# Patent

## Title
Methods for treating and preventing brain tumors based on bone morphogenetic proteins

## Abstract

The present invention is concerned with the therapy of neuroblastoma and related diseases. Specifically, the present invention relates to a method for treating or preventing neuroblastoma comprising administering to a subject a therapeutically effective amount of bone morphogenetic protein 4 (BMP4). Preferably, said BMP4 is applied together with chemotherapy and/or radiation therapy.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the National Phase of PCT/EP2009/051258 filed on Feb. 4, 2009, which claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application No. 61/028,371 filed on Feb. 13, 2008, all of which are hereby expressly incorporated by reference into the present application.


The present invention is concerned with the therapy of neuroblastoma and related diseases. Specifically, the present invention relates to a method for treating or preventing neuroblastoma comprising administering to a subject a therapeutically effective amount of bone morphogenetic protein 4 (BMP4). Preferably, said BMP4 is applied together with chemotherapy and/or radiation therapy.
The pediatric neuroblastoma in its advanced stage (stage 1V) is usually lethal. Although chemotherapy, surgery and radiation are available, statistically, some 70% of the affected children die. The therapies available so far often are accompanied with severe side effects resulting in lethality.
The current therapy of neuroblastoma pivotally comprises chemotherapy, radiation therapy, surgery, radionuclear therapeutic approaches as well as antibody or retinoic acid based therapies. In 70% to 90% of the cases, using a risk adopted therapeutic approach, localized stages of the diseases may be either treated by surgery alone or by a combination of surgery and radiation (Berthold, 2003, Cancer Lett 197: 11-17). Half of the children suffering from neuroblastoma, however, show upon diagnosis metastasis or a genetic amplification of the MYCNN oncogene. This group of patients has a poor prognosis and a (5 year period) survival rate of merely 33% even though treated by multiple approaches, such as surgery, chemotherapy, high dosage chemotherapy including stem cell transplantation, radiation, iodine-131 metaiodobenzylguanidine (131I-MIBG) therapy and anti-GD2 antibody therapy (Berthold loc. cit.). However, administration of the differentiation inducing agent retinoic acid could somewhat improve the prognosis for the patients of the risk group mentioned above (Matthay 1999, N Engl J Med 341: 1165-1173). BMP2, a member of the TGF-beta family of growth and differentiation factors, has been reported to induce growth arrest and neuronal differentiation in neuroblastoma-derived cell lines (Nakamura 2003, Biochemical and Biophysical Research Communications 307: 206-213).
A drawback of the current standard therapy for high risk neuroblastoma using surgery, chemotherapy, high dosage chemotherapy including stem cell transplantation, radiation and 131I-MIBG therapy is the poor efficacy accompanied by the high lethality rate of approx. 70%. Moreover, serious side effects have been regularly encountered including toxicity for the mucous membranes, damages of kidney, inner ear and heart as well as, most importantly, severe bone marrow depression resulting in the need of frequent transfusions and in fulminant infections leading to lethal sepsis.
Accordingly, there is a need for means and methods for reliably preventing or treating neuroblastoma and related diseases.
Thus, the present invention relates to a method for treating or preventing neuroblastoma comprising administering to a subject a therapeutically effective amount of bone morphogenetic protein 4 (BMP4).
The term “treating” as used herein refers to removing the neuroblastoma from the subject or at least ameliorating the neuroblastoma in size, weight or aggressiveness (i.e. abolishing further growth or progression from a benign to a malign stage). Moreover, the term also includes ameliorating the symptoms associated with the said neuroblastoma. It is to be understood that treatment will not be necessarily achieved in all (100%) of the subjects of a population to be treated. However, it is envisaged that at least a statistically significant portion of the subjects of a population can be treated. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test etc. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, it is envisaged that treatment occurs in at least the 75th percentile, at least the 80th percentile, at least the 90th percentile.
The term “preventing” as used herein refers to abolishing the de novo formation of neuroblastoma or the progression of an existing neuroblastoma. It is to be understood that prevention will not be necessarily achieved in all (100%) of the subjects of a population. However, it is envisaged that prevention occurs in at least a statistically significant portion of the subjects of a population. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools described above.
The term “neuroblastoma” encompasses all kinds of neuroblastomas. Specifically, all stages of a neuroblastoma are comprised by the term. Staging is carried out, preferably, according to the International Neuroblastoma Staging System (INSS) (Brodeur 1993, J Clin Oncol 11: 1466-1477). In principle, this surgical based staging distinguishes 5 basic stages of neuroblastoma: Stage I: Localized tumour confined to the area of origin. Complete gross resection with or without microscopic residual disease; identifiable ipsilateral and contralateral lymph node negative for tumour. Stage II: Unilateral tumour with incomplete gross resection; identifiable ipsilateral and contralateral lymph node negative for tumour (stage II a), with ipsilateral lymph node positive for tumour, identifiable contralateral lymph node negative for tumour (stage II b). Stage III: Tumour infiltrating across the midline with or without regional lymph node involvement; or unilateral tumour with contralateral lymph node involvement or midline tumour with bilateral lymph node involvement. Stage IV: Dissemination of tumour to distant lymph nodes, bone marrow, liver, or other organs except as defined in stage IVS. Stage IVS: Localized primary tumour as defined for stage 1 or 2 with dissemination limited to liver, skin, and bone marrow (<10% of nucleated marrow cells are tumor cells). Preferably, neuroblastomas as referred to herein are pediatric or childhood neuroblastomas which are in an advanced stage, i.e. stage IV. More preferably, the neuroblastomas to be treated in accordance with the use of the present invention are stage IV neuroblastomas or neuroblastomas having the MYCN oncogene amplified, most preferably stage IV neuroblastomas having the MYCN oncogene amplified. Whether a subject suffers from any one of the aforementioned neuroblastomas can be diagnosed by techniques well known in the art. For example, the neuroblastomas may be diagnosed based on accompanying symptoms which are described above. The following tests and procedures may be used to determine the stage: Bone marrow aspiration and biopsy: The removal of a small piece of bone and bone marrow by inserting a needle into the hipbone or tibia. A pathologist views both the bone and the bone marrow samples under a microscope to look for signs of cancer. Tumour and lymph node biopsy: The removal of all or part of a tumour or lymph node. A pathologist views the tissue under a microscope to look for cancer cells. One of the following types of biopsies may be done: Excisional biopsy, i.e. removal of an entire tumour or lymph node; Incisional biopsy or core biopsy, i.e. removal of part of a tumour or lymph node using a wide needle; Needle biopsy or fine-needle aspiration, i.e. removal of a sample of tissue or fluid from a tumour or lymph node using a thin needle; CT scan (CAT scan); MRI (magnetic resonance imaging) sometimes also called nuclear magnetic resonance imaging (NMRI); X-rays of the chest, bones, and abdomen; Ultrasound (sonogram); or Radionuclide scan: A procedure to find areas in the body where cells, such as cancer cells, are dividing rapidly. A very small amount of radioactive material is swallowed or injected into a vein and travels through the bloodstream. The radioactive material collects in the bones or other tissues and is detected by a radiation-measuring device. Given the strong association between MYCN oncogene amplification and poor clinical outcome, determination of MYCN oncogene status in neuroblastoma tumours prior to starting therapy has become an international standard and can be carried out as described in (Schwab 2003, Lancet Oncol, 4: 472-480. The MYCN amplification can be determined by standard molecular biology techniques such as PCR-based assays. The MYCN oncogene referred to herein is, preferably, the MYCNN oncogene.
The term subject as used herein, preferably, refers to a mammal and, more preferably, to a human.
The term “bone morphogenetic protein 4 (BMP4)” refers to a growth factor or cytokine of the TGF-beta family of growth factors which was originally due to its role in bone and cartilage formation as well as mesoderm patterning. BMP4 is transcribed as a preproprotein. The biologically active mature BMP4 is generated by proteolytic cleavage of from the said preproprotein. BMP4 as used encompasses to the mature BMP4 as well as to the proprotein or the preproprotein. The structure of BMP4 proteins from various species are well known in the art (Di Chen 2004, Growth Factors 22(4): 233-241; Granjeiro 2005, Brazilian Journal of Medical and Biological Research 38: 1463-1473; Leong 1996, Int J Biochem Cell Biol 28(12): 1293-1269). Preferably, BMP4 in accordance with the present invention BMP4 refers to human BMP4 as deposited in the NCBI database under accession numbers NM—001202.3 (GI:15727659), NM—130850.2 (GI:157276594) or NM—130851.2 (GI:157276596) or as having an amino acid sequence as shown in SEQ ID NO: 2 or being encoded by a nucleic acid sequence as shown in SEQ ID NO: 1. Moreover, variants of said sequences shall be encompassed which are at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 2 or to a protein encoded by the nucleic acid sequence as shown in SEQ ID NO: 1. The percent identity values are, preferably, calculated over the entire amino acid or nucleic acid sequence region. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins, CABIOS, 5 1989: 151-153) or the programs Gap and BestFit (Needleman 1970, J. Mol. Biol. 48; 443-453; Smith 1981, Adv. Appl. Math. 2; 482-489), which are part of the GCG software packet (Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711, 1991), are to be used. The sequence identity values recited above in percent (%) are to be determined, preferably, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments. Such homologous variants are, preferably, BMP4 orthologs from other species and, thus, exhibit the same biological properties as BMP4.
BMP4 as used herein refers, preferably, to the protein. The BMP4 protein can be either purified or obtained by recombinant manufacture. However, also envisaged by the present invention is the use of a nucleic acid encoding the BMP4 from which the BMP4 protein can be transcribed. Accordingly, the present invention by referring to BMP4 also encompasses expression polynucleotides comprising BMP4 encoding nucleic acids, preferably, expression cassettes which can be stably integrated into a host cell or expression vectors which allow for transient expression of BMP4 as well as for viral vectors to be used for gene therapeutic approaches. Such expression vectors may be introduced into host cells which are subsequently transferred into or placed adjacent to the neuroblastoma. Alternatively, the expression polynucleotides may be introduced in either cells of the neuroblastoma itself or other cells of the subject. It is to be understood that expression of BMP4 in such expression polynucleotides shall be, preferably, controlled by a promoter which is specifically active in the respective cell type, a constitutively active promoter or an inducible promoter. Suitable promoters are well known in the art and include, e.g., the human CMV promoter or tetracycline inducible promoters.
BMP4 will be applied in the method of the present invention as a pharmaceutical composition. It can be formulated as a pharmaceutically acceptable salt. Acceptable salts comprise acetate, methylester, HCl, sulfate, chloride and the like. The BMP4 comprising pharmaceutical compositions are, preferably, administered topically or systemically. Suitable routes of administration conventionally used for drug administration are oral, intravenous, or parenteral administration as well as inhalation.
The pharmaceutical composition comprising BMP4, preferably, further comprises a pharmaceutically acceptable carrier. The said carrier must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The pharmaceutical carrier employed may be either a solid, a gel or a liquid. Examples of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are phosphate buffered saline solution, syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
The pharmaceutical composition may also further comprise a diluent. The diluent shall not affect the pharmaceutical activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
A “therapeutically effective dose” as used herein refers to an amount of BMP4 to be used in a pharmaceutical composition of the present invention which prevents, ameliorates or treats the symptoms accompanying a disease or condition referred to in this specification. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
The dosage regimen for BMP4 in the method of the present invention will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the above described methods. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment. A typical dose can be, for example, in the range of 1 to 1000 μg; however, doses below or above this exemplary range are envisaged, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. BMP4 pharmaceutical compositions referred to herein are administered at least once in order to treat or ameliorate or prevent a disease or condition recited in this specification. However, the said pharmaceutical compositions may be administered more than one time, for example from one to four times daily up to a non-limited number of days.
Advantageously, it has been found in the studies underlying the present invention that BMP4 can be efficiently applied for the treatment of neuroblastoma. Specifically, neuroblastoma tumors were induced in nude mice. 21 days after BMP4 application, the neuroblastoma tumors found in BMP4-treated mice differed significantly in weight compared to those found in untreated control mice. BMP4 abolished by prevention or treatment neuroblastoma formation in nude mice. Thus, it was surprisingly found that a bone morphogenetic protein can act as an efficient anti-tumor drug. Moreover, neuroblastoma related tumors can also be treated by BMP4 using the method of the present invention. Preferably, said neuroblastoma related tumors are nephroblastoma, hepatoblastoma, retinoblastoma, sarcoma, PNET or ependymoma. Like neuroblastoma, these tumors are embryonic childhood tumors.
In the following, particular preferred embodiments of the method of the present invention are described. The definitions and explanations of the terms made above apply mutatis mutandis.
In a preferred embodiment of the method of the present invention, said BMP4 is applied together with chemotherapy. In a more preferred embodiment of the method of the present invention, said chemotherapy is selected from the group consisting of: paclitaxel, docetaxel, vincristine, vindesine, dacarbazine, ifosfamide, etoposide, topotecane, cyclophosphamide, TRAIL, methotrexate, epirubicine, doxurubicine, daunorubicin, idarubicin, epirubicin, mitoxantrone, ellipticine, camptothecin, cisplatin, carboplatin, taxotere, gemcitabine, epothilone B, all-trans retinoic acid, 13-cis retinoic acid, 5-aza-deoxycytidine, 5-aza-cytidine, valproic acid, SAHA, depsipeptide, MS-275 and other histone deacetylase inhibitors, trastuzumab, imatinib, bortezomib.
In a further preferred embodiment of the method of the present invention, said BMP4 is applied together with radiation therapy.
It is to be understood that the aforementioned preferred embodiments may also be combined with each other present, i.e. a combination of radiation therapy and chemotherapy may be additionally applied to the subject to which the method of the present invention shall be applied.
The present invention, in principle, also contemplates the use of BMP4 for the preparation of a pharmaceutical composition treating or preventing neuroblastoma or a neuroblastoma related disease as specified elsewhere in the description in a subject.
The definitions and explanations of the terms made above apply mutatis mutandis. 
Preferably, said BMP4 is to be administered in combination with a chemotherapeutic agent. More preferably, said chemotherapeutic agent is selected from the group consisting of paclitaxel, docetaxel, vincristine, vindesine, dacarbazine, ifosfamide, etoposide, topotecane, cyclophosphamide, TRAIL, methotrexate, epirubicine, doxurubicine, daunorubicin, idarubicin, epirubicin, mitoxantrone, ellipticine, camptothecin, cisplatin, carboplatin, taxotere, gemcitabine, gemcitabine, epothilone B, all-trans retinoic acid, 13-cis retinoic acid, 5-aza-deoxycytidine, 5-aza-cytidine, valproic acid, SAHA, depsipeptide, MS-275 and other histone deacetylase inhibitors, trastuzumab, imatinib, bortezomib.
Also preferably, said BMP4 is to be administered in combination with a radiation.
All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in the specification.



The FIGURE show:
FIG. 1: Tumor weight 21 days after treatment with BMP4 is compared to an untreated control (C).



The following Examples merely illustrate the present invention. They shall, whatsoever, not be construed as to limit its scope of protection.
EXAMPLE
BMP4 Represses Neuroblastoma Formation and Growth In Vivo
BE(2)-C neuroblastoma cells (ECACC) were pre-treated in vitro for 48 h with 100 ng/ml BMP4 (Acris), harvested and evaluated for number and viability by coulter counter measurement. 106 viable BE(2)-C were implanted subcutaneously into the flanks of nude mice. BMP4 delivery in vivo was polymer-based. Heparin acrylic beads (Sigma) were incubated in 0.1% BSA (Sigma) or 100 ng/ml BMP4 for 1 h at 37° C., rinsed and implanted. 21 d after implantation, both NB formation and growth were evaluated.
Tumor formation was observed in all BSA-treated animals (n=3). In BMP4-treated animals (n=2), tumor formation was observed in one animal. The tumor size was significantly higher in BSA-treated controls compared with BMP4-treated animals (5.6 g±0.8 versus 0.6 g±0.6; P=0.0009, unpaired Student's t-test performed with SigmaPlot 9.0). These results were confirmed by a second independently carried out experiment (FIGURE and data not shown). In conclusion, BMP4 represses neuroblastoma formation and growth in vivo.

## Claims

1. A method for treating or for abolishing the progression of neuroblastoma comprising administering to a subject a therapeutically effective amount of bone morphogenetic protein 4 (BMP4) protein.

2. The method of claim 1, wherein said BMP4 protein is administered together with chemotherapy.

3. The method of claim 1, wherein said BMP4 protein is administered together with paclitaxel, doxetaxel, vincristine, vindesine, dacarbazine, ifosfamide, etoposide, topotecane, cyclophosphamide, TRAIL, methotrexate, epirubicine, doxurubicine, daunrubicin, idarubicin, epirubicin, mitoxantrone, ellipticine, camptothecin, cisplatin, carboplatin, taxotere, gemcitabine, epothilone B, all-trans retinoic acid, 13-cis retinoic acid, 5-aza-deoxycytidine, 5-aza-cytidine, valproic acid, SAHA, depsipeptide, MS-275 and/or other histone deacetylase inhibitors, trastuzumab, imatinib, or bortezomib.

4. The method of claim 1, wherein said BMP4 protein is administered together with radiation therapy.

5. The method of claim 1, wherein said neuroblastoma is associated with MYCN gene amplification.

6. The method of claim 1, wherein said subject is a human.