WO2014049153A1 - Antagonists of the 5-ht2b receptor for use in the prevention or treatment of spasticity - Google Patents

Abstract

The present invention also relates to 5-HT_2B receptor antagonists or inhibitors of 5-HT_2B receptor gene expression for use in the prevention or treatment spasticity in a patient affected with a degenerative disorder of the CNS and to pharmaceutical compositions thereof.

Description

ANTAGONISTS OF THE 5-HT_2B RECEPTOR

FOR USE IN THE PREVENTION OR TREATMENT OF SPASTICITY

FIELD OF THE INVENTION:

The present invention relates to 5-HT_2B receptor antagonists or inhibitors of 5-HT_2B receptor gene expression for use in the prevention or treatment of spasticity in a patient affected with a degenerative disorder of the CNS. BACKGROUND OF THE INVENTION:

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative condition involving the loss of lower motor neurons (LMNs) in the brainstem and spinal cord, and of upper motor neurons (UMNs) in the motor cortex. It is characterized by progressive weakness, atrophy and spasticity, leading to paralysis and respiratory failure within five years of onset. Familial ALS accounts for 10% of all ALS cases; approximately 25% of these cases are due to mutations in the Cu/Zn superoxide dismutase gene (SOD1). SOD1 is a mainly cytoplasmic enzyme that catalyzes the breakdown of superoxide ions to oxygen and hydrogen peroxide, which in turn is degraded by glutathione peroxidase or catalase to form water. Several lines of evidence argue that the mutant SOD1 protein is neurotoxic through an acquired, adverse function that entails both oxidative pathology and protein aggregation, with secondary disturbances of glutamate metabolism, mitochondrial function, axonal transport and calcium homeostasis. Other genes have recently been linked to ALS, including TARDBP, FUS and C90RF72, but the mechanisms underlying the pathogenicity of their mutations await further investigations.

Spasticity is a symptom of many motor diseases that consists in velocity-dependent increase in muscle tone and exaggerated muscle responses to stretching. Spasticity develops either after trauma, in particular spinal cord injury (SCI), or in the course of degenerative diseases such as ALS, a fatal neurodegenerative disorder affecting upper and lower motor neurons (Kiernan et al, 2011). Spasticity represents the major phenotype of the upper motor neuron predominant subtype of ALS called primary lateral sclerosis (PLS), and might be underrecognized in other ALS patients since the physiological basis for detecting spasticity is disrupted by the degenerative process involving motor neurons of all classes (Swash, 2012). Spasticity is a painful and disabling symptom, and treatment options remain limited, especially in ALS and PLS patients (Ashworth et al, 2012).

Mechanisms of spasticity have been mostly studied after SCI. In the current view, SCI-associated spasticity arises from several mechanisms, a major one being injury to serotonergic axons. Indeed, serotonergic axons, descending from several brainstem serotonergic nuclei, densely innervate LMNs, and maintain motor neuron excitability through increased persistent calcium current (Heckman et al, 2009). After SCI, the transection of serotonergic axons leads to transient hypo excitability of LMNs. After a few weeks, LMNs compensate for loss of serotonin input through the production of constitutively active 5-HT_2B and 5-HT_2C receptors, leading to an intrinsic hyperexcitability and subsequent spasticity (Murray et al, 2011, Murray et al, 2010).

In ALS, degeneration of UMNs, whose axons form the cortico-spinal tract, is traditionally thought to cause spasticity as part of the "UMN syndrome" (Ivanhoe and Reistetter, 2004) but direct evidence linking UMNs and spasticity in ALS are lacking. Other hypotheses, in particular the implication of serotonin neurons, have not been explored so far. Indeed, studies on serotonergic involvement in ALS are scarce and limited. Early studies focusing on the quantification of serotonin and its metabolites yielded inconsistent results, most likely due to the very limited numbers of post-mortem brain tissues included (Bertel et al, 1991, Forrest et al, 1996, Sofic et al, 1991). More recent imaging studies have shown decreased binding of serotonin 1A (5-HT1A) ligands in ALS raphe and cortex (Turner et al, 2007, Turner et al, 2005). Recently, it was found that platelet serotonin levels were significantly decreased in ALS patients (in a cohort of 85 patients with ALS and a control group of 29 healthy subjects), and that higher platelet serotonin levels were positively correlated with increased survival of the patients (Dupuis et al, 2010), suggesting that serotonin might influence the course of ALS disease. However, investigation of a direct involvement of central serotonin in ALS has not been performed until now. To date, spasticity in degenerative diseases of the central nervous system, such as ALS or multiple sclerosis, is a condition which often resists current treatments and which can result in chronic pain and disability. Moreover, spasticity is only part of the spectrum of the symptoms of ALS, and up to now there is no treatment modifying disease progression in ALS. Thus, there is an unsatisfied need for drugs useful to prevent or treat degeneration of the motor neurons in ALS patients as well as drugs to prevent or treat spasticity in patients affected with a degenerative disorder of the CNS including ALS.

SUMMARY OF THE INVENTION:

The present invention relates to a 5-HT_2B receptor antagonist or an inhibitor of 5-HT_2B receptor gene expression for use in the prevention or treatment of spasticity in a patient affected with a degenerative disorder of the CNS.

The present invention also relates to a pharmaceutical composition for use in the prevention or the treatment of spasticity in a patient affected with a degenerative disorder of the CNS comprising a 5-HT_2B receptor antagonist or an inhibitor of 5-HT_2B receptor gene expression.

DETAILED DESCRIPTION OF THE INVENTION:

The present inventors have observed that central serotonin neurons degenerate during amyotrophic lateral sclerosis (ALS). From a functional point of view, animal studies also suggest that spasticity arise from serotonergic loss, i.e. serotonin depletion leads to overexpression of 5-HT_2B receptor and subsequent constitutive activity of this receptor during development of spasticity. They have further shown in SOD1 (G86R) mice that the use of antagonist or reverse agonist to the 5-HT_2B receptor represents a potential treatment for ALS and spasticity in patients affected with a degenerative disorder of the central nervous system including ALS, hereditary spastic paraplegia, primary lateral sclerosis and multiple sclerosis.

First, the inventors performed a pathology study in 7 ALS patients and 6 controls and observed that central serotonin neurons suffer from a degenerative process with prominent neuritic degeneration, and sometimes loss of cell bodies in ALS patients. Moreover, distal serotonergic projections to spinal cord motor neurons and hippocampus systematically degenerated in ALS patients.

Secondly, they showed in SOD1 mice, a transgenic model of ALS, serotonin levels were decreased in brainstem and spinal cord before onset of motor symptoms. Furthermore, there was noticable atrophy of serotonin neuronal cell bodies along with neuritic degeneration at disease onset. They showed in vivo that degeneration of serotonin neurons could underlie spasticity in ALS by using tail muscle spastic-like contractions in response to mechanical stimulation as a measure of spasticity. In SOD1 mice, tail muscle spastic-like contractions in response to mechanical stimulation as a measure of spasticity were observed at end-stage.

Thirdly and importantly, these contractions were abolished by 5HT_2B/_C inverse agonists. In line with this, 5HT_2B receptor expression was strongly increased at disease onset.

Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs.

According to the invention, the terms "serotonin" or "5-hydroxytryptamine" (5-HT) are used interchangeably and have their general meaning in the art. They refer to a monoamine neurotransmitter produced primarily in the gastrointestinal (GI) tract, platelets, and in the central nervous system (CNS) and acting on its receptors called serotonin receptors.

As used herein, the term "serotonin receptors" also known as 5-hydroxytryptamine receptors or 5-HT receptors, refers to a group of G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs) that activate an intracellular second messenger cascade to produce an excitatory or inhibitory response. The serotonin receptors are activated by the neurotransmitter serotonin, which acts as their natural ligand.

According to the invention, the term "5-HT_2B receptor" has its general meaning in the art and refers to the serotonin receptor type 2B (a subtype of the receptor 5-HT₂ with the subtypes 5-HT_2A and 5-HT_2A) . 5-HT_2B receptor can be from any source, but typically is a mammalian (e.g., human and non-human primate) 5-HT_2B receptor, particularly a human 5- HT_2B receptor. An exemplary native human 5-HT_2B receptor amino acid sequence is provided in UniProt database under accession number P41595 and an exemplary native human nucleotide sequence encoding for 5-HT_2B receptor is provided in GenBank database under accession number NM 000867.4.

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.

The term "5-HT_2B receptor antagonist" should be understood broadly and encompasses any substance able to prevent the action of serotonine (5-HT) on its receptor 5-HT_2B.

In the context of the invention, when 5-HT_2B receptor antagonists are small organic molecules, said antagonists are preferably selective for the 5-HT_2B receptor as compared with the other 5-HT receptors, including the 5-HT₂ receptor subtypes (i.e. 5-HT_2A and 5-HT_2C receptors). By "selective" it is meant that the affinity of the antagonist for the 5-HT_2B receptor is at least 10-fold, preferably 100-fold, more preferably 500-fold, still preferably 1000-fold higher than the affinity for the other 5-HT₂ receptors (5-HT_2A and 5-HT_2C receptors).

The affinity of an antagonist for 5-HT_2B receptor may be quantified by measuring the activity of 5-HT_2B receptor in the presence a range of concentrations of said antagonist in order to establish a dose-response curve. From that dose response curve, an IC₅₀ value may be deduced which represents the concentration of antagonist necessary to inhibit 50% of the response to an agonist in defined concentration. The IC₅₀ value may be readily determined by the one skilled in the art by fitting the dose-response plots with a dose-response equation as described by De Lean et al. (1979). IC₅₀ values can be converted into affinity constant (Ki) using the assumptions of Cheng and Prusoff (1973).

The term "small organic molecule" refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e.g., proteins, nucleic acids...). Preferred small organic molecules range in size up to about 5 kDa, more preferably up to 2 kDa, and most preferably up to about 1 kDa.

As used herein, the term "amyotrophic lateral sclerosis" (ALS) refers to a progressive, neurodegenerative condition involving the loss of motor neurons in the brainstem and spinal cord and of upper motor neurons in the motor cortex. It is characterized by progressive weakness, atrophy and spasticity, leading to paralysis and respiratory failure within five years of onset.

As used herein, the term "spasticity" refers to a common and disabling symptom observed in patients with central nervous system diseases, including ALS, a disease affecting both upper and lower motor neurons. It is a state of increased muscular tone with exaggeration of the tendon reflexes from an upper motor neuron (brain or spinal cord) injury in which spinal inhibitory processes are suppressed or lost. The result is chronic, severe spasm of the muscles of the extremities hindering function and causing pain. Therapeutic methods and uses

In a first aspect, the invention relates to a 5-HT_2B receptor antagonist for use in the prevention or treatment of spasticity in a patient affected with a degenerative disorder of central nervous system (CNS).

In a particular embodiment, such a degenerative disorder of the CNS is selected from the group consisting of amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegia, primary lateral sclerosis and multiple sclerosis.

In one embodiment, said 5-HT_2B receptor antagonist may be a low molecular weight antagonist, e. g. a small organic molecule.

In particular, suitable antagonists to the 5-HT_2B receptor can be specific to 5-HT_2B or not, and include reverse agonists . So, said 5-HT_2B receptor antagonist particularly encompasses classical 5-HT_2B receptor antagonist which are small organic molecules well known in the art. Said molecules may have an antagonist effect on different 5-HT receptor or on different subtypes of the 5-HT₂ receptor (i.e. 5-HT_2B/5-HT_2C antagonist or 5-HT_2B/5-HT_2A antagonist), or only on the 5 -HT_2B receptor subtype (selective 5 -HT_2B receptor).

In a particular embodiment of the invention, said 5-HT_2B receptor antagonist is a 5- HT_2B/5-HT_2C antagonist.

These antagonists include the dibenzo[a,e]cycloheptatriene described in US3,014,911, the condensed indole derivatives described in WO 94/04533, the indole derivatives described in WO 96/23783, the indolo(l,7-bc)(2b)-naphthyridine derivatives described in EP 0473550, the indole ureas described in WO 92/05170, the heterocyclic urea derivatives as described in WO 94/14801 and the indole and indoline derivatives as described in W096/11929.

Typically, the 5-HT_2B receptor antagonist of the invention may be selected from the group consisting of cyproheptadine, SB 206553 (5-HT_2B/5-HT_2C antagonist), SB 228357 (5- HT_2B/5-HT_2C antagonist), SB 221284 (5-HT_2B/5-HT_2C antagonist), SB 200646 (5-HT_2B/5- HT_2C antagonist), SDZ SER-082 (5-HT_2B/5-HT_2C antagonist), sarpogrelate (Anplag®) (5- HT_2A/5-HT_2B antagonist) and methysergide.

Particularly, said 5-HT_2B receptor antagonist is cyproheptadine (US3,014,911 and US3,851 ,059), described by the following formula:

In a particular embodiment, the antagonists are the condensed indole derivatives described in the International Patent Application WO 94/04533.

According to this particular embodiment, the antagonists are selected from those described in the International Patent Application WO 94/04533 and that are listed below: 5-Methyl- 1 -(3-pyridylcarbamoyl)-2,3-dihydropyrrolo[2,3-fJindole,

6-Methyl-3-(3-pyridylcarbamoyl)-2,3-dihydropyrrolo[3,2-e]indole,

5, 6-Dimethyl-l-(3-pyridylcarbamoyl)-2,3-dihydropyrrolo[2,3-fJ indole,

l-(3-Pyridylcarbamoyl)-2,3-dihydropyrrolo[2,3-fJ indole,

6-Methyl-3-(4-pyridylcarbamoyl)-2,3-dihydropyrrolo[3,2-e]indole,

6-Methyl-3-(2-pyridylcarbamoyl)-2,3-dihydropyrrolo[3,2-e]indole,

5-Methyl-l -(2-pyridylcarbamoyl)-2,3-dihydropyrrolo[2,3-fJindole,

5-Methyl-l-(4-pyridylcarbamoyl)-2,3-dihydropyrrolo[2,3-fJ indole,

5-Methyl-l-(3-pyridylcarbmaoyl)-2,3,6,7-tetrahydropyrrolo[2,3-fJindole,

5-Ethyl-l-(3-pyridylcarbamoyl)-2,3-dihydropyrrolo[2,3-fJ indole,

5-n-Propyl-l-(3-pyridylcarbamoyl)-2,3-dihydropyrrolo[2,3-fJindole,

5.6- Dimethyl-l-(3-pyridylcarbamoyl)-2,3-dihydropyrrolo[2,3-fJ indole,

6.7- Dimethyl-3-(3-pyridylcarbamoyl)-2,3-dihydropyrrolo[3,2-e]indole,

l-Methyl-N-(3-pyridyl)-5,6,7,8-tetrahydro-lH-pyrrolo[2,3-g]quinoline-5-carboxamide, 3-Methyl-N-(3-pyridyl)-6,7,8,9-tetrahydro-3H-pyrrolo[3,2-f]quinoline-6-carboxamide, 6-Methyl-3-(2-methyl-4-quinolinylcarbamoyl)-2,3-dihydropyrrolo[3,2-e]indole, 6-Methyl-3-(5-quinolinylcarbamoyl)-2,3-dihydro-pyrrolo[3,2-e]indole,

6-Methyl-3-(3-quinolinylcarbamoyl)-2,3-dihydropyrrolo[3,2-e]indole,

5- Methyl-l-(2-methyl-4-quinolinylcarbamoyl)-2,3-dihydropyrrolo[2,3-f] indole, 6,8-Dimethyl-3-(3-pyridylcarbamoyl)-2,3-dihydropyrrolo[3,2-e]indole,

6- Methyl-3-(3-pyridylcarbamoyl)-2,3,7,8-tetrahydropyrrolo[3,2-e]-indole,

5-Methyl-l-(2-pyrazinylcarbamoyl)-2,3-dihydropyrrolo[2,3-f] indole,

2,3-Dihydro-5-methyl-l-(3-methyl-5-isothiazolylcarbamoyl)-lH-pyrrolo[3,2-e]indole, 2,3-Dihydro-5-methyl-l-(3-methyl-5-isothiazolylcarbamoyl)-lH-pyrrolo[2,3-f]indol^ 2, 3-Dihydro-5-methyl-l-(5-quinolylcarbamoyl)-lH-pyrrolo[2,3-f] indole,

2,3-Dihydro-5-methyl-l-(3-methyl5-isoxazolylcarbamoyl)-lH-pyrrolo[2,3-f]lindole, N-(5-Isoquinolyl)-5-methyl-2,3-dihydropyrrolo[2,3-f]indole-l-carboxamide,

N-(6-Quinolyl)-5-methyl-2,3-dihydropyrrolo[2,3-f]indole-l-carboxamide,

or a pharmaceutically acceptable salts thereof.

Preferably, said 5-HT_2B receptor antagonist is the 5-methyl-l-(3-pyridylcarbamoyl)- l,2,3,5-tetrahydropyrrolo[2,3-fJindole (also known as SB206553) (International Patent Application WO 94/04533), described by the following formula:

In another particular embodiment of the invention, said 5-HT_2B receptor antagonist is a selective 5 -HT_2B receptor.

These antagonists include the substituted 2-oxazolamines described in WO03/068226 and WO2005/016338, the substituted aryl pyrimidines described in W097/4436, the substituted tetrahydro-beta-carbo lines described in WO95/24200 and W096/244351, the pyrazole-3-carboxamide derivatives described in WO2010/058858 and US2011/275628, the piperidine or acridine derivates described in US7,060,711 and US US7,511,064, the piperidinylamino-thieno[2,3-d]pyrimidine derivatives described in WO2004/089312 and US7,030,240 and the substituted methanones described in WO2010/080357.

Typically, the selective 5-HT_2B receptor antagonist of the invention may be selected from the group consisting of LY272015, LY266097, SB204741, SB215505, RS127445 and PRX08066.

In one embodiment, said selective HT_2B receptor antagonist is a beta-carboline derivative, in particular a tetrahydro-beta-carboline derivative. According to this embodiment, the antagonists are selected from those described in the

International Patent Application WO 95/24200 and that are listed below:

7-bromo-8-methyl-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole,

6-isopropyl-8-methoxy-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole,

5- chloro-8-ethoxy-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole,

6- chloro-7-methyl-8-fluoro-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole,

5- dimethylamino-8-hydroxy-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole,

6- nitro-8-butyl-l,2,3,4-tetrahydro-9H-pyrido[3,4b]- indole,

7- cyclohexyl-8-hydroxy-l,2,3,4-tetrahydro-9H- pyrido[3,4b]-indole,

6-[3-methyl-cyclohexyl]-8-methyl-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole, 6-benzyl-8-fluoro-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole,

5- cyclohexylmethyl-8-chloro-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole,

6- carboxyl-8-bromo-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole,

6-ethoxy-8-isopropyl-3-methyl-l,2,3,4-tetrahydro-9H-pyrido [3, 4b] -indole,

6,8-dichloro-4-naphthylmethyl-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole,

6,8-dimethyl-3,4-dimethyl-l,2,3,4-tetrahydro-9H-pyrido[3,4b]- indole,

7,8-difluoro-2(N)-methyl-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole,

6,8-dibutyl-2(N)-cyclopropylmethyl-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole, 6,8-dibromo-2(N)-cyclohexenylmethyl-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole,

8- chloro-2(N)-benzyl-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole, 8-fluoro-4-methyl-2(N)-cyclohexyl-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole,

6-methylamine-8-chloro-3-isopropyl-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole, and 6- chloromethyl-8-chloro-l,2,3,4-tetrahydro-9H-pyrido[3,4b]-indole;

or a pharmaceutical salt or solvate thereof.

Still according to this embodiment, said selective HT_2B receptor antagonist is the 1- [(3 ,4-dimethoxyphenyl)methy]-2,3 ,4,9-tetrahydro-6-methyl- 1 H-pyrido [3 ,4-b]indole (also known as LY272015) (Cohen et al, 1996), described by the following formula:

In another embodiment, said selective 5-HT_2B receptor antagonist is the N-(l-methyl- lH-indol-5-yl)-N'-(3-methylisothiazol-5-yl)urea (also known as SB204741) (Forbes et al. 1995), described by the following formula:

In another embodiment, said selective HT_2B receptor antagonist is an aryl pyrimidine derivative.

According to this particular embodiment, the antagonists are selected from those described in the International Patent Application WO 97/44326 and that are listed below:

2-amino-4-(2-methylnaphth- 1 -yl)-6-methylpyrimidine,

2-amino-4-(4-fluoronaphth- 1 -yl)-6-isopropylpyrimidine, 2-amino-4-(4-fluoronaphth-l-yl)-6-isopropylpyrimidine-l-N-oxide,

2-amino-4-(4-fluoronaphth-l-yl)-6-(2-methylpropyl)-pyrimidine,

2-amino-6-(tert-butyl)-4-(4-fluoronaphth- 1 -yl)pyrimidine,

2-amino-4-(2-methylnaphth- 1 -yl)-6-methylpyrimidine,

2-amino-4-(lH-indol-4-yl)-6-methylpyrimidine,

2-amino-4-(4-fluoronaphth-l-yl)-6-(l-fluoro-l-methyl-ethyl)-pyrimidine,

2-amino-4-(4-fluoronaphth-l-yl)-6-(l-hydroxy-l-methyl-ethyl)-pyrimidine,

2-amino-4-(4,6-difluoronaphth- 1 -yl)-6-( 1 -fluoro- 1 -methylethyl)-pyrimidine,

2-methylamino-4-(4-fluoronaphth-l-yl)-6-isopropyl-pyrimidine, and

2-amino-4-(4-fluoronaphth-l-yl)-6-(2-methylpropyl)-pyrimidine.

Particularly, said selective 5-HT_2B receptor antagonist is the 2-amino-4-(4- fluoronaphth-l-yl)-6-isopropylpyrimidine (also known as RS 127445) (International Patent Application WO 97/443626) described by the following formula:

In another embodiment, said 5-HT_2B receptor antagonist may be an antibody or antibody fragment that can partially or completely blocks the interaction between 5-HT and its receptor (i.e. 5-HT_2B receptor).

In particular, the 5-HT_2B receptor antagonist may consist in an antibody directed against serotonin or against 5-HT_2B receptor, in such a way that said antibody blocks the binding of serotonin on its receptor.

Antibodies directed against the serotonin or 5-HT_2B receptor can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies of the invention can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein; the human B-cell hybridoma technique and the EBV-hybridoma technique. Alternatively, techniques described for the production of single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies of the invention. 5-HT_2B receptor antagonist useful in practicing the present invention also include antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity.

Humanized antibodies and antibody fragments thereof can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

In a particular embodiment, said 5-HT_2B receptor antagonist is an anti-5-HT_2B receptor antibody. Furthermore, in another embodiment said 5-HT_2B receptor antagonist may be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena SD, 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods.

In another aspect, the invention relates to an inhibitor of 5-HT_2B receptor gene expression for use in the prevention or treatment of spasticity in patients affected with a degenerative disorder of the CNS.

As used herein, the term "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of a gene. Thus, an "inhibitor of 5-HT_2B receptor gene expression" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of the gene encoding the 5 -HT_2B receptor.

Inhibitors of 5-HT_2B receptor gene expression for use in the present invention may be based on anti-sense oligonucleotide constructs. Anti-sense oligonucleotides, including anti- sense R A molecules and anti-sense DNA molecules, would act to directly block the translation of 5-HT_2B receptor mR A by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of 5-HT_2B receptor, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding the 5-HT_2B receptor can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as inhibitors of 5-HT2B receptor gene expression for use in the present invention. 5-HT_2B receptor expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that 5-HT2B receptor gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (Elbashir SM et al, 2001; Tuschl T et al, 1999; Hannon GJ, 2002; Brummelkamp TR et al, 2002; McManus MT et al, 2002) U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836). shRNAs (short hairpin RNA) can also function as inhibitors of 5-HT_2B receptors gene expression for use in the present invention.

Ribozymes can also function as inhibitors of 5-HT_2B receptor gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of 5-HT_2B receptor mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable.

Both antisense oligonucleotides and ribozymes useful as inhibitors of 5-HT_2B receptor gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphorothioate chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a mean of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and preferably cells expressing 5-HT_2B receptor. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are well known in the art.

Preferred viruses for certain applications are the adenoviruses and adeno-associated (AAV) viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. Actually at least 12 different AAV serotypes (AAVl to 12) are known, each with different tissue tropisms. Recombinant AAV are derived from the dependent parvovirus AAV2. The adeno-associated virus type 1 to 12 can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion and most recombinant adenovirus are extrachromosomal.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC 18, pUC19, pRC/CMV, SV40, and pBlueScript, pSIREN. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parental, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

Another aspect relates to a method for preventing or treating of spasticity in a patient affected with a degenerative disorder of CNS comprising administering a subject in need thereof with a therapeutically effective amount of a 5-HT_2B receptor antagonist or an inhibitor 5-HT_2B receptor gene expression according the invention.

The term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a subject according to the invention is a human.

Preferably, said antagonist or inhibitor is administered in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of the antagonist or inhibitor to prevent and/or to treat ALS or spasticity at a reasonable benefit/risk ratio applicable to any medical treatment.

The antagonist or inhibitor may be administered in the form of a pharmaceutical composition, as defined below.

It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1 ,000 mg per adult per day. Preferably, the compositions contain 0.01 , 0.05, 0.1 , 0.5 , 1.0, 2.5 , 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. Pharmaceutical compositions

A further aspect of the invention relates to a pharmaceutical composition for use in the prevention or the treatment of spasticity in a patient affected with a degenerative disorder of CNS comprising a 5-HT_2B receptor antagonist or an inhibitor of 5-HT_2B receptor gene expression according the invention.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The 5-HT_2B receptor antagonist or a 5-HT_2B receptor gene expression may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The antagonist or inhibitor of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active substances in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

The antagonist or inhibitor of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.

In addition to the compounds of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Serotonergic neurons degenerate in SOD1 (G86R) mice.

A: mR A levels of 5-Htla receptor (5-Htla), serotonin transporter (Serf) and tryptophan hydroxylase 2 (Tph2) in SOD1 (G86R) mice at 75 days of age (Asymptomatic, NS) or at symptom onset (approximately 100 days of age,OS) and wild type littermates (Wt). Note that Sert and Tph2 gene expression levels are downregulated at symptom onset. N=7-10 per group. *, p<0.05 versus Wt, ANOVA followed by Newman- Keuls post-hoc test.

B: mean area of Tph2 positive neurons in SOD1 (G86R) mice at disease onset (OS) and wild type (Wt) littermates. The area of 200-800 neurons was measured per animal, with n=3 per group. p<0.0005 versus Wt, Student's t-test.

Figure 2: Decreased levels of serotonin in SOD1 (G86R) mice.

Serotonin (ng/mg tissue, A-C) and 5-HIAA/serotonin (D-F) in the brainstem (A,D), the spinal cord (B, E) and the cerebral cortex (C, F) of SOD1 (G86R) mice at 75 days of age (Asymptomatic, NS) or at symptom onset (approximately 100 days of age, OS) and wild type littermates (Wt) as measured using HPLC. *, p<0.05 versus Wt, **, p<0.01 versus Wt, p<0.001 versus Wt, #, p<0.05 versus NS, ##, p<0.01 versus NS. ANOVA followed by Newman- Keuls post-hoc test. N=5-10 per group.

Figure 3: Spasticity in SOD1 (G86R) mice is alleviated by 5-Ht2b/c inverse agonists.

A-C: representative recording of long-lasting reflexes using tail EMG in one diseased SOD1 (G86R) mouse before (left) and after injections of vehicle (Vh, A), SB206553 (SB206, B) or Cyproheptadine (Cypro, C). Spasticity was considered to be electrical activity above the baseline recorded 1 second after mechanical stimulation (arrowhead).

D: quantitative analysis. N= 5-6 mice per group and 2-3 EMGs were obtained before and after injection. A ratio between spasticity before and after injection and present the result as a percentage was calculated. *, p<0.05 versus before injection, ANOVA followed by Newman-Keuls post-hoc test.

E: mRNA levels of 5-Ht2b (5-Ht2b), and 5-Ht2c (5-Ht2c) receptors in SOD1 (G86R) mice at 75 days of age (Asymptomatic, NS) or at symptom onset (approximately 100 days of age,OS) and wild type littermates (Wt). Note that 5-Ht2b gene expression levels are heavily upregulated at symptom onset. N=12 per group. p<0.001 versus Wt, ANOVA followed by Newman-Keuls post-hoc test.

F: Levels of editing variants of the 5-Ht2c mRNA in the spinal cord of SOD 1 (G86R) mice at onset (OS) and wild type littermates (Wt). ABECD (mRNA variant with full editing of A, B, E, C and D sites), ABD (mRNA variant edited at A, B and D sites) and the non edited mRNA, that leads to the production of the constitutively active INI receptor were measured by Taqman assays. Levels of the non edited mRNA are decreased. N= 12 per group. *, p<0.05 versus Wt, Student's t-test.

EXAMPLE:

Material & Methods

Patient tissues: Autopsy samples from hippocampus, brainstem and spinal cord were obtained from 7 ALS patients and 6 controls. Case 2 had familial history of ALS but gene analysis demonstrated no pathogenic variations in the sodl gene. Hippocampus and brainstem samples were available for all patients. Spinal cord specimens were available for all ALS patients and control 6. Patients and/or families had provided written informed consent. Clinical details are presented in Supplemental Tables 1 and 2. ALS diagnosis was obtained using El Escorial criteria (Brooks et al, 2000) and was confirmed after autopsy. During autopsy, tissues were fixed in 4% formaldehyde and embedded in paraffin using standard protocols. Use of these tissues for research was declared at the French ministry for research and higher education (DC-2011-1433).

Supplemental table 1 : ALS patients included in the pathology study

5

Transgenic mice: Transgenic mice carrying the SOD1 (G86R) mutation (Dupuis et al, 2000, Ripps et al, 1995) and their non-transgenic littermates in FVB/N background were housed in our animal facility with unrestricted access to food and water. Mice were sacrificed at different stages of the disease to perform the studies using the following clinical scale: asymptomatic mice show normal gait and no paralysis and were scored 4. EMG is typically normal in these mice. Animals with a score of 3 showed a mildly abnormal gait or one hindlimb with paralysis. Score 3 typically occurs between 90 and 100 days of age, and is associated with already detectable EMG abnormalities, i.e. spontaneous muscle electrical activity, but no loss of motor neuron cell bodies (Halter et al, 2010). Frank paralysis of one limb is scored 2, and of both hindlimbs is scored 1. Profound weight loss and kyphosis are typical of score 0 and mice are euthanized at this stage. In this study, asymptomatic mice used were all scored 4, and had 75 days of age. Mice at disease onset were mice with a score of 3. These mice were followed daily and were sacrificed the 2nd day on which they showed a score of 3. End stage mice used in the EMG studies were scored 1 and thus showed frank paralysis of both hindlimbs. For ethical reasons, we did not use mice scored 0 in experiments but proceeded to their euthanasia.

For histology, brains were fixed by immersion in 4% formaldehyde in phosphate buffer 0.1M pH 7.4, and tissues were post-fixed 24 hours before paraffin embedding. For molecular biology, brainstem and lumbar spinal cord tissues were snap-frozen in liquid nitrogen. Animal experiments were performed under the supervision of authorized investigators (LD, FR), and approved by the local ethical committee for animal experiments (CREMEAS, agreement N° AL/01/02/02/ 12). Histology: Paraffin embedded tissues were cut in 4μιη sections using a HM 340E

Microtome (Micro m). Luxol Fast blue/Cresyl violet (Luxol FBV) stain was performed using a standard histological technique. Immunohistochemistry was performed in a Benchmark XT- automate using the Ventana NexES® software and EZ Prep Ventana Roche® reagent. Sections were heated, and endogenous peroxidases were inactivated using H202 Ventana Roche®. Primary and secondary antibodies were incubated during 2 hours at 37°C. Staining was performed using ultraview DAB Ventana Roche®. Human sections were counterstained with haematoxylin Ventana Roche®. Primary antibodies were as follows: rabbit polyclonal anti-ubiquitin (Dakocytomation, Netherlands, 1/200); rabbit polyclonal TDP-43 (Proteintech LTD, Manchester, UK, 1/800); rabbit polyclonal Tph2 (described in Gutknecht et al, 2009), 1/1000).

Quantification of TPH2 neurons in human samples: The number of Tph2 positive cell bodies in various regions of interest was evaluated semi-quantitatively in at least two sections of the considered nuclei identified. Number of neurons per section: Neg = 0-10, + = 11-20, ++ = 21-30, +++ = more than 30. The inventors systematically compared sections stained in parallel in matched regions. Regions of interest were identified in adjacent sections using Luxol FBV staining and counting of neurons were performed at 20x magnification in a blinded manner, on two sections of each region of interest.

Measurement of pericaryon size of Tph2 neurons in mouse: Sagittal brain sections (4μιη) were cut in series starting from the midline. In each animal, one of every five serial sections was sampled for Tph2 immunostaining. Using the 2nd edition of the mouse brain in stereotaxic coodinates atlas (Franklin and Paxinos, 1997), position of the dorsalis raphe nucleus (DRN) was determined on each section (medio-lateral: 0 to +0.48mm; anteroposterior: -4 to -5.3 mm from Bregma; dorso-ventral: +2.75 to +4mm). Images of the DRN were captured using a Nikon digital camera DXM1200 connected to an Nikon eclipseE800 microscope (Tokyo, Japan). Tph2 positive neurons were analyzed in 7-9 sections per animal in each group. The cell body area of all Tph2+ neurons with a visible nucleus in the DRN was measured using the NIH Image analysis software (ImageJ, version 1.45r), and 200-800 neurons were measured per animal.

RT-qPCR: Total RNA was extracted using Trizol (Invitrogen) and standard procedures. RT-qPCR was performed as previously described (Braunstein et al, 2010) using BIO-RAD iScript cDNA Synthesis Kit, iQ qPCR mix and a CFX95 thermocycler (BioRad). Data were normalized with the GeNorm software (Vandesompele et al, 2002) using geometric averaging of three internal standards (18S rRNA, Tata-box binding protein and RNA polymerase II subunit).

5-Ht2c editing: The inventors used the qPCR method developed by Lanfranco and collaborators to measure 5-Ht2c mRNA editing (Lanfranco et al, 2010, Lanfranco et al, 2009). This method is based on the use of Taqman probes selective for the various edited iso forms. They used the DNA templates provided by Lanfranco and collaborators to check for selectivity and specificity of the measurements, and obtained qPCR cycling conditions that discriminate fully between the different templates using the published Taqman probes.

HPLC: Serotonin and 5-HIAA were measured on tissue extracts using high- performance liquid chromatography with coulometric detection using a technique similar to (Alvarez et al, 1999). Results were standardized to initial wet weight of tissue.

Electromyographical evaluation of spasticity: Spasticity in tail muscles was measured with percutaneous EMG (electromyogram) wires inserted in segmental tail muscles at the midpoint of tail as described by Bennett and collaborators and adapted to mouse (Bennett et al, 2004). During EMG recording, muscle spasms were evoked with mechanical stimulation of the tail skin, and the tail was free to move. EMG was sampled at 5 kHz, rectified and averaged over a 4 s interval starting Is after stimulation. EMG over Is prior to stimulation was averaged for measure of background signal.

Statistical analysis: Statistical analysis was performed using GraphPad Prism software. For comparison between two groups, Student's t-test was used. For comparison between 3 or more groups, ANOVA followed by Newman-Keuls post-hoc test was applied. Significance level was set at p<0.05.

Results

Degeneration of serotonergic neurons in ALS: The inventors analyzed autoptic brains from 7 ALS patients, and 6 controls (Supplemental Tables 1 and 2). 3 ALS patients had a bulbar onset of symptoms, and 4 had spinal onset. They focused our studies on major serotonergic nuclei of the brainstem. Ubiquitin and TDP43 cytoplasmic aggregates, two pathological hallmarks of ALS (Kiernan et al, 2011, Neumann et al, 2006), were observed almost systematically in the raphe magnus and gigantocellular nuclei but more rarely in other nuclei studied One patient (patient 5) showed extensive ubiquitin and TDP43 pathology in all serotonergic nuclei studied. Serotonergic neurons were easily detected in the pons and rostral medulla nuclei of control patients using an antibody directed against tryptophan hydroxylase 2 (TPH2), the rate limiting enzyme in central serotonin synthesis. ALS patients showed loss of TPH2 positive cell bodies in serotonergic nuclei, although these nuclei were not uniformly affected in ALS patients. In many cases, cell bodies were still present, but loss of TPH2 positive neurites was obvious. Semi-quantitative analysis of TPH2 -positive cell bodies showed an heterogenous decrease in cell density in the studied serotonergic nuclei irrespective of the site of onset of disease, gender or age (Table 1). Patients 3 and 6 showed widespread serotonergic degeneration, while degeneration of serotonin cell bodies was more localized in patients 1 , 2 and 4. Patient 5, although displaying prominent ubiquitin and TDP43 pathology in these nuclei, and patient 7 appeared to show preserved neuronal counts. Analysis of serial sections revealed that the cells displaying TDP-43 or ubiquitin positive inclusions were not serotonergic neurons. Thus, serotonergic neurons suffer from a degenerative process with prominent neuritic degeneration, and sometimes cell body loss in ALS patients but do not show typical ALS pathology.

Table 1: semi-quantitative analysis of Tph2 positive cell bodies

in regions of interest of ALS patients

Number of neurons per section: Neg = 0-10, + = 11-20, ++ = 21-30, +++ = more than 30. Empty cells: tissue not available.

RPF: reticular pontine formation; RSCN: raphe superior central nucleus; GCN : giganto cellular nucleus; RM: raphe magnus; RLN: reticular lateral nucleus. Systematic degeneration of serotonergic projections in ALS:

The inventors next sought to determine whether projections of serotonergic neurons degenerated in ALS. TPH2-labelled projections of serotonergic neurons were readily detectable in the hippocampus of control patients but were almost absent in ALS patients. In the spinal cord of control 6, we observed spinal motor neurons densely innervated with TPH2 positive projections, as expected from the distribution of serotonin immunoreactivity in humans (Perrin et al, 2011). Contrasting with this, we occasionally observed isolated motor neurons with preserved serotonergic innervation, but neighbouring motor neurons were fully denervated. It should be noted that even in patients 5 and 7 with seemingly normal neuronal density, the inventors barely observed motor neurons innervated by serotonergic axons. In all, they observed a massive and generalized reduction of TPH2 -positive projections to spinal cord and hippocampus in ALS patients. Degeneration of serotonergic neurons and early serotonin depletion in SOD1

(G86R) mice: Studies in patients are hampered by the inaccessibility to presymptomatic period. To determine whether serotonergic neuron degeneration precedes motor symptoms, the inventors studied SOD1 (G86R) mice, a mutant mouse strain that overexpresses an ALS- linked mutant form of SOD1. This mouse strain, as well as other similar models, have been shown to demonstrate ALS-like disease, with both LMN and UMN degeneration (Gurney et al, 1994, Ozdinler et al, 2011, Ripps et al, 1995). At disease onset (score of 3, see Materials and methods for details on clinical evaluation of mice), mRNA levels of cell-specific markers of serotonergic neurons (Tph2, Serotonin transporter (Sert), 5-Htla receptor) were lower in the brainstem of SOD1 (G86R) mice, indirectly suggesting degeneration of these neurons (Figure 1 A). Direct visualization of serotonergic neurons in the dorsalis raphe nucleus using Tph2 immuno histochemistry revealed similar features in SOD1 (G86R) mice at symptom onset (score 3) to ALS patients. Semi-quantitative analysis revealed that the area of the cell body of Tph2 positive neurons was decreased of about one third in this nucleus (Figure IB). The inventors further observed fragmentation of Tph2 -positive neurites of SOD 1 (G86R) mice. Most importantly, levels of serotonin itself were decreased as compared with wild-type mice, in symptomatic (score 3) but also in non-symptomatic SOD1 (G86R) brainstem (score 4) (Figure 2A), spinal cord (Figure 2B) and cortex (Figure 2C). The ratio between 5-HIAA, the major serotonin metabolite depending of mono-amine oxidase A activity, and serotonin, represents an indirect measurement of local serotonin turnover (Shannon et al, 1986). In SOD1 (G86R) mice, the 5-HIAA/serotonin ratio was unchanged before symptoms in all three tissues tested (Figure 2D-F), and increased at disease onset in brainstem and cortex. This shows that serotonin depletion precedes increase in serotonin turnover suggesting that early loss of serotonin is due to decreased supply rather than to increased turnover. Thus, the development of ALS is associated with an early and general impairment of central serotonin function in an animal model of the disease.

Spasticity develops in SOD1 (G86R) mice, and is alleviated by 5-Ht2b/c inverse agonists: The inventors sought then to characterize whether serotonin depletion occurring early in SOD1 (G86R) had pathogenic consequences on motor neurons. Serotonin modulates excitability of motor neurons by allowing sustained entry of calcium (Heckman et al, 2009). In animal models of spinal cord injury, it was recently shown that serotonin depletion due to transection of serotonergic axons was overcompensated by motor neurons. More specifically, motor neurons produce constitutively active 5-Ht2b and 5-Ht2c receptors through still poorly defined mechanisms, decreased editing of the 5Ht2c mRNA being one of these (Murray et al., 2010). This constitutive activity of 5-Ht2b/c receptors is responsible for the occurence of spasticity upon spinal cord injury (Murray et al, 2010). Other serotonin receptors, including 5-HT1A, 2A, 3, 4, 5, 6 and 7 appear not involved in this event (Murray et al, 2011). By analogy, the inventors reasoned that the chronic loss of serotonergic innervation of lower motor neurons in ALS patients and SOD1 (G86R) mice could lead to spasticity. To explore this hypothesis, they used an electromyographical protocol adapted from Bennett and collaborators (Bennett et al, 2004). This objective and quantitative electromyographical method appears correlated with clinical evaluation of spasticity (Bennett et al, 2004) and allows to study the effect of classical anti-spastic drugs such as baclofen or clonidine (Li et al, 2004, Rank et al, 2011). They visually observed spasticity in the tail in some animals at disease onset (score 3), but this was difficult to distinguish from voluntary contractions in these mice that are not yet paralyzed. When disease progresses however, spasticity of the tail was systematically observed and strong in end-stage (stage 1) mice. Thus, the mechanisms for spasticity are already present at disease onset, and might help maintaining motor function, but are blurred by voluntary movements. For this reason, they used end-stage SOD1 (G86R) mice to adapt the EMG protocol initially developped for SCI injured rats. Under this experimental setup, spastic-like contractions of tail muscles were easily recorded (Figure 3A) providing objective evidence of spasticity as a component of ALS disease in this mouse model. They further studied involvement of constitutive activity of 5-Ht2b/c receptors in this measure of spasticity, and found that spasticity was strongly alleviated by injection of 5-Ht2b/c inverse agonists SB206553 (Figure 3B, D) and cyproheptadine (Figure 3C, D). In chronic spinal cord injured rats, spasticity is associated with increased production of the unedited isoform of the 5-Ht2c mR A leading to increased expression of the constitutively active INI-5-Ht2c receptor. In SOD 1 (G86R) mice, they observed, however, decreased production of this specific isoform and normal levels of total mRNA as well as of other various edited iso forms (Figure 3E-F) in the lumbar and sacral spinal cords at disease onset (score 3), i.e., the disease stage at which spasticity arises. Contrasting with this, the inventors observed a 10-fold increased expression of the 5-Ht2b receptor in the same animals (Figure 3E). In all, the present results suggest that serotonin depletion leads to overexpression of 5Ht2b receptors and subsequent constitutive activity of this receptor during development of spasticity. In turn, this constitutive activity likely leads to spasticity.

DISCUSSION:

The inventors have showed that ALS is associated with degeneration of central serotonin neurons, both in patients and animal model, and identify spasticity as a likely disease-relevant consequence of ALS-related serotonin deficiency. The first major result of this study is that central serotonergic neurons degenerate in

ALS patients and in an animal model. They observed a major decrease in serotonergic innervation in target regions, such as the spinal cord and the hippocampus, along with obvious decreased density of serotonergic neurites in the serotonergic nuclei. In some nuclei, this was also accompanied by shrinkage and loss of cell bodies. They found limited ubiquitin and TDP-43 pathology in most serotonergic nuclei studied but these inclusions were not in remaining cell bodies of serotonin neurons. This might reflect either high intrinsic capacity in clearing protein aggregates in serotonergic neurons, or low production of aggregate-prone proteins in this neuronal type or, on the contrary, extreme sensitivity leading to degeneration of neurites despite low levels of aggregates. Degeneration of serotonin neurons could be either independant of LMN degeneration, or be a secondary consequence of motoneuronal loss. However, they observed loss of serotonin in asymptomatic mice, as early as 75 days of age, an age that precedes from several weeks the onset of motor neuron degeneration. This suggests that degeneration of serotonin neurons occurs independently of motor neuron death, at least in animal models. The loss of serotonergic neurons causes loss of serotonin itself in regions of projections. In the animal model sudied, serotonin levels are decreased in the brainstem and the spinal cord long before motor symptoms arise. Previous studies on serotonin and 5-HIAA in ALS patients yielded conflicting results. Bertel and collaborators observed normal levels of serotonin and decreased levels of 5-HIAA in autopsy samples, while Forrest and collaborators observed normal serotonin levels but increased 5-HIAA (Bertel et al, 1991, Forrest et al, 1996). However, serotonin is a labile molecule that might be significantly altered in postmortem human samples with hours of delay before autopsy (Yoshimoto et al, 1993). The present study overcomes this problem by studying serotonergic neurons using TPH2 immuno staining in fixed tissues. In asymptomatic animals, the loss of serotonin was associated with normal serotonin turnover (unchanged 5-HIAA/serotonin ratio), strengthening the idea that decreased serotonin was due to decreased supply in serotonin rather than to increased degradation. Contrastingly, in end-stage mice, the inventors observed an increased serotonin turnover (increased 5-HIAA/serotonin ratio). This late increased serotonin turnover is likely due to increased release of serotonin by remaining nerve terminals. Indeed, this increased mobilization of residual serotonin in end-stage mice coincides with the loss of Sert expression, an event expected to limit serotonin reuptake and increase its turnover. The present study studies are consistent with imaging studies using the PET-SCAN ligand WAY100635 (Turner et al, 2007, Turner et al, 2005). This compound binds to the 5-HT1A receptor, that is broadly expressed in serotonergic neurons notably in brainstem and acts as an inhibitory somatodendritic autoreceptor in these neurons. The decreased binding potential of WAY 100635 in the raphe of ALS patients was hypothesized to be either due to decreased sensitivity of 5-HT 1A receptors to WAY 100635 or to loss of neurons that express this receptor. Since 5-HT1A receptor is strongly expressed in serotonin neurons in the raphe, the current results argue for the latter view.

The inventors next sought to delineate whether serotonin depletion had pathogenic consequences and focused on one of the potential consequences, the occurence of spasticity. Spasticity had been previously shown to occur in SCI as a late consequence of transection of serotonergic axons (Murray et al, 2011, Murray et al, 2010). Spasticity represents a symptom that is difficult to objectively measure in animals. They adapted an EMG technique measuring spastic-like contractions of tail muscles in response to a mechanical stimulation. Others had previously shown that this EMG method is clinically correlated with onset of spasticity in SCI injured rats and sensitive to classical anti-spastic drugs (Bennett et al, 2004, Li et al, 2004, Rank et al, 2011). This method thus represents a quantitative, observer-independent, measurement of spasticity. In the model studied, the inventors were able to almost abolish spasticity by the use of cyproheptadine, a broad 5-HT2 inverse agonist, and SB206553, a much more selective compound known to target 5-HT2B and C (Kennett et al, 1996), arguing for the involvement of one of these two receptors in ALS spasticity. Murray and collaborators observed increased production of the unedited 5-Ht2c mRNA in chronic spinal cord injured rats (Murray et al, 2010), while the present result in mSODl mice was opposite. It should be noted that recent work in another paradigm of SCI found unchanged levels of editing of the 5-ht2c mRNA in rats (Navarrett et al, 2012). This discrepancy might be due to species differences (rats vs mice), to the different kinetics of serotonin loss (abrupt in SCI but much slower in SOD1 (G86R) mice). They found a strong increase in spinal 5-Ht2b receptor expression when spasticity was obvious, which could underlie the constitutive activity observed. Indeed, 5-Ht2b receptor has intrinsic constitutive activity and the increase of concentration of a G-protein coupled receptor is on its own sufficient to further increase any constitutive activity (Seifert and Wenzel-Seifert, 2002). For instance, a 7-fold overexpression of the 5-Ht2b receptor in cardiomyocytes leads to a dramatic cardiac phenotype, suggesting that the overexpression of this receptor in the range we observed in SOD1 (G86R) mice is sufficient to induce strong constitutive activity (Nebigil et al, 2003). It should be kept in mind that our mouse model of ALS is based on transgenic overexpression of mutant SOD1. While these mouse models represent the only currently available model that display selective loss of both lower and upper motor neurons (Halter et al, 2010, Ozdinler et al, 2011), they only mimic the 20% of familial cases that display mutations in the sodl gene. Whether spasticity might also be alleviated by 5-HT2 inverse agonists in other, non-SODl ALS cases represents an open question. In all, the animal study suggests that spasticity, at least in SOD 1 -linked ALS, is due to constitutive activity of the 5-Ht2b receptor rather than 5-Ht2c.

How far can these mechanistic results be compared to the present pathology study in ALS patients? Among the patients included in this study, only case 5 showed the complete picture of UMN signs, in particular spasticity, while the other patients exhibited either increased reflexes and/or Babinski signs but not obvious spasticity (see Supplementary Table 1). The case 5 who displayed spasticity showed strong loss of serotonergic terminals on motor neurons and appeared thus indistinguishable from the other ALS patients in terms of loss of TPH2 projections. Interestingly however, case 5 was the single ALS case with widespread TDP43 pathology in serotonergic nuclei. Further work comparing autoptic material from patients with or without spasticity should be done to highlight potential correlations between serotonin loss and spasticity. Importantly, such study could also investigate other phenotypes potentially related with serotonin such as weight loss, depression or dementia.

The present work has potential clinical implications for the management of spasticity in those patients presenting such phenotype. This is especially true for patients with primary lateral sclerosis (PLS), a subtype of ALS with primary UMN involvement (Gordon et al, 2009, Singer et al, 2007). These patients develop prominent spasticity (Kuipers-Upmeijer et al, 2001, Le Forestier et al, 2001, Le Forestier et al, 2001) that is likely due to motor neuron hyperexcitability (Floeter et al, 2005). Spasticity is also sometimes associated with ALS but difficult to detect clinically since the tests used to assess spasticity rely on the integrity of alpha and gamma motor neurons, both degenerating during ALS (Swash, 2012). Spasticity in ALS and PLS has been very poorly studied and few clinical trials have been performed to treat this symptom (Ashworth et al, 2012). Only physical therapy was proven to be effective in a small trial (Drory et al, 2001), and current guidelines of the European Federation of Neurological Societies (EFNS) state that other anti-spastic medications display class IV level of evidence of efficacy and "may be tried" (Ashworth et al, 2006). A rigorous clinical trial assessing cyproheptadine in ALS spasticity is thus needed, although it should be noted that treatment of spasticity might also lead to worsening of motor function as observed in SCI.

Summarizing, the inventors showed that serotonin neurons degenerate in ALS, and that this is a likely cause of spasticity. Further research is needed to determine whether serotonergic degeneration has broader consequences on ALS pathophysiology.

REFERENCES:

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

Alvarez JC, Bothua D, Collignon I, Advenier C, Spreux-Varoquaux O. Simultaneous measurement of dopamine, serotonin, their metabolites and tryptophan in mouse brain homogenates by high-performance liquid chromatography with dual coulometric detection. Biomed Chromatogr. 1999 Jun;13(4):293-8.

Ashworth NL, Satkunam LE, Deforge D. Treatment for spasticity in amyotrophic lateral sclerosis/motor neuron disease. Cochrane Database Syst Rev. 2006(1):CD004156.

Ashworth NL, Satkunam LE, Deforge D. Treatment for spasticity in amyotrophic lateral sclerosis/motor neuron disease. Cochrane Database Syst Rev. 2012;2:CD004156.

Bennett DJ, Sanelli L, Cooke CL, Harvey PJ, Gorassini MA. Spastic long-lasting reflexes in the awake rat after sacral spinal cord injury. J Neurophysiol. 2004 May;91(5):2247-58.

Bertel O, Malessa S, Sluga E, Hornykiewicz O. Amyotrophic lateral sclerosis: changes of noradrenergic and serotonergic transmitter systems in the spinal cord. Brain Res. 1991 Dec 6;566(l-2):54-60.

Braunstein KE, Eschbach J, Rona-Voros K, Soylu R, Mikrouli E, Larmet Y, et al. A point mutation in the dynein heavy chain gene leads to striatal atrophy and compromises neurite outgrowth of striatal neurons. Hum Mol Genet. 2010 Nov 15;19(22):4385-98.

Brooks BR, Miller RG, Swash M, Munsat TL. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord. 2000 Dec;l(5):293-9.

Cohen et al. (1996) LY272015, a potent, selective and orally active 5-HT_2B receptor antagonist. J. Serotonin Res. 3 131.

Drory VE, Goltsman E, Reznik JG, Mosek A, Korczyn AD. The value of muscle exercise in patients with amyotrophic lateral sclerosis. J Neurol Sci. 2001 Oct 15;191(1- 2): 133-7.

Dupuis L, de Tapia M, Rene F, Lutz-Bucher B, Gordon JW, Mercken L, et al. Differential screening of mutated SODl transgenic mice reveals early up-regulation of a fast axonal transport component in spinal cord motor neurons. Neurobiol Dis. 2000 Aug;7(4):274- 85.

Dupuis L, Spreux-Varoquaux O, Bensimon G, Jullien P, Lacomblez L, Salachas F, et al. Platelet serotonin level predicts survival in amyotrophic lateral sclerosis. PLoS One. 2010;5(10):el3346.

Floeter MK, Zhai P, Saigal R, Kim Y, Statland J. Motor neuron firing dysfunction in spastic patients with primary lateral sclerosis. J Neurophysiol. 2005 Aug;94(2):919-27.

Forbes et al (1995) N-(l-Methyl-5-indolyl)-N'-(3-methyl-5-isothiazolyl)urea: a novel, high-affinity 5-HT2B receptor antagonist. J.Med.Chem. 38 855. Forrest V, Ince P, Leitch M, Marshall EF, Shaw PJ. Serotonergic neurotransmission in the spinal cord and motor cortex of patients with motor neuron disease and controls: quantitative autoradiography for 5-HTla and 5-HT2 receptors. J Neurol Sci. 1996 Aug; 139 Suppl:83-90.

Franklin K, Paxinos G. The Mouse Brain in Stereotaxic Coordinates: Academic Press, San Diego; 1997.

Gordon PH, Cheng B, Katz IB, Mitsumoto H, Rowland LP. Clinical features that distinguish PLS, upper motor neuron-dominant ALS, and typical ALS. Neurology. 2009 Jun 2;72(22): 1948-52.

Gurney ME, Pu H, Chiu AY, Dal Canto MC, Polchow CY, Alexander DD, et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science. 1994 Jun 17;264(5166): 1772-5.

Gutknecht L, Kriegebaum C, Waider J, Schmitt A, Lesch KP. Spatio-temporal expression of tryptophan hydroxylase isoforms in murine and human brain: convergent data from Tph2 knockout mice. Eur Neuropsychopharmacol. 2009 Apr;19(4):266-82.

Halter B, Gonzalez de Aguilar JL, Rene F, Petri S, Fricker B, Echaniz-Laguna A, et al. Oxidative stress in skeletal muscle stimulates early expression of Rad in a mouse model of amyotrophic lateral sclerosis. Free Radic Biol Med. 2010 Apr l;48(7):915-23.

Heckman CJ, Mottram C, Quinlan K, Theiss R, Schuster J. Motoneuron excitability:

The importance of neuromodulatory inputs. Clin Neurophysiol. 2009 Sep 26.

Ivanhoe CB, Reistetter TA. Spasticity: the misunderstood part of the upper motor neuron syndrome. Am J Phys Med Rehabil. 2004 Oct;83(10 Suppl):S3-9.

Kennett GA, Wood MD, Bright F, Cilia J, Piper DC, Gager T, et al. In vitro and in vivo profile of SB 206553, a potent 5-HT2C/5-HT2B receptor antagonist with anxiolytic- like properties. Br J Pharmacol. 1996 Feb;l 17(3):427-34.

Kiernan MC, Vucic S, Cheah BC, Turner MR, Eisen A, Hardiman O, et al. Amyotrophic lateral sclerosis. Lancet. 2011 Mar 12;377(9769):942-55.

Kuipers-Upmeijer J, de Jager AE, Hew JM, Snoek JW, van Weerden TW. Primary lateral sclerosis: clinical, neurophysiological, and magnetic resonance findings. J Neurol Neurosurg Psychiatry. 2001 Nov;71(5):615-20.

Lanfranco MF, Anastasio NC, Seitz PK, Cunningham KA. Quantification of RNA editing of the serotonin 2C receptor (5-HT(C)R) ex vivo. Methods Enzymol. 2010;485:311- 28. Lanfranco MF, Seitz PK, Morabito MV, Emeson RB, Sanders-Bush E, Cunningham KA. An innovative real-time PCR method to measure changes in RNA editing of the serotonin 2C receptor (5-HT(2C)R) in brain. J Neurosci Methods. 2009 May 15;179(2):247- 57.

Le Forestier N, Maisonobe T, Spelle L, Lesort A, Salachas F, Lacomblez L, et al.

Primary lateral sclerosis: further clarification. J Neurol Sci. 2001 Apr 1;185(2):95-100.

Le Forestier N, Maisonobe T, Piquard A, Rivaud S, Crevier-Buchman L, Salachas F, et al. Does primary lateral sclerosis exist? A study of 20 patients and a review of the literature. Brain. 2001 Oct;124(Pt 10): 1989-99.

Li Y, Li X, Harvey PJ, Bennett DJ. Effects of baclofen on spinal reflexes and persistent inward currents in motoneurons of chronic spinal rats with spasticity. J Neurophysiol. 2004 Nov;92(5):2694-703.

Murray KC, Stephens MJ, Ballou EW, Heckman CJ, Bennett DJ. Motoneuron excitability and muscle spasms are regulated by 5-HT2B and 5-HT2C receptor activity. J Neurophysiol. 2011 Feb;105(2):731-48.

Murray KC, Nakae A, Stephens MJ, Rank M, DAmico J, Harvey PJ, et al. Recovery of motoneuron and locomotor function after spinal cord injury depends on constitutive activity in 5-HT2C receptors. Nat Med. 2010 Jun;16(6):694-700.

Navarrett S, Collier L, Cardozo C, Dracheva S. Alterations of serotonin 2C and 2A receptors in response to T10 spinal cord transection in rats. Neurosci Lett. 2012 Jan 6;506(l):74-8.

Nebigil CG, Jaffre F, Messaddeq N, Hickel P, Monassier L, Launay JM, et al.

Overexpression of the serotonin 5-HT2B receptor in heart leads to abnormal mitochondrial function and cardiac hypertrophy. Circulation. 2003 Jul l;107(25):3223-9.

Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT, et al.

Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.

Science. 2006 Oct 6;314(5796): 130-3.

Ozdinler PH, Benn S, Yamamoto TH, Guzel M, Brown RH, Jr., Macklis JD.

Corticospinal motor neurons and related subcerebral projection neurons undergo early and specific neuro degeneration in hSODlG(9)(3)A transgenic ALS mice. J Neurosci. 201 1 Mar

16;31(l l):4166-77.

Perrin FE, Gerber YN, Teigell M, Lonjon N, Boniface G, Bauchet L, et al. Anatomical study of serotonergic innervation and 5-HT(l A) receptor in the human spinal cord. Cell Death Dis. 2011;2:e218. Rank MM, Murray KC, Stephens MJ, D'Amico J, Gorassini MA, Bennett DJ. Adrenergic receptors modulate motoneuron excitability, sensory synaptic transmission and muscle spasms after chronic spinal cord injury. J Neurophysiol. 2011 Jan;105(l):410-22.

Ripps ME, Huntley GW, Hof PR, Morrison JH, Gordon JW. Transgenic mice expressing an altered murine superoxide dismutase gene provide an animal model of amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A. 1995 Jan 31 ;92(3):689-93.

Seifert R, Wenzel-Seifert K. Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors. Naunyn Schmiedebergs Arch Pharmacol. 2002 Nov;366(5):381-416.

Shannon NJ, Gunnet JW, Moore KE. A comparison of biochemical indices of 5- hydroxytryptaminergic neuronal activity following electrical stimulation of the dorsal raphe nucleus. J Neurochem. 1986 Sep;47(3):958-65.

Singer MA, Statland JM, Wolfe GI, Barohn RJ. Primary lateral sclerosis. Muscle Nerve. 2007 Mar;35(3):291-302.

Sofic E, Riederer P, Gsell W, Gavranovic M, Schmidtke A, Jellinger K. Biogenic amines and metabolites in spinal cord of patients with Parkinson's disease and amyotrophic lateral sclerosis. J Neural Transm Park Dis Dement Sect. 1991 ;3(2): 133-42.

Swash M. Why are upper motor neuron signs difficult to elicit in amyotrophic lateral sclerosis? J Neurol Neurosurg Psychiatry. 2012 Jun;83(6):659-62.

Turner MR, Rabiner EA, Al-Chalabi A, Shaw CE, Brooks DJ, Leigh PN, et al.

Cortical 5-HT1A receptor binding in patients with homozygous D90A SOD1 vs sporadic ALS. Neurology. 2007 Apr 10;68(15): 1233-5.

Turner MR, Rabiner EA, Hammers A, Al-Chalabi A, Grasby PM, Shaw CE, et al.

[11C]- WAY 100635 PET demonstrates marked 5-HT1A receptor changes in sporadic ALS. Brain. 2005 Apr;128(Pt 4): 896-905.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology. 2002;3(7).

Yoshimoto K, Irizawa Y, Komura S. Rapid postmortem changes of rat striatum dopamine, serotonin, and their metabolites as monitored by brain microdialysis. Forensic Sci Int. 1993 Aug;60(3): 183-8.

Claims

CLAIMS:

1. A 5-HT2B receptor antagonist for use in the prevention or treatment of spasticity in a patient affected with a degenerative disorder of the central nervous system (CNS).

2. The antagonist for use according to claim 1, wherein the antagonist of the 5-HT_2B receptor antagonist is a 5-HT_2B/5-HT_2C antagonist.

3. The antagonist for use according to claim 2, wherein the 5-HT_2B/5-HT_2C antagonist is cyproheptadine or SB206553.

4. The antagonist for use according to claim 1, wherein the antagonist of the 5-HT_2B receptor antagonist is a selective 5-HT_2B receptor antagonist.

5. The antagonist for use according to claim 4, wherein the selective 5-HT_2B receptor antagonist is selected from the group consisting of LY272015, SB204741 or RSI 27445.

6. An inhibitor of 5-HT_2B receptor gene expression for use in the prevention or treatment of spasticity in patients affected with a degenerative disorder of the CNS.

7. The inhibitor of 5-HT_2B receptor gene expression for use according to claim 6, wherein the inhibitor of 5-HT_2B receptor expression is selected from the group consisting of a siRNA oligonucleotide, a ribozyme, or an antisense oligonucleotide.

8. The 5-HT_2B receptor antagonist for use according to claims 1 to 5 or the inhibitor of 5- HT_2B receptor gene expression for use according to claim 6 or 7, wherein the degenerative disorder of the CNS is selected from the group consisting of amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegia, primary lateral sclerosis or multiple sclerosis.

9. A pharmaceutical composition for use in the prevention or the treatment of spasticity in a patient affected with a degenerative disorder of the CNS comprising a 5-HT_2B receptor antagonist as defined in any of claims 1 to 5 or an inhibitor of 5-HT_2B receptor gene expression as defined in claims 6 to 7.