WO2009113436A1

WO2009113436A1 - Method for enhancing the anti-cancer activity of radioactive ray by using fgf33 gene inhibitor in combination with irradiation with the radioactive ray, and method for reducing adverse side effects of radioactive ray

Info

Publication number: WO2009113436A1
Application number: PCT/JP2009/054104
Authority: WO
Inventors: 丹沢秀樹
Original assignee: 国立大学法人千葉大学; 高信化学株式会社
Priority date: 2008-03-14
Filing date: 2009-02-26
Publication date: 2009-09-17
Also published as: JPWO2009113436A1

Abstract

The object is to enhance the anti-cancer activity of radiation therapy. A group of genes involved in the resistance to a radioactive ray are successfully identified. Attention is directed to a specific gene among the group of genes, and it is found that the therapeutic effect of radiation therapy can be enhanced and the adverse side effects of radiation therapy can be reduced by using an inhibitor of a product of the gene in combination with the radiation therapy. An enhancer of the therapeutic effect of radiation therapy comprises an inhibitor of fibroblast growth factor receptor-3 (FGFR3) as an active ingredient.

Description

Methods for enhancing the anticancer effects and reducing side effects of radiation using FGFR3 gene inhibitor in combination with radiation

The present invention relates to a method for enhancing the anticancer effect of radiation and a method for reducing side effects by using a FGFR3 gene inhibitor in combination with irradiation.

Background art

book

As part of cancer treatment, radiation therapy is known that uses the ionizing action of radiation such as X-rays and γ-rays to control malignant tumors. Radiation therapy aims to focus the cancer locally without complications by concentrating the dose locally on the cancer and reducing the dose to surrounding normal tissue. Radiation therapy is effective not only for radical radiation therapy but also for palliative and symptomatic treatment. In any case, in order to achieve the above objectives, an appropriate radiation treatment plan including the irradiation field, irradiation method, dose splitting method, combination therapy, etc. will be established for each case. When planning radiation therapy, the patient's age, general condition (PS), primary lesion, stage, histopathology, extent of lesion development, risk organ location, radical or symptomatic or palliative, Consider the details of treatment, complications, etc.

As side effects of radiation therapy, there are side effects during the period of radiation therapy (acute radiation damage) and side effects that occur after radiation therapy (spontaneous radiation damage). Specifically, although it varies from case to case, common side effects (acute radiation damage) include fatigue, skin changes, and loss of appetite. In addition, a common side effect (spontaneous radiation damage) is gastrointestinal bleeding when the gastrointestinal tract is included within the irradiated range.

By the way, the FGFR family 1 is classified in terms of connectivity and tissue distribution.

The FGFR protein is located in the extracellular region and consists of three immunoglobulin-like domains, a cytoplasmic tyrosine kinase, and a hydrophobic signal. Fibroblast growth factor receptor 3 (FGFR3) has been shown to inhibit chondrocyte proliferation and differentiation. It is. In addition, FGFR3 active mutations are known to develop soft bone dysplasia and lethal osteodysplasia, which are short stature chondrogenic dysplasias. There have also been reports of cases of skull fusion due to FGFR3 mutations.

In addition, the expression of FGFR3 gene has been reported to be increased in squamous cell carcinoma, bladder cancer and hepatocellular carcinoma, and when it is overexpressed in multiple myeloma, apoptosis is suppressed and proliferation is increased. Disclosure of the invention

However, there is no known correlation between the success of radiotherapy and the expression of the FGFR3 gene, and the effect on radiotherapy in the presence of FGFR3 inhibitors. Therefore, in view of the above-described circumstances, an object of the present invention is to provide a radiation therapy effect enhancing agent combined radiotherapy capable of enhancing an anticancer effect by radiation therapy.

As a result of intensive studies by the present inventors in order to achieve the above-mentioned object, the inventors succeeded in identifying a gene group involved in radiation resistance, and focusing on a specific gene from these groups, It has been found that the combined use of the inhibitor of the gene product enables enhancement of the radiotherapy effect and reduction of side effects, and the present invention has been completed.

The present invention includes the following.

(1) A radiation therapeutic effect enhancer comprising a fibroblast growth factor receptor 3 (FGFR3) inhibitor as an active ingredient.

(2) The radiotherapy effect enhancer according to (1), wherein the FGFR3 inhibitor is a substance that inhibits autophosphorylation of FGFR3.

(3) The above FGFR3 inhibitor is N-2-[[4- (Jetylamino) butyl] amino-6- (3,5-dimethoxyphenyl) pyrid [2,3_d] pyrimidine-7-yl] _N The agent for enhancing radiotherapeutic effect according to (1), which is' _ (1,1-dimethylethyl) urea.

The present invention also includes a method for treating cancer.

That is, the cancer treatment method according to the present invention includes a step of administering a composition comprising a fibroblast growth factor receptor 3 (FGFR3) inhibitor as an active ingredient to a radiation treatment target patient. Here, as the FGFR3 inhibitor, a substance that inhibits autophosphorylation of FGFR3 is preferably used. In particular, N-2 _ [[4- (Jetylamino) ptyl] amino-6- (3,5-dimethyl) It is more preferable to use toxiphenyl) pyrid [2,3-d] pyrimidine-7-yl] _] '-(1,1-dimethylethyl) urea. The administration method and administration route of the FGFR3 inhibitor are not particularly limited. In addition, the radiotherapy for the patient may be performed before the administration of the FGFR3 inhibitor, at the same time as the administration of the FGFR3 inhibitor, or after the administration of the FGFR3 inhibitor.

In the cancer treatment method according to the present invention, the cancer to be treated is not particularly limited. For example, lung cancer, ovarian cancer, pancreas cancer, stomach cancer, gallbladder cancer, kidney cancer, prostate cancer, breast cancer, esophageal cancer, liver cancer Oral cancer, colon cancer, colon cancer, uterine cancer, bile duct cancer, islet cell cancer, adrenal cortex cancer, bladder cancer, testicular cancer, testicular tumor, thyroid cancer, skin cancer, malignant carcinoid tumor, malignant melanoma, bone Examples include sarcomas, soft tissue sarcomas, neuroblastomas, Wilms tumors, retinoblastomas, melanomas, and daliomas.

This specification includes the contents described in the specification of Japanese Patent Application No. 2008-066443, which is the basis of the priority of the present application, and in the drawings or drawings. Brief Description of Drawings

FIG. 1 is a characteristic diagram showing the relationship between the survival rate and the X-ray irradiation dose of squamous cell carcinoma cell lines HSC2, HSC3, HSC4, Ca9-22, 0K92, and HO-1-u-1.

Figure 2 shows the results of a pathway analysis of 1 67 genes as a group of genes in which a 5-fold or higher expression was observed in the radiation resistant strain (HSC2) compared to the radiation sensitive strain (HSC3). It is a characteristic view which shows a network.

FIG. 3 is a characteristic diagram showing the network obtained as a result of further narrowing down the network shown in FIG. 2 by the cancer function.

FIG. 4 is a characteristic diagram showing the relationship between the irradiation dose and the elapsed time after irradiation and the expression level of the FGFR3 gene.

FIG. 5 is a characteristic diagram (A) showing the inhibition of FGFR3 gene expression by siRNA against the FGFR3 gene, and a photograph showing the protein expression level.

Figure 6 shows the results for the radiation-resistant strain when PD173074, an inhibitor of FGFR3, was administered. It is a characteristic view which shows the X-ray sensitivity enhancement effect.

FIG. 7 is a characteristic diagram showing the effect of enhancing X-ray sensitivity in a cell line showing moderate resistance to radiation when PD173074, an inhibitor of FGFR3, is administered.

FIG. 8 is a characteristic diagram showing the X-ray sensitivity enhancement effect in a radiation-sensitive strain when PD173074, an inhibitor of FGFR3, was administered.

FIG. 9 is a characteristic diagram showing the tumor cell growth inhibitory effect when combined with administration of PD173074, an FGFR3 inhibitor, and X-ray irradiation for mouse transplantation radiation-resistant cancer. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the radiotherapy effect enhancer according to the present invention will be described in detail. In the present invention, the radiotherapy capable of enhancing the radiotherapy effect includes, for example, X-ray, gamma ray, electron beam, proton beam, helium beam, carbon ion beam, neon ion beam, argon ion beam, silicon ion beam, A treatment method in which radiation such as negative pion beams or neutron beams is irradiated directly or in a divided manner (eg, several times a day, several times over a period of 1 to 2 months). It is.

In radiation therapy, the initial energy of radiation can be appropriately selected according to the size, condition, or location of the tumor of the patient, etc., or the surrounding conditions, but is usually about 100 to 500 Me V / n. Preferably about 200-300 Me VZn, most preferably about 29 OMe V, n. The radiation dose to the patient can be appropriately selected according to the size, state or location of the tumor of the patient, etc., or the surrounding conditions, but is usually about 0.1 to 100 Gy. , Preferably about 1 to 10 Gy, most preferably about 5 Gy, and the irradiation rate of radiation to a patient or the like is usually about 0.05 to 50 Gy / "min, preferably Is about 0.5-1 OGy / min, most preferably about 3 Gy / min, and the energy (linear energy transfer: LET) that the radiation gives to the tissue is the size and condition of the patient's tumor Or it can be appropriately selected according to the site, etc., but is usually about 50 to 70 ke VZ / m, preferably about 50 to 60 ke νΖμπι, and most preferably about 50 ke V m. The The tumor to be treated by radiation therapy is not particularly limited. For example, lung cancer, ovarian cancer, pancreas cancer, stomach cancer, gallbladder cancer, kidney cancer, prostate cancer, breast cancer, esophageal cancer, liver cancer, oral cancer, colon Cancer, colon cancer, uterine cancer, cholangiocarcinoma, knee cell carcinoma, adrenal cortical cancer, bladder cancer, testicular cancer, testicular tumor, thyroid cancer, skin cancer, malignant carcinoid tumor, malignant melanoma, osteosarcoma, soft tissue sarcoma, Neuroblastoma, Wilms tumor, retinoblastoma, melanoma or darioma.

The radiotherapeutic effect enhancer according to the present invention can increase the therapeutic effect in the radiotherapy as described above. Therefore, since the therapeutic agent for enhancing radiotherapy effect according to the present invention can increase the therapeutic effect in the radiotherapy as described above, a high therapeutic effect can be expected even when the radiation dose is reduced.

The radiotherapy effect-enhancing agent according to the present invention comprises a fibroblast growth factor receptor 1 3 (FGFR3) inhibitor as an active ingredient. This is because, in the patient group showing radiation resistance, an increase in the expression of the FGFR3 gene was observed compared to the patient group showing radiosensitivity, and the activity of FGFR3, which is the FGFR3 gene product, was inhibited. It is based on new knowledge that it can be enhanced. Furthermore, the present invention can further enhance the radiosensitivity by inhibiting the activity of the FGFR3 gene product, FGFR3, even in patients who have moderate tolerance to radiation and patients who are sensitive to radiation. Based on these new findings.

An FGFR3B inhibitor is a substance that significantly inhibits FGFR3 signaling. In the present invention, any substance may be used as the FGFR3B inhibitor as long as it can significantly reduce FGFR3B signal transduction. In the present invention, examples of the FGFR3 inhibitor include substances that reduce the expression level of the FGFR3 gene, substances that inhibit the mRNA of the FGFR3 gene, substances that inhibit the FGFR3 gene at the protein level, and the like. As the FGFR3 inhibitor, known substances that are currently clinically used as FGFR3 inhibitors can be used.

In particular, it is preferable to use a conventionally known FGFR3 inhibitor as the FGFR3 inhibitor in the present invention. Conventionally known FGFR3 inhibitors include, for example, N-2-[[4- (Jetylamino) butyl] amino-6- (3,5-dimethoxyphenyl) pyrid [2,3-d] pyrimidine-7- -N '-(1,1-dimethylethyl) urea, ie PD173074 The IC ₅ of PD173074. Is known to inhibit the cell cycle of G0 / G1 phase and to inhibit FGFR3 autophosphorylation in FGFR3-expressing cells. In addition, it is known that the selectivity of PD173074 for FGFR3 is about 100 times higher than VEGF receptor, IGF-1 receptor, and MAPKs.

PD173074 is known to be a cell-permeable pyridopyrimidine compound. PD173074 is known to act as a potent and reversible ATP competitive inhibitor for FGF and VEGF receptors (IC ₅₀ = 21.5 M for FGFR1). PD173074 also inhibits PDGFR and c_Src only at very high concentrations (IC ₅₀ = 17.6 / M and 19.8 μΜ, respectively) and 50 for EGFR, InsR, MEK and cPKC. It is known that even a high concentration of μΜ has little effect. In addition, PD173074 inhibits autophosphorylation of endogenous FGFRl (IC ₅ <5 μM) and overexpressed VEGFR2 (IC ₅₀ <200 μM) in vitro in ΝΙΗ3Τ3 cells, and FGF in mice in vivo. And have been shown to inhibit VEGF-induced angiogenesis. PD173074 is described in Koziczak, M., et al. 2004. Oncogene 23, 3501. Trudel, S., et al. 2004. Blood 103, 3521. Skaper, SD, et al. 2000. J. Neurochera. 75, 1520 Reference can be made to Mohammadi, M., et al. 1998. E B0 J. 17, 5896.

In addition to PD173074, conventionally known compounds such as CHIR258, SU5402, PRU-001, and PKC412 can be used as the FGFR3B inhibitor.

Examples of the FGFR3 inhibitor include siRNA (small interfering RNA), antisense RNA, ribozyme, and all vector DNAs capable of producing these RNAs for the FGFR3 gene. Such a nucleic acid can inhibit or suppress the expression of the FGFR3 gene.

The siRNA used as an FGFR3 inhibitor is a small double-stranded RNA containing a sequence that is complementary to the mRNA corresponding to the FGFR3 gene (that is, the mRNA encoded by the gene) or its alternatively spliced mRNA. The mRNA or its alternatively spliced mRNA is selectively processed through the formation of an RNA-nuclease complex (RNA induced silencing complex or RISC).

Alternatively, siRNA may be derived from its precursor double-stranded RNA (shRNA) through processing by Dicer, an intracellular RNase. shRNA is Double-stranded RNA having a loop between the sense strand sequence and the antisense strand sequence of siRNA, preferably 1-6, preferably 2-4 polyU overhangs at its 3 'end including. The shRNA is processed into siRNA by a dicer belonging to the RNase III family, and then siRNA is stranded, and its sense strand RNA forms a complex (RISC) with RNase H, thereby siRNA. The target mRNA having a sequence complementary to the sequence is cleaved, and as a result, the expression of the FGFR3 gene is suppressed.

Therefore, both the above siRNA and its precursor shRNA can be used as the FGFR3 inhibitor in the present invention.

When introducing the siRNA of the present invention into the body, it is preferable to directly inject the siRNA into the affected area or to use a vector capable of expressing the siRNA. Alternatively, siRNA or vectors can be complexed with and used with ribosomes such as lipofectamine, lipofectin, selfectin and other positively charged ribosomes (eg, positively charged cholesterol) or microcapsules (eg, Nakanishi Mamoru et al., Protein Nucleic Acid Enzymes, 44, 11, 48-54, 1999; Clinical Cancer research 59: 4325-4333, 1999; Wu et al., J. Biol. Chem. 262: 4429, 1987). Positively charged ribosomes are preferably used because the cell membrane is negatively charged, but it is assumed that the positively charged ribosome-nucleic acid complex is transferred into the cytoplasm or nucleus after being incorporated into the cell by endocytosis. Is done. Alternatively, the therapeutic nucleic acid is about

It can also be encapsulated in nanoparticles of 500 nm or less. Examples of nanoparticles include hollow nanoparticles formed from hepatitis B virus envelope L particles. Nucleic acids are encapsulated in the particles by an electrical mouth position. In this case, the nucleic acid-encapsulated particles are Can be delivered to the liver (T. Yamada et al.,

Nature Biotech 21 (8): 885-890, 2003). When encapsulating a nucleic acid in a liposome, the nucleic acid is treated with protamine sulfate to cause condensation to form a nucleic acid-protein complex, and then encapsulated in a positively charged lipid or polymer micelle.

Ribosome-nucleic acid complexes can be obtained, for example, by the reverse phase evaporation method (F. Szoka et al., Biochim. Biophys.

Acta, 601: 559, 1980), vortex shaking method, calcium fusion-EDTA chelate method (Yasushi Kaneda, Experimental Medicine Vol.22, No.14 (extra), 147-15 2 pages, 2004), etc. It can be produced by a known method.

Examples of the nucleic acid that can be used in the present invention include an expression vector containing a DNA sequence encoding the siRNA or a precursor thereof under the control of a promoter. One example of an expression vector is a hairpin vector. This vector includes DNA encoding a hairpin RNA in which the sense strand RNA sequence and the antisense strand RNA sequence are covalently linked via a single-stranded loop sequence, wherein the DNA is transcribed in a cell. Is a vector that forms the hairpin RNA and is processed by Dicer to form the siRNA. A poly-T sequence consisting of 1 to 6, preferably 1 to 5, T at the end of the hairpin DNA encoding siRNA, as a transcription stop signal sequence or for overhang, for example Four or five poly T sequences are linked. Short hairpin RNA (shRNA) as a siRNA precursor transcribed from vector DNA should have an overhang consisting of 2 to 4 U at the 3 ′ end of its antisense strand, and due to the presence of the overhang, Sense strand RNA and antisense strand RNA can increase stability against degradation by nucleases. There is one endogenous dicer in humans, which plays a role in converting long dsRNA and precursor micking RNA (miRNA) into siRNA and mature miRNA, respectively.

In general, a plasmid vector contains a drug resistance gene (for example, nemycin resistance gene, ampicillin resistance gene, puromycin resistance gene, hygromycin resistance gene), transcription, in addition to the siRNA-encoding DNA sequence and promoter. It can contain stop sequences, unique restriction sites or multiple cloning sites, replication origins, etc.

As the viral vector, for example, an adenovirus vector, an adeno-associated virus vector, a lentiwinores vector, a retroui / res vector (leukemia winores vector, etc.), a herpes virus vector and the like can be used. The virus vector is preferably of a type lacking self-replicating ability, for example, so as not to cause disease when used in humans. For example, in the case of an adenovirus vector, a self-replication ability-deficient adenovirus vector lacking the E1 gene and E3 gene (for example, pAdeno-X from Invitrogen) can be used. Virus vectors can be constructed using methods described in the literature (US Pat. No. 5,252,479, International publication W094 / 13788 etc.).

The plasmid vector of the present invention can be introduced into a patient's body in the form of a complex formed with a positively charged liposome such as lipophectamine, lipofectin, self-actin, positively charged cholesterol, etc. (Mr. Nakanishi et al., Supra). Wu et al., Supra). In addition, viral vectors can be introduced into cells by introducing them into affected areas and infecting cells (L. Zender et al., Proc. Natl. Acad. Sci. USA (2003), 100: 77797-7802; H. Xia et al., Nature Biotech. (2002), 20: 1006-1010; XF Qin et al., Proc. Natl. Acad. Sci. USA (2003), 100: 183-188; GM Barton et al., Pro Natl. Acad., Sci. USA (2002), 99: 14943-14945; JD Hommel et al., Nature Med. (2003), 9: 1539-1544). In particular, it has been confirmed that adenovirus vectors or adeno-associated virus vectors can introduce genes into various cell types with very high efficiency. This vector is also not integrated into the genome, so its effect is transient and safer than other viral vectors.

The antisense nucleic acid used as the FGFR3 inhibitor is either RNA or DNA containing a sequence of mRNA corresponding to the FGFR3 gene, or a sequence complementary to a partial sequence thereof. The partial sequence may include a sequence consisting of about 30 or more, 50 or more, 70 or more, 100 or more, 150 or more, 200 or more, or 250 or more and less than full length nucleotides in the sequence of FGFR3 gene or mRNA.

In addition to natural nucleotides, antisense nucleic acid nucleotides

Modified nucleotides having groups such as (fluorine, chlorine, bromine or iodine), methyl, carboxymethyl or thio groups can be included. Antisense nucleic acids are well-known

It can be synthesized using DNA / RNA synthesis technology or DNA recombination technology. When synthesizing by DNA recombination technology, vector DNA containing the base sequence of the FGFR3 gene is converted into a saddle shape and subjected to polymerase chain reaction (PCR) using primers that sandwich the sequence to be amplified, and then the target sequence. Can be amplified and cloned into a vector as necessary to produce antisense DNA. Alternatively, the thus obtained DNA having the amplified target sequence is inserted into a vector, the vector is introduced into a eukaryotic or prokaryotic cell, and antisense RNA can be obtained using the transcription system. Whether it is DNA or RNA, the antisense nucleic acid of the present invention can inhibit or suppress transcription or translation by binding to the FGFR3 gene or corresponding mRNA. In order to deliver the antisense nucleic acid of the present invention to a patient, the antisense nucleic acid may be encapsulated in a positively charged ribosome as described above, or the antisense nucleic acid may be, for example, a strong pol II or pol III promoter. It can be incorporated into a vector (the above mentioned plasmid or viral vector) as controlled. Furthermore, the ribozyme used as an FGFR3 inhibitor is an RNA having catalytic activity and has an activity of cleaving mRNA corresponding to the target FGFR3 gene. This cleavage inhibits or suppresses the expression of the FGFR3 gene. Ribozyme-cleavable target sequences are generally known to be sequences containing NUX (N = A, G, C, U; X = A, C, U), eg, GUC triplets. Ribozymes include hammerhead ribozymes. The hammerhead ribozyme is a nucleotide sequence that includes a nucleotide sequence that constitutes a sensor site, a nucleotide sequence that can form a cavity that can stably capture Mg ²⁺ ions only when RNA is bound to the sensor site, and a target RNA cleavage site. Nucleotide sequences comprising regions that are complementary to surrounding sequences can be included.

In order to deliver the ribozyme of the present invention to a patient, a drug delivery system is constructed by a method such as encapsulating the ribozyme in a ribosome (preferably, a positively charged ribosome) or incorporating it into a virus vector such as an adeno-associated virus. That's right.

A ribozyme can be incorporated into a vector so that it can be expressed. Promoters for expressing ribozymes include pol II or pol III promoters. Preferred promoters are pol III promoters, such as tRNA promoters from mammals, more preferably tRNAVal promoters.

Furthermore, an antibody used as an FGFR3 inhibitor means an antibody or a functional fragment thereof that inhibits or suppresses the in vivo function of the FGFR3 protein encoded by the FGFR3 gene or a variant thereof. Examples of antibodies against FGFR3 protein include monoclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, bispecific antibodies, and synthetic antibodies. In addition, functional fragments of the antibody include Fab fragments, F (ab ′) ₂ fragments, scFv, and the like. Preferred antibodies suitable for use in the present invention are human or humanized antibodies, particularly human or humanized monoclonal antibodies, that cause little or no side effects from anaphylaxis. The antibody class and subclass can be of any type. For example, IgG, IgM, IgE, IgD, and IgA are included as antibody classes, and IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2 are included as subclasses. Antibodies may also be derivatized by pegylation, acetylation, glycosylation, amidation, etc.

The human antibody can be obtained, for example, by a phage display library method (T. Thomas et al., Mol. Immunol. 33: 1389-1401, 1996) or a method using a human antibody-producing mouse (I. Ishida et al., Cloning Stem Cell 4: 91-102, 2002).

A human antibody-producing mouse, for example, introduces a human chromosome fragment containing a human antibody-producing gene into a human artificial chromosome, and then incorporates the artificial chromosome into, for example, the mouse embryonic stem cell genome using the microcell method. Transplant into the uterus, give birth to chimeric mice, and create a homozygous offspring mouse that contains the human antibody gene by mating male and female chimeric mice and can therefore produce human antibodies. Therefore, it can be produced. The human antibody-producing transgenic mouse is immunized with the FGFR3 protein as an antigen, and then the spleen is removed, and the spleen cells and mouse myeloma cells are fused to form a hybridoma, and the desired monoclonal antibody is selected. be able to.

Delivery of the antibody or fragment thereof to the patient can be accomplished either alone or in the form of the antibody or fragment thereof encapsulated in, for example, ribosomes (preferably positively charged ribosomes), microcapsules or nanoparticles, usually with a suitable carrier (shaped In combination with a non-oral route (for example, intravenous administration or topical administration).

The above FGFR3 inhibitors can significantly enhance the anti-cancer effect of radiation therapy when administered together with radiation therapy for patients diagnosed as requiring radiation therapy. Therefore, anticancer effects of radiation therapy can be expected even for carcinoma cases where the effect of radiation therapy has been low. In addition, FGFR3 inhibitors reduce the level of radiation resistance, so the dose of irradiated radiation has been reduced. It is possible to expect a therapeutic effect equivalent to or better than. Therefore, side effects caused by radiation therapy can be reduced, or it can be applied to non-indication cases of radiation therapy for reasons such as general condition, and an effective treatment method can be provided for the patient. The FGFR3 inhibitor is preferably administered to patients prior to radiation therapy. The FGFR3 inhibitor can be administered by any appropriate route. Suitable routes include oral, rectal, intranasal, topical (including buccal and sublingual), intravaginal, and parenteral (subcutaneous, intramuscular, intravenous, intradermal, intrathecal and dura mater) Including the outside). In addition, the FGFR3 inhibitor can be administered directly or locally to, for example, a tissue in which a tumor is present, or can be administered intravenously, artery, subcutaneously, intramuscularly or intraperitoneally. Alternatively, a tumor can be imaged using any device available in the art, such as nuclear magnetic resonance imaging or computed tomography, and administered directly, for example, by stereotaxic injection.

Here, in order to enhance the effect of radiotherapy by administering an FGFR3 inhibitor, the dose of FGFR3 inhibitor can be 10 to 100 mg / m ² (body surface area), preferably 10 to It can be 50 mg / tn ² (body surface area), more preferably ^{20 to} 50 mg / m ² (body surface area). .

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope which concerns on this invention is not limited to a following example.

Example 1

(Radiosensitivity test)

To select cell lines with different radiosensitivity, X-rays were irradiated at 2Gy, 4Gy and 6Gy against squamous cell carcinoma cell lines HSC2, HSC3, HSC4, Ca9-22, 0K92 and H0-l_u_l, and a survival curve was obtained. Created. As a result, HSC2 was selected as a radiation resistant strain and HSC3 was selected as a radiation sensitive strain. (Figure 1 )

<Comparison of gene expression status between radiation-sensitive and radiation-resistant cell lines> (Microarray analysis)

Microarray analysis using radiation-resistant and radiation-sensitive strains identified genes that specifically enhanced expression in resistant strains. As a microarray Affymetrix U133 Plus 2.0 on which 54675 types of probes were immobilized was used. For microarray analysis, GeneChip Operating Software 1.1 (Affymetrix) and GeneSpring 6.1 (Silicon Genetics) were used as analysis software.

As a result, 16.7 genes were identified as a gene group in which expression of 5 times or more was observed in the radiation resistant strain (HSC2) compared to the radiation sensitive strain (HSC3). These genes are genes that are specifically enhanced in radiation-resistant strains, and are considered to be radiation-resistance-related genes in squamous cell carcinoma cells.

(Analysis by Pathway analysis and Quantitative real-time RT-PCR method) Pathway analysis was performed on 1 6 7 genes identified as genes related to radiation resistance narrowed down by microarray analysis, and 1 6 7 genes Then, what kind of network was formed was examined. Specifically, the pathway analysis used software, Ingenuity Pathway Analysis (manufactured by Ingenuity systems). As a result, 40 genes out of 1 6 7 genes

It was found that these three networks formed one larger network (Fig. 2). On the other hand, if we classify 16 7 genes from a functional viewpoint, we can see that the genes related to cancer function are most significantly related. The 25 genes formed a network related to cancer function, and the 25 genes were all included in the 40 genes obtained from the previous pathway analysis. In other words, this 25 gene was considered to be a gene strongly associated with radiation resistance in squamous cell carcinoma cells (Fig. 3).

From the 25 genes selected by these microarray analysis and pathway analysis, the FGFR3 gene was selected as an apparent gene expression inhibitor gene. The raRNA expression status of the FGFR3 gene in the resistant strain (HSC2) and the susceptible strain (HSC3) was confirmed by the Quantitative real-time RT-PCR method with respect to the X-ray irradiation dose and the elapsed time after irradiation (Fig. 4). Fig. 4A shows the relationship between X-ray irradiation dose and mRNA expression level, and Fig. 4B shows the relationship between elapsed time after X-ray irradiation and mRNA expression level. Figure 4 Shown in A and B Thus, compared to the HSC3 strain, it was confirmed that the expression of the FGFR3 gene was always increased in the HSC2 resistant strain under X-ray irradiation conditions.

(Suppression of FGFR3 gene expression by iRNA)

Using a radiation-resistant strain HSC2 in which the FGFR3 gene is highly expressed, siRNA was introduced to examine changes in X-ray resistance. DhamaFECT (Dharmacon) was used as the introduction reagent. FGFR3 gene siRNA (Dharmacon) was introduced into HSC2, and after 96 hours, 200 / 4ml raediura / di sh (0, 2 or 4Gy), 400 / 4ml medium / di sh (6Gy), 800 / Cells were transferred to three 6cra dishes at a rate of 4ml medium / di sh. (8Gy) 24 hours later, irradiated with X-rays, incubated for 8-10 days, and then clonogenic survival assay. In the clonogenic survival assay, cells were stained and fixed with a crystal violet solution (25% methanol added) (Sigma Chemical Co.) on 6 cm dishes after incubation for 8-10 days. Count the number of cell colonies (consisting of more than 50 cells), and set the plating eff iciency to (average viable cell colony number) I (number of cells spread in one dish) of the cells in three dishes. Direct lighting, plating eff ic iency after shooting The value divided by the plating eff iciency of OGy was plotted in the graph as the survival rate (Clonogenic fract ions) The control cells were untreated, the cells to which only the introduction reagent without siRNAA was added (Vehicle), non -target s iRNA (siNT) was used Plots of survival rates are shown in Fig. 5 A. As shown in Fig. 5 A, cells transfected with s siRNA against the FGFR3 gene were included in the control group. In comparison, the resistance to X-rays was reduced, and it was considered that the sensitivity to X-rays was increased by suppressing the expression of the FGFR3 gene. This is shown in Fig. B. As can be seen from Fig. 5 B, it was confirmed that the expression of the FGFR3 gene protein was suppressed in the siRNA-transfected cells against the FGFR3 gene, which was considered to be a radiation resistance gene. .

(Enhancement of cancer killing ability by combined use of FGFR3 inhibitor and X-ray) In vitro, we examined whether PD173074 (sigma-aldrich), an inhibitor of FGFR3, can be used in combination with X-rays against radiation-resistant HSC2. We also examined the combined effects of PD173074 and X-rays on the radiation-sensitive strain HSC3 and Ca9-22 (see Fig. 1), which is moderately resistant to radiation.

As the inhibitor solvent, DMS0 was used to dissolve the inhibitor. In the experiment, the DMS0 concentration in the medium was adjusted to 0.1% or less, and the inhibitor was mixed. The inhibitor concentration was 5 nM, 10 nM, 25 nM, 50 nM or 又は ηΙΟΟ.

Experiments were carried out by using 200 radiation resistant strains HSC2, HSC3 or Ca9-22 in a 6cm dish / 4ml mediura / di sh (0, 2 or 4Gy), sputum / 4ml medium / di sh (6Gy), 800 / 4ml medium / dish (8Gy), and the medium was added the next day with inhibitors added at various concentrations. After 1-2 hours of irradiation, X-rays were irradiated and cultured for 8-10 hours to obtain a level by clonogenic survival assay. In the clonogenic survival assay, cells were stained and fixed with a crystal violet solution (25% methanol added) (Sigma Chemical) on 6 cm dishes of cells after 8-10 曰 coin-cuvette. Count the number of viable cell colonies (consisting of 50 or more cells) in each dish, and plating (average viable cell colony number) I (number of cells spread in one dish) of three dish cells The efficiency obtained by dividing the plating efficiency after irradiation with each dose by the plating efficiency of OGy was plotted as the survival fraction (Clonogenic fractions). Since the inhibitor was dissolved in 0.1% or less of DMS0, 0.1% DMS0 added and untreated HSC2, HSC3 or Ca9_22 cells were used as controls. The results of plotting the survival rate are shown in Figs. Fig. 6 shows the results for the radiation-resistant strain HSC2, Fig. 7 shows the results for Ca9-22, which shows moderate resistance to radiation, and Fig. 8 shows the results for the radiation-sensitive strain HSC3. is there. As can be seen from Fig. 6, when the function of FGFR3 was inhibited by administration of an FGFR3 inhibitor, it was considered that the sensitivity to X-rays in the radiation resistant strain increased. In addition, as shown in Fig. 7, it was considered that the sensitivity to X-rays also increased in cell lines that showed moderate resistance to radiation. Furthermore, as shown in Fig. 8, it was considered that the sensitivity to X-rays was also increased in the radiation-sensitive strain. <Radiation enhancement effect by FGFR3 gene expression inhibitor--in vivo>

We examined whether PD173074 (sigma-aldrich), an inhibitor of FGFR3, was used in combination with X-rays to show a combined action against radiation-resistant strain HSC2. Animal experiments were conducted in accordance with the standards of Canadian Council Animal Care (CCAC). All mice were premature, purchased at 4 weeks of age, and used for experiments from 6 weeks of age. First, the HSC2 strain was prepared to 5 × 10 ⁶ and inoculated subcutaneously into the right thigh of a mouse using a 29G needle (Myjector 1: TERUM0). The mice were bred while observing, and X-ray irradiation and administration of an FGFR3 inhibitor were started when the tumor diameter reached 8-9 mm.

FGFR3 inhibitor was administered intraperitoneally at 25 mg / kg. X-ray (4Gy) irradiation was started 2 hours after administration of the FGFR3 inhibitor. Administration of FGFR3 inhibitor and X-ray (4Gy) irradiation were performed once a day for 5 days. In addition, a group in which no treatment was performed, a group in which only FGFR3 inhibitor was administered, and a group in which only X-ray (4Gy) irradiation was performed were used as comparison targets.

The results are shown in FIG. In Fig. 9, "control" indicates the group that did not treat anything (n = 5), "PD173074" indicates the group that received only FGFR3 inhibitor administration (n = 5), and "4Gy" indicates X-ray. (4Gy) shows the group (n = 5) that received only irradiation, and “PD173074 + 4Gy” shows the group (n = 5) that combined administration of FGFR3 inhibitor and X-ray (4Gy) irradiation. From the results shown in Fig. 9, it can be seen that when FGFR3 inhibitor administration and X-ray (4Gy) irradiation are used in combination, compared with FGFR3 inhibitor administration or X-ray (4Gy) irradiation alone. It was revealed that the growth of tumor cells can be significantly suppressed. In particular, the effect of inhibiting the growth of tumor cells when FGFR3 inhibitor administration and X-ray (4Gy) irradiation are used in combination is the effect of tumor cells when FGFR3 inhibitor administration or X-ray (4Gy) irradiation is performed alone. It was so remarkable that it could not be predicted from the growth inhibitory effect.

First, from the results of (microarray analysis), we identified 167 highly reliable genes as genes related to cisbratin resistance. 167 genes selected by microarray analysis (pathway angle analysis and Quantitative real-time

Based on the results of RT-PCR analysis, 25 genes with higher reliability were selected as genes related to X-ray resistance. Quantitative for the 25 genes The mRNA expression was confirmed by real-time RT-PCR, and the FGFR3 gene that was highly expressed in the resistant strain and in which the inhibitor was present was selected.

In addition, we attempted (suppression of FGFR3 gene expression by siRNA) using a resistant strain HSC2 in which the FGFR3 gene was highly expressed. In resistant strains into which siRNA had been introduced, resistance to X-rays decreased compared to the control group. It was considered that the sensitivity to X-rays was increased by suppressing the expression of the FGFR3 gene.

In addition, the suppression of FGFR3 function by chemical substances was confirmed. In order to confirm the effect of FGFR3 inhibitors and X-rays on resistant strains, we first used PD173074, an inhibitor of the FGFR3 gene, and examined the combined effects of X-rays on resistant strains HSC2. As a result, the effect of improving the sensitivity to X-rays by administering an FGFR3 inhibitor was recognized. In addition, other squamous cell carcinoma cell lines that are not radiation-resistant strains were also effective in improving sensitivity to X-rays by administering FGFR3 inhibitors.

Furthermore, in vivo experimental results demonstrated that the suppression of tumor cell growth by the combined use of FGFR3 inhibitor and X-ray irradiation at the level of animal experiments.

These results revealed that the FGFR3 gene is an important X-ray resistance gene. Furthermore, it was confirmed that the action of X-rays was enhanced by using PD173074, an FGFR3 inhibitor, together with X-rays. Industrial applicability

According to the radiation therapy effect enhancer according to the present invention, the anticancer effect by radiation therapy can be enhanced. Therefore, by using the radiotherapy effect enhancer according to the present invention, for example, a radiotherapy can be performed with a low dose, and a treatment method that reduces the burden on the patient can be provided. Moreover, by using the radiotherapy effect enhancer according to the present invention, for example, radiotherapy with a low dose becomes possible, and side effects due to radiotherapy can be reduced. Furthermore, an effective therapeutic effect overcoming the radiation therapy resistance can be obtained by increasing the radiation therapy effect on the tumor having radiation resistance.

All publications, patents and patent applications cited in this specification are used as is for reference. Incorporated in this specification

Claims

The scope of the claims

1. A radiation therapeutic effect enhancer comprising a fibroblast growth factor receptor 3 (FGFR3) inhibitor as an active ingredient.

2. The radiotherapeutic effect enhancer according to claim 1, wherein the FGFR3 inhibitor is a substance that inhibits autophosphorylation of FGFR3.

3. The above FGFR3 inhibitor is N_2 _ [[4- (Jetylamino) butyl] amino _6_ (3,5-dimethoxif: nyl) pyrid [2,3-d] pyrimidine-7-yl]--' 2. The radiotherapeutic effect enhancer according to claim 1, which is (1,1-dimethylethyl) urea.