JP2015074643A - Neuroblastoma therapeutic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish an effective therapy for treating patients with a high risk of neuroblastoma because in spite of the strength treatment using surgical resection, chemotherapy, radiotherapy and the like, the prognosis is bad by causing frequently metastasis and relapse, the chemotherapeutic agent used for the conventional therapy for neuroblastoma, such as cisplatin, is ineffective for the relapse case, and an effective therapy thereof is not established at present.SOLUTION: The inventors estimated two or more molecular pathways (LMO1 molecular pathways) functioning oncogenically in a neuroblastoma by identifying the gene (LMO1 target gene) in which the expression is regulated by transcription factor LMO1 which is shown to function oncogenically in a neuroblastoma by preceding studies. Administering the molecule inhibitor inhibiting these molecular pathways to a neuroblastoma cultured cell showed that they inhibits proliferation of the neuroblastoma cell.

Description

The present invention relates to a therapeutic agent for neuroblastoma and a method for inhibiting cell growth.

Neuroblastoma is a common childhood cancer after leukemia and brain tumor. In recent years, treatments for neuroblastoma have made significant progress, and the 5-year survival rate for patients in the low and medium risk groups has exceeded 95%. However, in high-risk patients, metastasis / relapse often occurs (healed by surgery / chemotherapy but worsens again, despite intense treatment using surgical resection, chemotherapy, radiation therapy, etc.) The 5-year survival rate is still 50% or less (Non-patent Document 1). For relapse cases, chemotherapeutic agents conventionally used for the treatment of neuroblastoma such as cisplatin are ineffective, and no effective treatment has been established at present. Therefore, the discovery of a new therapeutic target molecule for developing a drug effective for relapse cases has become an important issue, and various molecules have been proposed as candidates (Non-patent document 2, Non-patent document). 3).

Recently, the latest genomic analysis that the present inventors have participated has shown that the transcription factor Lim domain only 1 (LMO1) is involved in the development of neuroblastoma, particularly high-risk groups within it. It has been clarified that LMO1 functions as an oncogene for tumor promotion in the development of blastoma (Non-patent Document 4).

Therefore, by comprehensively identifying the genes that regulate the expression of LMO1 in neuroblastoma cells (LMO1 target gene), an important tumor-promoting molecular pathway that functions in neuroblastoma has been elucidated. The development of inhibitors for molecular pathways or the application of existing inhibitors has led to the establishment of an effective treatment for recurrent cases in the high-risk group that currently has no treatment, leading to the present invention.

Maris, J.M. Recent advances in neuroblastoma. N. Engl. J. Med. 362, 22022211 (2010) Cole KA, et al. New strategies in refractory and recurrent neuroblastoma: translational opportunities to impact patient outcome.Clin. Cancer Res. 18, 2423-2428 (2012) Verissimo CS, et al. Neuroblastoma therapy: what is in the pipeline? Endocr. Relat. Cancer 18, R213-231 (2011) Wang K, et al. Integrative genomics identifies LMO1 as a neuroblastoma oncogene. Nature 469, 216220 (2011)

Neuroblastoma is a common childhood cancer after leukemia and brain tumor. In recent years, the treatment for neuroblastoma has made significant progress, and the 5-year survival rate for patients in the low-risk and medium-risk groups has exceeded 95%, but still remains below 50% in the high-risk group. Yes. This is due to the high frequency of metastasis and relapse in high-risk patients and the lack of effective treatment for relapse cases. Therefore, discovery of a new therapeutic target molecule for developing an effective drug for relapse cases has become an important issue. An object of the present invention is to provide a novel therapeutic target molecule for neuroblastoma, a novel therapeutic agent by inhibiting the same, and a method for inhibiting cell growth.

The present inventors identified a gene (LMO1 target gene) in which LMO1 regulates expression in neuroblastoma cells using the latest analysis technique called chromatin immunoprecipitation-sequence method (ChIP-Seq), Based on this, we clarified multiple tumor-promoting molecular pathways (LMO1 molecular pathway) in neuroblastoma cells. We focused on these three molecules, Src, RAS p21 protein activator 1 (RASA1), and integrin-linked kinase (ILK), related to the LMO1 molecular pathway.

In Src, RASA1, and ILK related to these LMO1 molecular pathways, the Src molecule inhibitor KX2-391 (N-Benzyl-2- [5- [4- [2- (4-morpholinyl) ethoxy] phenyl] pyridin-2- yl] acetamide), RASA1 molecule inhibitor Pluripotin (N- (3- (7- (1,3-dimethyl-1H-pyrazol-5-ylamino) -1-methyl-2-oxo-1,2-dihydropyrimido [4 , 5-d] pyrimidin-3 (4H) -yl) -4-methylphenyl) -3- (trifluoromethyl) benzamide), and ILK molecule inhibitor Cpd 22 (N-methyl-3- (1- (4- (piperazin 1-yl) phenyl) -5- (4 '-(trifluoromethyl) biphenyl-4-yl) -1H-pyrazol-3-yl) propanamide) is added to the neuroblastoma cell culture, It was found to be suppressed.

KX2-391 and Cpd 22 have been confirmed to be effective in animal models of prostate cancer, and Pluripotin has been proposed to be useful in maintaining pluripotency such as embryonic stem cells (ES cells) under culture conditions However, it is not known to use inhibitors against these molecules in the treatment of neuroblastoma. This finding presents a novel therapeutic molecular target and inhibitor for neuroblastoma, and proposes a novel therapeutic method.

The screening method of the present invention comprises a step of comparing the activity of Src in the presence and absence of a test compound and analyzing the presence or absence of Src inhibitory activity. The compound having Src inhibitory activity obtained by the above configuration can be used for the development of a therapeutic agent for neuroblastoma.

The screening method of the present invention is characterized by comprising the steps of comparing the activity of RASA1 in the presence and absence of the test compound and analyzing for the presence or absence of RASA1 inhibitory activity. The compound having RASA1 inhibitory activity obtained by the above configuration can be used for the development of a therapeutic agent for neuroblastoma.

Furthermore, the screening method of the present invention is characterized by comprising the steps of comparing the activity of ILK in the presence and absence of a test compound and analyzing the presence or absence of ILK inhibitory activity. The compound having ILK inhibitory activity obtained by the above configuration can be used for the development of a therapeutic agent for neuroblastoma.

A protein encoded by the KHDRBS2 gene, which is one of the LMO1 target genes, is a molecule that functions in a mitogenic manner, and the Src molecule is known to activate its function by phosphorylating this protein. The Src molecule inhibitor can suppress the growth of cancer cells by inhibiting the activation of KHDRBS2 protein by the Src molecule or by inhibiting phosphorylation of other proteins by the Src molecule.

A protein encoded by the RASA1 gene, which is one of the LMO1 target genes, has a function of inhibiting the function of a DLC1 molecule (known as a cancer suppressor gene) that functions repressively with respect to the ability of cancer cells to proliferate. RASA1 molecule inhibitors can suppress the growth of cancer cells by suppressing the inhibitory action of RASA1 on this tumor suppressor gene and by inhibiting the functions of other phosphorylases.

A protein encoded by the LIMS1 gene, which is one of the LMO1 target genes, is a molecule that forms a complex with ILK molecules and functions mitogenically in cancer cells. Therefore, an ILK molecule inhibitor can suppress the growth of cancer cells by inhibiting the function of the LIMS1-ILK complex.

In patients with high risk of neuroblastoma, despite the frequent occurrence of relapse, an effective treatment for relapse cases has not been established, so the prognosis is poor and the 5-year survival rate is 50% or less. Yes. LMO1 is a molecule that functions in high-risk group neuroblastoma, and inhibitors of the molecular pathways that this molecule regulates are likely to be effective in treating relapse cases and treat these molecular inhibitors It can be used to improve the prognosis of high-risk group patients. The method for inhibiting cell proliferation of neuroblastoma of the present invention has the effect of promoting new application of existing preparations, drug discovery of novel molecular inhibitors and their clinical research for neuroblastoma treatment.

Since the three molecular inhibitors of the present invention inhibit cell proliferation of cultured neuroblastoma cells, it can be expected to inhibit neuroblastoma cell proliferation in vivo. The Src molecule inhibitor KX2-391 has been evaluated in animal models for prostate cancer, an adult cancer, and is currently undergoing clinical trials (Phase II) in humans. It has been confirmed that it can be administered to humans, and clinical trials are also possible for application to the treatment of neuroblastoma.

ILK molecule inhibitor Cpd 22 has been confirmed to be effective by oral administration, and has a tumor reduction effect in mouse models transplanted with prostate cancer cultured cells and no significant adverse events were observed at this time Therefore, the possibility of administration to humans is considered, and clinical studies on neuroblastoma and other cancers can be considered.

The RASA1 molecule inhibitor Pluripotin has only been confirmed to be useful in maintaining the pluripotency of fetal stem cells (ES cells) and other cells under culture conditions. However, for the first time in the present invention, the effectiveness for cancer was revealed.

Inhibition of neuroblastoma cell proliferation by three molecular inhibitors. KX2-391 (Src molecule inhibitor), Pluripotin (RASA1 molecule inhibitor), Cpd 22 (ILK molecule inhibitor) were added to the culture solution at different concentrations, and three types of neuroblastoma cultured cells (SK-N-BE) (2), SK-N-SH, LA-N-5) were cultured for 72 hours, and cell growth was suppressed in a concentration-dependent manner compared to the control to which only the solvent DMSO (dimethyl sulfoxide) was added. It was.

Hereinafter, embodiments of the present invention will be described in detail.

The therapeutic agent for neuroblastoma of the present invention contains a substance that inhibits the function of any one of Src, RASA1, and ILK related to LOM1 signaling as an active ingredient. Inhibiting the function of any of Src, RASA1, and ILK means that the presence of the substance suppresses phosphorylation, a characteristic of Src, RASA1, and ILK, and inhibits signal transduction that proceeds in the direction of canceration. it can.

As Src inhibitors, KX2-391, PP1 (CAS # 172889-26-8: 4-amino-5- (4-methylphenyl) -7- (t-butyl) pyrazolo-d-3,4-pyrimidine), Src Kinase Inhibitor I (CAS # 179248-59-0: 4- (4′-Phenoxyanilino) -6,7-dimethoxyquinazoline), TX-1123 (CAS # 157397-06-3: 2-((3,5-di- tert-Butyl-4-hydroxyphenyl) -methylene) -4-cyclopentene-1,3-dione), but is not limited thereto.

RASA1 inhibitors include, but are not limited to, Pluripotin.

Examples of ILK inhibitors include, but are not limited to, Cpd 22, DRM02, QLT0267, KP-392, T315 and the like.

The agent of the present invention may be in the form of a substance that inhibits the functions of Src, RASA1, and ILK as it is, or may be in the form of a pharmaceutical composition containing a pharmaceutically acceptable additive.

The form of the pharmaceutical composition containing the drug of the present invention is not particularly limited, and is, for example, a solid form such as a powder, a granule, or a tablet; a liquid such as a solution, emulsion, or dispersion; or It can adjust to arbitrary forms, such as semi-solid forms, such as paste form. As oral preparations, dosage forms such as powders, granules, fine granules, tablets, pills, troches, capsules (including soft capsules and hard capsules), chewables, and solutions can be used. Moreover, as a parenteral formulation, it can be set as dosage forms, such as an injection, an instillation, an external medicine, an inhalant, or a suppository.

The dosage and dosage form of the drug of the present invention may be appropriately selected depending on the subject, pathological condition and its progress, administration route, dosage form, and other conditions, but is usually 0.001-100 mg / kg body weight per day. Preferably, it is 0.01-10 mg / kg per day.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

(Experimental material)
Three types of neuroblast culture cells were prepared and administration experiments were performed. SK-N-BE (2) cells were purchased from the European Collection of Cell Cultures (ECACC), with EMEM (Wako Pure Chemicals) supplemented with fetal calf serum (15%) and non-essential amino acids (0.5%, life technology). The cells were cultured in a 1: 1 mixture of Ham's F12 (Wako Pure Chemical Industries). SK-N-SH cells were purchased from ECACC and cultured in DMEM (Wako Pure Chemicals) culture medium supplemented with fetal bovine serum (10%). LA-N-5 cells were transferred from RIKEN BioResource Center and cultured in RPMI1640 (life technology) culture medium supplemented with fetal bovine serum (10%). The relative number of cells was measured by Cell Counting Kit-8 (Dojin Chemical) by the MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) method. Src molecule inhibitor KX2-391 (Selleckchem), ILK molecule inhibitor Cpd 22 (Calbiochem), RASA1 molecule inhibitor Pluripotin (Sigma) dissolved in dimethyl sulfoxide (DMSO, Merck) And added to the culture broth.

(experimental method)
The cell growth suppression experiment was performed according to the method described in the package insert of Cell Counting Kit-8 (Dojindo Laboratories).
Three types of neuroblastic cultured cells were each seeded in a 96-well plate (2000 cells / well), and three concentrations of molecular inhibitors prepared by serial dilution the next day were added to each well. After culturing for 72 hours, Cell Counting Kit-8 reagent was added and reacted for 2 hours, and then the absorbance at 450 nm was measured. For each concentration of each molecular inhibitor, an experiment was carried out as a set of 5 wells, and the average value and standard deviation were obtained.

(result)
Each of the three molecular inhibitors suppressed the cell proliferation of SK-N-BE (2) cells, SK-N-SH cells and LA-N-5 cells in a concentration-dependent manner (FIG. 1).

Claims (8)

A therapeutic agent for neuroblastoma comprising as an active ingredient a substance that inhibits the function of any of Src, RASA1, and ILK related to LOM1 signaling. The therapeutic agent for neuroblastoma according to claim 1, wherein the substance that inhibits Src is KX2-391. The therapeutic agent for neuroblastoma according to claim 1, wherein the substance that inhibits RASA1 is Pluripotin. The therapeutic agent for neuroblastoma according to claim 1, wherein the substance that inhibits ILK is Cpd22. A method for screening a therapeutic agent for neuroblastoma, comprising a step of comparing the activity of Src in the presence and absence of a test compound and analyzing the presence or absence of Src inhibitory activity. A method for screening a therapeutic agent for neuroblastoma, comprising a step of comparing the activity of RASA1 in the presence and absence of a test compound and analyzing the presence or absence of RASA1 inhibitory activity. A method for screening a therapeutic agent for neuroblastoma, comprising a step of comparing the activity of ILK in the presence and absence of a test compound and selecting a compound having ILK inhibitory activity. A method for inhibiting neuroblastoma cell proliferation, which comprises inhibiting the function of any of Src, RASA1, and ILK related to LOM1 signaling.

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