Activated Ras as a therapeutic and diagnostic target for neurofibromatosis and its uses

Abstract

The present invention relates to a biomarker composition for predicting sensitivity to a neurofibromatosis therapeutic agent that utilizes Ras activity, and a pharmaceutical composition for preventing or treating neurofibromatosis, which comprises a Ras activity inhibitor. The biomarker composition can accurately diagnose sensitivity to a neurofibromatosis therapeutic agent through a simple method of measuring the expression levels of RAS (Ras-GTP) protein activated by binding of GTP and activated p-ERK protein or the gene encoding same in an individual's biological sample, and the pharmaceutical composition inhibits Ras protein activity, and will therefore be useful for preventing or treating neurofibromatosis in which Ras protein activity is increased.

Description

[Technical field]

[0001] Activated Ras and its uses as a therapeutic and diagnostic target for neurofibromatosis. [Background technology]

[0002] Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder with a relatively high incidence rate of 1 in 3,000 people worldwide. It is caused by NF1 gene mutations and is characterized by multiple cafe au lait spots and cutaneous neurofibromas, and various levels of nervous system, skeletal system and neoplastic manifestations can significantly affect patients. NF1 patients are at risk of developing central and peripheral nervous system tumors, which is 1,000 times higher than the general population.

[0003] The only current treatments for neurofibromatosis type 1 are mass therapies such as chemotherapy, radiation therapy, and surgical resection, but surgery is often not possible when the disease occurs multiple times in multiple organs.

[0004] Recently, MEK inhibitor selumetinib was approved by the FDA and as an orphan drug in Korea as a non-invasive treatment for patients with neurofibromatosis type 1. Another treatment, tyrosine kinase inhibitor carbozantinib, has been reported to be safe and effective in tumor suppression in clinical trials, but it has limitations in that it cannot shrink or completely remove various tumors. In addition, patients with neurofibromatosis type 1 do not respond to all of the above treatments, so more treatments are needed.

[0005] Meanwhile, a survey of the types of gene mutations in Korean neurofibromatosis patients showed that about 30% of patients had stop codon mutations. In addition, Ataluren (PTC THERAPEUTICS, USA), which reads through premature stop codons, has been undergoing clinical research in recent years as a muscle disease treatment and has been mentioned as a treatment for neurofibromatosis patients with stop codon mutations, but there has been no research on its efficacy. Summary of the Invention [Problem to be solved by the invention]

[0006] One aspect is to provide a biomarker composition that can predict the sensitivity of neurofibromatosis patients to a compound that reads through a premature stop codon by utilizing Ras activity or p-ERK activity.

[0007] Another aspect is to provide a kit for predicting the sensitivity of a neurofibromatosis patient to the compound, comprising the composition.

[0008] Another aspect is to provide a method that utilizes the biomarker composition to provide information necessary to predict the sensitivity of a neurofibromatosis patient to the compound.

[0009] Another aspect is to provide a pharmaceutical composition for preventing or treating neurofibromatosis, comprising a compound capable of inhibiting Ras activity.

[0010] Another aspect provides a method for screening a therapeutic agent for neurofibromatosis, comprising the steps of contacting a biological sample having an NF1 gene mutation with a test substance, and measuring, in the contacted biological sample, the expression levels of (1) any one or more proteins selected from the group consisting of RAS (Ras-GTP) protein activated by binding with GTP and activated p-ERK protein, and (2) genes encoding the proteins.

[0011] Another aspect provides a use of a biomarker comprising a preparation capable of measuring the expression level of (1) any one or more proteins selected from the group consisting of GTP-bound activated RAS (Ras-GTP) protein and activated p-ERK protein, or (2) a gene encoding said protein, for predicting sensitivity to a neurofibromatosis therapeutic agent.

[0012] Another aspect provides a method for predicting the sensitivity of a neurofibromatosis patient to a neurofibromatosis therapeutic agent, the method comprising the steps of measuring, in a biological sample obtained from an individual, the expression levels of (1) any one or more proteins selected from the group consisting of RAS (Ras-GTP) protein activated by binding with GTP and activated p-ERK protein, and (2) genes encoding the proteins.

[0013] Another aspect is to provide a method for preventing or treating neurofibromatosis comprising the step of administering to an individual a composition comprising the compound or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0014] Another aspect provides a use of a composition comprising the compound or a pharma- ceutical acceptable salt thereof as an active ingredient for the manufacture of a therapeutic agent for use in the prevention or treatment of neurofibromatosis. [Means for solving the problem]

[0015] In order to achieve the above-mentioned object, one embodiment provides a biomarker composition for predicting sensitivity to a therapeutic agent for neurofibromatosis, comprising a preparation for measuring the expression level of (1) any one or more proteins selected from the group consisting of RAS (Ras-GTP) protein activated by binding with GTP and activated p-ERK protein, and (2) a gene encoding the protein.

[0016] The term "RAS protein" refers to a type of small GTP (guanosine triphoshate) binding protein, which has isoforms such as K-Ras protein, H-Ras protein, and N-Ras protein. Ras protein mediates signals such as cell proliferation, differentiation, and adhesion caused by various extracellular stimuli from upstream tyrosine kinase receptors to downstream effectors.

[0017] Ras protein is active when bound to GTP (guanosine triphosphate) (Ras-GTP) and inactive when bound to GDP (guanosine diphosphate) (Ras-GDP), and can regulate cell proliferation and other functions by changing between the active and inactive states. On the other hand, it is known that Ras protein mutates frequently in human cancers, and that this mutation causes Ras protein to always be in the active form.

[0018] Activated Ras with transforming activity has amino acid mutations important for the interaction with the guanine ring or phosphate group of GDP or GTP, and can show a decrease in intrinsic GTP hydrolysis activity and an increase in the GDP dissociation rate. Its expression increases the ratio of activated Ras bound to GTP in cells, which can cause abnormalities in cell proliferation control. Activated Ras bound to GTP can directly regulate the stepwise chain of serine-threonine phosphorylation enzymes from Raf to MAP kinase by binding to Raf.

[0019] The ERK may be one of the Ser / Thr protein kinases regulated by Ras proteins. Inactivated ERK can be in the cytoplasm, whereas activated ERK can move into the nucleus. Furthermore, activated ERK can phosphorylate target proteins or activate other protein kinases to regulate signal transduction pathways.

[0020] The term "sensitivity" can mean whether a specific drug shows a therapeutic effect for an individual neurofibromatosis patient. It is known that even a type of therapeutic agent recognized as effective for the treatment of neurofibromatosis may or may not show an effect depending on the individual patient. Whether or not a therapeutic agent shows an effect for such an individual patient's neurofibromatosis can be called sensitivity to the therapeutic agent. The above-mentioned "sensitivity" can be used interchangeably with "therapeutic response", and "for predicting sensitivity" can be used interchangeably with "for predicting therapeutic response" or "for companion diagnostics".

[0021] The term "Companion Diagnosis" can refer to a diagnostic technique for predicting a patient's response to treatment.

[0022] The companion diagnostic composition may be intended to provide information regarding the need for administration of a neurofibromatosis therapeutic agent, the possibility of development of drug resistance to the neurofibromatosis therapeutic agent, sensitivity to the neurofibromatosis therapeutic agent, and prognosis prediction of treatment with the neurofibromatosis therapeutic agent.

[0023] The term "biomarker" can also be used as a companion diagnostic marker and can refer to a substance in a biological sample that can predict in advance the responsiveness of a patient to a specific drug treatment. It can include organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, and sugars (e.g., monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase or decrease in biological samples taken from neurofibromatosis patients compared to biological samples obtained from normal individuals.

[0024] In one embodiment, the biomarker for predicting sensitivity to a neurofibromatosis therapeutic agent may be a GTP-bound activated RAS (Ras-GTP) protein or a gene encoding the same, and when the expression level is high, it can be determined that the sensitivity to a neurofibromatosis therapeutic agent is high, and conversely, when the expression level is low, it can be determined that the sensitivity to a neurofibromatosis therapeutic agent is low.

[0025] In one embodiment, the biomarker for predicting sensitivity to a neurofibromatosis therapeutic agent may be an activated p-ERK protein or a gene encoding the same, and a high expression level of the activated p-ERK protein or a gene encoding ...

[0026] The therapeutic agent for neurofibromatosis may be a biomarker composition for predicting sensitivity to a therapeutic agent for neurofibromatosis, which is a compound represented by the following Chemical Formula 1 or a pharma- ceutical acceptable salt thereof.

[0027] [ka] In the above formula Z is halogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, or substituted or unsubstituted heterocycloalkyl; R can be hydrogen or halogen.

[0028] The term "halogen" can mean F, Cl, Br or I, unless otherwise specified.

[0029] The term "hydroxyl," unless otherwise specified, can refer to a monovalent atomic group consisting of one hydrogen atom and one oxygen atom and having the chemical formula --OH.

[0030] The term "alkyl" can mean, unless otherwise specified, a linear or branched saturated hydrocarbon residue. For example, "C 1~10 "Alkyl" refers to an alkyl group having a skeleton of 1 to 10 carbon atoms. 1~10 Alkyl can include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, i-pentyl, t-pentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like.

[0031] The term "alkoxy", unless otherwise specified, can refer to a group having the formula -O-alkyl, in which an alkyl group, as defined above, is attached to the parent compound through an oxygen atom. The alkyl portion of the alkoxy group can have 1 to 20 carbon atoms (i.e., C 1~ C 20 alkoxy), 1 to 12 carbon atoms (i.e., C 1~ C 12 alkoxy), or 1 to 6 carbon atoms (i.e., C 1~ C alkoxy). Examples of suitable alkoxy groups include methoxy (-O-CH or -OMe), ethoxy (-OCHCH or -OEt), t-butoxy (-OC(CH) or -O-tBu), and the like.

[0032] The term "aryl" can refer to an aromatic hydrocarbon radical derived by removing one hydrogen atom from six carbon atoms of a parent aromatic ring system. For example, an aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms.

[0033] The term "cycloalkyl" refers to a saturated monocycle or polycycle containing only carbon atoms in the ring. Cycloalkyls can have from 3 to 7 carbon atoms as monocycles, from 7 to 12 carbon atoms as bicycloalkyls, and up to about 20 carbon atoms as polycycles.

[0034] The term "heterocycle" refers to an aromatic or non-aromatic ring having one or more heteroatoms, which may be saturated or unsaturated, and may be monocyclic or polycyclic. For example, a "4-10 membered heterocycle" refers to a heterocycle consisting of a total of 4 to 10 nuclear frameworks including heteroatoms and carbon atoms. Specifically, 4-10 membered heterocycles include azetidine, diazetidine, pyrrolidine, pyrrole, imidazolidine, imidazole, pyrazolidine, pyrazole, oxazolidine, oxazole, isoxazolidine, isoxazole, thiazolidine, thiazole, isothiazolidine, isothiazole, piperidine, pyridine, piperazine, diazine, moporine, thiomoporine, azepane, diazepane, etc.

[0035] The term "heteroaryl" refers to an aromatic heterocyclyl having at least one or more heteroatoms in the ring. Non-limiting examples of heteroaryl include pyridinyl, pyrrolyl, oxazolyl, indolyl, isoindolyl, purinyl, furanyl, thienyl, benzofuranyl, benzothiophenyl, carbazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, quinolyl, isoquinolyl, pyridazyl, pyrimidyl, pyrazyl (which may have one or more substituents on the ring), and the like.

[0036] The term "heterocycloalkyl" can refer to a non-aromatic heterocyclyl having one or more heteroatoms in the ring. Heterocycloalkyl can have one or more carbon-carbon double bonds or carbon-heteroatom double bonds in the ring, to the extent that the presence of the double bonds does not render the ring aromatic. Non-limiting examples of heterocycloalkyl include azetidinyl, aziridinyl, pyrrolidinyl, piperidinyl, piperazinyl, homopiperazinyl, mopolino, thiomopolino, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, and the like, which may have one or more substituents in the ring.

[0037] The term "heteroatom" means an atom other than carbon (C), and may specifically be a nitrogen (N), oxygen (O) or sulfur (S) atom.

[0038] The term "substitution" can refer to the replacement of hydrogen atoms in a molecular structure with a substituent such that the atom on the specified atom does not exceed (Valence) and the compound is chemically stable from such substitution. For example, "group A is substituted with substituent B" can mean that a hydrogen atom bonded to an atom such as carbon that constitutes the backbone of group A is replaced with substituent B, and group A and substituent B form a covalent bond.

[0039] The Z may be any of p-tolyl, (4-chloromethyl-phenyl), (2-chloro-pyridin-3-yl), (2-fluoro-phenyl), (3,4-difluoro-phenyl), (4-methoxy-phenyl), benzo[1,3]dioxol-yl, (4-ethyl-phenyl), o-tolyl, (2-chloro-phenyl), (3-methyl-thiophen-2-yl), benzo[b]thiophen-2-yl, (3-fluoro-phenyl), (4-tert-butyl-phenyl), and the like. methyl-phenyl), (2-methoxy-phenyl), (2,difluoro-phenyl), thiophen-2-yl, (2,4-difluoro-phenyl), (3-chloro-phenyl), m-tolyl, (4-trifluoromethyl-phenyl), (4-fluoro-phenyl), (3-methoxy-phenyl), phenyl, (2,6-difluoro-phenyl), (2,dimethyl-furan-3-yl), (4-pyrrol-1-yl-phenyl), (3-dimethylamino -phenyl), biphenyl-4-yl, (4-dimethylamino-phenyl), benzo[1,2,5]oxadiazol-yl, m-tolyl, (2-trifluoromethyl-phenyl), (6-chloro-pyridin-3-yl), (3,bis-trifluoromethyl-phenyl), ran-2-yl, (4-nitro-phenyl), (3,4-dimethoxy-phenyl), (3-trifluoromethoxy-phenyl), naphthalen-1-yl, cyclohexyl, pyridin-3-yl, pyridin-4-yl, cyclopentyl, cyclopropyl, (4-pentyloxy-phenyl), (3,4,trimethoxy-phenyl), (4-isobutyl-phenyl), cyclobutyl, (1-acetyl-piperidin-4-yl), isoxazol-yl, [2-chloro-6-fluoro-phenyl)-methyl-isoxazol-4-yl] or [2-chloro-phenyl)-methyl-isoxazol-4-yl]. Specifically, Z can be (2-fluoro-phenyl), (3,4-difluoro-phenyl), (2-chloro-phenyl), (3-fluoro-phenyl), (2,5-difluoro-phenyl), (2,4-difluoro-phenyl), (3-chloro-phenyl), (4-fluoro-phenyl), or (2,6-difluoro-phenyl).

[0040] The compound represented by Chemical Formula 1 can read-through premature stop codons.

[0041] The compound represented by the formula 1 may be 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid. The compound is represented by the formula 2 below.

[0042] [ka] The compound represented by the above formula 2 is also called Ataluren, PCT124, or Translarna, and is capable of reading through premature stop codons.

[0043] The therapeutic agent for neurofibromatosis may include a pharma- ceutical acceptable salt of the compound. The pharma-ceutical acceptable salt must have low toxicity to humans and may not negatively affect the biological activity and physicochemical properties of the parent compound.

[0044] For example, the pharma- ceutically acceptable salt may be an alkali metal salt (such as a sodium salt) or an alkaline earth metal salt (such as a potassium salt), which may be obtained, for example, by dissolving the compound in a solution of excess alkali metal hydroxide or alkaline earth metal hydroxide, filtering the undissolved compound salt, and then evaporating and drying the filtrate.

[0045] Additionally, the compounds may contain chiral carbon centers and therefore may exist in the form of R or S isomers, racemates, individual enantiomers or mixtures, individual diastereomers or mixtures, and all such stereoisomers and mixtures thereof are intended to be within the scope of the present invention.

[0046] Furthermore, the compound may include hydrates and solvates of the compound. The hydrates and solvates may be produced using known methods, and are preferably non-toxic and water-soluble. In particular, the hydrates and solvates may each be formed by combining 1 to 5 molecules of water and alcoholic solvent (especially ethanol, etc.).

[0047] Measuring the expression levels of the Ras-GTP protein and / or activated p-ERK protein is a process of confirming the presence of the protein and the degree of expression of the protein in an individual's biological sample in order to predict sensitivity to a neurofibromatosis therapeutic agent, and may involve confirming the amount of the protein using a molecule that specifically binds to the protein.

[0048] The preparation capable of measuring the expression level of the protein may be any one or more selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), an aptamer, and a nanoparticle, but is not limited thereto.

[0049] The analytical methods used for this purpose include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket, immunoelectrophoresis, immunoprecipitation assay, immunohistochemical analysis, complement fixation assay, fluorescence activated cell sorter (FACS), protein chip, two-dimensional electrophoresis, protein mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry), MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, and SELDI-TOF (Sulface Examples of analytical methods include, but are not limited to, Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry (ESF) analysis.

[0050] According to one embodiment, the preparation capable of measuring the expression level of the Ras-GTP protein may be one that specifically binds to the Ras-GTP protein and does not bind to the Ras-GDP protein.

[0051] According to one embodiment, the preparation capable of measuring the expression level of the activated p-ERK protein may specifically bind to the p-ERK protein.

[0052] Measuring the expression level of a gene encoding the protein is a process of confirming the presence or absence of a gene encoding the protein and the degree of expression of the gene in an individual's biological sample in order to predict sensitivity to a neurofibromatosis therapeutic agent, and may involve confirming the amount of the gene using a molecule that specifically binds to the gene.

[0053] The preparation capable of measuring the expression level of the gene encoding the protein may be any one or more selected from the group consisting of a primer pair, a probe, and an antisense nucleotide that specifically binds to the gene, but is not limited thereto.

[0054] Analytical methods for this purpose include, but are not limited to, reverse transcriptase-polymerase chain reaction (RT-PCR), real time-polymerase chain reaction, RNase protection assay (RPA), Northern blotting, and DNA chips.

[0055] The neurofibromatosis may have a stop codon mutation in the NF1 gene.

[0056] The term "stop codon mutation" may refer to a mutation that causes a portion of a base sequence to become one of the stop codons UAG, UAA, or UAG, thereby interrupting polypeptide synthesis.

[0057] The stop codon mutation may be a nonsense mutation, which is a point mutation in which a DNA base is replaced with another base, or a frameshift mutation.

[0058] The term "nonsense mutation" refers to a mutation in which, when a DNA base sequence is converted into a protein, part of the base sequence is converted into a stop codon (one of UAG, UAA, or UGA), preventing further protein synthesis.

[0059] The term "frameshift mutation" refers to a mutation that results in the insertion or deletion of one, two, or a number of nucleotides that is not a multiple of three in a DNA base sequence, resulting in a significant change in the amino acid sequence of a protein produced through the transcription and translation processes of a gene.

[0060] In one embodiment, the stop codon mutation is one of the following: i) the 5242nd base, cytidine (C), in the DNA base sequence of the NF1 gene is replaced with thymine (T), converting the 1748th amino acid, arginine (Arg, R), constituting the NF1 protein, into a stop codon; ii) the 2560th base, cytidine (C), in the DNA base sequence of the NF1 gene is replaced with thymine (T), converting the 854th amino acid, glutamine (Gln, Q), constituting the NF1 protein, into a stop codon; or iii) the 4537th base, cytidine (C), in the DNA base sequence of the NF1 gene is replaced with thymine (T), converting the 854th amino acid, glutamine (Gln, Q), constituting the NF1 protein, into a stop codon. In the DNA sequence of the NF1 gene, thymine (T), the 2978th ​​base, is replaced by guanine (G), converting the 993rd amino acid leucine (Leu, L) that constitutes the NF1 protein into a stop codon; or in the DNA sequence of the NF1 gene, cytidine (C), the 7486th base, is replaced by thymine (T), converting the 2496th amino acid arginine (Arg, R) that constitutes the NF1 protein into a stop codon.

[0061] In another embodiment, among neurofibromatosis patients, patients with stop codon mutations in the NF1 gene are selected, and Ras-GTP activity is measured in the fibroblasts of the selected patients. As a result, the expression of Ras-GTP and p-ERK protein is significantly decreased in the patient group in which Ras-GTP expression is significantly increased compared to healthy subjects, thereby confirming that ataluren has a therapeutic effect on neurofibromatosis. Therefore, among neurofibromatosis patients with stop codon mutations in the NF1 gene, sensitivity to ataluren can be predicted depending on whether the expression of activated Ras (Ras-GTP) and / or activated p-ERK protein is increased or decreased.

[0062] Another aspect provides a kit for predicting sensitivity to a therapeutic agent for neurofibromatosis, comprising the biomarker composition.

[0063] The kit can predict sensitivity to the therapeutic agent for neurofibromatosis using a biological sample isolated from an individual. Specifically, the kit can predict sensitivity to the therapeutic agent for neurofibromatosis using fibroblasts collected from an individual.

[0064] The kit may include not only a preparation for measuring the expression level of the protein or gene, but also tools, reagents, and the like that are commonly used in immunological analysis.

[0065] Examples of the tool or reagent may include, but are not limited to, a suitable carrier, a labeling substance capable of generating a detectable signal, a chromophore, a dissolving agent, a detergent, a buffer, a stabilizer, etc. When the labeling substance is an enzyme, a substrate capable of measuring the enzyme activity and a reaction stopper may be included. The carrier may be a soluble carrier or an insoluble carrier. An example of a soluble carrier is a physiologically acceptable buffer solution known in the art, such as PBS, and an example of an insoluble carrier may be a polymer such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, polysaccharide, latex plated with metal, other paper, glass, metal, agarose, and combinations thereof.

[0066] The biomarker composition is as described above.

[0067] Another aspect provides a method for providing information necessary for predicting sensitivity to a therapeutic agent for neurofibromatosis, comprising measuring, in a biological sample obtained from an individual, (1) any one or more proteins selected from the group consisting of RAS (Ras-GTP) protein activated by binding with GTP and activated p-ERK protein, and (2) the expression level of a gene encoding the protein.

[0068] The method may further include a step of determining that the subject has high sensitivity to a therapeutic agent for neurofibromatosis if the expression level of the activated Ras-GTP protein or the gene encoding it is increased compared to the expression level measured in a biological sample obtained from a normal individual, and conversely, determining that the subject has low sensitivity to a therapeutic agent for neurofibromatosis if the expression level is low.

[0069] The method may further include a step of determining that the subject has high sensitivity to the therapeutic agent for neurofibromatosis when the expression level of the activated p-ERK protein or the gene encoding it is increased compared to the expression level measured in a biological sample obtained from a normal individual, and conversely, determining that the subject has low sensitivity to the therapeutic agent for neurofibromatosis when the expression level is low.

[0070] The term "biological sample" may refer to any sample obtained from an individual in which the expression level of a Ras-GTP protein or the gene encoding it and / or the expression level of a p-ERK protein or the gene encoding it according to one embodiment can be measured.

[0071] The biological sample may be, but is not limited to, blood, plasma, serum, urine, saliva, sputum, cerebrospinal fluid, cells, cell culture fluid, tissue extract or tumor tissue. Specifically, the biological sample may be fibroblast cells taken from an individual. The biological sample may be processed and prepared by methods commonly used in the art.

[0072] In another embodiment, there is provided a pharmaceutical composition for preventing or treating neurofibromatosis, comprising a compound represented by the following chemical formula 1, or a pharma- ceutically acceptable salt thereof, as an active ingredient:

[0073] [ka] In the above formula Z is halogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, or substituted or unsubstituted heterocycloalkyl; R can be hydrogen or halogen.

[0074] The compound represented by Chemical Formula 1 and its pharma- ceutically acceptable salts are as described above.

[0075] The compound represented by Chemical Formula 1 may be 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid.

[0076] The neurofibromatosis may have a stop codon mutation in the NF1 gene.

[0077] The neurofibromatosis may be caused by increased activity of the Ras protein.

[0078] The neurofibromatosis is as described above.

[0079] In one embodiment, patients with stop codon mutation in the NF1 gene are selected from neurofibromatosis patients, and Ras-GTP activity is measured in the fibroblasts of the selected patients. As a result of treatment with ataluren, the expression of Ras-GTP and p-ERK protein is significantly decreased in the patient group in which Ras-GTP activity is significantly increased compared to healthy subjects, thereby confirming that ataluren has a therapeutic effect on neurofibromatosis. Therefore, ataluren can be used for the treatment of neurofibromatosis patients with stop codon mutation in the NF1 gene in which the expression of active Ras (Ras-GTP) is increased.

[0080] The pharmaceutical composition may contain the compound as an active ingredient in an amount of about 0.1% by weight to about 90% by weight, specifically about 0.5% by weight to about 75% by weight, and more specifically about 1% by weight to about 50% by weight, based on the total weight of the composition.

[0081] The pharmaceutical composition may include common non-toxic pharma- ceutical acceptable additives that are formulated into formulations according to conventional methods. For example, the pharmaceutical composition may further include a pharma- ceutical acceptable carrier, diluent, or excipient.

[0082] Examples of additives used in the pharmaceutical composition may include sweeteners, binders, solvents, solubilizers, wetting agents, emulsifiers, isotonicity agents, absorbents, disintegrants, antioxidants, preservatives, lubricants, lubricants, fillers, flavorings, etc. For example, the additives may include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, talc, stearic acid, stearin, magnesium stearate, magnesium aluminosilicate, starch, gelatin, tragacanth alginate, sodium alginate, methylcellulose, sodium carboxymethylcellulose, agar, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, vanilla flavor, etc.

[0083] The pharmaceutical compositions can be formulated in a variety of preparation forms for oral administration (e.g., tablets, pills, powders, capsules, syrups or emulsions) or parenteral administration (e.g., intramuscular, intravenous or subcutaneous injection).

[0084] Specifically, the pharmaceutical composition can be formulated as a preparation for oral administration, and the additives used in this case can include cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, magnesium stearate, calcium stearate, gelatin, talc, surfactants, suspending agents, emulsifiers, diluents, etc. Examples of the glidant include colloidal silicon dioxide and magnesium silicate. Examples of the diluent include microcrystalline cellulose, lactose anhydrous, lactose monohydrate, and silicified MCC HD90. Examples of the disintegrant include croscarmellose sodium and crospovidone. Examples of the lubricant include magnesium stearate, sodium lauryl sulfate, and stearic acid.

[0085] In addition, examples of liquid preparations for oral administration include suspensions, emulsions, syrups, etc., and may contain various excipients other than water and liquid paraffin, which are commonly used simple diluents, such as wetting agents, sweeteners, flavorings, preservatives, etc.

[0086] Furthermore, the formulation for parenteral administration includes sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories. For non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used. For the base of the suppository, witepsol, macrogol, twin 61, cacaodi, laurindi, glycerogelatin, etc. may be used. On the other hand, for the injection, conventional additives such as a solubilizer, an isotonic agent, a suspending agent, an emulsifier, a stabilizer, and a preservative may be included.

[0087] The pharmaceutical compositions may be administered to a patient in a therapeutically effective amount or a medicamentally effective amount.

[0088] The term "therapeutically effective amount" or "pharmaceutical effective amount" refers to an amount of a compound or composition effective in preventing or treating a target disease, and is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to any medical treatment and without causing side effects. The level of the effective amount can be determined by factors including the patient's health condition, type and severity of the disease, activity of the drug, sensitivity to the drug, method of administration, administration time, route of administration and excretion rate, duration of treatment, co-administered or co-used drugs, and other factors well known in the medical field.

[0089] The pharmaceutical compositions can be administered as individual therapeutic agents or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. Taking into account all of the above factors, it is important to administer an amount that can obtain maximum effect at a minimum amount without side effects, which can be easily determined by one skilled in the art.

[0090] Specifically, the effective amount of the compound in the pharmaceutical composition may vary depending on the age, sex, and weight of the patient, and may generally be administered at about 0.1 mg to about 1,000 mg, or about 5 mg to about 200 mg per kg of body weight, daily or in one to three divided doses per day. However, the range is not limited thereto, since the amount may increase or decrease depending on the route of administration, severity of the disease, sex, weight, age, etc.

[0091] Furthermore, the pharmaceutical composition may be administered for tumor therapy in combination with chemotherapy, radiotherapy, immunotherapy, hormonal therapy, bone marrow transplantation, stem cell replacement therapy, other biological therapy, surgical intervention, or a combination thereof, for example, as an adjuvant therapy with other therapeutic strategies that are developed over time, or to maintain tumor regression in severely ill patients or the patient's condition after chemopreventive therapy.

[0092] Another aspect provides a method for screening for a therapeutic agent for neurofibromatosis, comprising the steps of: (a) contacting a biological sample having an NF1 gene mutation with a test substance; and (b) measuring, in the contacted biological sample, (1) any one or more proteins selected from the group consisting of RAS (Ras-GTP) protein activated by binding with GTP and activated p-ERK protein, and (2) the expression level of a gene encoding the protein.

[0093] The biological sample may be a cell or an animal model. The sample may be a sample isolated from a neurofibromatosis patient.

[0094] According to one specific example, the screening method can include a step of determining that the test substance is a therapeutic agent for neurofibromatosis if the expression level of Ras-GTP protein or the gene encoding it in a biological sample after contact with the test substance is decreased compared to before contact with the test substance.

[0095] According to one embodiment, the screening method may include a step of determining that the test substance is a therapeutic agent for neurofibromatosis if the expression level of activated p-ERK protein or a gene encoding the same in a biological sample after contact with the test substance is decreased compared to before contact with the test substance.

[0096] The screening method may include a step of comparing the results of measuring the expression level of Ras-GTP protein or the gene encoding it in a biological sample contacted with the test substance with the results measured from a biological sample not contacted with the test substance.

[0097] The screening method may include a step of comparing the results of measuring the expression level of activated p-ERK protein or a gene encoding the same in a biological sample contacted with the test substance with the results measured from a biological sample not contacted with the test substance.

[0098] Another aspect provides use of a biomarker comprising (1) any one or more proteins selected from the group consisting of RAS (Ras-GTP) protein activated by binding with GTP and activated p-ERK protein, and (2) a preparation capable of measuring the expression level of a gene encoding the protein, for predicting sensitivity to a neurofibromatosis therapeutic agent.

[0099] Another aspect provides a method for predicting the sensitivity of a neurofibromatosis patient to a neurofibromatosis therapeutic agent, comprising steps of measuring, in a biological sample obtained from an individual, (1) any one or more proteins selected from the group consisting of RAS protein activated by binding to GTP (Ras-GTP) protein and activated p-ERK protein, and (2) the expression level of a gene encoding the protein.

[0100] Another aspect provides a method for preventing or treating neurofibromatosis, comprising the step of administering to an individual a composition comprising, as an active ingredient, a compound represented by the following chemical formula 1, or a pharma- ceutical acceptable salt thereof:

[0101] [ka] In the above formula Z is halogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, or substituted or unsubstituted heterocycloalkyl; R can be hydrogen or halogen.

[0102] Another aspect provides a use of a composition comprising, as an active ingredient, a compound represented by the following chemical formula 1, or a pharma- ceutical acceptable salt thereof, for the manufacture of a therapeutic agent for use in the prevention or treatment of neurofibromatosis.

[0103] [ka] In the above formula Z is halogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, or substituted or unsubstituted heterocycloalkyl; R can be hydrogen or halogen.

[0104] Duplicate content will be omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have the meanings commonly used in the technical field to which this invention pertains. Effect of the Invention

[0105] By using a biomarker composition for predicting sensitivity to a neurofibromatosis therapeutic agent according to one embodiment, sensitivity to a neurofibromatosis therapeutic agent can be accurately diagnosed through a simple method of measuring the expression levels of RAS (Ras-GTP) protein activated by GTP binding or the gene encoding same and / or activated p-ERK protein or the gene encoding same in an individual's biological sample.

[0106] The pharmaceutical composition for preventing or treating neurofibromatosis according to one embodiment can be used to suppress the activity of Ras protein, and can therefore be useful for preventing or treating neurofibromatosis in which the activity of Ras protein is increased. [Brief description of the drawings]

[0107] [Figure 1] FIG. 1 is a diagram showing stop codon mutation information of neurofibromatosis patients screened for the presence of stop codon mutations in the NF1 gene according to one embodiment. [Diagram 2] FIG. 2 shows the results of measuring the expression level of RAS (Ras-GTP) protein activated by binding with GTP in a neurofibromatosis patient screened for the presence of a stop codon mutation in the NF1 gene according to one embodiment. [Diagram 3] Figure 3 shows the results of measuring the change in Ras-GTP expression levels before and after administration of 30 μg / ml of ataluren (PTC) to fibroblasts from a patient with neurofibromatosis who was selected in one embodiment as having a stop codon mutation in the NF1 gene and had high Ras-GTP expression levels. [Figure 4] Figure 4 shows the results of electrophoresis to compare the expression levels of p-ERK before and after administration of 20 μg / ml or 30 μg / ml of ataluren (PTC-124) to fibroblasts from a patient with neurofibromatosis who was selected in one embodiment as having a stop codon mutation in the NF1 gene and had high levels of Ras-GTP expression. [Diagram 5]Figure 5 shows the results of measuring the expression ratio of ERK and p-ERK before and after administration of 20 μg / ml or 30 μg / ml of ataluren (PTC-124) to fibroblasts from a patient with neurofibromatosis who was selected in one embodiment as having a stop codon mutation in the NF1 gene and had high levels of Ras-GTP expression. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0108] The present invention will be described in more detail below with reference to the following embodiments. However, these embodiments are merely for illustrative purposes and the scope of the present invention is not limited to these embodiments.

[0109] Embodiment 1: Selection of patients with neurofibromatosis who have a stop codon mutation in the NF1 gene The NF1 gene, which is the causative gene of neurofibromatosis, was amplified using polymerase chain reaction (PCR) from DNA extracted from patients with neurofibromatosis, and then Sanger sequencing was performed. Based on the analysis results, 22 patients with stop codon mutations in the NF1 gene were selected. The stop codon mutation information in the NF1 gene of the selected patients is shown in Table 1 below (Figure 1).

[0110] [Table 1] Embodiment 2: Measurement of RAS protein activity in selected neurofibromatosis patients The activity of RAS protein was compared between skin fibroblasts from healthy subjects and patients with neurofibromatosis selected in embodiment 1 by the following method.

[0111] The donated skin fibroblasts from healthy subjects and neurofibromatosis patients were cultured in DMEM + 10% FBS + 1% Penicillin-Streptomycin medium. After culturing until the cell density reached 70-80% of the container, the medium was replaced with one without FBS, and the cells were cultured and starvation process was performed for 24 hours. Next, the cells were transferred to a medium containing FBS, and treated with EGF (epidermal growth factor), a Ras activator, diluted to a concentration of 100ng / ml and reacted at 37℃ for 5 minutes to prepare each sample. For the activity analysis of RAS protein, we used Cytoskeleton's G-LISA Ras activation Assay Biochem Kit (Absorbance Based) (Cytoskeleton, Cat No. BK131), which can measure many samples at the same time as a small amount of cells, complementing the shortcomings of the conventional measurement method, Pull-down assay.

[0112] Briefly, the method for analyzing the activity of RAS protein was as follows: After EGF treatment, cells were lysed using the cell lysis solution included in the kit, and only the supernatant was obtained, and the protein in the supernatant was quantified. The same amount of protein from all samples was placed in the wells of a plate bound to Ras-GTP binding protein, and reacted at 4°C for 30 minutes while stirring at 300 rpm on a stirrer. After washing three times with a washing solution, unbound proteins were removed, and anti-Ras antibody was added and reacted at room temperature for 45 minutes on a stirrer at 300 rpm, followed by three washing processes. After completely removing the washing solution, the secondary antibody was added and reacted at room temperature for 45 minutes on a stirrer at 400 rpm in the same manner as the primary antibody reaction process, followed by three washing processes. Through these processes, the active state of Ras bound to GTP (GDP-bound Ras) protein remains, while the inactive state of Ras bound to GDP (GDP-bound Ras) protein is removed during the washing process. Next, HRP detection reagent is added and reacted at room temperature for 15 minutes, and the absorbance is measured at 490 nm to measure the activity of Ras protein through the amount of Ras-GTP present.

[0113] As a result, we confirmed that Ras protein activity was increased in fibroblasts derived from most neurofibromatosis patients compared to healthy controls, based on the fact that high Ras-GTP signals were measured (Figure 2).

[0114] Example 3: Measurement of changes in RAS protein activity by treatment with Ataluren The donated skin fibroblasts of healthy subjects and neurofibromatosis patients were treated with ataluren at concentrations of 20 μg / ml and 30 μg / ml in a DMEM+10% FBS+1% Penicillin-Streptomycin medium, respectively, and then cultured for 3 days. After culturing until the cell density reached 70-80% of the container, the medium was replaced with one without FBS, and the cells were cultured and subjected to a starvation process for 24 hours. Next, the cells were transferred to a medium containing FBS, and then treated with EGF (epidermal growth factor), a Ras activator, diluted to a concentration of 100 ng / ml, and reacted at 37°C for 5 minutes to prepare each sample. The activity of Ras protein in each sample was measured in the same manner as described in embodiment 2.

[0115] As a result, it was confirmed that when ataluren was applied to skin fibroblasts from neurofibromatosis patients, in which Ras protein activity is increased compared to healthy individuals, the Ras-GTP signal was significantly reduced (Figure 3).This demonstrated that ataluren has a therapeutic effect on neurofibromatosis by reducing the activity of Ras protein.

[0116] Example 4: Measurement of changes in p-ERK / ERK protein expression levels following treatment with ataluren The same proteins as those used in measuring the Ras activity in Example 3 were subjected to electrophoresis using NuPAGE™ 4-12% Bris-Tris Gel (Invitrogen).

[0117] As a result, in fibroblasts from healthy individuals, there was no change in the expression of activated phosphorylated ERK (p-ERK) upon treatment with ataluren, but it was confirmed that p-ERK expression was decreased in fibroblasts from neurofibromatosis patients, in which the activity of Ras protein was reduced by treatment with ataluren (Figure 4).

[0118] To compare ERK activity more accurately, the proteins electrophoresed above were transferred to a hybond-ECL membrane, and the membrane was blocked using 3% skim milk. The membrane was then incubated with ERK and p-ERK antibodies for 24 hours at 4℃, and then washed three times with TBST. The secondary antibody was incubated for 1 hour at room temperature, and then washed three times with TBST. The expression ratio of ERK and p-ERK was measured using the washed membrane.

[0119] As a result, we found that in fibroblasts from healthy individuals, the expression level of p-ERK protein increased slightly after treatment with ataluren, but in fibroblasts from neurofibromatosis patients, in which the activity of Ras protein was reduced after treatment with ataluren, p-ERK expression decreased (Figure 5).

Claims

1. (1) one or more proteins selected from the group consisting of a RAS protein activated by binding to GTP (Ras-GTP) protein and an activated p-ERK protein; (2) A biomarker composition for predicting sensitivity to a therapeutic agent for neurofibromatosis, comprising a preparation for measuring the expression level of a gene encoding the protein.

2. The biomarker composition for predicting sensitivity to a therapeutic agent for neurofibromatosis according to claim 1, wherein the therapeutic agent for neurofibromatosis is a compound represented by the following chemical formula 1, or a pharma- ceutical acceptable salt thereof. 【Chemistry 1】 In the above formula Z is halogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, or substituted or unsubstituted heterocycloalkyl; R is hydrogen or halogen.

3. The biomarker composition for predicting sensitivity to a therapeutic agent for neurofibromatosis according to claim 2, wherein the compound represented by Chemical Formula 1 is 3-[5-(2-fluoro-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid.

4. The biomarker composition for predicting sensitivity to a neurofibromatosis therapeutic agent according to claim 1, wherein the preparation capable of measuring the expression level of the protein is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), an aptamer, and a nanoparticle, which specifically bind to the protein.

5. 2. The biomarker composition for predicting sensitivity to a therapeutic agent for neurofibromatosis according to claim 1, wherein the preparation capable of measuring the expression level of the gene encoding the protein is selected from the group consisting of a primer pair, a probe, and an antisense nucleotide that specifically binds to the gene.

6. The biomarker composition for predicting sensitivity to a therapeutic agent for neurofibromatosis according to claim 1, wherein the neurofibromatosis has a stop codon mutation in the NF1 gene.

7. A kit for predicting sensitivity to a therapeutic agent for neurofibromatosis, comprising the biomarker composition according to any one of items 1 to 6.

8. In a biological sample obtained from an individual, (1) one or more proteins selected from the group consisting of a RAS protein activated by binding to GTP (Ras-GTP) protein and an activated p-ERK protein; (2) A method for providing information necessary for predicting sensitivity to a therapeutic agent for neurofibromatosis, comprising a step of measuring the expression level of a gene encoding the protein.

9. A pharmaceutical composition for preventing or treating neurofibromatosis, comprising a compound represented by the following chemical formula 1, or a pharma- ceutical acceptable salt thereof, as an active ingredient: 【Chemistry 2】 In the above formula Z is halogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, or substituted or unsubstituted heterocycloalkyl; R is hydrogen or halogen.

10. The pharmaceutical composition for preventing or treating neurofibromatosis according to claim 9, wherein the compound represented by Chemical Formula 1 is 3-[5-(2-fluorophenyl)-[1,2,4]oxadiazol-3-yl]-benzoic acid.

11. The pharmaceutical composition for preventing or treating neurofibromatosis according to claim 9, wherein the neurofibromatosis has a stop codon mutation in the NF1 gene.

12. The pharmaceutical composition for preventing or treating neurofibromatosis according to claim 9, wherein the neurofibromatosis is caused by increased activity of Ras protein.

13. For predicting sensitivity to neurofibromatosis therapeutic agents, (1) one or more proteins selected from the group consisting of a RAS protein activated by binding to GTP (Ras-GTP) protein and an activated p-ERK protein; (2) Use of a biomarker comprising a preparation capable of measuring the expression level of a gene encoding the protein.

14. In a biological sample obtained from an individual, (1) one or more proteins selected from the group consisting of a RAS protein activated by binding to GTP (Ras-GTP) protein and an activated p-ERK protein; (2) A method for predicting the sensitivity of a neurofibromatosis patient to a therapeutic agent for neurofibromatosis, comprising a step of measuring the expression level of a gene encoding the protein.

15. A method for preventing or treating neurofibromatosis, comprising the step of administering to an individual a composition comprising, as an active ingredient, a compound represented by the following chemical formula 1, or a pharma- ceutical acceptable salt thereof: 【Chemistry 3】 In the above formula Z is halogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, or substituted or unsubstituted heterocycloalkyl; R is hydrogen or halogen.

16. 1. Use of a composition comprising, as an active ingredient, a compound represented by the following chemical formula 1, or a pharma- ceutical acceptable salt thereof, in the manufacture of a therapeutic agent for use in the prevention or treatment of neurofibromatosis: 【Chemistry 4】 In the above formula Z is halogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycle, or substituted or unsubstituted heterocycloalkyl; R is hydrogen or halogen.

17. (a) contacting a biological sample having an NF1 gene mutation with a test substance; (b) a method for screening a therapeutic agent for neurofibromatosis, comprising a step of measuring, in the contacted biological sample, the expression levels of (1) any one or more proteins selected from the group consisting of RAS (Ras-GTP) protein activated by binding to GTP and activated p-ERK protein, and (2) a gene encoding the protein.

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