WO2017170981A1 - Prophylactic or therapeutic agent for fgfr3 disease - Google Patents

Prophylactic or therapeutic agent for fgfr3 disease Download PDF

Info

Publication number
WO2017170981A1
WO2017170981A1 PCT/JP2017/013524 JP2017013524W WO2017170981A1 WO 2017170981 A1 WO2017170981 A1 WO 2017170981A1 JP 2017013524 W JP2017013524 W JP 2017013524W WO 2017170981 A1 WO2017170981 A1 WO 2017170981A1
Authority
WO
WIPO (PCT)
Prior art keywords
ggti
inhibitor
ips cells
fti
drug
Prior art date
Application number
PCT/JP2017/013524
Other languages
French (fr)
Japanese (ja)
Inventor
範行 妻木
Original Assignee
国立大学法人京都大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人京都大学 filed Critical 国立大学法人京都大学
Priority to JP2018509644A priority Critical patent/JP6908283B2/en
Publication of WO2017170981A1 publication Critical patent/WO2017170981A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/223—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of alpha-aminoacids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to a preventive or therapeutic agent for fibroblast growth factor receptor 3 (FGFR3) disease.
  • FGFR3 fibroblast growth factor receptor 3
  • FGFR3 fibroblast growth factor receptor 3
  • Non-Patent Document 1 Jonquoy et al. By tyrosine kinase inhibitors (Non-Patent Document 1), Rauchenberger et al. By FGFR3 neutralizing antibody (Non-Patent Document 2), and Yasoda et al. By c-type natriuretic peptide (CNP) (Non-patent Document 1).
  • Patent Document 3 reports a method of inhibiting excessive signal transduction from FGFR3. Some of these methods actually restore bone growth in model mice with cartilage dysplasia associated with FGFR3.
  • the present inventors searched for therapeutic drug candidates using iPS cells prepared using somatic cells derived from patients with FGFR3 disease, and statins that are hyperlipidemic drugs are candidates for therapeutic drugs for FGFR3 disease. (Patent Literature 1 and Non-Patent Literature 4).
  • An object of the present invention is to provide a therapeutic and / or prophylactic agent for fibroblast growth factor receptor 3 (FGFR3) disease.
  • FGFR3 fibroblast growth factor receptor 3
  • the present inventor obtained metformin, a Farnesyl transferase inhibitor, It was found that Geranylgeranyl transferase inhibitor and Rock inhibitor promote differentiation induction from iPS cells derived from FGFR3 disease patients to chondrocytes.
  • metformin a Farnesyl transferase inhibitor
  • a pharmaceutical for treatment and / or prevention of FGFR3 disease comprising as an active ingredient at least one drug selected from the group consisting of a Farnesyl transferase inhibitor, a Geranylgeranyl transferase inhibitor and a Rock inhibitor.
  • a drug selected from the group consisting of a Farnesyl transferase inhibitor, a Geranylgeranyl transferase inhibitor and a Rock inhibitor.
  • the Farnesyl transferase inhibitor is Lonafarnib, Chaetomellic acid A, FPT Inhibitor I, FPT Inhibitor II, FPT Inhibitor III, FTase Inhibitor I, FTase Inhibitor II, FTI-276 trifluoroacetate salt, FTI-277 trifluoroacetate salt, L- 744,832
  • the medicament according to [1] which is a drug selected from the group consisting of Dihydrochloride, Manumycin A, Tipifarnnib, and ⁇ -hydroxy Farnesyl Phosphonic Acid.
  • the Geranylgeranyl transferase inhibitor is selected from the group consisting of GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297 and GGTI-298
  • the medicament according to [1] which is a medicinal agent.
  • the Rock inhibitor is a drug selected from the group consisting of Y-27632, Fasudil / HA1077, SR3677, GSK269962, H-1152 and Wf-536.
  • FGFR3 disease is lethal osteodysplasia (TD) and / or achondroplasia (ACH).
  • TD lethal osteodysplasia
  • ACH achondroplasia
  • a method for treating and / or preventing FGFR3 disease comprising administering at least one drug selected from the group consisting of metformin, a Farnesyl transferase inhibitor, a Geranylgeranyl transferase inhibitor, and a Rock inhibitor. Including.
  • the Farnesyl transferase inhibitor is Lonafarnib, Chaetomellic acid A, FPT Inhibitor I, FPT Inhibitor II, FPT Inhibitor III, FTase Inhibitor I, FTase Inhibitor II, FTI-276 trifluoroacetate salt, FTI-277 trifluoroacetate salt, L- 744,832
  • the method according to [7] which is a drug selected from the group consisting of Dihydrochloride, Manumycin A, Tipifarnnib and ⁇ -hydroxy Farnesyl Phosphonic Acid.
  • the Geranylgeranyl transferase inhibitor is selected from the group consisting of GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297 and GGTI-298 [7] The method according to [7], wherein [11] The method according to [7], wherein the Rock inhibitor is an agent selected from the group consisting of Y-27632, Fasudil / HA1077, SR3677, GSK269962, H-1152 and Wf-536.
  • FGFR3 disease is lethal osteodysplasia (TD) and / or achondroplasia (ACH).
  • TD lethal osteodysplasia
  • ACH achondroplasia
  • [13] Use of at least one drug selected from the group consisting of metformin, a Farnesyl transferase inhibitor, a Geranylgeranyl transferase inhibitor and a Rock inhibitor in the manufacture of a medicament for the treatment and / or prevention of FGFR3 disease.
  • the drug is metformin.
  • the Farnesyl transferase inhibitor is Lonafarnib, Chaetomellic acid A, FPT Inhibitor I, FPT Inhibitor II, FPT Inhibitor III, FTase Inhibitor I, FTase Inhibitor II, FTI-276 trifluoroacetate salt, FTI-277 trifluoroacetate salt, L- 744,832
  • the Geranylgeranyl transferase inhibitor is selected from the group consisting of GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297 and GGTI-298
  • the Rock inhibitor is a drug selected from the group consisting of Y-27632, Fasudil / HA1077, SR3677, GSK269962, H-1152 and Wf-536.
  • FGFR3 disease is lethal osteodysplasia (TD) and / or achondroplasia (ACH).
  • the Farnesyl transferase inhibitor is Lonafarnib, Chaetomellic acid A, FPT Inhibitor I, FPT Inhibitor II, FPT Inhibitor III, FTase Inhibitor I, FTase Inhibitor II, FTI-276 trifluoroacetate salt, FTI-277 trifluoroacetate salt, L- 744,832
  • the drug according to [19] which is a drug selected from the group consisting of Dihydrochloride, Manumycin A, Tipifarnnib and ⁇ -hydroxy Farnesyl Phosphonic Acid.
  • the Geranylgeranyl transferase is selected from the group consisting of GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297 and GGTI-298
  • the Rock inhibitor is a drug selected from the group consisting of Y-27632, Fasudil / HA1077, SR3677, GSK269962, H-1152 and Wf-536.
  • chondrocytes induced by differentiation from healthy individual-derived iPS cells and undifferentiated healthy individual-derived iPS cells (iPSC) are also shown.
  • Vehicle base
  • Rosuva Rosuvastatin
  • TD714 using a low cholesterol medium The results of Safranin O-fast green-iron hematoxylin staining of particles induced from iPS cells are shown.
  • TD714-derived iPS cells ACH8858-derived iPS cells, or healthy individual-derived iPS cells using a medium supplemented with vehicle (base), 1 ⁇ M Rosuvastatin (rosuvastatin) and metformin (Met) at each concentration (0.2 mM or 2 mM) 409B2) shows the results of Safranin O-fast green-iron hematoxylin staining of particles induced to differentiate into cartilage.
  • TD714-derived iPS cells TD714-derived iPS cells, ACH8858-derived iPS cells, or healthy individuals-derived iPS using a medium supplemented with vehicle (base), 1 ⁇ M Rosuvastatin (rosuvastatin) and metformin (Met) at each concentration (0.2 mM, 2 mM or 5 mM)
  • base 1 ⁇ M Rosuvastatin (rosuvastatin) and metformin (Met) at each concentration (0.2 mM, 2 mM or 5 mM)
  • grains which induced differentiation from the cell (409B2) to the cartilage by real-time PCR is shown.
  • FGFR3 disease refers to any bone morphogenetic disease in which bone dysplasia is caused by having a mutation in FGFR3.
  • FGFR3 disease preferably means a series of diseases belonging to the FGFR3 disease group described in the international classification of bone system diseases (Warman et al., Am J Med Genet 155A (5): 943-68 (2011)).
  • TD lethal osteodysplasia
  • ACH achondroplasia
  • CASHL hearing loss syndrome
  • Crouzon-like craniosynostosis with acanthosis nigricans Crouzonodermoskeletal
  • Examples include early skull fusion.
  • the mutation occurring in FGFR3 may be either a gain-of-function type mutation or a function-deficient type mutation, but may preferably be a gain-of-function type mutation.
  • the therapeutic and / or prophylactic agent for a disease having a mutation in FGFR is at least one selected from the group consisting of metformin, Farnesyl transferase inhibitor, Geranylgeranyl transferase inhibitor, and Rock inhibitor. It is a medicine containing a drug as an active ingredient.
  • the active ingredient may be at least one drug selected from the group consisting of metformin and a Farnesyl transferase inhibitor, and may be metformin.
  • Metformin (chemical name: 1,1-Dimethylbiguanide, CAS number: 657-24-9, C 4 H 11 N 5 ) is known as a biguanide hypoglycemic drug. Metformin hydrochloride is formulated and marketed.
  • the Farnesyl transferase inhibitor in the present invention is not particularly limited as long as it can suppress the function of Farnesyl transferase.
  • Lonafarnib (CAS number: 193275-84-2, C 27 H 31 Br 2 ClN 4 O 2 , for example, Shen M et al., Drug Discov Today 20, 267-276 (2015))
  • Chaetomellic acid A (CAS number: 148796-51-4, C 19 H 32 O 4 2Na, eg, JB Gibbs, et al .; J. Biol. Chem. 268, 7617 (1993), F. Tamanoi; Trends Biochem. Sci.
  • FPT Inhibitor I C 19 H 29 NO 6 P 3Na, eg, Manne, V. , et al. 1995. Drug Development Res. 34, 121, Patel, DV, et al. 1995. J. Med. Chem. 38, 2906
  • FPT Inhibitor II C 17 H 28 NO 5 P 2Na, eg, Manne, V., et al. 1995. Drug Development Res. 34, 121
  • FPT Inhibitor III C 23 H 39 NO 7 P Na, eg, Wang, D., et al. 1998. J. Biol. Chem) 273, 33027, Manne, V., et al. 1995. Drug Development Res.
  • FTase Inhibitor I (CAS number: 149759-96-6, C 22 H 38 N 4 O 3 S 2 , eg , Cox, A D., et al., 1994 The Journal of biological chemistry. 269 (30): 19203-6, Garcia, A M., et al., 1993 The Journal of biological chemistry. 268 (25): 18415-8 ), FTase Inhibitor II (CAS number: 156707-43-6, C 15 H 21 N 3 O 4 S 2 , eg, Song, Jia L., et al., 2003 Microbiology (Reading, England).
  • FTI-276 trifluoroacetate salt (CAS number: 170006-72-1 (free base), C 21 H 27 N 3 O 3 S 2 C 2 HF 3 O 2 , eg, Cohen-Jonathan, E., et al., 1999 Radiation research. 152 (4): 404-11), FTI-277 trifluoroacetate salt (CAS number: 170006-73-2 (free base), C 22 H 29 N 3 O 3 S 2 x C 2 HF 3 O 2 , eg, Cohen-Jonathan, E., et al., 1999 Radiation research.
  • Tipifarnib (CAS number: 192185-72-1, C 27 H 22 Cl 2 N 4 O, see eg Shen M et al., Drug Discov Today 20, 267-276 (2015)) and ⁇ -hydroxy Farnesyl Phosphonic Acid ( CAS number: 148796-53-6, C 15 H 27 O 4 P, for example, see Pompliano, DL, Rands, E., Schaber, MD, et al., Biochemistry 31 3800-3807 (1992)) It is not limited to these.
  • the Geranylgeranyl transferase inhibitor in the present invention is not particularly limited as long as it can suppress the function of Geranylgeranyl transferase.
  • GGTI-2418 (CAS number: 171744-11-9, C 23 H 31 N 3 O 3 S, for example) , Shen M et al, Drug Discov Today 20, see 267-276 (2015)
  • GGTI- 2133 (CAS number:. 191102-79-1, C 27 H 28 N 4 O 3 C 2 HF 3 O 2, example , Shen M et al., Drug Discov Today 20, 267-276 (2015))
  • GGTI-2147 CAS number: 191102-87-1, C 28 H 30 N 4 O 3 , eg, Bernot, D., et al. 2003.
  • GGTI-2154 (CAS number: 251577-10-3, eg, Shen M et al., Drug Discov Today 20, 267-276 (2015)).
  • GGTI-2166 (Nikkaji number: J1.244.741H, C 25 H 30 N 4 O 3 )
  • GGTI-286 (CAS number: 171744-11-9, C 23 H 31 N 3 O 3 S, for example)
  • GGTI-287 (C 22 H 29 N 3 O 3 S, eg, Shen M et al.
  • GGTI-297 (CAS Number:. 181045-83-0, C 26 H 31 N 3 O 3 S C 2 HF 3 O 2, example, Sun, J., et al 1998. Oncogene 16, 1467 reference) and GGTI-298 (CAS number: 180977-44-0, C 27 H 33 N 3 O 3 S CF 3 CO 2 H, eg, McGuire, T F., et al., 1996 The Journal of biological chemistry. 271 (44 ): 27402-7), but not limited to.
  • the Rock inhibitor in the present invention is not particularly limited as long as it can suppress the function of Rho-kinase (ROCK).
  • ROCK Rho-kinase
  • Y-27632 eg, Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325,273-284 (2000)
  • Fasudil / HA1077 eg, Uenata et al., Nature 389: 990-994 (1997)
  • SR3677 eg, Feng Y et al., J Med Chem. 51: 6642-6645 (2008)
  • GSK269962 eg, StavengerStRA et al., J Med Chem.
  • H- 1152 eg, Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)
  • Wf-536 eg Nakajima et al., Cancer Chemother Pharmacol. 52 (4): 319-324 (2003)
  • antisense nucleic acids to ROCK eg, siRNA
  • RNA interference inducing nucleic acids eg, siRNA
  • dominant negative mutants eg, and their expression vectors.
  • Other known low-molecular compounds can also be used as ROCK inhibitors (for example, US Patent Application Publication Nos. 2005/0209261, 2005/0192304, 2004/0014755, 2004/0002508).
  • ROCK inhibitors may be used.
  • metformin, Farnesyl transferase inhibitor, Geranylgeranyl transferase inhibitor or Rock inhibitor of the present invention includes pharmacologically acceptable salts thereof (preferably hydrochloride, sodium salt or calcium salt).
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like.
  • compositions for parenteral administration for example, injections, suppositories and the like are used, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like.
  • Dosage forms may be included. These formulations include excipients (eg sugar derivatives such as lactose, sucrose, sucrose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; Gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate and magnesium metasilicate aluminate; phosphates such as calcium hydrogen phosphate; Carbonates such as calcium; inorganic excipients such as sulfates such as calcium sulfate), lubricants (eg, stearic acid metal salts such as stearic acid, calcium stearate, magnesium stearate; talc; Colloidal silica; like bead wax, gay wax Boric acid; adipic acid; sulfate such as sodium sul
  • the dose of the drug of the present invention to a patient varies depending on the type of pathological condition to be treated, symptoms and severity of the disease, patient age, sex or body weight, administration method, etc. By determining in consideration of the above situation, an appropriate dose can be determined as appropriate.
  • the lower limit is 0.1 mg (preferably 0.5 mg) and the upper limit is 1000 mg (preferably 500 mg).
  • the lower limit is 0.01 mg (preferably 0.05 mg) and an upper limit of 100 mg (preferably 50 mg) can be administered to adults 1 to 6 times per day. The dose may be increased or decreased depending on the symptoms.
  • Example 1 Generation of iPS cells All the following experiments were conducted with the approval of the clinical trial review committee, animal experiment committee and institutional biosafety committee, and Kyoto University.
  • HDF human dermal fibroblasts derived from TD patients (TD714, TD10749, TD315H and TD329N) were obtained from the Coryell Medical Research Institute and Saitama Children's Medical Center. Sequence analysis of genomic DNA extracted from these human dermal fibroblasts (HDF) confirmed a heterozygous mutation (Arg248Cys) in the FGFR3 gene in all TD patients. As HDFs from ACH patients, HDFs (ACH8857 and ACH8858) of two ACH patients with a Gly380Arg heterozygous mutation in the FGFR3 gene, and HDF (ACHhomo-8859) showing more severe chondrogenesis than ACH Obtained from the Coryell Medical Institute.
  • IPS cells were prepared from the HDF derived from each patient by the method described below.
  • As control iPS cells a healthy individual-derived iPS cell line (409B2) obtained from K. Okita and S. Yamanaka (Kyoto University iPS Cell Laboratory) (Okita, K., et al. Nature methods 8, 409-412 (2011)).
  • iPS cells were produced by the following method. Specifically, each obtained human fibroblast (HDF) was cultured in DMEM (Sigma) supplemented with 10% FBS (Invitrogen), 50 U / ml penicillin and 50 ⁇ g / ml streptomycin. Subsequently, episomal plasmid vectors (Mixture Y4: OCT3 / 4, SOX2, KLF4, L-MYC, LIN28 and p53 shRNA) were electroporated into each HDF by Neon transfection system (Invitrogen) (Okita, K ., et al. Nature methods 8, 409-412 (2011)).
  • Neon transfection system Invitrogen
  • Example 2 Differentiation from each iPS cell was induced to chondrocytes according to the method described below, which was modified from a method reported before cartilage induction (Oldershaw, RA, et al., Nat Biotechnol 28, 1187-1194 (2010)).
  • Each iPS cell was seeded on a Matrigel (Invitrogen) coated dish, a medium supplemented with 50 units / ml penicillin and 50 ⁇ g / ml streptomycin was added to Essential 8 medium (Life Technologies), and cultured under feeder-free conditions.
  • colonies consisting of 1-2 ⁇ 10 5 cells were transformed into mesodermal differentiation medium (10 ng / ml Wnt3A (R & D), 10 ng / ml activin A (R & D), 1% insulin for DMEM / F12) -Transferrin-sodium selenite (Invitrogen), 1% fetal bovine serum, 50 units / ml penicillin, 50 ⁇ g / ml prepared by mixing streptomycin) (Day 0 of differentiation induction).
  • mesodermal differentiation medium (10 ng / ml Wnt3A (R & D), 10 ng / ml activin A (R & D), 1% insulin for DMEM / F12) -Transferrin-sodium selenite (Invitrogen), 1% fetal bovine serum, 50 units / ml penicillin, 50 ⁇ g / ml prepared by mixing streptomycin) (Day 0 of differentiation induction).
  • each drug (1 ⁇ M rosuvastatin (BioVision, 1995-5), each concentration (0.1 ⁇ M, 1 ⁇ M or 10 ⁇ M) of Farnesyl transferase inhibitor (FTI-277) (Sigma) (hereinafter, FTI), Geranylgeranyl transferase inhibitor (GGTI-287) (Sigma) (hereinafter referred to as GGTI) at each concentration (0.1 ⁇ M, 1 ⁇ M or 10 ⁇ M), Rock inhibitor at each concentration (0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M or 100 ⁇ M) (Y27632) (Wako) (hereinafter referred to as Rocki)) cartilage differentiation medium (50 ⁇ g / ml ascorbic acid, 10 ng / ml BMP2 (Osteopharma), 10 ng / ml TGF ⁇ (Pepro Tech), 10 ng / ml GDF5, 1% Insulin-transferrin-sodium
  • FTI-277 Sigma
  • Lonafarnib Lona
  • Tipifarnib LCL
  • the culture medium was replaced with a cartilage differentiation medium added at 0.01 ⁇ M or 0.1 ⁇ M, the cells were peeled off from the dish on the 14th day of induction of differentiation, and cultured for 28 days in suspension culture using the same medium. It was collected.
  • the expression level of mRNA of the genes encoding chondrocyte marker SOX9 and cartilage matrix protein (type II collagen (COL2A1) and aggrecan (Acan)) was examined by real-time PCR.
  • the obtained particles were stained with safranin O to confirm the presence of safranin O-positive cartilage tissue and to determine the ratio of safranin O-positive regions.
  • Fig. 1 shows a medium containing TD714-derived iPS cells supplemented with Farnesyl transferase inhibitor (FTI-277), Geranylgeranyl transferase inhibitor (GGTI-287), or Rock inhibitor (Y27632).
  • FTI-277 Farnesyl transferase inhibitor
  • GGTI-287 Geranylgeranyl transferase inhibitor
  • Rock inhibitor Y27632
  • Fig. 5 shows the results of real-time PCR measurement of chondrocyte markers in chondrocytes induced by differentiation from TD714-derived iPS cells using media supplemented with three types of Farnesyl transferase inhibitors (FTI-277, Lonafarnib and Tipifarnib). Indicated. (A) is the result of SOX9, (B) is the result of COL2A1, and (C) is the result of Acan. Even when any Farnesyl transferase inhibitor was used, it was confirmed that the expression of each gene was increased as compared with Vehicle (base).
  • FTI-277 Farnesyl transferase inhibitors
  • FIG. 6 and FIG. 7 show the results of staining with Safranin O for particles induced to differentiate from TD714-derived iPS cells using media supplemented with three types of Farnesyl transferase inhibitors (FTI-277, Lonafarnib and Tipifarnib), respectively. . Safranin O-positive cartilage tissue was confirmed when any Farnesyl transferase inhibitor was used.
  • FTI-277 Farnesyl transferase inhibitors
  • Fig. 10 shows the results of real-time PCR measurement of chondrocyte markers in chondrocytes induced to differentiate from TD329N-derived iPS cells using media supplemented with three types of Farnesyl transferase inhibitors (FTI-277, Lonafarnib and Tipifarnib). Indicated. (A) is the result of SOX9, (B) is the result of COL2A1, and (C) is the result of Acan. Even when any Farnesyl transferase inhibitor was used, it was confirmed that the expression of each gene was increased as compared with Vehicle (base).
  • FTI-277 Farnesyl transferase inhibitors
  • FIG. 11 and FIG. 12 show the addition of Farnesyl transferase inhibitor (FTI-277), Geranylgeranyl transferase inhibitor (GGTI-287), or Rock inhibitor (Y27632) from ACH8858-derived iPS cells.
  • FTI-277 Farnesyl transferase inhibitor
  • GGTI-287 Geranylgeranyl transferase inhibitor
  • Rock inhibitor Y27632
  • FIG. 13 shows the results of real-time PCR measurement of chondrocyte markers in chondrocytes induced to differentiate from ACH8858-derived iPS cells using media supplemented with three types of Farnesyl transferase inhibitors (FTI-277, Lonafarnib and Tipifarnib), respectively. Indicated. (A) is the result of SOX9, (B) is the result of COL2A1, and (C) is the result of Acan. Even when any Farnesyl transferase inhibitor was used, it was confirmed that the expression of each gene was increased as compared with Vehicle (base).
  • FTI-277 Farnesyl transferase inhibitors
  • FIG. 14 and FIG. 15 show that from ACHhomo-8859-derived iPS cells, a Farnesyl transferase inhibitor (FTI-277), Geranylgeranyl transferase inhibitor (GGTI-287), or Rock inhibitor. (Y27632) The results of staining with Safranin O for the particles induced to differentiate using the added medium are shown. Safranin O-positive cartilage tissue was confirmed when any drug was used.
  • FIG. 16 shows real-time PCR for chondrocyte markers in chondrocytes induced to differentiate from ACHhomo-8859-derived iPS cells using media supplemented with three types of Farnesyl transferase inhibitors (FTI-277, Lonafarnib and Tipifarnib). Results are shown.
  • FTI-277 Farnesyl transferase inhibitors
  • B is the result of COL2A1
  • C is the result of Acan. Even when any Farnesyl transferase inhibitor was used, it was confirmed that the expression of each gene was increased as compared with Vehicle (base).
  • Example 3 Except for changing the test drug to metformin, chondrocytes were induced from TD714-derived iPS cells and ACH8858-derived iPS cells in the same manner as in Example 2. As a control iPS cell, 409B2 derived from a healthy individual was used. The concentration of metformin (1,1-Dimethylbiguanide hydrochloride (sigma)) was 0.2 mM, 2 mM or 5 mM.
  • FIG. 17 shows the result of Safranin O staining of particles induced to differentiate using a medium supplemented with metformin. Safranin O-positive cartilage tissue was confirmed in both particles induced to differentiate from TD714-derived iPS cells and ACH8858-derived iPS cells.
  • FIG. 18 shows the results of real-time PCR measurement of chondrocyte markers (COL2A1, Acan, SOX9) in chondrocytes induced to differentiate using a medium supplemented with metformin.
  • the expression level of each gene was expressed as a relative value, assuming that the expression level when TD714-derived iPS cells were induced to differentiate with 1 ⁇ M rosuvastatin was 1. It was confirmed that the addition of metformin increases the expression of each gene.
  • metformin By using metformin, Farnesyl transferase inhibitor, Geranylgeranyl transferase inhibitor or Rock inhibitor, cartilage tissue can be formed even from iPS cells derived from TD patients and iPS cells derived from ACH patients It was confirmed that it was possible. Therefore, it was suggested that metformin, Farnesyl® transferase inhibitor, Geranylgeranyl® transferase inhibitor and Rock inhibitor can be therapeutic agents for lethal osteodysplasia and achondroplasia.
  • the present invention provides a substance that promotes differentiation into chondrocytes obtained as a result of screening, and the substance can be used as a new therapeutic agent and / or preventive agent for FGFR3 disease.

Abstract

The present invention provides a therapeutic and/or prophylactic agent for FGFR3 disease, which contains at least one chemical substance selected from the group consisting of metformin, a farnesyl transferase inhibitor, a geranylgeranyl transferase inhibitor and a Rock inhibitor as an active ingredient.

Description

ＦＧＦＲ３病の予防または治療剤Preventive or therapeutic agent for FGFR3 disease
　本発明は、線維芽細胞増殖因子受容体3（FGFR3）病の予防または治療剤に関する。 The present invention relates to a preventive or therapeutic agent for fibroblast growth factor receptor 3 (FGFR3) disease.
　致死性骨異形成症 (TD)や軟骨無形成症 (ACH)などの骨形成異常に関する疾患は、一般に線維芽細胞増殖因子受容体3（FGFR3）病と呼ばれており、FGFR3中の機能獲得型突然変異により引き起こされる疾患であると考えられている。したがって、これらの疾患の治療を行う上での分子標的として、FGFR3およびその下流のシグナル伝達経路に係る種々の分子が注目されており、FGFR3からの過剰なシグナル伝達を阻害する様々な方法が試みられてきた。 Diseases related to bone dysplasia such as lethal osteodysplasia TD (TD) and achondroplasia A (ACH) are commonly called fibroblast growth factor receptor 3 (FGFR3) disease, and gain function in FGFR3. It is considered a disease caused by type mutations. Therefore, various molecules related to FGFR3 and its downstream signal transduction pathway are attracting attention as molecular targets for the treatment of these diseases, and various methods for inhibiting excessive signal transduction from FGFR3 have been tried. Has been.
　例えば、Jonquoyらは、チロシンキナーゼ阻害剤（非特許文献１）により、Rauchenbergerらは、FGFR3の中和抗体（非特許文献２）により、およびYasodaらは、c型ナトリウム利尿ペプチド（CNP）（非特許文献３）により、FGFR3からの過剰なシグナル伝達を阻害する方法を報告している。これらの方法のうちいくつかは、実際に、FGFR3に関連する軟骨異形成のモデルマウスにおける骨成長を回復させている。しかしながら、変異型FGFR3を導入した形質転換細胞での確認に留まり、適切なヒト細胞モデルを用いての有効性は確認されておらず、FGFR3病に十分な治療が行い得るかについては不明な点も多いため、更なる新規治療薬の開発が切望されている。 For example, Jonquoy et al. By tyrosine kinase inhibitors (Non-Patent Document 1), Rauchenberger et al. By FGFR3 neutralizing antibody (Non-Patent Document 2), and Yasoda et al. By c-type natriuretic peptide (CNP) (Non-patent Document 1). Patent Document 3) reports a method of inhibiting excessive signal transduction from FGFR3. Some of these methods actually restore bone growth in model mice with cartilage dysplasia associated with FGFR3. However, it has been confirmed only in transformed cells into which mutant FGFR3 has been introduced, its effectiveness has not been confirmed using an appropriate human cell model, and it is unclear whether sufficient treatment can be performed for FGFR3 disease. Therefore, the development of further new therapeutic agents is eagerly desired.
　本発明者らは、FGFR3病患者由来の体細胞を用いて作製したiPS細胞を利用して治療薬の候補を探索し、高脂血症治療薬であるスタチン類がFGFR3病の治療薬の候補となり得ることを報告している（特許文献１および非特許文献４）。 The present inventors searched for therapeutic drug candidates using iPS cells prepared using somatic cells derived from patients with FGFR3 disease, and statins that are hyperlipidemic drugs are candidates for therapeutic drugs for FGFR3 disease. (Patent Literature 1 and Non-Patent Literature 4).
WO2015/083582WO2015 / 083582
　本発明の課題は、線維芽細胞増殖因子受容体3（FGFR3）病の治療剤および／または予防剤を提供することにある。 An object of the present invention is to provide a therapeutic and / or prophylactic agent for fibroblast growth factor receptor 3 (FGFR3) disease.
　本発明者は上記の課題を解決すべくFGFR3病の治療剤および／または予防剤を見出すべくFGFR3病患者の体細胞由来のiPS細胞を用いてスクリーニングを行った結果、メトホルミン、Farnesyl transferase阻害剤、Geranylgeranyl transferase阻害剤およびRock阻害剤が当該FGFR3病患者由来のiPS細胞から軟骨細胞への分化誘導を促進することを見出した。本発明はそのような知見に基づいて完成されたものである。 As a result of screening using iPS cells derived from somatic cells of FGFR3 disease patients in order to find a therapeutic agent and / or preventive agent for FGFR3 disease in order to solve the above problems, the present inventor obtained metformin, a Farnesyl transferase inhibitor, It was found that Geranylgeranyl transferase inhibitor and Rock inhibitor promote differentiation induction from iPS cells derived from FGFR3 disease patients to chondrocytes. The present invention has been completed based on such findings.
　すなわち、本発明は次に記載の事項を提供するものである。
[１]　有効成分としてFarnesyl transferase阻害剤、Geranylgeranyl transferase阻害剤およびRock阻害剤から成る群より選択される少なくとも１つの薬剤を含む、FGFR3病の治療および／または予防用医薬。
[２]　前記薬剤がメトホルミンである、[１]に記載の医薬。
[３]　前記Farnesyl transferase阻害剤が、Lonafarnib、Chaetomellic acid A、FPT Inhibitor I、FPT Inhibitor II、FPT Inhibitor III、FTase Inhibitor I、FTase Inhibitor II、FTI-276 trifluoroacetate salt、FTI-277 trifluoroacetate salt、L-744,832 Dihydrochloride、Manumycin A、Tipifarnibおよびα-hydroxy Farnesyl Phosphonic Acidから成る群より選択される薬剤である、[１]に記載の医薬。
[４]　前記Geranylgeranyl transferase阻害剤が、GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297およびGGTI-298から成る群より選択される薬剤である、[１]に記載の医薬。
[５]　前記Rock阻害剤が、Y-27632、Fasudil/HA1077、SR3677、GSK269962、H-1152およびWf-536から成る群より選択される薬剤である、[１]に記載の医薬。
[６]　前記FGFR3病が、致死性骨異形成症 (TD) および／または軟骨無形成症 (ACH)である、[１]から[５]のいずれか一つに記載の医薬。
[７]　FGFR3病を治療および／または予防するための方法であって、メトホルミン、Farnesyl transferase阻害剤、Geranylgeranyl transferase阻害剤およびRock阻害剤から成る群より選択される少なくとも１つの薬剤を投与することを含む、方法。
[８]　前記薬剤がメトホルミンである、[７]に記載の方法。
[９]　前記Farnesyl transferase阻害剤が、Lonafarnib、Chaetomellic acid A、FPT Inhibitor I、FPT Inhibitor II、FPT Inhibitor III、FTase Inhibitor I、FTase Inhibitor II、FTI-276 trifluoroacetate salt、FTI-277 trifluoroacetate salt、L-744,832 Dihydrochloride、Manumycin A、Tipifarnibおよびα-hydroxy Farnesyl Phosphonic Acidから成る群より選択される薬剤である、[７]に記載の方法。
[１０]　前記Geranylgeranyl transferase阻害剤が、GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297およびGGTI-298から成る群より選択される薬剤である、[７]に記載の方法。
[１１]　前記Rock阻害剤が、Y-27632、Fasudil/HA1077、SR3677、GSK269962、H-1152およびWf-536から成る群より選択される薬剤である、[７]に記載の方法。
[１２]　前記FGFR3病が、致死性骨異形成症 (TD) および／または軟骨無形成症 (ACH)である、[７]から[１１]のいずれか一つに記載の方法。
[１３]　FGFR3病の治療および／または予防用医薬の製造におけるメトホルミン、Farnesyl transferase阻害剤、Geranylgeranyl transferase阻害剤およびRock阻害剤から成る群より選択される少なくとも１つの薬剤の使用。
[１４]　前記薬剤がメトホルミンである、[１３]に記載の使用。
[１５]　前記Farnesyl transferase阻害剤が、Lonafarnib、Chaetomellic acid A、FPT Inhibitor I、FPT Inhibitor II、FPT Inhibitor III、FTase Inhibitor I、FTase Inhibitor II、FTI-276 trifluoroacetate salt、FTI-277 trifluoroacetate salt、L-744,832 Dihydrochloride、Manumycin A、Tipifarnibおよびα-hydroxy Farnesyl Phosphonic Acidから成る群より選択される薬剤である、[１３]に記載の使用。
[１６]　前記Geranylgeranyl transferase阻害剤が、GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297およびGGTI-298から成る群より選択される薬剤である、[１３]に記載の使用。
[１７]　前記Rock阻害剤が、Y-27632、Fasudil/HA1077、SR3677、GSK269962、H-1152およびWf-536から成る群より選択される薬剤である、[１３]に記載の使用。
[１８]　前記FGFR3病が、致死性骨異形成症 (TD) および／または軟骨無形成症 (ACH)である、[１３]から[１７]のいずれか一つに記載の使用。
[１９]　FGFR3病を治療および／または予防するために使用されるメトホルミン、Farnesyl transferase阻害剤、Geranylgeranyl transferase阻害剤およびRock阻害剤から成る群より選択される少なくとも１つの薬剤。
[２０]　前記薬剤がメトホルミンである、[１９]に記載の薬剤。
[２１]　前記Farnesyl transferase阻害剤が、Lonafarnib、Chaetomellic acid A、FPT Inhibitor I、FPT Inhibitor II、FPT Inhibitor III、FTase Inhibitor I、FTase Inhibitor II、FTI-276 trifluoroacetate salt、FTI-277 trifluoroacetate salt、L-744,832 Dihydrochloride、Manumycin A、Tipifarnibおよびα-hydroxy Farnesyl Phosphonic Acidから成る群より選択される薬剤である、[１９]に記載の薬剤。
[２２]　前記Geranylgeranyl transferaseが、GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297およびGGTI-298から成る群より選択される薬剤である、[１９]に記載の薬剤。
[２３]　前記Rock阻害剤が、Y-27632、Fasudil/HA1077、SR3677、GSK269962、H-1152およびWf-536から成る群より選択される薬剤である、[１９]に記載の薬剤。
[２４]　前記FGFR3病が、致死性骨異形成症 (TD) および／または軟骨無形成症 (ACH)である、[１９]から[２３]のいずれか一つに記載の薬剤。 That is, the present invention provides the following matters.
[1] A pharmaceutical for treatment and / or prevention of FGFR3 disease, comprising as an active ingredient at least one drug selected from the group consisting of a Farnesyl transferase inhibitor, a Geranylgeranyl transferase inhibitor and a Rock inhibitor.
[2] The medicament according to [1], wherein the drug is metformin.
[3] The Farnesyl transferase inhibitor is Lonafarnib, Chaetomellic acid A, FPT Inhibitor I, FPT Inhibitor II, FPT Inhibitor III, FTase Inhibitor I, FTase Inhibitor II, FTI-276 trifluoroacetate salt, FTI-277 trifluoroacetate salt, L- 744,832 The medicament according to [1], which is a drug selected from the group consisting of Dihydrochloride, Manumycin A, Tipifarnnib, and α-hydroxy Farnesyl Phosphonic Acid.
[4] The Geranylgeranyl transferase inhibitor is selected from the group consisting of GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297 and GGTI-298 The medicament according to [1], which is a medicinal agent.
[5] The medicament according to [1], wherein the Rock inhibitor is a drug selected from the group consisting of Y-27632, Fasudil / HA1077, SR3677, GSK269962, H-1152 and Wf-536.
[6] The medicament according to any one of [1] to [5], wherein the FGFR3 disease is lethal osteodysplasia (TD) and / or achondroplasia (ACH).
[7] A method for treating and / or preventing FGFR3 disease, comprising administering at least one drug selected from the group consisting of metformin, a Farnesyl transferase inhibitor, a Geranylgeranyl transferase inhibitor, and a Rock inhibitor. Including.
[8] The method according to [7], wherein the drug is metformin.
[9] The Farnesyl transferase inhibitor is Lonafarnib, Chaetomellic acid A, FPT Inhibitor I, FPT Inhibitor II, FPT Inhibitor III, FTase Inhibitor I, FTase Inhibitor II, FTI-276 trifluoroacetate salt, FTI-277 trifluoroacetate salt, L- 744,832 The method according to [7], which is a drug selected from the group consisting of Dihydrochloride, Manumycin A, Tipifarnnib and α-hydroxy Farnesyl Phosphonic Acid.
[10] The Geranylgeranyl transferase inhibitor is selected from the group consisting of GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297 and GGTI-298 [7] The method according to [7], wherein
[11] The method according to [7], wherein the Rock inhibitor is an agent selected from the group consisting of Y-27632, Fasudil / HA1077, SR3677, GSK269962, H-1152 and Wf-536.
[12] The method according to any one of [7] to [11], wherein the FGFR3 disease is lethal osteodysplasia (TD) and / or achondroplasia (ACH).
[13] Use of at least one drug selected from the group consisting of metformin, a Farnesyl transferase inhibitor, a Geranylgeranyl transferase inhibitor and a Rock inhibitor in the manufacture of a medicament for the treatment and / or prevention of FGFR3 disease.
[14] The use according to [13], wherein the drug is metformin.
[15] The Farnesyl transferase inhibitor is Lonafarnib, Chaetomellic acid A, FPT Inhibitor I, FPT Inhibitor II, FPT Inhibitor III, FTase Inhibitor I, FTase Inhibitor II, FTI-276 trifluoroacetate salt, FTI-277 trifluoroacetate salt, L- 744,832 The use according to [13], which is a drug selected from the group consisting of Dihydrochloride, Manumycin A, Tipifarnnib and α-hydroxy Farnesyl Phosphonic Acid.
[16] The Geranylgeranyl transferase inhibitor is selected from the group consisting of GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297 and GGTI-298 The use according to [13], which is a drug to be used.
[17] The use according to [13], wherein the Rock inhibitor is a drug selected from the group consisting of Y-27632, Fasudil / HA1077, SR3677, GSK269962, H-1152 and Wf-536.
[18] The use according to any one of [13] to [17], wherein the FGFR3 disease is lethal osteodysplasia (TD) and / or achondroplasia (ACH).
[19] At least one drug selected from the group consisting of metformin, a Farnesyl transferase inhibitor, a Geranylgeranyl transferase inhibitor, and a Rock inhibitor used to treat and / or prevent FGFR3 disease.
[20] The drug according to [19], wherein the drug is metformin.
[21] The Farnesyl transferase inhibitor is Lonafarnib, Chaetomellic acid A, FPT Inhibitor I, FPT Inhibitor II, FPT Inhibitor III, FTase Inhibitor I, FTase Inhibitor II, FTI-276 trifluoroacetate salt, FTI-277 trifluoroacetate salt, L- 744,832 The drug according to [19], which is a drug selected from the group consisting of Dihydrochloride, Manumycin A, Tipifarnnib and α-hydroxy Farnesyl Phosphonic Acid.
[22] The Geranylgeranyl transferase is selected from the group consisting of GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297 and GGTI-298 The drug according to [19], which is a drug.
[23] The drug according to [19], wherein the Rock inhibitor is a drug selected from the group consisting of Y-27632, Fasudil / HA1077, SR3677, GSK269962, H-1152 and Wf-536.
[24] The drug according to any one of [19] to [23], wherein the FGFR3 disease is lethal osteodysplasia (TD) and / or achondroplasia (ACH).
　本発明により、FGFR3病の治療剤および／または予防剤を提供することが可能となる。 According to the present invention, it is possible to provide a therapeutic and / or preventive agent for FGFR3 disease.
Vehicle（基剤）、Rosuva（ロスバスタチン）、各濃度（0.1μM、1μMまたは10μM）のFTI、GGTIまたはRockiを添加してTD714由来iPS細胞から分化誘導した軟骨細胞、または低コレステロール培地を用いてTD714由来iPS細胞から分化誘導した軟骨細胞（low chole）における、(A)SOX9、(B)COL2（II型コラーゲン）および(C)Acan（アグリカン）の発現をリアルタイムPCRで測定した結果を示す。対照として、健常個体由来iPS細胞から分化誘導した軟骨細胞（WT）および未分化の健常個体由来iPS細胞（iPSC）における同mRNAの発現を測定した結果も示す。Vehicle (base), Rosuva (rosuvastatin), chondrocytes induced to differentiate from TD714-derived iPS cells by adding FTI, GGTI or Rocki at each concentration (0.1 μM, 1 μM or 10 μM), or TD714 using low cholesterol medium The result of having measured the expression of (A) SOX9, (B) COL2 (type II collagen) and (C) Acan (aggrecan) in the chondrocytes (low chole) induced to differentiate from the derived iPS cells by real-time PCR is shown. As a control, the results of measuring the expression of the mRNA in chondrocytes (WT) induced by differentiation from healthy individual-derived iPS cells and undifferentiated healthy individual-derived iPS cells (iPSC) are also shown. Vehicle（基剤）、Rosuva（ロスバスタチン）、各濃度（0.1μM、1μM、10μMまたは100μM）のRockiを添加した培地を用いてTD714由来iPS細胞から分化誘導したパーティクル、および低コレステロール培地を用いてTD714由来iPS細胞から分化誘導したパーティクルのSafranin O-fast green-iron hematoxylin stainingの結果を示す。Vehicle (base), Rosuva (rosuvastatin), particles induced to differentiate from TD714-derived iPS cells using a medium containing Rocki at various concentrations (0.1 μM, 1 μM, 10 μM or 100 μM), and TD714 using a low cholesterol medium The results of Safranin O-fast green-iron hematoxylin staining of particles induced from iPS cells are shown. Vehicle（基剤）、Rosuva（ロスバスタチン）、各濃度（0.1μM、1μM、または10μM）のRocki、FTIまたはGGTIを添加した培地を用いてTD714由来iPS細胞から分化誘導したパーティクル、および低コレステロール培地を用いてTD714由来iPS細胞から分化誘導したパーティクルのSafranin O-fast green-iron hematoxylin stainingの結果を示す。Vehicle (base), Rosuva (rosuvastatin), particles induced to differentiate from TD714-derived iPS cells using a medium containing Rocki, FTI or GGTI at each concentration (0.1 μM, 1 μM, or 10 μM), and a low cholesterol medium The results of Safranin O-fast green-iron hematoxylin staining of particles induced to differentiate from TD714-derived iPS cells are shown. Vehicle（基剤）、Rosuva（ロスバスタチン）、各濃度（0.1μM、1μM、または10μM）のRocki、FTIまたはGGTIを添加した培地を用いてTD714由来iPS細胞から分化誘導したパーティクル、および低コレステロール培地を用いてTD714由来iPS細胞から分化誘導したパーティクルにSafranin O-fast green-iron hematoxylin stainingを施し、Safranin O陽性領域の比率を比較した結果を示す。Vehicle (base), Rosuva (rosuvastatin), particles induced to differentiate from TD714-derived iPS cells using a medium containing Rocki, FTI or GGTI at each concentration (0.1 μM, 1 μM, or 10 μM), and a low cholesterol medium The results are shown in which Safranin-O-fast green-iron hematoxylin staining was applied to particles induced to differentiate from TD714-derived iPS cells, and the ratio of Safranin O positive regions was compared. Vehicle（基剤）、各濃度（0.0001μM、0.001μM、0.01μMまたは0.1μM）のFTI-277(FTI)、Lonafarnib(Lona)またはTipifarnib(Tipi)を添加した培地を用いてTD714由来iPS細胞から分化誘導した軟骨細胞における、(A)SOX9、(B)COL2（II型コラーゲン）および(C)Acan（アグリカン）の発現をリアルタイムPCRで測定した結果を示す。From TD714-derived iPS cells using vehicle (base), medium supplemented with FTI-277 (FTI), Lonafarnib (Lona) or Tipifarnnib (Tipi) at each concentration (0.0001μM, 0.001μM, 0.01μM or 0.1μM) The result of having measured the expression of (A) SOX9, (B) COL2 (type II collagen) and (C) Acan (aggrecan) in the differentiation-induced chondrocytes by real-time PCR is shown. 各濃度（0.0001μM(0.1nM)、0.001μM(1nM)、0.01μMまたは0.1μM）のFTI-277を添加した培地を用いてTD714由来iPS細胞から分化誘導したパーティクルのSafranin O-fast green-iron hematoxylin stainingの結果（上段）、およびSafranin O陽性領域の比率を比較した結果（下段）を示す。Safranin O-fast green-iron of particles induced to differentiate from TD714-derived iPS cells using medium supplemented with FTI-277 at each concentration (0.0001μM (0.1nM), 0.001μM (1nM), 0.01μM or 0.1μM) The results of hematoxylin staining (upper) and the results of comparison of the ratio of Safranin O positive regions (lower) are shown. 各濃度（0.0001μM(0.1nM)、0.001μM(1nM)、0.01μMまたは0.1μM）のLonafarnib(Lona)またはTipifarnib(Tipi)を添加した培地を用いてTD714由来iPS細胞から分化誘導したパーティクルのSafranin O-fast green-iron hematoxylin stainingの結果（上段）、およびSafranin O陽性領域の比率を比較した結果（下段）を示す。Safranin of particles induced to differentiate from TD714-derived iPS cells using medium containing Lonafarnib (Lona) or Tipifarnib (Tipi) at each concentration (0.0001 μM (0.1 nM), 0.001 μM (1 nM), 0.01 μM or 0.1 μM) The results of O-fast green-iron hematoxylin staining (upper) and the results of comparison of the ratio of Safranin O positive regions (lower) are shown. Vehicle（基剤）、Rosuva（ロスバスタチン）、各濃度（0.1μM、1μM、または10μM）のRocki、FTIまたはGGTIを添加した培地を用いてTD329N由来iPS細胞から分化誘導したパーティクル、および低コレステロール培地を用いてTD329N由来iPS細胞から分化誘導したパーティクルのSafranin O-fast green-iron hematoxylin stainingの結果を示す。Vehicle (base), Rosuva (rosuvastatin), particles induced to differentiate from TD329N-derived iPS cells using medium containing Rocki, FTI or GGTI at each concentration (0.1 μM, 1 μM, or 10 μM), and low cholesterol medium The results of Safranin O-fast green-iron hematoxylin staining of particles induced to differentiate from TD329N-derived iPS cells are shown. Vehicle（基剤）、Rosuva（ロスバスタチン）、各濃度（0.1μM、1μM、または10μM）のRocki、FTIまたはGGTIを添加した培地を用いてTD329N由来iPS細胞から分化誘導したパーティクル、および低コレステロール培地を用いてTD329N由来iPS細胞から分化誘導したパーティクルにSafranin O-fast green-iron hematoxylin stainingを施し、Safranin O陽性領域の比率を比較した結果を示す。Vehicle (base), Rosuva (rosuvastatin), particles induced to differentiate from TD329N-derived iPS cells using medium containing Rocki, FTI or GGTI at each concentration (0.1 μM, 1 μM, or 10 μM), and low cholesterol medium The results of using Safranin O-fast green-iron hematoxylin staining on particles induced to differentiate from TD329N-derived iPS cells and comparing the proportions of Safranin O positive regions are shown. Vehicle（基剤）、各濃度（0.0001μM、0.001μM、0.01μMまたは0.1μM）のFTI-277(FTI)、Lonafarnib(Lona)またはTipifarnib(Tipi)を添加した培地を用いてTD329N由来iPS細胞から分化誘導した軟骨細胞における、(A)SOX9、(B)COL2（II型コラーゲン）および(C)Acan（アグリカン）の発現をリアルタイムPCRで測定した結果を示す。From TD329N-derived iPS cells using vehicle (base), medium supplemented with FTI-277 (FTI), Lonafarnib (Lona) or Tipifarnib (Tipi) at each concentration (0.0001μM, 0.001μM, 0.01μM or 0.1μM) The result of having measured the expression of (A) SOX9, (B) COL2 (type II collagen) and (C) Acan (aggrecan) in the differentiation-induced chondrocytes by real-time PCR is shown. Vehicle（基剤）、Rosuva（ロスバスタチン）、各濃度（0.1μM、1μM、または10μM）のRocki、FTIまたはGGTIを添加した培地を用いてACH8858由来iPS細胞から分化誘導したパーティクル、および低コレステロール培地を用いてACH8858由来iPS細胞から分化誘導したパーティクルのSafranin O-fast green-iron hematoxylin stainingの結果を示す。Vehicle (base), Rosuva (rosuvastatin), particles induced to differentiate from ACH8858-derived iPS cells using medium containing Rocki, FTI or GGTI at each concentration (0.1 μM, 1 μM, or 10 μM), and low cholesterol medium The results of Safranin O-fast green-iron hematoxylin staining of particles induced by differentiation from ACH8858-derived iPS cells are shown. Vehicle（基剤）、Rosuva（ロスバスタチン）、各濃度（0.1μM、1μM、または10μM）のRocki、FTIまたはGGTIを添加した培地を用いてACH8858由来iPS細胞から分化誘導したパーティクル、および低コレステロール培地を用いてACH8858由来iPS細胞から分化誘導したパーティクルにSafranin O-fast green-iron hematoxylin stainingを施し、Safranin O陽性領域の比率を比較した結果を示す。Vehicle (base), Rosuva (rosuvastatin), particles induced to differentiate from ACH8858-derived iPS cells using medium containing Rocki, FTI or GGTI at each concentration (0.1 μM, 1 μM, or 10 μM), and low cholesterol medium The results of using Safranin O-fast green-iron hematoxylin staining to particles differentiated from ACH8858-derived iPS cells and comparing the proportion of Safranin O positive regions are shown. Vehicle（基剤）、各濃度（0.0001μM、0.001μM、0.01μMまたは0.1μM）のFTI-277(FTI)、Lonafarnib(Lona)またはTipifarnib(Tipi)を添加した培地を用いてACH8858由来iPS細胞から分化誘導した軟骨細胞における、(A)SOX9、(B)COL2（II型コラーゲン）および(C)Acan（アグリカン）の発現をリアルタイムPCRで測定した結果を示す。From ACH8858-derived iPS cells using vehicle (base), medium supplemented with FTI-277 (FTI), Lonafarnib (Lona) or Tipifarnib (Tipi) at each concentration (0.0001μM, 0.001μM, 0.01μM or 0.1μM) The result of having measured the expression of (A) SOX9, (B) COL2 (type II collagen) and (C) Acan (aggrecan) in the differentiation-induced chondrocytes by real-time PCR is shown. Vehicle（基剤）、Rosuva（ロスバスタチン）、各濃度（0.1μM、1μM、または10μM）のRocki、FTIまたはGGTIを添加した培地を用いてACHhomo-8859由来iPS細胞から分化誘導したパーティクル、および低コレステロール培地を用いてACHhomo-8859由来iPS細胞から分化誘導したパーティクルのSafranin O-fast green-iron hematoxylin stainingの結果を示す。Vehicle (base), Rosuva (rosuvastatin), particles induced by differentiation from ACHhomo-8859-derived iPS cells using media containing various concentrations (0.1 μM, 1 μM, or 10 μM) of Rocki, FTI, or GGTI, and low cholesterol The results of Safranin O-fast green-iron hematoxylin staining of particles induced to differentiate from ACHhomo-8859-derived iPS cells using a medium are shown. Vehicle（基剤）、Rosuva（ロスバスタチン）、各濃度（0.1μM、1μM、または10μM）のRocki、FTIまたはGGTIを添加した培地を用いてACHhomo-8859由来iPS細胞から分化誘導したパーティクル、および低コレステロール培地を用いてACHhomo-8859由来iPS細胞から分化誘導したパーティクルにSafranin O-fast green-iron hematoxylin stainingを施し、Safranin O陽性領域の比率を比較した結果を示す。Vehicle (base), Rosuva (rosuvastatin), particles induced by differentiation from ACHhomo-8859-derived iPS cells using media containing various concentrations (0.1 μM, 1 μM, or 10 μM) of Rocki, FTI, or GGTI, and low cholesterol The results of Safranin O-fast green-iron hematoxylin staining were applied to particles induced to differentiate from ACHhomo-8859-derived iPS cells using a medium, and the ratios of Safranin O positive regions were compared. Vehicle（基剤）、各濃度（0.0001μM、0.001μM、0.01μMまたは0.1μM）のFTI-277(FTI)、Lonafarnib(Lona)またはTipifarnib(Tipi)を添加した培地を用いてACHhomo-8859由来iPS細胞から分化誘導した軟骨細胞における、(A)SOX9、(B)COL2（II型コラーゲン）および(C)Acan（アグリカン）の発現をリアルタイムPCRで測定した結果を示す。ACHhomo-8859-derived iPS using a medium supplemented with vehicle (base), each concentration (0.0001μM, 0.001μM, 0.01μM or 0.1μM) of FTI-277 (FTI), Lonafarnib (Lona) or Tipifarnib (Tipi) The results of measuring the expression of (A) SOX9, (B) COL2 (type II collagen) and (C) Acan (aggrecan) in chondrocytes induced to differentiate from cells by real-time PCR are shown. Vehicle（基剤）、1μMのRosuvastatin（ロスバスタチン）、各濃度（0.2mMまたは2mM）のメトホルミン（Met）を添加した培地を用いて、TD714由来iPS細胞、ACH8858由来iPS細胞または健常個体由来iPS細胞（409B2）から軟骨へ分化誘導したパーティクルのSafranin O-fast green-iron hematoxylin stainingの結果を示す。TD714-derived iPS cells, ACH8858-derived iPS cells, or healthy individual-derived iPS cells using a medium supplemented with vehicle (base), 1 μM Rosuvastatin (rosuvastatin) and metformin (Met) at each concentration (0.2 mM or 2 mM) 409B2) shows the results of Safranin O-fast green-iron hematoxylin staining of particles induced to differentiate into cartilage. Vehicle（基剤）、1μMのRosuvastatin（ロスバスタチン）、各濃度（0.2mM、2mMまたは5mM）のメトホルミン（Met）を添加した培地を用いて、TD714由来iPS細胞、ACH8858由来iPS細胞または健常個体由来iPS細胞（409B2）から軟骨へ分化誘導したパーティクルにおける、COL2（II型コラーゲン）、Acan（アグリカン）およびSOX9の発現をリアルタイムPCRで測定した結果を示す。TD714-derived iPS cells, ACH8858-derived iPS cells, or healthy individuals-derived iPS using a medium supplemented with vehicle (base), 1 μM Rosuvastatin (rosuvastatin) and metformin (Met) at each concentration (0.2 mM, 2 mM or 5 mM) The result of having measured the expression of COL2 (type II collagen), Acan (aggrecan), and SOX9 in the particle | grains which induced differentiation from the cell (409B2) to the cartilage by real-time PCR is shown.
FGFRの変異を有する疾患（例えば、FGFR3病）の治療剤および／または予防剤
　本明細書において、「FGFR3病」は、FGFR3中に突然変異を有することにより骨形成異常を生じたあらゆる骨形成疾患を意味する。FGFR3病は、好ましくは、骨系統疾患の国際分類（Warman et al., Am J Med Genet 155A(5):943-68 (2011)）に記載されたFGFR3病群に属する一連の疾患を意味し、例えば、致死性骨異形成症 (TD) 、軟骨無形成症 (ACH)、軟骨低形成症、Camptodactyly, tall stature, and hearing loss syndrome (CATSHL)、Crouzon-like craniosynostosis with acanthosis nigricans (Crouzonodermoskeletal)、頭蓋骨早期癒合症などが挙げられる。FGFR3中に生じる突然変異は、機能獲得型もしくは機能欠損型のいずれの変異であってもよいが、好ましくは、機能獲得型の突然変異であり得る。 Therapeutic and / or preventive agent for a disease having a mutation in FGFR (for example, FGFR3 disease) In the present specification, “FGFR3 disease” refers to any bone morphogenetic disease in which bone dysplasia is caused by having a mutation in FGFR3. Means. FGFR3 disease preferably means a series of diseases belonging to the FGFR3 disease group described in the international classification of bone system diseases (Warman et al., Am J Med Genet 155A (5): 943-68 (2011)). For example, lethal osteodysplasia (TD), achondroplasia (ACH), hypochondral dysplasia, Camptodactyly, tall stature, and hearing loss syndrome (CATSHL), Crouzon-like craniosynostosis with acanthosis nigricans (Crouzonodermoskeletal), Examples include early skull fusion. The mutation occurring in FGFR3 may be either a gain-of-function type mutation or a function-deficient type mutation, but may preferably be a gain-of-function type mutation.
　本発明において、FGFRの変異を有する疾患（例えば、FGFR3病）の治療および／または予防剤は、メトホルミン、Farnesyl transferase阻害剤、Geranylgeranyl transferase阻害剤およびRock阻害剤から成る群より選択される少なくとも１つの薬剤を有効成分として含む医薬である。有効成分はメトホルミンおよびFarnesyl transferase阻害剤から成る群より選択される少なくとも１つの薬剤であってもよく、メトホルミンであってもよい。 In the present invention, the therapeutic and / or prophylactic agent for a disease having a mutation in FGFR (for example, FGFR3 disease) is at least one selected from the group consisting of metformin, Farnesyl transferase inhibitor, Geranylgeranyl transferase inhibitor, and Rock inhibitor. It is a medicine containing a drug as an active ingredient. The active ingredient may be at least one drug selected from the group consisting of metformin and a Farnesyl transferase inhibitor, and may be metformin.
　メトホルミン（化学名：1,1-Dimethylbiguanide、CAS番号：657-24-9、C4H11N5）は、ビグアナイド系の血糖降下薬として知られている。メトホルミン塩酸塩が製剤化され上市されている。 Metformin (chemical name: 1,1-Dimethylbiguanide, CAS number: 657-24-9, C 4 H 11 N 5 ) is known as a biguanide hypoglycemic drug. Metformin hydrochloride is formulated and marketed.
　本発明におけるFarnesyl transferase阻害剤はFarnesyl transferaseの機能を抑制できるものである限り特に限定されず、例えば、Lonafarnib（CAS番号：193275-84-2、C27H31Br2ClN4O2、例、Shen M et al., Drug Discov Today 20, 267-276 (2015)参照）、Chaetomellic acid A（CAS番号：148796-51-4、C19H32O4 2Na、例、J.B. Gibbs, et al.; J. Biol. Chem. 268, 7617 (1993)、 F. Tamanoi; Trends Biochem. Sci. 18, 349 (1993) 参照）、FPT Inhibitor I（C19H29NO6P 3Na、例、Manne, V., et al. 1995. Drug Development Res. 34, 121、Patel, D.V., et al. 1995. J. Med. Chem. 38, 2906参照）、FPT Inhibitor II（C17H28NO5P 2Na、例、Manne, V., et al. 1995. Drug Development Res. 34, 121参照）、FPT Inhibitor III（C23H39NO7P Na、例、Wang, D., et al. 1998. J. Biol. Chem. 273, 33027、Manne, V., et al. 1995. Drug Development Res. 34, 121参照）、FTase Inhibitor I（CAS番号：149759-96-6、C22H38N4O3S2、例、Cox, A D., et al., 1994 The Journal of biological chemistry. 269(30): 19203-6、Garcia, A M., et al., 1993 The Journal of biological chemistry. 268(25): 18415-8参照）、FTase Inhibitor II（CAS番号：156707-43-6、C15H21N3O4S2、例、Song, Jia L., et al., 2003 Microbiology (Reading, England). 149(Pt 1): 249-59参照）、FTI-276 trifluoroacetate salt（CAS番号：170006-72-1 (free base) 、C21H27N3O3S2 C2HF3O2、例、Cohen-Jonathan, E., et al., 1999 Radiation research. 152(4): 404-11参照）、FTI-277 trifluoroacetate salt（CAS番号：170006-73-2 (free base)、C22H29N3O3S2 xC2HF3O2、例、Cohen-Jonathan, E., et al., 1999 Radiation research. 152(4): 404-11参照）、L-744,832 Dihydrochloride（CAS番号：1177806-11-9、C26H45N3O6S2 2HCl、例、R.E. Barrington et al. Mol. Cell. Biol. 1998 18 85 2、L. Sepp-Lorenzo and N. Rosen J. Biol. Chem. 1998 273 20243 3、M.M. Moasser et al. Proc. Natl. Acad. Sci. USA 1998 95 1369参照）、Manumycin A（CAS番号：52665-74-4、C31H38N2O7、例、Hara, M., et al. 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 2281-2285参照）、Tipifarnib（CAS番号：192185-72-1、C27H22Cl2N4O、例、Shen M et al., Drug Discov Today 20, 267-276 (2015)参照）およびα-hydroxy Farnesyl Phosphonic Acid（CAS番号：148796-53-6、C15H27O4P、例、Pompliano, D.L., Rands, E., Schaber, M.D., et al., Biochemistry 31 3800-3807 (1992) 参照）を含むが、これらに限定されない。 The Farnesyl transferase inhibitor in the present invention is not particularly limited as long as it can suppress the function of Farnesyl transferase. For example, Lonafarnib (CAS number: 193275-84-2, C 27 H 31 Br 2 ClN 4 O 2 , for example, Shen M et al., Drug Discov Today 20, 267-276 (2015)), Chaetomellic acid A (CAS number: 148796-51-4, C 19 H 32 O 4 2Na, eg, JB Gibbs, et al .; J. Biol. Chem. 268, 7617 (1993), F. Tamanoi; Trends Biochem. Sci. 18, 349 (1993)), FPT Inhibitor I (C 19 H 29 NO 6 P 3Na, eg, Manne, V. , et al. 1995. Drug Development Res. 34, 121, Patel, DV, et al. 1995. J. Med. Chem. 38, 2906), FPT Inhibitor II (C 17 H 28 NO 5 P 2Na, eg, Manne, V., et al. 1995. Drug Development Res. 34, 121), FPT Inhibitor III (C 23 H 39 NO 7 P Na, eg, Wang, D., et al. 1998. J. Biol. Chem) 273, 33027, Manne, V., et al. 1995. Drug Development Res. 34, 121), FTase Inhibitor I (CAS number: 149759-96-6, C 22 H 38 N 4 O 3 S 2 , eg , Cox, A D., et al., 1994 The Journal of biological chemistry. 269 (30): 19203-6, Garcia, A M., et al., 1993 The Journal of biological chemistry. 268 (25): 18415-8 ), FTase Inhibitor II (CAS number: 156707-43-6, C 15 H 21 N 3 O 4 S 2 , eg, Song, Jia L., et al., 2003 Microbiology (Reading, England). 149 (Pt 1 ): 249-59), FTI-276 trifluoroacetate salt (CAS number: 170006-72-1 (free base), C 21 H 27 N 3 O 3 S 2 C 2 HF 3 O 2 , eg, Cohen-Jonathan, E., et al., 1999 Radiation research. 152 (4): 404-11), FTI-277 trifluoroacetate salt (CAS number: 170006-73-2 (free base), C 22 H 29 N 3 O 3 S 2 x C 2 HF 3 O 2 , eg, Cohen-Jonathan, E., et al., 1999 Radiation research. 152 (4): 404-11), L-744,832 Dihydrochloride (CAS number: 1177806-11-9, C 26 H 45 N 3 O 6 S 2 2HCl, e.g., RE Barrington et al. Mol. Cell. Biol. 1998 18 85 2, L. Sepp-Lorenzo and N. Rosen J. Biol. Chem. 1998 273 20243 3, MM Moasser et al. Proc. Natl. Acad. Sci. USA 1998 95 1369), Man umycin A (CAS number: 52665-74-4, C 31 H 38 N 2 O 7 , eg Hara, M., et al. 1993. Proc. Natl. Acad. Sci. USA 90: 2281-2285), Tipifarnib (CAS number: 192185-72-1, C 27 H 22 Cl 2 N 4 O, see eg Shen M et al., Drug Discov Today 20, 267-276 (2015)) and α-hydroxy Farnesyl Phosphonic Acid ( CAS number: 148796-53-6, C 15 H 27 O 4 P, for example, see Pompliano, DL, Rands, E., Schaber, MD, et al., Biochemistry 31 3800-3807 (1992)) It is not limited to these.
　本発明におけるGeranylgeranyl transferase阻害剤はGeranylgeranyl transferaseの機能を抑制できるものである限り特に限定されず、例えば、GGTI-2418（CAS番号：171744-11-9、C23H31N3O3S、例、Shen M et al., Drug Discov Today 20, 267-276 (2015)参照）、GGTI-2133（CAS番号：191102-79-1、C27H28N4O3 C2HF3O2、例、Shen M et al., Drug Discov Today 20, 267-276 (2015)参照）、GGTI-2147（CAS番号：191102-87-1、C28H30N4O3、例、Bernot, D., et al. 2003. J. Cardiovasc. Pharmacol. 41, 316 参照）、GGTI-2154（CAS番号：251577-10-3、例、Shen M et al., Drug Discov Today 20, 267-276 (2015)参照）、GGTI-2166（日化辞番号：J1.244.741H、C25H30N4O3）、GGTI-286（CAS番号：171744-11-9、C23H31N3O3S、例、Lerner, E C., et al., 1995 The Journal of biological chemistry. 270(45): 26770-3 参照）、GGTI-287（C22H29N3O3S、例、Shen M et al., Drug Discov Today 20, 267-276 (2015)参照）、GGTI-297（CAS番号：181045-83-0、C26H31N3O3S C2HF3O2、例、Sun, J., et al. 1998. Oncogene 16, 1467参照）およびGGTI-298（CAS番号：180977-44-0、C27H33N3O3S CF3CO2H、例、McGuire, T F., et al., 1996 The Journal of biological chemistry. 271(44): 27402-7 参照）を含むが、これらに限定されない。 The Geranylgeranyl transferase inhibitor in the present invention is not particularly limited as long as it can suppress the function of Geranylgeranyl transferase. For example, GGTI-2418 (CAS number: 171744-11-9, C 23 H 31 N 3 O 3 S, for example) , Shen M et al, Drug Discov Today 20, see 267-276 (2015)), GGTI- 2133 (CAS number:. 191102-79-1, C 27 H 28 N 4 O 3 C 2 HF 3 O 2, example , Shen M et al., Drug Discov Today 20, 267-276 (2015)), GGTI-2147 (CAS number: 191102-87-1, C 28 H 30 N 4 O 3 , eg, Bernot, D., et al. 2003. See J. Cardiovasc. Pharmacol. 41, 316), GGTI-2154 (CAS number: 251577-10-3, eg, Shen M et al., Drug Discov Today 20, 267-276 (2015)). ), GGTI-2166 (Nikkaji number: J1.244.741H, C 25 H 30 N 4 O 3 ), GGTI-286 (CAS number: 171744-11-9, C 23 H 31 N 3 O 3 S, for example) Lerner, E C., et al., 1995 The Journal of biological chemistry. 270 (45): 26770-3), GGTI-287 (C 22 H 29 N 3 O 3 S, eg, Shen M et al. , Drug Discov Today 20, 267-276 (2015) Irradiation), GGTI-297 (CAS Number:. 181045-83-0, C 26 H 31 N 3 O 3 S C 2 HF 3 O 2, example, Sun, J., et al 1998. Oncogene 16, 1467 reference) and GGTI-298 (CAS number: 180977-44-0, C 27 H 33 N 3 O 3 S CF 3 CO 2 H, eg, McGuire, T F., et al., 1996 The Journal of biological chemistry. 271 (44 ): 27402-7), but not limited to.
　本発明におけるRock阻害剤は、Rho-キナーゼ（ROCK）の機能を抑制できるものである限り特に限定されず、例えば、Y-27632（例、Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000)；Narumiya et al., Methods Enzymol. 325,273-284 (2000)参照）、Fasudil/HA1077（例、Uenata et al., Nature 389: 990-994 (1997)参照）、SR3677（例、Feng Y et al., J Med Chem. 51: 6642-6645(2008)参照）、GSK269962（例、Stavenger RA et al., J Med Chem. 50: 2-5 (2007)またはWO2005/037197参照）、H-1152（例、Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)参照）、Wf-536（例、Nakajima et al., Cancer Chemother Pharmacol. 52(4): 319-324 (2003)参照）およびそれらの誘導体、ならびにROCKに対するアンチセンス核酸、RNA干渉誘導性核酸（例、siRNA）、ドミナントネガティブ変異体、およびそれらの発現ベクターが挙げられる。また、ROCK阻害剤としては他の公知の低分子化合物も使用できる（例えば、米国特許出願公開第2005/0209261号、同第2005/0192304号、同第2004/0014755号、同第2004/0002508号、同第2004/0002507号、同第2003/0125344号、同第2003/0087919号、及び国際公開第2003/062227号、同第2003/059913号、同第2003/062225号、同第2002/076976号、同第2004/039796号参照）。本発明では、１種または２種以上のROCK阻害剤が使用され得る。 The Rock inhibitor in the present invention is not particularly limited as long as it can suppress the function of Rho-kinase (ROCK). For example, Y-27632 (eg, Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325,273-284 (2000)), Fasudil / HA1077 (eg, Uenata et al., Nature 389: 990-994 (1997)), SR3677 (eg, Feng Y et al., J Med Chem. 51: 6642-6645 (2008)), GSK269962 (eg, StavengerStRA et al., J Med Chem. 50: 2-5 (2007) or WO2005 / 037197), H- 1152 (eg, Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (eg Nakajima et al., Cancer Chemother Pharmacol. 52 (4): 319-324 (2003) And derivatives thereof, as well as antisense nucleic acids to ROCK, RNA interference inducing nucleic acids (eg, siRNA), dominant negative mutants, and their expression vectors. Other known low-molecular compounds can also be used as ROCK inhibitors (for example, US Patent Application Publication Nos. 2005/0209261, 2005/0192304, 2004/0014755, 2004/0002508). 2004/0002507, 2003/0125344, 2003/0087919, and International Publications 2003/062227, 2003/059913, 2003/062225, 2002/076976 No., 2004/039796). In the present invention, one or more ROCK inhibitors may be used.
　なお、本発明のメトホルミン、Farnesyl transferase阻害剤、Geranylgeranyl transferase阻害剤またはRock阻害剤は、その薬理学的に許容される塩（好ましくは、塩酸塩、ナトリウム塩またはカルシウム塩）を包含する。 The metformin, Farnesyl transferase inhibitor, Geranylgeranyl transferase inhibitor or Rock inhibitor of the present invention includes pharmacologically acceptable salts thereof (preferably hydrochloride, sodium salt or calcium salt).
　メトホルミン、Farnesyl transferase阻害剤、Geranylgeranyl transferase阻害剤またはRock阻害剤をFGFR3病の治療剤および／または予防剤として使用する場合、常套手段に従って製剤化することができる。例えば、経口投与のための組成物としては、固体または液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤等が挙げられる。一方、非経口投与のための組成物としては、例えば、注射剤、坐剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含しても良い。これらの製剤は、賦形剤（例えば、乳糖、白糖、葡萄糖、マンニトール、ソルビトールのような糖誘導体；トウモロコシデンプン、バレイショデンプン、α澱粉、デキストリンのような澱粉誘導体；結晶セルロースのようなセルロース誘導体；アラビアゴム；デキストラン；プルランのような有機系賦形剤；および、軽質無水珪酸、合成珪酸アルミニウム、珪酸カルシウム、メタ珪酸アルミン酸マグネシウムのような珪酸塩誘導体；燐酸水素カルシウムのような燐酸塩；炭酸カルシウムのような炭酸塩；硫酸カルシウムのような硫酸塩等の無機系賦形剤である）、滑沢剤（例えば、ステアリン酸、ステアリン酸カルシウム、ステアリン酸マグネシウムのようなステアリン酸金属塩；タルク；コロイドシリカ；ビーズワックス、ゲイ蝋のようなワックス類；硼酸；アジピン酸；硫酸ナトリウムのような硫酸塩；グリコール；フマル酸；安息香酸ナトリウム；ＤＬロイシン；ラウリル硫酸ナトリウム、ラウリル硫酸マグネシウムのようなラウリル硫酸塩；無水珪酸、珪酸水和物のような珪酸類；および、上記澱粉誘導体である）、結合剤（例えば、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドン、マクロゴール、および、前記賦形剤と同様の化合物である）、崩壊剤（例えば、低置換度ヒドロキシプロピルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、内部架橋カルボキシメチルセルロースナトリウムのようなセルロース誘導体；カルボキシメチルスターチ、カルボキシメチルスターチナトリウム、架橋ポリビニルピロリドンのような化学修飾されたデンプン・セルロース類である）、乳化剤（例えば、ベントナイト、ビーガムのようなコロイド性粘土；水酸化マグネシウム、水酸化アルミニウムのような金属水酸化物；ラウリル硫酸ナトリウム、ステアリン酸カルシウムのような陰イオン界面活性剤；塩化ベンザルコニウムのような陽イオン界面活性剤；および、ポリオキシエチレンアルキルエーテル、ポリオキシエチレンソルビタン脂肪酸エステル、ショ糖脂肪酸エステルのような非イオン界面活性剤である）、安定剤（メチルパラベン、プロピルパラベンのようなパラオキシ安息香酸エステル類；クロロブタノール、ベンジルアルコール、フェニルエチルアルコールのようなアルコール類；塩化ベンザルコニウム；フェノール、クレゾールのようなフェノール類；チメロサール；デヒドロ酢酸；および、ソルビン酸である）、矯味矯臭剤（例えば、通常使用される、甘味料、酸味料、香料等である）、希釈剤等の添加剤を用いて周知の方法で製造される。 When using metformin, Farnesyl transferase inhibitor, Geranylgeranyl transferase inhibitor or Rock inhibitor as a therapeutic and / or prophylactic agent for FGFR3 disease, it can be formulated according to conventional means. For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like. On the other hand, as a composition for parenteral administration, for example, injections, suppositories and the like are used, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. Dosage forms may be included. These formulations include excipients (eg sugar derivatives such as lactose, sucrose, sucrose, mannitol, sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; Gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate and magnesium metasilicate aluminate; phosphates such as calcium hydrogen phosphate; Carbonates such as calcium; inorganic excipients such as sulfates such as calcium sulfate), lubricants (eg, stearic acid metal salts such as stearic acid, calcium stearate, magnesium stearate; talc; Colloidal silica; like bead wax, gay wax Boric acid; adipic acid; sulfate such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL leucine; lauryl sulfate such as sodium lauryl sulfate and magnesium lauryl sulfate; anhydrous silicic acid, silicic acid hydrate Such as silicic acids; and the above-mentioned starch derivatives), binders (for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, and compounds similar to the excipients), disintegrants ( For example, low-substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, cellulose derivatives such as internally crosslinked sodium carboxymethylcellulose; carboxymethyl starch, carboxymethyl starch sodium, Chemically modified starches and celluloses such as cross-linked polyvinyl pyrrolidone), emulsifiers (eg, colloidal clays such as bentonite and bee gum; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; sodium lauryl sulfate Anionic surfactants such as calcium stearate; cationic surfactants such as benzalkonium chloride; and nonionic interfaces such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, sucrose fatty acid esters Activators), stabilizers (paraoxybenzoates such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenol, Phenols such as alcohol; thimerosal; dehydroacetic acid; and sorbic acid), flavoring agents (for example, commonly used sweeteners, acidulants, fragrances, etc.), additives such as diluents Is manufactured by a well-known method.
　本発明の薬剤の患者への投与量は、治療すべき病態の種類、症状および疾患の重篤度、患者年齢、性別もしくは体重、投与法などにより異なるので一義的には言えないが、医師が前記状況を考慮して判断することにより、適宜適当な投与量を決定することができる。 The dose of the drug of the present invention to a patient varies depending on the type of pathological condition to be treated, symptoms and severity of the disease, patient age, sex or body weight, administration method, etc. By determining in consideration of the above situation, an appropriate dose can be determined as appropriate.
　経口投与の場合には、例えば、１回あたり下限0.1mg（好ましくは、0.5mg）、上限1000mg（好ましくは、500mg）を、非経口的投与の場合には、１回あたり下限0.01mg（好ましくは、0.05mg）、上限100mg（好ましくは、50mg）を、成人に対して１日あたり１から６回投与することができる。症状に応じて増量もしくは減量してもよい。 In the case of oral administration, for example, the lower limit is 0.1 mg (preferably 0.5 mg) and the upper limit is 1000 mg (preferably 500 mg). In the case of parenteral administration, the lower limit is 0.01 mg (preferably 0.05 mg) and an upper limit of 100 mg (preferably 50 mg) can be administered to adults 1 to 6 times per day. The dose may be increased or decreased depending on the symptoms.
　本発明を以下の実施例でさらに具体的に説明するが、本発明の範囲はそれら実施例に限定されないものとする。 The present invention will be described more specifically in the following examples, but the scope of the present invention is not limited to these examples.
実施例１
iPS細胞の作製
　以下のすべての実験は、治験審査委員会、動物実験委員会および施設内生物安全委員会、ならびに京都大学の承認を得て行われた。 Example 1
Generation of iPS cells All the following experiments were conducted with the approval of the clinical trial review committee, animal experiment committee and institutional biosafety committee, and Kyoto University.
　TD患者由来のヒト皮膚線維芽細胞(TD714, TD10749, TD315HおよびTD329N) を、コリエル医学研究所および埼玉県立小児医療センターから入手した。これらのヒト皮膚線維芽細胞（HDF）から抽出したゲノムDNAを配列解析したところ、すべてのTD患者においてFGFR3遺伝子のヘテロ接合突然変異（Arg248Cys）が確認された。ACH患者由来のHDFとして、FGFR3遺伝子においてGly380Argのヘテロ接合突然変異を有する２人のACH患者のHDF（ACH8857およびACH8858）、およびACHよりも重篤な軟骨形成不全を示すHDF（ACHhomo-8859）をコリエル医学研究所より入手した。各患者由来のHDFから以下に記載する方法でiPS細胞を作製した。対照iPS細胞として、K. OkitaおよびS. Yamanaka（京都大学iPS細胞研究所）から入手した健常な個体由来のiPS細胞株(409B2)（Okita, K., et al. Nature methods 8, 409-412 (2011))を用いた。 Human skin fibroblasts derived from TD patients (TD714, TD10749, TD315H and TD329N) were obtained from the Coryell Medical Research Institute and Saitama Children's Medical Center. Sequence analysis of genomic DNA extracted from these human dermal fibroblasts (HDF) confirmed a heterozygous mutation (Arg248Cys) in the FGFR3 gene in all TD patients. As HDFs from ACH patients, HDFs (ACH8857 and ACH8858) of two ACH patients with a Gly380Arg heterozygous mutation in the FGFR3 gene, and HDF (ACHhomo-8859) showing more severe chondrogenesis than ACH Obtained from the Coryell Medical Institute. IPS cells were prepared from the HDF derived from each patient by the method described below. As control iPS cells, a healthy individual-derived iPS cell line (409B2) obtained from K. Okita and S. Yamanaka (Kyoto University iPS Cell Laboratory) (Okita, K., et al. Nature methods 8, 409-412 (2011)).
　iPS細胞の製造は、次の方法によって行った。詳細には、入手したそれぞれのヒト線維芽細胞（HDF）を、10% FBS (Invitrogen), 50 U/ml penicillinおよび50 μg/ml streptomycinを添加したDMEM (Sigma)で培養した。続いて、Neon transfection system (Invitrogen)により、エピソーマルプラスミドベクター(Mixture Y4: OCT3/4, SOX2, KLF4, L-MYC, LIN28およびp53 shRNA)を各HDFにエレクトロポレーションで導入した(Okita, K., et al. Nature methods 8, 409-412 (2011))。１週間後に、フィーダー細胞をあらかじめ播種しておいた100 mmディッシュに1×105個の当該ベクターを導入したHDFを播種した。次いで、hiPSC培地（20% KSR（Invitrogen）, 2mM L-グルタミン（Invitrogen）, 1×10-4M 非必須アミノ酸（Invitrogen）, 1×10-4M 2-メルカプトエタノール（Invitrogen）, 50units/ml ペニシリン（Invitrogen）, 50μg/ml ストレプトマイシン（Invitrogen）, 4ng/ml bFGF（WAKO）を添加したDMEM/F12 (Sigma)）で培養した。得られた各iPS細胞は、抗SSEA4抗体(Santa Cruz, sc-5279)および抗-TRA1-60抗体(Abcam, ab16287)を用いた免疫組織化学により評価した。 iPS cells were produced by the following method. Specifically, each obtained human fibroblast (HDF) was cultured in DMEM (Sigma) supplemented with 10% FBS (Invitrogen), 50 U / ml penicillin and 50 μg / ml streptomycin. Subsequently, episomal plasmid vectors (Mixture Y4: OCT3 / 4, SOX2, KLF4, L-MYC, LIN28 and p53 shRNA) were electroporated into each HDF by Neon transfection system (Invitrogen) (Okita, K ., et al. Nature methods 8, 409-412 (2011)). One week later, 1 × 10 5 HDFs into which the vector was introduced were seeded on 100 mm dishes in which feeder cells had been seeded in advance. Next, hiPSC medium (20% KSR (Invitrogen), 2 mM L-glutamine (Invitrogen), 1 × 10 −4 M non-essential amino acid (Invitrogen), 1 × 10 −4 M 2-mercaptoethanol (Invitrogen), 50 units / ml The cells were cultured in penicillin (Invitrogen), 50 μg / ml streptomycin (Invitrogen), 4 ng / ml bFGF (WAKO) -added DMEM / F12 (Sigma)). Each obtained iPS cell was evaluated by immunohistochemistry using an anti-SSEA4 antibody (Santa Cruz, sc-5279) and an anti-TRA1-60 antibody (Abcam, ab16287).
　その結果、作製されたすべてのiPSC株はES細胞マーカー（SSEA4およびTRA1-60）を発現しており、三胚葉を含むテラトーマを形成することが確認された。 As a result, it was confirmed that all iPSC strains produced expressed ES cell markers (SSEA4 and TRA1-60) and formed teratomas containing three germ layers.
mRNAの測定
　RNAは、RNeasy Mini Kit (Qiagen)を用いて、各細胞から単離した。得られた全RNAのうち500 ngを鋳型として、ReverTra Ace (TOYOBO)を用いて逆転写によりcDNAを合成した。定量PCR（リアルタイムPCR）のための標準曲線を作成して解析を行った。リアルタイムPCR解析は、KAPA SYBR FAST qPCR kit Master Mix ABI prism (KAPA BIOSYSTEMS)を用いて、Step One system (ABI)により測定した。mRNAの発現量は、b-ACTINの発現量で校正した。使用したプライマーの配列を表１に示す。 Measurement of mRNA RNA was isolated from each cell using RNeasy Mini Kit (Qiagen). CDNA was synthesized by reverse transcription using ReverTra Ace (TOYOBO) using 500 ng of the obtained total RNA as a template. A standard curve for quantitative PCR (real-time PCR) was prepared and analyzed. Real-time PCR analysis was performed by Step One system (ABI) using KAPA SYBR FAST qPCR kit Master Mix ABI prism (KAPA BIOSYSTEMS). The expression level of mRNA was calibrated with the expression level of b-ACTIN. The primer sequences used are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
実施例２
軟骨誘導
　以前に報告された方法（Oldershaw, R.A., et al., Nat Biotechnol 28, 1187-1194 (2010)）を修正した以下に記載の方法に従って、各iPS細胞から軟骨細胞に分化誘導した。 Example 2
Differentiation from each iPS cell was induced to chondrocytes according to the method described below, which was modified from a method reported before cartilage induction (Oldershaw, RA, et al., Nat Biotechnol 28, 1187-1194 (2010)).
　各iPS細胞をマトリゲル（Invitrogen）コートディッシュ上に播種し、Essential 8培地（Life Technologies）に50units/ml ペニシリンおよび50μg/ml ストレプトマイシンを添加した培地を加え、フィーダーフリー条件下で未分化維持培養した。播種後10～15日目に、1～2×105細胞から成るコロニーを中胚葉分化培地（DMEM/F12に対し10ng/ml Wnt3A（R&D）, 10ng/ml アクチビンA（R&D）, 1% インスリン-トランスフェリン-亜セレン酸ナトリウム（Invitrogen）, 1% 牛胎児血清, 50units/ml ペニシリン, 50μg/ml ストレプトマイシンを混合して調製）に交換した（分化誘導０日目）。３日後（分化誘導３日目）、各薬剤（1μMのロスバスタチン（BioVision, 1995-5）、各濃度（0.1μM、1μMまたは10μM）のFarnesyl transferase阻害剤（FTI-277）（Sigma）（以下、FTIという）、各濃度（0.1μM、1μMまたは10μM）のGeranylgeranyl transferase阻害剤（GGTI-287）（Sigma）（以下、GGTIという）、各濃度（0.1μM、1μM、10μMまたは100μM）のRock阻害剤（Y27632）（Wako）（以下、Rockiという））を添加した軟骨分化培地（50μg/mlアスコルビン酸、10ng/ml BMP2（Osteopharma）、10ng/ml TGFβ（Pepro Tech）、10ng/ml GDF5、1% インスリン-トランスフェリン-亜セレン酸ナトリウム、1% FBS、50units/ml ペニシリンおよび 50μg/ml ストレプトマイシンを含有するDMEM/F12）または低コレステロール軟骨分化培地（上記軟骨分化培地のFBSを低コレステロールFBSへ置換した培地）に交換し、１１日後（分化誘導１４日目）にディッシュから細胞を引き剥がした。同じ培地を用いて浮遊培養により２８日間培養し、パーティクルを回収した（分化誘導４２日目）。分化誘導中は、２～７日ごとに培地を交換した。 Each iPS cell was seeded on a Matrigel (Invitrogen) coated dish, a medium supplemented with 50 units / ml penicillin and 50 μg / ml streptomycin was added to Essential 8 medium (Life Technologies), and cultured under feeder-free conditions. 10-15 days after seeding, colonies consisting of 1-2 × 10 5 cells were transformed into mesodermal differentiation medium (10 ng / ml Wnt3A (R & D), 10 ng / ml activin A (R & D), 1% insulin for DMEM / F12) -Transferrin-sodium selenite (Invitrogen), 1% fetal bovine serum, 50 units / ml penicillin, 50 μg / ml prepared by mixing streptomycin) (Day 0 of differentiation induction). Three days later (differentiation induction day 3), each drug (1 μM rosuvastatin (BioVision, 1995-5), each concentration (0.1 μM, 1 μM or 10 μM) of Farnesyl transferase inhibitor (FTI-277) (Sigma) (hereinafter, FTI), Geranylgeranyl transferase inhibitor (GGTI-287) (Sigma) (hereinafter referred to as GGTI) at each concentration (0.1 μM, 1 μM or 10 μM), Rock inhibitor at each concentration (0.1 μM, 1 μM, 10 μM or 100 μM) (Y27632) (Wako) (hereinafter referred to as Rocki)) cartilage differentiation medium (50 μg / ml ascorbic acid, 10 ng / ml BMP2 (Osteopharma), 10 ng / ml TGFβ (Pepro Tech), 10 ng / ml GDF5, 1% Insulin-transferrin-sodium selenite, 1% FBS, DMEM / F12 containing 50 units / ml penicillin and 50 μg / ml streptomycin or low cholesterol cartilage differentiation medium (medium in which FBS in the above cartilage differentiation medium is replaced with low cholesterol FBS 11 days later (differentiation invitation) The cells were peeled off from the dish on day 14). The particles were collected by floating culture using the same medium for 28 days (differentiation induction day 42). During differentiation induction, the medium was changed every 2 to 7 days.
　別途、上記と同じ方法で、３種類のFarnesyl transferase阻害剤を用いて各iPS細胞から軟骨細胞に分化誘導した。すなわち、分化誘導３日目にFTI-277（Sigma）（以下、FTIという）、Lonafarnib（Cayman）（以下、Lonaという）およびTipifarnib（LCL）（以下、Tipiという）をそれぞれ濃度0.0001μM、0.001μM、0.01μMまたは0.1μMで添加した軟骨分化培地に交換し、分化誘導１４日目にディッシュから細胞を引き剥がし、同じ培地を用いて浮遊培養により２８日間培養し、分化誘導４２日目にパーティクルを回収した。 Separately, differentiation was induced from each iPS cell to a chondrocyte using three types of Farnesyl transferase inhibitors in the same manner as described above. That is, on the third day of differentiation induction, FTI-277 (Sigma) (hereinafter referred to as FTI), Lonafarnib (Cayman) (hereinafter referred to as Lona) and Tipifarnib (LCL) (hereinafter referred to as Tipi) at concentrations of 0.0001 μM and 0.001 μM, respectively. The culture medium was replaced with a cartilage differentiation medium added at 0.01 μM or 0.1 μM, the cells were peeled off from the dish on the 14th day of induction of differentiation, and cultured for 28 days in suspension culture using the same medium. It was collected.
　得られたパーティクル内に存在する軟骨細胞における、軟骨細胞マーカーであるSOX9および軟骨マトリックスタンパク質をコードする遺伝子（II型コラーゲン（COL2A1）およびアグリカン（Acan））のmRNAの発現量をリアルタイムPCRで調べた。さらに、得られたパーティクルをサフラニンO染色し、サフラニンO陽性の軟骨組織の存在を確認すると共に、サフラニンO陽性領域の比率を求めた。 In the chondrocytes present in the obtained particles, the expression level of mRNA of the genes encoding chondrocyte marker SOX9 and cartilage matrix protein (type II collagen (COL2A1) and aggrecan (Acan)) was examined by real-time PCR. . Furthermore, the obtained particles were stained with safranin O to confirm the presence of safranin O-positive cartilage tissue and to determine the ratio of safranin O-positive regions.
（1）TD714由来iPS細胞の結果
　図１にTD714由来iPS細胞から、Farnesyl transferase阻害剤（FTI-277）、Geranylgeranyl transferase阻害剤（GGTI-287）、またはRock阻害剤（Y27632）を添加した培地を用いて分化誘導した軟骨細胞における軟骨細胞マーカーをリアルタイムPCRで測定した結果を示した。(A)がSOX9、(B)がCOL2A1、(C)がAcanの結果である。いずれの薬剤を用いた場合でも、Vehicle（基剤）と比較して各遺伝子の発現を上昇させることが確認された。 (1) Results of TD714-derived iPS cells Fig. 1 shows a medium containing TD714-derived iPS cells supplemented with Farnesyl transferase inhibitor (FTI-277), Geranylgeranyl transferase inhibitor (GGTI-287), or Rock inhibitor (Y27632). The results of measuring the chondrocyte markers in the chondrocytes induced to differentiate by real-time PCR are shown. (A) is the result of SOX9, (B) is the result of COL2A1, and (C) is the result of Acan. In any case, it was confirmed that the expression of each gene was increased as compared with Vehicle (base).
　図２、図３および図４に、TD714由来iPS細胞から、Farnesyl transferase阻害剤（FTI-277）、Geranylgeranyl transferase阻害剤（GGTI-287）、またはRock阻害剤（Y27632）を添加した培地を用いて分化誘導したパーティクルをサフラニンO染色した結果を示した。いずれの薬剤を用いた場合でも、サフラニンO陽性の軟骨組織が確認された。 2, 3, and 4, using a medium supplemented with a Farnesyl transferase inhibitor (FTI-277), Geranylgeranyl transferase inhibitor (GGTI-287), or Rock inhibitor (Y27632) from TD714-derived iPS cells. The results of the differentiation-induced particles were stained with safranin O. Safranin O-positive cartilage tissue was confirmed when any drug was used.
　図５にTD714由来iPS細胞から、３種類のFarnesyl transferase阻害剤（FTI-277、LonafarnibおよびTipifarnib）をそれぞれ添加した培地を用いて分化誘導した軟骨細胞における軟骨細胞マーカーをリアルタイムPCRで測定した結果を示した。(A)がSOX9、(B)がCOL2A1、(C)がAcanの結果である。いずれのFarnesyl transferase阻害剤を用いた場合でも、Vehicle（基剤）と比較して各遺伝子の発現を上昇させることが確認された。 Fig. 5 shows the results of real-time PCR measurement of chondrocyte markers in chondrocytes induced by differentiation from TD714-derived iPS cells using media supplemented with three types of Farnesyl transferase inhibitors (FTI-277, Lonafarnib and Tipifarnib). Indicated. (A) is the result of SOX9, (B) is the result of COL2A1, and (C) is the result of Acan. Even when any Farnesyl transferase inhibitor was used, it was confirmed that the expression of each gene was increased as compared with Vehicle (base).
　図６および図７に、TD714由来iPS細胞から、３種類のFarnesyl transferase阻害剤（FTI-277、LonafarnibおよびTipifarnib）をそれぞれ添加した培地を用いて分化誘導したパーティクルをサフラニンO染色した結果を示した。いずれのFarnesyl transferase阻害剤を用いた場合でも、サフラニンO陽性の軟骨組織が確認された。 FIG. 6 and FIG. 7 show the results of staining with Safranin O for particles induced to differentiate from TD714-derived iPS cells using media supplemented with three types of Farnesyl transferase inhibitors (FTI-277, Lonafarnib and Tipifarnib), respectively. . Safranin O-positive cartilage tissue was confirmed when any Farnesyl transferase inhibitor was used.
（2）TD329N由来iPS細胞の結果
　図８および図９に、TD329N由来iPS細胞から、Farnesyl transferase阻害剤（FTI-277）、Geranylgeranyl transferase阻害剤（GGTI-287）、またはRock阻害剤（Y27632）を添加した培地を用いて分化誘導したパーティクルをサフラニンO染色した結果を示した。いずれの薬剤を用いた場合でも、サフラニンO陽性の軟骨組織が確認された。 (2) Results of TD329N-derived iPS cells In FIGS. 8 and 9, a Farnesyl transferase inhibitor (FTI-277), a Geranylgeranyl transferase inhibitor (GGTI-287), or a Rock inhibitor (Y27632) is obtained from TD329N-derived iPS cells. The results of staining with Safranin O for the particles induced to differentiate using the added medium are shown. Safranin O-positive cartilage tissue was confirmed when any drug was used.
　図１０にTD329N由来iPS細胞から、３種類のFarnesyl transferase阻害剤（FTI-277、LonafarnibおよびTipifarnib）をそれぞれ添加した培地を用いて分化誘導した軟骨細胞における軟骨細胞マーカーをリアルタイムPCRで測定した結果を示した。(A)がSOX9、(B)がCOL2A1、(C)がAcanの結果である。いずれのFarnesyl transferase阻害剤を用いた場合でも、Vehicle（基剤）と比較して各遺伝子の発現を上昇させることが確認された。 Fig. 10 shows the results of real-time PCR measurement of chondrocyte markers in chondrocytes induced to differentiate from TD329N-derived iPS cells using media supplemented with three types of Farnesyl transferase inhibitors (FTI-277, Lonafarnib and Tipifarnib). Indicated. (A) is the result of SOX9, (B) is the result of COL2A1, and (C) is the result of Acan. Even when any Farnesyl transferase inhibitor was used, it was confirmed that the expression of each gene was increased as compared with Vehicle (base).
（3）ACH8858由来iPS細胞の結果
　図１１および図１２に、ACH8858由来iPS細胞から、Farnesyl transferase阻害剤（FTI-277）、Geranylgeranyl transferase阻害剤（GGTI-287）、またはRock阻害剤（Y27632）添加した培地を用いて分化誘導したパーティクルをサフラニンO染色した結果を示した。いずれの薬剤を用いた場合でも、サフラニンO陽性の軟骨組織が確認された。 (3) Results of ACH8858-derived iPS cells FIG. 11 and FIG. 12 show the addition of Farnesyl transferase inhibitor (FTI-277), Geranylgeranyl transferase inhibitor (GGTI-287), or Rock inhibitor (Y27632) from ACH8858-derived iPS cells. The results are shown in which the particles induced to differentiate using the prepared medium were stained with safranin O. Safranin O-positive cartilage tissue was confirmed when any drug was used.
　図１３にACH8858由来iPS細胞から、３種類のFarnesyl transferase阻害剤（FTI-277、LonafarnibおよびTipifarnib）をそれぞれ添加した培地を用いて分化誘導した軟骨細胞における軟骨細胞マーカーをリアルタイムPCRで測定した結果を示した。(A)がSOX9、(B)がCOL2A1、(C)がAcanの結果である。いずれのFarnesyl transferase阻害剤を用いた場合でも、Vehicle（基剤）と比較して各遺伝子の発現を上昇させることが確認された。 FIG. 13 shows the results of real-time PCR measurement of chondrocyte markers in chondrocytes induced to differentiate from ACH8858-derived iPS cells using media supplemented with three types of Farnesyl transferase inhibitors (FTI-277, Lonafarnib and Tipifarnib), respectively. Indicated. (A) is the result of SOX9, (B) is the result of COL2A1, and (C) is the result of Acan. Even when any Farnesyl transferase inhibitor was used, it was confirmed that the expression of each gene was increased as compared with Vehicle (base).
（4）ACHhomo-8859由来iPS細胞の結果
　図１４および図１５に、ACHhomo-8859由来iPS細胞から、Farnesyl transferase阻害剤（FTI-277）、Geranylgeranyl transferase阻害剤（GGTI-287）、またはRock阻害剤（Y27632）添加した培地を用いて分化誘導したパーティクルをサフラニンO染色した結果を示した。いずれの薬剤を用いた場合でも、サフラニンO陽性の軟骨組織が確認された。 (4) Results of ACHhomo-8859-derived iPS cells FIG. 14 and FIG. 15 show that from ACHhomo-8859-derived iPS cells, a Farnesyl transferase inhibitor (FTI-277), Geranylgeranyl transferase inhibitor (GGTI-287), or Rock inhibitor. (Y27632) The results of staining with Safranin O for the particles induced to differentiate using the added medium are shown. Safranin O-positive cartilage tissue was confirmed when any drug was used.
　図１６にACHhomo-8859由来iPS細胞から、３種類のFarnesyl transferase阻害剤（FTI-277、LonafarnibおよびTipifarnib）をそれぞれ添加した培地を用いて分化誘導した軟骨細胞における軟骨細胞マーカーをリアルタイムPCRで測定した結果を示した。(A)がSOX9、(B)がCOL2A1、(C)がAcanの結果である。いずれのFarnesyl transferase阻害剤を用いた場合でも、Vehicle（基剤）と比較して各遺伝子の発現を上昇させることが確認された。 FIG. 16 shows real-time PCR for chondrocyte markers in chondrocytes induced to differentiate from ACHhomo-8859-derived iPS cells using media supplemented with three types of Farnesyl transferase inhibitors (FTI-277, Lonafarnib and Tipifarnib). Results are shown. (A) is the result of SOX9, (B) is the result of COL2A1, and (C) is the result of Acan. Even when any Farnesyl transferase inhibitor was used, it was confirmed that the expression of each gene was increased as compared with Vehicle (base).
実施例３
　被験薬剤をメトホルミンに変更した以外は実施例２と同じ方法で、TD714由来iPS細胞およびACH8858由来iPS細胞から軟骨細胞に誘導した。対照iPS細胞として、健常個体由来の409B2を用いた。メトホルミン（1,1-Dimethylbiguanide hydrochloride(sigma)）の濃度は、0.2mM, 2mMまたは5mMとした。 Example 3
Except for changing the test drug to metformin, chondrocytes were induced from TD714-derived iPS cells and ACH8858-derived iPS cells in the same manner as in Example 2. As a control iPS cell, 409B2 derived from a healthy individual was used. The concentration of metformin (1,1-Dimethylbiguanide hydrochloride (sigma)) was 0.2 mM, 2 mM or 5 mM.
　図１７にメトホルミンを添加した培地を用いて分化誘導したパーティクルをサフラニンO染色した結果を示した。TD714由来iPS細胞およびACH8858由来iPS細胞から分化誘導したパーティクルには、どちらももサフラニンO陽性の軟骨組織が確認された。 FIG. 17 shows the result of Safranin O staining of particles induced to differentiate using a medium supplemented with metformin. Safranin O-positive cartilage tissue was confirmed in both particles induced to differentiate from TD714-derived iPS cells and ACH8858-derived iPS cells.
　図１８にメトホルミンを添加した培地を用いて分化誘導した軟骨細胞における軟骨細胞マーカー（COL2A1、Acan、SOX9）をリアルタイムPCRで測定した結果を示した。各遺伝子の発現量は、TD714由来iPS細胞を1μMのロスバスタチンで分化誘導したときの発現量を1として、相対値で示した。メトホルミンの添加により、各遺伝子の発現を上昇させることが確認された。 FIG. 18 shows the results of real-time PCR measurement of chondrocyte markers (COL2A1, Acan, SOX9) in chondrocytes induced to differentiate using a medium supplemented with metformin. The expression level of each gene was expressed as a relative value, assuming that the expression level when TD714-derived iPS cells were induced to differentiate with 1 μM rosuvastatin was 1. It was confirmed that the addition of metformin increases the expression of each gene.
　以上の結果から、メトホルミン、Farnesyl transferase阻害剤、Geranylgeranyl transferase阻害剤またはRock阻害剤を用いることで、TD患者由来のiPS細胞およびACH患者由来のiPS細胞からであっても軟骨組織を形成させることができることが確認された。従って、メトホルミン、Farnesyl transferase阻害剤、Geranylgeranyl transferase阻害剤およびRock阻害剤は、致死性骨異形成症および軟骨無形成症の治療剤となり得ることが示唆された。 From the above results, by using metformin, Farnesyl transferase inhibitor, Geranylgeranyl transferase inhibitor or Rock inhibitor, cartilage tissue can be formed even from iPS cells derived from TD patients and iPS cells derived from ACH patients It was confirmed that it was possible. Therefore, it was suggested that metformin, Farnesyl® transferase inhibitor, Geranylgeranyl® transferase inhibitor and Rock inhibitor can be therapeutic agents for lethal osteodysplasia and achondroplasia.
　本発明は、スクリーニングの結果得られた軟骨細胞への分化を促進する物質を提供するものであり、当該物質は、FGFR3病の新たな治療剤および／または予防剤として利用可能である。 The present invention provides a substance that promotes differentiation into chondrocytes obtained as a result of screening, and the substance can be used as a new therapeutic agent and / or preventive agent for FGFR3 disease.

Claims (6)

  1. 　有効成分としてメトホルミン、Farnesyl transferase阻害剤、Geranylgeranyl transferase阻害剤およびRock阻害剤から成る群より選択される少なくとも１つの薬剤を含む、FGFR3病の治療および／または予防用医薬。 A pharmaceutical for the treatment and / or prevention of FGFR3 disease, comprising as an active ingredient at least one drug selected from the group consisting of metformin, Farnesyl transferase inhibitor, Geranylgeranyl transferase inhibitor and Rock inhibitor.
  2. 　前記薬剤がメトホルミンである、請求項１に記載の医薬。 The medicament according to claim 1, wherein the drug is metformin.
  3. 　前記Farnesyl transferase阻害剤が、Lonafarnib、Chaetomellic acid A、FPT Inhibitor I、FPT Inhibitor II、FPT Inhibitor III、FTase Inhibitor I、FTase Inhibitor II、FTI-276 trifluoroacetate salt、FTI-277 trifluoroacetate salt、L-744,832 Dihydrochloride、Manumycin A、Tipifarnibおよびα-hydroxy Farnesyl Phosphonic Acidから成る群より選択される薬剤である、請求項１に記載の医薬。 The Farnesyl transferase inhibitor is Lonafarnib, Chaetomellic acid A, FPT Inhibitor I, FPT Inhibitor II, FPT Inhibitor III, FTase Inhibitor I, FTase Inhibitor II, FTI-276 trifluoroacetate salt, FTI-277 trifluoroacetate 744alt, The medicament according to claim 1, which is a drug selected from the group consisting of ManumycinyA, Tipifarnib, and α-hydroxy Farnesyl Phosphonic Acid.
  4. 　前記Geranylgeranyl transferase阻害剤が、GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297およびGGTI-298から成る群より選択される薬剤である、請求項１に記載の医薬。 The Geranylgeranyl transferase inhibitor is selected from the group consisting of GGTI-2418, GGTI-2133, GGTI-2147, GGTI-2154, GGTI-2166, GGTI-286, GGTI-287, GGTI-297 and GGTI-298. The medicament according to claim 1, wherein
  5. 　前記Rock阻害剤が、Y-27632、Fasudil/HA1077、SR3677、GSK269962、H-1152およびWf-536から成る群より選択される薬剤である、請求項１に記載の医薬。 The medicament according to claim 1, wherein the Rock inhibitor is a drug selected from the group consisting of Y-27632, Fasudil / HA1077, SR3677, GSK269962, H-1152 and Wf-536.
  6. 　前記FGFR3病が、致死性骨異形成症 (TD) および／または軟骨無形成症 (ACH)である、請求項１から請求項５のいずれか一つに記載の医薬。 The medicament according to any one of claims 1 to 5, wherein the FGFR3 disease is lethal bone dysplasia (TD) and / or achondroplasia (ACH).
PCT/JP2017/013524 2016-04-01 2017-03-31 Prophylactic or therapeutic agent for fgfr3 disease WO2017170981A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2018509644A JP6908283B2 (en) 2016-04-01 2017-03-31 Preventive or therapeutic agents for FGFR3 disease

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2016074721 2016-04-01
JP2016-074721 2016-04-01

Publications (1)

Publication Number Publication Date
WO2017170981A1 true WO2017170981A1 (en) 2017-10-05

Family

ID=59965978

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2017/013524 WO2017170981A1 (en) 2016-04-01 2017-03-31 Prophylactic or therapeutic agent for fgfr3 disease

Country Status (2)

Country Link
JP (1) JP6908283B2 (en)
WO (1) WO2017170981A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019093389A1 (en) * 2017-11-08 2019-05-16 公益財団法人 東京都医学総合研究所 Therapeutic agent for mental retardation or autism
JP2020115771A (en) * 2019-01-22 2020-08-06 国立大学法人京都大学 Method for producing cartilage tissue from pluripotent stem cell
WO2024061474A1 (en) * 2022-09-23 2024-03-28 Norwegian University Of Science And Technology (Ntnu) Combination therapy for the treatment or prevention of neurological disorders

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001516572A (en) * 1997-09-19 2001-10-02 マガイニン ファーマシューティカルズ インク． Asthma-related factors as targets for treating atopic allergy, including asthma and related diseases
WO2004091660A1 (en) * 2003-04-17 2004-10-28 Kowa Co., Ltd. Lklf/klf2 gene expression promoter
JP2009513660A (en) * 2005-10-26 2009-04-02 旭化成ファーマ株式会社 Fasudil in combination therapy for the treatment of pulmonary arterial hypertension
WO2015083582A1 (en) * 2013-12-02 2015-06-11 国立大学法人京都大学 Prophylactic and therapeutic agent for fgfr3 diseases and method for screening same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001516572A (en) * 1997-09-19 2001-10-02 マガイニン ファーマシューティカルズ インク． Asthma-related factors as targets for treating atopic allergy, including asthma and related diseases
WO2004091660A1 (en) * 2003-04-17 2004-10-28 Kowa Co., Ltd. Lklf/klf2 gene expression promoter
JP2009513660A (en) * 2005-10-26 2009-04-02 旭化成ファーマ株式会社 Fasudil in combination therapy for the treatment of pulmonary arterial hypertension
WO2015083582A1 (en) * 2013-12-02 2015-06-11 国立大学法人京都大学 Prophylactic and therapeutic agent for fgfr3 diseases and method for screening same

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019093389A1 (en) * 2017-11-08 2019-05-16 公益財団法人 東京都医学総合研究所 Therapeutic agent for mental retardation or autism
JPWO2019093389A1 (en) * 2017-11-08 2020-11-19 公益財団法人東京都医学総合研究所 Remedies for intellectual disability or autism
US11642337B2 (en) 2017-11-08 2023-05-09 Tokyo Metropolitan Institute Of Medical Science Therapeutic agent for mental retardation or autism
JP2020115771A (en) * 2019-01-22 2020-08-06 国立大学法人京都大学 Method for producing cartilage tissue from pluripotent stem cell
JP7269620B2 (en) 2019-01-22 2023-05-09 国立大学法人京都大学 Method for producing cartilage tissue from pluripotent stem cells
WO2024061474A1 (en) * 2022-09-23 2024-03-28 Norwegian University Of Science And Technology (Ntnu) Combination therapy for the treatment or prevention of neurological disorders

Also Published As

Publication number Publication date
JPWO2017170981A1 (en) 2019-02-07
JP6908283B2 (en) 2021-07-21

Similar Documents

Publication Publication Date Title
Hombach-Klonisch et al. Glioblastoma and chemoresistance to alkylating agents: Involvement of apoptosis, autophagy, and unfolded protein response
US8691829B2 (en) Treatment of liver disorders with PI3K inhibitors
Matthiesen et al. Muscarinic receptors mediate stimulation of human lung fibroblast proliferation
Tomasi et al. S‐adenosyl methionine regulates ubiquitin‐conjugating enzyme 9 protein expression and sumoylation in murine liver and human cancers
WO2019084271A1 (en) Papd5 inhibitors and methods of use thereof
EP1458395B1 (en) Medical use of gsk3-inhibiting oxindole derivatives
WO2017170981A1 (en) Prophylactic or therapeutic agent for fgfr3 disease
US20060217368A1 (en) Drug for nerve regeneration
CN103638029A (en) MPO inhibitors for the treatment of huntington's disease and multiple system atrophy
US20170319611A1 (en) Methods and compositions comprising akt inhibitors and/or phospholipase d inhibitors
JPWO2016104574A1 (en) Ectopic ossification preventive / therapeutic agent and screening method thereof
WO2008075201A2 (en) Epigallocatechin-3-gallate compositions for cancer therapy and chemoprotection
Geleta et al. Cyclic dependent kinase (CDK): role in cancer pathogenesis and as drug target in cancer therapeutics
WO2016159301A1 (en) Therapeutic agent for chronic myeloid leukemia
Zhu et al. Epigenetic regulation of cancer stem cell and tumorigenesis
Mourlevat et al. Prevention of dopaminergic neuronal death by cyclic AMP in mixed neuronal/glial mesencephalic cultures requires the repression of presumptive astrocytes
Osawa et al. Lipopolysaccharide-induced sensitization of adenylyl cyclase activity in murine macrophages
JP2023154067A (en) Prophylactic and/or therapeutic agent for amyotrophic lateral sclerosis
Libertini et al. Aurora A and B kinases-targets of novel anticancer drugs
US20150231109A1 (en) Combinations (catechins and methotrexate) for use in the treatment of melanomas
Zhang et al. Autophagy-related proteins in genome stability: autophagy-dependent and independent actions
US20050171182A1 (en) Methods and compositions for use in the treatment of mutant receptor tyrosine kinase driven cellular proliferative diseases
TW590772B (en) Combination preparation for use in dementia
EP2873419B1 (en) Pharmaceutical composition for the prevention or treatment of brain tumor or temodal-resistant glioblastoma multiform comprising azathioprine as active ingredient
US11472807B2 (en) Purine derivatives for use as medicament and for use in treating neurodegenerative or neuro-inflammatory disorders

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 2018509644

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17775513

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17775513

Country of ref document: EP

Kind code of ref document: A1