U.S. patent application number 11/598034 was filed with the patent office on 2007-05-31 for novel chemical entities affecting neuroblastoma tumor-initiating cells.
This patent application is currently assigned to The Hospital For Sick Children. Invention is credited to Alessandro Datti, David R. Kaplan, Kristen M. Smith.
Application Number | 20070123448 11/598034 |
Document ID | / |
Family ID | 38229028 |
Filed Date | 2007-05-31 |
United States Patent
Application |
20070123448 |
Kind Code |
A1 |
Kaplan; David R. ; et
al. |
May 31, 2007 |
Novel chemical entities affecting neuroblastoma tumor-initiating
cells
Abstract
Disclosed are neuroblastoma tumor-initiating cell inhibiting
compositions comprising chemical entities capable of affecting
neuroblastoma tumor-initiating cells. Pharmaceutical preparations
that include these chemical entities are also provided for the
treatment of neuroblastoma. These pharmaceutical preparations are
suitable for the treatment of humans, and are particularly suited
for the treatment of children of 12 years of age or younger having
neuroblastoma. The compositions and pharmaceutical preparations
posses reduced normal cell cytotoxicity. The compositions and
pharmaceutical preparations may be used alone or together with
other conventional neuroblastoma preparations as part of a clinical
regimen in the treatment and management of neuroblastoma.
Inventors: |
Kaplan; David R.; (Toronto,
CA) ; Smith; Kristen M.; (Toronto, CA) ;
Datti; Alessandro; (Toronto, CA) |
Correspondence
Address: |
JAGTIANI + GUTTAG
10363-A DEMOCRACY LANE
FAIRFAX
VA
22030
US
|
Assignee: |
The Hospital For Sick
Children
Toronto
ON
M5G 1X8
|
Family ID: |
38229028 |
Appl. No.: |
11/598034 |
Filed: |
November 13, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60739337 |
Nov 25, 2005 |
|
|
|
Current U.S.
Class: |
514/26 ; 514/171;
514/19.3; 514/243; 514/261.1; 514/27; 514/283; 514/332; 514/410;
514/449; 514/624; 514/682 |
Current CPC
Class: |
A61K 31/522 20130101;
G01N 33/5058 20130101; A61K 31/53 20130101; A61K 31/704 20130101;
A61K 31/4745 20130101; A61K 31/7048 20130101; C12N 2503/02
20130101; C12N 5/0695 20130101; A61K 31/56 20130101; A61K 31/444
20130101; A61K 31/519 20130101; G01N 33/5011 20130101; A61P 35/00
20180101 |
Class at
Publication: |
514/002 ;
514/026; 514/027; 514/449; 514/332; 514/171; 514/283; 514/410;
514/261.1; 514/243; 514/624; 514/682 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 31/7048 20060101 A61K031/7048; A61K 31/704
20060101 A61K031/704; A61K 31/522 20060101 A61K031/522; A61K 31/53
20060101 A61K031/53; A61K 31/56 20060101 A61K031/56; A61K 31/519
20060101 A61K031/519; A61K 31/4745 20060101 A61K031/4745; A61K
31/444 20060101 A61K031/444 |
Claims
1. A neuroblastoma inhibiting composition comprising a chemical
entity that selectively affects neuroblastoma tumor-initiating
cells, said composition comprising one or more active ingredients
comprising: 2.3-Dimethoxy-1.4-naphthoquinone, Aklavine
Hydrochloride, Amodiaquin dihydrochloride dehydrate; Amsacrine
Hydrochloride; Azaguanine-8; beta-peltatin; Camptothecine (S.+);
CGP-74514A hydrochloride; Chelerythrine chloride;
Cholestan-3beta.5alpha.6beta-Triol; Ciclopirox Olamine;
Clofazimine; Colchicine; Convallatoxin; Crassin Acetate; Crinamine;
Dequalinium analog. C-14 linker; Dequalinium dichloride; Digitoxin;
Digoxigenin; Dihydrogambogic acid; Dihydroouabain; Erysolin;
Gambogic acid; Mechlorethamine; Meclizine hydrochloride; MG 624;
Mitoxanthrone Hydrochloride; Ouabain; Oxybendazole; Oxybendazole;
Paclitaxel; Parthenolide; Patulin; Periplocymarin; Peruvoside;
Primaquine diphosphate; Quinacrine dihydrochloride; Sanguinarine
chloride; or Tomatine.
2. The neuroblastoma-inhibiting composition of claim 1 further
comprising ancitabine hydrochloride, doxorubicin hydrochloride,
etoposide, vincristine sulfate, or a combination thereof.
3. The neuroblastoma inhibiting composition of claim 1 further
defined as having reduced non-neuroblastoma tumor-initiating cell
cytotoxicity.
4. The neuroblastoma inhibiting composition of claim 1 further
defined as essentially free of non-neuroblastoma tumor cell
inhibiting activity.
5. A pharmaceutical formulation for the inhibition of neuroblastoma
comprising an effective amount of a neuroblastoma tumor-initiating
cell inhibiting composition, said composition comprising one or
more active ingredients comprising:
2.3-Dimethoxy-1.4-naphthoquinone, Aklavine Hydrochloride,
Amodiaquin dihydrochloride dehydrate; Amsacrine Hydrochloride;
Azaguanine-8; beta-peltatin; Camptothecine (S.+); CGP-74514A
hydrochloride; Chelerythrine chloride;
Cholestan-3beta.5alpha.6beta-Triol; Ciclopirox Olamine;
Clofazimine; Colchicine; Convallatoxin; Crassin Acetate; Crinamine;
Dequalinium analog. C-14 linker; Dequalinium dichloride; Digitoxin;
Digoxigenin; Dihydrogambogic acid; Dihydroouabain; Erysolin;
Gambogic acid; Mechlorethamine; Meclizine hydrochloride; MG 624;
Mitoxanthrone Hydrochloride; Ouabain; Oxybendazole; Oxybendazole;
Paclitaxel; Parthenolide; Patulin; Periplocymarin; Peruvoside;
Primaquine diphosphate; Quinacrine dihydrochloride; Sanguinarine
chloride; or Tomatine.
6. The pharmaceutical formulation of claim 5 further comprising
ancitabine hydrochloride, doxorubicin hydrochloride, etoposide,
vincristine sulfate, or a combination thereof.
7. The pharmaceutical preparation of claim 6 further comprising a
pharmaceutically acceptable carrier solution.
8. A method for inhibiting neuroblastoma tumor-initiating cells
comprising administering an effective amount of a composition
comprising a neuroblastoma tumor-initiating cell inhibiting
ingredient.
9. The method of claim 8 wherein said neuroblastoma
tumor-initiating cell inhibiting ingredient comprises one or more
active ingredients comprising: 2.3-Dimethoxy-1.4-naphthoquinone,
Aklavine Hydrochloride, Amodiaquin dihydrochloride dehydrate;
Amsacrine Hydrochloride; Azaguanine-8; beta-peltatin; Camptothecine
(S.+); CGP-74514A hydrochloride; Chelerythrine chloride;
Cholestan-3beta.5alpha.6beta-Triol; Ciclopirox Olamine;
Clofazimine; Colchicine; Convallatoxin; Crassin Acetate; Crinamine;
Dequalinium analog. C-14 linker; Dequalinium dichloride; Digitoxin;
Digoxigenin; Dihydrogambogic acid; Dihydroouabain; Erysolin;
Gambogic acid; Mechlorethamine; Meclizine hydrochloride; MG 624;
Mitoxanthrone Hydrochloride; Ouabain; Oxybendazole; Oxybendazole;
Paclitaxel; Parthenolide; Patulin; Periplocymarin; Peruvoside;
Primaquine diphosphate; Quinacrine dihydrochloride; Sanguinarine
chloride; or Tomatine.
10. The method of claim 8 wherein the effective amount of the
neuroblastoma tumor initiating cell inhibiting ingredient is an
amount effective to arrest growth of neuroblastoma tumor-initiating
cells.
11. The method of claim 8 wherein the composition further comprises
ancitabine hydrochloride, doxorubicin hydrochloride, etoposide,
vincristine sulfate, or a combination thereof.
12. The method of claim 8 wherein the neuroblastoma
tumor-initiating cells are in an animal having neuroblastoma.
13. The method of claim 8 wherein the composition has a reduced
non-neuroblastoma tumor-initiating cell cytotoxicity.
14. The method of claim 8 wherein the composition is essentially
free of non-neuroblastoma tumor cell inhibiting activity.
15. A method for inhibiting neuroblastoma in an animal comprising
administering an effective amount of a composition comprising a
neuroblastoma tumor-initiating cell inhibiting ingredient.
16. The method of claim 15 wherein said neuroblastoma
tumor-initiating cell inhibiting ingredient comprises one or active
ingredients comprising: 2.3-Dimethoxy-1.4-naphthoquinone, Aklavine
Hydrochloride, Amodiaquin dihydrochloride dehydrate; Amsacrine
Hydrochloride; Azaguanine-8; beta-peltatin; Camptothecine (S.+);
CGP-74514A hydrochloride; Chelerythrine chloride;
Cholestan-3beta.5alpha.6beta-Triol; Ciclopirox Olamine;
Clofazimine; Colchicine; Convallatoxin; Crassin Acetate; Crinamine;
Dequalinium analog. C-14 linker; Dequalinium dichloride; Digitoxin;
Digoxigenin; Dihydrogambogic acid; Dihydroouabain; Erysolin;
Gambogic acid; Mechlorethamine; Meclizine hydrochloride; MG 624;
Mitoxanthrone Hydrochloride; Ouabain; Oxybendazole; Oxybendazole;
Paclitaxel; Parthenolide; Patulin; Periplocymarin; Peruvoside;
Primaquine diphosphate; Quinacrine dihydrochloride; Sanguinarine
chloride; or Tomatine.
17. The method of claim 15 wherein the composition further
comprises ancitabine hydrochloride, doxorubicin hydrochloride,
etoposide, vincristine sulfate, or a combination thereof.
18. The method of claim 15 wherein the animal is a human of 12
years of age or younger.
19. The method of claim 15 wherein the composition is essentially
free of non-neuroblastoma tumor-initiating cell inhibiting
activity.
20. The method of claim 15 wherein the effective amount of the
composition is further described as an amount effective to arrest
growth of neuroblastoma tumor-initiating cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application makes reference to the following co-pending
U.S. patent application. The application is U.S. App. No.
60/739,337, entitled "Cancer Stem Cells and Uses Thereof," filed
Nov. 23, 2005. The entire disclosure and contents of the above
application is hereby incorporated by reference.
BACKGROUND
[0002] 1. Field of the Invention
[0003] The present invention relates to the field of
pharmacologically active chemical compositions useful in affecting
neuroblastoma tumor-initiating cells, and the use of such
compositions in the treatment of neuroblastoma and related
conditions.
[0004] 2. Related Art
[0005] Neuroblastoma (NB) is the most common extracranial solid
tumor in children, with poor survival rates in children with
metastatic disease. NB is estimated to be responsible for about 15%
of cancer-related deaths in children (1,2). The survival rate for
metastatic NB is estimated to be less than 30%. In the majority of
these cases, conventional cancer therapies have been
ineffective.
[0006] Little is reported concerning the precise molecular
alterations that give rise to NB, its cell of origin, or why NB
cells metastasize and become resistant to chemotherapeutic agents.
Unfortunately, genetic mutations that contribute to the origin and
progression of 98% of NB cases have not been identified.
[0007] One identifiable hallmark of NB is the appearance of
proliferating cells with characteristics of neural crest-derived
sympathetic neuronal precursors (neuroblasts). NB tumors also
frequently contain other neural crest cell types, including
neuroendocrine and Schwann cells. Moreover, NB appears in tissues
that developmentally derive from the neural crest including
sympathoadrenal precursors which differentiate into both
sympathetic neurons and adrenal chromaffin cells, the paravertebral
and preaortic sympathetic ganglia, and the adrenal gland.
[0008] The clinical behavior of NB is unique. Tumors that arise in
children under one year of age may spontaneously regress by
differentiation or apoptosis, even after arising in or
metastasizing to liver and skin. In contrast, NB tumors in children
over one-year-old often grow aggressively, disseminate to the bone
and bone marrow, and are fatal in the vast majority of cases.
[0009] Mass screening of infants showed that NB is much more
frequent than previously thought. Many of these tumors regress
without clinical diagnosis. Regressing or favorable-prognosis
tumors have been reported to express high levels of the TrkA/NGF
receptor and display phenotypes of differentiated peripheral neural
cells, while malignant or unfavorable-prognosis tumors resemble
proliferating sympathoadrenal precursors, often expressing TrkB,
amplified N-myc, and many genes involved in neural crest
development.
[0010] The only reported germline NB predisposition gene is Phox2b,
which is mutated in many familial cases of NB, and is required for
proper differentiation of sympathetic neurons from neural crest
precursors (NCPs) (3,4). In the regressive form of the disease, the
transformed precursors ultimately differentiate or die, while in
older children, these molecular transformations instead result in a
population of persistently proliferating and highly migratory
transformed neuroblasts.
[0011] The concept of tumor-initiating cells (TIC) (also called
tumor or cancer stem cells) postulates that only rare cells in
tumors are endowed with tumorigenic potential, and was initially
developed to explain why (i) most tumors are comprised of both
undifferentiated proliferating progenitors and post mitotic
differentiated cells, (ii) only a very small fraction of tumor
cells form colonies after plating in vitro, and (iii) large numbers
of tumor cells are required to seed the growth of a new tumor in
mice (4-10).
[0012] Dick et al. and others reported that clonally-derived tumor
cells of acute myelogenous leukemia (AML) patients could be
physically separated into tumorigenic and non-tumorigenic fractions
(11,12). Brain and breast tumors have also been reported to contain
a subpopulation of TICs (13,14). Thus, in solid tumors, a rare
tumor cell population may fuel tumor growth and seed metastasis.
This hypothesis has major implications for treating cancer
patients. For example, many current therapies kill the bulk of
proliferating tumor cells, but these cells may not be intrinsically
tumorigenic, and in many cases the TICs may escape the effects of
the therapeutic agents, leading to tumor relapse. Thus, it is
essential to identify and characterize TICs from various tumors in
order to develop and target therapies against this critical cell
type.
[0013] TICs have also been shown to share phenotypic
characteristics with stem cells derived from their tissue of
origin. For example, for a given tissue, the tissue stem cells and
TICs both (i) self-renew, (ii) express common phenotypic markers,
(iii) grow in a similar fashion in response to mitogens, and (iv)
yield tissue-appropriate progeny (13,14). However, whereas tissue
stem cells generate mature differentiated cell types,
differentiation of TICs is generally arrested at the level of one
or more tissue progenitor cells resulting in tumors comprising a
hierarchy of progenitors and some differentiated progeny (4).
[0014] Many pediatric and adult tissues contain resident stem cells
(4). It is currently unknown if TICs originate by transformation of
tissue stem cells. Observations have been made that oncogenic
mutations commonly affect genes required for normal stem cell
renewal and differentiation (4). This may be particularly relevant
for children's tumors, since developing tissues contain a higher
proportion of tissue stem cells than do adult tissues.
[0015] Tumor initiating cells from some solid tissue tumors, such
as breast and brain tumors, have been described. However, a tumor
initiating cell population from tumor tissue in a patient with
neuroblastoma has not been isolated. One reported observation in
some infantile forms of NB (called stage 4S) is that large tumors
are frequently found in skin (15). It was previously assumed that
skin was a preferred metastatic target for NB. However, a
population of tumor initiating cells from such solid tumor tissue
has not yet been reported.
[0016] The above and other observations in the field reveal a
continuing medical need continues to exist in the art to determine
why and in which cell type NB arises, and why some neuroblastoma
tumors spontaneously regress and others are fatal. In addition, new
effective drug targets and therapeutics tailored to identifying and
treating specific forms and stages of neuroblastoma are needed.
SUMMARY
[0017] The above and other long-felt needs in the art are met in
the present invention.
Compositions/Pharmaceutical Preparations:
[0018] In one aspect, the invention provides compositions
comprising novel chemical entities that are capable of affecting
neuroblastoma. In some embodiments, these chemical entities may be
described as compounds that specifically kill neuroblastoma
tumor-initiating cells, or that arrest the growth of neuroblastoma
tumor-initiating cells. In other aspects, these chemical entities
and compositions containing one or more of them may be described as
having specifically cytostatic or cytotoxic activity toward
neuroblastoma tumor-initiating cells.
[0019] In some embodiments, the anti-neuroblastoma composition may
be described as comprising one or more active ingredients
comprising:
[0020] 2.3-Dimethoxy-1.4-naphthoquinone,
[0021] Aklavine Hydrochloride,
[0022] Amodiaquin dihydrochloride dehydrate;
[0023] Amsacrine Hydrochloride;
[0024] Azaguanine-8;
[0025] beta-peltatin;
[0026] Camptothecine (S.+);
[0027] CGP-74514A hydrochloride;
[0028] Chelerythrine chloride;
[0029] Cholestan-3beta.5alpha.6beta-Triol;
[0030] Ciclopirox Olamine;
[0031] Clofazimine;
[0032] Colchicine;
[0033] Convallatoxin;
[0034] Crassin Acetate;
[0035] Crinamine;
[0036] Dequalinium analog. C-14 linker;
[0037] Dequalinium dichloride;
[0038] Digitoxin;
[0039] Digoxigenin;
[0040] Dihydrogambogic acid;
[0041] Dihydroouabain;
[0042] Erysolin;
[0043] Gambogic acid;
[0044] Mechlorethamine;
[0045] Meclizine hydrochloride;
[0046] MG 624;
[0047] Mitoxanthrone Hydrochloride;
[0048] Ouabain;
[0049] Oxybendazole;
[0050] Oxybendazole;
[0051] Paclitaxel;
[0052] Parthenolide;
[0053] Patulin;
[0054] Periplocymarin;
[0055] Peru voside;
[0056] Primaquine diphosphate;
[0057] Quinacrine dihydrochloride;
[0058] Sanguinarine chloride; or
[0059] Tomatine,
[0060] In some embodiments, the chemical entities of the invention
may be described as compounds that possess specific cytostatic or
cytotoxic activity toward neuroblastoma tumor-initiating cells. In
other embodiments, the neuroblastoma-inhibiting composition further
comprises ancitabine hydrochloride, doxorubicin hydrochloride,
etoposide, vincristine sulfate, or a combination thereof. In yet
other embodiments, the neuroblastoma inhibiting composition may be
further described as having reduced non-neuroblastoma
tumor-initiating cell cytotoxicity.
[0061] In some embodiments, the chemical entities may be described
as possessing potent anti-neuroblastoma tumor-initiating cell
activity, and a reduced cytotoxicity to normal, non-neuroblastoma
tumor-initiating cells, relative to conventional neuroblastoma
treatment preparations. In some embodiments, the compositions are
described as essentially free of non-neuroblastoma tumor cell
inhibiting activity.
Methods of Inhibiting Neuroblastoma Tumor Initiating Cells/Methods
of Treating and/or Inhibiting Neuroblastoma in an Animal
[0062] In yet another aspect, the invention provides methods for
inhibiting neuroblastoma tumor-initiating cells. In some
embodiments, the method comprises administering an effective amount
of a composition comprising a neuroblastoma tumor-initiating cell
inhibiting ingredient. In some embodiments, the neuroblastoma
tumor-initiating cell inhibiting ingredient comprises one or more
active ingredients comprising:
[0063] 2.3-Dimethoxy-1.4-naphthoquinone,
[0064] Aklavine Hydrochloride,
[0065] Amodiaquin dihydrochloride dehydrate;
[0066] Amsacrine Hydrochloride;
[0067] Azaguanine-8;
[0068] beta-peltatin;
[0069] Camptothecine (S.+);
[0070] CGP-74514A hydrochloride;
[0071] Chelerythrine chloride;
[0072] Cholestan-3beta.5alpha.6beta-Triol;
[0073] Ciclopirox Olamine;
[0074] Clofazimine;
[0075] Colchicine;
[0076] Convallatoxin;
[0077] Crassin Acetate;
[0078] Crinamine;
[0079] Dequalinium analog. C-14 linker;
[0080] Dequalinium dichloride;
[0081] Digitoxin;
[0082] Digoxigenin;
[0083] Dihydrogambogic acid;
[0084] Dihydroouabain;
[0085] Erysolin;
[0086] Gambogic acid;
[0087] Mechlorethamine;
[0088] Meclizine hydrochloride;
[0089] MG 624;
[0090] Mitoxanthrone Hydrochloride;
[0091] Ouabain;
[0092] Oxybendazole;
[0093] Oxybendazole;
[0094] Paclitaxel;
[0095] Parthenolide;
[0096] Patulin;
[0097] Periplocymarin;
[0098] Peruvoside;
[0099] Primaquine diphosphate;
[0100] Quinacrine dihydrochloride;
[0101] Sanguinarine chloride; or
[0102] Tomatine.
[0103] In some embodiments, the effective amount of the
neuroblastoma tumor initiating cell inhibiting ingredient is an
amount effective to arrest the growth of and/or kill neuroblastoma
tumor-initiating cells, or effective to induce differentiation of
said cells to cell types that no longer proliferate. In other
embodiments, the method may further comprise administering a
composition further comprising ancitabine hydrochloride,
doxorubicin hydrochloride, etoposide, vincristine sulfate, or a
combination thereof.
[0104] In some embodiments, the composition may further include a
pharmaceutically acceptable carrier solution.
[0105] In yet other embodiments, the neuroblastoma tumor-inhibiting
cells are in an animal having neuroblastoma. In some embodiments,
the animal is a human. In some embodiments, the human is 12 years
of age or younger. That is, it is anticipated that the invention is
particularly useful in the treatment of children afflicted with
neuroblastoma, and will have a profound effect on reducing the high
rate of mortality in this population of neuroblastoma patients.
[0106] The method may be further described as administering a
composition that has a reduced non-neuroblastoma tumor-initiating
cell cytotoxicity. It is expected that the methods and compositions
of the present invention will provide fewer and/or less pronounce
undesirable side affect in the treatment of a patient as a result.
In some embodiments, the composition employed in the method is
essentially free of non-neuroblastoma tumor cell inhibiting
activity.
[0107] The compositions may be described as comprising a mixture of
any or all of the compounds listed below: TABLE-US-00001 TABLE 1
2.3-Dimethoxy-1.4-naphthoquinone AKLAVINE HYDROCHLORIDE Amodiaquin
dihydrochloride dihydrate AMSACRINE HYDROCHLORIDE ANCITABINE
HYDROCHLORIDE Azaguanine-8 beta-PELTATIN Camptothecine (S.+)
CGP-74514A hydrochloride Chelerythrine chloride
CHOLESTAN-3beta.5alpha.6beta-TRIOL CICLOPIROX OLAMINE Clofazimine
Colchicine CONVALLATOXIN CRASSIN ACETATE CRINAMINE Dequalinium
analog. C-14 linker Dequalinium dichloride Digitoxin Digoxigenin
Digoxin DIHYDROGAMBOGIC ACID Dihydroouabain ERYSOLIN Etoposide
GAMBOGIC ACID Idarubicin MECHLORETHAMINE MECLIZINE HYDROCHLORIDE MG
624 MITOXANTHRONE HYDROCHLORIDE OUABAIN OXYBENDAZOLE Paclitaxel
Parthenolide PATULIN PERIPLOCYMARIN PERUVOSIDE Podophyllotoxin
Primaquine diphosphate Quinacrine dihydrochloride Sanguinarine
chloride TENIPOSIDE TOMATINE Vinblastine sulfate salt Vincristine
sulfate
BRIEF DESCRIPTION OF THE DRAWINGS
[0108] FIG. 1, according to one embodiment of the invention,
presents a diagrammatic flow chart demonstrating the design of the
high throughput, dual-cell (Normal or Tumor cells) screening assay
employed in the selection of candidate test compounds that target
neuroblastoma tumor-initiating cells. Normal or tumor-initiating
cell spheres are dissociated; 3,000 single cells/well are plated in
96 well plates; candidate test compound is added; cell
proliferation assayed by Alamar Blue signal. Blue/nonfluorescent
compound is converted to a red/fluorescent compound under reducing
conditions such as those produced by live cells. The magnitude of
the fluorescent signal is proportional to the metabolic activity of
the cell sample.
[0109] FIG. 2, according to one embodiment of the invention,
presents the results from a study wherein FS90 (normal human SKPs,
passage 3) cells were treated with the LOPAC library of chemical
compounds. Alamar Blue was added after 30 hours and fluorescence
intensity read after an additional 24 hours. The hit cutoff is
indicated in the graph by the thick line across the graph at the Y
axis value of about 69.00% Control Alamar Blue Signal (which
corresponds to 3 standard deviations from the mean of all test
samples). Nine compounds whose Alamar Blue signals fall below this
line were identified as primary hits in this study. (X axis
presents the Compound ID number (n=80.times.8 plates); Y axis
presents the % Control Alamar Blue Signal).
[0110] FIG. 3A-3C, according to one embodiment of the invention,
presents the study results from primary screens of the chemical
libraries examined. 3A presents the results of the primary screen
in Venn diagram form. The Venn diagrams depict the primary hits
from each library. Compounds in the gray-bordered circles (left
circle) affected the tumor-initiating cells, while compounds in the
black-bordered circles (right circle) affected normal cells.
Compounds that affected both cell types lie in the overlap region.
Note that there is some compound redundancy between the libraries.
3B presents the confirmed primary hits in Venn diagram form.
Primary hits were retested against NB12, FS90 and FS105 (normal
human SKPs). 87% of the primary hits were confirmed in this step,
yielding 54 unique compounds that target tumor-initiating cells, 4
unique compounds that target normal cells, and 46 compounds that
have activity against both normal and tumor cells (overlap region).
3C presents in a pie-format the classification of primary hits by
mechanism of action. (Solid light gray area=DNA damaging
agents/cell cycle inhibitors; Solid dark gray area=Na+/K+ ATPase
inhibitors; Diagonal striped area=Neuronal receptor effectors;
Vertical striped area=Other; Solid white area=Metabolic inhibitor;
Checkerboard area=Neuronal channel effectors; Dotted area=Specific
protein effectors).
[0111] FIG. 4A-4E, according to one embodiment of the invention,
presents the IC50 values that were determined for the 64 selected
candidate compounds. Compounds were chosen for further testing
based on differential cell type selectivity, mechanism of action,
and pharmacological interest. Tumor-initiating cells and normal
cells were treated with 10 serial dilutions of compounds (5 .mu.M
to 9 nM). Representative graphs are shown in FIG. 4A (Complete
Response), 4B (Partial Response), and 4C (Threshold Effect).
Compounds that affected the tumor-initiating cells at a much lower
dose than normal cells (4D graph, left) or compounds that had a
greater effect on the tumor-initiating cells than normal cells (4E,
right graph), were selected for secondary in vitro screens in
addition to those compounds that only affected tumor-initiating
cells. (FS90=normal cells; NB12=tumor-initiating cells).
[0112] FIG. 5A-5C, according to one embodiment of the invention,
presents the results from secondary screens of the candidate
compounds. Compounds of interest are being tested against
additional normal primary cultures (FS89, FS105), a stage 1V
neuroblastoma primary culture (NB25), and a neuroblastoma cell line
(KCNR) using a sphere formation assay. 5A presents a flow diagram
of the secondary in vitro screen. The candidate compound is added
at 0 days and at 3 days. Spheres are counted at 7 days. FIG. 5B
presents a dose response curve of various cell lines (FS89, FS90,
FS105, NB12, NB25 and KCNR) to amsacrine. FIG. 5C presents a dose
response curve of various cell lines (FS89, FS90, FS105, NB12, NB25
and KCNR) to MG624.
[0113] FIG. 6A-6FF, according to one embodiment of the invention,
presents IC.sub.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 .mu.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.
[0114] 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 .mu.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.
DETAILED DESCRIPTION
Definitions
[0115] Where the definition of terms departs from the commonly used
meaning of the term, applicant intends to utilize the definitions
provided below, unless specifically indicated.
[0116] For the purposes of the present invention, "a", "an" and
"the" include reference to the plural unless the context as herein
presented clearly indicates other wise.
[0117] For purposes of the present invention, the term "active
agent" is defined as a chemical entity, group of chemical entities
or compound that is capable of providing an affect on neuroblastoma
tumor initiating cells or neuroblastoma cells in vitro or in vivo.
The affect of the active agent may be a reduction in cytotoxicity
relative to the level of cytotoxicity demonstrated in the absence
of the active agent under similar conditions, or a cytostatic
affect on neuroblastoma or on neuroblastoma tumor initiating cells
that results in a reduced rate of neuroblastoma or neuroblastoma
tumor-initiating cell proliferation and/or growth, or a reduction
of the rate or occurrence of differentiation into neuroblastoma
cell types, precursors, or any other cell type that is related to
the progression of a neuroblastoma pathology, or to an increase in
the inducement of the differentiation of neuroblastoma
tumor-initiating cells into cell types (for example, neurons) that
no longer proliferate (for example, retinoic acid is a common
differentiation therapy for neuroblastoma that is used as an
adjunct therapy after removal of a tumor, differentiation
therapy).
[0118] For purposes of the present invention, the term "enriched"
is defined as containing at least 50% of the identified biological
moiety, such as a cancer stem cell.
[0119] For purposes of the present invention, the term
"neuroblastoma tumor initiating cell" (NB TIC) is defined as a cell
that is capable of giving rise to neuroblastoma or a tumor cell
that is identifiable with a condition of neuroblastoma, such as a
tumor cell that may be identified to have particular identifiable
cell surface markers associated with neuroblastoma (such as NB84,
CD44, TrkA, GD2, CD34, p75NTR, and/or versican) and/or is without
cell surface markers that are characteristic of tumor cells that
are not from neuroblastoma (such as CD133, TrkB, and/or CD31).
[0120] For purposes of the present invention, the term
"neuroblastoma tumor-initiating cell inhibiting activity" is
defined as an activity for affecting neuroblastoma tumor-initiating
cell survival, proliferation, or that promotes cell differentiation
into benign cell types.
[0121] For purposes of the present invention, the term, "effective
amount" is defined as an amount of a compound that will inhibit
and/or reduce neuroblastoma tumor initiating cell survival,
proliferation, or that will promote the differentiation of
neuroblastoma tumor-initiating cells into benign cell types.
Description
EXAMPLES
Example I Materials and Methods
[0122] The present example provides a description of the screening
method used to identify the chemical entities capable of affecting
neuroblastoma cells reported in the present series of studies.
[0123] Malignant neuroblastoma (NB) is the most common
extra-cranial solid tumor in children. Survival of patients older
than 1 year remains less than 30% with conventional therapies.
Candidate NB tumor-initiating cells (TICs) were isolated, and it
was hypothesized that TICs are related to SKPs (SKin-derived
Precursors). Both SKPs and TICs originate from the neural crest,
express similar neural crest markers, and differentiate in vitro
into similar cell types. The availability of two neural crest stem
cell sources, one from the NB tumor and the other from the skin of
the same patient, affords us a unique opportunity for therapeutic
target discovery.
Study 1 Screen:
Materials and Methods:
[0124] To identify compounds that suppress the growth and survival
of neuroblastoma (NB) tumor-initiating cells and not nontransformed
normal cells (SKPs), a cell-based assay was established and used in
which NB tumor-initiating cells from a multiple relapse NB patient
(NB12, passage 6-17) and normal SKPs (FS90, passage 2-5) were
tested in parallel to detect specific alterations of cell
viability/proliferation. For each cell type, cells were passaged 5
days prior to screening. Three thousand (3000) cells in 100
.quadrature.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).
[0125] The robustness of the screen was initially evaluated by
using a collection of 1280 bioactive compounds (LOPAC library,
Sigma). For both normal SKPs and NB tumor-initiating cells,
variability of signals was low, with CV values ranging between
3.5-4.5% across the plates, and the dimensionless, statistical
parameters Z' and Z factors were >0.5, suggesting an excellent
assay quality. "Hits" were defined as the compounds whose signals
were shifted away by at least 3.times. standard deviations (99.73%
confidence interval) from the mean of the general sample
population.
Results:
[0126] The screen of the LOPAC library at 5 .mu.M 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-US-00002 TABLE 2
13 compounds that affect both normal and NB cells: Ancitabine
hydrochloride Brefeldin A from Penicillium brefeldianum
Calmidazolium chloride CGP-74514A hydrochloride Dihydroouabain
Diphenyleneiodonium chloride Emetine dihydrochloride hydrate
Idarubicin Mitoxantrone Ouabain Quinacrine dihydrochloride Ammonium
pyrrolidinedithiocarbamate Sanguinarine chloride
[0127] TABLE-US-00003 TABLE 3 18 compounds that selectively target
NB cells. Loratadine MG 624 Melphalan Podophyllotoxin Ro 25-6981
hydrochloride Rotenone DL-Stearoylcarnitine chloride Taxol
Vincristine sulfate Vinblastine sulfate salt Chelerythrine chloride
Colchicine Cytosine-1-beta-D-arabinofuranoside hydrochloride
Dequalinium dichloride (S)-(+)-Camptothecin Dequalinium analog,
C-14 linker 2,3-Dimethoxy-1,4-naphthoquinone Etoposide
[0128] TABLE-US-00004 TABLE 4 4 compounds selectively target normal
cells: 8-Methoxymethyl-3-isobutyl-1-methylxanthine Oligomycin A
Sphingosine Thapsigargin
Study 2 Screen: Materials and Methods
[0129] The Prestwick library was screened at 5 .mu.M using FS90 and
NB12 and at 1 .mu.M using NB12 only due to the high number of
"hits" at 5 .mu.M. This screen identified 9 compounds that
selectively target NB12 and 15 compounds that affect both NB12 and
FS90.
[0130] Results: TABLE-US-00005 TABLE 5 9 compounds that selectively
target NB12: Azaguanine-8 Paclitaxel Camptothecine (S.+) Colchicine
Etoposide Doxorubicin hydrochloride Lanatoside C Podophyllotoxin
Proscillaridin A
[0131] TABLE-US-00006 TABLE 6 15 compounds that affect both NB12
and FS90: Disulfiram Mitoxantrone dihydrochloride Anisomycin
Cephaeline dihydrochloride heptahydrate Digitoxigenin Digoxin
Strophantine octahydrate Puromycin dihydrochloride Daunorubicin
hydrochloride Emetine dihydrochloride Methyl benzethonium chloride
Strophanthidin Cycloheximide Thonzonium bromide Sanguinarine
Study 3 Screen: Methods:
[0132] The results from the LOPAC and Prestwick screens were
confirmed using FS90, FS105, and NB12. Thirty-six (36) compounds
were confirmed that specifically affect NB12 and 29 compounds that
affect FS90/105 and NB12. Thirty-two (32) compounds were selected
for IC.sub.50 determinations using FS90, FS105, and NB12. IC.sub.50
for the remaining compounds of interest will be tested at a later
date (in combination with hits from additional libraries).
[0133] Results: TABLE-US-00007 TABLE 7 36 compounds that
specifically affect NB12 (S)-(+)-Camptothecin
2.3-Dimethoxy-1.4-naphthoquinone Ancitabine hydrochloride Antimycin
A Azaguanine-8 Benzethonium chloride Camptothecine (S.+)
Chelerythrine chloride Ciclopirox ethanolamine Clofazimine
Colchicine Colchicine Cycloheximide
Cytosine-1-beta-D-arabinofuranoside hydrochloride Dequalinium
analog. C-14 linker Dequalinium dichloride Dequalinium dichloride
Digoxigenin Diphenyleneiodonium chloride DL-Stearoylcarnitine
chloride Doxorubicin hydrochloride Etoposide Etoposide MG 624
Mycophenolic acid Paclitaxel Parthenolide Podophyllotoxin
Podophyllotoxin Primaquine diphosphate Quinacrine dihydrochloride
Quinacrine dihydrochloride dihydrate Scoulerine Taxol Vinblastine
sulfate salt Vincristine sulfate
[0134] TABLE-US-00008 TABLE 8 29 compounds that affect FS90/105 and
NB12 Alexidine dihydrochloride Ammonium pyrrolidinedithiocarbamate
Amodiaquin dihydrochloride dihydrate Anisomycin Brefeldin A from
Penicillium brefeldianum Calmidazolium chloride Cephaeline
dihydrochloride heptahydrate CGP-74514A hydrochloride Daunorubicin
hydrochloride Digitoxigenin Digoxin Dihydroouabain Disulfiram
Emetine dihydrochloride Emetine dihydrochloride hydrate Idarubicin
Lanatoside C Methyl benzethonium chloride Mitoxantrone Mitoxantrone
dihydrochloride Ouabain Proscillaridin A Puromycin dihydrochloride
Sanguinarine Sanguinarine chloride Strophanthidin Strophantine
octahydrate Terfenadine Thonzonium bromide
[0135] TABLE-US-00009 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 .mu.M:
[0136] The Spectrum collection was screened using the same
protocol. At 5 .mu.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
.mu.M and 1 .mu.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.sub.50
determinations for an additional 32 compounds were performed.
TABLE-US-00010 TABLE 10 34 hits that affect NB12 and FS90:
3-METHYLORSELLINIC ACID 5alpha-CHOLESTAN-3beta-OL-6-ONE
5-AZACYTIDINE AKLAVINE HYDROCHLORIDE CETRMONIUM BROMIDE CHELIDONINE
(+) COLCHICEINE COLCHICINE CYTARABINE DACTINOMYCIN DEOXYSAPPANONE B
7.3'-DIMETHYL ETHER DIGITOXIN DIHYDROGAMBOGIC ACID DISULFIRAM
EMETINE GENTIAN VIOLET JUGLONE LANATOSIDE C LYCORINE MITOMYCIN C
OXYPHENBUTAZONE PATULIN PERIPLOCYMARIN PERUVOSIDE PHENYLMERCURIC
ACETATE PUROMYCIN HYDROCHLORIDE PYRITHIONE ZINC PYRROMYCIN
RETUSOQUINONE SANGUINARINE SULFATE SARMENTOGENIN STROPHANTHIDIN
THIMEROSAL TOMATINE
[0137] TABLE-US-00011 TABLE 11 33 hits that specifically target
NB12: 10-HYDROXYCAMTOTHECIN 4'-DEMETHYLEPIPODOPHYLLOTOXIN
ANDROGRAPHOLIDE AMODIAQUINE DIHYDROCHLORIDE AMSACRINE HYDROCHLORIDE
ANCITABINE HYDROCHLORIDE BENZALKONIUM CHLORIDE BENZETHONIUM
CHLORIDE BEPRIDIL HYDROCHLORIDE beta-PELTATIN CAMPTOTHECIN
CETYLPYRIDINIUM CHLORIDE CHOLESTAN-3beta.5alpha.6beta-TRIOL
CICLOPIROX OLAMINE CONVALLATOXIN CRASSIN ACETATE CRINAMINE DIGOXIN
ERYSOLIN GAMBOGIC ACID IMIDACLOPRIDE LIMONIN MECHLORETHAMINE
MECLIZINE HYDROCHLORIDE OUABAIN OXYBENDAZOLE PACLITAXEL
PARAROSANILINE PAMOATE PARTHENOLIDE PODOPHYLLOTOXIN ACETATE
STROPHANTHIDINIC ACID LACTONE ACETATE TENIPOSIDE VINBLASTINE
SULFATE
[0138] TABLE-US-00012 TABLE 12 32 Compounds selected for IC50
Determinations: Aklavine hydrochloride AMSACRINE HYDROCHLORIDE
ANCITABINE HYDROCHLORIDE ANDROGRAPHOLIDE BEPRIDIL HYDROCHLORIDE
beta-PELTATIN CGP-74514A hydrochloride
CHOLESTAN-3beta.5alpha.6beta-TRIOL CICLOPIROX OLAMINE CONVALLATOXIN
CRASSIN ACETATE CRINAMINE DIHYDROGAMBOGIC ACID ERYSOLIN Gambogic
Acid IMIDACLOPRIDE JUGLONE LIMONIN MECHLORETHAMINE MECLIZINE
HYDROCHLORIDE Mitomycin C Mitoxantrone hydrochloride OUABAIN
OXYBENDAZOLE PARAROSANILINE PAMOATE PERIPLOCYMARIN PERUVOSIDE
Prenyletin PYRITHIONE ZINC TENIPOSIDE Tomatidine hydrochloride
TOMATINE
[0139] These results suggest that patient-specific therapeutics, as
well as the molecular and biochemical alterations that lead to
neuroblastoma, can be identified using this assay.
Example 2--Identified Compounds that Affect Normal, NB, or NB and
Normal Cells
[0140] The present example provides a description of the screening
method used to identify and select chemical entities capable of
affecting (i.e., reducing and/or inhibiting) neuroblastoma cells.
The screening method is used here with the LOPAC compound
collection. (LOPAC library, Sigma).
[0141] Candidate NB tumor-initiating cells (TICs) were isolated.
These TICs were used in the screening assay for the identification
of these kinds of compounds because they are related to SKPs
(SKin-derived Precursors). For example, both SKPs and TICs
originate from the neural crest, express similar neural crest
markers, and differentiate in vitro into similar cell types. The
availability of two neural crest stem cell sources, one from the NB
tumor and the other from the skin of the same patient, affords an
approach for the therapeutic target discovery provided here.
Materials and Methods:
Methods:
[0142] To identify compounds that specifically target neuroblastoma
TICs, a cell-based assay in which TICs from a NB patient and normal
SKPs were tested in parallel. Cells were treated with test compound
prior to incubation with a cell viability dye. For both cell
sources, signal variability was low and the Z' and Z factors were
>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
[0143] From 3 libraries of compounds, the LOPAC collection, the
Prestwick Collection and the Spectrum Collection, 46 compounds were
found to affect both normal and NB cells. These 46 compounds are
listed in Table 13. TABLE-US-00013 TABLE 13 Normal and
Neuroblastoma Hits 3-METHYLORSELLINIC ACID
5alpha-CHOLESTAN-3beta-OL-6-ONE 5-AZACYTIDINE AKLAVINE
HYDROCHLORIDE Alexidine dihydrochloride Ammonium
pyrrolidinedithiocarbamate Anisomycin Brefeldin A from Penicillium
brefeldianum Calmidazolium chloride Cephaeline dihydrochloride
heptahydrate CETRMONIUM BROMIDE CHELIDONINE (+) COLCHICEINE
DACTINOMYCIN Daunorubicin hydrochloride DEOXYSAPPANONE B
7.3'-DMETHYL ETHER Digitoxigenin Digoxin DIHYDROGAMBOGIC ACID
Dihydroouabain Disulfiram EMETINE GENTIAN VIOLET JUGLONE LANATOSIDE
C LYCORINE Methyl benzethonium chloride MITOMYCIN C Mitoxantrone
OXYPHENBUTAZONE PATULIN PERIPLOCYMARIN PERUVOSIDE PHENYLMERCURIC
ACETATE Proscillaridin A Puromycin dihydrochloride PYRITHIONE ZINC
PYRROMYCIN RETUSOQUINONE Sanguinarine SARMENTOGENIN Strophanthidin
Terfenadine THIMEROSAL Thonzonium bromide TOMATINE
Table 13: 54 Identified Compounds that Affect NB Cells
[0144] Fifty-four (54) compounds selected from the LOPAC
collection, Prestwick Collection and the Spectrum Collection, were
found to selectively target NB cells. These 56 compounds appear in
Table 14. TABLE-US-00014 TABLE 14 Neuroblastoma Specific Hits
10-HYDROXYCAMTOTHECIN 2.3-Dimethoxy-1.4-naphthoquinone
4'-DEMETHYLEPIPODOPHYLLOTOXIN Amodiaquin dihydrochloride dihydrate
AMSACRINE HYDROCHLORIDE Ancitabine hydrochloride ANDROGRAPHOLIDE
Antimycin A Azaguanine-8 BENZALKONIUM CHLORIDE Benzethonium
chloride BEPRIDIL HYDROCHLORIDE beta-PELTATIN Camptothecin (S.+)
CETYLPYRIDINIUM CHLORIDE CGP-74514A hydrochloride Chelerythrine
chloride CHOLESTAN-3beta.5alpha.6beta-TRIOL Ciclopirox ethanolamine
Clofazimine CONVALLATOXIN CRASSIN ACETATE CRINAMINE Cycloheximide
Cytosine-1-beta-D-arabinofuranoside hydrochloride Dequalinium
analog. C-14 linker Dequalinium dichloride Diphenyleneiodonium
chloride DL-Stearoylcarnitine chloride Doxorubicin hydrochloride
ERYSOLIN Etoposide GAMBOGIC ACID Idarubicin IMIDACLOPRIDE LIMONIN
Loratadine MECHLORETHAMINE MECLIZINE HYDROCHLORIDE MG 624
Mycophenolic acid Ouabain OXYBENDAZOLE Paclitaxel PARAROSANILINE
PAMOATE Parthenolide Podophyllotoxin Primaquine diphosphate
Quinacrine dihydrochloride Scoulerine Taxol TENIPOSIDE Vinblastine
sulfate salt Vincristine sulfate
[0145] Four (4) compounds selected from the LOPAC collection,
Prestwick Collection and the Spectrum Collection, were found to
successfully treat a NB patient and were selected as NB specific
hits according to the assay criteria provided herein. These
compounds serve as positive controls in the selection and screening
methods. These results emphasize the validity of the assay in
identifying active agents for treating neuroblastoma. These 4
compounds are listed in Table 15.
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
[0146] These results suggest that patient-specific therapeutics as
well as novel molecular effectors of neuroblastoma can be
identified using this assay.
Example 3--Cumulative Screening Assay Selection Results
[0147] The present example presents the tabulated data obtained
with the various chemical library screens conducted. TABLE-US-00015
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
[0148] The present example demonstrates the utility of the present
invention for providing a composition suitable for the inhibition
of neuroblastoma, and for the treatment of neuroblastoma.
[0149] Forty-seven (47) compounds were selected based on
differential cell toxicity and compound mechanism of action. Forty
are novel compounds for the treatment of neuroblastoma. None of
these 40 compounds have been used clinically in neuroblastoma
therapy nor have they been examined in clinical trials. Seven
compounds have been previously used for neuroblastoma treatment
(marked with asterisk), and serve as positive controls in the
selection and screening process of new chemical entities that may
be used in the treatment of neuroblastoma according the present
invention. TABLE-US-00016 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
[0150] The standard of care for poor prognosis neuroblastoma tumors
is intensive induction chemotherapy with cisplatin, etoposide,
cyclophosphamide, and doxorubicin, high-dose myeloablative therapy
with bone marrow transplant, surgery, radiation therapy, and
biologic or maintenance therapy to eradicate minimal residual
disease. The chemotherapy regimen is designed to induce massive
genomic damage and subsequent cell death in proliferating cells.
This strategy results in the death of both tumor and normal cells
and is extremely debilitating to young patients. Additionally, this
strategy does not target the tumor-initiating cells (TICs). The
survival of NB TICs may contribute to tumor relapse.
[0151] The compounds identified in the present invention target
cellular pathways specific to neuroblastoma tumor-initiating cells
while having little or no effect on normal cells (FIG. 5).
Therefore, the compositions and pharmaceutical preparations of the
present invention will provide a treatment method for neuroblastoma
having fewer and/or less intense or pronounced toxic side effects
in patients.
[0152] All documents, patents, journal articles and other materials
cited in the present application are hereby incorporated by
reference.
[0153] Although the present invention has been fully described in
conjunction with several embodiments thereof with reference to the
accompanying drawings, it is to be understood that various changes
and modifications may be apparent to those skilled in the art. Such
changes and modifications are to be understood as included within
the scope of the present invention as defined by the appended
claims, unless they depart therefrom.
BIBLIOGRAPHY
[0154] The references listed below as well as the references cited
throughout the specification are incorporated herein by reference
to the extent that they supplement, explain, provide a background
for or teach methodology, techniques and/or compositions employed
herein. [0155] 1. Brodeur G M. (2003), Nat Rev Cancer, 3:203-16.
[0156] 2. Maris J M. (2005), Curr Opin Pediatr, 17:7-13. [0157] 3.
van Limpt V., et al. (2005), Cancer Lett, 228:59-63. [0158] 4.
Pardal, R., et al. (2003), Nat Rev Cancer, 3:895-902. [0159] 5.
Beachy, P. A., et al. (2004), Nature, 432:324-31. [0160] 6. Warner,
J. K., et al. (2004), Oncogene, 23:7164. [0161] 7. Hamburger, A.
& Salmon, S. E. (1997), J Clin Invest, 60:846-54. [0162] 8.
Heppner, G. H. (1984), Cancer Res, 44:2259-65. [0163] 9. Singh, S.
K., et al. (2004), Oncogene, 23:7267-73. [0164] 10. Al-Hajj, M.
& Clarke, M. F. (2004), Oncogene, 23:7274-82. [0165] 11.
Lapidot T, et al. (1994), Nature, 367:645-8. [0166] 12. Bonnet D,
Dick J E. (1997), Nat Med, 3:730-7. [0167] 13. Al-Hajj, M., (2003),
Proc Natl Acad Sci USA, 100:3983-8. [0168] 14. Singh, S. K. et al.
(2004), Nature, 432:396-401. [0169] 15. van Noesel M M, et al.
(1997), Cancer, 80:834-43. [0170] 16. Toma, J. G. et al. (2001),
Nat Cell Biol, 3:778-84. [0171] 17. Fernandes K J, et al. (2004),
Nat Cell Biol, 6:1082-93. [0172] 18. Toma J G, et al. (2005), Stem
Cells, 23:727-37. [0173] 19. Christiansen J H, (2000), Curr Opin
Cell Biol, 12:719-24. [0174] 20. Ambros P F, et al. (2003), Cancer
Lett, 197:29-34. [0175] 21. Miettinen M, et al. (1998), Am J Surg
Pathol, 22:327-32. [0176] 22. LaBrosse E H, et al. (1976), J Natl
Cancer Inst, 57:633-8. [0177] 23. Barnabe-Heider F, Miller F D.
(2003), J Neurosci, 23:5149-60. [0178] 24. Marsh H N, et al.
(2003), J Cell Biol, 163:999-1010. [0179] 25. Lo Piccolo M S,
Cheung N K, Cheung I Y. (2001), Cancer, 92:924-31. [0180] 26.
Fernandes K J, et al. (2006), Exp Neurol, 201:32-48. [0181] 27.
Hafer R, et al. (1999), J Neuroimmunol, 96:201-6. [0182] 28. Khanna
C, et al. (2002), In Vivo, 16:77-85. [0183] 29. Nakagawara A, Ohira
M. (2004), Cancer Lett, 204:213-24. [0184] 30. Ohira M, et al.
(2005), Cancer Cell, 7:337-50. [0185] 31. Weiss W A, et al. (1997),
EMBO J, 16:2985-95. [0186] 32. ElShamy W M, Fridvall L K, Emfors P.
(1998), Neuron, 21:1003-15. [0187] 33. Lasorella A, et al (2002)
Cancer Res, 62:301-6. [0188] 34. Valsesia-Wittmann S, et al.
(2004), Cancer Cell, 6:625-30. [0189] 35. Dubreuil V, et al.
(2000), Development, 127:5191-201. [0190] 36. Pattyn A, (2000), Mol
Cell Neurosci, 15:235-43. [0191] 37. Pozniak C D, (2000), Science,
289:304-6. [0192] 38. Casciano I, et al. (2002), Cell Death Differ,
9:246-51. [0193] 39. Matsumoto K, et al. (1995), Cancer Res,
55:1798-806 [0194] 40. Jaboin J, et al. (2002), Cancer Res,
62:6756-63. [0195] 41. Kaplan D R, et al. (1993), Neuron,
11:321-31. [0196] 42. Lucarelli E, et al. (1997), Eur J Cancer,
33:2068-70. [0197] 43. Lavoie J F, et al. (2005), J Biol. Chem.,
280:29199-207. [0198] 44. Wartiovaara K, et al. (2002), J Neurosci,
22:815-24. [0199] 45. Atwal J K, et al (2000), Neuron, 27:265-77.
[0200] 46. Toma J G, et al. (2000), J Neurosci, 20:7648-56. [0201]
47. Ellis J, Yao S. (2005), Curr Gene Ther, 5:367-73. [0202] 48.
Ellis J., (2005), Human Gene Ther, 16:1241-6. [0203] 49. Mckenzie I
A, et al. (2006), J Neurosci, 26:6651-60. [0204] 50. Torkin R, et
al. (2005), Mol Cancer Ther, 4: 1-11. [0205] 51. Barnabe-Heider F,
et al. (2005), Neuron, 48: 253-65. [0206] 52. U.S. Pat. No.
6,787,355--Miller, et al. (2004). [0207] 53. Guzman, M L., et al.
(2005), Blood, 105(11): 4163-9.
* * * * *