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.
  • FIG. 1 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 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).
  • Y axis presents the % Control Alamar Blue Signal).
  • FIG. 3A-3C 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).
  • 3C presents in a pie-format the classification of primary hits by mechanism of action.
  • FIG. 4A-4E 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 ⁇ M to 9 nM). Representative graphs are shown in FIG. 4A (Complete Response), 4B (Partial Response), and 4C (Threshold Effect).
  • FIG. 5A-5C 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 - 6 FF 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 ⁇ M 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 - 7 FF 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 ⁇ M 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.
  • 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 neuroblasto
  • enriched is defined as containing at least 50% of the identified biological moiety, such as a cancer stem cell.
  • NB TIC neuroblastoma tumor initiating cell
  • NB TIC neuroblastoma tumor initiating cell
  • a tumor 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).
  • 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.
  • 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.
  • 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.
  • NB Malignant neuroblastoma
  • TICs NB tumor-initiating cells
  • Both SKPs and TICs originate from the neural crest, express similar neural crest markers, and differentiate in vitro into similar cell types.
  • NB tumor-initiating cells 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 Prestwick library was screened at 5 ⁇ M using FS90 and NB12 and at 1 ⁇ M using NB12 only due to the high number of “hits” at 5 ⁇ M. This screen identified 9 compounds that selectively target NB12 and 15 compounds that affect both NB12 and FS90.
  • 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
  • the Spectrum collection was screened using the same protocol. At 5 ⁇ M, 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 ⁇ M and 1 ⁇ M 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.
  • 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).
  • TICs Candidate NB tumor-initiating cells
  • 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.
  • TICs 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.
  • 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
  • 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
  • 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,
  • DLCs delocalized lipophilic cations
  • 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
  • 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.
  • 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.
  • 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.

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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
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Claims (20)

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.
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