WO2010000675A1 - Polypeptides, cyclic polypeptides and pharmaceutical compositions comprising thereof for inhibiting activity of trkb - Google Patents

Abstract

The present invention provides polypeptides, cyclic polypeptides and pharmaceutical compositions which selectively inhibit tropomyosin-related kinase B (TrkB) receptor activity and are thus suitable for the prevention and/or treatment of disorders related to TrkB. The polypeptide of the invention may comprise an amino acid sequence consisting in CNPMGYTKEGC.

Description

POLYPEPTIDES, CYCLIC POLYPEPTIDES AND PHARMACEUTICAL COMPOSITIONS COMPRISING THEREOF FOR INHIBITING ACTIVITY OF TRKB

FIELD OF THE INVENTION:

The present invention provides polypeptides, cyclic polypeptides and pharmaceutical compositions which selectively inhibit tropomyosin-related kinase B receptor (TrkB), a member of family receptor tyrosine kinases, and are thus suitable for the prevention and/or treatment of disorders related to TrkB receptor.

BACKGROUND OF THE INVENTION:

Neurotrophins belong to a class of growth factors, secreted proteins, which induce the survival of neurons and are known as neurotrophic factors. Neurotrophic factors are secreted by target tissue and act by preventing the associated neuron from initiating programmed cell death; thus allowing the neurons to survive.

Each neurotrophin interacts with a preferred tropomyosin-related kinase (Trk) receptor. Thus, nerve growth factor (NGF) binds preferentially to TrkA, brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) bind to TrkB, and neurotrophin-3 (NT-3) binds to TrkC, whereas the non-selective p75NTR receptor interacts with all the neurotrophins with similar affinity.

More particularly, BDNF regulates neuronal development and survival by interacting with two classes of cell surface receptors, TrkB receptors and the nonselective p75NTR receptor (1 ). Binding of BDNF to TrkB triggers receptor dimerisation and subsequent autophosphorylation on tyrosine residues. In addition, TrkB receptors can be transactivated in absence of BDNF through different mechanisms (2-5). Although BDNF was initially regarded to be involved in development and maintenance of central and peripheral nervous system, more recent evidences have implicated BDNF in the regulation of synaptic strength and long-term memory processes (6). Given its trophic effects on neurons and its central role in high-order cognitive functions, BDNF has rapidly emerged as a key element in the pathophysiology of numerous brain disorders, including neurological disorders (e.g. epileptogenesis (7)), neurodegenerative diseases (e.g. amyotrophic lateral sclerosis (8), Huntington (9), Alzheimer's and Parkinson's diseases (10)) and psychiatric disorders (e.g. depression (11 ), addiction (12) and schizophrenic psychosis (13)), thus representing a promising new therapeutic target. In addition, recent reports have strongly implicated TrkB in the formation of tumors and metastases in various types of cancers (28). However, due to the lack of specific TrkB modulators, the precise implication of BDNF and TrkB in these pathologies and in high-order cognitive functions still remains elusive.

Indeed, contrary to NGF and TrkA, very few structural data are available for the BDNF/TrkB complex, hampering the development of a virtual screening of compounds in a specific TrkB binding pocket; even if numerous biochemical studies have highlighted specific regions, variable among neurotrophins, implicated in binding specificity and/or activation of their cognate Trk receptors (14).

Thus, until now, TrkB receptor activity inhibitors are, for example, non-peptidic molecules such as K-252 compounds (glycosylated indole carbazole alkaloids) (15) and peptidic molecules such as BDNF loop 2 derived peptides (17). However, these inhibitors are not selective TrkB receptor inhibitors and show also unsatisfied affinity.

Moreover, their in vivo use remains uncertain.

Therefore such inhibitors may induce unwanted effects a lack of sensitivity since they block others neurotrophic receptors such TrkA or TrkC. Compared to current available inhibitors, selective inhibitors TrkB receptor represents a challenge and a field of interest for treating different disorders including neurological disorders.

There is a need in the art for better understanding the biological function of TrkB pathway and also for providing a selective and potent inhibitor of TrkB receptor. In other respects, recent studies, disclosed in the international patent publication WO2007/051477, suggest that different peptides derived from neurotrophic factors such as BDNF may be useful as modulators and more particularly as agonists of neurotrophin receptor and thus useful for the treatment or prevention of many disorders such as cancer, neurological and neurodegenerative disorders.

SUMMARY OF THE INVENTION:

It has now been found by the inventors that a particular fragment derived from BDNF as well as a cyclic polypeptide derived from such fragment act as a potent and selective inhibitor of TrkB receptor. Accordingly, the present invention provides an isolated polypeptide derived from Brain-Derived Neurotrophic Factor (BDNF) comprising an amino acid sequence consisting of:

X1 -P-M-G-Y-T-X2-E-G (SEQ ID NO:1 )

wherein amino acid X1 is H, K, N or R; and X2 is D, E, H, K or R.

The invention also relates to a cyclic polypeptide wherein the polypeptide according to the invention is cyclised via a disulfide bound between two cysteine residues.

The invention relates to the use of a polypeptide or a cyclic polypeptide according to the invention for the prevention and/or treatment of disorders related to tropomyosin-related kinase B receptor (TrkB).

DETAILED DESCRIPTION OF THE INVENTION:

Definitions:

A "receptor" or "receptor molecule" is a soluble or membrane bound/associated protein or glycoprotein comprising one or more domains to which a ligand binds to form a receptor-ligand complex. By binding the ligand, which may be an agonist or an antagonist, the receptor is activated or inactivated and may initiate or block pathway signalling.

The term "TrkB" or "TrkB receptor" are used interchangeably and refers to the tropomyosin-related kinase B receptor. An exemplary native TrkB receptor amino acid sequence is provided in GenPept database under accession number Q16620.1

(Homo sapiens) and an exemplary native TrkB receptor sequence is provided in

GenBank database under accession number NM 006J₁₁SO (Homo sapiens).

The term "BNDF" refers to the Brain-Derived Neurotrophic Factor. An exemplary native BNDF amino acid sequence is provided in GenPept database under accession number P23560.1 (Homo sapiens) and an exemplary native BDNF sequence is provided in GenBank database under accession number NM_J301709.3 or NP 001700.2 (Homo sapiens).

As used herein, the term "fragment" of a reference sequence refers to a fragment that is shorter than the reference sequence of BDNF. Said fragment may have a length of e.g. 9, 10, 15, 20 or 25 amino acids.

As used herein, the term "derivatives" refers to an amino acid sequence having a percentage of identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with amino acid sequence consisting of SEQ ID NO: 1 and having the ability to selectively inhibit TrkB receptor activity. By a polypeptide having an amino acid sequence at least, for example, 95%

"identical" to amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to said reference sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid.

Methods for comparing the identity and homology of two or more sequences are well known in the art. Thus for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux et al., 1984), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % homology between two polypeptide sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (1981 ) and finds the best single region of similarity between two sequences. Other programs for determining identity and/or similarity between sequences are also known in the art, for instance the BLAST family of programs (Altschul et al., 1990), accessible through the home page of the NCBI at world wide web site ncbi.nim.nih.gov) and FASTA (Pearson and Lipman, 1988; Pearson, 1990).

Polypeptides consisting of an amino acid sequence "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence may comprise mutations such as deletions, insertions and/or substitutions compared to the reference sequence. In case of substitutions, the substitution preferably corresponds to a conservative substitution. In a preferred embodiment, the polypeptide consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence only differs from the reference sequence by conservative substitutions. In another preferred embodiment, the peptide consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence corresponds to a naturally-occurring allelic variant of the reference sequence.

By an "isolated" peptide, it is intended that the peptide is not present within a living organism, e.g. within human body.

The term "cell penetrating peptide" (CPP) is defined as a carrier peptide that is capable of crossing biological membrane or a physiological barrier. Cell penetrating peptides are also called cell-permeable peptides, protein-transduction domains (PTD) or membrane-translocation sequences (MTS).

By "receptor antagonist" is meant a natural or synthetic compound that has a biological effect opposite to that of a receptor agonist. The term is used indifferently to denote a "true" antagonist and an "inverse" agonist of a receptor. A "true" receptor antagonist is a compound which binds the receptor and blocks the biological activation of the receptor, and thereby the action of the receptor agonist, for example, by competing with the agonist for said receptor. An inverse agonist is a compound which binds to the same receptor as the agonist but exerts the opposite effect.

Inverse agonists have the ability to decrease the constitutive level of receptor activation in the absence of an agonist.

Within the meaning of the invention, the terms "inhibitor of TrkB receptor activity inhibitor", "inhibitor of TrkB activity" or "TrkB receptor antagonist" are used interchangeably and are intended to mean a compound able to reduce or suppress a biological activity associated with activation of the TrkB receptor in the patient, including any of the downstream biological effects otherwise resulting from the binding to TrkB receptor of its natural ligand BNDF or many transactivations processes (such as GPCR-, zinc-, endocannabinoids-mediated transactivations).

The antagonistic activity of compounds towards the TrkB receptor may be determined using various methods. Thus, the affinity of an antagonist for TrkB receptor may be assayed by determining the ability of said antagonist to block the phosphorylation level on appropriate tyrosine residues of TrkB.

In the context of the present invention, polypeptides and cyclic polypeptides according to the invention are selective for the TrkB receptor as compared with the TrkA and TrkC receptors. By "selective" it is meant that the affinity of the antagonist for the TrkB receptor is at least 50-fold, preferably 100-fold, more preferably 500-fold, still preferably 1000-fold higher than the affinity for the TrkA and TrkC receptors.

Within the meaning of the invention, the term "disorders related to TrkB" or

"disorders related to the TrkB receptor activity" shall include diseases associated with or implicating TrkB activity, and more precisely hyperactivity of TrkB, and conditions that accompany these diseases. Such hyperactivity may the consequence of BDNF- dependent TrkB activation, BDNF-independent TrkB activation (also called basal activity of TrkB) or both and results in an activation of different signalling pathways following to the phosphorylation of TrkB on appropriate tyrosine residues. Examples of disorders related to TrkB include neurological disorders, neurodegenerative disorders, psychiatric disorders, angiogenesis and metastasis in cancer.

Within the meaning of the invention, "pharmaceutical composition" is intended to mean any substance or composition intended to be administered to an individual, human or animal, prevent, reduce, relieve and/or treat a disorder or a sign associated with said disorder and/or to make a diagnostic of a disorder.

As used herein, the term "treatment" refers to inhibiting the disorder or condition, i.e. arresting its development; relieving the disorder or condition, i.e. causing regression of the disorder; or relieving the conditions caused by the disease, i.e. symptoms of the disease.

As used herein, the term "prevention" refers to preventing the disorder or condition from occurring in a subject who has not yet been diagnosed as having it.

POLYPEPTIDES AND CYCLIC POLYPEPTIDES An object of the invention relates to an isolated polypeptide derived from

Brain-Derived Neurotrophic Factor (BDNF) comprising an amino acid sequence consisting of:

X1 -P-M-G-Y-T-X2-E-G (SEQ ID NO:1 )

wherein amino acid X1 is H, K, N or R; amino acid X2 is D, E, H, K or R. In a particular embodiment, the polypeptide of the invention comprises an amino acid consisting of NPMGYTKEG (SEQ ID NO: 2).

It should be recalled that in the description as a whole, "amino acid" is understood to mean the amino acids in the L form which can be found in natural proteins, that is to say alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V). In a particular embodiment, polypeptides of the invention can be conformationally constrained to enable the polypeptides to bind TrkB receptor with a better affinity.

Cyclisation is well known in the art and generally involves the introduction of a disulfide bound between two cysteine residues. Typically, the cycle is formed through a side chain to side chain ring involving a monosulfide or disulfide bridge between pairs of cysteines, penicillamines, homocysteines, combinations of the foregoing, or other pairs of amino acids in which the side chains are linked with either one or two sulfur atoms. Methods for the synthesis of disulfide cyclic polypeptide are well known in the art and are described for example in US3,929,758, US4.216,141 ; and US4,102,877. Polypeptides of the invention may thus comprise cysteine residues at terminal ends to allow the cyclisation of the polypeptide.

Accordingly, polypeptides of the invention may comprise an amino acid sequence consisting of:

C-X1 -P-M-G-Y-T-X2-E-G-C (SEQ ID NO: 3)

wherein amino acid X1 is H, K, N or R; amino acid X2 is D, E, H, K or R.

In a particular embodiment, the polypeptide of the invention comprises an amino acid sequence consisting of CNPMGYTKEGC (SEQ ID NO: 4) In another particular embodiment, the polypeptides as above described are cyclised via a disulfide bound between the two cysteine residues of the polypeptides.

Accordingly, the cyclic polypeptides of the invention may comprise an amino acid sequence having the formula (SEQ ID NO: 5):

C-X1 -P-M-G-Y-T-X2-E-G-C

wherein amino acid X1 is H, K, N or R; amino acid X2 is D, E, H, K or R.

In a preferred embodiment, the cyclic polypeptide named "cyclotraxin-B" has the following formula (SEQ ID NO: 6):

CNPMGYTKEGC

Cyclisation can also be performed by peptide bonds between the amino- terminal of the first amino acid and the carboxy-terminal of the last amino acid. Other methods of cyclization are contemplated by the invention. For example, those methods include but are not limited by those described by Marlowe (1993, Biorg. Med. Chem. Lett. 3:437-44) who describes peptide cyclization on TFA resin using trimethylsilyl (TMSE) ester as an orthogonal protecting group; PaIMn and Tarn (1995, J. Chem. Soc. Chem. Comm. 2021 -2022) who describe the cyclization of unprotected peptides in aqueous solution by oxime formation; Algin et al (1994, Tetrahedron Lett. 35:9633-9636) who disclose solid-phase synthesis of head-to-tail cyclic peptides via lysine side-chain anchoring; Kates et al (1993, Tetrahedron Lett. 34:1549-1552) who describe the production of head-to-tail cyclic peptides by three- dimensional solid phase strategy; Tumelty et al (1994, J. Chem. Soc. Chem. Comm. 1067-1068) who describe the synthesis of cyclic peptides from an immobilized activated intermediate, wherein activation of the immobilized peptide is carried out with N-protecting group intact and subsequent removal leading to cyclization; McMurray et al (1994, Peptide Res. 7:195-206) who disclose head-to-tail cyclization of peptides attached to insoluble supports by means of the side chains of aspartic and glutamic acid; Hruby et al (1994, Reactive Polymers 22:231 -241 ) who teach an alternate method for cyclizing peptides via solid supports; and Schmidt and Langer (1997, J. Peptide Res. 49:67-73) and those described by Davies JS (The cyclisation of peptides and depsipeptides J Pept Sci 2003,8:471 -501 ); and Li and Roller (PPCyclisation strategies in peptide derived drug design. Curr. Tp Med. Chem. 2002, 3:325-41 ).

The polypeptides of the invention may optionally comprise chemical modifications. Chemical modifications aim at obtaining peptides with increased protection of the peptides against enzymatic degradation in vivo, and/or increased capacity to cross membrane barriers, thus increasing its half-life and maintaining or improving its biological activity. Any chemical modification known in the art can be employed according to the present invention. In a particular embodiment, polypeptides or cyclic polypeptides of the invention may comprise a cell penetrating peptide sequence at its terminal ends to allow the addressing to the brain of the polypeptides.

Such cell penetrating peptide sequence may be derived from proteins and their peptide derivatives which have been found to possess cell internalization properties including but not limited to the Human Immunodeficiency Virus type 1 (HIV-I) Tat-carrier peptide, the herpes virus tegument protein VP22, the homeotic protein of Drosophila melanogaster Antennapedia (the CPP is called Penetratin) and the proteghn 1 (PG-I) anti-microbial peptide SynB.

Thus, the polypeptides and cyclic polypeptides according to the present invention may be fused with Tat peptide having the amino acid sequence consisting in YGRKKQRRR (SEQ ID NO: 10).

Accordingly, in a particular embodiment, the polypeptides of the invention may comprise an amino acid sequence having the formula (SEQ ID NO: 7):

Y-G-R-K-K-R-R-Q-R-R-R-C-X1 -P-M-G-Y-T-X2-E-G-C

wherein amino acid X1 is H, K, N or R; amino acid X2 is D, E, H, K or R. In another particular embodiment, the cyclic polypeptides of the invention may comprise an amino acid sequence having the formula (SEQ ID NO: 8):

Y-G-R-K-K-R-R-Q-R-R-R-C-X1 -P-M-G-Y-T-X2-E-G-C

wherein amino acid X1 is H, K, N or R; amino acid X2 is D, E, H, K or R.

In a preferred embodiment, the cyclic polypeptide of the invention consists in a fusion peptide between peptide Tat and cyclotraxin-B having the formula (SEQ ID NO: 9):

YGRKKRRQRRRCNPMGYTKEGC ' '

Such fusion peptide allows its delivery to the brain after intravenous injection resulting in the inhibition of brain TrkB receptor activity in vivo.

Polypeptides according to the invention may be prepared by any well-known procedure in the art, such as solid phase synthesis, liquid phase synthesis or genetic engineering. As a solid phase synthesis, for example, the amino acid corresponding to the C-terminus of the peptide to be synthesized is bound to a support which is insoluble in organic solvents, and by alternate repetition of reactions, one wherein amino acids with their amino groups and side chain functional groups protected with appropriate protective groups are condensed one by one in order from the C- terminus to the N-terminus, and one where the amino acids bound to the resin or the protective group of the amino groups of the peptides are released, the peptide chain is thus extended in this manner. After synthesis of the desired peptide, it is subjected to the deprotection reaction and cut out from the solid support. Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptides, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer's instructions.

Alternatively, the polypeptides of the invention can be synthesized by recombinant DNA techniques as it is now well-known in the art. For example, these polypeptides can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired polypeptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well- known techniques.

Therefore, a further object of the invention relates to an isolated nucleic acid molecule encoding for a polypeptide of the invention.

Typically, said nucleic acid molecule is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. The terms "vector", "cloning vector" and

"expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.

So, a further object of the invention relates to a vector comprising a nucleic acid molecule of the invention.

Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said polypeptide upon administration to a subject. The vectors may further comprise one or several origins of replication and/or selectable markers. The promoter region may be homologous or heterologous with respect to the coding sequence, and provide for ubiquitous, constitutive, regulated and/or tissue specific expression, in any appropriate host cell, including for in vivo use. Examples of promoters include bacterial promoters (17, pTAC, Trp promoter, etc.), viral promoters (LTR, TK, CMV- IE, etc.), mammalian gene promoters (albumin, PGK, etc), and the like. Examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv÷ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861 ,719, US 5,278,056 and WO 94/19478.

A further object of the present invention relates to a cell which has been transfected, infected or transformed by a nucleic acid and/or a vector according to the invention. The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed".

The nucleic acids of the invention may be used to produce a recombinant polypeptide of the invention in a suitable expression system. The term "expression system" means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.

Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Kluyveromyces or Saccharomyces yeasts, and mammalian cell lines (e.g., Vera cells, CHO cells, 3T3 cells, COS cells, etc.). The present invention also relates to a method for producing a recombinant host cell expressing a polypeptide according to the invention, said method comprising the steps consisting of: (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and optionally secrete said polypeptide. Such recombinant host cells can be used for the production of the polypeptides according to the present invention, as previously described.

The invention further relates to a method of producing a polypeptide of the invention, which method comprises the steps consisting of: (i) culturing a transformed host cell according to the invention under conditions suitable to allow expression of said polypeptide; and (ii) recovering the expressed polypeptide.

Polypeptides of the invention may be used in an isolated (e.g., purified) form or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome). Polypeptides and cyclic polypeptides according to the invention have the biological activity of exerting a potent and selective inhibition of TrkB activity and thus constitutes powerful research tool for the evaluation of therapeutic potential of TrkB signalling pathway in animal models of neurodegenerative and psychiatric disorders.

THERAPEUTIC METHODS AND USES

Therefore, in a particular embodiment, the invention relates to a polypeptide or a cyclic polypeptide according to the present invention for the prevention and/or treatment of disorders related to TrkB.

Another object of the invention relates to a method for the prevention and/or treatment of a disease condition mediated by TrkB activity comprising at least the step of administering to an individual in need thereof at least an effective amount of at least a polypeptide or a cyclic polypeptide as described above.

The disorders related to TrkB may be chosen among the group consisting of neurological disorders, psychiatric disorders, tumour formation and metastasis in cancer.

Examples of neurological disorders include neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease, dementia, neuro-muscular degeneration, senile degeneration of brain, spinocerebellar disease, cerebellar ataxia, neuropathy, primary cerebellar degeneration such as spinal muscular atrophy, motor neuron disease, amyotrophic lateral sclerosis, progressive bulbar palsy, multiple sclerosis, pain, epilepsy.

Examples of psychiatric disorders include psychoses, schizophrenia, bipolar disorders, affective disorders, neurotic disorders, paranoid personality disorder, mania, addictive behaviours and drug abuse, feeding troubles, autism, stress, anxiety and depression.

Examples of cancer include cancer of neural system, neuroblastoma, Wilm's tumours, glial tumours, myeloma, Kaposi's sarcoma, leukemias, melanoma, prostate adenocarcinomas, liver and pancreatic adenocarcinomas, but also angiogenesis, tumour formation and metastasis. PHARMACEUTICAL COMPOSITIONS

The polypeptide or a cyclic polypeptide according to the present invention may be administered in the form of a pharmaceutical composition, as defined below. Preferably, said compound is administered in a therapeutically effective amount.

The term "pharmaceutical" or "medicament", used herein interchangeably, refers to an agent or mixture of agents that is primarily intended to treat and/or ameliorate and/or prevent a disease or a disorder or to diagnostic a disease or a disorder. The term "pharmaceutically acceptable" means that which is useful in preparing no a pharmaceutical composition that is generally safe, non-toxic, and neither biologically or otherwise undesirable and includes what is acceptable for veterinary as well as human pharmaceutical use.

An "effective amount" means an amount sufficient to induce a positive modification in the condition to be regulated or treated, but low enough to avoid serious side effects. An effective amount may vary with the cosmetic or pharmaceutical effect to obtain or the particular condition being treated, the age and physical condition of the end user, the severity of the condition being treated/prevented, the duration of the treatment, the nature of other treatments, the specific compound or product/composition employed, the route of administration, and like factors.

The term "subject" or "individual" are used interchangeably herein and means mammals and non-mammals. Examples of mammals include, but are not limited to: humans; non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and the like. Examples of non-mammals include, but are not limited to, birds, and the like. The term "subject" or "individual" does not denote a particular age or sex.

The invention will further be illustrated in view of the following figure and examples. FIGURES:

Figure 1 : Cyclotraxin-B is a highly potent allosteric inhibitor of TrkB receptor:

Three-dimensional representation of a BDNF monomer (variable regions are noted). Boxed is the highly variable region III that served as a basis for the design of cyclotraxin-B.

EXAMPLES: Material & Methods

Plasmid constructs and cell lines: Plasmid constructs were generated by using standard cloning techniques and were confirmed by sequencing. The human recombinant TrkB receptor was fluorescently tagged at its C-terminus with the Enhanced cyan fluorescent protein (rhTrkB-ECFP) by cloning the full human TrkB sequence (S1) into the pECFP-N1 expression vector (Clontech). For inducible expression, rhTrkB-ECFP was subcloned into the tetracycline-responsive (TetOn) vector pTRE2 (Clontech). Chinese Hamster Ovary cells stably expressing rhTrkB- ECFP (TetOn-rhTrkB cells), were produced by transfection of the pTRE2-rhTrkB- ECFP plasmid into CHO-K1 TetOn cells (Clontech) followed by clone selection in 0.25 mg/ml hygromycin B (Invitrogen) and 0.1 mg/ml geneticin (G418; Invitrogen). Resistant cells were selected based on both fluorescence intensity and KIRA-ELISA profile after an overnight incubation in presence of 1000 ng/ml doxycycline (Clontech). Cortical neurons were prepared and cultured from E16 mouse embryos as described previously {S2). The nnr5 PC12-TrkA, nnr5 PC12-TrkB and nnr5 PC12- TrkC cells are NGF non-responding mutant PC12 cells stably transfected with TrkA, TrkB and TrkC cDNA, respectively {S3).

Peptides design and synthesis: Peptides were designed using a method that rapidly produces and isolates mimetic peptides by direct proteolysis of mature recombinant BDNF (Peprotech). Fragments were obtained using endoproteinase GIu-C V8 (Sigma), purified and identified using a HPLC system connected to a mass spectrometer (LC/ESI-MS, Thermo Electron). HPLC fractions were assessed in presence of BDNF in TetOn-rhTrkB cells using KIRA-ELISA. Best candidates were chosen for further chemical synthesis. Cyclotraxin-B, L2-8, faf-cyclotraxin-B and tat- empty were purchased from BioS&T. Peptide sequences were as follows: cyclotraxin-B, CNPMGYTKEGC (cvclized by disulfide bridge between the two terminal cysteine residues); L2-8, CVPVSKGQLC (see {17)); faf-cyclotraxin-B, YGRKKRRQRRRCNPMGYTKEGC; faf-empty, YGRKKRRQRRR. Peptides were purified to the highest grade by reverse-phase high performance liquid chromatography (> 98.5%).

KIRA-ELISA: TrkB activity was quantified using the previously described KIRA-ELISA assay (16) with slight modifications. Briefly, cortical neurons were seeded on polyornith in-coated flat-bottom 96-well culture plates (12 x 10⁴ cells per well) and cultured eight days at 37°C in 5% CO2. TetOn-rhTrkB cells were seeded on flat-bottom 96-well culture plates (4 x 1 O⁴ CeIIs per well) and incubated overnight with 1000 ng/ml doxycycline except when indicated. Fluorescence levels in TetOn-rhTrkB cells were verified before each assay. Both cell types were carefully washed four times with Dulbecco's modified Eagle's medium (DMEM, Invitrogen) before being pre-treated for 30 min with ligands and stimulated for 20 min with 4 nM BDNF, in DMEM containing 0.5% BSA and 25 mM Hepes (pH 7.4). Assay was stopped by removing the medium on ice and membranes were solubilized. The following combination of antibodies was used for phosphorylation assays: TrkB capture with polyclonal anti-GFP (1 :5000, Clontech) for TetOn-rhTrkB, polyclonal anti-TrkB (1 μg/ml, Upstate) for cultured neurons and brain slices; tyrosine phosphorylation quantification with biotinylated anti-phosphotyrosine (4G10, 0.5 μg/ml; Upstate) and a streptavidin-peroxydase system (1 :4000; Amersham Biosciences). The following combination was used for total TrkB quantification : TrkB capture with monoclonal anti-GFP (1 :3000, Chemicon) for TetOn-rhTrkB, monoclonal anti-TrkB (1 :1000, BD Biosciences) for cultured neurons and brain slices; total TrkB quantification with polyclonal anti-GFP (1 :5000, Clontech) for TetOn-rhTrkB, polyclonal anti-TrkB (1 μg/ml, Upstate) for cultured neurons and brain slices followed by appropriate secondary antibodies and a peroxydase system. Absorbance was read at 450 nm after addition of 3,3',5',5-tetramethyl-benzidine substrate (Sigma) and acidification with 1 N hydrochloric acid.

lmmunoblot analysis: Cell lysates were prepared in boiling SDS (2%, w/v) as described previously (S4) and subjected to SDS-PAGE. elF4E and MAPK phosphorylation were detected and analysed using anti-total and -phospho-elF4E antibodies (1 :1000, Cell Signaling) or anti-total and -phospho-MAPK antibodies (1 :1000, Cell Signaling), respectively. For TrkB expression analysis, cortical neurons were cultured for eight days and TetOn-rhTrkB cells were incubated overnight with doxycycline. Cells were solubilized as for KIRA-ELISA assays and lysates were centhfuged at 14,000 x g for 20 min at 4°C. Cleared supernatant was collected and proteins were subjected to SDS-PAGE. Detection of rhTrkB-ECFP and neuronal mouse TrkB were performed using anti-GFP polyclonal antibody (1 :5000, Clontech) and anti-rodent TrkB polyclonal antibody (1 μg/ml, Upstate), respectively. Proteins were visualized using appropriate HRP-conjugated secondary antibodies and films were quantified by densitometry.

BDNF quantification: Endogenous BDNF was quantified in cortical neuron cultures by using the BDNF Emax immunoassay system (Promega).

Fluorescence: ECFP fluorescence quantification was read at 485 nm using a Mithras LB 940 counter (Berthold Technologies).

Immunofluorescence: Cortical neurons seeded on polyornith in-coated cover slips were fixed in 4% paraformaldehyde for 20 min and washed in glycine buffer (0.1

M, pH 7.4). Detection was performed using anti-rodent TrkB polyclonal antibody (1 μg/ml, Upstate) or anti-rodent MAP-2 monoclonal antibody (1 :500, Millipore) in saponin buffer (0.05%) supplemented with 0.2% BSA and 10% heat-inactivated fetal bovine serum and revealed using Alexa488-conjugated (1 :100, Interchim) or Cy3- conjugated (1 :200, Jackson) appropriate secondary antibodies.

Immunohistochemistry: Mice were injected twice with biotinylated tat- cyclotraxin-B. Three hours after the first injection, animals were deeply anesthetized with pentobarbital and perfused intracardiacally with a solution of saline followed by 4% paraformaldehyde. Brains were postfixed for 1 h in 4% paraformaldehyde and cut in 40-μm sections with a vibratome. Sections were rinsed in PBS and incubated in TBS containing 0.05% Tween-20 with VectaStain ABC Elite kit (Vector Laboratories). Signal was revealed using 3,3' diaminobenzidine (DAB). Non-injected mice were used as negative controls for endogenous biotin. Neurite outgrowth assessment: nnr5 PC12 cells were prepared as described elsewhere (S5). nnr5 PC12-TrkB, -TrkA and -TrkC cells were treated with BDNF (1 nM), NGF (2 nM) and NT-3 (10 nM. The number of cells bearing neurites longer than 2 cells in diameter was microscopically determined in each counting field (two fields per well, three wells per condition). Counting was performed blind each 24h for three days.

Metabolic labeling with [³⁵S]-methionine: Measurement of [³⁵S]-methionine incorporation was mainly performed as described previously (S4). Briefly, cortical neurons were washed twice and incubated with DMEM containing 0.5% BSA and 25 mM Hepes for 5-6 h. Neurons were incubated for 20 min with 4nM BDNF in the presence of 4 μCi/ml of [³⁵S]-methionine (1000 Ci/mmol, Amersham Biosciences).

Cyclotraxin-B (100 nM) and K252a (10 μM) were added for 30 min prior to BDNF stimulation. Neurons were washed twice and protein were precipitated with trichloroacetic acid (TCA; 10%, w/v). Amino acid uptake into neurons and incorporation into proteins were estimated by counting the radioactivity in the supernatant and the pellet, respectively. [³⁵S]-Methionine incorporation was calculated as the ratio of TCA-precipitable to TCA-soluble radioactivity. None of the treatments altered [³⁵S]-methionine uptake into neurons.

Assessment of cell injury: Neurotoxicity experiments were performed on cortical neurons (25 x 10⁴ cells per well in 24-well plates) cultured in presence of glial cells in Neurobasal medium supplemented with B27 (Gibco) for 10-14 days. Control medium, faf-cyclotraxin-B (200 nM) or BDNF (4 nM) were added to culture medium each 24h for 72h, alone or in combination. Neuronal necrosis was determined by measuring the leakage of LDH with respect to total LDH using the kit of Sigma, as described by the manufacturer. Cell survival was quantified by measuring the reduction of MTT into a blue formazan precipitate that is subsequently solubilized in dimethyl sulphoxide and read at 560 nm.

[¹²⁵I]-BDNF binding studies: Two micrograms of recombinant human BDNF (Peprotech) were dissolved in 50 mM phosphate buffer (pH 7.0). lodination was allowed by incubation with 0.5 mCi [¹²⁵I] and chloramine T and reaction was stopped by addition of sodium disulfite. The labeled BDNF was purified by PD-10 desalting columns (Amersham Biosciences) and fractions containing the [¹²⁵I]-BDNF were pooled (63.3μCi/μg). Competition binding studies were done mainly as previously described (S6). Briefly, 2.5-3 x 10⁵ cells or neurons were carefully washed with ice- cold binding buffer (DMEM containing 0.5% BSA and 25 mM Hepes) before being treated. Cells were then washed four times with ice-cold binding buffer and lyzed with 1 N NaOH. Radioactivity was counted in a 1260 MultiGammall counter (LKB Wallac).

Animals: Adult male Sprague-Dawley rats or Swiss mice (2-3 months-old and 4-6 weeks, respectively; Janvier) were used. All experimental procedures were carried out in accordance with the INSERM committee guidelines and European

Communities Council Directive (86/609/EEC, 24 November 1986) regarding the care and use of animals.

Electrophysiological recordings: Hippocampal slices (400 μm) were prepared as previously described (S7). Cyclotraxin-B was directly diluted in medium (1 μM) and slices were incubated for at least 30 min. Slices were then transferred to a submersion-type recording chamber equilibrated with 95% O2, 5% CO2, and submerged in a stream of medium containing 1 μM cyclotraxin-B for ~ 20 min prior to I/O curves and PPF recordings or to LTP induction. Extracellular recordings were performed at room temperature in apical dendritic layers of CA1 area using glass micropipettes filled with 2 M NaCI with a resistance of 2-6 MΩ. Field excitatory postsynaptic potentials (fEPSPs) were evoked by electrical stimulation of CA1 afferent Schaffer collaterals and commissural fibers in the stratum radiatum. Test stimuli (100 μs duration) were adjusted to get 30% of maximum fEPSP slope and applied every 15 seconds.

Long-term potentiation (LTP): LTP was induced by applying high frequency stimulation (HFS) to Schaffer collaterals (two 1 -sec trains at 100 Hz separated by 20 seconds) after baseline recording (at least 15 minutes or until stable). The magnitude of fEPSPs was determined by measuring the slope of fEPSPs. Three successive fEPSPs were averaged (each fEPSP was normalized to the mean of fEPSPs recorded before tetanus) and plotted across time using Pclamp9 software (DIPSI). Recording after single pulse testing was performed for at least 60 minutes following tetanus. Input/Output (I/O): Curves were constructed to assess the responsiveness of AMPA/Kainate glutamate receptor subtypes-dependent responses to electrical stimulation in slices. Slopes of three averaged presynaptic fiber volleys (PFVs) and fEPSPs were plotted as a function of stimulation intensity (400-2200 μA). Paired-pulse facilitation (PPF): PPF of synaptic transmission induced by paired-pulse stimulation was monitored at 40 ms inter-stimulus intervals. PPF was quantified by normalizing the slope of the second fEPSP by the slope of the first one.

Intravenous delivery of cyclotraxin-B and in vivo KIRA-ELISA analysis: Adult Swiss mice (20-25 g) received a double 90-min-interval i.v. injection of saline buffer, faf-cyclotraxin-B (2 x 200 μg) or faf-empty (2 x 200 μg). Three hours after the first injection, mice were decapitated, brains quickly removed on ice, 300-μm coronal sections were prepared with a vibratome at three levels (bregma 0.90 mm : caudate putamen / nucleus accumbens level; bregma -1.60 mm : dorsal hippocampus level; bregma -2.70 mm : ventral hippocampus level) and placed in a holding chamber for one hour. Slices were then incubated in Ringer's solution supplemented with tat- cyclotraxin-B or faf-empty (1 μM) in holding chamber saturated with 95% O₂, 5% CO2. After 45 min incubation, BDNF (4 nM) was added for 20 min. Experiments were stopped by quick removing of the medium on ice and addition of KIRA-ELISA solubilization buffer. Slices were homogenized, membranes solubilized overnight at 4°C and then stored at -20⁰C before determination of protein concentration and KIRA-ELISA assays.

Behavioral testing: Mice were treated as described above before being subjected to behavioral studies. Anxiety-related behavior was measured using the elevated plus-maze (EPM) test. Antidepressant-like action of tat-cyclotraxin-B was assessed with the forced-swim test (FST). Effects of acute treatment with tat- cyclotraxin-B on spontaneous locomotion were tested using locomotor activity assessment procedures. Each mouse received a double 90-interval i.v. injection of tat-cyclotraxin-B or saline buffer before being placed in EPM for 5 min to assess anxiety-related behaviors (26 cm high, 5 cm-wide x 36 cm-long arms). The diazepam control group consisted of saline buffer double-treated mice that further received a 30-min i.p. injection of diazepam (1 mg/kg). Mice were assessed for number of entries and amount of time spent in open versus closed arms. The FST procedure was applied to treated mice as described by Porsolt et al.,1977. Mice were dropped individually into glass cylinder (height, 25 cm; diameter, 10 cm) containing 10 cm water height, maintained at 23-25°C. Animals were tested for a total of 6 min. The total time of swimming, the number of climbing attempts and the time of immobility was recorded during the last 4 min of the session, after 2 min of habituation. The paroxetine control group consisted of saline buffer double-treated mice that further received a 30-min i.p. injection of paroxetine (2 mg/kg). Behaviors were recorded on videotape and scored blind. Spontaneous locomotor activity was assessed for 30 min by numbering infrared crossed beams in an actimeter (Imetronic), consisting in individual boxes placed in a quiet room.

Statistical analysis: Concentration-response and competition curves were obtained using an iterative least-squares method derived from that of Parker & Waud (1971 ). Eadie-Hofstee plotting of the data provided estimates for BDNF EC₅O in presence or not of cyclotraxin-B and IC₅O values of peptides L2-8, cyclotraxin-B and faf-cyclotraxin-B. KIRA-ELISA competition studies, protein synthesis, elF4E phosphorylation, HFS-induced LTP and survival assessments (MTT and LDH release methods) were analyzed using one-way ANOVA followed by Newmann-Keuls multiple comparison post-hoc test. Concentration-response curves and Eadie- Hofstee plots were analyzed using two-way ANOVA. Significance over time in locomotor activity studies was given using two-way ANOVA followed by Bonferroni's post-hoc test. Cyclotraxin-B reversibility and 30-min cumulative counts in locomotor activity were analyzed using Student's t test. One-way ANOVA followed by post-hoc Dunnett's test was used to evaluate statistical significance compare to saline group in EPM tests, n represents the number of independent experiments.

Results

After having designed several BDNF-dehved mimetic peptides which were tested for their ability to inhibit TrkB, the inventors have described the inhibitory properties of a small polypeptide mimicking the reverse turn structure of the variable region III that protrudes from the core of BDNF.

More particularly, the inventors underlined that a cyclic polypeptide behaves as a highly potent and specific allosteric antagonist of TrkB receptor. The main feature of said cyclic polypeptide lies in its capacity to inhibit both basal and BDNF- induced TrkB activity. Such cyclic polypeptide inhibits TrkB-dependent molecular and cellular events such as long-term potentiation, cap-dependent protein translation, elF4E phosphorylation and neurite outgrowth. Thus, in order to evaluate the effect of cyclotraxin-B on TrkB, the inventors developed a modified version of KIRA-ELISA (16), a rapid, sensitive and high- capacity assay that quantifies phospho-tyrosine level of TrkB. In a first set of experiments, cyclotraxin-B was assayed using doxycycline-inducible TetOn cells expressing recombinant human TrkB receptors (TetOn-rhTrkB). Cyclotraxin-B was found to inhibit BDNF-induced TrkB activity through an uncompetitive mechanism with a very high potency (< nanomolar), that is three-order of magnitude higher than that of the previously described peptide L2-8 (17). Moreover, binding of [¹²⁵I]-BDNF was not altered by cyclotraxin-B, suggesting further that cyclotraxin-B is an allosteric modulator of TrkB receptors. When cyclotraxin-B was tested on native TrkB receptors from mouse cortical neurons, both amplitude of inhibition and potency were enhanced (1.6-fold and one-order of magnitude higher, respectively), while remaining uncompetitive. The enhancement of inhibition observed in neurons may result from slight sequence differences between mouse and human forms of TrkB. This may also be explained by the presence of p75^NTR in cortical neurons (18) but not in TetOn- rhTrkB, which is known to alter Trks conformation and provide greater discrimination for its cognate ligand (19), as illustrated by the difference in both affinity and activity observed between BDNF and NT-3 in neurons but not in TetOn-rhTrkB cells. Surprisingly, contrary to L2-8, cyclotraxin-B also decreased a BDNF-independent (basal) TrkB activity in both recombinant and neuronal cells with the same relative inhibition as in presence of BDNF. Endogenously synthesized BDNF was not involved in this apparent basal activity since neutralizing anti-BDNF antibody did not alter cyclotraxin-B effects and BDNF was not detectable by ELISA. This spontaneous activity may result from transactivations by G-protein coupled receptors (GPCR) (2, 3), zinc (5), endocannabinoids (4) but also from increases in TrkB density that lead to spontaneous autophosphorylation, as already proposed for TrkA (20). Taking advantage of the inducible heterologous TetOn-rhTrkB system, the inventors investigated the effect of cyclotraxin-B among TrkB densities. As expected, a threshold in TrkB expression was needed to detect a basal TrkB activity and, as soon as a basal activity was measurable, the relative inhibition of cyclotraxin-B was stable at all TrkB densities. Same results were observed in the presence of BDNF. Together, these data suggest that cyclotraxin-B interfere with TrkB conformation, thereby hampering proper activation of the receptor. Next, we analyzed the reversibility of inhibitory effects of cyclotraxin-B. The t_/2 of cyclotraxin-B was ~3 hours in TetOn-rhTrkB cells and ~6 hours in neurons, demonstrating long-lasting effects of cyclotraxin-B after its withdrawal. These relative high values may reflect the great potencies of cyclotraxin-B on both cell types. Similarly, the significant differences in t_/2 observed between both cell types may be explained by differences in potencies.

The inventors next extended the evaluation of cyclotraxin-B effects to TrkB- related cellular processes. BDNF can promote neurite outgrowth, notably in nnr5 PC12-TrkB cells (21), through activation of the MAP-Kinase pathway. This increase in neurite length and number of branch points was lowered by cyclotraxin-B with similar pharmacological properties to that of neurons in KIRA-ELISA assays. This effect was linked to the dramatic decrease in BDNF-induced MAP-Kinase phosphorylation. As no neurite outgrowth and phospho-MAPK were detected in absence of BDNF, no cyclotraxin-B effect could be assessed in this experimental condition. Cyclotraxin-B was specific for TrkB receptors since concentrations up to 1 μM did not affect NGF- nor NT-3-induced neurite outgrowth in nnr5 PC12-TrkA and - TrkC, respectively. Moreover, morphological analysis ruled out toxicity effects of cyclotraxin-B and no cell death was observed even 72 hours after exposure to 10 μM cyclotraxin-B.

Induction and maintenance of the late phase of long-term potentiation have been linked to BDNF-induced protein synthesis in neurons, eliciting in part its role in the long-term modulation of synaptic efficacy and plasticity (6). This effect has been linked to cap-dependent translation in neurons through phosphorylation of the eukaryotic initiation factor 4E (elF4E) (6). The BDNF-induced increase in newly synthesized protein level, reflected by a higher [³⁵S]-methionine incorporation in cortical neurons, was significantly reduced by co-treatment with cyclotraxin-B. Of interest is the decrease in basal rate of protein synthesis by cyclotraxin-B but also by the non selective Trk inhibitor K252a. These results combined with the observation that neither BDNF nor cyclotraxin-B showed any effect on non-induced TetOn-rhTrkB cells suggest that a part of basal rate of protein synthesis depends on basal TrkB activity. Similarly, cyclotraxin-B significantly reduced both BDNF-induced and basal phosphorylation of elF4E in cultured cortical neurons. As expected, K252a deeply inhibited the phosphorylation of the initiation factor. In fact, cyclotraxin-B revealed a linear correlation between elF4E phosphorylation level and the rate of protein synthesis, strongly suggesting a direct link between these two cellular events and TrkB activation. Since long-term potentiation (LTP) at the Schaffer collateral-CA1 synapses requires BDNF derived from presynaptic CA3 neurons (22), attempts were made to determine whether cyclotraxin-B was able to hamper tetanus-induced LTP in this neural network. High frequency stimulation (HFS) applied to hippocampal slices induced LTP in both control and cyclotraxin-B-treated slices LTP (P < 0.001 for control; P < 0.01 for cyclotraxin-B, 50-60 min after HFS). This potentiation was significantly decreased in presence of cyclotraxin-B even one hour after HFS (P < 0.01 ). As previously observed by Kang and colleagues using function-blocking TrkB antiserum (23), cyclotraxin-B did not alter either synaptic strength or presynaptic functions, as evaluated using input-output curves and paired-pulse facilitation, respectively. This corroborates the idea that basal synaptic transmission is not modified by acute manipulation of TrkB function. Altogether, these results indicate that cyclotraxin-B counteracts TrkB-dependent LTP without hampering normal synaptic transmission at Schaffer collateral-CA1 synapses.

Recent reports have suggested that sustained activation of TrkB receptors could lead to enhanced neuronal vulnerability to toxic insults (24). Transgenic mice overexpressing BDNF in noradrenergic neurons have smaller anterior cortex than that of wild-types animals and an elevated TrkB activation in this area (25). Moreover, other studies proposed that susceptibility of motor neurons to excitotoxic insults in amyotrophic lateral sclerosis (ALS) may be due to both BDNF- and A2a- induced TrkB activation (8). These topical observations suggest that BDNF/TrkB hyperactivity is related to atrophy of many brain structures in neuropsychiatric disorders but also to motoneurons degeneration in ALS. In this context, cyclotraxin-B demonstrated protective effects against neuronal necrosis induced in vitro by repeated exposure to BDNF, as quantified by lactate dehydrogenase (LDH) release in culture medium. Addition of cyclotraxin-B alone to culture medium did not alter basal neuron survival, demonstrating that basal TrkB activity is not sufficient to promote neurons survival in our culture conditions. Similar conclusions were drawn from cell survival experiments using MTT (methylthiazolyldiphenyl-tetrazolium bromide) method. While prolonged exposure to BDNF led to a loss of 34.3 ± 4.7 % of total neurons (P < 0.001 ), co-treatments with cyclotraxin-B fully prevented neuronal death (113.4 ± 7.8 %; P < 0.001 ). Long exposure to high concentrations of BDNF may lead to activation-dependent down-regulation of its receptor which in turn results in cell desensitization to TrkB-dependent survival. In fact, immunostaining revealed a TrkB signal disappearance, mostly confined to neurites, in cortical neurons treated for prolonged times with BDNF. This phenomenon was not observed in neurons co- treated with cyclotraxin-B, suggesting that cyclotraxin-B can protect neurons from BDNF-induced necrosis by preventing excessive down-regulation of TrkB receptors. Moreover, as cyclotraxin-B is a partial antagonist, it preserves a minimal level of activated TrkB receptors necessary to the survival of neurons.

Finally, the inventors fused the transduction domain of the tat protein from the HIV type 1 to cyclotraxin-B (faf-cyclotraxin-B) in order to allow its delivery to the brain after intravenous injection (26). KIRA-ELISA analyzes in TetOn-rhTrkB cells and in cortical neurons showed that pharmacological properties of faf-cyclotraxin-B were similar to that of cyclotraxin-B. However, in brain slices, faf-cyclotraxin-B inhibited TrkB receptors with a better efficiency than cyclotraxin-B (cyclotraxin-B, -29.8 ± 5.6 %, n = 9; faf-cyclotraxin-B, -51.2 ± 3.6 %, n = 12; P < 0.05), while faf-empty did not produce any effect. This may reflect the plasma membrane permeability of the tat- fused cyclotraxin-B that facilitates its penetration into the slice. Moreover, tat- cyclotraxin-B may also target intracellular active TrkB²⁷. Four hours after the last intravenous injection to adult mice, faf-cyclotraxin-B was found in many brain structures and brain TrkB phosphorylation was dramatically decreased as compared to saline or faf-empty-treated animals. Similar results were found in all tested brain regions (caudate putamen/nucleus accumbens, dorsal or ventral hippocampus). Remarkably, even with very different administration kinetics (minutes for incubation; hours for injection), the decrease in TrkB activity was not significantly different when faf-cyclotraxin-B was injected to animals or directly added to the slice. This latter observation illustrates the long-lasting effects of faf-cyclotraxin-B in vivo.

Then, the inventors focused the study of adult mice injected with tat- cyclotraxin-B on anxiety-like and depression-like behaviors. First, analyzing spontaneous locomotor activity revealed a significant hypolocomotor effect of tat- cyclotraxin-B at the dose used (15-20 mg/kg), as compared with saline (F₁J₂S = 27.85, P<0.0001 ). Post hoc analysis showed a significant decrease in the locomotion of animals treated with tat-cyclotraxin-B in the first ten minutes while the cumulative measurement for 30 min showed a 40% decrease in the overall spontaneous activity. Mice were also subjected to the forced swim test, a putative model for depression. As for many antidepressant compounds, mice treated with paroxetine demonstrated significant decreased time of immobility and increased climbing attempts. This escape behavior was not observed for mice that received saline solution or tat- cyclotraxin-B, suggesting that cyclotraxin-B does not possess antidepressant-like activity. Noteworthy, swimming capabilities were not affected in mice treated with tat- cyclotraxin-B, suggesting that the hypolocomotor effect previously observed was not due to some peripheral alterations but rather to central effects. Effects of acute treatment with tat-cyclotraxin-B were also assessed on anxiety-related behaviors using the elevated-plus maze procedure. Remarkably, mice that received tat- cyclotraxin-B exhibited a significant increase in the time spent in open arms similar to that of diazepam, a benzodiazepine commonly used to treat anxiety in humans, suggesting an anxiolytic-like effect of cyclotraxin-B. Contrary to diazepam, there was no difference in the number of entries in open arms for tat-cyclotraxin-B mice, leading to a greater time spent/number of entries ratio (2.2-fold increase compare to saline; P<0.01 ). This observation may be related only in part to the hypolocomotor effect of tat-cyclotraxin-B since these mice did not spend more time in closed arms.

Overall, these behavioral studies further demonstrated the specificity of central effects elicited by tat-cyclotraxin-B after intravenous injections and confirmed its pharmacological potential in vivo.

Since the discovery of the first antidepressant and neuroleptic agents, very few new therapeutic agents have been proposed for the treatment of psychiatric or neurodegenerative diseases. Increasing topical literature now proposes BDNF and TrkB as promising targets for the treatment of various brain disorders {7-13) but also for the treatment of angiogenesis, tumor formation and metastasis in cancer (28). Thus, as the first potent and specific modulator of TrkB receptors that can be easily administered in vivo, cyclotraxin-B leads to new prospects for innovative therapeutic strategies. As well, its ability to inhibit basal activity will help to investigate thoroughly TrkB transactivation processes in physiological functions but also in the etiology of neurological pathologies, such as epileptogenesis (7) and ALS (8), without hampering other GPCR effects.

In most cases, the involvement of BDNF or TrkB in these diseases is based on the observation of an excess or a lack of the BDNF/TrkB coupling either in human post-mortem tissues or in genetic animal models. These data are especially confused for some psychiatric diseases, such as schizophrenic psychoses (13), in which the effect of a modulator of TrkB activity may be difficult to predict. In this context, cyclotraxin-B may represent a great tool to evaluate the interest of a systemic pharmacological intervention on TrkB receptors in pathologies with altered BDNF/TrkB signaling.

The sustained effect elicited by cyclotraxin-B after transient application both in vitro and in vivo is warranted by its high affinity and slow reversibility but also by its short size that reduces protease-sensitivity. However, long-term treatments with TrkB modulators may have unexpected consequences on receptor internalization and recycling. Such a modulation of activity may alter the level of plasma membrane- bound TrkB receptors, which will be sensitive to the newly-released BDNF, making risky any prediction of long-term effects.

As a partial allosteric antagonist, cyclotraxin-B should avoid deleterious effects of a full inhibitor. Indeed, BDNF and its receptor have important trophic and protective effects on neuronal cells so that a minimal level of TrkB activity must be preserved. Moreover, in some conditions in which a BDNF/TrkB hyperactivity increases neurons vulnerability to excitotoxic insults, cyclotraxin-B behaves as a neuroprotective agent by preventing excessive activation-induced TrkB endocytosis while maintaining them, however, in a minimal activation state. Altogether, these observations suggest that cyclotraxin-B represents a promising tool for the study of the BDNF/TrkB signaling in physiological and pathophysiological functions and will serve as a lead compound for the development of new therapeutic strategies.

The pharmacology of recombinant human and neuronal mouse TrkB receptors was analyzed using KIRA-ELISA assay. As expected, recombinant TrkB in TetOn- rhTrkB cells indistinctively responded to BDNF and NT-3 (ratio signal/background = 3.9 for BDNF and 3.8 for NT-3). In TrkB + p75NTR co-expressing cortical neurons, NT-3 behaved as a partial agonist (ratio signal/background = 2.1 for BDNF and 1.7 for NT-3). Similarly, whereas EC50 of BDNF and NT-3 remained almost the same in recombinant TetOn-rhTrkB cells (BDNF, 614 ± 123 pM; NT-3, 702 ± 134 nM), neuronal TrkB receptors were significantly more selective to BDNF than to NT-3 (BDNF, 208 ± 80 pM; NT-3, 716 ± 167 nM; P = 0.0247). NGF did not produce any signal neither in recombinant nor in neuronal cells. BDNF signal was fully reversed by the non-selective tyrosine kinase inhibitor K252a. REFERENCES:

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Claims

CLAIMS:

1. An isolated polypeptide derived from Brain-Derived Neurotrophic Factor (BDNF) comprising an amino acid sequence consisting of:

X1 -P-M-G-Y-T-X2-E-G (SEQ ID NO:1 )

wherein amino acid X1 is H, K, N or R; and

X2 is D, E, H, K or R.

2. The polypeptide according to claim 1 comprising an amino acid sequence consisting of NPMGYTKEG (SEQ ID NO: 2).

3. The polypeptide according to claim 1 comprising cysteine residues at terminal ends of the amino sequence of said polypeptide.

4. The polypeptide according to claim 2 comprising an amino acid sequence consisting of CNPMGYTKEGC (SEQ ID NO: 4).

5. A cyclic polypeptide wherein the polypeptide according to claim 2 or claim 3 is cyclised via a disulfide bound between the two cysteine residues.

6. The cyclic polypeptide according to claim 4 having the formula (SEQ ID NO: 6) of:

CNPMGYTKEGC

7. The cyclic polypeptide according to claim 4 or claim 5 comprising a cell penetrating peptide sequence.

8. The cyclic polypeptide according to claim 7 having the formula (SEQ ID NO: 9) of:

YGRKKRRQRRRCNPMGYTKEGC

9. An isolated nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 3.

10. A vector comprising a nucleic acid according to claim 7.

11.A host cell, which has been transformed by a nucleic acid according to claim 7 or a vector according to claim 8.

12.A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 4 or a cyclic polypeptide according to any one of claims 5 to 8 eventually associated with a pharmaceutically acceptable vehicle.

13. A polypeptide according to any one of claims 1 to 4 or a cyclic polypeptide according to any one of claims 5 to 8 or a pharmaceutical composition according to claim 12 for the prevention and/or treatment of disorders related to tropomyosin-related kinase B receptor (TrkB).

14. The polypeptide or the cyclic polypeptide or the pharmaceutical composition according to claim 13, wherein said disorder is chosen among the group consisting of neurological disorders, psychiatric disorders and cancer.