WO2016046375A1

WO2016046375A1 - Theobroma cacao extract for use in the treatment or prevention of receptor tyrosine kinases related disorders

Info

Publication number: WO2016046375A1
Application number: PCT/EP2015/072129
Authority: WO
Inventors: Laurence LEGEAI-MALLET; Antonio CARRETERO
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm); Universite Paris Descartes; Universidad De Granada
Priority date: 2014-09-25
Filing date: 2015-09-25
Publication date: 2016-03-31

Abstract

The present invention relates to the treatment or prevention of receptor tyrosine kinases-related disorders. More particularly, the present invention concerns a Theobroma cacao extract for use in the treatment of receptor tyrosine kinases related disorders and a pharmaceutical composition comprising such extract.

Description

Theobroma cacao extract for use in the treatment or prevention of receptor tyrosine kinases related disorders

The present invention relates to the treatment or prevention of receptor tyrosine kinases related disorders. More particularly, the present invention concerns a Theobroma cacao extract for use in the treatment or prevention of receptor tyrosine kinases related disorders and a pharmaceutical composition comprising such extract.

Background Numerous diseases are due to constitutive activation of downstream signaling pathways that impair proliferation and differentiation of cells.

Receptor tyrosine kinases (RTKs), cell surface receptors, play pivotal roles in the development, tissue repair, normal cellular homeostasis and mediate cellular responses to a broad array of extracellular signals involved in the regulation of cell proliferation, migration, differentiation and survival signaling.

Aberrant expression or signaling patterns of these kinases are linked to the progression of a diversity of diseases, including rare diseases (e.g. skeletal disorders, neurocristopathy, RASopathy), cancer, atherosclerosis, asthma, and fibrosis.

Normal and pathological activation of RTKs initiates a cascade of events, including receptor homodimerization, activation of intrinsic kinase activity, intermolecular tyrosine trans-phosphorylation, associated with phosphorylation of substrates, signal-transducing proteins and their signaling pathways (Lemmon MA and Schlessinger J, Cell. 2010, 141 : 1 1 17-1 134).

Current therapies of the RTK-linked disorders are not efficient due to a high toxicity and low specificity.

Consequently, there is a great need for new therapeutic approaches for diseases driven by activated RTKs, especially for those driven by the fibroblast Growth Factor Receptor 3 (FGFR3), a RTK associated with skeletal disorders (chondrodysplasias and craniosynostoses) (Horton WA, Curr Opin Pediatr 1997, 9:437-442, BonaventureJ, El Ghouzzi V, Expert Rev Mol Med. 2003; 29: 1 -17), cancers (Bladder tumor, myeloma multiple) and skin benign tumors (Chesi M et al Curr Opin Hematol 2002;9:288-93, Bernard-Pierrot I et al Carcinogenesis. 2006 27: 740-747.).

Description of the invention In an attempt to find a new therapeutic approach for RTK-related disorders, the inventors have surprisingly found that a Theobroma cacao can be used efficiently.

Cocoa, a product derived from the beans of the Theobroma cacao ( T cacao) tree, and consumed by pre-Columbian American civilizations, was introduced to Europe by Spaniards in the 16^th century (Lanaud, Motamayor, & Sounigo, 2003). The cocoa market has remained stable over the last few years (Ellam & Williamson, 2013), and scientific interest in this product is growing. Indeed, a large number of studies support the health benefits of cocoa consumption (Ellam & Williamson, 2013; Ramiro-Puig & Castell, 2009; Smith, 2013), being attributed mainly to the flavanol content (Payne, Hurst, Miller, Rank, & Stuart, 2010; Quinones, Sanchez, Muguerza, Miguel, & Aleixandre, 201 1 ; Ramiro-Puig & Castell, 2009; Smith, 2013).

Cocoa flavanols consist of monomeric (+)-catechin and (-)-epicatechin, and oligomeric flavanols (procyanidins) ranging from dimers to decamers. Concerning the interflavanoid linkage (IFL) nature, B-type procyanidins [C-4 (upper unit)→C-6 or C-8 (lower unit)] are more abundant than A-type procyanidins, which present an additional ether-type bond [C-2 (upper unit)→0→C-5 or C-7 (lower unit)], as well as IFL can be either a or β type. This list of combinations together with the occurrence of glycosylated and methylated derivatives explains the high diversity of this family and the wide range of biological and biochemical activities in plants (He, Pan, Shi, & Duan, 2008). In addition, processing could lead to the formation of the diasteroisomers (+)-epicatechin and (-)- catechin (Payne et al., 2010). On the other hand, theobromine (a methylxanthine alkaloid), minor amounts of quercetin derivatives and the less known /V-phenylpropenoyl-L-amino acids have also been reported (Andres-Lacueva et al., 2008; Ortega et al., 2008; Sanbongi et al., 1998; Stark & Hofmann, 2005a; Tomas-Barberan et al., 2007), and thus their contribution to the beneficial effects of cocoa should not be ruled out (Ellam & Williamson, 2013; Zeng et al., 201 1 ).

The present invention thus relates to a Theobroma cacao extract for use in the treatment or prevention of receptor tyrosine kinases related disorders. By "receptor tyrosine kinases related disorders" is meant disorders linked to aberrant expression or signaling patterns of these kinases i.e disorders caused by an abnormal activation of these kinases.

The man skilled in the art is able to determine which disorders fall under this definition based on its general knowledge.

In particular, the present invention thus relates to a Theobroma cacao extract for use in the treatment or prevention of a disorder caused by an abnormal activation of the receptor tyrosine kinases.

By "abnormal activation" is meant an activation which is not in the normal ranges of activity of the receptor tyrosine kinases such as an increased activation of the receptor tyrosine kinases. The man skilled in the art is perfectly able to determine these normal ranges based on its general knowledge.

In particular, the present invention thus relates to a Theobroma cacao extract for use according to the invention, wherein said disorder is caused by an increased activation of the receptor tyrosine kinases.

By "increased activation" is meant an activation which is above the normal ranges of activity of the receptor tyrosine kinases. The man skilled in the art is perfectly able to determine these normal ranges based on its general knowledge.

More particularly, said Theobroma cacao extract is effective by inhibiting said receptor tyrosine kinases.

The present invention thus also concerns a Theobroma cacao extract for use to inhibit the receptor tyrosine kinases.

It also relates to the use of a Theobroma cacao extract to inhibit the receptor tyrosine kinases.

It further relates to a Theobroma cacao extract for use according the invention, wherein said Theobroma cacao extract inhibits the receptor tyrosine kinases.

As used herein, the term "inhibit" or "inhibiting" refers to a compound that inhibits the receptor tyrosine kinases biological activity. It can be easily determined by a man skilled in the art, for example by following the methods detailed in the experimental part of the present application.

In the scope of the present invention, it has to be understood that "a Theobroma cacao extract for use" is equivalent to "the use of a Theobroma cacao extract for" and in particular that "a Theobroma cacao extract for use in the treatment or prevention of" is equivalent to "the use of a Theobroma cacao extract for the treatment or prevention of" and to "the use of a Theobroma cacao extract for the manufacture of a medicament intended for the treatment or prevention of". Receptor tyrosine kinase or RTKs, as mentioned above, are high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. They have been shown not only to be key regulators of normal cellular processes but also to have a critical role in the development and progression of many types of cancer. RTKs are part of the larger family of protein tyrosine kinases, encompassing the receptor tyrosine kinase proteins which contain a transmembrane domain, as well as the nonreceptor tyrosine kinases which do not possess transmembrane domains.

In particular, said receptor tyrosine kinases are fibroblast growth factor receptors (FGFRs). FGFRs are, as their name implies, receptors that bind to members of the fibroblast growth factor family of proteins. FGFRs consist of a cellular ligand domain composed of three immunoglobulin-like domains, a single transmembrane helix domain, and an intracellular domain with tyrosine kinase activity. These receptors bind fibroblast growth factors, members of the largest family of growth factor ligands, comprising 22 members. Five distinct membrane FGFRs have been identified in vertebrates and all of them belong to the tyrosine kinase superfamily (FGFR1 , FGFR2, FGFR3, FGFR4 and FGFR5).

In particular, fibroblast growth factor receptors (FGRFs) according to the invention relate to FGFR1 , FGFR2 and FGFR3.

More particularly, said FGFRs are FGFR3. The disorders in the scope of the present invention can thus be chosen from skeletal disorders, neurocristopathy, RASopathy, cancer, atherosclerosis, asthma, fibrosis, skin benign tumors, developed by patients that display increased activation of the fibroblast growth factor receptor 3 (FGFR3), in particular by expression of a constitutively activated mutant of FGFR3; in particular from skeletal disorders chosen from chondrodysplasias such as achondroplasia, hypochondroplasia and thanatophoric dysplasia, and craniosynostoses (e.g: Muenke syndrome, Crouzon syndrome with acanthosis nigricans, Apert syndrome and Pfeiffer syndrome), neurocristopathy, RASopathy, fibrosis, skin benign tumors; and more particularly from skeletal disorders chosen from chondrodysplasias such as achondroplasia, hypochondroplasia and thanatophoric dysplasia, and craniosynostoses (e.g: Muenke syndrome, Crouzon syndrome with acanthosis nigricans, Apert syndrome and Pfeiffer syndrome), fibrosis, skin benign tumors. By "neurocristopathy", is meant the normal definition known by a man skilled in the art. In particular, it relates to a class of pathologies that may arise from defects in the development of tissues containing cells commonly derived from the embryonic neural crest cell lineage. More particularly, it relates to the following disorders: piebaldism, Waardenburg syndrome, Hirschsprung disease, Ondine's curse (congenital central hypoventilation syndrome), pheochromocytoma, paraganglioma, Merkel cell carcinoma, multiple endocrine neoplasia, neurofibromatosis type I, CHARGE syndrome, familial dysautonomia, DiGeorge syndrome, Axenfeld-Rieger syndrome, Goldenhar syndrome (a.k.a. hemifacial microsomia), craniofrontonasal syndrome, congenital melanocytic nevus, melanoma.

By "RASopathy", is meant the normal definition known by a man skilled in the art. In particular, it relates to syndromes caused by germline mutations in genes that alter the Ras subfamily and Mitogen-activated protein kinases that control signal transduction. More particularly, it relates to the following disorders: Capillary malformation-AV malformation syndrome, Autoimmune lymphoproliferative syndrome, Cardiofaciocutaneous syndrome CFC syndrome, Hereditary Gingival fibromatosis type 1 , Neurofibromatosis type 1 , Noonan syndrome, Costello syndrome, Legius syndrome, LEOPARD syndrome.

More particularly, the disorders in the scope of the present invention are chosen from skeletal disorders, even more particularly from chondrodysplasias such as achondroplasia, hypochondroplasia and thanatophoric dysplasia, and craniosynostoses (e.g: Muenke syndrome, Crouzon syndrome with acanthosis nigricans, Apert syndrome and Pfeiffer syndrome).

The present invention thus relates to a Theobroma cacao extract for use according to the invention, wherein said disorders are chosen from skeletal disorders, neurocristopathy, RASopathy, cancer, atherosclerosis, fibrosis and skin benign tumors, in particular fom skeletal disorders, preferably from chondrodysplasias and craniosynostoses.

A Theobroma cacao extract according to the invention can be obtained by the man skilled in the art by applying currently known methods in the field of extraction of chemical and natural products or can be obtained from commercially available extracts. Extracts according to the invention are prepared from the seeds of the cacao pod. For example, after depulping unfermented beans extracted from the fruit, they are blanched with water at 85 ⁵C during 5 minutes. Then, the beans are dried at 60 ⁵C. Dried and blanched unfermented beans are defatted using an extruder press at 45 ⁵C, resulting in the called "defatted cake". Finally, polyphenolic-enriched cocoa extract is obtained from the defatted cake using a column packed with Amberlite™ FPX66 resin as selective adsorbent. The purified extract fraction is distilled and the aqueous extract is dried under vacuum. In particular, the extract for use according to the invention comprises at least one compound chosen from flavonols, flavon-3-ols, sweroside, hexenyl xylopyranosyl glucopyranoside and derivatives thereof.

More particularly, said at least one flavonol or derivative thereof can be chosen in the context of the present invention from quercitin and derivatives thereof and even more particularly from quercetin, quercetin glucuronide, quercetin hexose, quercetin arabinoside and isomers thereof.

In particular, said flavon-3-ol or derivative thereof can be chosen from procyanidin, catechin, cinchonain and derivatives thereof and even more particularly from proanthocyanidin A, (epi)catechin, (epi)catechin dimer hexose, arabinopyranosyl- (epi)catechin-(epi)catechin, (epi)gallocatechin, (epi)catechin glucopyranoside, catechin diglucopyranoside, cinchonain I, (epi)catechin tetramer, (epi)catechin pentamer, (epi)catechin hexamer, (epi)catechin methyl dimer, (epi)catechin ethyl dimer, procyanidin A, procyanidin B, procyanidin C and isomers thereof.

Said Theobroma cacao extract for use according to the invention and as mentioned above, can also further comprise at least one 3,4-N-phenylpropenoyl-L-aminoacid or derivative thereof, for example chosen from /V-caffeoyl-L-aspartate, L-Aspartic acid, N-[3- (4-hydroxyphenyl)-1 -oxo-2-propenyl], L-Aspartic acid, A/-[3-(4-hydroxy-3-methoxyphenyl)- 1 -oxo-2-propenyl], trans-clovamide (A/-[(2E)-3-(3,4-dihydroxyphenyl)-1 -oxo-2-propen-1 -yl]- 3-hydroxy-L-tyrosine), deoxyclovamide (A/-[(2E)-3-(3,4-dihydroxyphenyl)-1 -oxo-2-propen- 1 -yl]-L-tyrosine) and derivatives thereof.

In one embodiment of the present invention, the Theobroma cacao extract for use according to the invention and as mentioned above, comprises at least one compound chosen from:

- procyanidin B;

(epi)catechin;

(epi)catechin tetramer; - (epi)catechin pentamer;

- hexenyl xylopyranosyl glucopyranoside;

- (epi)catechin dimer hexose;

- arabinopyranosyl-(epi)catechin-(epi)catechin;

- procyanidin C;

- proanthocyanidin A;

- (epi)catechin ethyl dimer;

- quercetin;

- quercetin hexose; cinchonain I;

- procyanidin A; and

- sweroside.

By at least one, it is meant one of these compounds or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or 14 of these compounds or a combination of the totality of these compounds.

In particular, the Theobroma cacao extract for use according to the invention and as mentioned above, comprises at least two compounds chosen from:

- procyanidin B, procyanidin C, (epi)catechin tetramer, (epi)catechin pentamer, (epi)catechin, (epi)catechin dimer hexose, arabinopyranosyl-(epi)catechin- (epi)catechin and procyanidin A;

- procyanidin B, (epi)catechin, (epi)catechin dimer hexose and arabinopyranosyl-(epi)catechin-(epi)catechin; or

- procyanidin B, (epi)catechin, quercetin hexose, hexenyl xylopyranosyl glucopyranoside, cinchonain I, procyanidin A, sweroside, quercetin, (epi)catechin ethyl dimer and proanthocyanidin A.

More particulalrly, the Theobroma cacao extract for use according to the invention and as mentioned above, comprises: - procyanidin B, procyanidin C, (epi)catechin tetramer, (epi)catechin pentamer, (epi)catechin, (epi)catechin dimer hexose, arabinopyranosyl-(epi)catechin- (epi)catechin and procyanidin A;

In another embodiment, the Theobroma cacao extract for use according to the invention and as mentioned above comprises one or more of the following compounds: sucrose; tri-O-methylsucrose; (epi)catechin tetramer; procyanidin C; procyanidin B;

(epi)catechin glucopyranoside; N-caffeoyl-L-aspartate; (epi)catechin pentamer ; (epi)catechin hexamer; (epi)gallocatechin ;

L-Aspartic acid, N-[3-(4-hydroxyphenyl)-1 -oxo-2-propenyl]; (epi)Catechin ;

L-Aspartic acid, N-[3-(4-hydroxy-3-methoxyphenyl)-1 -oxo-2-propenyl]; catechin diglucopyranoside; trans-Clovamide (N-[(2E)-3-(3,4-Dihydroxyphenyl)-1 -oxo-2-propen-1 -yl]-3- hydroxy-L-tyrosine); (epi)catechin dimer hexose; arabinopyranosyl-(epi)catechin-(epi)catechin; proanthocyanidin A; quercetin glucuronide; deoxyclovamide (N-[(2E)-3-(3,4-Dihydroxyphenyl)-1 -oxo-2-propen-1 -yl]-L- tyrosine); quercetin hexose; hexenyl xylopyranosyl glucopyranoside; quercetin arabinoside; cinchonain I;

(epi)catechin methyl dimer; sweroside; quercetin; theobromine; caffeine; and

(epi)catechin ethyl dimer.

By one or more, it is meant any one of these compounds or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28 or 29 of these compounds or a combination of the totality of these compounds.

In still another embodiment, the Theobroma cacao extract for use according to the invention and as mentioned above comprises one or more of the following compounds: sucrose (isomer 1 ); sucrose (isomer 2); tri-O-methylsucrose;

(epi)catechin tetramer (isomer 1 ); procyanidin C (isomer 1 );

procyanidin B (isomer 1 );

procyanidin C (isomer 2);

procyanidin B (isomer 2);

(epi)catechin tetramer (isomer 2);

procyanidin C (isomer 3);

procyanidin C (isomer 4);

(epi)catechin tetramer (isomer 3);

(epi)catechin glucopyranoside (isomer 1 );

(epi)catechin glucopyranoside (isomer 2);

procyanidin B (isomer 3);

N-caffeoyl-L-aspartate;

(epi)catechin tetramer (isomer 4);

procyanidin C (isomer 5);

procyanidin C (isomer 6);

(epi)catechin tetramer (isomer 5);

(epi)catechin pentamer (isomer 1 );

(epi)catechin hexamer;

(epi)gallocatechin (isomer 1 );

(epi)gallocatechin (isomer 2);

L-Aspartic acid, N-[3-(4-hydroxyphenyl)-1 -oxo-2-propenyl];

(epi)Catechin (isomer 2);

L-Aspartic acid, N-[3-(4-hydroxy-3-methoxyphenyl)-1 -oxo-2-propenyl]; procyanidin B (isomer 5); catechin diglucopyranoside; trans-Clovamide (N-[(2E)-3-(3,4-Dihydroxyphenyl)-1 -oxo-2-propen-1 -yl]-3 hydroxy-L-tyrosine);

(epi)catechin dimer hexose; arabinopyranosyl-(epi)catechin-(epi)catechin (isomer 2); procyanidin B (isomer 6); procyanidin C (isomer 7); proanthocyanidin A (isomer 1 );

(epi)catechin pentamer (isomer 2);

(epi)catechin tetramer (isomer 6); quercetin glucuronide; deoxyclovamide (N-[(2E)-3-(3,4-Dihydroxyphenyl)-1 -oxo-2-propen-1 -yl]-L tyrosine); quercetin hexose (isomer 1 ); quercetin hexose (isomer 2); hexenyl xylopyranosyl glucopyranoside (isomer 1 ); quercetin arabinoside; hexenyl xylopyranosyl glucopyranoside (isomer 2); cinchonain I;

(epi)catechin methyl dimer (isomer 2); sweroside; quercetin; theobromine; caffeine; (epi)catechin (isomer 1 ); procyanidin B (isomer 4) ; arabinopyranosyl-(epi)catechin-(epi)catechin (isomer 1 ); procyanidin A (isomer 1 );

(epi)catechin ethyl dimer (isomer 1 ); and proanthocyanidin A (isomer 2).

By one or more, it is meant any one of these compounds or a combination of 2 to 55 of these compounds or a combination of the totality of these compounds.

Particular combinations of these compounds are detailed below: sucrose, procyanidin C, tri-O-methylsucrose, procyanidin B, theobromine, (epi)catechin and N-caffeoyl-L-aspartate; sucrose, procyanidin C, tri-O-methylsucrose, procyanidin B, theobromine, (epi)catechin and (epi)catechin glucopyranoside; procyanidin C, (epi)catechin tetramer, procyanidin B, theobromine and (epi)catechin; procyanidin C, (epi)catechin tetramer, procyanidin B, (epi)catechin and (epi)gallocatechin; procyanidin C, (epi)catechin tetramer, procyanidin B, (epi)catechin pentamer and (epi)catechin; procyanidin C, (epi)catechin tetramer, procyanidin B, (epi)catechin pentamer, (epi)catechin and (epi)gallocatechin; procyanidin B, procyanidin C, (epi)catechin tetramer, (epi)catechin pentamer, (epi)catechin, (epi)catechin dimer hexose, arabinopyranosyl-(epi)catechin- (epi)catechin and procyanidin A; - procyanidin B, (epi)catechin, (epi)catechin dimer hexose and arabinopyranosyl-(epi)catechin-(epi)catechin; procyanidin B, procyanidin C, (epi)catechin, (epi)catechin dimer hexose, arabinopyranosyl-(epi)catechin-(epi)catechin, quercetin hexose, deoxyclovamide (N-[(2E)-3-(3,4-Dihydroxyphenyl)-1 -oxo-2-propen-1 -yl]-L- tyrosine);proanthocyanidin A and procyanidin A; procyanidin B, procyanidin C, (epi)catechin, (epi)catechin dimer hexose, arabinopyranosyl-(epi)catechin-(epi)catechin, quercetin hexose and deoxyclovamide (N-[(2E)-3-(3,4-Dihydroxyphenyl)-1 -oxo-2-propen-1 -yl]-L- tyrosine); procyanidin B, procyanidin C, (epi)catechin, quercetin hexose, hexenyl xylopyranosyl glucopyranoside and quercetin arabinoside; procyanidin B, procyanidin C, (epi)catechin, quercetin hexose, hexenyl xylopyranosyl glucopyranoside, (epi)catechin tetramer and quercetin arabinoside; procyanidin B, (epi)catechin, quercetin hexose, hexenyl xylopyranosyl glucopyranoside and quercetin arabinoside; procyanidin B, (epi)catechin, quercetin hexose, hexenyl xylopyranosyl glucopyranoside, cinchonain I and quercetin arabinoside; procyanidin B, (epi)catechin, cinchonain I and quercetin arabinoside;

- procyanidin B, (epi)catechin, quercetin hexose, hexenyl xylopyranosyl glucopyranoside, cinchonain I, procyanidin A, sweroside, quercetin, (epi)catechin ethyl dimer and proanthocyanidin A; procyanidin B, (epi)catechin, (epi)catechin methyl dimer, procyanidin A, quercetin, sweroside, (epi)catechin ethyl dimer and proanthocyanidin A; procyanidin B and (epi)catechin; procyanidin B, (epi)catechin, (epi)catechin methyl dimer, procyanidin A, quercetin and sweroside; procyanidin B, (epi)catechin, (epi)catechin ethyl dimer, procyanidin A, quercetin, sweroside and proanthocyanidin A; procyanidin B, (epi)catechin, quercetin and sweroside; procyanidin B, (epi)catechin, (epi)catechin methyl dimer, quercetin and sweroside; and procyanidin B, (epi)catechin, quercetin arabinoside and hexenyl xylopyranosyl glucopyranoside.

The present invention also relates to a pharmaceutical composition comprising a Theobroma cacao extract as defined above.

In particular, said pharmaceutical composition further comprises a pharmaceutically acceptable excipient. As used herein, a "pharmaceutically acceptable excipient" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a human. A pharmaceutically acceptable excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Pharmaceutical compositions of the invention may be prepared by a variety of synthetic routes. The reagents and starting materials are commercially available, or readily synthesized by well-known techniques by one of ordinary skill in the arts.

In one embodiment, the pharmaceutical compositions according to the invention further comprise specific tyrosine kinase antibodies, Mapkinase inhibitors, CNP analog, tyrosine kinase inhibitors and/or growth hormone.

The present invention also relates to a method of treatment or prevention of one of the previously mentioned disorders, comprising the administration to a subject in need thereof of an effective amount of a Theobroma cacao extract as defined above.

In particular, the method of treatment or prevention of one of the previously mentioned disorders, comprise the following steps:

- determining if a subject is afflicted or has the potential to be afflicted by one the previously mentioned disorders, and

- consequently administering to a subject in need thereof an effective amount of a Theobroma cacao extract as defined above. In one embodiment of the method according to the invention, the Theobroma cacao extract is administered together with specific tyrosine kinase antibodies, Mapkinase inhibitors, CNP analog, tyrosine kinase inhibitors and/or growth hormone.

In one embodiment, the present invention also relates to a method for inhibiting the receptor tyrosine kinases, in particular FGFR3, comprising the administration to a subject in need thereof of an effective amount of a Theobroma cacao extract as defined above.

In particular said method is carried out to treat or prevent the above mentioned disorders.

By "subjects", it is meant a human, a male or female, which is afflicted, or has the potential to be afflicted with one or more disorders described herein.

As used herein, a "therapeutically effective amount" refers to an amount which is effective in reducing, eliminating, treating or controlling the symptoms of the herein- described disorders. The term "controlling" is intended to refer to all processes wherein there may be a slowing, interrupting, arresting, or stopping of the progression of the disorders described herein, but does not necessarily indicate a total elimination of all disorders, and is intended to include prophylactic treatment and chronic use.

The identification of the subjects who are in need of treatment of herein-described disorders is well within the ability and knowledge of one skilled in the art. A clinician skilled in the art can readily identify, by the use of clinical tests, physical examination and medical/family history, those subjects who are in need of such treatment.

A therapeutically effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining the therapeutically effective amount, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of subject; its size, age, and general health; the specific disease involved; the degree of involvement or the severity of the disease; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristic of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

The amount of Theobroma cacao extract used in the context of the present invention, which is required to achieve the desired biological effect, will vary depending upon a number of factors, including the chemical characteristics (e.g. hydrophobicity) of the compound employed, the potency of the compound, the type of disorder, the diseased state of the patient, and the route of administration.

For example, the amount of Theobroma cacao extract used in the context of the present invention is from 1 to 1000 μg/ml, more particularly from 1 to 100 mg/day of Theobroma cacao extract.

Examples of modes of administration include parenteral (e.g., subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intravenous, intradermal, intraperitoneal, intraportal, intra-arterial, intrathecal, transmucosal, intra-articular, and intrapleural), transdermal (e.g., topical), epidural, and mucosal (e.g. intranasal) injection or infusion, as well as oral, inhalation, pulmonary, and rectal administration.

In one embodiment, the Theobroma cacao extract used in the context of the present invention is intended for oral administration.

As described herein, a Theobroma cacao extract according to the invention can be formulated into pharmaceutical compositions, in particular for a use in the previously mentioned methods of treatment or prevention, by admixture with one or more pharmaceutically acceptable excipients. Such compositions may be prepared for use in oral administration, particularly in the form of tablets or capsules, in particular orodispersible (lyoc) tablets; or parenteral administration, particularly in the form of liquid solutions, suspensions or emulsions; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, for example, topically or via trans-dermal patches or ocular administration, or intravaginal or intra-uterine administration, particularly in the form of pessaries or by rectal administration.

The tablets, pills, powders, capsules, troches and the like can contain one or more of any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, or gum tragacanth; a diluent such as starch or lactose; a disintegrant such as starch and cellulose derivatives; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, or methyl salicylate. Capsules can be in the form of a hard capsule or soft capsule, which are generally made from gelatin blends optionally blended with plasticizers, as well as a starch capsule. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric agents. Other oral dosage forms syrup or elixir may contain sweetening agents, preservatives, dyes, colorings, and flavorings. In addition, the Theobroma cacao extract may be incorporated into fast dissolve, modified-release or sustained-release preparations and formulations, and wherein such sustained-release formulations are preferably bi-modal. Liquid preparations for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The liquid compositions may also include binders, buffers, preservatives, chelating agents, sweetening, flavoring and coloring agents, and the like. Non-aqueous solvents include alcohols, propylene glycol, polyethylene glycol, acrylate copolymers, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include mixtures of alcohols and water, hydrogels, buffered media, and saline. In particular, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of the Theobroma cacao extract. Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.

Alternative modes of administration include formulations for inhalation, which include such means as dry powder, aerosol, or drops. They may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for buccal administration include, for example, lozenges or pastilles and may also include a flavored base, such as sucrose or acacia, and other excipients such as glycocholate. Formulations suitable for rectal administration are preferably presented as unit-dose suppositories, with a solid based carrier, and may include a salicylate.

The invention will be further illustrated by the following figures and examples.

FIGURES

Figure 1 : Base peak chromatogram of the Theobroma cacao extract obtained by HPLC-ESI-QTOF-MS

Figure 2: FGFR3 signalling pathway Figure 3: Effect of Theobroma cacao extract on the Mapkinase pathway: A- results on cell lines expressing FGFR3-WT, FGFR3-K650N and FGFR3-G380R and B- results on Human TD primary chondrocytes (R248C mutation)

Figure 4: Effect of Theobroma cacao extract on the phosphorylation of PLC-gamma, GSK3alpha/beta and beta-catenin, on cell lines expressing FRGFR3-WT, FGFR3-K650N and FGFR3-G380R: A- results on PCL-gamma pathway, B- results on GSK3alpha-beta pathway and C- results on beta-catenin pathway

Figure 5: Effect of Theobroma cacao extract on the phosphorylation of PLC-gamma, GSK3 alp ha/beta and beta-catenin, on Human TD primary chondrocytes (R248C mutation)

EXAMPLES

Material and methods

1. Chemicals

All chemicals were of HPLC-MS and used as received. Acetic acid and methanol for HPLC were purchased from Fluka (Sigma-Aldrich, Steinheim, Germany) and Lab-Scan (Gliwice, Sowinskiego, Poland), respectively. Dimethyl sulfoxide (DMSO) and sodium acetate were purchased from Panreac (Barcelona, Spain). Water was purified by a Milli-Q system from Millipore (Bedford, MA, USA).

2. Sample preparation A concentrated Theobroma cacao (Ivory Coast origin) extract was used. The polyphenols from whole cocoa matrix were analytically characterized using a solution of cocoa extract of 10 mg/mL. Briefly, 10 mg of cocoa extract were dissolved in 1 mL of DMSO. The sample was sonicated for 5 min, vortexed for 1 min, and then centrifuged for 5 min at 10,000 rpm and the supernatant was filtered through a 0.25 mm filter before the HPLC analysis.

For the purification of polyphenols from cocoa extract, a solution stock of 75 mg/mL was prepared by dissolving the appropriate amount of cocoa extract in DMSO. The sample was sonicated for 5 min, vortexed for 1 min and then was centrifuged for 5 min at 10,000 rpm. The supernatant of this solution stock was filtered through a 0.25 mm filter before the preparative HPLC analysis. 3. Instrumentation

The polyphenols from the Theobroma cacao extract were fractionated using a Gilson preparative HPLC system (Gilson, Middleton, USA) equipped with a binary pump (model 331/332), automated liquid handling solutions (model GX-271 ) and UV-Vis detector (model UV-Vis 156).

Theobroma cacao extract and isolated fractions were analytically characterized using an Agilent 1200 series rapid-resolution LC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a binary pump, an autosampler and a diode-array detector (DAD). The HPLC system was coupled to a quadrupole time-of-flight mass spectrometer (QTOF) (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization (ESI) interface (model G1607A from Agilent Technologies, Palo Alto, CA).

4. Fractionation of polyphenols from Theobroma cacao extract

The compounds from Theobroma cacao were fractionated at room temperature. An Ascentis C18 column (10 μηι, 250x21 2 mm) was used to separate the compounds. The mobile phases consisted of acetic acid 0.5% (A) and methanol (B). The following multi-step linear gradient was applied: 0 min, 0% B; 10 min, 20% B; 15 min, 25% B; 25 min, 35% B; 35 min, 60% B; 70 min, 60% B; 75 min, 70% B; 78 min, 80% B; 80 min, 100% B; 82 min, 0% B. The initial conditions were held for 15 min. The injection volume was 1 ml_. The flow rate used was set at 15 mL/min. The compounds separated were monitored with UV-Vis (220-280 nm). Fraction-collection step consisted of UV-based purification, determining the elution time window for collecting each fraction. Finally, a total of 40 fractions were collected and the solvent was evaporated under vacuum. The residue of each fraction was weighted and dissolved with an appropriate volume of DMSO at concentration level of 100 μg/mL. Finally, all fractions were filtered through a 0.25 μηι filter before the HPLC analysis.

5. Chromatographic, UV, and spectrophotometry conditions

The compounds from the Theobroma cacao and fractions were separated at room temperature using a Zorbax Eclipse Plus C18 column (1 .8 μηι, 1 50x4.6 mm). The mobile phases consisted of acetic acid 0.5% (A) and methanol (B). The following multi-step linear gradient was applied: 0 min, 0% B; 5 min, 25% B; 15 min, 35% B; 20 min, 39% B; 38 min, 60% B; 40 min, 70% B; 42 min, 80% B; 44 min, 1 00% B; 46 min, 0% B; 48 min, 0% B. The initial conditions were held for 1 0 min. The injection volume was 1 0 μί. The flow rate used was set at 0.3 mL/min. The DAD coupled to the HPLC system was set in spectrum range starting at 190 nm and ending at 950 nm.

6. ESI-QTOF-MS detection

The HPLC system was coupled to a QTOF mass spectrometer equipped with an ESI interface operating in negative ion mode using a capillary voltage of +3.5 kV. The other optimum values of the source parameters were: drying gas temperature, 220°C; drying gas flow, 9 L/min; and nebulizing gas pressure, 2.5 bar. The detection was performed considering a mass range of 50-1200 m/z.

The accurate mass data of the molecular ions were processed through the software DataAnalysis 4.0 (Bruker Daltonics), which provided a list of possible elemental formulas using Generate Molecular Formula Editor.

During the development of the HPLC method, external instrument calibration was performed using a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, IL, USA) directly connected to the interface, with a sodium acetate cluster solution passing through containing 5 mM sodium hydroxide and 0.2% acetic acid in water:isopropanol 1 :1 v/v.

The calibration solution was injected at the beginning of each run and all the spectra were calibrated prior to the compound identification.

7. Evaluation of the properties of Cocoa with cell lines and primary chondrocytes expressing FGFR3 The extracts of Cocoa were evaluated in vitro using cell lines expressing FGFR3.

Stable cell lines (NIH3T3) expressing FGFR3-WT and FGFR3-mutants have been generated. The mutant cell lines (NIH3T3-K650N, NIH3T3-G380R, NIH3T3-R248C) express mutations that cause achondroplasia, hypochondroplasia, thanatophoric dysplasia, cancers and benign skin tumors. In addition, an analysis of the extract of Cocoa on primary chondrocytes isolated from human fetal cartilage with thanatophoric dysplasia (TD) expressing an heterozygous FGFR3 mutations (R248C) was done.

After activation of the FGFR3, with ligand (Fibroblast Growth Factor), the canonical pathways such as Mapkinase (ERK1/2), PLC-gamma, beta-Catenin, GSK3-beta and STATs were studied by Western blotting. Results

1. Characterization of Theobroma cacao bv HPLC-ESI-QTOF-MS

The base peak chromatogram of the Theobroma cacao extract obtained by HPLC- ESI-QTOF-MS is shown in Fig. 1 . The compounds characterized are presented in the Table 1 below, numbered according to their elution order.

Table 1 :

Peak Compound RT Ion Measured Calculated Error mSigma Fragmentation Molecular Fr

(min) m/z m/z (ppm) pattern Formula

1 Sucrose (isomer 1) 5,11 [M-H]^" 341,1099 341,1089 2,9 3,5 non fragmented C12H22O11 F1,

2 Sucrose (isomer 2) 5,31 [M-H]^" 341,1107 341,1089 5,3 6,9 non fragmented C12H22O11 F1,

3 unknown 8,62 [M-H]^" 265,0936 265,0929 2,6 24,6 187,1429 CioHi80s

4 Tri-O-methylsucrose 9,23 [M-H]^" 383,1563 383,1559 1,2 18,7 non fragmented C15H28O11

5 unknown 9,71 [M-H]^" 442,156 442,1566 1,4 3,6 395,1454 C16H29NO13

6 (Epi)catechin tetramer (isomer 1) 9,88 [M- 576,1258 576,1273 2,6 21,6 407,0789; 289,0737 C60H50O24

2H]^2"

7 Procyanidin C (isomer 1) 10,26 [M-H]^" 865,1983 865,1985 0,3 19,3 577,1142; 289,0756 C45H38O18

8 Procyanidin B (isomer 1) 10,88 [M-H]^" 577,1342 577,1351 1,6 29,7 451,1243; 425,0750; C30H26O12

289,0756

9 Procyanidin C (isomer 2) 11,02 [M-H]^" 865,1981 865,1985 0,5 14,8 577,1337; 432,0929 C45H38O18

10 Procyanidin B (isomer 2) 11,27 [M-H]^" 577,1339 577,1351 2,2 32,2 289,0756 C30H26O12

11 (Epi)catechin tetramer (isomer 2) 11,72 [M- 576,1257 576,1273 2,9 30,9 451,1231; 432,0928; C60H50O24

2H]^2" 289,0755

12 Procyanidin C (isomer 3) 11,87 [M-H]^" 865,1959 865,1985 1 19,7 577,1139; 451,1227; C45H₃₈0i8

433,0716; 289,0647

13 Procyanidin C (isomer 4) 12,36 [M-H]^" 865,201 865,1985 2,9 22,7 577,1291; 432,0952 C45H38O18

14 (Epi)catechin tetramer (isomer 3) 12,59 [M- 576,126 576,1273 2,3 51,1 432,0926 C60H50O24

2H]^2"

15 (Epi)catechin glucopyranoside 13,48 [M-H]^" 451,1244 451,1246 0,5 20 289,0783 C21H24O11 (isomer 1)

16 (Epi)catechin glucopyranoside 13,7 [M-H]^" 451,1236 451,1246 2,2 9,3 433,1111; 289,0753 C21H24O11 (isomer 2)

17 Procyanidin B (isomer 3) 14,62 [M-H]^" 577,1367 577,1351 2,8 50,7 289,072 C30H26O12

18 /V-caffeoyl-L-aspartate 15,19 [M-H]^" 294,0631 294,0619 4,1 5,3 179,0283 C₁₃H₁₃N0₇

19 (Epi)catechin tetramer (isomer 4) 15,71 [M- 576,126 576,1273 2,4 24,6 432,0922; 289,0643 C60H50O24

2H]^2"

20 Procyanidin C (isomer 5) 15,91 [M-H]^" 865,2002 865,1985 1 ,9 15,6 577,1335; 432,0953; C45H38O18

289,0751

21 Procyanidin C (isomer 6) 16,28 [M-H]^" 865,2022 865,1985 4,2 46,5 432,0951 ; 289,0751 C45H38O18 F6,

F9,

22 (Epi)catechin tetramer (isomer 5) 16,53 [M- 576,1256 576,1273 2,9 16 432,0921 ; 289,0751 C60H50O24 F6,

2H]^2" F9,

23 (Epi)catechin pentamer (isomer 1 ) 17,01 [M- 720,1586 720,159 577,1283 C75H62O30 F8,

2H]^2"

24 (Epi)catechin hexamer 17,23 [M- 864,191 1 864,1907 720,1582; 577,1283 C90H74O36

2H]^2"

25 (Epi)gallocatechin (isomer 1 ) 17,95 [M-H]^" 305,0707 305,0667 9 28,9 289,075 Ci5H₁₄07

26 (Epi)gallocatechin (isomer 2) 18,42 [M-H]^" 305,0717 305,0667 12,8 28,2 289,0749 Ci5H₁₄07

27 L-Aspartic acid, Λ/-[3-(4- 18,94 [M-H]^" 278,0681 278,067 4 17,5 non fragmented C₁₃H₁₃N0₆

hydroxyphenyl)-1 -oxo-2-propenyl]

28 (Epi)Catechin (isomer 2) 19,84 [M-H]^" 289,0747 289,0718 10,2 35 245,0824 Ci5H₁₄06

29 L-Aspartic acid, A/-[3-(4-hydroxy-3- 20,58 [M-H]^" 308,0787 308,0776 3,6 9,9 non fragmented C₁₄H₁₅N0₇

methoxyphenyl)-1 -oxo-2-propenyl]

30 Procyanidin B (isomer 5) 20,96 [M-H]^" 577,1349 577,1351 0,4 14,3 289,0726 C30H26O12

31 Catechin diglucopyranoside 21 ,87 [M-H]^" 593,1507 593,1512 0,8 6,6 451 ,1053; 289,0715 C27H30O15

32 trans-Clovamide (Λ/-[(2Ε)-3-(3,4- 23,01 [M-H]^" 358,0944 358,0932 3,3 17,9 178,0501 C₁₈H₁₇N0₇

Dihydroxyphenyl)-1 -oxo-2-propen-1 - yl]-3-hydroxy-L-tyrosine)

33 (Epi)catechin dimer hexose 23,47 [M-H]^" 737,1744 737,1723 2,8 7,9 289,0744 C36H34O17 F1

F

34 Arabinopyranosyl-(epi)catechin- 24,23 [M-H]^" 707,164 707,1618 3,1 5,1 289,0743 C35H32O16 F1 (epi)catechin (isomer 2) F

35 Procyanidin Β (isomer 6) 25,05 [M-H]^" 577,1362 577,1351 1 ,8 26,2 289,0742 C30H26O12 F1

F1 F15,

36 Procyanidin C (isomer 7) 25,99 [M-H]^" 865,1997 865,1985 1 ,3 32,1 432,0948 C45H38O18 F1

F15,

37 Proanthocyanidin (Type A) (isomer 1 ) 26,57 [M-H]^" 591 ,1494 591 ,1508 2,4 9,6 439,1005; 289,0737 C31 H28O12

38 (Epi)catechin pentamer (isomer 2) 27,73 [M- 720,1583 720,159 575, 1 197;451 , 1030 C75H62O30

2H]^2"

39 (Epi)catechin tetramer (isomer 6) 28,61 [M- 576,1257 576,1273 2,8 29,6 437,2017; 245,0926 C60H50O24 F

2H]^2"

40 Quercetin glucuronide 29,4 [M-H]^" 477,067 477,0675 1 14,8 431 ,0979 C2l H₁₈0l3

41 Deoxyclovamide (Λ/-[(2Ε)-3-(3,4- 31 ,73 [M-H]^" 326,1045 326,1045 3,4 17,8 282,1 137 C₁₈H₁₇N0₅ F Dihydroxyphenyl)-1 -oxo-2-propen-1 - yl]-L-tyrosine)

42 Quercetin hexose (isomer 1 ) 33,57 [M-H]^" 463,0879 463,0882 OJ 5 300,0277; 285,0401 C21 H20O12 F1

F15,

43 Quercetin hexose (isomer 2) 33,84 [M-H]^" 463,0882 463,0882 0,1 25 300,0274 C21 H20O12 F18,

44 Hexenyl xylopyranosyl 34,77 [M-H]^" 393,177 393,1766 1 22,3 249,1337 C17H30O10 F1 glucopyranoside (isomer 1 ) F17.

45 Quercetin arabinoside 35,38 [M-H]^" 433,0788 433,0776 2,6 20,5 300,0276 C20H18O11 F1

F17.

F

46 Hexenyl xylopyranosyl 35,9 [M-H]^" 393,1769 393,1766 0,8 14,5 249,1357 C17H30O10 F18, glucopyranoside (isomer 2)

47 (Epi)catechin derivative 36,15 [M-H]^" 427,161 1 427,161 0,3 25,6 289,0703 C20H28O10 F19,

48 Cinchonain 1 36,33 [M-H]^" 451 ,1026 451 ,1026 1 ,9 8,4 341 ,0673; 217,0146 C24H20O9 F19,

49 Procyanidin A (isomer 2) 37,44 [M-H]^" 575,1 197 575,1 195 0,3 18,2 289,0698; 285,0421 C30H24O12 F

F

50 unknown 37,64 [M-H]^" 516,2454 516,245 0,8 73,6 non fragmented C24H39NO11 F1

F

51 (Epi)catechin methyl dimer (isomer 2) 37,69 [M-H]^" 605,1652 605,1664 2 6,6 453,1210; 315,0834; C32H30O12 F

289,0725

52 Sweroside 38,19 [M-H]^" 357,1227 357,1 191 10 25 non fragmented C_{l 6}H₂₂0g F

F

53 Quercetin 43,01 [M-H]^" 301 ,0361 301 ,0354 2,3 5 non fragmented Cl5H_{1 0}O7 F

F

54^* Theobromine 21 ,7 [M+H]⁺ 181 ,0771 181 ,0760 5,1 14,8 non fragmented C₇H₈N₄0₂ F3

55^* Caffeine 32,3 [M+H]⁺ 195.087 195,0877 3,2 14 non fragmented C₈H₁₀N₄O2

nd, compounds not detected in fractions.

"Compounds in positive mode.

This table includes the retention time, experimental m/z, MS/MS fragments, molecular formulas, errors and σ values for all of the compounds detected in the samples analyzed. All the compounds were characterized by the interpretation of their mass spectra determined by the QTOF mass analyzer while taking into account the information provided by the literature and databases.

2. Isolation of Theobroma cacao by semi-preparative HPLC and characterization of fractions by HPLC-ESI-QTOF-DAD-MS

The compounds in the Theobroma cacao extract were isolated by semi- preparative HPLC. Afterwards, the composition of each fraction was established by the detailed HPLC-ESI-QTOF-DAD-MS method. This complements the characterization of the Theobroma cacao extract. All of these compounds were correctly separated using the semi-preparative HPLC technique according to their elution order (Table 1 ).

At least seven new identified compounds were detected only in isolated fractions (Table 2).

Table 2:

Compound RT Ion Measured Calculated Error mSigma Molecular Fractions

(min) m/z m/z (ppm) Formula

(Epi)catechin (isomer 1 ) 19,09 [M- 289,0726 289,0718 2,9 7 Ci5H₁₄06 F3

H]-

Procyanidin B (isomer 4) 20,31 [M- 577,1302 577,1351 8,7 37,1 C30H26O12 F9

H]-

Arabinopyranosyl-(epi)catechin- 22,34 [M- 707,153 707,1618 12,5 7,4 C35H32016 F1 1 (epi)catechin (isomer 1 ) H]-

Procyanidin A (isomer 1 ) 28,02 [M- 575,1 138 575,1 195 10 37,5 C30H24O12 F1 1 , F13

H]-

Procyanidin B (isomer 6) 34,03 [M- 577,1319 577,1351 5,7 17,8 C30H26O12 F17, F21

H]-

(Epi)catechin ethyl dimer (isomer 1 ) 39,67 [M- 605,1672 605,1664 1 ,3 35,3 C32H30O12 F21 , F22, F25

H]-

Proanthocyanidin (Type A) (isomer 2) 40,72 [M- 591 ,1519 591 ,1508 1 ,9 14,9 C31 H28012 F21 , F22, F25

H]-

Most of these compounds were characterized as new isomers of (epi)catechin, procyanidin B, arabinopyranosyl-(epi)catechin-(epi)catechin, procyanidin A, (epi)catechin ethyl dimer, and proanthocyanidin A. Moreover, fraction 3, 4 and 5 yielded a new compound, which had not been detected in the analysis of the complex extract.

3. Inhibition of the canonical Ras-MapK pathway with Theobroma cocoa extract

Normal and pathological activation of FGFR3 initiates a cascade of events, including activation of several signaling pathways (Fig. 2). The impact of the Theobroma cacao extract on the signaling pathway downstream FGFR3, was evaluated on several cellular models. On one hand, NIH3T3 cell lines expressing normal or mutant FGFR3 have been tested and fractions 1 1 , 12 and 21 , for example, showed a total inhibition of the phosphorylation of ERK1 /2 in NIH3T3 cell line lysates. These fractions inhibit FGFR3 wild type activated with the FGF2 ligand and mutant FGFR3 expressing G380R (achondroplasia) and K650N (hypochondroplasia) mutations (Fig. 3A). On the other hand, the Theobroma cacao extract has been tested on chondrocytes isolated from human cartilage and an inhibition of the phosphorylation of ERK1 /2 has been observed in primary TD chondrocytes with fractions 1 1 and 12, for example (Fig. 3B).

4. Inhibition of PLCg, GSK3b, bCatenin, Stat pathway with cocoa extract The inhibiting properties of the Theobroma cacao extract have also been evaluated in others pathways and an inhibition of the phosphorylation of PLC-gamma in cell lines (3T3-WT, 3T3-G380R, 3T3-K650N) was observed. Fractions 1 1 and 12, for example, reduced the phosphorylation of GSK3-beta and beta-Catenin (Fig. 4A, B and C). The inhibiting properties of the extract have further been tested in primary TD chondrocytes. An important inhibition of the phosphorylated PLC-gamma proteins and a reduced phosphorylation of GSK3-beta, beta-Catenin and Stat have been observed with fractions 1 1 and 12 for example, in primary chondrocytes (Fig. 5). REFERENCES

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Claims

1 . A Theobroma cacao extract for use in the treatment or prevention of receptor tyrosine kinases related disorders.

2. A Theobroma cacao extract for use according to claim 1 , wherein said disorder is caused by an abnormal activation of the receptor tyrosine kinases.

3. A Theobroma cacao extract for use according to claim 1 or claim 2, wherein said disorder is caused by an increased activation of the receptor tyrosine kinases.

4. A Theobroma cacao extract for use according to any one of claims 1 to 3, wherein said Theobroma cacao extract inhibits the receptor tyrosine kinases.

5. A Theobroma cacao extract for use to inhibit the receptor tyrosine kinases.

6. A Theobroma cacao extract for use according to any of one the preceding claims, wherein said receptor tyrosine kinases are fibroblast growth factor receptors (FGRFs).

7. A Theobroma cacao extract for use according to any one of the preceding claims, for the treatment or prevention of skeletal disorders, neurocristopathy, RASopathy, cancer, atherosclerosis, asthma, fibrosis and skin benign tumors.

8. A Theobroma cacao extract for use according to any one of claims 1 to 6, wherein said FGFRs are FGFR3.

9. A Theobroma cacao extract for use according to claim 8, for the treatment or prevention of skeletal disorders, preferably chondrodysplasias and craniosynostoses.

10. A Theobroma cacao extract for use according to any of the preceding claims, wherein said extract comprises at least one compound chosen from flavonols, flavon-3-ols, sweroside, hexenyl xylopyranosyl glucopyranoside and derivatives thereof.

1 1 . A Theobroma cacao extract for use according to claim 10, wherein said at least one flavonol or derivative thereof is chosen from quercitin and derivatives thereof and/or said at least one flavon-3-ol or derivative thereof is chosen from procyanidin, catechin, cinchonain and derivatives thereof.

12. A Theobroma cacao extract for use according to any of claims 10 to 1 1 , further comprising at least one 3,4-N-phenylpropenoyl-L-aminoacid or derivative thereof chosen from /V-caffeoyl-L-aspartate, L-Aspartic acid, A/-[3-(4-hydroxyphenyl)-1 - oxo-2-propenyl], L-Aspartic acid, A/-[3-(4-hydroxy-3-methoxyphenyl)-1 -oxo-2- propenyl], trans-clovamide (A/-[(2E)-3-(3,4-dihydroxyphenyl)-1 -oxo-2-propen-1 -yl]- 3-hydroxy-L-tyrosine), deoxyclovamide (A/-[(2E)-3-(3,4-dihydroxyphenyl)-1 -oxo-2- propen-1 -yl]-L-tyrosine) and derivatives thereof.

13. A Theobroma cacao extract for use according to claim 1 1 , wherein said at least one quercetin or derivative thereof is chosen from quercetin, quercetin glucuronide, quercetin hexose, quercetin arabinoside and isomers thereof and/or said at least one procyanidin, catechin, cinchonain and derivative thereof is chosen from proanthocyanidin A, (epi)catechin, (epi)catechin dimer hexose, arabinopyranosyl-(epi)catechin-(epi)catechin, (epi)gallocatechin, (epi)catechin glucopyranoside, catechin diglucopyranoside, cinchonain I, (epi)catechin tetramer, (epi)catechin pentamer, (epi)catechin hexamer, (epi)catechin methyl dimer, (epi)catechin ethyl dimer, procyanidin A, procyanidin B, procyanidin C and isomers thereof.

14. A Theobroma cacao extract for use according to any of the preceding claims, wherein said extract comprises at least one compound chosen from:

- procyanidin B;

- (epi)catechin;

- (epi)catechin tetramer;

- (epi)catechin pentamer;

- hexenyl xylopyranosyl glucopyranoside;

- (epi)catechin dimer hexose;

- arabinopyranosyl-(epi)catechin-(epi)catechin;

- procyanidin C;

- proanthocyanidin A;

- (epi)catechin ethyl dimer;

- quercetin;

- quercetin hexose; - cinchonain I;

- procyanidin A; and

- sweroside.

15. A Theobroma cacao extract for use according to any of the preceding claims, wherein said extract comprises at least two compounds chosen from:

16. A Theobroma cacao extract for use according to any of the preceding claims, wherein said extract comprises:

17. A pharmaceutical composition comprising a Theobroma cacao as defined in any of the preceding claims.

18. A method of treatment or prevention of receptor tyrosine kinases related disorders, comprising the administration to a subject in need thereof of an effective amount of a Theobroma cacao extract as defined in any of the preceding claims.