U.S. patent application number 16/662126 was filed with the patent office on 2022-02-03 for 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.
The applicant listed for this patent is ASSISTANCE PUBLIQUE-HOPITAUX DE PARIS, CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS), IMAGINE, INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM), UNIVERSITE DE PARIS. Invention is credited to Florent BARBAULT, Patricia BUSCA, Laurence LEGEAI-MALLET, Arnold MUNNICH.
Application Number | 20220031696 16/662126 |
Document ID | / |
Family ID | |
Filed Date | 2022-02-03 |
United States Patent
Application |
20220031696 |
Kind Code |
A9 |
LEGEAI-MALLET; Laurence ; et
al. |
February 3, 2022 |
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, at 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.
Inventors: |
LEGEAI-MALLET; Laurence;
(Paris, FR) ; MUNNICH; Arnold; (Paris, FR)
; BUSCA; Patricia; (Paris, FR) ; BARBAULT;
Florent; (Paris, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
(INSERM)
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS)
ASSISTANCE PUBLIQUE-HOPITAUX DE PARIS
UNIVERSITE DE PARIS
IMAGINE |
Paris
Paris
Paris
Paris
Paris |
|
FR
FR
FR
FR
FR |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20200246337 A1 |
August 6, 2020 |
|
|
Appl. No.: |
16/662126 |
Filed: |
October 24, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15872307 |
Jan 16, 2018 |
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16662126 |
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14844041 |
Sep 3, 2015 |
9931341 |
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15872307 |
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14364320 |
Jun 11, 2014 |
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PCT/EP2012/075294 |
Dec 12, 2012 |
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14844041 |
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International
Class: |
A61K 31/506 20060101
A61K031/506; C08L 25/06 20060101 C08L025/06; B32B 27/30 20060101
B32B027/30; C08L 51/04 20060101 C08L051/04; C08J 5/18 20060101
C08J005/18; B29C 48/00 20060101 B29C048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 12, 2011 |
IB |
PCT/IB2011/003253 |
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
FIELD OF THE INVENTION
[0001] The present invention relates to the treatment or prevention
of skeletal disorders, in particular skeletal diseases and
craniosynostosis, 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.
BACKGROUND
[0002] Skeletal development in humans is regulated by numerous
growth factors. Among them Fibroblast Growth Factor Receptor 3
(FGFR3) has been described as both a negative and a positive
regulator of endochondral ossification.
[0003] The FGFR3 gene, which is located on the distal short arm of
chromosome 4, encodes a 806 amino acid protein precursor
(fibroblast growth factor receptor 3 isoform 1 precursor; SEQ ID
NO: 1).
[0004] The FGFR3 protein belongs to the receptor-tyrosine kinase
family. This family comprises receptors FGFR1, FGFR2, FGFR3 and
FGFR4 that respond to fibroblast growth factor (FGF) ligands. These
structurally related proteins exhibit an extracellular domain
composed of three immunoglobin-like domains which form the
ligand-binding domain, an acid box, a single transmembrane domain
and an intracellular split tyrosine kinase domain. Although to date
the physiological ligand(s) for FGFR3 is (are) not known, like
other FGFRs, it is activated by FGF ligands. Binding of one of the
22 FGFs induces receptor dimerization and autophosphorylation of
tyrosine residues in the cytoplasmic domain. The phosphorylated
tyrosine residues are required for activation of the signaling
pathways. The most relevant tyrosines are Y648, Y647, located in
the activation loop.
[0005] Several signaling pathways have been described downstream of
FGFR3 activation, including the ERK and p38 MAP kinase pathways
(Legeai-Mallet et al., J Biol Chem, 273: 13007-13014, 1998;
Murakami et al., Genes Dev, 18: 290-305, 2004; Matsushita et al.,
Hum Mol Genet, 18: 227-240, 2009; Krejci et al., J Cell Sci, 121:
272-281, 2008) and the signal transducer and activation of
transcription (STAT) pathway (Su, W. C. et al., Nature, 386:
288-292, 1997; Legeai-Mallet et al., Bone, 34: 26-3, 2004; Li, C.
et al., Hum Mol Genet, 8: 35-44, 1999). Others pathways in
endochondral bone growth have been identified such as the
phosphoinositide 3 kinase-AKT (Ulici, V. et al., Bone, 45:
1133-1145, 2009) and protein kinase C pathways. The degradation of
mutant receptors is disturbed as demonstrated by higher levels of
FGFR3 mutant receptors at the cell surface (Monsonego-Ornan et al.,
Mol Cell Biol, 20: 516-522, 2000; Monsonego-Ornan et al., FEBS
Lett, 528: 83-89, 2002; Delezoide et al., Hum Mol Genet, 6:
1899-1906, 1997), and disruption of c-Cbl-mediated ubiquitination
(Cho, J. Y. et al., Proc Natl Acad Sci USA, 101: 609-614, 2004).
FGFR3 mutations disrupt the formation of glycosylated isoforms of
the receptor and impeded its trafficking (Gibbs et al., Biochim
Biophys Acta, 1773: 502-512, 2007; Bonaventure et al., FEBS J, 274:
3078-3093, 2007).
[0006] While long bone development involves endochondral
ossification, craniofacial development is dependent on both
endochondral and membranous ossification.
[0007] In skull vault, activated FGFR3 induces craniosiosynostosis.
This disease consists of premature fusion of one or more of the
cranial sutures. Two FGFR3 mutations cause specific
craniosynostoses, Muenke syndrome and Crouzon syndrome with
acanthosis nigricans. These diseases are an autosomal dominant
hereditary disorder.
[0008] In long bone, FGFR3, when activated, exerts a negative
regulatory influence mainly in the growth phase, in which it
reduces the turnover necessary for bone elongation, the rate of
cartilage template formation and disrupts chondrocyte proliferation
and differentiation.
[0009] Abnormal FGFR3 overactivation or constitutive activation of
FGFR3 leads to a severe disorganization of the growth plate
cartilage. Gain of function mutants of FGFR3 (also called
"constitutively active mutants of FGFR3") disrupt endochondral
ossification in a spectrum of skeletal dysplasias which include
achondroplasia (ACH), the most common form of human dwarfism,
hypochondroplasia (HCH), and thanatophoric dysplasia (TD), the most
common form of lethal skeletal dysplasia. On the contrary, it has
been shown that FGFR3 knock-out mice and humans without functional
FGFR3 demonstrate skeletal overgrowth.
[0010] Therefore, FGFR3-related skeletal diseases (e.g.
FGFR3-related skeletal dysplasias and FGFR3-related
craniosiosynostosis) are the result of increased signal
transduction from the activated receptor.
[0011] Among skeletal dysplasias, achondroplasia is of particular
interest since it is one of the most common congenital diseases
responsible for dwarfism, disorder characterized by short limbs
relative to trunk. It is diagnosed by growth failure in the major
axes of the long bones of extremities and typical physical features
such as a large frontally projecting cranium and a short nose. This
disease is an autosomal dominant hereditary disorder, but most of
cases are found to be sporadic. Hypochondroplasia is also
characterized by short stature with disproportionately short arms
and legs. The skeletal features are very similar to achondroplasia
but usually tend to be milder.
[0012] Current therapies of achondroplasia and hypochondroplasia
include orthopedic surgeries such as leg lengthening and growth
hormone therapy. However, leg lengthening inflicts a great pain on
patients, and growth hormone therapy increases body height by means
of periodic growth hormone injections starting from childhood.
Further, growth ceases when injections are stopped.
[0013] Consequently, it is desirable to develop a new
achondroplasia and hypochondroplasia therapy and to identify
molecules suitable for treating achondroplasia and
hypochondroplasia, as well as other FGFR3-related skeletal diseases
such as FGFR3-related craniosiosynostosis.
DESCRIPTION OF THE INVENTION
[0014] In an attempt to find a new treatment for skeletal diseases,
the inventors succeeded in restoring bone growth by administering
tyrosine kinase inhibitors, more particularly inhibitors which are
able to inhibit auto-phosphorylation of FGFR3. Indeed, the
inventors have shown in an ex vivo model (consisting of culturing
femurs of embryonic dwarf mice which displays impaired endochondral
ossification) that tyrosine kinase inhibitors (in particular those
which prevent ATP from binding to the "ATP binding site" of FGFR3)
restore a normal growth of the bones. Further, the inventors have
shown in vivo in an animal model that administration of tyrosine
kinase inhibitors (e.g. compounds that belong to the
pyrido[2,3-d]pyrimidine class and to the N-aryl-N'-pyrimidin-4-yl
urea class) improves dwarfism condition by increasing growth of
bones.
[0015] Consequently, inhibitors of FGFR3 are useful for treating
FGFR3-related skeletal diseases.
[0016] Therefore, the present invention provides a method for
treating or preventing FGFR3-related skeletal diseases which
comprises the step of administering at least one antagonist of the
FGFR3 tyrosine kinase receptor, or a composition comprising such an
antagonist, to a subject in need thereof.
[0017] The invention also relates to an antagonist of the FGFR3, or
a composition comprising such an antagonist, for use in the
treatment or prevention of FGFR3-related skeletal diseases.
[0018] As used herein, the terms "FGFR3". "FGFR3 tyrosine kinase
receptor" and "FGFR3 receptor" are used interchangeably throughout
the specification and refer to all of the naturally-occurring
isoforms of FGFR3.
[0019] In particular, an antagonist of a FGFR3 tyrosine kinase
receptor refers to an antagonist capable of inhibiting or blocking
the activity of: [0020] a) a FGFR3 polypeptide comprising or
consisting of the amino acid sequence shown in NCBI reference
NP_000133 and in UniProt reference P22607 (sequence SEQ ID NO: 1);
and/or [0021] b) a FGFR3 corresponding to the mature isoform of the
a FGFR3 polypeptide of (a) (i.e. obtained after cleavage of the
signal peptide); and/or [0022] c) an allelic variant of a FGFR3 of
(a) or (b); and/or [0023] d) a splice variant of a FGFR3 of (a),
(b) or (c); and/or [0024] e) a constitutively active mutant of a
FGFR3 of (a), (b), (c) or (d). [0025] f) an isoform obtained by
proteoiytic processing of a FGFR3 of (a), (b), (c), (d) or (e).
[0026] As used herein, the expressions "constitutively active FGFR3
receptor variant", "constitutively active mutant of the FGFR3" or
"mutant FGFR3 displaying a constitutive activity" are used
interchangeably and refer to a mutant of said receptor exhibiting a
biological activity (i.e. triggering downstream signaling) in the
absence of FGF ligand stimulation, and/or exhibiting a biological
activity which is higher than the biological activity of the
corresponding wild-type receptor in the presence of FGF ligand.
[0027] A constitutively active FGFR3 variant according to the
invention is in particular chosen from the group consisting of
(residues are numbered according to their position in the precursor
of fibroblast growth factor receptor 3 isoform 1-806 amino acids
long-):
[0028] a mutant wherein the serine residue at position 84 is
substituted with lysine (named herein below S84L);
[0029] a mutant wherein the arginine residue at position 248 is
substituted with cysteine (named herein below R200C);
[0030] a mutant wherein the arginine residue at position 248 is
substituted with cysteine (named herein below R248C);
[0031] a mutant wherein the serine residue at position 249 is
substituted with cysteine (named herein below S249C);
[0032] a mutant wherein the proline residue at position 250 is
substituted with arginine (named herein below P250R);
[0033] a mutant wherein the asparagine residue at position 262 is
substituted with histidine (named herein below N262H);
[0034] a mutant wherein the glycine residue at position 268 is
substituted with cysteine (named herein below G268C);
[0035] a mutant wherein the tyrosine residue at position 278 is
substituted with cysteine (named herein below Y278C)
[0036] a mutant wherein the serine residue at position 279 is
substituted with cysteine (named herein below S279C);
[0037] a mutant wherein the glycine residue at position 370 is
substituted with cysteine (named herein below G370C);
[0038] a mutant wherein the serine residue at position 371 is
substituted with cysteine (named herein below S371C);
[0039] a mutant wherein the tyrosine residue at position 373 is
substituted with cysteine (named herein below Y373C);
[0040] a mutant wherein the glycine residue at position 380 is
substituted with arginine (named herein below G380R);
[0041] a mutant wherein the valine residue at position 381 is
substituted with glutamate (named herein below V381E);
[0042] a mutant wherein the alanine residue at position 391 is
substituted with glutamate (named herein below A391E);
[0043] a mutant wherein the asparagine residue at position 540 is
substituted with Lysine (named herein below N540K);
[0044] a mutant wherein the termination codon is eliminated due to
base substitutions. in particular the mutant wherein the
termination codon is mutated in an arginine, cysteine, glycine,
serine or tryptophane codon (named herein below X807R, X807C,
X807G, X807S and X807W, respectively);
[0045] a mutant wherein the lysine residue at position 650 is
substituted with another residue, in particular with methionine,
glutamate, asparagine or glutamine (named herein below K650M,
K650E, K650N and K650Q).
[0046] Preferably, a constitutively active FGFR3 variant according
to the invention is K650M, K650E or Y373C mutant.
[0047] In the context of the present invention, the term
"FGFR3-related skeletal disease" is intended to mean a skeletal
disease that is caused by an abnormal increased activation of
FGFR3, in particular by expression of a constitutively active
mutant of the FGFR3 receptor, in particular a constitutively active
mutant of the FGFR3 receptor as described above.
[0048] The FGFR3-related skeletal diseases are preferably
FGFR3-related skeletal dysplasias and FGFR3-related
craniosynostosis.
[0049] The FGFR3-related skeletal dysplasias according to the
invention may correspond to an inherited or to a sporadic
disease.
[0050] As used herein, the term "FGFR3-related skeletal dysplasias"
includes but is not limited to thanatophoric dysplasia type I,
thanatophoric dysplasia type II, hypochondroplasia, achondroplasia
and SADDAN (severe achondroplasia with developmental delay and
acanthosis nigricans).
[0051] In a preferred embodiment, the FGFR3-related skeletal
dysplasia is caused by expression in the subject of a
constitutively active FGFR3 receptor variant such as defined
above.
[0052] In a preferred embodiment, the FGFR3-related skeletal
dysplasia is a achondroplasia caused by expression of the G380R
constitutively active mutant of the FGFR3 receptor.
[0053] In a preferred embodiment, the FGFR3-related skeletal
dysplasia is a hypochondroplasia caused by expression of the N540K,
K650N, K650Q, S84L, R200C, N262H, G268C, Y278C, S279C, V381E,
constitutively active mutant of the FGFR3 receptor.
[0054] In a preferred embodiment, the FGFR3-related skeletal
dysplasia is a thanatophoric dysplasia type I caused by expression
of a constitutively active mutant of the FGFR3 receptor chosen from
the group consisting of R248C, S248C, G370C, S371C; Y373C, X807R,
X807C, X807G, X807S, X807W and K650M FGFR3 receptors.
[0055] In a preferred embodiment, the FGFR3-related skeletal
dysplasia is a thanatophoric dysplasia type II caused by expression
of the K650E constitutively active mutant of the FGFR3
receptor.
[0056] In a preferred embodiment, the FGFR3-related skeletal
dysplasia is a severe severe achondroplasia with developmental
delay and acanthosis nigricans caused by expression of the K650M
constitutively active mutant of the FGFR3 receptor
[0057] The FGFR3-related craniosynostosis according to the
invention may correspond to an inherited or to a sporadic
disease.
[0058] In a preferred embodiment, the FGFR3-related
craniosynostosis Muenke syndrome caused by expression of the P250R.
constitutively active mutant of the FGFR3 receptor or Crouzon
syndrome with acanthosis nigricans caused by expression of the
A391G constitutively active mutant of the FGFR3 receptor.
[0059] As used herein the term "antagonist" refers to an agent
(i.e. a molecule) which inhibits or blocks the activity of FGFR3.
For instance, an antagonist of FGFR3 refers to a molecule which
inhibits or blocks the activity of the FGFR3 receptor. Preferably,
the FGFR3 antagonists according to the invention act through direct
interaction with the FGFR3 receptor.
[0060] The antagonists of the present invention act by blocking or
reducing FGFR3 receptor functional activation. This may for example
be achieved by interfering with FGF ligand binding to FGFR3
receptor or with ATP binding to "ATP binding site" of FGFR3
receptor for preventing phosphorylation of tyrosine residues
located towards the cytoplasmic domain (activation loop), i.e. on
Tyr.sup.648 and Tyr.sup.647.
[0061] Alternatively, this may be achieved by reducing or
preventing expression of FGFR3 receptor.
[0062] The term "expression" when used in the context of expression
of a gene or nucleic acid refers to the conversion of the
information, contained in a gene, into a gene product. A gene
product can be the direct transcriptional product of a gene (e.g.,
mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any
other type of RNA) or a protein FGFR3) produced by translation of a
mRNA.
[0063] Both options ultimately result in blocking or reducing
signal transduction, hence in blocking or reducing receptors
functional activity.
[0064] The antagonists according to the invention are capable of
inhibiting or eliminating the functional activation of the FGFR3
receptor in vivo and/or in vitro. The antagonist may inhibit the
functional activation of the FGFR3 receptor by at least about 10%,
preferably by at least about 30%, preferably by at least about 50%,
preferably by at least about 70, 75 or 80%, still preferably by 85,
90, 95, or 100%.
[0065] Preferably, the antagonists according to the invention are
more specific for FGFR3 versus FGFR1, 2 and 4, for instance the
inhibitor constant "KI" of the antagonists for FGFR3 is at least 2,
preferably 5, more preferably 10, times lower than the KI for at
least one of FGFR1, 2 and 4.
[0066] Antagonists for FGFR3 receptor are well-known to those
skilled in the art and include, e.g., anti-FGFR3 antibodies, for
instance the antibodies described by Rauchenberger, R. et al. (J.
Biol. Chem. 2003 Oct. 3; 278(40):38194-205.),
Martinez-Torrecuadrada, J., et al. (Clin. Cancer Res. 2005 Sep. 1;
11(17):6280-90), Trudel S., et al., (Blood 2006 May 15;
107(10):4039-46.), Qing J. et al. (J. Clin. Invest. 2009,
119(5):1216-29), the anti-FGFR3 antibodies disclosed in
IN2011CN02023, WO2010/111367, US 2010/0098696, WO2010/02862,
WO2007/144893, WO2002/102973.
[0067] Antagonists for FGFR3 receptor also include small chemical
molecules, for instance those disclosed in WO2010/22169 (e.g. the
compound of general formula 1 corresponding to
4,4',4'',4'''-[carbonyl-bis[imino-5, 1,3-benzenetriyl
bis-{carbonylimino}]3tetrakis-{benzene-1,3-disulfonic acid}),
WO2007/26251, WO2005/47244, US2005/261307, as well as nucleic acid
compounds for regulating/inhibiting FGFR3 expression described in
WO2003/23004, US2007/049545 and WO2011/139843.
[0068] Functional activation of the FGFR3 receptor may be readily
assessed by the one skilled in the art according to known methods.
Indeed, since activated FGFR3 receptor is phosphorylated on
tyrosine residues located towards the cytoplasmic domain, i.e. on
Tyr.sup.648 and Tyr.sup.647, functional activation of the FGFR3
receptor may for example be assessed by measuring its
phosphorylation.
[0069] For instance, analysis of ligand-induced phosphorylation of
the FGFR3 receptor can be preformed as described in Le Corre et al.
(Org. Biomol. Chem., 8: 2164-2173, 2010).
[0070] Alternatively, receptor phosphorylation in cells can be
readily detected by immunocytochemistry, immunohistochemistry
and/or flow cytometry using antibodies which specifically recognize
this modification. For instance phosphorylation of FGFR3 on the
Tyr.sup.648 and Tyr.sup.647 residues can be detected by
immunocytochemistry, immunohistochemistry and/or flow cytometry
using monoclonal or polyclonal antibodies directed against
phosphorylated Tyr.sup.648 and Tyr.sup.647-FGFR3.
[0071] Functional activation of the FGFR3 receptor may also be
tested by using FGFR3-dependent cell lines (for instance BaF3 cell
line). The FGFR3 antagonist activity of a compound is determined by
measuring its ability to inhibit the proliferation of a
FGFR3-dependent cell line (see methods described by Vito Guagnano
et al., Journal of Medicinal Chemistry, 54: 7066-7083, 2011).
[0072] Further, FGFR3, when associated with its ligand, mediates
signaling by activating the ERK and p38 MAP kinase pathways, and
the STAT pathway. Therefore activation of FGFR3 receptor can also
be assessed by determining the activation of these specific
pathways as described by Horton et al. (lancet, 370: 162-172,
2007)
[0073] Accordingly, an antagonist may be identified as a molecule
which reduces the level of phosphorylation of the receptor to be
tested upon stimulation with its specific ligand of a cell
expressing said receptor, as compared with the level of receptor
phosphorylation measured in the cell when stimulated with its
specific ligand in the absence of the antagonist.
[0074] The antagonists according to the present invention include
those which specifically bind to the FGFR3 receptor, thereby
reducing or blocking signal transduction. Antagonists of this type
include antibodies (in particular the antibodies as disclosed
above) or aptamers which bind to FGFR3, fusion polypeptides,
peptides, small chemical molecules which bind to FGFR3, and
peptidomimetics.
[0075] The term "small chemical molecule" refers to a molecule,
preferably of less than 1,000 daltons, in particular organic or
inorganic compounds. Structural design in chemistry should help to
find such a molecule.
[0076] According to a preferred embodiment, the small chemical
molecule prevents binding of ATP to the "ATP binding site" of
FGFR3. In a more preferred embodiment, the small chemical molecule
which prevents binding of ATP to the "ATP binding site" of FGFR3
belongs to the pyrido[2,3-d]pyrimidine class.
[0077] More preferably, the small chemical molecule which prevents
binding of ATP to the "ATP binding site" of FGFR3 is selected from
the group consisting of the compounds PD173074, 18, 19a to 19 m,
22b, 22c, 23b to 23f disclosed in table below (as well as in FIG.
2a and Scheme 3 of the article by Le Corre et al., Org. Biomol.
Chem., 8: 2164-2173, 2010).
TABLE-US-00001 Compose Structure PD173074 ##STR00001## 19a
##STR00002## 19b ##STR00003## 19c ##STR00004## 19d ##STR00005## 19e
##STR00006## 19f ##STR00007## 19g ##STR00008## 19h ##STR00009## 19i
##STR00010## 19j ##STR00011## 19k ##STR00012## 19l ##STR00013## 19m
##STR00014## 22b ##STR00015## 22c ##STR00016## 23b ##STR00017## 23c
##STR00018## 23d ##STR00019## 23e ##STR00020## 23f ##STR00021##
[0078] Advantageously, the small chemical molecule which prevents
binding of ATP to the "ATP binding site" of FGFR3 is the compound
PD173075 or the compound 19g, corresponding to compound "A31"
disclosed in FIG. 1A of the present application and in Table A.
[0079] In another more preferred embodiment, the small chemical
molecule which prevents binding of ATP to the "ATP binding site" of
FGFR3 belongs to the N-aryl-N'-pyrimidin-4-yl urea class.
[0080] More preferably, the small chemical molecule which prevents
binding of ATP to the "ATP binding site" of FGFR3 is selected from
the group consisting of the compounds 1a to 1n disclosed in Table B
below (as well as in Table 1 of the article by Vito Guagnano et
al., Journal of Medicinal Chemistry, 54: 7066-7083, 2011).
[0081] Advantageously, the small chemical molecule which prevents
binding of ATP to the "ATP binding site" of FGFR3 is the compound
1h (also named BGJ-398), disclosed in the Table B below.
TABLE-US-00002 TABLE B Compound Structure 1a ##STR00022## 1b
##STR00023## 1c ##STR00024## 1d ##STR00025## 1e ##STR00026## 1f
##STR00027## 1g ##STR00028## 1h ##STR00029## 1i ##STR00030## 1j
##STR00031## 1k ##STR00032## 1l ##STR00033## 1m ##STR00034## 1n
##STR00035##
[0082] As used herein the term "polypeptide" refers to any chain of
amino acids linked by peptide bonds, regardless of length or
post-translational modification. Polypeptides include natural
proteins, synthetic or recombinant polypeptides and peptides (i.e.
polypeptides of less than 50 amino acids) as well as hybrid,
post-translationally modified polypeptides, and peptidomimetic.
[0083] As used herein, the term "amino acid" refers to the 20
standard alpha-amino acids as well as naturally occurring and
synthetic derivatives. A polypeptide may contain L or D amino acids
or a combination thereof.
[0084] As used herein the term "peptidomimetic" refers to
peptide-like structures which have non-amino acid structures
substituted but which mimic the chemical structure of a
peptide.
[0085] The term "antibody" refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e.,
molecules that contain an antigen binding site that
immunospecifically binds an antigen. As such, the term antibody
encompasses not only whole antibody molecules, but also antibody
fragments as well as variants (including derivatives) of antibodies
and antibody fragments.
[0086] In particular, the antibody according to the invention may
correspond to a polyclonal antibody, a monoclonal antibody (e.g. a
chimeric, humanized or human antibody), a fragment of a polyclonal
or monoclonal antibody or a diabody.
[0087] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fv, Fab,
F(ab').sub.2, Fab', Fd, dAb, dsFv, scFv, sc(Fv).sub.2, CDRs,
diabodies and multi-specific antibodies formed from antibodies
fragments.
[0088] Antibodies according to the invention may be produced by any
technique known in the art, such as, without limitation, any
chemical, biological, genetic or enzymatic technique, either alone
or in combination. The antibodies of this invention can be obtained
by producing and culturing hybridomas.
[0089] According to a preferred embodiment, the antagonist is an
antibody which specifically recognizes and binds to the FGFR3
receptor and prevents binding of ATP to the ATP binding site of
FGFR3.
[0090] In another embodiment, the antagonist is an antibody which
prevents functional oligomerization of the receptor.
[0091] "Aptamers" are a class of molecule that represents an
alternative to antibodies in term of molecular recognition.
Aptamers are oligonucleotide or oligopeptide sequences with the
capacity to recognize virtually any class of target molecules with
high affinity and specificity. Such ligands may be isolated through
Systematic Evolution of Ligands by EXponential enrichment (SELEX)
of a random sequence library, as described in Tuerk C. and Gold L.,
Science, 1990, 249(4968):505-10. The random sequence library is
obtainable by combinatorial chemical synthesis of DNA. In this
library, each member is a linear oligomer, eventually chemically
modified, of a unique sequence. Possible modifications, uses and
advantages of this class of molecules have been reviewed in
Jayasena S. D., Clin. Chem., 1999, 45(9):1828-50. Peptide aptamers
consists of a conformationally constrained antibody variable region
displayed by a platform protein, such as E. coli Thioredoxin A that
are selected from combinatorial libraries by two hybrid methods
(Colas et al., Nature, 1996,380, 548-50).
[0092] In order to target the antagonist of the invention
specifically to growth plate chondrocytes, the antagonist may be
tagged with molecules that possess affinity for cartilage or
chondrocytes. Such molecules are for instance described by
Rothenfluh et al. (Nat Mater 7: 248-254, 2008), and Laroui H et al.
(Biomacromolecules, 8: 1041-1021, 2007).
[0093] The antagonist of the invention can be used in combination
with growth hormones and/or substances activating guanylyl cyclase
B (such as the substances disclosed in application US
2003/0068313).
[0094] The antagonists comprises in the combination are intended to
be administered simultaneously or sequentially.
[0095] Thus, the present invention also relates to a combination of
at least one antagonist of the invention and at least one other
agent such as growth hormones and/or substances activating guanylyl
cyclase B, for sequential or simultaneous use in the treatment or
prevention of a FGFR3-related skeletal dysplasia.
[0096] The antagonist or combination used in the above recited
method or use are provided in a pharmaceutically acceptable
carrier, exipient or diluent which is not prejudicial to the
patient to be treated.
[0097] Pharmaceutically acceptable carriers and exipient that may
be used in the compositions of this invention include, but are not
limited to, ion exchangers, alumina, aluminium stearate, lecithin,
self-emulsifying drug delivery systems (SEDDS) such as
d-a-tocopherol polyethyleneglycol 1000 succinate, surfactants used
in pharmaceutical dosage forms such as Tweens or other similar
polymeric delivery matrices, serum proteins, such as human serum
albumin, buffer substances such as phosphates, glycine, sorbic
acid, potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat.
[0098] As appreciated by skilled artisans, compositions are
suitably formulated to be compatible with the intended route of
administration. Examples of suitable routes of administration
include parenteral route, including for instance intramuscular,
subcutaneous, intravenous, intraperitoneal or local intratumoral
injections. The oral route can also be used, provided that the
composition is in a form suitable for oral administration, able to
protect the active principle from the gastric and intestinal
enzymes.
[0099] Further, the amount of antagonist or combination used in the
above recited method or use is a therapeutically effective amount.
A therapeutically effective amount of antagonist is that amount
sufficient to achieve growth of bones or cartilages, or to treat a
desired disease without causing overly negative effects in the
subject to which the antagonist or the combination is administered.
The exact amount of antagonist to be used and the composition to be
administered will vary according to the age and the weight of the
patient being treated, the type of disease, the mode of
administration, the frequency of administration as well as the
other ingredients in the composition which comprises the
antagonist. Generally, the antagonist for use in the treatment or
prevention of FGFR3-related skeletal dyspiasias may be administered
in the rage from about 100 .mu.g/kg to 1 mg/kg, alternatively from
about 1 mg to about 10 mg/Kg, alternatively from about 10 mg to
about 100 mg/Kg. Effective doses will also vary depending on route
of administration, as well as the possibility of co-usage with
other agents.
[0100] When the antagonist belongs to the pyrido[2,3-d]pyrimidine
class, it is preferably administered in the range from about 1
mg/kg to about 10 mg/Kg. Typically, antagonist PD173074 is
administered from about 1 mg/kg to about 10 mg/Kg, preferably from
2 mg/kg to about 8 mg/Kg, more preferably 4 mg/kg to about 6 mg/Kg.
When the antagonist belongs to the N-aryl-N'-pyrimidin-4-yl urea
class, it is preferably administered in the range from about 1
mg/kg to about 10 mg/Kg. Typically, antagonist BGJ-398 is
administered from about 1 mg/kg to about 10 mg/Kg, preferably from
2 mg/kg to about 8 mg/Kg, more preferably 4 mg/kg to about 6 mg/Kg.
Advantageously, BGJ-398 is administered to 1.66 mg/kg.
[0101] As used herein, the term "subject" denotes a human or
non-human mammal, such as a rodent, a feline, a canine, or a
primate. Preferably, the subject is a human being, more preferably
a child (i.e. a child who is growing up). Preferably, when the
subject to be treated is a child, the antagonist is administered
during all or part of child growth period.
[0102] In the context of the invention, the term "treating" is used
herein to characterize a therapeutic method or process that is
aimed at (1) slowing down or stopping the progression, aggravation,
or deterioration of the symptoms of the disease state or condition
to which such term applies; (2) alleviating or bringing about
ameliorations of the symptoms of the disease state or condition to
which such term applies; and/or (3) reversing or curing the disease
state or condition to which such term applies.
[0103] As used herein, the term "preventing" intends characterizing
a prophylactic method or process that is aimed at delaying or
preventing the onset of a disorder or condition to which such term
applies.
[0104] Throughout the present application, the references to
entries of public databases refer to the entries in force on Nov.
23, 2011. Further, throughout this application, various references
are cited. The disclosures of these references are hereby
incorporated by reference into the present disclosure.
[0105] The present invention will be further illustrated by the
additional description which follows, which refer to examples which
show that administration tyrosine kinase inhibitors, in particular
a compound which belong to the pyrido[2,3-d]pyrimidine class or to
the N-aryl-N'-pyrirnidin-4-yl urea class, restores bone growth in
ex vivo and in vivo models. It should be understood however that
the invention is defined by the claims, and that these examples are
given only by way of illustration of the invention and do not
constitute in anyway a limitation thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0106] FIG. 1. A31 prevents the kinase activity of FGFR3.
[0107] (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.
[0108] FIG. 2. A31 inhibits the constitutive activation of
FGFR3.
[0109] 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.
[0110] FIG. 3. A31 restores longitudinal bone growth of
Fgfr3Y367C/+ femurs.
[0111] (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 .mu.m; WT, 1247.+-.227 .mu.m;
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 .mu.m; WT, 1863.+-.255 .mu.m; ***p<10.sup.-19 versus
untreated controls). The experiments were performed 6 times and
bone length is shown as mean +/-s.d.
[0112] FIG. 4. A31 modifies the size of the growth plate and
chondrocyte morphology.
[0113] (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.
[0114] FIG. 5. A31 decreases Fgfr3 overexpression in Fgfr3Y367C/+
femurs.
[0115] 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.
[0116] FIG. 6. A31 reduces proliferation and cell cycle regulator
expression in growth plates.
[0117] (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.
[0118] FIG. 7. PD173074 restores longitudinal bone growth of
Fgfr3Y367C/+ femurs.
[0119] (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).
[0120] (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.
[0121] FIG. 8. PD173074 attenuates the dwarfism phenotype of
Fgfr3.sup.Y367C/+ mice.
[0122] 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
Fgfr3Y367C/+ 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
.mu.M 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. Bal-398 attenuates the dwarfism phenotype of
Fgfr3.sup.Y367C/+ mice.
[0128] 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
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 19 g) 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.degree. C. before use.
Computational Analyses
[0130] The kinase domain structure of FGFR3 was predicted by
homology modelling 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.times.20.times.33 .ANG. 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 .ANG.. The inventors selected the conformation,
which presented the lowest docking free energy of binding in the
most populated cluster.
Ex Vivo Experiments
[0131] 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.degree. 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 24 h. 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.
Histological, In Situ Hybridization and Immunohistochemical
Analyses
[0132] Limb explants were fixed after culture in 4%
paraformaldehyde at 4.degree. 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.
[0133] 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-KI67
(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-IX2-UCB microscope.
Quantification of PCNA Expression
[0134] 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.
Immunoprecipiation, Immunoblotting and Immunocytochemistry
Experiments
[0135] 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).
[0136] 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.degree. 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.degree. 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).
[0137] 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.RTM.488 goat
antirabbit and Alexa Fluor.RTM.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.
Proliferation Studies
[0138] 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).
In Vivo Experiments
[0139] The effectiveness of PD173074 and BGJ-398 in attenuating the
dwarfism phenotype of Fgfr3.sup.Y367C/+ mice was assessed in viva.
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
[0140] 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 silica
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 Fdfr3 Cell Ones
[0141] 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.+/+).
[0142] 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.
[0143] 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).
[0144] 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.
[0145] 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 .mu.M 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
[0146] 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.
[0147] 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 (FIG. 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 .mu.m; WT,
1247.+-.226 .mu.m; 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 .mu.m; WT, 1863.+-.255
.mu.m; 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.
[0148] 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.
[0149] 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.
[0150] Thus, embryonic femur explants were co-incubated with 150 nM
of PD173074 for 5 days.
[0151] 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)).
[0152] Similar experiments were conducted with an antagonist which
belongs to the N-aryl-N'-pyrirnidin-4-yl urea class, i.e. the
tyrosine kinase inhibitor "BGJ-398".
[0153] Embryonic femur explants were co-incubated 100 nM
(10.sup.-7M) or 1 .mu.M (10.sup.-6M) of BGJ-398 for 6 days.
[0154] A concentration-dependent increase in femur size was
observed for BGJ-398 concentrations ranging from 100 nM to 1 .mu.M,
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.+/+).
[0155] Histological examinations using HES staining (FIG. 10A) and
type X collagen labeling (FIG. 10B) were also carried out.
[0156] 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).
[0157] 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.
[0158] 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
[0159] 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
[0160] 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
KI67 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 KI67 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, KI67) 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
[0161] 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.
[0162] 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 vehicule ("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 vehicule-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.
[0163] 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
[0164] Seven days old mice received daily subcutaneous
administrations of 1.66 mg/kg of BGJ-398 for 10 days.
[0165] 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 vehicule
("mock" experiment)--mouse on the right side of the figure).
Sequence CWU 1
1
11806PRTHomo sapiens 1Met Gly Ala Pro Ala Cys Ala Leu Ala Leu Cys
Val Ala Val Ala Ile1 5 10 15Val Ala Gly Ala Ser Ser Glu Ser Leu Gly
Thr Glu Gln Arg Val Val 20 25 30Gly Arg Ala Ala Glu Val Pro Gly Pro
Glu Pro Gly Gln Gln Glu Gln 35 40 45Leu Val Phe Gly Ser Gly Asp Ala
Val Glu Leu Ser Cys Pro Pro Pro 50 55 60Gly Gly Gly Pro Met Gly Pro
Thr Val Trp Val Lys Asp Gly Thr Gly65 70 75 80Leu Val Pro Ser Glu
Arg Val Leu Val Gly Pro Gln Arg Leu Gln Val 85 90 95Leu Asn Ala Ser
His Glu Asp Ser Gly Ala Tyr Ser Cys Arg Gln Arg 100 105 110Leu Thr
Gln Arg Val Leu Cys His Phe Ser Val Arg Val Thr Asp Ala 115 120
125Pro Ser Ser Gly Asp Asp Glu Asp Gly Glu Asp Glu Ala Glu Asp Thr
130 135 140Gly Val Asp Thr Gly Ala Pro Tyr Trp Thr Arg Pro Glu Arg
Met Asp145 150 155 160Lys Lys Leu Leu Ala Val Pro Ala Ala Asn Thr
Val Arg Phe Arg Cys 165 170 175Pro Ala Ala Gly Asn Pro Thr Pro Ser
Ile Ser Trp Leu Lys Asn Gly 180 185 190Arg Glu Phe Arg Gly Glu His
Arg Ile Gly Gly Ile Lys Leu Arg His 195 200 205Gln Gln Trp Ser Leu
Val Met Glu Ser Val Val Pro Ser Asp Arg Gly 210 215 220Asn Tyr Thr
Cys Val Val Glu Asn Lys Phe Gly Ser Ile Arg Gln Thr225 230 235
240Tyr Thr Leu Asp Val Leu Glu Arg Ser Pro His Arg Pro Ile Leu Gln
245 250 255Ala Gly Leu Pro Ala Asn Gln Thr Ala Val Leu Gly Ser Asp
Val Glu 260 265 270Phe His Cys Lys Val Tyr Ser Asp Ala Gln Pro His
Ile Gln Trp Leu 275 280 285Lys His Val Glu Val Asn Gly Ser Lys Val
Gly Pro Asp Gly Thr Pro 290 295 300Tyr Val Thr Val Leu Lys Thr Ala
Gly Ala Asn Thr Thr Asp Lys Glu305 310 315 320Leu Glu Val Leu Ser
Leu His Asn Val Thr Phe Glu Asp Ala Gly Glu 325 330 335Tyr Thr Cys
Leu Ala Gly Asn Ser Ile Gly Phe Ser His His Ser Ala 340 345 350Trp
Leu Val Val Leu Pro Ala Glu Glu Glu Leu Val Glu Ala Asp Glu 355 360
365Ala Gly Ser Val Tyr Ala Gly Ile Leu Ser Tyr Gly Val Gly Phe Phe
370 375 380Leu Phe Ile Leu Val Val Ala Ala Val Thr Leu Cys Arg Leu
Arg Ser385 390 395 400Pro Pro Lys Lys Gly Leu Gly Ser Pro Thr Val
His Lys Ile Ser Arg 405 410 415Phe Pro Leu Lys Arg Gln Val Ser Leu
Glu Ser Asn Ala Ser Met Ser 420 425 430Ser Asn Thr Pro Leu Val Arg
Ile Ala Arg Leu Ser Ser Gly Glu Gly 435 440 445Pro Thr Leu Ala Asn
Val Ser Glu Leu Glu Leu Pro Ala Asp Pro Lys 450 455 460Trp Glu Leu
Ser Arg Ala Arg Leu Thr Leu Gly Lys Pro Leu Gly Glu465 470 475
480Gly Cys Phe Gly Gln Val Val Met Ala Glu Ala Ile Gly Ile Asp Lys
485 490 495Asp Arg Ala Ala Lys Pro Val Thr Val Ala Val Lys Met Leu
Lys Asp 500 505 510Asp Ala Thr Asp Lys Asp Leu Ser Asp Leu Val Ser
Glu Met Glu Met 515 520 525Met Lys Met Ile Gly Lys His Lys Asn Ile
Ile Asn Leu Leu Gly Ala 530 535 540Cys Thr Gln Gly Gly Pro Leu Tyr
Val Leu Val Glu Tyr Ala Ala Lys545 550 555 560Gly Asn Leu Arg Glu
Phe Leu Arg Ala Arg Arg Pro Pro Gly Leu Asp 565 570 575Tyr Ser Phe
Asp Thr Cys Lys Pro Pro Glu Glu Gln Leu Thr Phe Lys 580 585 590Asp
Leu Val Ser Cys Ala Tyr Gln Val Ala Arg Gly Met Glu Tyr Leu 595 600
605Ala Ser Gln Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Val Leu
610 615 620Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe Gly Leu
Ala Arg625 630 635 640Asp Val His Asn Leu Asp Tyr Tyr Lys Lys Thr
Thr Asn Gly Arg Leu 645 650 655Pro Val Lys Trp Met Ala Pro Glu Ala
Leu Phe Asp Arg Val Tyr Thr 660 665 670His Gln Ser Asp Val Trp Ser
Phe Gly Val Leu Leu Trp Glu Ile Phe 675 680 685Thr Leu Gly Gly Ser
Pro Tyr Pro Gly Ile Pro Val Glu Glu Leu Phe 690 695 700Lys Leu Leu
Lys Glu Gly His Arg Met Asp Lys Pro Ala Asn Cys Thr705 710 715
720His Asp Leu Tyr Met Ile Met Arg Glu Cys Trp His Ala Ala Pro Ser
725 730 735Gln Arg Pro Thr Phe Lys Gln Leu Val Glu Asp Leu Asp Arg
Val Leu 740 745 750Thr Val Thr Ser Thr Asp Glu Tyr Leu Asp Leu Ser
Ala Pro Phe Glu 755 760 765Gln Tyr Ser Pro Gly Gly Gln Asp Thr Pro
Ser Ser Ser Ser Ser Gly 770 775 780Asp Asp Ser Val Phe Ala His Asp
Leu Leu Pro Pro Ala Pro Pro Ser785 790 795 800Ser Gly Gly Ser Arg
Thr 805
* * * * *