Patent Application
20070123448
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.
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:
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
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.
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:
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:
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.
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.
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: 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.
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.
The present example presents the tabulated data obtained with the various chemical library screens conducted.
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
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.
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 (
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.
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.
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.
| Number | Date | Country | |
|---|---|---|---|
| 60739337 | Nov 2005 | US |
