Compositions for treating FGFR3-associated cognitive disorders using catechins

Abstract

The present inventors provide strong evidence that FGFR3 gain-of-function mutations expressed in the brain induce cognitive and behavioral disorders. To provide evidence that the constitutive activation of FGFR3 and its downstream signaling pathways are responsible for these behavioral disorders, the present inventors used subcutaneous injections of catechin isomers for at least 7 days to investigate the effects of FGFR3 on cognitive and behavioral disorders. A385E / + Furthermore, the present inventors treated mice with Fgfr3 N534K / + Mice are treated with bottle treatment. Treatment restores abnormalities in short-term learning and coping strategies.Therefore, the present invention relates to a method for treating FGFR3-related cognitive impairment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one catechin.

Description

[Technical field]

[0001] Field of the invention: The present invention is in the field of medicine, particularly in the field of skeletal disorders and neurology.

[0002] Background of the invention: The fibroblast growth factor receptor (FGFR) family plays important roles in bone development and skeletal diseases. Missense mutations in FGFR1, FGFR2, and FGFR3 are responsible for a range of syndromic craniosynostosis characterized by premature fusion of the craniosynostosis (Robin et al., 1993; Twigg and Wilkie, 2015). Two specific FGFR3 dominant mutations are responsible for the rare syndromic craniosynostosis, Crouzon syndrome with acanthosis nigricans (CAN[MIM612247]), and the most common syndromic craniosynostosis, Muenke syndrome (MS[MIM602849]) (Wilkie et al., 2010). Patients with Crouzon syndrome with acanthosis nigricans (CAN) present with acanthosis nigricans but otherwise resemble patients with FGFR2-associated Crouzon syndrome: they are characterized by premature synostosis of the coronal sutures of the skull, brachycephaly, midface hypoplasia, and craniovertebral junction abnormalities (Arnaud-Lopez et al., 2007; Di Rocco et al., 2011; Meyers et al., 1995; Mir A et al., 2013). Crouzon syndrome with acanthosis nigricans (CAN) is defined by a single point mutation (p.Ala391Glu) localized within the transmembrane domain of FGFR3 (Li et al., 2006; Meyers et al., 1995). Potential consequences of abnormal cranial vault, base, and facial growth include elevated intracranial pressure, hearing and vision impairment, impaired cerebral blood flow, hindbrain malformation, syringomyelia, sleep apnea, and other developmental delays. Several studies have reported cognitive impairments in FGFR3-associated craniosynostosis, characterized by impaired memory capacity, attention, anxiety, and emotional control (Kruszka et al., 1993; Maliepaard et al., 2014; Yarnell et al., 2015). FGFR3 is widely recognized as a key regulator of endochondral ossification. However, the biological functions of sutural growth and its role in membranous ossification are less known. The role of the p.Ala391Glu mutation in Crouzon syndrome with acanthosis nigricans remains to be explored.We present the first mouse model of CAN expressing the dominant p.Ala385Glu mutation (Fgfr. A385E / + ) was created. Fgfr3 A385E / + The mice exhibited normal craniocerebral ratios with no craniosynostosis.In the central nervous system, FGFR3 is highly expressed in the hippocampus, a brain structure essential for cognitive mechanisms.

[0003] FGFR3-related chondrodysplasias, such as achondroplasia, severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), and hypochondroplasia nigricans (HCH), are characterized by short limbs, skull base abnormalities, macrocephaly, deafness, midfacial hypoplasia, mandibular prognathism, and less frequent but significant clinical features of mild to moderate intellectual disability and learning disabilities. We have described the first HCH mouse model (Fgfr3 N534Lys / + ) was created. Fgfr3 A385E / + Mice exhibited mild dwarfism, absence of craniosynostosis and normal craniocerebral ratios, and memory impairment.

[0004] However, the effects of gain-of-function mutations in FGFR3 on cognitive behavior have never been investigated.

[0005] Summary of the invention: The present invention is defined by the claims. In particular, the present invention relates to methods and compositions for the treatment of FGFR3-associated cognitive disorders.

[0006] Detailed description of the invention: A gain-of-function mutation (p.Ala391Glu) in fibroblast growth factor receptor 3 (FGFR3) is responsible for a rare form of craniosynostosis: Crouzon syndrome with acanthosis nigricans (CAN). CAN patients are characterized by premature synostosis of the coronal sutures of the skull, midface hypoplasia, acanthosis nigricans, and neurological disorders. FGFR3 has been defined as a negative regulator of long bone growth. However, the role of the CAN mutation p.Ala391Glu in the biological function of cranial sutural growth remains to be explored. We observed that the CAN mutation induced receptor hyperactivation and perturbed protein maturation independently of the ligand. We have demonstrated the first CAN mouse model expressing a dominant p.Ala385Glu mutation (Fgfr3 A385E / + ), and a HCH mouse model expressing the dominant p.Asn540Lys mutation (Fgfr3 N534Lys / + ) was created. Fgfr3 A385E / + Mice showed an absence of craniosynostosis and normal craniocerebral ratios. However, by analyzing the CAN and hippocampus of these hypochondroplasia adult mice, the inventors showed that FGFR3 hyperactivation was associated with reduced proliferation and neurogenesis of progenitor cells in the dentate gyrus. Consequently, behavioral tests demonstrated that Fgfr3 A385E / + This was carried out in mice and hippocampal-dependent memory impairment and abnormal coping strategies were observed. Finally, using catechin, the inventors demonstrated that Fgfr3 A385E / + Inhibits overactivation of FGFR3 in mouse brain, thereby inhibiting Fgfr3 A385E / + The mice recovered from behavioral and cognitive deficits, highlighting the first behavioral abnormalities associated with Fgfr3 hyperactivation in the brain, which allows for a better understanding of the role played by FGFR3 in learning processes and emotional responses in craniosynostosis and chondrodysplasia.

[0007] Therefore, a first object of the present invention relates to a method for treating FGFR3-associated cognitive disorders in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one catechin.

[0008] As used herein, the term "subject" or "patient" refers to a subject who is or will be suffering from a cognitive disorder associated with FGFR3. In some embodiments, the patient is a child. In some embodiments, the patient is a premature infant. In some embodiments, the patient is under 15 years of age. In some embodiments, the patient is under 10 years of age. In some embodiments, the patient is under 7 years of age. In some embodiments, the patient is under 5 years of age. In some embodiments, the patient is under 3 years of age. In some embodiments, the patient is an adult. In some embodiments, the subject is over 15 years of age. In some embodiments, the subject is over 20 years of age. In some embodiments, the subject is over 25 years of age. In some embodiments, the subject is over 30 years of age. In some embodiments, the subject is over 35 years of age. In some embodiments, the patient is elderly.

[0009] In some embodiments, the subject has a constitutively active FGFR3 receptor mutant, hi some embodiments, the subject has an FGFR3 gain-of-function mutation.

[0010] The term "fibroblast growth factors" (FGFs) as used herein refers to a family of cell signaling proteins produced by macrophages; they are involved in a wide variety of processes, most notably as essential components for normal development in animal cells. Irregularities in their function lead to a range of developmental abnormalities. These growth factors typically act as systemically or locally circulating molecules of extracellular origin that activate cell surface receptors. A defining property of FGFs is that they bind to heparin and heparin sulfate. Thus, some are trapped in the extracellular matrix of tissues containing heparin sulfate proteoglycans and are released locally upon injury or tissue remodeling.

[0011] The term "fibroblast growth factor receptor" (FGFR) as used herein relates to receptors that bind members of the fibroblast growth factor (FGF) protein family. Some of these receptors are involved in pathological conditions. Distinct membrane-based FGFRs have been identified in vertebrates, all of which belong to the tyrosine kinase superfamily: FGFR1 (see also fibroblast growth factor receptor 1) (=CD331), FGFR2 (see also fibroblast growth factor receptor 2) (=CD332), FGFR3 (see also fibroblast growth factor receptor 3) (=CD333), FGFR4 (see also fibroblast growth factor receptor 4) (=CD334), FGFRL1 (see also fibroblast growth factor receptor-like 1) and FGFR6.

[0012] As used herein, the terms "FGFR3", "FGFR3 tyrosine kinase receptor" and "FGFR3 receptor" are used synonymously throughout the specification and refer to all naturally occurring isoforms of FGFR3. An exemplary human amino acid sequence of FGFR3 is provided by SEQ ID NO:1. [ka]

[0013] As used herein, the terms "FGFR3 gain-of-function mutation," "constitutively active FGFR3 receptor mutant," "constitutively active FGFR3 mutant," or "FGFR3 mutant exhibiting constitutive activity" are used synonymously and refer to a mutant of the receptor that exhibits biological activity (i.e., triggers downstream signaling) and / or exhibits greater biological activity in the presence of an FGF ligand than the biological activity of the corresponding wild-type receptor. Constitutively active FGFR3 mutants according to the invention are in particular (residues numbered according to their position in the precursor of fibroblast growth factor receptor 3 isoform 1 (806 amino acids long)): a mutant in which the serine residue at position 84 has been replaced by a lysine (hereinafter designated S84L); a mutant in which the arginine residue at position 200 has been replaced by a cysteine ​​(hereinafter designated R200C); a mutant in which the arginine residue at position 248 has been replaced by a cysteine ​​(hereinafter designated R248C); a mutant in which the serine residue at position 249 has been replaced by a cysteine ​​(hereinafter designated S249C); a mutant in which the proline residue at position 250 has been replaced by an arginine (hereinafter designated P250R); an asparagine residue at position 262 has been replaced by an arginine (hereinafter designated P250R); a mutant in which the residue is replaced by a histidine (hereinafter designated N262H); a mutant in which the glycine residue at position 268 is replaced by a cysteine ​​(hereinafter designated G268C); a mutant in which the tyrosine residue at position 278 is replaced by a cysteine ​​(hereinafter designated Y278C); a mutant in which the serine residue at position 279 is replaced by a cysteine ​​(hereinafter designated S279C); a mutant in which the glycine residue at position 370 is replaced by a cysteine ​​(hereinafter designated G370C); a mutant in which the serine residue at position 371 is replaced by a cysteine ​​(hereinafter designated S371C); a mutant in which the tyrosine residue at position 373 is replaced by a cysteine ​​(hereinafter designated Y373C);Mutants in which the glycine residue at position 380 has been replaced by arginine (hereinafter designated G380R); mutants in which the valine residue at position 381 has been replaced by glutamate (hereinafter designated V381E); mutants in which the alanine residue at position 391 has been replaced by glutamate (hereinafter designated A391E); mutants in which the asparagine residue at position 540 has been replaced by lysine (hereinafter designated N540K); mutants in which the stop codon has been eliminated due to a base substitution, in particular mutants in which the stop codon has been mutated in the codon for arginine, cysteine, glycine, serine or tryptophan (hereinafter designated X807R, X807C, X807G, X807S and X807R, respectively). a mutant in which the lysine residue at position 650 is replaced by another residue, in particular methionine, glutamate, asparagine or glutamine (hereinafter designated K650M, K650E, K650N and K650Q); a mutant in which the methionine residue at position 528 is replaced by isoleucine (hereinafter designated M528I); a mutant in which the isoleucine residue at position 538 is replaced by valine (hereinafter designated I538V); a mutant in which the asparagine residue at position 540 is replaced by serine (hereinafter designated N540S); a mutant in which the asparagine residue at position 540 is replaced by threonine (hereinafter designated N540T). Typically, the constitutively active FGFR3 mutant according to the present invention is an N540K, K650N, K650Q, M528I, I538V, N540S, N540T or A391E mutant.

[0014] The term "cognitive disorder" as used herein refers to a set of symptoms, including depression, memory, perception, slowness and intractable problems.Cognitive disorders may be present as symptoms in some mental disorders (psychosis, mood disorders, anxiety), but they are mainly synonymous with brain damage.The term "FGFR3-related cognitive disorder" as used herein is intended to mean cognitive disorder caused by abnormal overactivation of FGFR3 in the brain, particularly by the expression of constitutively active mutants of FGFR3 receptors, particularly the constitutively active mutants of FGFR3 receptors as described above.

[0015] In some embodiments, the subject has or will have a cognitive and behavioral disorder selected from the group consisting of episodic memory impairment, antidepressant effects, abnormalities in learning, and stress response.

[0016] In particular, constitutively active FGFR3 receptor mutants induce brain morphological abnormalities, learning and memory impairments, and / or reduced neurogenesis. The term "neurogenesis" as used herein has its general meaning in the art and relates to the process by which new neurons are formed in the brain. Neurogenesis is important during embryo development, but 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 connections. For example, the hippocampus, a brain region that plays an important role in memory and spatial navigation, alone has at least 27 different types of neurons. The incredible diversity of neurons in the brain results from regulated neurogenesis during embryonic development. During the process, neural stem cells differentiate, i.e., they become any one of many specialized cell types at specific times and regions in the brain.

[0017] In some embodiments, the subject has an FGFR3-associated skeletal disease.

[0018] The term "FGFR3-associated skeletal disease" as used herein is intended to mean a skeletal disease caused by abnormally increased activation of FGFR3, in particular by expression of a mutant constitutively active FGFR3 receptor, in particular a mutant constitutively active FGFR3 receptor as described above.

[0019] In some embodiments, the FGFR3-related skeletal disease is preferably FGFR3-related chondrodysplasia and FGFR3-related craniosynostosis.

[0020] 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).

[0021] In particular, the FGFR3-related skeletal disease is short stature.

[0022] As used herein, the term "dwarfism" has its ordinary meaning in the art and refers to short stature resulting from genetic or medical conditions. Dwarfism is generally defined as an adult height of 147 cm or less.

[0023] In particular, an FGFR3-associated skeletal disease is hypochondroplasia (HCH).

[0024] As used herein, the term "hypoplasia achondroplasia" (HCH) has its common meaning in the art and refers to disproportionately short sutures, shortened limbs, and a head that appears large compared to the underdeveloped parts of the body, and mild to moderate intellectual disability.

[0025] In some embodiments, the FGFR3-associated chondrodysplasia is hypochondroplasia caused by expression of a constitutively active FGFR3 receptor mutant of N540K, K650N, K650Q, M528I, I538V, N540S or N540T.

[0026] In particular, an FGFR3-related skeletal disease is achondroplasia (ACH).

[0027] As used herein, the term "achondroplasia" (ACH) has its common meaning in the art and relates to a genetic defect in which the hands and feet are short but the torso is typically of normal length, with an enlarged head and prominent forehead.

[0028] In particular, the FGFR3-related skeletal disease is thanatophoric dysplasia (TD).

[0029] 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, very short limbs, and excess skin folds on the limbs.

[0030] 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.

[0031] In particular, FGFR3-associated craniosynostosis is Muenke syndrome, which is caused by expression of a constitutively active mutant form of the FGFR3 receptor, P250R.

[0032] In particular, FGFR3-associated craniosynostosis is Crouzon syndrome with acanthosis nigricans (CAN), which is caused by expression of a constitutively active A391E mutant of the FGFR3 receptor.

[0033] As used herein, the term "craniosynostosis" has its common meaning in the art and relates to a condition in which one or more of the fibrous sutures in a patient's skull fuse prematurely by turning into bone (ossification), thereby altering the growth pattern of the skull. "Crouzon syndrome with acanthosis nigricans" (CAN) is a very rare form of craniosynostosis.

[0034] The term "acanthosis nigricans" as used herein refers to brown to black, poorly defined, velvety textured pigmentation of the skin.

[0035] The term "treatment" or "treating" as used herein refers to both prophylactic or preventative treatments, as well as curative and improving a patient's condition or disease-modifying treatments (including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition), including the suppression of clinical recurrence. Treatments may be administered to patients with a medical disorder or who may eventually develop a disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or to extend the patient's survival beyond the expected survival in the absence of such treatment. By "therapeutic regimen" is meant a pattern of treatment of a disease, such as a dosing pattern used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a 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. An induction regimen may (partially or entirely) use 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 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) that is used for the maintenance of a patient during disease treatment, for example, to keep the patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous therapy (e.g., administering a drug at regular intervals, such as once a day, once a week, once a month, once a year, etc.) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon reaching a certain predetermined criterion (e.g., symptoms of disease, etc.).

[0036] The term "preventing" as used herein is intended to characterize a prophylactic method or process, the purpose of which is to delay or prevent the onset of the disorder or condition to which such term applies.

[0037] The term "expression" when used in the context of gene or nucleic acid expression refers to the conversion of the information contained in a gene into a gene product. A gene product may 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 produced by translation of an mRNA (i.e., FGFR3).

[0038] As used herein, the term "catechin" refers to a compound having the general formula (I): [ka] Flavan-3-ol is a type of natural phenol and antioxidant.

[0039] Catechin is a secondary metabolite of plants and belongs to the group of flavan-3-ols (or simply flavanols), which are part of the flavonoid chemical family. Catechin has two benzene rings (called A-ring and B-ring) and a dihydropyran heterocycle (C-ring) with a hydroxyl group on C3. The A-ring resembles the resorcinol moiety, while the B-ring resembles the catechol moiety. There are two asymmetric centers on C2 and 3 of the molecule. Catechin therefore has four diastereoisomers: two of the isomers are in the trans configuration and are called catechin, and the other two are in the cis configuration and are called epicatechin. The most common catechin isomer is (+)-catechin. The other stereoisomers are (-)-catechin or ent-catechin. The most common epicatechin isomer is (-)-epicatechin (also known as L-epicatechin, epicatechol, (-)-epicatechol, 1-acacatechin, 1-epicatechol, epi-catechin, 2,3-cis-epicatechin, or (2R,3R)-(-)-epicatechin). Methods for producing, synthesizing, or extracting catechin are well known to those of skill in the art.

[0040] In some embodiments, the catechin isomer is (+)-catechin (2R,3S). As used herein, the term "(+)-catechin" has its ordinary meaning in the art and is represented by the following formula: [ka] has.

[0041] In some embodiments, the catechin isomer is (-)-catechin (2S,3R). As used herein, the term "(-)-catechin" has its ordinary meaning in the art and is represented by the following formula: [ka] has.

[0042] In some embodiments, the epicatechin isomer is (+)-epicatechin (2R,3S). As used herein, the term "(+)-epicatechin" has its ordinary meaning in the art and is represented by the following formula: [ka] has.

[0043] In some embodiments, the epicatechin isomer is (-)-epicatechin (2R,3R). As used herein, the term "(-)-epicatechin" has its general meaning in the art and refers to (2R,3R)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol and has the following formula: [ka] It has.

[0044] In some embodiments, the (-)-epicatechin is substantially pure (-)-epicatechin. The term "substantially pure" as used herein refers to the total absence or near complete absence of impurities, such as related substance impurities. For example, when a (-)-epicatechin composition is said to be substantially pure, there is no detectable related substance impurity, or if a single related substance impurity is detected, it is present in an amount of 0.1% by weight or less, or if multiple related substance impurities are detected, they are present in aggregates in an amount of 0.6% by weight or less. The substantially pure (-)-epicatechin according to the present invention can inhibit or eliminate the functional activation of downstream signaling pathways of FGFR3 in vivo and / or in vitro. Substantially pure (-)-epicatechin may inhibit functional activation of downstream signaling pathways of FGFR3 by at least about 10%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably at least about 70, 75 or 80%, more preferably 85, 90, 95 or 100%, or preferably at least about 300% or preferably at least about 500%.

[0045] In some embodiments, the subject is administered a pharmaceutical composition comprising a therapeutically effective amount of a catechin.

[0046] The term "administering" or "administration" as used herein refers to the act of delivering a substance (as it exists outside the body) (e.g., catechin) to a patient, for example, by oral delivery (e.g., via bottle treatment), intracerebroventricular, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When a disease or a symptom thereof is treated, administration of the substance is typically performed after the onset of the disease or its symptoms. When a disease or a symptom thereof is prevented, administration of the substance is typically performed before the onset of the disease or its symptoms.

[0047] According to the present invention, catechin is administered to a patient using any suitable method that allows catechin to reach the brain. In some embodiments, catechin is administered to a patient orally (i.e., via bottle treatment) or systemically (i.e., via systemic administration). Thus, in some embodiments, catechin is administered to a patient so that it enters the circulatory system and is distributed throughout the body. In some embodiments, catechin is administered to a patient by local administration, for example, by local administration to the hypothalamus.

[0048] "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 catechin may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of catechin to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of catechin are outweighed by the therapeutically beneficial effects. The effective dose and dosage regimen for a drug depends on the disease or condition to be treated and can be determined by one of ordinary skill in the art. A physician of ordinary skill in the art can determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician can start the dose of the drug used in the pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. In general, the appropriate dose of the composition of the present invention will be the amount of the compound that is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend 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. Those skilled in the art will be able to determine such amounts based on such factors as the patient's size, the severity of the patient's symptoms, and the specific composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of catechin is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, such as about 0.1-20 mg / kg, such as about 0.1 mg-10 mg / kg, such as about 0.5, such as about 0.3, about 1, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg. The dosage regimen in the above methods of treatment and use is 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 the treatment is monitored during the treatment, for example, at predetermined time points.As a non-limiting example, the treatment according to the present invention may be carried out in an amount of about 0.1 to 100 mg / kg per day, e.g., 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, 1, 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, or 1, 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 1 A single or divided dose may be provided every 24, 12, 8, 6, 4 or 2 hours, or any combination thereof, on at least one day of the 5th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th or 40th day, or alternatively for at least one week of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th or 20th week, or any combination thereof.

[0049] In some embodiments, the patient is administered a pharmaceutical composition comprising a therapeutically effective amount of catechin as an active pharmaceutical ingredient and at least one pharma- ceutically acceptable excipient.

[0050] As used herein, the terms "active drug substance" or "active ingredient" are used synonymously. As used herein, the term "pharmaceutical composition" refers to a composition described herein or a pharma- ceutically acceptable salt thereof, which comprises other substances, such as carriers and / or excipients. Pharmaceutical compositions as provided herein typically include a pharma- ceutically acceptable carrier.

[0051] As used herein, the term "pharmaceutical acceptable carrier" includes any and all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, 16th Edition, E. W. Martin (Mack Publishing, Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation.

[0052] A pharma- ceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulant material, or formulation auxiliary. Typically, pharmaceutical compositions contain vehicles that are pharma- ceutically acceptable for injectable formulations. These may be, in particular, isotonic, sterile saline solutions (such as monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or mixtures of such salts), or dried, in particular lyophilized, compositions that allow reconstitution of an injectable solution upon addition of sterile water or saline, as the case may be. Pharmaceutical dosage forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Sterile injectable solutions are prepared by incorporating the agent of the present invention in the required amount in a suitable solvent together with some other ingredients as listed above as required, followed by sterilization filtration.Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other ingredients as listed above as required.In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of active ingredient and any additional desired ingredient from its solution that has been previously sterilized and filtered.

[0053] In some embodiments, the subject is administered a food composition comprising a therapeutically effective amount of catechins.

[0054] Typically, the food composition comprises or consists of a food product, food additive, food ingredient, functional food, medical food, dietary supplement, nutraceutical, nutritional supplement, or oral preparation comprising an effective amount of catechin. In some embodiments, the food composition is selected from a complete food composition, a food supplement, a nutraceutical composition, and the like. The composition of the present invention may be used as a food ingredient and / or a feed ingredient. The food ingredient may be in a liquid or solid dosage form depending on the use and / or the mode of application and / or the mode of administration. The food composition of the present invention may be prepared using known methods, such as adding an effective amount of catechin to a food base. The composition comprising the food product, food additive, food ingredient, functional food, medical food, dietary supplement, nutraceutical, nutritional supplement, or oral preparation of the present invention may be solid, semi-solid, or liquid. It may be in the form of a food product or food supplement, such as a tablet, gel, powder, capsule, drink, bar, bottle treatment, and the like. For example, the composition may be in the form of a powder packaged in a sachet, which can be dissolved in water, fruit juice, milk, or another beverage.

[0055] The term "food" as used herein refers to liquid (i.e. drink), solid, or semi-solid dietary compositions, especially compositions that are entirely food (food substitutes), that do not require additional nutritional intake or food supplement compositions. Food supplement compositions do not completely replace the intake of nutrients by other means. The terms "food product", "food additive", "food ingredient" or "feed ingredient" as used herein include preparations that are functional foods or foodstuffs or can be added as nutritional supplements to functional foods or foodstuffs. By "nutritional food" or "nutraceutical" or "functional food" we mean foodstuffs that contain ingredients that have a beneficial effect on health or can improve physiological functions. By "food supplement" we mean foodstuffs that have the purpose of completing a normal food diet. Food supplements are concentrated sources of nutrients or other substances that have nutritional or physiological effects when they are taken alone or in combination in small amounts. The term "functional food" as used herein summarizes foodstuffs and the corresponding products that have been recently developed, the importance of which is due to their value not only for nutrition and taste, but also for the substances of certain components. According to the invention, the medium or long-term maintenance and promotion of health is important. In this respect, non-therapeutic applications are preferred. However, the preventive aspects and promotion of health, as well as the dietary character of the product, are best revealed by the term functional foods. In many cases, these concern products that are increasingly accumulated through assortment and selection (as is the case in the present invention), purification, concentration, additions. They do not include isolated active substances, especially in tablet or pill form. Although there is no legal definition of functional foods, the majority of patients interested in this area agree that they are foods that are marketed as having a specific health effect beyond the basic nutritional effect. Thus, functional foods are ordinary foods that incorporate into them constituents or ingredients (such as those described herein) that confer a specific function on the food, for example a medical or physiological benefit other than a purely nutritional effect.

[0056] In some embodiments, the food composition is a beverage, which is a functional or therapeutic beverage, a thirst quenching beverage, or a normal beverage.For example, the composition of the present invention can be used in soft drinks, fruit juices, or beverages containing whey protein as a component, health teas, cocoa beverages, milk beverages, and lactobacillus beverages, yogurt and drinking yogurt, cheese, ice cream, shaved ice, and desserts, confectionery, biscuits, cakes, and cake mixes, snack foods, balanced foods and beverages, fruit fillings, care glazes, chocolate bread fillings, cheesecake flavored fillings, fruit flavored cake fillings, cake and donut icings, instant bread filling creams, fillings for cookies, ready-to-use bread fillings, reduced calorie fillings, adult nutritional beverages, acidified soy / juice beverages, aseptic / retort chocolate beverages, bar mixes, powdered beverages, calcium-fortified soy / plain and chocolate milk, calcium-fortified coffee beverages.

[0057] The food composition, including the food product, food additive, food ingredient, functional food, medical food, dietary supplement, nutraceutical, nutritional supplement, or oral preparation of the present invention, typically includes a carrier or vehicle. "Carrier" or "vehicle" refers to a substance suitable for administration, including any such substance known in the art, such as any liquid, gel, solvent, liquid diluent, solubilizer, etc. that is non-toxic and does not adversely interact with any of the components of the composition. Examples of nutritionally acceptable carriers include, for example, water, salt solution, alcohol, silicone, wax, yellow petrolatum, vegetable oil, polyethylene glycol, propylene glycol, liposome, sugar, gelatin, lactose, amylose, magnesium stearate, talc, surfactant, silicic acid, viscous paraffin, flavor oil, fatty acid monoglyceride and diglyceride, petroethral fatty acid ester, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc. In some embodiments, the compositions comprising the food products, food additives, food ingredients, functional foods, medical foods, dietary supplements, nutraceuticals, nutritional supplements, or oral preparations of the present invention contain protective hydrocolloids (e.g., gums, proteins, modified starches), binders, film formers, encapsulating agents / materials, wall / shell materials, matrix compounds, coating agents, emulsifiers, surfactants, solubilizing agents (oils, fats, waxes, lecithin, etc.), adsorbents, carriers, fillers, co-complexes, dispersing agents, wetting agents, processing aids (solvents), flow agents, taste masking agents, bulking agents, gelling agents, gel forming agents, antioxidants, and antimicrobial agents. The compositions may also contain conventional pharmaceutical additives and adjuvants, excipients and diluents, including, but not limited to, water, gelatin of any origin, vegetable gums, lignin sulfonates, talc, sugars, starches, gum arabic, vegetable oils, polyalkylene glycols, flavorings, preservatives, stabilizers, emulsifiers, buffers, lubricants, coloring agents, wetting agents, fillers, etc. In all cases such additional ingredients will be selected with regard to their suitability for the intended recipient.

[0058] The present invention will be further illustrated by the following figures and examples, which should not, however, be construed as limiting the scope of the present invention in any way. [Brief description of the drawings]

[0059] [Figure 1A] Learning impairment and antidepressant behavior in a hypochondroplasia (HCH) mouse model. Forced swimming test (FST) performed in 4-month-old male animals in two independent groups. FST was performed on two consecutive days. Immobility periods were assessed for both days 1 and 2 (Fgfr3+ / +, n=15; Fgfr3N534K / +, n=9, FgfrN534K / ++(-)-epicatechin, n=10). [Figure 1B] Learning impairment and antidepressant behavior in a hypochondroplasia (HCH) mouse model. The tail suspension test (TST) was performed on two consecutive days in 4-month-old male animals and immobility periods were assessed for both (Fgfr3+ / +, n=15; FgfrN534K / +, n=9; FgfrN534K / ++(-)-epicatechin, n=10). [Figure 2A] Learning impairment is reversed by a single daily subcutaneous injection of (-)-epicatechin in a hypochondroplasia (HCH) mouse model. Novel object location recognition (NOL) was performed in 4-month-old male animals. After 24 hours of the training period, the discrimination index was measured to evaluate the memory performance of FgfrN534K / + and their control littermates. [Figure 2B] Learning impairment is reversed by a single daily subcutaneous injection of (-)-epicatechin in a mouse model of hypochondroplasia (HCH). Novel object recognition (NOR) was performed in 4-month-old male animals. Twenty-four hours after the training period, discrimination index was measured to evaluate the memory performance of FgfrN534K / ++(-)-epicatechin, Fgfr3N534K / + and their control littermates. NOR and NOL were performed on FgfrN534K / + (n=9), Fgfr3N534K / ++(-)-epicatechin (n=10) and their controls. [Figure 3A]Learning impairment and antidepressant behavior in acanthosis nigricans-Crouzon syndrome (CAN) mouse model. Forced swim test (FST) performed in 4-month-old male animals in two independent groups. FST was performed on two consecutive days. Immobility periods were assessed for both days 1 and 2 (Fgfr3+ / +, n=9; Fgfr3A385E / +, n=4; Fgfr3A385E / ++(-)-epicatechin, n=5). [Figure 3B] Learning impairment and antidepressant behavior in acanthosis nigricans-Crouzon syndrome (CAN) mouse model. The tail suspension test (TST) was performed on two consecutive days in 4-month-old male animals and immobility periods were assessed for both (Fgfr3+ / +, n=9; Fgfr3A385E / +n=4, FgfrA385E / ++(-)-epicatechin, n=5). [Figure 4A] Learning impairment is reversed by a single daily subcutaneous injection of (-)-epicatechin in acanthosis nigricans-Crouzon syndrome (CAN) mouse model. Novel object location recognition (NOL) was performed in 4-month-old male animals. After 24 hours of the training period, discrimination indices were measured to assess memory performance in Fgfr3A385E / + and their control littermates. [Figure 4B] Learning impairment is reversed by a single daily subcutaneous injection of (-)-epicatechin in acanthosis nigricans-Crouzon syndrome (CAN) mouse model. Novel object recognition (NOR) was performed in 4-month-old male animals. Twenty-four hours after the training period, discrimination index was measured to evaluate the memory performance of Fgfr3A385E / ++(-)-epicatechin, Fgfr3A385E / + and their control littermates. NOR and NOL were performed for Fgfr3+ / +, n=9; Fgfr3A385E / +, n=4, Fgfr3A385E / ++(-)-epicatechin, n=5. [Figure 5A] Learning impairment is reversed by once-daily bottle treatment of (-)-epicatechin in a hypochondroplasia (HCH) mouse model.Tail suspension test (TST) was assessed in Fgfr3+ / +, n=15; FgfrN534K / +, n=12, FgfrN534K / ++(-)-epicatechin, n=13. [Figure 5B] Learning impairment is reversed by once-daily bottle-fed treatment of (-)-epicatechin in a hypochondroplasia (HCH) mouse model. The forced swim test (FST) was assessed in Fgfr3+ / +, n=15; Fgfr3N534K / +, n=12, Fgfr3N534K / ++(-)-epicatechin, n=13. [Figure 5C] Learning impairment is reversed by once-daily bottle-dose treatment of (-)-epicatechin in a hypochondroplasia (HCH) mouse model. Spatial memory (NOL) was assessed in Fgfr3+ / +, n=15; Fgfr3N534K / +, n=12, Fgfr3N534K / ++(-)-epicatechin, n=13. [Figure 5D] Learning impairment is reversed by once-daily bottle-dose treatment of hypochondroplasia (HCH) mouse models with (-)-epicatechin.Episodic memory (NOR) was assessed in Fgfr3+ / +, n=15; Fgfr3N534K / +, n=12, Fgfr3N534K / ++(-)-epicatechin, n=13. [Figure 5E] Learning impairment is reversed by once-daily bottle-fed treatment of (-)-epicatechin in a hypochondroplasia (HCH) mouse model. Spatial memory (MWM) was assessed in Fgfr3+ / +, n=9; Fgfr3N534K / +, n=7; Fgfr3N534K / ++(-)-epicatechin, n=8). [Figure 5F] Learning impairment is reversed by once-daily bottle treatment of (-)-epicatechin in a hypochondroplasia (HCH) mouse model. Barbeam: Locomotor activity and fine coordination were assessed in Fgfr3+ / +, n=14; FgfrN534K / +, n=11; FgfrN534K / ++(-)-epicatechin, n=11. [Figure 5G] Learning impairment is reversed by once-daily bottle treatment of (-)-epicatechin in a hypochondroplasia (HCH) mouse model. Barbeam: Locomotor activity and fine coordination were assessed in Fgfr3+ / +, n=14; FgfrN534K / +, n=11; FgfrN534K / ++(-)-epicatechin, n=11. [Figure 5H] Learning impairment is reversed by once-daily bottle-fed treatment of hypochondroplasia (HCH) mouse models with (-)-epicatechin. Associative memory (CFC) was assessed in Fgfr3+ / +, n=14; Fgfr3N534K / +, n=10, Fgfr3N534K / ++(-)-epicatechin, n=12

[0060] Definition: As used herein, "FGFR3 A385E / + The term relates to the CAN mouse model, in which memory impairment was observed.

[0061] As used herein, "FGFR3 N534K / + The term "hypoplasia" refers to a mouse model of hypochondroplasia. Mutant mice display clinical features of hypochondroplasia, including growth retardation, growth plate abnormalities, partial loss of synchondros, and lordosis.

[0062] As used herein, "FGFR3 Y367C / + The term relates to a mouse model that recapitulates the human achondroplasia (ACH) phenotype and clinical features of achondroplasia, such as short stature associated with a reduced size of the foramen magnum, mandibular hypoplasia, hearing loss, intervertebral disc abnormalities, and impaired ciliogenesis (Pannier et al. 2009, 2010, Mugniery et al. 2012, Di Rocco et al. 2014, Biosse Duplan et al. 2016, Komla Ebri et al. 2016, Martin et al. 2018).

[0063] Example 1: Methods All procedures were approved by the French Animal Experimentation Committee. Genomic DNA was isolated from the tail using the NucleoSpin Tissue kit (Machler-Nagel) according to the manufacturer's instructions. Mice were genotyped using the following primers for wild-type and mutant mouse isolates: 5'-GTGGGGGTTCTGCGGTTGG-3' (SEQ ID NO: 2) and 5'-TGACAGGCTTGGCAGTACGG-3' (SEQ ID NO: 3). For all analyses, wild-type littermates were used as controls.

[0064] Brain Magnetic Resonance Nuclear magnetic resonance imaging of mouse brains was performed at a resolution of 100 μm using a 4.7-T small animal nuclear magnetic resonance instrument (Biospec 47 / 40; Bruker, Billerica, MA, USA) on a small animal imaging platform (Sorbonne-Université Paris-City Medical School, Paris, France). A385E / + Mice and 5 Fgfr3 N534K / + Male mice and five 4-month-old control littermates were anesthetized with inhalation of isoflurane gas during imaging. 3D reconstructions and measurements were performed using Imaris (Bitplane).

[0065] Surgery and medical treatment Fgfr3 at 4 months of age N534K / + Mouse and Fgfr3 A385E / + Mice received subcutaneous administration of (-)-epicatechin (0.15 mg / kg) or vehicle (HCl 3.5 mM, DMSO 2%) once a day for at least 7 days. For habituation and NOR and NOL testing, injections were performed 1 h before the test period. Injection times for the different tests: NOL on day 7, NOR on day 14, TST on day 22, and FST on day 24.

[0066] Schematic of novel object recognition (NOR) We used a modified NOR task as described in Glatigny et al., 2019. Mice were transported a short distance from the mouse housing facility to the testing room in their home cages and left undisturbed for at least 1 h before the start of the test. The testing room was illuminated with two 60 W incandescent light bulbs and behavioral activity periods were recorded with a camera above the testing arena (gray plastic box (60 × 40 × 32 cm)). Mice could not contact or see each other during exposure. The light intensity was equal in all parts of the arena (approximately 20 lux). Two different objects were used, available in triplicates. The objects were a blue ceramic pot (diameter 6.5 cm, maximum height 7.5 cm) and a transparent plastic funnel (diameter 8.5 cm, maximum height 8.5 cm). The objects serving as novel objects and as object left / right localization were counterbalanced within each group. The objects elicited a comparable level of exploration as determined in pilot experiments and training periods. Between exposures, mice were housed individually in standard cages, objects and activity areas were cleaned with phagosphore, and bedding was changed.

[0067] The NOR schematic consists of three periods (over 3 days): habituation, training, and testing. Mice were always placed in the center of the arena at the beginning of each exposure. Day 1: During the habituation period, mice were given 5 min to explore the arena without any objects and were then returned to their home cage. During the training period on day 2, mice were allowed 10 min to explore two identical objects aligned in symmetrically opposite positions from the center of the arena and were then transferred to their home cage. During the testing period on day 3, mice were given 15 min to explore two objects (old and new) in the same arena while maintaining the same object positions.

[0068] The following behaviors were judged as object exploration: sniffing, licking, or touching the object with the nose or forepaws, or pointing the nose to the object at a distance of 1 cm or less. No exploration was scored if the mouse was on top of the object or completely immobile. The discrimination index was calculated as (time spent exploring the new object - time spent exploring the existing object) / (total time spent exploring both objects). As a control, the orientation index for the (right / left) object location or object A vs. B during the training period of novel object recognition (NOR) was measured in all groups of mice exposed to the test. We confirm here that no initial preference for any exposed object (A or B) or any direction (right / left) was observed in any group. Locomotor activity was assessed for each mouse. Behavior was video-scored by two observers blinded to treatment, and the total exploration time of the object was quantified during the test period.

[0069] Novel object location recognition (NOL) For the novel object location recognition task, all procedures were identical to the novel object recognition task, except that during the testing period, rather than presenting a novel object, mice encountered both existing objects (one object located at a different location within the arena). The time and frequency of exploration of the novel / relocated object are measured as an index of memory. Behavior was video-scored by two observers blinded to treatment, and the total time of exploring the objects during the testing period was quantified.

[0070] Light / dark transition test (D / LT) The test is based on rodents' innate aversion to brightly illuminated areas and their spontaneous exploratory behavior in response to the stressor represented by the 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 lux). Naïve mice were individually placed in the center of the dark area in the test chamber, facing away from the entrance to the illuminated compartment. Mice were tested for 10 min and two parameters were recorded: time spent in the illuminated compartment and number of movements between compartments, which are indicative of anxiety-related behavior and exploratory activity. Behavior was scored using an infrared light beam activity monitor using actiMot2 software (PhenoMaster software, TSE).

[0071] Open Field Test (OFT) This test exploits rodents' aversion to brightly lit areas. Each mouse was placed in the center of the OFT chamber (43 x 43 cm chamber) and allowed to explore for 30 min. Mice were monitored throughout each test session by an infrared light beam activity monitor using actiMot2 software (PhenoMaster Software, TSE). Overall locomotor activity was quantified as the total distance traveled (ambulatory activity). Anxiety was quantified by measuring the time and distance spent in the center vs. periphery of the open field chamber.

[0072] Tail suspension test (TST) This test is based on the observation that rodents, when placed in an unavoidable stressful condition, develop an immobile posture after an initial avoidance-type behavior. Each mouse is suspended by its tail in an uncontrollable manner 25 cm above the floor. The mouse is tested for 5 min and the time spent immobile is quantified.

[0073] Forced swim test (FST) This test is based on observations similar to those of the TST. Each mouse is placed in a cylinder (height: 25 cm, diameter: 10 cm) filled with water (23-25 ​​°C). The mouse is tested for 5 min and the time spent immobile (behavioral despair) is quantified.

[0074] Morris Water Maze The Morris Water Maze (MWM) is a spatial learning test for mice that relies on distant cues to determine the location of a submerged avoidance platform proceeding from a start location. Spatial learning is assessed over 8 days of repeated testing, and reference memory is determined by the location of the platform area.

[0075] Bar Beam The bar beam is a test to measure fine motor coordination and balance in mice by assessing whether the mice maintain an upright posture and walk across an elevated narrow beam to a safe platform. This test was performed on two consecutive days, and we measured the time it took the mice to cross the beam and the maximum distance they crossed.

[0076] Example 2: Hypochondroplasia model Clinical Features of Hypochondroplasia Existing literature indicates that people with hypochondroplasia are at increased risk for learning disabilities and mental retardation. Learning disabilities may be present in an estimated 50% of people and may not be noticeable until the child reaches school age. Cognitive impairments are present in 10-12% of children. There are multiple areas of possible structural brain abnormalities, including enlarged temporal lobes, malformation of the hippocampus, and maldevelopment of the temporal lobes (Bober MB et al., 2013., Linnankivi T et al., 2012; Philpott CM et al., 2013). Patients with hypochondroplasia exhibit maldevelopment of the brain lobes (Kannu et al. 2005) and abnormal hippocampal placement (Linnankivi et al. 2012). Furthermore, patients with hypochondroplasia exhibit learning disabilities, mild intellectual disability, global developmental delay, and occasional seizures and epilepsy (Linnankivi et al. 2012).

[0077] Fgfr3 N534K / + Mice exhibit abnormal brain morphology The present inventors developed a hypochondroplasia mouse model, Fgfr3, expressing the most common hypochondroplasia mutation, p.Asn540Lys, located in the FGFR3 tyrosine kinase I domain. Asn534Lys / + In the central nervous system, FGFR3 is highly expressed in the hippocampus, a brain structure essential for memory consolidation mechanisms. N534K / + We have described mice that exhibit cranial abnormalities with large skulls and premature fusion of the cranial base synchondroses. Brain abnormalities and developmental abnormalities of the cerebral lobes have been reported to be characteristic of Fgfr3 gain-of-function mutant mouse models. Therefore, we propose that Fgfr3 N534K / + We performed nuclear magnetic resonance and three-dimensional reconstructions of the brains of mice and their control littermates. We did not observe any significant abnormalities of the hippocampus (p=0.6905) and total brain volume (p=0.3810) in hypochondroplasia mice (data not shown). Nuclear magnetic resonance analysis showed alterations in brain shape compared to controls (data not shown).

[0078] Fgfr3 N534K / + Learning and memory impairments in mice Adult neurogenesis is known to play an important role in maintaining hippocampal memory function. Asn534Lys / + Mice and their control littermates were subjected to a battery of behavioral tests measuring spatial learning (novel object location recognition, NOL) and episodic learning (novel object recognition, NOR) and memory. In NOR, we found that hypochondroplasia mutant mice explored the novel object significantly less (p<<0.0001) than their control littermates during the test period (data not shown). The same impairment was observed when spatial memory was analyzed through the NOL test. Indeed, we found that Fgfr3 N534K / +We found that mice explored the relocated objects significantly less during the test period than control littermates (p<0.0001) (data not shown).

[0079] Stress and memory behavioral tests We performed a test related to hippocampal associative memory (one-trial contextual fear conditioning, CFC) on 4-month-old male mice. This test allows us to understand the ability of mice to recall events related to memory consolidation. During this behavioral test, we evaluated the freezing time of the mice, and the baseline of the mutant mice was similar to that of control littermates during the training period (p=0.2303). However, during the test day, Fgfr3 Asn534Lys / + Mice showed increased contextually induced freezing times compared to control mice (p=0.0466), indicating that conditioned fear memory was impaired in mutant mice (data not shown).

[0080] Antidepressant effects of FGFR3 The FGF pathway may be involved in depression. Therefore, we performed the tail suspension test (TST) and forced swimming test (FST) to evaluate the Fgfr3 N534K / + The present inventors conducted a study in mice (Figures 1A and 1B). N534K / + Mice were treated for 7 days. Animals that endured the short-term and unavoidable stress of being suspended by their tails or forced to swim would develop immobile postures characteristic of depression-related behaviors, which would be scored. By performing these tests, we demonstrated that Fgfr3 Asn534Lys / + They found that the mice exhibited a reduced immobile posture, suggesting that gain-of-function mutations in FGFR3 may have antidepressant effects.

[0081] Treatment with (-)-epicatechin inhibited Fgfr3 Y367C / + is sufficient to reverse the cognitive impairment observed in mice Subcutaneous injection of (-)-epicatechin improved the phenotype of Fgfr3 mice (Fgfr3 Y367C / + (De la Luz Cadiz-Gurrea M et al. 2019, Martin L et al. 2021). To confirm the effect of FGFR3 gain-of-function in learning disorders, the inventors used at least (-)-epicatechin to inhibit Fgfr3. N534K / + Mice were treated for 7 days. As a result, the inventors demonstrated that subcutaneous injection of (-)-epicatechin inhibited Fgfr N534K / + We found that FGFR3 was sufficient to restore the spatial (NOL) (Figure 2A) and episodic memory impairments (NOR) (Figure 2B) observed in mice. In fact, injected mutant mice were able to explore the rearranged objects (NOL) (p=0.9538) and novel objects (NOR) (p=0.8697) at the same level as control littermates (Figures 2A and 2B). Altogether, these results confirm the importance of FGFR3 in regulating learning and memory. The reversal of these behavioral abnormalities with (-)-epicatechin confirmed our hypothesis and supported that FGFR3 hyperactivation was involved in the cognitive phenotype of FGFR3-associated chondrodysplasia patients.

[0082] Example 3: CAN model Clinical features of Crouzon syndrome associated with acanthosis nigricans CAN syndrome associated with FGFR3 mutations shows a skeletal phenotype similar to Crouzon syndrome [MIM123500] caused by FGFR2 mutations (Coll et al., 2018, 2016; Di Rocco et al., 2011): ocular-orbital disproportion, mandibular prognathism, midface hypoplasia (data not shown), and brachycephaly secondary to premature synostosis of the coronal and sagittal sutures (to various degrees) (data not shown). Calvarial abnormalities cause mechanical pressure on the brain and increase the risk of increased cranial pressure (Al-Namnam et al., 2019) (data not shown). In addition, brain MRI revealed mild temporal abnormalities in three unrelated CAN patients. Affected cases presented with thickened parahippocampal sulci with altered angulation of the structures (data not shown). One case presenting with a cloverleaf-shaped skull could not be interpreted.

[0083] Skull base abnormalities in both FGFR2- and FGFR3-related Crouzon syndrome patients account for anterior to midface hypoplasia and posterior to the craniovertebral junction abnormalities. Premature fusion of the spheno-occipital synchondrosis is associated with a shortened skull base, whereas premature fusion of the intraoccipital synchondrosis is associated with a narrowed foramen magnum in patients with CAN (data not shown).

[0084] Skeletal phenotype in the CAN mouse model Fgfr3 A385E / + are mildly affected in To assess the effect of the CAN mutation on the skeleton, we generated a mouse model expressing the ubiquitous p.Ala385Glu missense mutation, which corresponds to the p.Ala391Glu human mutation (data not shown). FGFR3 p.Ala385Glu transcripts were detected in fibroblasts and osteoblasts of the parietal bone (data not shown). Consistent with the human disease, a distinct shortened limb phenotype was observed in Fgfr3 A385E / +No significant differences were observed in the prenatal and neonatal stages (data not shown) or adult stages (data not shown) of mice. Body weight, nose-anus length, and femur and tibia lengths were significantly increased in mice with Fgfr3 A385E / + Mouse and Fgfr3 + / + The results were similar in mice (data not shown). Normal femoral growth demonstrated a well-organized growth plate without hypertrophic zone abnormalities as revealed by staining for collagen type X. A385E / + This was confirmed by evaluation of cartilage in mice (data not shown). Both structural parameters were evaluated to confirm the absence of an abnormal phenotype. Microcomputed tomography images of femurs at 3 months of age revealed Fgfr3 A385E / + The mice were found to have normal cancellous and cortical bone structure (data not shown).

[0085] The craniofacial phenotype was assessed using skull microcomputed tomography. A385E / + The mice exhibited normal craniofacial features (data not shown); the coronal sutures and synchondrosis at the base of the skull were patent at postnatal day 21, similar to control mice (data not shown). A385E / + Mouse and Fgfr3 + / + Mouse landmark-based geometric morphometry (Heuze et al., 2010) showed no differences in skull shape (d=0.0148; p=0.0940). However, mandibular shape was significantly different between the two mouse groups (d=0.0159; P<0.01) (data not shown). To confirm the absence of premature closure of the frontal-parietal suture, we examined the parietal Fgfr3 expression levels. A385E / + The in vitro function of osteoblasts from mice was evaluated. Significant differences in mineralization capacity, proliferation, and mitogen-activated protein kinase (MAPK) activation were observed in mice with Fgfr3 compared to controls. A385E / +None were 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, as a consequence, did not affect craniofacial development (data not shown). These data support the conclusion that Fgfr3 A385E / + We describe the absence of a craniosynostosis phenotype in mice. Regarding the skin, there are no signs of hyperkeratosis, nor of epidermal thickening or alterations in pigmentation, in the Fgfr3 A385E / + It was not detected in mice (data not shown).

[0086] Fgfr3 A385E / + Mouse models show dentate gyrus has reduced neurogenesis Structural brain abnormalities have been described in 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 lobes. It is well known that premature fusion of cranial sutures alters the shape of the skull and impairs normal brain growth, causing functional problems such as elevated intracranial pressure, visual impairment, hearing loss, and cognitive impairment (Di Rocco et al., 2011). Previous studies have shown that Muenke syndrome patients presented with impaired adaptive and executive functions. The behavioral phenotype included deficits in working memory, attention deficit hyperactivity disorder, emotion regulation, and anxiety (Yarnell et al., 2015). These neurological disorders suggest the influence of FGFR3 in the brain on the control of cognitive functions. Therefore, we have demonstrated that Fgfr3 A385E / + Magnetic resonance imaging (MRI) was performed in mice to measure the volumes of various brain regions.

[0087] Fgfr3 A385E / +No volume alteration or any compression was observed in various brain regions of mice, suggesting normal embryonic brain development (data not shown). However, FGFs and FGFRs are known to be involved in the proliferation and differentiation of neural stem and 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 and 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 mouse brain A385E We hypothesized that the mutation may affect adult neurogenesis. The absence of craniofacial abnormalities provides a good opportunity to evaluate neurogenesis, especially during adulthood. To explore the role of Fgfr3 in adult neurogenesis, we examined the expression of Fgfr3 by immunofluorescence (data not shown) and Western blot (data not shown). A385E / + and Fgfr3 + / + We observed a similar expression of FGFR3 in the mouse hippocampus. However, the canonical MAPK pathway activated by FGFR3 has been found to be deregulated in both Fgfr knock-in (Komla-Ebri et al., 2016) and FGFR3 knock-out (Zhou et al., 2015) mouse models. Indeed, we observed a significantly increased expression of phosphorylated Erk1 / 2 in lysates of adult hippocampus (data not shown), thus supporting the regulation of Fgfr3 in the brain. A385 The mutation was confirmed to activate the MAPK pathway. FGFR3 plays an important role in the proliferation of progenitor cells in the dentate gyrus and in the differentiation of neural cells (Inglis-Broadgate et al., 2005; Kang and Hebert, 2015; Moldrich et al., 2011; Thomson et al., 2009). A385EUsing the NeuN marker, we showed that the mature neuronal area of ​​the granular layer of the dentate gyrus was significantly reduced in the hippocampus of mice compared to controls (data not shown). We assessed whether this reduced neuronal population was caused by reduced proliferation of progenitor cells. The number of cells positive for the cell cycle marker K167 was significantly increased by Fgfr3 A385E / + In mice, Fgfr3 expression was significantly decreased in the subgranular zone of the dentate gyrus (data not shown). In the subgranular zone, the rate of neuronal differentiation, as determined by doublecortin (DCX) immunolabeling, was slightly decreased (data not shown). Altogether, these data strongly suggested that the gain-of-function mutation in Fgfr3 mainly affects the proliferation and thus the maturation of neurons in the dentate gyrus.

[0088] Fgfr3 A385E / + Mouse models show impaired learning ability and antidepressant effects Reduced size of hippocampal structures and reduced proliferation have been shown to be associated with altered memory and cognition in humans and mice (Kitamura and Inokuchi, 2014). Therefore, we hypothesized that Fgfr3 A385E / + Mice and their control littermates were subjected to a battery 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) and spatial learning (Morris water maze, MWM) and memory (Figures 4A and 4B). In contextual fear conditioning, mutant mice did not show any difference in baseline freezing time. However, Fgfr3 gain-of-function mutations resulted in a decrease in contextually induced freezing time during the test period compared to their control littermates, indicating that fear conditioning memory is impaired in mutant mice (data not shown).

[0089] Next, we used a modified NOR conceptual scheme (Denny et al., 2012; Ennaceur and Delacour, 1988), which measures rodents' ability to recognize novel objects in the environment. Wild-type mice were able to distinguish novel objects from existing objects and tended to explore the novel objects for longer. As shown in Figures 4A and 4B, 4-month-old Fgfr3 mice A385E / + Mice explored significantly fewer novel objects than controls. However, no impairment was observed when memory was analyzed through the Morris water maze task, which assesses spatial learning and memory in rodents. Notably, Fgfr3 A385E / + Mice and controls performed equally in the open field test (OFT) and the light-dark paradigm (L / DT) (data not shown), indicating that their locomotor activity and anxiety state were unchanged.

[0090] Next, we evaluated the coping strategies against unavoidable stress using the forced swimming test (FST) and tail suspension test (TST). As shown in Fig. 3A, Fgfr3 mice at 4 months of age A385E / + The mutant mice spent significantly less time immobile during the forced swim test than did control mice. The same impairment was observed during the tail suspension test. Indeed, the mutant mice spent significantly less time immobile than their control littermates (Figure 3B).

[0091] Altogether, these data demonstrate that gain-of-function mutations in Fgfr3 significantly affect hippocampal-dependent episodic and fear associative memory acquisition, affecting coping strategies against unavoidable stress.

[0092] Importantly, these data support the notion that Fgfr3 A385E The mutations recapitulated previously described behavioral deficits in human patients, including deficits in working memory and emotion regulation, and attention-deficit hyperactivity disorder ( Yarnell et al., 2015 ).

[0093] Subcutaneous injection of (-)-epicatechin inhibits Fgfr3 A385E / + Reversing cognitive impairment in mice Fgfr3 A385E / + To confirm that the cognitive impairment in mice was due to increased phosphorylation of the receptor and activation of some downstream signaling pathways, we decided to treat mice with (-)-epicatechin (De la Luz Cadiz-Gurrea M et al. 2019, Martin L et al. 2021). A385E / + Mice and their control littermates were subcutaneously injected with at least (-)-epicatechin or vehicle solution for at least 7 days and subjected to two behavioral tests (novel object recognition and forced swimming test; Figures 4B and 3A). Injection of (-)-epicatechin inhibited Fgfr3 A385E / + Fgfr3 reversed the memory impairment observed in a novel object recognition paradigm in mice (Figure 4B), and re-established the coping strategies against inescapable stress observed in the forced swim test (Figure 3A) compared to control littermates. A385E / + The absence of craniocerebral imbalance, and thus potentially increased intracranial hypertension, in mice allows us to hypothesize that the reported behavioral abnormalities observed are due to a direct effect of Fgfr3 on the brain. A385E It can be concluded that these abnormalities were due to a direct effect of the mutation. The reversal of these behavioral abnormalities with (-)-epicatechin confirmed our hypothesis and supported the fact that FGFR3 hyperactivation was involved in the cognitive phenotype of patients with FGFR3-related craniosynostosis.

[0094] Example 4: Treatment by bottle (oral) Oral administration of (-)-epicatechin inhibits Fgfr3 N534K Reversing cognitive impairment in mice Fgfr3 N534K / +To confirm the direct involvement of brain FGFR3 in the cognitive impairment observed in mice, we decided to treat mice with (-)-epicatechin bottle treatment. Animals were bottle treated with (-)-epicatechin (3mg / L) for 4 weeks before behavioral testing. (-)-epicatechin was selected due to its highest binding specificity to FGFR3.

[0095] Here, adult Fgfr3 N534K / + Treatment with (-)-epicatechin in bottles in mice (hypoplasia mouse model) showed recovery of impairments in spatial memory (novel object location recognition; Morris water maze, Figures 5C-5E), episodic and associative memory (CFC, Figure 5H), antidepressant effects (tail suspension test, forced swimming test, Figures 5A and 5B), and locomotor activity and fine coordination (bar beam, Figures 5F and 5G). These data demonstrated the direct impact of Fgfr3 gain-of-function mutations in the brain on cognitive performance. We also demonstrated that FGFR3 played a key role in hippocampal adult neurogenesis, and we established a direct link between hippocampal abnormalities and learning and stress response.

[0096] These results highlight the interest of catechin for the treatment of cognitive impairment in subjects with FGFR3 gain-of-function mutations (FGFR-related disorders = achondroplasia, hypochondroplasia, Crouzon syndrome with acanthosis nigricans, and Muenke syndrome) and diseases associated with memory impairment.

[0097] Consideration CAN syndrome is a very rare syndromic craniosynostosis associated with a specific p.Ala391Glu gain-of-function mutation in FGFR3 (Meyers et al., 1995). The p.Ala391Glu mutation was previously disclosed to have the effect of causing hyperactivation of FGFR3 (Chen et al., 2013, 2011; Li et al., 2006).

[0098] Fgfr3 A385E / +The CAN mouse model showed the absence of major craniofacial skeletal phenotypes. Interestingly, Fgfr3 A385E / + The phenotype of Fgfr3 is a mouse model of 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 the coronal suture fused in a few individuals ( Laurita et al., 2011 ; Twigg et al., 2008 ).

[0099] In CAN and Muenke, patients had elevated intracranial pressure due to premature fusion of the calvarial sutures combined with premature fusion of the synchondroses at the base of the skull associated with narrowing of the foramen magnum (Di Rocco et al., 2011). Premature fusion of the calvarial vault has also been associated with abnormalities in brain structure, including abnormal hippocampal development (Grosso et al., 2003; Gurbuz et al., 2016; Okubo et al., 2017).

[0100] Hypochondroplasia patients (14600MIM) are characterized by short-limbed dwarfism, mild macrocephaly, midfacial hypoplasia, short, quadrate ilia, and occasionally acanthosis nigricans (Blomberg et al. 2010). The most common hypochondroplasia mutation (p.Asn540Lys) is localized to the tyrosine kinase 1 domain of FGFR3 (Bonaventure et al. 1996; Rousseau et al. 1994).

[0101] Patients with hypochondroplasia exhibit abnormal development of the brain lobes (Kannu et al. 2005) and abnormal hippocampal configuration (Linnankivi et al. 2012). In addition, patients with hypochondroplasia exhibit learning disabilities, mild intellectual disability, global developmental delay, and occasionally seizures and epilepsy (Linnankivi et al. 2012).

[0102] Fgfr3 mouse model associated with thanatophoric dysplasia, ubiquitous and under the nestin promoter + / K644E Previous studies of the mutations have demonstrated severe cerebral and cortical overgrowth, whereas Fgfr3 - / - Mice presented with an underdeveloped neocortex (Inglis-Broadgate et al., 2005; Moldrich et al., 2011; Thomson et al., 2009, 2007). The premature fusion of cranial sutures observed in craniosynostosis is generally recognized to alter brain morphology and be associated with cognitive impairment via chronically elevated intracranial pressure (Aldridge et al., 2010; Arnaud-Lopez 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 brain did not exhibit severe morphological alterations, thus indicating that Fgfr3 A385E We show that the mutation had a moderate effect on embryonic brain neurogenesis. In contrast, analysis of adult hippocampal neurogenesis showed reduced proliferation of progenitor cells in the dentate gyrus, which was supported by a reduced granular zone in the dentate gyrus.

[0103] Interestingly, previous studies reported decreased progenitor cell proliferation in loss-of-function mutations of FGFR1, 2, and 3, whereas loss-of-function mutations of FGFR3 (Fgfr3 TD11K650E ) promotes the differentiation of increased progenitor cells in the dentate gyrus (Kang and Hebert, 2015). Our results are in contrast to these observations. We show that overactivation of FGFR3 promotes the differentiation of increased progenitor cells in the dentate gyrus (Kang and Hebert, 2015). A385E / + We observed that this resulted in decreased cell proliferation in mice. The phosphorylation level of the receptor appeared to interfere with neurogenesis: Fgfr3. TD11K650EMutations result in excessive receptor activation levels, whereas Fgfr3 A385E The mutation resulted in a more moderate degree of hyperactivation. These data suggested that the regulation of hippocampal neurogenesis by FGFRs is related to the activation level of FGFR3.

[0104] Next, the inventors investigated the effect of Fgfr3 on brain cognitive function. A385E and Fgfr3 N534K The effects of the mutations in Fgfr3 A385E / + Mouse and Fgfr3 N534K / + We observed that mice exhibited severe impairments in working and episodic memory functions without any locomotor activity, anxiety-related behavioral phenotypes, or spatial memory phenotypes. Although the effect of FGFR signaling on memory is unclear, to date, our study is the first to link Fgfr3 mutations to cognitive abnormalities in mice. Mechanistically, the impairments in learning and memory performance may be linked, at least in part, to the reduced neurogenesis of the hippocampus observed in our mutant mice. Interestingly, deletion of Fgfr2 in embryonic and adult mice shows reduced proliferation and differentiation of progenitor cells in the dentate gyrus, exerting a specific negative effect on associative and spatial memory performance (Stevens et al., 2012). In summary, our data demonstrate that Fgfr3 gain-of-function mutations result in severe learning and memory impairments and reduced adult neurogenesis in the hippocampus.

[0105] In addition, a decrease in coping strategies for unavoidable stress (previously named "depressive behavior") is associated with Fgfr3 A385E / + Mouse and Fgfr3 N534K / +This was observed in mice. The role of this FGFR was previously reported in major depression in humans with downregulation of the receptor (Evans et al., 2004). Furthermore, patients with FGFR3-associated craniosynostosis syndrome showed impaired emotional control and anxious behavior (de Jong et al., 2010; Maliepaard et al., 2014; Yarnell et al., 2015). To date, no studies have reported depression or mood disorders in cases of craniosynostosis. Animal model studies have also demonstrated antidepressant and anxiolytic effects of FGF2 or exogenous FGF2 injection in FGF2 knockouts (Elsayed et al., 2012; Salmaso et al., 2016). In contrast to FGF2, FGF9 plays an antidepressant effect in mice (Aurbach et al., 2015). Both FGF2 and FGF9 are major FGFR3 ligands and can also bind to other FGFRs. These observations are consistent with our data and may involve a complex combination of binding of various FGFs to FGFR1, 2, and 3.

[0106] 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 (-)-epicatechin by selective brain injection. (-)-epicatechin was selected for its highest binding specificity to FGFR3, and previous studies have shown the efficacy of (-)-epicatechin in skeletal abnormalities in FGFR3-associated achondroplasia mouse models (Martin et al., 2021).

[0107] Here, adult Fgfr3 A385E / + Mouse and Fgfr3 N534K / +Subcutaneous injection of (-)-epicatechin into mice showed recovery of working and episodic memory impairments and antidepressant effects. These data demonstrated the direct effect of Fgfr3 gain-of-function mutations in the brain on cognitive performance. We also demonstrated that FGFR3 played a key role in adult hippocampal neurogenesis, and we established a direct link between hippocampal abnormalities and learning and stress response.

[0108] These results highlight the interest of catechin for the treatment of cognitive impairment in subjects with FGFR3 gain-of-function mutations.

[0109] References: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into this disclosure.

[0110] [Table 1] TIFF2024546856000008.tif242165 TIFF2024546856000009.tif241165 TIFF2024546856000010.tif236165 TIFF2024546856000011.tif244165 TIFF2024546856000012.tif249165 TIFF2024546856000013.tif250165 TIFF2024546856000014.tif140165

Claims

1. A method for treating FGFR3 (fibroblast growth factor receptor 3)-associated cognitive impairment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one catechin.

2. The method of claim 1 , wherein the subject is a child or an adult.

3. The method of claim 1 , wherein the subject has a gain-of-function mutation of FGFR3.

4. 4. The method of claim 3, wherein the gain-of-function mutation of FGFR3 is a N540K, K650N, K650Q, M528I, I538V, N540S or N540T mutation.

5. The method of claim 3, wherein the FGFR3 gain-of-function mutation is an A391E mutation.

6. The method of claim 1, wherein said catechin is (+)-catechin.

7. The method of claim 1, wherein said catechin is (-)-catechin.

8. The method of claim 1, wherein said catechin is (+)-epicatechin.

9. The method of claim 1, wherein said catechin is (-)-epicatechin.

10. The method of claim 1 , wherein the subject is suffering from an FGFR3-associated skeletal disease.

11. 11. The method of claim 10, wherein the FGFR3-related skeletal disease is selected from the group consisting of hypochondroplasia (HCH), achondroplasia (ACH), thanatophoric dysplasia (TD), craniosynostosis, and short stature.

12. 12. The method of claim 11, wherein the FGFR3-associated skeletal disease is hypochondroplasia (HCH).

13. 12. The method of claim 11, wherein the FGFR3-associated skeletal disease is achondroplasia (ACH).

14. 12. The method of claim 11, wherein the FGFR3-associated skeletal disease is craniosynostosis.

15. 15. The method of claim 14, wherein the craniosynostosis is Crouzon syndrome with acanthosis nigricans (CAN).

16. The method of claim 1, wherein the subject is administered a pharmaceutical composition comprising a therapeutically effective amount of catechin as an active pharmaceutical ingredient and at least one pharma- ceutical acceptable excipient.

17. The method of claim 1 , wherein the subject is administered a food composition containing a therapeutically effective amount of catechins.

Images