Methods and pharmaceutical compositions for the treatment of FGFR3-associated cognitive deficits

Abstract

The present invention relates to a method and pharmaceutical composition for treating FGFR3-related cognitive deficits.The present inventors provide strong evidence that FGFR3 gain-of-function mutations expressed in the brain induce cognitive and behavioral deficits, regardless of skull abnormalities.To provide evidence that constitutive activation of FGFR3 is involved in these behavioral disorders, the present inventors investigated Fgfr3 A385E / + Mice were treated with a tyrosine kinase inhibitor, BGJ398, via intracerebroventricular injection for 7 days, which rescued abnormalities in short-term learning and coping strategies. The present invention relates to a method for treating an FGFR3-associated cognitive deficit in a subject suffering from an FGFR3-associated skeletal disease in need thereof, comprising administering to the subject a therapeutically effective amount of an FGFR3 inhibitor.

Description

[Technical Field]

[0001] Field of the invention:

[0002] The present invention relates to methods and pharmaceutical compositions for treating FGFR3-associated cognitive deficits.

[0003] Background of the invention:

[0004] The fibroblast growth factor receptor (FGFR) family plays an important role in bone development and skeletal diseases. Missense mutations in FGFR1, FGFR2, and FGFR3 are involved in a range of syndromic craniosynostosis characterized by premature fusion of cranial sutures (Robin et al., 1993; Twigg and Wilkie, 2015). Two specific dominant mutations in FGFR3 cause Crouzon syndrome with acanthosis nigricans (CAN [MIM ​​612247]), a rare syndromic craniosynostosis, and Muenke syndrome (MS [MIM ​​602849]), the most common syndromic craniosynostosis (Wilkie et al., 2010). Patients with CAN present with acanthosis nigricans but otherwise resemble patients with FGFR2-related Crouzon syndrome: they are characterized by premature craniosynostosis, brachycephaly, central facial hypoplasia, and craniocervical junction involvement (Arnaud-Lepez et al., 2007; Di Rocco et al., 2011; Meyers et al., 1995; Mir A et al., 2013). CAN is defined by a single point mutation (p.Ala391Glu) localized in the transmembrane domain of FGFR3 (Li et al., 2006; Meyers et al., 1995). Potential consequences of abnormal cranial vault, skull base, and facial growth include increased intracranial pressure, hearing and visual impairment, impaired cerebral blood flow, hindbrain malformations, syringomyelia, sleep apnea, and multifactorial developmental delay. Several studies have reported cognitive deficits in FGFR3-related craniosynostosis, characterized by impairments in memory, attention, anxiety, and emotional regulation (Kruszka et al., 1993; Maliepaard et al., 2014; Yarnell et al., 2015). FGFR3 is widely recognized as a key regulator of endochondral ossification. However, its role in sutural biology and endochondral ossification is less known. The role of the p.Ala391Glu CAN mutation remains unexplored. We have developed the first CAN mouse model (Fgfr3 ) expressing the dominant p.Ala385Glu mutation. A385E / + ) was created. Fgfr3 A385E / +The mice showed an absence of craniosynostosis and normal craniocerebral ratios. In the central nervous system, FGFR3 is highly expressed in the hippocampus, a brain structure essential for cognitive mechanisms. We hypothesized that the p.Ala385Glu mutation may affect adult neurogenesis and cognitive abilities. To test this hypothesis and define the effects of FGFR3 gain-of-function mutations on behavior, we examined neurogenesis in the hippocampus and examined Fgfr3 expression. A385E / + Mice showed reduced proliferation associated with a reduced size of the granular layer in the dentate gyrus. Furthermore, Fgfr3 A385E / + Mice exhibited hippocampal-dependent learning and memory impairments and abnormal coping strategies to inescapable stress.

[0005] We also developed an Hch mouse model Fgfr3 expressing the most common HCH mutation, p.Asn540Lys, located in the FGFR3 tyrosine kinase I domain. N534Ks / + To test this hypothesis and define the impact of FGFR3 gain-of-function mutations on behavior, we investigated the effects of Fgfr3 N534K / + They performed a series of behavioral tests on the mice and their control littermates, and found that Fgfr3 N534Ks / + We found that mice exhibit hippocampal-dependent memory impairment and abnormal coping strategies against inescapable stress.

[0006] Here, we provide strong evidence that brain-expressed FGFR3 gain-of-function mutations induce cognitive and behavioral deficits independent of cranial abnormalities. To provide evidence that constitutive activation of FGFR3 is involved in these behavioral disorders, we used intracerebroventricular injection of BGJ398 for 7 days to investigate the role of FGFR3 in the behavioral disorders. A385E / + Mouse and Fgfr N53Ks / + Mice were treated with tyrosine kinase inhibitors, which rescued the abnormalities in learning and memory, as well as in coping strategies. Thus, targeting FGFR3 offers a new and effective therapeutic prospect for treating cognitive impairment in chondrodysplasia and craniosynostosis.

[0007] Summary of the Invention:

[0008] The present invention relates to methods and pharmaceutical compositions for the treatment of FGFR3-associated cognitive deficits. In particular, the invention is defined by the claims.

[0009] Detailed description of the invention:

[0010] A fibroblast growth factor receptor 3 (FGFR3) gain-of-function mutation (p.Ala391Glu) is involved in a rare form of craniosynostosis: Crouzon syndrome with acanthosis nigricans (CAN). Patients with CAN are characterized by premature synostosis of the coronal sutures of the skull, central facial hypoplasia, acanthosis nigricans, and neurological impairment. FGFR3 is defined as a negative regulator of long bone growth. However, the role of the p.Ala391Glu CAN mutation in cranial suture growth biology remains unexplored. We observed that the CAN mutation induces receptor hyperactivation independent of ligand and prevents protein maturation. We developed the first CAN mouse model (Fgfr3) expressing a dominant p.Ala385Glu mutation. A385E / + ) and a HCH mouse model expressing the dominant p.Asn540Lys mutation (Fgfr3 Asn534Lys / + ) was created. Fgfr3 A385E / + Mice showed an absence of craniosynostosis and normal craniocerebral ratios. However, by analyzing the adult hippocampus of these mice, we showed that FGFR3 hyperactivation was associated with reduced dentate gyrus progenitor cell proliferation and neurogenesis. Consequently, behavioral tests were performed on Fgfr3 A385E / + This study was carried out in mice, and hippocampal-dependent memory impairment and abnormal coping strategies were observed. Finally, using a specific FGFR3 inhibitor (BGJ398), the inventors demonstrated that Fgfr3 A385E / +Inhibiting FGFR3 hyperactivation in the mouse brain thus reversed behavioral and cognitive deficits, highlighting for the first time behavioral abnormalities associated with FGFR3 hyperactivation in the brain, thereby enabling a better understanding of the role FGFR3 plays in learning processes and emotional responses in craniosynostosis.

[0011] Accordingly, the present invention relates to a method for treating an FGFR3-associated cognitive deficit in a subject suffering from an FGFR3-associated skeletal disease in need thereof, comprising administering to the subject a therapeutically effective amount of an FGFR3 inhibitor.

[0012] The present invention also relates to FGFR3 inhibitors for use in the treatment of FGFR3-associated cognitive deficits in subjects suffering from an FGFR3-associated skeletal disease.

[0013] As used herein, the term "subject" refers to a mammal, such as a rodent, cat, dog, and primate. In particular, a subject according to the present invention is a human. As used herein, the term "subject" encompasses a "patient."

[0014] In some embodiments, a subject of the present invention is or will be suffering from a cognitive deficit.

[0015] In some embodiments, subjects of the present invention are or will be affected by a brain-expressed FGFR3 gain-of-function mutation that causes cognitive and behavioral deficits.

[0016] In some embodiments, subjects of the present invention have or will have episodic memory deficits, antidepressant effects, abnormalities in learning, and stress response.

[0017] As used herein, the terms "FGFR3," "FGFR3 tyrosine kinase receptor," and "FGFR3 receptor" are used interchangeably throughout this specification and refer to all naturally occurring isoforms of FGFR3. An exemplary human amino acid sequence of FGFR3 is represented by SEQ ID NO:1.

change

[0018] As used herein, the expressions "constitutively active FGFR3 receptor variant," "constitutively active mutant of FGFR3," or "mutant FGFR3 exhibiting constitutive activity" are used interchangeably and refer to a mutant of the receptor that exhibits biological activity (i.e., induces downstream signaling) and / or exhibits greater biological activity than the corresponding wild-type receptor in the presence of an FGF ligand. Constitutively active FGFR3 variants according to the invention are in particular selected from the group consisting of (residues numbered according to their position in the precursor of fibroblast growth factor receptor 3 isoform 1 to 806 amino acids in length): a variant in which the serine residue at position 84 is substituted with a lysine (hereinafter designated S84L); a variant in which the arginine residue at position 200 is substituted with a cysteine ​​(hereinafter designated R200C); a variant in which the arginine residue at position 248 is substituted with a cysteine ​​(hereinafter designated R248C); a variant in which the serine residue at position 249 is substituted with a cysteine ​​(hereinafter designated S249C); a variant in which the proline residue at position 250 is substituted with an arginine (hereinafter designated P250R); a mutant in which the asparagine residue at position 268 is substituted with a histidine (hereinafter designated N262H); a mutant in which the glycine residue at position 268 is substituted with a cysteine ​​(hereinafter designated G268C); a mutant in which the tyrosine residue at position 278 is substituted with a cysteine ​​(hereinafter designated Y278C); a mutant in which the serine residue at position 279 is substituted with a cysteine ​​(hereinafter designated S279C); a mutant in which the glycine residue at position 370 is substituted with a cysteine ​​(hereinafter designated G370C); a mutant in which the serine residue at position 371 is substituted with a cysteine ​​(hereinafter designated S371C); a mutant in which the tyrosine residue at position 373 is substituted with a cysteine ​​(hereinafter designated Y373C);a variant in which the glycine residue at position 380 is substituted with arginine (hereinafter designated G380R); a variant in which the valine residue at position 381 is substituted with glutamic acid (hereinafter designated V381E); a variant in which the alanine residue at position 391 is substituted with glutamic acid (hereinafter designated A391E); a variant in which the asparagine residue at position 540 is substituted with lysine (hereinafter designated N540K); a variant in which the stop codon has been removed due to a base substitution, in particular a variant in which the stop codon has been mutated at an arginine, cysteine, glycine, serine, or tryptophan codon (hereinafter designated X807R, X807C, X807G, X807S, and X807R, respectively). a mutant in which the lysine residue at position 650 is substituted with another residue, in particular methionine, glutamic acid, asparagine, or glutamine (hereinafter referred to as K650M, K650E, K650N, and K650Q); a mutant in which the methionine residue at position 528 is substituted with isoleucine (hereinafter referred to as M528I); a mutant in which the isoleucine residue at position 538 is substituted with valine (hereinafter referred to as I538V); a mutant in which the asparagine residue at position 540 is substituted with serine (hereinafter referred to as N540S); a mutant in which the asparagine residue at position 540 is substituted with threonine (hereinafter referred to as N540T). Typically, the constitutively active FGFR3 variant according to the present invention is an N540K, K650N, K650Q, M528I, I538V, N540S, N540T, or A391E mutant;

[0019] As used herein, the term "cognitive deficits" refers to a range of symptoms, including depression, memory, perception, slowness, and problem-solving difficulties. Cognitive deficits may be present as a symptom in some psychiatric disorders (psychosis, mood disorders, anxiety disorders), but they are primarily synonymous with brain damage.

[0020] As used herein, the term "FGFR3-associated cognitive deficit" is intended to mean a cognitive deficit caused by abnormal overactivation of FGFR3 in the brain, in particular by expression of a constitutively active mutant of the FGFR3 receptor, in particular the constitutively active mutant of the FGFR3 receptor described above.

[0021] In some embodiments, the subject with an FGFR3-associated cognitive deficit is afflicted with an FGFR3-associated skeletal disease.

[0022] As used herein, the term "neurogenesis" has its general meaning in the art and refers to the process by which new neurons are formed in the brain. Neurogenesis is important during embryonic development, but also continues in specific brain regions after birth and throughout life. The mature brain has many specialized functional areas and neurons that differ in structure and connectivity. The hippocampus, for example, is a brain region that plays an important role in memory and spatial navigation and alone contains at least 27 types of neurons. The incredible diversity of neurons in the brain results from regulated neurogenesis during embryonic development. During this process, neural stem cells differentiate, i.e., they become any one of a number of specialized cell types at specific times and regions in the brain.

[0023] As used herein, the term "FGFR3-associated skeletal disease" is intended to mean a skeletal disease caused by increased abnormal 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.

[0024] In some embodiments, the FGFR3-related skeletal disease is preferably FGFR3-related chondrodysplasia and FGFR3-related craniosynostosis.

[0025] As used herein, "FGFR3-associated chondrodysplasia" includes, but is not limited to, short stature disorders such as hypochondroplasia (HCH), thanatophoric dysplasia (TD) type I, thanatophoric dysplasia type II, achondroplasia (ACH), and SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans).

[0026] In particular, the FGFR3-related skeletal disorder is short stature.

[0027] As used herein, the term "dwarfism" has its ordinary meaning in the art and refers to short stature resulting from a genetic or medical condition. Dwarfism is generally defined as an adult height of 147 cm or less.

[0028] In particular, an FGFR3-related skeletal disease is achondroplasia (HCH).

[0029] As used herein, the term "achondroplasia" (HCH) has its general meaning in the art and is associated with disproportionately short stature, micromelia, and a head that appears large compared to the underdeveloped parts of the body.

[0030] In some embodiments, the FGFR3-associated chondrodysplasia is hypochondroplasia caused by expression of a constitutively active N540K, K650N, K650Q, M528I, I538V, N540S, or N540T mutant of the FGFR3 receptor.

[0031] In particular, an FGFR3-related skeletal disease is achondroplasia (ACH).

[0032] As used herein, the term "achondroplasia" (ACH) has its common meaning in the art and relates to a genetic defect associated with short arms and legs, a typically normal-length trunk, and an enlarged head and prominent forehead.

[0033] In particular, an FGFR3-related skeletal disease is thanatophoric dysplasia (TD).

[0034] As used herein, the term "thanatophoric dysplasia" (TD) has its common meaning in the art and relates to severe skeletal defects characterized by a disproportionately small rib cage, extremely short limbs, and excess skin folds on the arms and legs.

[0035] In some embodiments, the FGFR3-associated skeletal disease is FGFR3-associated craniosynostosis. In some embodiments, the FGFR3-associated craniosynostosis corresponds to a genetic or sporadic disease.

[0036] In particular, the FGFR3-associated craniosynostosis is Muenke syndrome, which is caused by expression of a constitutively active mutant of the FGFR3 receptor P250R.

[0037] In particular, an FGFR3-related craniosynostosis is Crouzon syndrome with acanthosis nigricans (CAN), which is caused by expression of the A391E constitutively active mutant of the FGFR3 receptor.

[0038] As used herein, the term "craniosynostosis" has its common meaning in the art and relates to a condition in which one or more fibrous sutures in a subject's skull fuse prematurely by turning into bone (ossification), thereby altering the growth pattern of the skull. "Crouzon syndrome with acanthosis nigricans" (CAN) is an extremely rare form of craniosynostosis.

[0039] As used herein, the term "acanthosis nigricans" refers to a brown to black, poorly defined, velvety hyperpigmentation of the skin.

[0040] As used herein, the term "FGFR3 Y367C / +This relates to a mouse model that recapitulates the human ACH phenotype and is associated with clinical features of ACH (e.g., short stature, reduced size of the foramen magnum, mandibular hypoplasia, hearing loss, and intervertebral disc abnormalities) (Pannier et al. 2009, 2010; Mugniery et al. 2012; Di Rocco et al. 2014; Komla Ebri et al. 2016).

[0041] As used herein, the term "FGFR3 N534K / + " relates to a mouse model of HCH. Mutant mice exhibit clinical features of HCH, including growth failure, growth plate abnormalities, partial loss of synchondroses, and lordosis.

[0042] As used herein, the term "FGFR3 A385E / + " relates to the CAN mouse model in which defective memory was observed.

[0043] As used herein, the term "treatment" or "treating" refers to prophylactic or preventative treatment, as well as therapeutic, patient-condition-improving, disease-modifying treatment (including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition), including the suppression of clinical recurrence. The treatment may be administered to a subject who has a medical deficiency or who may eventually acquire a deficiency to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the deficiency or recurrent deficiency, or to prolong the subject's survival beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant a pattern of disease treatment, e.g., a pattern of medication used during treatment. A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to the patient during the initial period of the treatment regimen. The induction regimen may use (partially or entirely) a "loading regimen," which may involve administering a higher dose of drug than the physician would use during a maintenance regimen, administering a drug more frequently than the physician would administer a drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient during disease treatment, for example, to keep a patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous treatment (administering a drug at regular intervals, for example, daily, weekly, monthly, yearly, etc.) or intermittent treatment (e.g., discontinued treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of certain predetermined criteria (e.g., disease manifestation, etc.)).

[0044] As used herein, the term "prevent" is intended to characterize a prophylactic method or process that aims to delay or prevent the onset of the defect or condition to which such term applies.

[0045] The term "expression," when used in the context of gene or nucleic acid expression, refers to the conversion of the information contained in the gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein (i.e., FGFR3) produced by translation of an mRNA.

[0046] As used herein, the term "inhibitor" includes not only drugs for inhibiting the activity of a target molecule, but also drugs for inhibiting the expression of a target molecule.

[0047] The inhibitors according to the present invention can inhibit or eliminate the functional activation of the FGFR3 receptor in vivo and / or in vitro, and can inhibit the functional activation of the FGFR3 receptor by at least about 10%, preferably 20%, at least about 30%, preferably at least about 50%, preferably at least about 70, 75, or 80%, and more preferably 85, 90, 95, or 100%.

[0048] Inhibitors according to the present invention include inhibitors that specifically bind to the FGFR3 receptor, thereby reducing or blocking signal transduction. Antagonists of this type include antibodies (particularly those disclosed above) or aptamers that bind to FGFR3, fusion polypeptides that bind to FGFR3, peptides, small chemical molecules, and peptidomimetics.

[0049] 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 hybrids, post-translationally modified polypeptides, and peptidomimetics.

[0050] As used herein, the term "amino acid" refers to the 20 standard alpha-amino acids and their natural and synthetic derivatives. Polypeptides can contain L- or D-amino acids or combinations thereof.

[0051] As used herein, the term "peptidomimetic" refers to a peptide-like structure that has substituted non-amino acid structures but that mimics the chemical structure of a peptide.

[0052] 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 to an antigen. As such, the term "antibody" encompasses whole antibody molecules as well as antibody fragments and variants (including derivatives) of antibodies and antibody fragments.

[0053] In particular, the antibody according to the present 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. An "antibody fragment" comprises 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')2, Fab', Fd, dAb, dsFv, scFv, sc(Fv)2, CDR, diabody, and multispecific antibodies formed from antibody fragments.

[0054] The antibodies according to the present invention may be produced by any technique known in the art, including but not limited to any chemical, biological, genetic, or enzymatic technique, either alone or in combination. The antibodies of the present invention can be obtained by producing and culturing hybridomas.

[0055] "Aptamers" are a class of molecules that represent an alternative to antibodies for molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the ability to recognize virtually any class of target molecules with high affinity and specificity. Such ligands can be isolated through systematic evolution of ligands by exponential enrichment of random sequence libraries (SELEX), as described in Tuerk C. and Gold L., Science, 1990, 249(4968):505-10. Random sequence libraries are available through combinatorial chemical synthesis of DNA. In this library, each member is ultimately chemically modified to form a linear oligomer of unique sequence. The possible modifications, uses, and advantages of this class of molecules are reviewed in Jayasena SD, Clin. Chem., 1999, 45(9):1628-50. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by platform proteins such as Escherichia coli thioredoxin A that are selected from combinatorial libraries by the two-hybrid method (Colas et al., Nature, 1996, 380, 548-50).

[0056] The term "small chemical molecule" preferably refers to a molecule of less than 1,000 daltons, especially an organic or inorganic compound. Structural design in chemistry should help to find such molecules.

[0057] In some embodiments, the FGFR3 inhibitor is a tyrosine kinase inhibitor.

[0058] The present invention relates to a method for treating FGFR3-associated cognitive deficits in a subject suffering from an FGFR3-associated skeletal disease in need thereof, comprising administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor (TKI).

[0059] The present invention also relates to a tyrosine kinase inhibitor for use in the treatment or prevention of FGFR3-associated cognitive deficits in a subject suffering from an FGFR3-associated skeletal disease.

[0060] As used herein, the term "tyrosine kinase inhibitor" (TKI) refers to a compound (natural or synthetic) that is effective in inhibiting tyrosine kinase activity. Inhibitors with specific activity against tyrosine kinases may also be preferred.

[0061] Examples of tyrosine kinase inhibitors include PD173074 (CAS No. 219580-11-7), AZD4547 (CAS No. 1035270-39-3), BGJ398 (CAS No. 872511-34-7), AP24534 (CAS No. 943319-70-8), BIBF1120 (CAS No. 656247-17-5), JNJ-42756493 (CAS No. 1346242-81-6), TKI-258 (CAS No. 405169-16-6), PHA-739358 (CAS No. 827318-97-8), BMS-540215 (CAS No. 649735-46-6), TKI-258 dilactic acid (CAS No.852433-84-2), MK-2461(CAS No.917879-39-1), BMS-582664(CAS No.649735-63-7), SSR128129E(CAS No.848318-25-2), PRN1371(CAS No.1802929-43-6), PD166866(CAS No.192705-79-6), BLU554(CAS No.1707289-21-1), S49076(CAS No.1265965-22-7), SU5402(CAS No.215543-92-3), BLU9931(CAS No.1538604-68-0), FIN-2(CAS Examples of suitable CAS numbers include, but are not limited to, TKI-258 lactic acid (CAS No. 1633044-56-0), TKI-258 lactic acid (CAS No. 915769-50-5), CH5183284 (CAS No. 1265229-25-1), LY2874455 (CAS No. 1254473-64-7), or ASP5878 (CAS No. 1453208-66-6). As is well recognized, the Chemical Abstracts Service (CAS) number assigned to each molecule is a unique identifier for each compound.

[0062] In certain embodiments, the tyrosine kinase inhibitor is BGJ398 (a potent inhibitor of the FGFR family). As used herein, the term "BGJ398" has its general meaning in the art and refers to 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-[6-[4-(4-ethylpiperazin-1-yl)anilino]pyrimidin-4-yl]-1-methylurea. This term is also known as infigratinib, NVP-BGJ398, or BGJ-398.

[0063] As used herein, the term "administering" or "administration" refers to the act of injecting or otherwise physically delivering an exogenous substance (e.g., an FGFR3 inhibitor) to a subject, such as by intracerebroventricular, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or its symptoms. When a disease or a symptom thereof is being prevented, administration of the substance typically occurs before the onset of the disease or its symptoms.

[0064] A "therapeutically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic result. The therapeutically effective amount of a drug can vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the drug to elicit a desired response in an individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the FGFR3 inhibitor are outweighed by the therapeutically beneficial effects. The effective dosage and dosing regimen for a drug depends on the disease or condition being treated and can be determined by one of ordinary skill in the art. A physician with ordinary skill in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, a physician can start a dose of the drug used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally, the appropriate dose of the composition of the present invention is the amount of the compound that is the lowest dose effective to produce a therapeutic effect according to a particular dosing regimen. Such an effective dose generally depends on the factors described above. For example, a therapeutically effective amount for therapeutic use can be measured by its ability to stabilize the progression of a disease. One of ordinary skill in the art would be able to determine such amounts based on factors such as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of a drug is about 0.1 to 100 mg / kg, such as about 0.1 to 50 mg / kg, for example, about 0.1 to 20 mg / kg, such as about 0.1 to 10 mg / kg, for example, about 0.5, about 0.3, about 1, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg. Administration may be, for example, intracerebroventricular, intravenous, intramuscular, intraperitoneal, or subcutaneous, e.g., administered proximal to the target site. Dosage regimens in the above methods and uses of treatment are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the effectiveness of treatment is monitored during therapy, eg, at predefined time points.As a non-limiting example, treatment according to the present invention may be carried out in an amount of about 0.1 to 100 mg / kg per day, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg per day, as a daily dose of the agent of the present invention. , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or alternatively, at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks after initiation of treatment, or any combination thereof, using single or divided doses every 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, or 2 hours, or any combination thereof.

[0065] Thus, the subject is administered a pharmaceutical composition comprising an FGFR3 inhibitor as an active ingredient and at least one pharmaceutically acceptable excipient. As used herein, the terms "active ingredient" and "active ingredient" are used interchangeably. The active ingredient is used to alleviate, treat, or prevent a medical condition or disease. The term "pharmaceutically acceptable excipient" herein refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient and is not excessively toxic to the host at the concentration at which it is administered. The excipient is selected from conventional excipients known to those skilled in the art depending on the pharmaceutical form and the desired method of administration. In some embodiments, the pharmaceutical composition of the present invention does not contain a second active ingredient.

[0066] The present invention also provides therapeutic applications in which the FGFR3 inhibitor of the present invention is used in combination with at least one additional therapeutic agent, for example, to treat cognitive deficits.Such administration can be simultaneous, separate, or sequential.For simultaneous administration, the agents can be administered as one composition or as separate compositions, as appropriate.The additional therapeutic agent is typically related to the deficit to be treated.

[0067] As used herein, the term "combination" is intended to refer to all forms of administration that provide a first drug together with an additional (second, third, ...) drug. The drugs may be administered simultaneously, separately, or sequentially, in any order. According to the present invention, a drug is administered to a subject using any suitable method that allows the drug to reach the brain. In some embodiments, a drug is administered to a subject systemically (i.e., via systemic administration). Thus, in some embodiments, a drug is administered to a subject so that it enters the circulatory system and is distributed throughout the body. In some embodiments, a drug is administered to a subject by local administration, for example, by local administration to the hypothalamus.

[0068] As used herein, the terms "combination treatment," "combination therapy," or "combination therapy" refer to treatment using more than one medication. Combination treatment can be dual therapy or bitherapy.

[0069] As used herein, the term "co-administration" refers to the administration of two active ingredients by the same route at the same time or substantially the same time. The term "separate administration" refers to the administration of two active ingredients by different routes at the same time or substantially the same time. The term "sequential administration" refers to the administration of two active ingredients at different times, where the routes of administration are the same or different.

[0070] The present invention is further illustrated by the following figures and examples, which, however, should not be construed in any way as limiting the scope of the present invention. [Brief explanation of the drawings]

[0071] [Figure 1] Learning impairment and antidepressant-like behavior in the CAN model. A. Novel object recognition (NOR) was performed in 4-month-old male animals. Discrimination indices were measured 24 hours after the training phase to assess memory performance in Fgfr3A385E / + and their control littermates. NOR was performed in two independent groups of Fgfr3A385E / + (n = 17) and their control (n = 14). B. Contextual fear conditioning (CFC) was performed in 4-month-old male animals (Fgfr3+ / +, n = 13; Fgfr3A385E / +, n = 12). Percent freezing time was recorded for the training phase (to control for basal levels) and the test phase (to assess memory performance). C. Forced swim test (FST) was performed in two independent groups of 4-month-old male animals. The FST was performed over two consecutive days. Immobility periods were assessed on both days 1 and 2 (Fgfr3+ / +, n=17; Fgfr3A385E / +, n=14). D. The tail suspension test (TST) was performed on 4-month-old male animals on two consecutive days, and immobility periods were assessed on both days (Fgfr3+ / +, n=17; Fgfr3A385E / +, n=14). All behavioral analyses were performed on two independent cohorts of mice and two independent experiments for each group. [Figure 2]Downregulation of Fgfr3A385E / + phosphorylation in the hippocampus rescued memory deficits and antidepressant-like behavior. A. Local intracerebroventricular injections of either BGJ398 or vehicle were given once daily for 7 days to 4-month-old male animals. The NOR training session occurred 1 hour after the last injection. B. NOR was performed in groups injected with vehicle or BGJ398. Injections and NOR were performed in two independent groups under each of three conditions (Fgfr3+ / ++ vehicle, n = 15; Fgfr3A385E / ++ vehicle, n = 14; Fgfr3A385E / ++BGJ398, n = 14). C. Local intracerebroventricular injections of either BGJ398 or vehicle were given once daily for 7 days to 4-month-old male animals. One FST test session occurred 1 hour after the last injection. The immobility period was assessed during testing. D. FST performed on groups injected with vehicle or BGJ398. Injections and FST were performed on two independent groups with three conditions each (Fgfr3+ / ++vehicle, n=15; Fgfr3A385E / ++vehicle, n=14; Fgfr3A385E / ++BGJ398, n=14). *P<0.05, **P<0.01, ***P<0.001. NS: Not significant by ANOVA with Tukey's multiple comparison post-hoc test and Student's unpaired t-test. [Figure 3] The learning impairment was reversed by a daily subcutaneous injection of the FGFR3 tyrosine kinase inhibitor BGJ398. A. Novel object location (NOL) was performed in 4-month-old male animals. Discrimination indices were measured 24 hours after the training phase to assess the memory performance of Fgfr3Asn534Lys / + and their control littermates. B. Novel object recognition (NOR) was performed in 4-month-old male animals. Discrimination indices were measured 24 hours after the training phase to assess the memory performance of Fgfr3Asn534Lys / ++BGJ398, Fgfr3Asn534Lys / +, and their control littermates. NOR and NOL were performed in two independent groups: Fgfr3Asn534Lys / + (n = 12), Fgfr3Asn534Lys / ++BGJ398 (n = 14), and their control littermates (n = 14).

[0072] Materials and Methods.

[0073] All procedures were approved by the French Animal Care and Use Committee. Genomic DNA was isolated from the tail using the NucleoSpin Tissue kit (Macherey-Nagel) according to the manufacturer's instructions. Mice were genotyped using the following primers: 5'-GTGGGGGTTCTGCGGTTGG-3' (SEQ ID NO: 2) and 5'-TGACAGGCTTGGCAGTACGG-3' (SEQ ID NO: 3) (to isolate WT and mutant mice). For all analyses, wild-type littermates were used as controls.

[0074] Brain MRI.

[0075] Mouse brain MRI was acquired at 100 μm resolution using a small animal 4.7-T MR imaging unit (Biospec47 / 40; Bruker, Billerica, MA, USA). A385E / + mice and five Fgfr3 Asn534Lys / + Male mice and five 4-month-old control littermates were anesthetized with isoflurane gas inhalation during acquisition. Three-dimensional reconstruction and measurements were performed using Imaris (Bitplane).

[0076] Surgery and drug treatments.

[0077] BGJ398 was purchased from LC Laboratories, Woburn, MA, USA. For BGJ398 injections, 4-month-old Fgfr3 mice were used. Asn534Lys / + Mouse and Fgfr3 A385E / + Mice received daily subcutaneous injections of BGJ398 (2 mg / kg) or vehicle (HCl 3.5 mM, DMSO 2%) for 6 days. For habituation and testing of NOR and NOL, injections were performed 1 h before the test phase.

[0078] behavioral testing (Commons et al. 2017; Glatigny et al. 2019).

[0079] 3 Footshock contextual fear conditioning (CFC).

[0080] Mice were transported a short distance in their home cages from the mouse facility to the testing room and left there for at least 1 h before the start of testing. Conditioning chambers were obtained from Bioseb (France) and had internal dimensions of 25 × 25 × 25 cm. Each chamber was placed within a larger insulated plastic cabinet (67 × 55 × 50 cm, Bioseb, France) that provided protection from external light and noise, and mice were tested individually in the conditioning boxes. The chamber floor consisted of 27 stainless steel bars wired to a shock generator with a scrambler for footshock delivery. Signals generated by mouse movement were recorded and analyzed via a highly sensitive weight transducer system. Analog signals were transmitted via a load cell unit to a freezing software module for recording purposes, and analysis of activity / immobility time (freezing) was performed. The CFC procedure was performed over two consecutive days. On day 1, mice were placed in the conditioning chamber and received three foot shocks (1.5 seconds, 0.5 mA), which were administered 60, 120, and 180 seconds after placing the animals in the chamber. They were returned to their home cages 60 seconds after the last shock. Contextual fear memory was assessed 24 hours after conditioning by returning the mice to the conditioning chamber and measuring freezing behavior during a 4-minute retention test. Freezing was automatically scored and analyzed using Packwin 2.0 software (Bioseb, France). Freezing behavior was considered to have occurred if the animal froze for at least 2 seconds. Behavior was scored by the freezing software.

[0081] Novel object recognition paradigm (NOR).

[0082] We used a modified version of the NOR task, as described in Glatigny et al., 2019. Mice were transported a short distance from the mouse facility to the testing room in their home cages and left there for at least 1 h before the start of testing. The testing room was illuminated with two 60-W light bulbs, and behavioral sessions were recorded with a camera from above the test arena (a gray plastic box (60 × 40 × 32 cm)). Mice were not allowed to touch or see each other during the illumination. Light intensity was equal in all parts of the arena (approximately 20 lx). Two different objects were used, available in triplicate. The objects were a blue ceramic pot (6.5 cm diameter, 7.5 cm maximum height) and a clear plastic funnel (8.5 cm diameter, 8.5 cm maximum height). The object serving as the novel object, as well as the left / right localization of the objects, were counterbalanced within each group. The objects elicited the same level of exploration as determined in the pilot experiment and training phase. Between exposures, mice were housed individually in standard cages, the objects and arena were cleaned with phagophore, and the bedding was changed.

[0083] The NOR paradigm consisted of three phases (over three days): a habituation phase, a training phase, and a test phase. Mice were always placed in the center of the arena at the beginning of each exposure. Day 1: Habituation phase, mice were given 5 minutes to explore the arena without any objects and then returned to their home cage. Day 2, Training phase, mice explored two identical objects placed symmetrically opposite the center of the arena for 10 minutes and then transported to their home cage. Day 3, Test phase, mice were given 15 minutes to explore two objects: a familiar object and a novel object in the same arena, with the same object localization maintained.

[0084] The following behaviors were considered object exploration: sniffing, licking, or touching the object with the nose or forepaws, or pointing the nose toward the object at a distance of ≤1 cm. Investigations were not scored if the mouse was on the object or completely immobile. Discrimination indices were calculated as (time spent exploring the novel object - time spent exploring the familiar object) / (total time spent exploring both objects). As controls, preference indices for object location (right / left) or object A vs. B during the novel object recognition (NOR) training phase were measured in all groups of mice exposed to the test. We confirm herein that no initial preference for any exposed object (A or B) or any direction (right / left) was observed in any group. Locomotion was assessed for each mouse. Behavior was video-scored by two observers blinded to treatment, and the total time exploring the objects was quantified during the test phase.

[0085] New object location (NOL).

[0086] For the novel object location task, all procedures were identical to those for the novel object recognition task, except that during the test phase, rather than presenting a novel object, mice encountered both familiar objects, with one object located in a different location in the arena. The duration and frequency of exploration of the novel / relocated object were measured as an index of memory. Behavior was video-scored by two observers blinded to treatment, and the total time spent exploring the objects was quantified during the test phase.

[0087] Light-dark transition test (D / LT).

[0088] This test is based on rodents' innate aversion to brightly illuminated areas and their spontaneous exploratory behavior in response to the stressor represented by light. The test apparatus consisted of a dark safe compartment and an illuminated aversive compartment. The illuminated compartment was brightly illuminated with an 8W fluorescent tube (1000 lx). Naive mice were placed individually in the test chamber in the center of the dark area facing away from the doorway to the illuminated compartment. Mice were tested for 10 min, and two parameters were recorded: time spent in the illuminated compartment and number of transitions between compartments, indices of anxiety-related behavior and exploratory activity. Behavior was scored using an infrared light activity monitor using actiMot2 Software (PhenoMaster Software, TSE).

[0089] Open field test (OFT).

[0090] This test exploits rodents' aversion to bright areas. Each mouse was placed in the center of an OFT chamber (43 x 43 cm chamber) and allowed to explore for 30 minutes. Mice were monitored throughout each test session with an infrared light beam activity monitor using actiMot2 Software (PhenoMaster Software, TSE). Overall motor activity was quantified as total distance traveled (ambulatory activity). Anxiety was quantified by measuring the time and distance spent in the center versus periphery of the open field chamber.

[0091] Tail suspension test (TST).

[0092] This test is based on the observation that rodents, after an initial escape-oriented movement, develop an immobile posture when placed in an unavoidable stressful situation. Each mouse is suspended uncontrollably by its tail 25 cm above the floor. Mice are tested for 5 minutes, and the time spent immobile is quantified.

[0093] Forced swim test (FST).

[0094] This test is based on similar observations to the TST. Each mouse is placed in a cylinder (height: 25 cm, diameter: 10 cm) filled with water (23-25°C). Mice are tested for 5 minutes, and the time spent immobile (behavioral despair) is quantified.

[0095] Example 1: CAN model.

[0096] Clinical features of Crouzon syndrome with acanthosis nigricans.

[0097] CAN syndrome is associated with FGFR3 mutations and exhibits a skeletal phenotype similar to Crouzon syndrome [MIM123500] caused by FGFR2 mutations (Coll et al., 2018, 2016; Di Rocco et al., 2011): orbital disproportion, mandibular prognathism, central facial hypoplasia (data not shown), and brachycephaly secondary to premature coronal and sagittal suture fusion (variably different) (data not shown). The calvarial abnormality exerts mechanical pressure on the brain, increasing the risk of elevated intracranial pressure (Al-Namnam et al., 2019) (data not shown). Additionally, brain MRI revealed mild temporal abnormalities in three unrelated CAN patients. Affected cases presented with thickened parahippocampal sulci and altered structural angles (data not shown). One case presenting with a cloverleaf-shaped skull was uninterpretable.

[0098] Skull base abnormalities in patients with Crouzon syndrome involve both FGFR2 and FGFR3, contributing anteriorly to central facial hypoplasia and posteriorly to craniovertebral junction abnormalities. Premature fusion of the sphenooccipital synchondrosis is associated with a shortened cranial base, while premature fusion of the intraoccipital synchondrosis is associated with a narrowed foramen magnum in patients with CAN (data not shown).

[0099] Skeletal phenotypes are observed in the CAN mouse model Fgfr3 A385E / + It is mildly affected in

[0100] To assess the impact of CAN mutations on the skeleton, we generated a mouse model expressing the ubiquitous p.Ala385Glu missense mutation, corresponding to the p.Ala391Glu human mutation (data not shown). FGFR3 p.Ala385Glu transcripts were detected in fibroblasts and calvarial osteoblasts (data not shown). Similar to the human disease, the pronounced short-limbed phenotype persists throughout the prenatal and neonatal stages (data not shown) and into adulthood. A385E / + This was not observed in mice (data not shown). Body weight, nose-anus, and femur and tibia lengths were significantly increased in Fgfr3 mice. A385E / + Mouse and Fgfr3 + / + The results were similar in mice (data not shown). A385E / + This was confirmed by cartilage evaluation in 3-month-old rats, which showed well-organized growth plates without abnormalities in the hypertrophic zone as revealed by type X collagen staining (data not shown). To confirm the absence of an abnormal phenotype, bone structure parameters were evaluated. Micro-CT images of femurs at 3 months of age revealed Fgfr3 expression. A385E / + Normal structures of trabecular and cortical bone were evident in the mice (data not shown).

[0101] Craniofacial phenotypes were assessed using micro-CT skull acquisition. A385E / + The mice exhibited normal craniofacial features (data not shown); the coronal sutures and cranial base synchondrosclerosis were patent at postnatal day 21, similar to control mice (data not shown). A385E / + Mouse and Fgfr3 + / + Landmark-based geometric morphometry of mice (Heuze et al., 2010) did not reveal any differences in skull shape (d = 0.0148; p = 0.0940). However, mandibular shape was significantly different between the two groups of mice (d = 0.0159; p < 0.01) (data not shown). To confirm the absence of premature closure of the fronto-parietal suture, we analyzed calvarial Fgfr3 A385E / +In vitro osteoblast function was assessed. Significant differences in mineralization capacity, proliferation, and mitogen-activated protein kinase (MAPK) activation were observed in Fgfr3-treated osteoblasts compared to controls. A385E / + This was not observed in mouse osteoblasts (data not shown). All these data allowed us to conclude that the p.Ala385Glu mutation expressed in osteoblasts was not active and, consequently, did not affect craniofacial development (data not shown). These data support the notion that Fgfr3 A385E / + This explains the absence of a craniosynostosis phenotype in mice. Regarding the skin, signs of hyperkeratosis or changes in epidermal thickness and pigmentation are not associated with Fgfr3 A385E / + It was not detected in mice (data not shown).

[0102] Fgfr3 A385E / + Mouse models show that the dentate gyrus reduces neurogenesis.

[0103] Structural brain abnormalities have been described in patients with CAN (Gurbuz et al., 2016) (data not shown) and Muenke patients (Abdel-Salam et al., 2011; Grosso et al., 2003; Okubo et al., 2017). These abnormalities include abnormal morphology of the hippocampus and temporal lobe. It is well known that premature fusion of cranial sutures alters the shape of the skull, impairs normal brain growth, and leads to functional problems such as increased intracranial pressure, visual impairment, hearing loss, and cognitive impairment (Di Rocco et al., 2011). Previous studies have shown that patients with MS exhibit deficits in adaptive and executive function. This behavioral phenotype included working memory deficits, attention deficit hyperactivity disorder, emotional regulation, and anxiety (Yarnell et al., 2015). These neurological disorders suggest the influence of FGFR3 in the brain on the regulation of cognitive function. Therefore, we investigated the effects of Fgfr3 on 4-month-old mice. A385E / + Magnetic resonance imaging (MRI) was performed on the mice to measure the volumes of various brain regions.

[0104] Volume changes and any compression can affect Fgfr3 A385E / + However, FGFs and FGFRs are known to be involved in the proliferation and differentiation of neural stem cells and neural progenitor cells in the central nervous system (Huang et al., 2017; Kang and Hebert, 2015; Moldrich et al., 2011; Ohkubo et al., 2004; Stevens et al., 2012). Therefore, we hypothesized that Fgfr3 is involved in the proliferation and differentiation of neural stem cells and neural progenitor cells in the central nervous system (CNS) (Huang et al., 2017; Kang and Hebert, 2015; Moldrich et al., 2011; Ohkubo et al., 2004; Stevens et al., 2012). A385E / + Fgfr3 expressed in the mouse brain A385E We hypothesized that the mutation may affect adult neurogenesis. The absence of craniofacial abnormalities provides a good opportunity to specifically assess neurogenesis during adulthood. To explore the role of Fgfr3 in adult neurogenesis, we analyzed Fgfr3 by immunofluorescence (data not shown) and Western blotting (data not shown). A385E / + and Fgfr3 + / + We observed similar expression of FGFR3 in the mouse hippocampus. However, the canonical MAPK pathway activated by FGFR3 has been found to be dysregulated in both FGFR3 knock-in (Komla-Ebri et al., 2016) and FGFR3 knock-out (Zhou et al., 2015) mouse models. Indeed, we observed significantly increased expression of phosphorylated Erk1 / 2 in adult hippocampal lysates (data not shown), thus supporting the role of FGFR3 in the brain. A385E The MAPK pathway was activated by the mutation. FGFR3 plays an important role in the proliferation of progenitor cells in the dentate gyrus and neuronal differentiation (Inglis-Broadgate et al., 2005; Kang and Hebert, 2015; Moldrich et al., 2011; Thomson et al., 2009). A385EIn the hippocampus, using the NeuN marker, we demonstrated that the mature neuron area in the dentate granular layer was significantly reduced compared to controls (data not shown). We assessed whether this reduced neuronal population was caused by reduced progenitor cell proliferation. The number of cells positive for the cell cycle marker KI67 was significantly higher than that of Fgfr3. A385E / + In mice, Fgfr3 expression was significantly reduced in the subgranular zone of the dentate gyrus (data not shown). In the subgranular zone, the rate of neuronal differentiation, as revealed by doublecortin (DCX) immunolabeling, was slightly reduced (data not shown). Taken together, these data strongly suggested that Fgfr3 gain-of-function mutations primarily affect proliferation and, thus, the maturation and differentiation of neurons in the dentate gyrus.

[0105] Fgfr3 A385E / + Mouse models show reduced learning ability and antidepressant effects.

[0106] Reduced size and proliferation of the hippocampal structure have been shown to be associated with altered memory and cognition in humans and mice (Kitamura and Inokuchi, 2014). Therefore, we investigated the role of Fgfr3 in 4-month-old mice. A385E / + Mice and their control littermates were subjected to a series of behavioral tests, believed to reflect hippocampal-related behavioral functions, measuring associative (one-trial contextual fear conditioning, CFC) and episodic (novel object recognition test, NOR) learning and memory, as well as spatial (Morris water maze, MWM) learning and memory (Figures 1A-1D). In CFC, mutant mice showed no difference in baseline freezing time. However, Fgfr3 gain-of-function mutations resulted in reduced context-evoked freezing times during the test phase compared with their control littermates, indicating that contextual fear memory was impaired in mutant mice (Figure 1B).

[0107] Next, we used a modified version of the NOR paradigm (Denny et al., 2012; Ennaceur and Delacour, 1988), which measures rodents' ability to recognize novel objects in their environment. Wild-type mice were able to distinguish novel objects from familiar ones and tended to explore the novel objects for longer periods of time. As shown in Figure 1A, 4-month-old Fgfr3 mice were able to distinguish novel objects from familiar ones and tended to explore the novel objects for longer periods of time. A385E / + Mice explored the novel object significantly less than controls, but no impairment was observed when memory was analyzed through the MWM task (which assesses spatial learning and memory in rodents). A385E / + Mice and controls had comparable performance in the open field test (OFT) and the light / dark paradigm (L / DT) (data not shown), indicating that their locomotion and anxiety state were intact.

[0108] Next, we evaluated coping strategies against inescapable stress using the forced swim test (FST) and tail suspension test (TST). As shown in Figure 1C, 4-month-old Fgfr3 mice were significantly more susceptible to stress than controls. A385E / + Mutant mice spent significantly less time immobile during the FST than control mice. The same impairment was observed during the TST. Indeed, mutant mice spent significantly less time immobile than control littermates (Figure 1D).

[0109] Together, these data demonstrate that Fgfr3 gain-of-function mutations significantly affect hippocampus-dependent episodic and associative fear memory acquisition, affecting coping strategies against unavoidable stress.

[0110] Importantly, these data support the notion that Fgfr3 A385E The mutations reproduced working memory deficits and behavioral deficits previously described in human patients, including emotion regulation disorders and attention deficit hyperactivity disorder (Yarnell et al., 2015).

[0111] Intracerebroventricular injection of BGJ398 inhibited Fgfr3 A385E / + Cognitive impairment in mice is rescued.

[0112] Fgfr3 A385E / + To confirm that the cognitive impairment in mice was due to increased phosphorylation of the receptor, we decided to treat mice with the specific tyrosine kinase inhibitor BGJ398 (infigratinib) (Gudernova et al., 2015; Komla-Ebri et al., 2016). A385E / + Mice and their control littermates received intracerebroventricular injections of BGJ398 or vehicle solution for 7 days and were subjected to two behavioral tests (NOR and FST; Figures 2A to 2C). BGJ398 injection significantly reduced Fgfr3 expression compared to control littermates. A385E / + The memory deficits observed in the NOR paradigm in mice were reversed (Figure 2B), and the coping strategies for inescapable stress observed in the FST were reestablished (Figure 2D). A385E / + The absence of craniocerebral imbalance in mice, and thus the lack of potentially increased intracranial pressure, allows us to hypothesize that the observed and reported behavioral abnormalities are due to the increased uptake of Fgfr3 relative to the brain. A385E We can conclude that these behavioral abnormalities were due to a direct effect of the mutation. Rescue of these behavioral abnormalities with the FGFR3 inhibitor BGJ398 confirmed our hypothesis and supported the fact that FGFR3 hyperactivation is involved in the cognitive phenotype of patients with FGFR3-related craniosynostosis.

[0113] Example 2: HCH model.

[0114] Fgfr3 Asn534Lys / + The mice exhibit morphological abnormalities in the brain.

[0115] The present inventors have investigated the role of Fgfr3 Asn534Lys / +We previously described mice that exhibit cranial abnormalities with large skull bones and premature fusion of the cranial base synchondroses (Loisay et al., manuscript in preparation). Brain abnormalities and lobe hypoplasia were reported to be characteristic of Fgfr3 gain-of-function mutant mouse models. Therefore, we investigated the role of Fgfr3 in the pathogenesis of Fgfr3. Asn534Lys / + MRI and 3D reconstruction of the brains of Hch mice and their control littermates were performed. We did not observe any significant abnormalities in the hippocampus (p=0.6905) or total brain volume (p=0.3810) in Hch mice (data not shown), while MRI analysis showed changes in brain shape when compared with controls (data not shown).

[0116] Fgfr3 Asn534Lys / + Learning and memory deficits in mice.

[0117] Adult neurogenesis is known to play an important role in maintaining hippocampal memory. Asn534Lys / + Mice and their control littermates were subjected to a battery of behavioral tests measuring spatial (novel object location, NOL) and episodic (novel object recognition, NOR) learning and memory. In NOR, we found that Hch mutant mice explored the novel object significantly less than control littermates during the test phase (p<<0.0001) (data not shown). The same impairment was observed when spatial memory was analyzed through the NOL test. Indeed, we found that Fgfr3 Asn534Lys / + We found that mice explored the relocated objects significantly less during the test phase than control littermates (p<0.0001) (data not shown).

[0118] FGFR3 tyrosine kinase inhibitor treatment inhibits Fgfr3 Asn534Lys / + This is sufficient to reverse the cognitive impairment observed in mice.

[0119] Subcutaneous injection of the tyrosine kinase inhibitor BGJ398 (infigratinib) is sufficient to abolish FGFR3 hyperactivation and rescue the chondrodysplasia phenotype (Komla-Ebri et al. 2016). To confirm the impact of FGFR3 gain-of-function on learning disabilities, we used BGJ398 to inhibit FGFR3 Asn534Lys / + The mice were treated for 6 days. As a result, the present inventors found that injection of BGJ398 inhibited Fgfr3 Asn534Lys / + We found that the FGFR3-mediated signaling pathway was sufficient to reverse the spatial (NOL) (Figure 3A) and episodic memory deficits (NOR) (Figure 3B) observed in mice. Indeed, injected mutant mice were able to explore the relocated object (NOL) (p=0.9538) and novel object (NOR) (p=0.8697) to the same level as control littermates (p=0.9538) (Figures 3A and 3B). Together, these results confirm the importance of FGFR3 in regulating learning and memory.

[0120] Stress and memory behavioral tests.

[0121] We next performed single-trial contextual fear conditioning (CFC) in 4-month-old male mice to assess associative memory. Asn534Lys / + Although the mice did not show any impairment in baseline freezing time (p = 0.2303), surprisingly, the mutant mice showed a significant increase in contextually evoked freezing time during the test phase compared to control mice (p = 0.0466), strongly suggesting that contextual fear memory is impaired in the mutant mice.

[0122] Antidepressant effects of FGFR3.

[0123] The FGF pathway may be involved in depressive disorders. Therefore, we investigated the role of Fgfr3 Asn534Lys / +Tail suspension test (TST) and forced swimming test (FST) were performed in mice (data not shown). Animals subjected to short-term, unavoidable stress, such as being suspended by their tails or being forced to swim, developed immobility, characteristic of depression-related behavior, which was scored. By performing these tests, the inventors demonstrated that Fgfr3 Asn534Lys / + They found that the mice exhibited a reduction in immobility, suggesting that gain-of-function mutations in FGFR3 may have antidepressant effects.

[0124] Fgfr3 Asn534Lys / + No effect on anxiety behavior in mice.

[0125] Mutant and WT mice had comparable performance in the open field test (OFT) (data not shown) and the light / dark paradigm (L / DT) (data not shown), indicating that their locomotion and anxiety state were unaffected. Asn534Lys / + Due to the dwarf phenotype of the mice, mutant mice moved slower than control littermates (p=0.0003) and traveled less total distance during OF (p=0.0003). Consequently, OFT data analysis was performed considering the % distance (median distance / total distance x 100). Collectively, these results indicate that FGFR3 does not play an important role in anxiety and confirm that the cognitive deficits observed in learning and memory in our mutant mice are independent of any anxiety- or exploration-related behavioral deficits.

[0126] Consideration.

[0127] CAN syndrome is an extremely rare syndromic craniosynostosis associated with a specific p.Ala391Glu gain-of-function mutation in FGFR3 (Meyers et al., 1995). The effect of the p.Ala391Glu mutation has previously been described as causing hyperactivation of FGFR3 (Chen et al., 2013, 2011; Li et al., 2006).

[0128] Mouse Fgfr3 A385E / + The CAN model showed the absence of major craniofacial skeletal phenotypes. Interestingly, Fgfr3 A385E / + The phenotype of Fgfr3 is a mouse model for Muenke syndrome. P244R / P244R The phenotype was similar to that observed in mice. P244R / P244R It was found to be mildly affected in mutants, with fused coronal sutures in a few individuals (Laurita et al., 2011; Twigg et al., 2008).

[0129] In CAN and Muenke, premature fusion of the calvarial sutures combined with premature fusion of the cranial base synchondroses associated with narrowing of the foramen magnum leads to increased intracranial pressure in patients (Di Rocco et al., 2011). Premature fusion of the calvaria is also associated with structural brain abnormalities, including abnormal hippocampal development (Grosso et al., 2003; Gurbuz et al., 2016; Okubo et al., 2017).

[0130] HCH patients (14600MIM) are characterized by proximal segmental dwarfism, mild macrocephaly, central facial hypoplasia, short, squared ilia, and, in some cases, acanthosis nigricans (Blomberg et al. 2010). The most common HCH mutation (p.Asn540Lys) is localized in the tyrosine kinase 1 domain of FGFR3 (Bonaventure et al. 1996; Rousseau et al. 1994).

[0131] Patients with HCH exhibit lobar hypoplasia (Kannu et al. 2005) and abnormal hippocampal organization (Linnankivi et al. 2012). In addition, patients with HCH exhibit learning disabilities, mild intellectual disability, global developmental delay, and occasional seizures and epilepsy (Linnankivi et al. 2012).

[0132] Fgfr3 mouse models are associated with thanatophoric bone dysplasia and express Fgfr3 both ubiquitously and under the nestin promoter. + / K644E Previous studies of the mutation have shown severe overgrowth of the cerebrum and cortex, while Fgfr3 - / - Mice exhibited an underdeveloped neocortex (Inglis-Broadgate et al., 2005; Moldrich et al., 2011; Thomson et al., 2009, 2007). It is generally accepted that the premature fusion of cranial sutures observed in craniosynostosis alters brain morphology and is associated with cognitive impairment via a chronic increase in intracranial pressure (Aldridge et al., 2010; Arnaud-Lepez et al., 2007; Gurbuz et al., 2016; Martinez-Abadias et al., 2011). Here, Fgfr3 A385E / + Taking advantage of the absence of an abnormal skull phenotype in mice, we analyzed the role of activating Fgfr3 gain-of-function mutations in the brain. A385E / + The mouse brains did not exhibit severe morphological changes, thus indicating that Fgfr3 A385E This indicates that the mutation had a moderate effect on embryonic neurogenesis in the brain. In contrast, analysis of adult hippocampal neurogenesis showed reduced progenitor proliferation in the dentate gyrus, supported by reduced granular zones in the dentate gyrus.

[0133] Interestingly, previous studies reported decreased progenitor cell proliferation in FGFR1, 2, and 3 loss-of-function mutations, whereas FGFR3 (Fgfr3 TDIIK650E ) promotes increased progenitor cell differentiation in the dentate gyrus (Kang and Hebert, 2015). Our results contrast with these observations. We show that overactivation of FGFR3 promotes increased progenitor cell differentiation in the dentate gyrus. A385E / + They observed that this led to decreased cell proliferation in mice. The level of phosphorylation of the receptor, Fgfr3, appeared to impede neurogenesis. TDIIK650EMutations led to excessive receptor activation levels, whereas Fgfr3 A385E The mutation led to a more moderate hyperactivation. These data suggest that FGFR regulation of hippocampal neurogenesis is related to the level of FGFR3 activation.

[0134] Next, the inventors investigated the role of Fgfr3 in brain cognitive function. A385E Mutations of Fgfr3 N534K The effects of the mutations were analyzed. A385E / + Mouse Fgfr3 N534K / + We observed that mice exhibited severe performance impairments and episodic memory function without any locomotion, anxiety-related behavior, or spatial memory phenotype. While the effect of FGFR signaling on memory is unclear, our study is the first to link Fgfr3 mutations to cognitive abnormalities in mice to date. Mechanistically, the deficits in learning and memory performance may be related, at least in part, to the reduced hippocampal neurogenesis observed in our mutant mice. Interestingly, deletion of Fgfr2 in embryonic and adult mice showed reduced progenitor cell proliferation and differentiation in the dentate gyrus, with specific negative effects on associative and spatial memory capacity (Stevens et al., 2012). Collectively, our data demonstrate that Fgfr3 gain-of-function mutations lead to severe learning and memory deficits and lower adult neurogenesis in the hippocampus.

[0135] Also, decreased coping strategies against unavoidable stress (previously termed "depressive-like behavior") are associated with Fgfr3 A385E / + Mouse and Fgfr3 N534K / +This role of FGFR3 was previously reported in humans in major depressive disorder, associated with receptor downregulation (Evans et al., 2004). Furthermore, patients with FGFR3-related craniosynostosis syndromes exhibited impaired emotional regulation and anxiety behavior (de Jong et al., 2010; Maliepaard et al., 2014; Yarnell et al., 2015). To date, no studies have reported depressive or mood disorders in cases of craniosynostosis. Animal model studies have also demonstrated the antidepressant and anxiolytic effects of FGF2 in FGF2 knockout or exogenous FGF2 injection (Elsayed et al., 2012; Salmaso et al., 2016). In contrast to FGF2, FGF9 exerts depressant effects in mice (Aurbach et al., 2015). Both FGF2 and FGF9 are among the major FGFR3 ligands and can also bind to other FGFRs. These observations are consistent with our data and may involve complex combinations of different FGFs and FGFR1, 2, and 3 binding. However, Fgfr3 A385E / + Mouse and Fgfr3 N534K / + It is unclear how the antidepressant phenotype observed in mice can be translated to the human condition.

[0136] Fgfr3 A385E / + Mouse and Fgfr3 N534K / + To confirm the direct involvement of brain FGFR3 in the cognitive impairment observed in mice, we decided to treat mice with BGJ398, a tyrosine kinase inhibitor, following selective brain injection. BGJ398 was selected for its highest binding specificity for FGFR3, and previous studies have shown its efficacy in treating skeletal abnormalities in a mouse model of FGFR3-related achondroplasia (Komla-Ebri et al., 2016). Here, adult FGFR3 A385E / + Mouse and Fgfr3 N534K / +Intracerebroventricular injection of BGJ398 in mice showed rescue of working memory and episodic memory deficits and antidepressant effects. These data demonstrate the direct impact of FGFR3 gain-of-function mutations in the brain on cognitive ability. We also demonstrated that FGFR3 plays a key role in hippocampal adult neurogenesis, and we demonstrated a direct link between hippocampal abnormalities and learning and stress response.

[0137] These results highlight the existence of cognitive impairment without a craniosynostosis phenotype in mouse models expressing Fgfr3 gain-of-function mutations. Our data suggest that the brains of patients with FGFR-related craniosynostosis can be affected by this mutation independently of skull abnormalities.

[0138] References:

[0139] Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are hereby incorporated by reference into this disclosure. [Table 1] TIFF0007757322000003.tif246165 TIFF0007757322000004.tif250165 TIFF0007757322000005.tif250165 TIFF0007757322000006.tif239165 TIFF0007757322000007.tif243165 TIFF0007757322000008.tif242165 TIFF0007757322000009.tif242165 TIFF0007757322000010.tif170165

Claims

1. A pharmaceutical composition for treating FGFR3-associated cognitive deficits in a subject suffering from an FGFR3-associated skeletal disease, comprising an FGFR3 inhibitor, wherein the FGFR3 inhibitor is BGJ398.

2. 2. The pharmaceutical composition according to claim 1, wherein the FGFR3-related skeletal disease is hypochondroplasia (HCH), achondroplasia (ACH), lethal dysplasia (TD), craniosynostosis, or dwarfism.

3. The pharmaceutical composition according to claim 2, wherein the FGFR3-related skeletal disease is hypochondroplasia (HCH).

4. The pharmaceutical composition according to claim 2, wherein the FGFR3-related skeletal disease is achondroplasia (ACH).

5. The pharmaceutical composition according to claim 2, wherein the FGFR3-related skeletal disease is craniosynostosis.

6. The pharmaceutical composition according to claim 5, wherein the craniosynostosis is Crouzon syndrome with acanthosis nigricans (CAN).

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the FGFR3-associated skeletal disease is caused by expression of a constitutively active FGFR3 mutant.

8. 8. The pharmaceutical composition of claim 7, wherein the constitutively active FGFR3 mutant is an N540K, K650N, K650Q, M528I, I538V, N540S, or N540T mutant.

9. The pharmaceutical composition of claim 7, wherein the constitutively active FGFR3 mutant is the A391E mutant.

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