ANTAGONIST OF THE FIBROBLAST GROWTH FACTOR RECEPTOR 3 (FGFR3) FOR USE IN THE TREATMENT OR THE PREVENTION OF SKELETAL DISORDERS LINKED WITH ABNORMAL ACTIVATION OF FGFR3

Abstract

The present invention relates to the treatment or prevention of skeletal disorders, in particular skeletal diseases, developed by patients that display abnormal increased activation of the fibroblast growth factor receptor 3 (FGFR3), in particular by expression of a constitutively activated mutant of FGFR3.

Claims

1. A method for treating or preventing a FGFR3-related skeletal disease which comprises the step of administering at least one antagonist of the FGFR3 tyrosine kinase receptor of formula: ##STR00036## or a composition comprising such an antagonist, to a subject in need thereof.

2. The method according to claim 1, wherein the FGFR3-related skeletal disease is selected from the group consisting of thanatophoric dysplasia type I, thanatophoric dysplasia type II, severe achondroplasia with developmental delay and acanthosis nigricans, hypochondroplasia, achondroplasia and FGFR3-related craniosynostosis such as Muenke syndrome and Crouzon syndrome with acanthosis nigricans.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0107] FIG. 1. A31 prevents the kinase activity of FGFR3.

[0108] (A) Molecular scheme of the A31 compound. (B) Overall structure showing docking conformation of A31 inside the FGFR3 binding pocket. A31 is represented with rods. (C) Overall structure showing A31 in the ATP binding site.

[0109] FIG. 2. A31 inhibits the constitutive activation of FGFR3.

[0110] Immunoblots showing FGFR3 overexpression in transfected cells (HEK) with WT human-cDNA (FGFR3.sup.+/+) and 3 human mutant cDNA constructs (FGFR3.sup.Y373C, FGFR3.sup.K650E, FGFR3.sup.K650M). FGFR3 is immunoprecipitated (IP) and immunoblotted (IB) with anti-FGFR3 and antiphosphotyrosine antibodies (Ptyr). Ptyr immunoblot showing constitutive phosphorylation of FGFR3 in transfected cells with mutant cDNA constructs. FGFR3 immunoblots showing three isoforms of the protein (105, 115 and 130 kDa) in WT (FGFR3.sup.+/+) and one mutant (FGFR3.sup.Y373C/+). Two isoforms of FGFR3 protein (105 kDa and 115 kDa) were present in cells transfected with mutant constructs (FGFR3.sup.K650M and FGFR3.sup.K650E). A31 reduces the constitutive phosphorylation of FGFR3.

[0111] FIG. 3. A31 restores longitudinal bone growth of Fgfr3Y367C/+ femurs.

[0112] (A) Fgfr3.sup.Y367C/+ mouse embryo at E16.5 shows a dome-shape skull. (B) Fgfr3.sup.Y367C/+ femur is broader with a shorter diaphysis at E16.5 (C) Alizarin red and alcian blue staining show the small size of Fgfr3.sup.Y367C/+ femurs. A31 increases the size of the Fgfr3.sup.Y367C/+ femurs after 5 days of culture. (D) Bone length measurements showing a reduced longitudinal growth in Fgfr3.sup.Y367C/+ femurs compared with WT (Fgfr3.sup.Y367C/+, 461±119 μm; WT, 1247±227 μm; p<10.sup.−10). A31 enhances longitudinal growth in Fgfr3.sup.Y367C/+ femurs, the bone growth is greater in Fgfr3.sup.Y367C/+ femurs compared with controls (Fgfr3.sup.Y367C/+, 1880±558 μm; WT, 1863±255 μm; ***p<10.sup.−19 versus untreated controls). The experiments were performed 6 times and bone length is shown as mean+/−s.d.

[0113] FIG. 4. A31 modifies the size of the growth plate and chondrocyte morphology.

[0114] (A) HES staining showing the reduced size of the Fgfr3.sup.Y367C/+ growth plate. A31 induces an increase in the size of the growth plate of the Fgfr3.sup.Y367C/+ mice. (B) In situ hybridization of type X collagen showing a markedly reduced hypertrophic zone (see the size of “H” symbolized by the size of the double-headed arrows) of Fgfr3.sup.Y367C/+ growth plates compared with WT. A31 induces enhanced type X collagen expression in Fgfr3.sup.Y367C/+ growth plates.

[0115] FIG. 5. A31 decreases Fgfr3 overexpression in Fgfr3Y367C/+ femurs.

[0116] Costal primary chondrocytes were examined by western-blot with anti-FGFR3. Fgfr3 protein level is higher in Fgfr.sup.3Y367C/+ chondrocytes compared with WT. A31 reduced this overexpression.

[0117] FIG. 6. A31 reduces proliferation and cell cycle regulator expression in growth plates.

[0118] (A) Quantification of PCNA-positive cells in proliferative (P), prehypertrophic (PH) and hypertrophic zones (H) showing a higher level of PCNA positive cells in Fgfr3.sup.Y367C/+ growth plates (73% (PH) and 43% (H), **p<0,005 versus WT) compared with WT (31% (PH) and 18% (H)). A31 induces a strong decrease of PCNA expression in PH and H zones of Fgfr3.sup.Y367C/+ growth plates (20% (PH) and 18% (H), ***p<10-4 versus untreated femurs). The experiments were performed six times and three observers counted positive cells. % PCNA positive cells are shown as mean+/−s.d. (B) Immunoblot showing a higher cyclin D1 expression in costal primary Fgfr3.sup.Y367C/+ chondrocytes compared with WT. A31 reduces the expression of cyclin D1 in Fgfr3.sup.Y367C/+ chondrocytes. Actin is included as loading control.

[0119] FIG. 7. PD173074 restores longitudinal bone growth of Fgfr3Y367C/+ femurs.

[0120] (A) Alizarin red and alcian blue staining show that after 5 days of culture PD173074 increases the size of the Fgfr3.sup.Y367C/+ femurs (left panel). The effect of PD173074 on femur growth is similar to that of A31 (right panel).

[0121] (B) PD173074 enhances longitudinal growth in Fgfr3.sup.Y367C/+ femurs (see bar “PD173074” vs bar “no treatment”), and the bone growth of PD173074 treated femurs is analogous to that observed when Fgfr3.sup.Y367C/+ femurs are treated with A31.

[0122] FIG. 8. PD173074 attenuates the dwarfism phenotype of Fgfr3.sup.Y367C/+ mice. Fgfr3.sup.Y367C/+ mice seven days old received daily subcutaneous administration of 4.00 mg/kg PD173074 for 10 days. Effect of the treatment on the skeleton and body growth was assessed by an X-rays analysis. On panels (A) and (B), PD173074 treated Fgfr3.sup.Y367C/+ mouse is on the left, vehicle treated Fgfr3.sup.Y367C/+ mouse is on the right).

[0123] FIG. 9. BGJ-398 restores longitudinal bone growth of Fgfr3.sup.Y367C/+ femurs.

[0124] Bone length measurements showing a reduced longitudinal growth in Fgfr3.sup.Y367C/+ femurs compared with WT (Fgfr3.sup.+/+). Concentration of BGJ-398 ranging from 100 nM to 1 μM enhances longitudinal growth in Fgfr3.sup.Y367C/+ femurs: the bone growth is greater in Fgfr3.sup.Y367C/+ femurs compared with controls (Fgfr3.sup.+/+).

[0125] FIG. 10. BGJ-398 modifies the size of the growth plate and chondrocyte morphology.

[0126] (A) HES staining showing the reduced size of the Fgfr3.sup.Y367C/+ growth plate. BGJ-398 induces an increase in the size of the growth plate of the Fgfr3.sup.Y367C/+ mice. (B) In situ hybridization of type X collagen showing a markedly reduced hypertrophic zone (symbolized by the size of the double-headed arrows) of Fgfr3.sup.Y367C/+ growth plates compared with WT (Fgfr3.sup.+/+). BGJ-398 induces enhanced type X collagen expression in Fgfr3.sup.Y367C/+ growth plates.

[0127] FIG. 11. BGJ-398 attenuates the dwarfism phenotype of Fgfr3.sup.Y367C/+ mice. Fgfr3.sup.Y367C/+ mice seven days old received daily subcutaneous administration of 1.66 mg/kg BGJ-398 for 10 days. Effect of the treatment on the skeleton and body growth was assessed by an X-rays analysis (BGJ-398 treated Fgfr3.sup.Y367C/+ mouse is on the left, vehicle treated Fgfr3.sup.Y367C/+ mouse is on the right).

EXAMPLE 1: Materials and Methods

[0128] Chemical Compound.

[0129] A series of inhibitors was previously designed and synthesized as PD173074 (Miyake et al., J Pharmacol Exp Rher., 332: 797-802, 2010) analogues bearing various N-substituents. Of 27 analogues synthesized, A31 (refers to 19g) was selected in the course of preliminary cellular assays for its ability to inhibit FGFR3 phosphorylation (Le Corre et al., Org Biomol Chem, 8: 2164-2173, 2010.). This compound competes with ATP binding and can inhibit autophosphorylation of FGFR3, with an IC50 value of approximately 190 nM. As a control, the inventors used the commercial FGFR TKI, PD173074. TKIs were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. The stock solution was stored at −20° C. before use.

[0130] Computational Analyses.

[0131] The kinase domain structure of FGFR3 was predicted by homology modeling with the Esypred3D software (Lambert et al., Bioinformatics, 18: 1250-1256, 2002) using a recent X-ray structure of the highly homologous FGFR1 protein (pdb code 3JS2) (Ravindranathan et al., J Med Chem, 53: 1662-1672, 2010). The inventors used AMBER software (Case, D. A., Darden, T. A., Cheatham, T. E., Simmerling, C. L., Wang, J., Duke, R. E., Luo, R., Merz, K. M., Pearlman, D. A., Crowley, M. et al. (2006). University of California, San Francisco) according to a previously published protocol (Luo, Y. et al., J Mol Model, 14: 901-910, 2008). The inventors built A31 compound using the Sybyl software package version 11.0 (SYBYL. Tripos Inc., 1699 South Hanley Rd., St Louis, Mo., 63144 USA). Two states of the asymmetric carbon (R, S) and two different protonation states of the neighboring amino moiety (neutral and +1) were considered. Four distinct chemical structures were obtained. Energy minimizations of these four A31 structures were performed (Hu, R., Barbault, F., Delamar, M. and Zhang, R., Bioorg Med Chem, 17: 2400-2409, 2009). Docking calculations were carried out with version 4.2 of the program AutoDock (Morris et al., J Comput Chem., 19: 1639-1662, 1998.). Kollman's united atomic charges were computed. A grid box of 23×20×33 Å was constructed in, respectively, the x,y and z axes around the binding cavity. All ligand torsion angles were allowed to rotate during docking, leading to a complete flexibility. One hundred cycles of calculations of Lamarckian Genetic Algorithm were performed to complete the conformational search. 100 resulting docking structures were clustered into conformation families according to a RMSD lower than 2.0 Å. The inventors selected the conformation, which presented the lowest docking free energy of binding in the most populated cluster.

[0132] Ex Vivo Experiments.

[0133] Heterozygous Fgfr3.sup.Y367C/+ mice ubiquitously expressing the Y367C mutation and exhibiting a severe dwarfism were used (Pannier et al., Biochim Biophys Acta, 1792: 140-147, 2009). Six sets of ex vivo experiments were performed. Femur embryos at day E16.5 from WT (n=6) and Fgfr3.sup.Y367C/+ (n=6) mice were used and incubated for 5 days in DMEM medium with antibiotics and 0.2% BSA (Sigma) supplemented with A31 or PD173074 (as control) at a concentration of 2 mM. Right femur was cultured in supplemented medium and compared with the left one cultured in control medium. Rib cage from E16.5 WT and Fgfr3.sup.Y367C/+ mice embryos were isolated and stripped of all soft tissues. Primary chondrocytes were obtained from rib cages. The ribs were incubated in a pronase solution (Roche; 2 mg/ml) followed by a digestion in Collagenase A (Roche; 3 mg/ml) at 37° C. Isolated chondrocytes were plated out at a density of 2.105 cells in 6-well plates containing DMEM supplemented with 10% FCS and antibiotics, and were allowed to reach subconfluency. Cultures were supplemented with A31 or PD173074 (as control) at a concentration of 2 mM. Cells were treated with A31 (2 mM) PD173074 (as control) in serum-free DMEM supplemented with 0.2% BSA and harvested after 24h. To establish the effect of the inhibitors, the right femur was cultured in supplemented medium and compared with the left one cultured in control medium The bone length was measured at the beginning (before treatment) and at the end of time course. Each experiment was repeated at least three times. The genotype of WT, Fgfr3.sup.Y367C/+ and Fgfr3.sup.−/− mice were determined by PCR of tail DNA as previously described (Pannier et al., Biochim Biophys Acta, 1792: 140-147, 2009). All experimental procedures and protocols were approved by the Animal Care and Use Committee.

[0134] Histological, In Situ Hybridization and Immunohistochemical Analyses.

[0135] Limb explants were fixed after culture in 4% paraformaldehyde at 4° C., and placed in a staining solution for 45-60 minutes (0.05% Alizarin Red, 0.015% Alcian Blue, 5% acetic acid in 70% ethanol) or embedded in paraffin. Serial mm?sections of 5 were stained with Hematoxylin-Eosin using standard protocols for histological analysis or were subjected to in situ hybridization or immunohistochemical staining.

[0136] In situ hybridization using [S35]-UTP labeled antisense ribopropes for collagen X was carried out as previously described (Delezoide et al., Hum Mol Genet, 6, 1899-1906, 1997). Sections were counterstained with Hematoxylin. For immunohistochemistry, sections were stained with antibodies specific to FGFR3 (1:250 dilution; Sigma), anti PCNA (1:1000 dilution; Abcam), anti-K167 (1:300; Abcam), anti-cyclin D1 (1:80 dilution; Santa Cruz) and anti-p57 (1:100 dilution; Santa Cruz) using the Dako Envision kit. Images were captured with an Olympus PD70-1X2-UCB microscope.

[0137] Quantification of PCNA Expression.

[0138] Three observers counted PCNA-positive and negative chondrocytes in proliferative (H), prehypertrophic (PH) and hypertrophic (H) zones of the growth plate. A Student's t-test was used to compare treated (A31) and untreated femurs. Imagine software cellSens (Olympus) was used for counting cells. A p-value<0.05 is considered significant.

[0139] Immunoprecipiation, Immunoblotting and Immunocytochemistry Experiments.

[0140] Human Embryonic Kidney (HEK) cells and human chondrocyte lines (Benoist-Lasselin et al., FEBS Lett, 581: 2593-2598, 2007.) were transfected transiently with FGFR3 human constructs (FGFR.sup.3Y373C, FGFR3.sup.K650M, FGFR3.sup.K650E) (Gibbs, L. and Legeai-Mallet, L. Biochim Biophys Acta, 1773: 502-512, 2007) using Fugene 6 (Roche). A31 (31) or PD173074 (Parke Davies) were added at a concentration of 2 mM overnight. Transfected cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.5% NP40, 0.25% sodium deoxycholate, supplemented with protease and phosphatase inhibitors).

[0141] Immunoprecipitation were performed by incubating 3 mL rabbit anti-FGFR3 (Sigma)/500 mg protein with protein A-agarose (Roche). Immunoprecipitated proteins were subjected to SDS-polyacrylamide gel electrophoresis on NuPAGE 4-12% bis-tris acrylamide gels (Invitrogen). Immunoprecipitated proteins were subjected to SDSpolyacrilamide gels electrophoresis on NuPAGE 4-12% bis-tris acrylamide gels (Invitrogen). Blots were hybridized overnight at 4° C. with anti-FGFR3 polyclonal antibody (1:1,000 dilution; Sigma), or anti-phosphotyrosine monoclonal antibody (1:400 dilution; Cell Signaling). Lysates of primary murine chondrocytes (E16.5) were subjected to SDS-polyacrylamide gel electrophoresis and were hybridized overnight at 4° C. with anti-cyclin D1 monoclonal antibody (1:100 dilution; Santa Cruz). A secondary antibody, anti-rabbit or anti-mouse coupled to peroxidase, was used at a dilution of 1:10,000 (Amersham). Bound proteins were detected by chemiluminescence (ECL, Amersham). The blots were rehybrididized with an antipan-actin antibody for quantification (Millipore).

[0142] For immunocytochemistry, the inventors used the following primary antibodies: anti-FGFR3 antibodies (1:400 dilution; Sigma) and anti-phosphotyrosine antibodies (1:200 dilution; Cell Signaling) and secondary antibodies Alexa Fluor® 488 goat antirabbit and Alexa Fluor® 568 goat anti-mouse (1:400 dilution; Molecular Probes). Cells were covered with Faramount Aquaeous Mounting Medium (Dako) and analyzed using an Olympus PD70-IX2-UCB microscope.

[0143] Proliferation Studies.

[0144] NIH-3T3 clones stably expressing FGFR3.sup.+/+ (WT) and FGFR3.sup.Y373C, FGFR3K650M (human constructs) were used. The stable clones were selected with G418. NIH-3T3 clones were incubated for 8 h in 10% newborn calf serum DMEM supplemented or not with A31 (2 mM). [3H] thymidine was added at a concentration of 10 mCi/ml and incubated for 16 hours. The cells were harvested on glass fiber filter paper and assayed for radioactivity by liquid scintillation counting. The inventors used Top Count Microplates scintillation counter (Perkin Elmer).

[0145] In Vivo Experiments.

[0146] The effectiveness of PD173074 and BGJ-398 in attenuating the dwarfism phenotype of Fgfr3.sup.Y367C/+ mice was assessed in vivo. The mice were seven days of age at treatment initiation and received daily subcutaneous administration of 4.00 mg/kg PD173074 or of 1.66 mg/kg BGJ-398 for 10 days.

EXAMPLE 2: Strong Interaction Between the Tyrosine Kinase Domain of FGFR3 and A31

[0147] Computational analyses were used to estimate interactions between FGFR3 and A31, a synthetic compound of the pyrido-[2,3-d] pyrimidine class, as a novel FGFR3 tyrosine kinase inhibitor (TKI) (FIG. 1A). To date, attempts to determine the experimental Xray structure of the FGFR3 kinase domain have failed. To overcome this drawback, the inventors predicted the structure of FGFR3 in silico by using a new crystal structure of the highly homologous FGFR1 (Ravindranathan et al., J Med Chem, 53: 1662-1672, 2010). The resulting 3D structure of FGFR3 showed a low global energy and negative electrostatic and Van der Waals components indicating a high level of confidence for this prediction. Docking calculations were used to find the optimal position of A31 in the binding pocket of FGFR3. The interactions between the FGFR3 kinase domain and A31 are depicted in FIG. 1B. The aromatic group carrying the two methoxy moieties and the biphenyl ring induce strong interactions between the FGFR3 kinase domain and A31. Two hydrogen bonds locate the biphenyl ring at the adenine position of ATP, filling the FGFR3 active site and, in this way, A31 competes directly with the substrate. The cyclic amino tail of A31 is deeply nestled inside the FGFR3 cavity in the vicinity of a protein salt bridge. As a consequence, the salt bridge is disrupted, thus preventing the kinase activity of FGFR3. These in-silico data suggest that A31 specifically inhibits FGFR3 kinase activity.

EXAMPLE 3: A31 Inhibits FGFR3 Phosphorylation and Proliferation of Mutant Fgfr3 Cell Lines

[0148] The inventors evaluated the ability of A31 to inhibit the constitutive phosphorylation of FGFR3 in human chondrocyte lines (Gibbs, L. and Legeai-Mallet, L. Biochim Biophys Acta, 1773: 502-512, 2007) transiently expressing activated forms of FGFR3 (FGFR3.sup.Y373C or FGFR3.sup.K650E (TD), FGFR3.sup.K650M (SADDAN) or FGFR3.sup.+/+).

[0149] Immunoprecipitation and Western blotting showed the presence of a 130 kDa mature isoform in the WT and FGFR3.sup.Y373C cell lysates, whereas only an 115 kDa immature form was present in FGFR3.sup.K650M and FGFR3.sup.K650E lysates (FIG. 2). A31, abolished receptor phosphorylation in all cells expressing FGFR3 mutations (FIG. 2). Similar results were found with a commercial TKI inhibitor (PD173074) (FIG. 2). This inhibition was confirmed by immunocytochemistry in transfected cells expressing FGFR3 mutations (data not shown). The inventors observed a complete inhibition of FGFR3 phosphorylation by A31.

[0150] This data confirmed the ability of A31 to inhibit constitutive FGFR3 phosphorylation in transfected cells. To determine whether A31 modulates the mitogenic activity of activated FGFR3, the inventors measured [3H]-thymidine incorporation in FGFR3.sup.Y373c and FGFR3.sup.K650M transfected NIH3T3 cells. The mitogenic activity was increased in cells expressing FGFR3 mutations compared to WT (FGFR3.sup.Y373C, 9927±2921 cpm; FGFR3.sup.K650M, 15048±5251 cpm; WT, 7499±1667 cpm; p<10.sup.−5 versus WT).

[0151] A31 treatment strongly reduced DNA synthesis of all mutant cell lines (FGFR3.sup.Y373C, 3144±1201 cpm; FGFR3.sup.K650M, 6281±2699 cpm; **p<10.sup.−10, ***p<10.sup.−20 versus DMSO). These results demonstrate that A31 decreases the mitogenic activity of FGFR3 mutants.

[0152] To confirm these results, the ability of BGJ-398 (also designated as compound 1 h in Table B), another tyrosine kinase inhibitor, to inhibit the constitutive phosphorylation of FGFR3 in cells (HEK-293) transiently expressing activated forms of FGFR3 (i.e. FGFR3Y373C, FGFR3K650E , FGFR3K650M, FGFR3G380R) was also tested. It was found that 10 μM of BGJ-398 abolished receptor phosphorylation in all cells expressing FGFR3 mutations (data not shown).

EXAMPLE 4: Rescue of the Fgfr3.SUP.Y367C/+ femur growth defect by A31 and BGJ-398

[0153] A31 was tested on a gain of function Fgfr3.sup.Y367C/+ mouse model (Pannier et al., Biochim Biophys Acta, 1792: 140-147, 2009). It is to be noted that mutation Y367C in mouse FGFR3 corresponds to mutation Y373C in human FGFR3.

[0154] Fgfr3.sup.Y367C/+ mice display reduced length of long bones, broad femurs, a narrow trunk, short ribs and a slightly dome-shaped skull, closely resembling achondroplasia (FIGS. 3A and B). The inventors analyzed the effects of A31 on endochondral ossification in Fgfr3.sup.Y367C/+ mice by using an ex vivo culture system for embryonic day 16.5 (E16.5) limb explants. Mutant femurs cultured without A31 had a significantly reduced longitudinal growth compared to WT (Fgfr3.sup.Y367C/+, 461±119 μm; WT, 1247±226 μm; p<10.sup.−10) (FIGS. 3C and D). A31 was able to induce and fully restore limb growth in Fgfr3.sup.Y367C/+ femurs (Fgfr3.sup.Y367C/+, gain of 1880±558 μm; WT, 1863±255 μm; p<10.sup.−19) (FIGS. 3C and D). After 5 days of culture, the increase in length of the treated mutant femurs was 2.6 times more than for that of WT.

[0155] Histological examinations using HES staining (FIG. 4A) and type X collagen labeling (FIG. 4B), revealed a reduction in size of the hypertrophic zone of the Fgfr3.sup.Y367C/+ mouse growth plate (FIG. 4B), with abnormally small chondrocytes resembling prehypertrophic rather than hypertrophic cells. The inventors evaluated the impact of A31 on the growth plate (FIG. 4A). Interestingly, A31 induced a marked expansion of the hypertrophic zone, with marked modifications of the shape of proliferative and hypertrophic cells. A31-treated chondrocytes appeared enlarged and more spherical, resembling to hypertrophic chondrocytes (data not shown). Therefore, these results suggest that A31 increased the size of mutant growth plates by restoring the disrupted chondrocyte maturation process.

[0156] To confirm the results obtained with tyrosine kinase inhibitor “A31”, another FGFR3 belonging to the pyrido[2,3-d]pyrimidine class, i.e. the tyrosine kinase inhibitor “PD173074”, was also tested.

[0157] Thus, embryonic femur explants were co-incubated with 150 nM of PD173074 for 5 days.

[0158] As illustrated by the gain in femur length, PD173074 for 5 days is sufficient for correcting the difference in length and normalized the size of the epiphyses PD173074 enhances longitudinal growth in Fgfr3.sup.Y367C/+ femurs (gain of 77%; see FIGS. 7(A) and (B); mutant femurs cultured without PD173074 had a reduced longitudinal growth compared to WT femurs (Fgfr3.sup.+/+). The effect of PD173074 on femur growth is similar to that of A31 (see FIGS. 7(A) and (B)).

[0159] Similar experiments were conducted with an antagonist which belongs to the N-aryl-N′-pyrimidin-4-yl urea class, i.e. the tyrosine kinase inhibitor “BGJ-398”.

[0160] Embryonic femur explants were co-incubated 100 nM (10.sup.−7M) or 1 μM (10.sup.−6M) of BGJ-398 for 6 days.

[0161] A concentration-dependent increase in femur size was observed for BGJ-398 concentrations ranging from 100 nM to 1 μM, as illustrated by the gain in femur length. 100 nM of BGJ-398 for 6 days is sufficient for correcting the difference in length and normalized the size of the epiphyses. A gain of 71.86% is observed in treated Fgfr3.sup.Y367C/+ femurs (FIG. 9; mutant femurs cultured without BGJ-398 had a reduced longitudinal growth compared to WT femurs (Fgfr3.sup.+/+).

[0162] Histological examinations using HES staining (FIG. 10A) and type X collagen labeling (FIG. 10B) were also carried out.

[0163] HES staining of WT (Fgfr3.sup.+/+) and Fgfr3.sup.Y367C/+ mice showed that growth plate from Fgfr3.sup.Y367C/+ mice have smaller mutant chondrocytes, whereas cells are larger and more spherical when femurs are cultured in the presence of 10.sup.−6M of BGJ-398 (FIG. 10A).

[0164] FIG. 10B shows that BGJ-398 induces enhanced type X collagen expression in Fgfr3.sup.Y367C/+ growth plate and that the size of the hypertrophic zone of the femur explants of Fgfr3.sup.Y367C/+ mice increases.

[0165] Taken together, these results showed histological changes (increased chondrocyte proliferation and differentiation) when femurs from Fgfr3.sup.Y367C/+ mice are cultivated in the presence of BGJ-398.

EXAMPLE 5: Effect of A31 on Fgfr3 Protein Expression

[0166] The inventors evaluated the level of Fgfr3 protein expression by immunohistochemical staining and found an overexpression of Fgfr3 in Fgfr3.sup.Y367C/+ growth plates. A31 induced a large decrease of Fgfr3 expression in mutant femurs (data not shown). These results were confirmed by Western blotting on primary chondrocytes isolated from E16.5 ribs (FIG. 5). A higher level of Fgfr3 was revealed in untreated Fgfr3.sup.Y367C/+ chondrocytes, whereas this level was similar to WT after addition of A31. These data indicate that inhibition of the constitutive phosphorylation of Fgfr3 by A31 rescues the turnover of the receptor.

EXAMPLE 6: A31 Modulates the Expression of Cell Cycle Regulator Genes

[0167] Analysis of expression of Proliferating Cell Nuclear Antigen (PCNA), an Sphase marker, revealed abnormally high levels of PCNA in the prehypertrophic (PH) (73% of total cells positive; p<0.005) and hypertrophic (H) areas of Fgfr3.sup.Y367C/+ mouse growth plates (43% of total cells positive; p<0.005). A31 strongly decreased PCNA expression in the corresponding areas of mutant growth plates (20% and 18% for PH and H areas, respectively, ***p<10.sup.−4) (FIG. 6A). Likewise, higher expression levels of K167 were observed in the PH and H areas of Fgfr3.sup.Y367C/+ mouse growth plates compared to controls (data not shown). A31 also decreased this expression in Fgfr3.sup.Y367C/+ mouse growth plates (data not shown). The inventors noted a higher expression of PCNA than K167 in the mutant growth plate. Furthermore, the inventors investigated whether the presence of mutated Fgfr3 caused an impairment of expression of cell cycle regulators. In fact, activated Fgfr3 induced a significant overexpression of cyclin D1 in the proliferative and PH chondrocytes of Fgfr3.sup.Y367C/+ mice. Interestingly, A31 returned cyclin D1 expression to control levels in mutant femurs (data not shown). Consistent with this Western blots showed a reduced level of cyclin D1 in A31-treated murine chondrocytes isolated from E16.5 ribs compared to untreated chondrocytes (FIG. 6B). The inventors further analyzed the level of CDK inhibitors (CDKIs) negatively regulating the cell cycle, particularly p57, a member of the Cip/Kip family. Activated Fgfr3 induced a higher expression of p57 predominantly in late proliferative and PH chondrocytes. A31 reduced expression of p57 protein particularly in the PH zone and enabled PH chondrocytes to properly differentiate into H chondrocytes (data not shown). The inventors conclude that activated FGFR3 leads to over-expression of markers of proliferation (PCNA, K167) and cell cycle regulators (cyclin D1 and p57) particularly in the prehypertrophic zone. These data highlight the dysregulation of the cell cycle in this skeletal pathology.

EXAMPLE 7: Effect of PD173074 in a Dwarfism Mouse Model

[0168] The effectiveness of PD173074 in attenuating the dwarfism phenotype of Fgfr3.sup.Y367C/+ mice was assessed in vivo. The mice were seven days of age at treatment initiation and received daily subcutaneous administrations of 4.00 mg/kg of PD173074 for 10 days.

[0169] Results of this experiment are disclosed in FIG. 8 which is an X-rays analysis of Fgfr3.sup.Y367C/+ mice administered with PD173074 or with a vehicle (“mock” experiment). Amelioration in key relevant achondroplasia clinical features including bowed femur and tibia, anterior crossbite and domed skull was observed (see FIGS. 8A and B; compare PD173074-treated mouse on the left side of panels A and B vs vehicle-administered mouse on the right side of panels A and B). Indeed, dramatic phenotypic changes are observed, including larger paws and digits, and longer and straightened tibia and femurs in Fgfr3.sup.Y367C/+ mouse treated with PD173074.

[0170] Therefore, improvement in the dwarfism was obvious after 10 days of treatment in animals given 4.00 mg/kg PD173074 and included an overall increase in body size with longer tail and snout.

EXAMPLE 8: Effect of BGJ-398 in a Dwarfism Mouse Model

[0171] Seven days old mice received daily subcutaneous administrations of 1.66 mg/kg of BGJ-398 for 10 days.

[0172] Dramatic phenotypic changes are observed, including larger paws and digits, and longer and straightened tibia and femurs in Fgfr3.sup.Y367C/+ mouse treated with BGJ-398 (see FIG. 11 which is an X-rays of Fgfr3.sup.Y367C/+ mice administered with BGJ-398—mouse on the left side of the figure—or with a vehicle (“mock” experiment)—mouse on the right side of the figure).