FIELD OF INVENTION The present invention relates to the treatment of Parkinson disease (PD) and other diseases, disorders or conditions related to dysfunction or death of dopamine (DA) neurons in the substantia nigra (SN). More specifically, the present invention relates to a composition comprising depolarizing agents such as, for example, the Orexin peptides, and modulators of the nicotinic cholinergic pathway acting on a receptor expressed by DA neurons of the SN, The composition of the invention may be used, for protecting DA neurons in PD, in particular in patients developing REM sleep behavior disorder (RBD), a frequent prodrome of PD and related disorders.

BACKGROUND OF INVENTION Parkinson Disease (PD) is a neurodegenerative disease of the central nervous system, resulting from the death of dopamine (DA) -generating cells in the substantia nigra (SN). PD is more common in the elderly, with most cases occurring after the age of 50.

PD patients experience motor symptoms, such as, for example, bradykinesia, tremor at rest, rigidity or stiffness, shaking, and postural instability; as well as non-motor neuron symptoms, including, without limitation, autonomic dysfunction, cognitive (impairment of cognitive and executive performances) and behavioral problems leading sometimes to dementia, emotional problems (mostly depression), sensory problems and sleep disorders. Indeed, PD is usually preceded or accompanied by daytime sleep attacks, nocturnal insomnia, REM sleep behavior disorder (RBD), hallucinations and depression. These symptoms are frequently as troublesome for the patient's life as the motor symptoms of PD. Therefore, in addition to treatments acting on motor and behavioral impairments, there is also a need for specific treatments allowing restoring normal sleep in PD patients. Orexin-A (OXA) and orexin-B (OXB) (also known as hypocretin-1 and -2) are small neuropeptides that are derived from the cleavage of a common precursor prepro-orexin, produced in the lateral hypothalamus. The orexin peptides are highly conserved between humans and mice, with identical OXA sequences and just two amino acid substitutions in OXB (Sakurai et al, Cell, 1998). These peptides operate through activation of two G-protein-coupled receptors, the 0X1 and 0X2 receptors (OX1R and OXR2, respectively). One of the key functions of orexin peptides is to regulate normal wakefulness, as loss of orexin-producing neurons causes narcolepsy in humans and rodents (Scammell and Winrow, Annu. Rev. Pharmacol. Toxicol. 2011). A loss of orexin-containing neurons has been reported in the hypothalamus of PD patients (Fronczek et al, Brain, 2007; Thannickal et al, Brain, 2007) which may explain why a number of symptoms present in narcolepsy, i.e., daytime sleep attacks, nocturnal insomnia, hallucinations, REM sleep behavior disorder (RBD) are often associated to PD (Thannickal et al, Brain, 2007). Most interestingly, longitudinal studies revealed that among patients diagnosed for idiopathic RBD, 46% will develop PD within 5 years (Arnulf, Mov Disord, 2012).

Therefore, it could be assumed that the molecular mechanism that causes sleep disorders in PD may be linked to some extent to that causing DA cell demise in the SN of PD patients and that orexin peptides may be involved, directly or indirectly, in this process. Therefore, some treatments of sleep disorders comprising the administration of orexin peptides were suggested in the art. For example, the patent application WOO 1/74162 describes a method of treating a sleep disorder in a patient, especially of narcolepsy, wherein said method comprises administering to the patient an agonist of the hypocretin receptor, preferably OXA. By studying the effect of orexin peptides in PD, the Inventors surprisingly showed that administration of orexin peptides provides neuroprotection to DA neurons in a model system of midbrain cultures in which these neurons degenerate progressively and selectively as they mature (Toulorge et al, Faseb J, 2011). In this model, the depolarizing alkaloid nicotine (NIC) was shown to be protective for DA neurons and to exert survival-promoting effect, but only in presence of concurrent depolarizing treatments (Toulorge et al, Faseb J, 2011). The Inventors herein surprisingly showed a synergy between nicotine (NIC) and orexin peptides to promote the survival of DA neurons.

The present invention thus relates to a composition comprising orexin peptides and a modulator of nicotinic acetylcholine receptors (nAChRs), such as, for example, NIC for the treatment of PD associated dysfunction/degeneration of DA neurons in the SN, in particular at an early stage of the disease when patients develop prodromal signs such as RBD. The experimental results obtained by the Inventors also support the use of such a composition for treating neuropsychiatric disorders wherein DA neurons of the SN and/or neighboring DA neurons of the ventral tegmental area are also dysfunctional.

SUMMARY

One object of the invention is a composition comprising a depolarizing agent acting on a receptor expressed by the dopamine (DA) neurons of the substantia nigra (SN), and a modulator of the nicotinic cholinergic pathway, wherein said receptor expressed by the DA neurons of the SN is an orexin receptor, a purinergic receptor or a neurokinin receptor.

In one embodiment, said depolarizing agent is an agonist of an orexin receptor.

In another embodiment, the agonist of an orexin receptor is a selective agonist of the orexin receptor OX2R.

In another embodiment, the agonist of an orexin receptor is an orexin peptide, preferably OXB.

In another embodiment, said depolarizing agent is an agonist of a purinergic receptor, preferably P2XR. In another embodiment, said depolarizing agent is an agonist of a neurokinin receptor, preferably NK1 and/or NK3. In another embodiment, the modulator of the nicotinic cholinergic pathway is an agonist of a nAChR, more preferably an agonist of the cc7-nAChR.

In another embodiment, said agonist of the cc7-nAChR is selected from the group comprising NIC, PNU 282,987, AR-RI7779, SSRI80711, JN403, ABBF, TC-5619, PHA-543613, SENI2333, TC-5619, GTS-21/DMXB-A, MEM3454, AZD0328, A- 582941, and mixtures thereof; preferably the agonist of the cc7 nAChRs is NIC or PNU 282,987.

Another object of the invention is a pharmaceutical composition comprising a composition as described here above and at least one pharmaceutically acceptable excipient.

Another object of the invention is a medicament comprising a composition as described here above.

Another object of the invention is a composition as described here above, a pharmaceutical composition as described here above or a medicament as described here above for providing neuroprotection to a subject in need thereof.

Another object of the invention is a composition as described here above, a pharmaceutical composition as described here above or a medicament as described here above for treating a disease, disorder or condition related to the dysfunction of the DA neurons of the SN in a subject in need thereof. In one embodiment, said disease, disorder or condition related to the dysfunction of the DA neurons of the SN is selected from the list comprising PD or other neurodegenerative disorders with parkinsonian syndromes, preferably PD, sleep disorders, deregulations of the sleep wake cycle, and neuropsychiatric diseases, disorders or conditions. Another object of the invention is a composition as described here above, a pharmaceutical composition as described here above or a medicament as described here above for treating sleep disorders or deregulations of the sleep wake cycle associated with PD or another neurodegenerative disorder with parkinsonian syndromes, preferably PD.

In one embodiment, the subject is affected with, is diagnosed with or is at risk of developing PD or another neurodegenerative disorder with parkinsonian syndromes, preferably PD.

DEFINITIONS

In the present invention, the following terms have the following meanings:

"Synergy" defines the interaction of two or more agents acting together in a positive way to produce an effect that neither could produce alone. An "additive synergy" defines a synergy wherein the combined effect of the agents is equal to the sum of the effects of each agent alone. When the combined effect is greater than the sum of the effects of each agent operating by itself, the synergy is referred as a "potentiating effect". In one embodiment, the synergy is an additive synergy. In another embodiment, the synergy is a potentiating effect.

"Modulator" refers to a compound that modulates the activity of the nicotinic cholinergic pathway. Preferably, a modulator is a compound whose administration leads to an activation of this pathway. The said modulator may act on the expression and/or on the activity of a nAChR, preferably, the modulator acts on the activity of an nAChR.

"Agonist" refers to a compound that binds to a receptor on a cell, thereby triggering a response by that cell. An agonist usually mimics the action (i.e, induces a response that is of the same type, but not necessarily of the same order) of a naturally occurring substance that naturally binds to said receptor. In one embodiment, the binding of an agonist of a receptor on said receptor activates said receptor.

"Treatment" refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the disease, disorder or condition related to the dysfunction of the DA neurons of the SN. Those in need of treatment include those already with the disease, disorder or condition related to the dysfunction of the DA neurons of the SN as well as those prone to develop the disease, disorder or condition related to the dysfunction of the DA neurons of the SN or those in whom the disease, disorder or condition related to the dysfunction of the DA neurons of the SN is to be prevented. A subject or mammal is successfully "treated" for a disease, disorder or condition related to the dysfunction of the DA neurons of the SN if, after receiving a therapeutic amount of a composition according to the invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the progression of dopaminergic deficits, such as, for example, using brain imaging approaches with specific radioligands; and/or relief to some extent, of one or more of the clinical symptoms associated with the disease, disorder or condition related to the dysfunction of the DA neurons in the SN, such as, for example, using a clinical rating scale to assess the spectrum of PD related symptoms; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by procedures familiar to research clinicians and/or physicians.

"Therapeutically effective amount" refers to the level or amount of agent that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a disease, disorder or condition related to the dysfunction of the DA neurons of the SN; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of a disease, disorder or condition related to the dysfunction of the DA neurons of the SN; (3) bringing about ameliorations of the symptoms of a disease, disorder or condition related to the dysfunction of the DA neurons of the SN; (4) reducing the severity or incidence of a disease, disorder or condition related to the dysfunction of the DA neurons of the SN; or (5) curing a disease, disorder or condition related to the dysfunction of the DA neurons of the SN. An effective amount may be administered prior to the onset of a disease, disorder or condition related to the dysfunction of the DA neurons of the SN, for a prophylactic or preventive action. Alternatively or additionally, the effective amount may be administered after initiation of a disease, disorder or condition related to the dysfunction of the DA neurons of the SN, for a therapeutic action.

"Neuroprotection" refers to preventing or slowing the development of neurologic disorders such as, for example, diseases, disorders or conditions related to the dysfunction of the DA neurons of the SN. In one embodiment, neuroprotection may aim at stopping or slowing down the loss of neurons related to these diseases.

"About" preceding a figure means plus or less 10% of the value of said figure.

DETAILED DESCRIPTION The present invention relates to a composition comprising a depolarizing agent acting on a receptor expressed by the DA neurons of the SN, preferably an agonist of a receptor expressed by the DA neurons of the SN, and a modulator of the nicotinic cholinergic pathway.

Examples of depolarizing agents acting on a receptor expressed by the DA neurons of the SN include, but are not limited to, agonists of an orexin receptor (OXR); agonists of a purinergic receptor, preferably of the P2X receptor (P2XR); and agonists of a neurokinin receptor, preferably of the NK1 and/or NK3 receptors.

In one embodiment of the invention, the depolarizing agent is not a K+-channel blocker. Methods for determining if a compound operates as a K+ channel blocker are well- known from the skilled artisan. Examples of such methods include, but are not limited to the demonstration that said compound possesses the molecular/structural requirements for K+ channel blockade; the demonstration by electrophysiological recordings that said compound modifies the activation profile of K+ channels overexpressed in Xenopus oocytes or in mammalian cell lines; the demonstration through binding studies that said compound displaces radioactive forms of known blockers of K+ channels. Examples of K+-channel blocker include, but are not limited to synthetic compounds such as tetraethylammonium (TEA), and 4-aminopyridine (4- AP) and small peptides such as charybdotoxin (CTX), and apamin (APA). Methods for determining if a compound is an agonist of a given receptor or receptor/channels are well-known in the art. Examples of such methods include, but are not limited to, the demonstration that well know agonists of the said receptor produce the same biological effect as the test compound using a cell line overexpressing this receptor; the demonstration that known antagonists of the said receptor reduce or prevent the biological effects of the test compound using a cell line overexpressing this receptor.

Methods for determining if a compound is a modulator of the nicotinic cholinergic pathway are well-known in the art. Examples of such methods include, but are not limited to the demonstration that well know agonists of the said receptors, including the alkaloid NIC, produce the same biological effects as the test compound using a cell line overexpressing this receptor; the demonstration that specific antagonists of a given nAChR prevent the biological effects of the test compound using a cell line overexpressing this receptor. In a first embodiment, the present invention relates to a composition comprising an agonist of an orexin receptor and a modulator of the nicotinic cholinergic pathway.

In one embodiment, the agonist of an orexin receptor is an agonist of the 0X1 and/or 0X2 receptor(s). In one embodiment, the agonist of an orexin receptor is a selective agonist of the OX2R. The selectivity for OX2R over OX1R can be assessed by comparing the potency of a given compound to elevate intracellular calcium in stably expressing OX2R and/or OX1R cell lines, such as, for example, PC12 (human pheochromocytoma) or Neuro-2a (murine neuroblastoma) cell lines transfected with cDNA for OX1R and/or OX2R. Examples of methods for measuring intracellular calcium levels include, but are not limited to, Ca ²⁺ imaging (such as, for example, fura-2 Ca ²⁺ imaging) (Holmqvist et al, FEBS Lett, 2002).

In one embodiment, the selective agonist of the OX2R exhibits at least about 10-fold selectivity for OX2R over OX1R, preferably at least about 50-fold selectivity, more preferably at least about 100-fold selectivity for OX2R over OX1R. In one embodiment, the agonist of an orexin receptor is an orexin peptide, preferably OXA (human OXA CAS number 205640-90-0), OXB (human OXB CAS number 205640-91-1) or a mixture of OXA and OXB. Advantageously, the agonist of an orexin receptor is OXB. In another embodiment, the agonist of an orexin receptor is not a selective agonist of the orexin receptor OX1R.

In one embodiment, the selective agonist of the orexin receptor OX2R has an affinity for OX2R at least 10-fold, 25-fold, 50-fold, 75-fold, 80-fold, 90-fold, 95 fold, 100-fold, 125-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, preferably 500-fold higher than the affinity for OX1R.

In another embodiment, the agonist of an orexin receptor is not the OXA peptide.

Examples of orexin agonists include but are not limited to agonists described in the following patents applications incorporated herein: US2010150840; US2014051700; WO2006081522.

In one embodiment, the orexin agonist is selected from the list of agonists for type-2 orexin receptor described in US2010/150840 and US2014/051700, such as, for example, compounds of formula:

wherein X is CI and Y is CI, or X is CI and Y is H, or X is H and Y is H, or X is OMe and Y is H, or X is H and Y is Br, or X is H and Y is N0 ₂ ; or compounds of formula:

wherein R _{1 ;} R ₂ and R ₄ are as described in the Table below: no./salt Rl R2 R4

1 /hydrochloride -CH20(4-C1C6H4) benzyl H

(4-chlorophenoxy-methyl)

2/hydrochloride 3 ,4-dichlorophenyl benzyl H

3/hydrochloride -CH20C6H5(phenoxymethyl) benzyl H

4/hydrochloride phenoxymethyl 4-methylbenzyl H

5/hydrochloride phenyl benzyl H

6/hydrochloride 2-thiophenyl benzyl H

7/hydrochloride 4-chiorophenyl benzyl H

8/hydrochloride phenoxymethyl n-butyl H

9/hydrochloride 3 ,4-dichlorophenyl ethyl H

10/hydrochloride 4-chlorophenoxy-n-butyl n-butyl H

11 /hydrochloride 3 ,4-dichlorophenyl 4-methylbenzyl H

12/hydrochloride phenoxymethyl 4-t-butylbenzyl H

13/hydrochloride 3 ,4-dichlorophenyl methyl H

14/hydrochloride phenoxymethyl 4-chlorobenzyl H

15/hydrochloride 4-chlorophenoxy-4- 4-chlorobenzyl H chlorobenzyl

16/hydrochloride 2-thienyl 2-(l -morpholino)- H ethyl

17/hydrochloride 2-thienyl ethyl H

18/hydrochloride phenyl n-butyl H

19/hydrochloride phenoxymethyl ethyl H

20/hydrochloride phenoxymethyl n-propyl H

21 /hydrochloride p-chlorophenoxy-methyl ethyl H

22/hydrochloride phenoxymethyl ( 1 -piperido)ethyl H

23/hydrochloride 1-adamantyl methyl H

24/hydrochloride methyl benzyl H

25/hydrochloride 2-furyl 2-(l -morpholino)- H ethyl

26/hydrochloride t-butyl benzyl H

27/hydrochloride 4-methoxyphenyl methyl H

28/hydrochloride 4-methylphenyl 2-(N,N-diethyl- H amino)ethyl

29/hydrochloride 4-chlorophenoxy-methyl n-propyl H

30/hydrochloride phenyl 2-( 1 -piperido)ethyl H /hydrochloride 4-dimethoxyphenyl 2-(N,N-diethyl- H amino)ethyl

/hydrochloride 1-naphthyl 2-( 1 -piperido)ethyl H/hydrochloride phenoxymethyl 2-(l- H morpholino)ethyl/hydrochloride 2-thienyl Methyl H/hydrochloride 4-chlorophenoxy-methyl 2-(l- H morpholino)ethyl/hy drochloride t-butyl allyl H/hydrochloride 4-ethoxyphenyl 2-( 1 -piperido)ethyl H/hy drochloride 2-thienyl 2-( 1 -piperido)ethyl H/hydrochloride 4-bromophenyl allyl H/hydrochloride 4-chlorophenoxy-methyl 2-( 1 -piperido)ethyl H /hydrochloride phenoxymethyl allyl H/hydrochloride 4-chlorophenoxy-n-propyl 2-(N,N-diethyl- H amino)ethyl

/hydrochloride phenyl n-propyl H/hydrochloride phenoxymethyl methyl H/hydrochloride 4-methoxyphenyl 2-( 1 -piperido)ethyl H/hydrochloride 4-chlorophenoxy-methyl allyl H/hydrochloride 4-methoxyphenyl benzyl H/hydrochloride 4-chlorophenyl n-propyl H/hydrochloride 4-ethoxyphenyl allyl H/hydrochloride 4-methylphenyl allyl H/hydrochloride 3 ,4-dichlorophenyl 2-(N,N-diethyl- H amino)ethyl

/hydrochloride 4-chlorophenoxy-methyl 4-methylbenzyl H/hydrochloride 4-chlorophenyl n-butyl H/hydrochloride 4-chlorophenyl methyl H/hydrochloride phenoxymethyl 2-(N,N-diethyl- H amino)ethyl

/hy drochloride 4-chlorophenoxy-methyl 2-(N,N-diethyl- H methyl-aminoethyl)/hydrochloride 4-chlorophenoxy-methyl methyl H/hy drochloride 1-naphthyl methyl H/hydrochloride t-butyl 4-chlorobenzyl H/hydrochloride 4-methylphenyl n-propyl H /hydrochloride methyl n-propyl H/hydrochloride 4-bromophenyl n-butyl H/hydrochloride 4-bromophenyl benzyl H/hydrochloride p-chlorophenyl allyl H/hydrochloride phenyl methyl H/hydrochloride methyl methyl H/hydrochloride 2-furyl benzyl H/hy drochloride 2-furyl 4-chlorobenzyl H/hydrochloride t-butyl 2-( 1 -piperido)ethyl H/hydrochloride phenoxymethyl 4-fluorobenzyl H /hydrochloride -CH ₂ OCH ₂ C ₆ H ₅ benzyl H

(benzyloxymethyl)

/hydrochloride phenoxymethyl n-octyl H 73/hydrochloride phenoxymethyl Methylene- 1- H

naphthyl

74/hydrochloride phenoxymethyl n-undecyl H

75/hydrochloride phenoxymethyl benzyl -C0 ² C ² H ^s

76/hydrochloride phenoxymethyl 2-(N,N-dibenzyl- H

amino)ethyl

77/hydrochloride benzyl benzyl H

78/hydrochloride phenoxymethyl 4-methoxybenzyl H

In one embodiment, the orexin agonist is selected from the list of orexin agonists described in WO2006/081522 such as orexin A, orexin B, [Ala ⁿ ,D-Leu ¹⁵ ]-Orexin B.

In one embodiment, the selective agonist of the orexin receptor OX2R is [Ala ^u ,D- Leu ¹⁵ ]-Orexin B as described in Asahi S et al. Bioorg Med Chem Lett. 2003 Jan 6; 13(l): l l l-3 and in US2004044031 incorporated herein.

In a second embodiment, the present invention relates to a composition comprising an agonist of a purinergic receptor, preferably of the P2X receptor and a modulator of the acetylcholine (ACh) pathway.

Examples of agonists of the P2X receptor include, but are not limited to, ATP; ATP disodium salt; ATPyS tetralithium salt; α,β-Methyleneadenosine 5 '-triphosphate lithium salt; α,β-meATP (α,β-methyleneadenosine 5 '-triphosphate trisodium salt); 2- methylthioadenosine triphosphate (2-ME-SATP) tetrasodium salt; 8-Bromoadenosine 5 '-triphosphate sodium salt; BzATP triethylammonium salt; β,γ-Methyleneadenosine 5'- triphosphate disodium salt; Cytidine 5 '-triphosphate disodium salt; 5-bromouridine 5- triphosphate, 3'-0-(4-benzoylbenzoyl)-ATP, zinc and mixtures thereof.

Examples of agonists of the P2X receptor include but are not limited to agonists described in the following patents incorporated herein: EP0760850 and WO2007/139775.

In one embodiment, the P2X receptor agonist is selected from the list of P2X receptor agonists described in EP0760850: ATP, 2meSATP, 2-chloro-ATP, α,β-MeATP, ADP, BzATP, AP ₅ A, ATP- γ -S, D-1-β, γ-ΑΤΡ, adenosine, AMP and UTP. In one embodiment, the P2X receptor agonist is selected from the list of P2X receptor agonists described in WO2007/139775: N-methanocarba derivatives of AMP, having the following formula:

wherein Ri is hydrogen, alkyl, alkoxy, amino, mono- or di-alkylamino, mono or bicyclic cycloalkyl, cycloalkyloxy, aryl, arylalkyl, acyl, sulfonyl, arylsulfonyl, or a mono- or bicyclic thiazolyl group; R ₂ is hydrogen, halogen, thiol, cyano, alkyl, alkenyl, alkynyl, alkylthio, alkylsulfmyl, alkylsulfonyl, aryl, arylamino, or aryloxy; R ₃ is hydrogen, halogen, methyl, or ethyl; and R ₄ and R5 are independently hydrogen, methyl, or methoxy; and in particular the compound MRS2339:

In a third embodiment, the present invention relates to a composition comprising an agonist of a neurokinin receptor (also known as tachykinin receptor), preferably of the NK1 and/or NK3 receptor and a modulator of the nicotinic cholinergic pathway. Examples of agonists of the NK1 receptor includes, but are not limited to, C14TKL-1; GR 73632; Hemokinin 1 (human); Hemokinin 1 (mouse); [Sar ⁹ ,Met(0 ₂ ) ⁿ ]-Substance P; substance P; Ranakinin; compounds of the 3-Quinolinecarboxamides family; and mixtures thereof. Examples of agonists of the NK3 receptor includes, but are not limited to, Senktide; PG-KII; [MePhe(7)] -neurokinin B ( [MePhe(7 ) ] -NKB ) ; SR142801; and mixtures thereof.

In one embodiment, the modulator of the nicotinic cholinergic pathway is a modulator of a nAChR, preferably an agonist of a nAChR, even more preferably an agonist of a neuronal nAChR and still even more preferably a modulator of the cc7-nAChR.

Examples of agonists of the cc7-ACh receptor include, but are not limited to, nicotine (NIC), PNU 282,987, AR-RI7779, SSRI80711, JN403, ABBF, TC-5619, PHA-543613, SENI2333, TC-5619, GTS-21/DMXB-A, MEM3454, AZD0328, A-582941, and mixtures thereof. In one embodiment, the composition of the invention comprises an orexin peptide, preferably orexin B and NIC.

In another embodiment, the composition of the invention comprises an orexin peptide, preferably orexin B and PNU 282,987.

The present invention also relates to a pharmaceutical composition comprising a depolarizing agent acting on a receptor expressed by the DA neurons of the SN, preferably an agonist of a receptor expressed by the DA neurons of the SN, and a modulator of the nicotinic cholinergic pathway, and at least one pharmaceutically acceptable excipient.

As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

In one embodiment, the pharmaceutical composition of the invention comprises a composition as hereinabove described. The present invention also relates to a medicament comprising a depolarizing agent acting on a receptor expressed by the DA neurons of the SN, preferably an agonist of a receptor expressed by the DA neurons of the SN, and a modulator of the nicotinic cholinergic pathway.

In one embodiment, the medicament of the invention comprises a composition as hereinabove described.

Examples of suitable excipients that may be included in the composition, pharmaceutical composition or medicament of the invention include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and solutions of ethanol, glucose, sucrose, dextran, mannose, mannitol, sorbitol, polyethylene glycol (PEG), phosphate, acetate, gelatin, collagen, Carbopol®, vegetable oils, and the like. In one embodiment, the composition, pharmaceutical composition or medicament of the invention may additionally include suitable preservatives, stabilizers, antioxidants, antimicrobials, and buffering agents, such as, for example, BHA, BHT, citric acid, ascorbic acid, tetracycline, and the like. Other examples of pharmaceutically acceptable excipients that may be used in the composition, pharmaceutical composition or medicament of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat. In one embodiment, the composition pharmaceutical composition or medicament of the invention may comprise some excipients, such as, for example, surfactants (e.g. hydroxypropylcellulose); suitable carriers, such as, for example, solvents and dispersion media containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils, such as, for example, peanut oil and sesame oil; isotonic agents, such as, for example, sugars or sodium chloride; coating agents, such as, for example, lecithin; agents delaying absorption, such as, for example, aluminum monostearate and gelatin; preservatives, such as, for example, benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal and the like; buffers, such as, for example, boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium biphosphate and the like; tonicity agents, such as, for example, dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride; antioxidants and stabilizers, such as, for example, sodium bisulfite, sodium metabisulfite, sodium thiosulfite, thiourea and the like; nonionic wetting or clarifying agents, such as, for example, polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol; viscosity modifying agents, such as, for example dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose; and the like.

According to an embodiment, the composition, the pharmaceutical composition or the medicament of the invention is injected, preferably systemically injected. Examples of formulations adapted to systemic injections include, but are not limited to, liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection. Examples of systemic injections include, but are not limited to, intravenous, subcutaneous, intramuscular, intradermal and intraperitoneal injection, and perfusion. According to an embodiment, when injected, the composition for use, the pharmaceutical composition or the medicament of the invention is sterile. Methods for obtaining a sterile pharmaceutical composition include, but are not limited to, GMP synthesis (GMP stands for "Good manufacturing practice"). According to another embodiment, the composition, the pharmaceutical composition or the medicament of the invention is orally administered. Examples of formulations adapted to oral administration include, but are not limited to, solid forms, liquid forms and gels. Examples of solid forms adapted to oral administration include, but are not limited to, pill, tablet, capsule, soft gelatin capsule, hard gelatin capsule, caplet, compressed tablet, cachet, wafer, sugar-coated pill, sugar coated tablet, orodispersing/orodisintegrating tablet, powder, solid forms suitable for solution in, or suspension in, liquid prior to oral administration and effervescent tablet. Examples of liquid form adapted to oral administration include, but are not limited to, solutions, suspensions, drinkable solutions, elixirs, sealed phial, potion, drench, syrup and liquor.

Other examples of administration routes include, but are not limited to, nasal, buccal, rectal, vaginal, topical, intratracheal, endoscopic, transdermal, transmucosal, and percutaneous administration or administration using an aerosol.

The Inventors herein showed an unexpected synergy, which is a potentiating effect, between modulators of the nicotinic cholinergic pathway (NIC or PNU 282,987) and the orexin peptide OXB, on neuroprotection (see Examples). This effect of a composition of the invention on neuroprotection was particularly unexpected as NIC or PNU 282,987 alone does not have any neuroprotective effect. Without willing to be linked by any theory, the Inventors suggest that the depolarizing agent of the composition of the invention, and in particular the agonist of the Orexin receptor may have an unmasking effect for neuroprotection mediated by modulators of the nicotinic cholinergic pathway, such as, for example, NIC or PNU 282,987.

The present invention thus relates to a composition comprising a depolarizing agent acting on a receptor expressed by the DA neurons of the SN, preferably an agonist of a receptor expressed by the DA neurons of the SN, and a modulator of the nicotinic cholinergic pathway, for, or for use in, neuroprotection.

The present invention thus also relates to a composition, a pharmaceutical composition or a medicament as hereinabove described, for, or for use in, neuroprotection. The present invention also relates to a composition comprising a depolarizing agent acting on a receptor expressed by the DA neurons of the SN, preferably an agonist of a receptor expressed by the DA neurons of the SN, and a modulator of the nicotinic cholinergic pathway, for, or for use in, treating a disease, disorder or condition related to the dysfunction of the DA neurons of the SN.

The present invention thus also relates to a composition, a pharmaceutical composition or a medicament as hereinabove described, for, or for use in, treating a disease, disorder or condition related to the dysfunction of the DA neurons of the SN.

Examples of diseases, disorders or conditions related to the dysfunction of the DA neurons of the SN include, but are not limited to, PD or other related neurodegenerative disorders with parkinsonian syndromes, restless legs syndrome, sleep disorders, disorders with deregulations of the sleep-wake cycle, and neuropsychiatric diseases, disorders or conditions.

Examples of neuropsychiatric diseases, disorders or conditions include, but are not limited to, Alzheimer's disease, attention-deficit/hyperactivity disorder, Tourette's syndrome and schizophrenia.

As used herein, the term "Parkinsonian syndrome" relates to a vast number of symptoms which are usually associated with degenerative disorders involving the dopaminergic system such as PD. Symptoms of a parkinsonian syndrome may include, without limitation, tremor at rest; akinesia and rigidity, such as, for example, slowness of movements, amimia, micrographia, loss of arm swing, difficulties in walking, sensation of stiffness; joint pain, dystonia, swallowing disorders, abnormal tiredness, trembling sensation, bradykinesia, action tremor, tremors, dysarthria, dysautonomia, dysphagia, dystonia, eye apraxia, limb apraxia, myoclonus, oculomotor tremors, night tremor, gait and posture impairment, sleep disorders...

Neurodegenerative disorders with Parkinsonian Syndromes include, but are not limited to, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration or Lewy body dementia. In one embodiment, the composition, the pharmaceutical composition or the medicament of the invention is for treating PD or another neurodegenerative disorder with parkinsonian syndromes.

In one embodiment, the composition, the pharmaceutical composition or the medicament is for treating sleep disorders associated with PD or with another neurodegenerative disorder with parkinsonian syndromes, preferably PD and/or for treating deregulations of the sleep wake cycle associated with PD or with another neurodegenerative disorder with parkinsonian syndromes, preferably with PD.

Examples of sleep disorders or deregulations of the sleep wake cycle that may be treated by the composition of the invention, in particular sleep disorders associated with PD or with another neurodegenerative disorder with parkinsonian syndromes, preferably PD, include, but are not limited to, daytime sleep attacks, narcolepsy, cataplexy, nocturnal insomnia, REM sleep behavior disorder (RBD), sleep apnea, hallucinations periodic leg movements in sleep (PLMS) and restless legs syndrome, circadian rhythm disorder, hypersomnia.

In one embodiment, the sleep disorders or deregulation of the sleep wake cycle that may be treated by the composition of the invention comprises daytime sleep attacks, hallucinations, nocturnal insomnia and REM sleep behavior disorder (RBD).

In one embodiment, in the meaning of the present invention, sleep disorders or deregulations of the sleep wake cycle do not include insomnia. In one embodiment, the sleep disorders or deregulation of the sleep wake cycle that may be treated by the composition of the invention, in particular sleep disorders associated with PD, is selected from the list comprising daytime sleep attacks, narcolepsy, cataplexy, REM sleep behavior disorder (RBD), sleep apnea, hallucinations, periodic leg movements in sleep (PLMS) and restless legs syndrome (RLS), circadian rhythm disorder, hypersomnia. Preferably, the sleep disorders or deregulation of the sleep wake cycle that may be treated by the composition of the invention is selected from the list comprising daytime sleep attacks, hallucinations, and REM sleep behavior disorder (RBD). In one embodiment, the composition, the pharmaceutical composition or the medicament of the invention is for preventing the onset of sleep disorders in a PD subject or in a subject affected with another neurodegenerative disorder with parkinsonian syndromes, preferably PD, or for preventing the onset of sleep disorders in a subject at risk of developing PD or another neurodegenerative disorder with parkinsonian syndromes, preferably PD.

In one embodiment of the invention, the composition, the pharmaceutical composition or the medicament of the invention further comprises another therapeutic agent useful for treating PD. Examples of such therapeutic agents include, but are not limited to dopamine agonists, such as, for example, bromocriptine, cabergoline, pergolide, pramipexole, fenoldopam, ropinirole, rotigotine, quinagolide and apomorphine; monoamine oxidase inhibitors, such as, for example, benmoxin, hydralazine, iproclozide, iproniazid, isocarboxazid, isoniazid, mebanazine, nialamide, octamoxin, phenelzine, pheniprazine, phenoxypropazine, pivalylbenzhydrazine, Pivalylbenzhydrazine, Procarbazine, Safrazine, Caroxazone, Echinopsidine, Furazolidone, Linezolid, Tranylcypromine, Brofaromine, Metralindole, Minaprine, Moclobemide, Pirlindole, Toloxatone, Lazabemide, Pargyline, Rasagiline, Selegiline; or other drugs with antiparkinsonian effects other than levodopa, for example methylphenidate, anticholinergic drugs. In one embodiment, the composition, the pharmaceutical composition or the medicament of the invention further comprises Levodopa.

In one embodiment, the composition, the pharmaceutical composition or the medicament of the invention comprises a therapeutically effective amount of a depolarizing agent acting on a receptor expressed by the DA neurons of the SN, and a therapeutically effective amount of a modulator of the nicotinic cholinergic pathway.

In one embodiment of the invention, the therapeutically effective amount of a depolarizing agent ranges from about 0.01 mg to about 500 mg per kg, preferably from about 0.05 mg to about 100 mg per kg, more preferably from about 0.1 mg to about 10 mg per kg. In another embodiment of the invention, the therapeutically effective amount of a modulator of the nicotinic cholinergic pathway ranges from about 0.01 mg to about 500 mg per kg, preferably from about 0.05 mg to about 100 mg per kg, more preferably from about 0.1 mg to about 10 mg per kg.

According to one embodiment of the invention, the composition, the pharmaceutical composition or the medicament of the invention is administered at least once a month, preferably at least once a week, more preferably at least once a day.

In one embodiment of the invention, the modulator is administered in a sustained- release form. In one embodiment of the invention, the composition comprises a delivery system that controls the release of the modulator. Examples of suitable carriers for sustained or delayed release include, but are not limited to, gelatin; Arabic gum; xanthane polymers; thermoplastic resins such as, for example polyvinyl halides, polyvinyl esters, polyvinylidene halides and halogenated polyolefins; elastomers such as, for example, brasiliensis, polydienes, and halogenated natural and synthetic rubbers; and flexible thermoset resins such as polyurethanes, epoxy resins; biodegradable polymers and the like.

In one embodiment of the invention, the subject is a mammal, preferably a human. In one embodiment of the invention, the subject is a female. In another embodiment of the invention, the subject is a male.

In one embodiment, the subject is affected, preferably is diagnosed with a disease, disorder or condition related to the dysfunction of the DA neurons of the SN.

In another embodiment, the subject is at risk of developing a disease, disorder or condition related to the dysfunction of the DA neurons of the SN. Examples of risks include, but are not limited to, familial history of disease, disorder or condition related to the dysfunction of the DA neurons of the SN; genetic predisposition to a disease, disorder or condition related to the dysfunction of the DA neurons of the SN; occurrence of REM sleep behavior disorder; exposure to environmental factors and the like.

In one embodiment of the invention, the subject is affected, preferably is diagnosed with PD. In one embodiment, an onset of a symptom of PD has been diagnosed in the subject. In another embodiment, no onset of a symptom of PD has been diagnosed in the subject. Examples of symptoms of PD include, but are not limited to, tremor at rest; akinesia and rigidity, such as, for example, slowness of movements, amimia, micrographia, loss of arm swing, difficulties in walking, sensation of stiffness; joint pain, dystonia, swallowing disorders, abnormal tiredness, trembling sensation, bradykinesia, action tremor, tremors, dysarthria, dysautonomia, dysphagia, dystonia, eye apraxia, limb apraxia, myoclonus, oculo-motor tremors, night tremor, gait and posture impairment, sleep disorders and the like. In another embodiment, the subject is at risk of developing PD. In one embodiment, the subject has a genetic or familial predisposition to PD. In one embodiment of the invention, the subject presents a non-genetic predisposition to PD. Non-genetic risk factors for developing PD include, but are not limited to, exposure to heavy metals, such as, for example, Lead, Manganese or Copper; exposure to pesticides such as, for example, rotenone or paraquat; exposure to pollutants; exposure to herbicides such as, for example, Substance Orange; exposure to toxic substances, such as, for example, MPTP...

The present invention also relates to a method for providing neuroprotection to a subject in need thereof, wherein said method comprises administering to the subject a therapeutically effective amount of a depolarizing agent acting on a receptor expressed by the DA neurons of the SN, preferably of an agonist of a receptor expressed by the DA neurons of the SN, and a therapeutically effective amount of a modulator of the nicotinic cholinergic pathway.

The present invention also relates to a method for, or for use in, treating a disease, disorder or condition related to the dysfunction of the DA neurons of the SN to a subject in need thereof, wherein said method comprises administering to the subject a therapeutically effective amount of a depolarizing agent acting on a receptor expressed by the DA neurons of the SN, preferably of an agonist of a receptor expressed by the DA neurons of the SN, and a therapeutically effective amount of a modulator of the nicotinic cholinergic pathway. Figure 1 is a concentration-response curve of the protective effects of OXB for DA neurons. Counts of DA (tyrosine hydroxylase positive, TH+) neurons at 10 days in vitro (DIV) as a function of OXB concentrations (0.1-10 nM). GDNF was used as positive control at a concentration of 20 ng/ml. Each data point is the mean value of 3 independent experiments. CON: Control.

Figure 2 is a bar-graph showing that NIC-mediated protection of DA neurons is unmasked by OXB. Number of TH+ neurons in 10-DIV cultures chronically exposed to OXB (3 nM) only, or to OXB and NIC (1 μΜ) in the absence or presence of the nonselective nAChR antagonist mecamylamine (MECA, 50 μΜ), the cc7 nAChR antagonist bungarotoxin (BgTx, 1 μΜ) or the dual orexin receptor antagonist TCS-1102 (1 nM). The trophic peptide GDNF (20 ng/ml) was used as a reference treatment to protect DA neurons. *P <0.05 vs. control; #P <0.05 vs. OXB + NIC treatment. §P <0.05 vs. other cultures treated with OXB, only. Each bar is the mean value of 3 independent experiments. Comparisons by one-way ANOVA followed by Student- Newman-Keuls test.

Figure 3 is an illustration that shows the presence of the two orexin receptors OXIR and OX2R in DA neurons in midbrain cultures. Primary antibodies against OXIR and OX2R were revealed using secondary antibodies carrying a green fluorescent dye. White arrows point to DA neurons detected by immunodetection of TH (red fluorescent signal). Insert shows that when OX1R/OX2R antibodies were omitted, no significant green fluorescent signal was detectable in the cultures. Scale bar: 25 μιη.

Figure 4 is a bar-graph showing the rescuing effects of treatments that combine the OXB peptide with NIC or the cc7 nAChR agonist PNU 282,987. Midbrain cultures were processed for the evaluation of DA cell survival at 10 DIV. OXB was used at 3nM and concentrations of NIC and PNU 282,987 are given in the figure. *P <0.05 vs. control. ^# P <0.05 vs. cultures treated with OXB, only. Each bar is the mean value of 4 independent experiments. Comparisons by one-way ANOVA followed by Dunnett's test.

Figure 5 is a bar-graph showing that protective effects of OXB are mediated by OX2R. Counts of dopamine neurons (tyrosine hydroxylase+) in midbrain cultures exposed continuously for 10 days to OXB (10 nM) in the presence or not of TCS-1102 (Ν-[1,Γ- Biphenyl] -2-yl- 1 - [2- [( 1 -methyl- 1 H-benzimidazol-2-yl)thio] acetyl-2- pyrrolidinedicarboxamide, 1 nM), a dual OXR antagonist, SB408124 (N-(6,8-Difluoro- 2-methyl-4-quinolinyl)-N'-[4-(dimethylamino)phenyl]urea, 1 μΜ), a selective OX1R antagonist or EMPA (N-Ethyl-2-[(6-methoxy-3-pyridinyl)[(2- methylphenyl)sulfonyl]amino]-N-(3-pyridinylmethyl)acetamide, 100 nM), a selective OX2R antagonist. Some cultures were also exposed to the modified OXB peptide, [Alal l, D-Leu] OXB (10 nM), a highly potent and selective OX2R agonist that displays a 400-fold selectivity over OXlRs. The effects of [Alal l, D-Leu] OXB were tested in the presence or not of EMPA. * p<0.05, vs control cultures; # p<0.05, vs corresponding OXB- or [Alal l,D-Leu] OXB-treated cultures.

Figure 6 is a bar-graph showing that OXB-mediated protection of dopamine neurons requires activation of intracellular calcium-release channels. Counts of dopamine neurons (tyrosine hydroxylase-i-) in midbrain cultures exposed continuously for 10 days to OXB (10 nM) in the presence or not of 2-APB (2-AminoethoxydiPhenylBorane, 10 μΜ), an IP3 receptor antagonist or dantrolene (30 μΜ), a blocker of ryanodine receptor channels. * p<0.05, vs control cultures; # p<0.05, vs OXB -treated cultures.

Figure 7 is a bar-graph showing that OXA provides limited protection to dopamine neurons in comparison to OXB. Counts of dopamine neurons (tyrosine hydroxylase-i-) in midbrain cultures exposed continuously for 10 days to OXB or OXA, each at 10 nM. The effect of OXA was sensitive to the OX2R antagonist EMPA. * p<0.05, vs control cultures; # p<0.05, vs OXA-treated cultures.

EXAMPLES

The present invention is further illustrated by the following examples. Example 1: Survival promoting effects of the composition of the invention

In this example, we tested the survival promoting effects of OXB in a model system of midbrain cultures in which DA neurons degenerate progressively and selectively as they mature (Michel and Agid, J Neurochem, 1996; Toulorge et al, Faseb J, 2011). We were particularly interested in determining whether the alkaloid NIC could act synergistically with the orexin peptides to promote the survival of DA neurons.

Materials and methods

Cultures were prepared from the ventral mesencephalon of embryos (gestational age 15.5) harvested from Wistar female rats (Janvier Breeding Center, Le Genest St Isle, France). The protocol for producing and maintaining these cultures has been reported previously in detail (Salthun-Lassalle et al, 2004; Toulorge et al, Faseb J, 2011). The cultures were fed daily by replacing 70% of the medium and pharmacological treatments were applied concurrently. The evaluation of the survival of DA neurons was performed by counting cells immunopositive for TH as described (Toulorge et al, Faseb J, 2011).

Results

Figure 1 shows that a chronic exposure to OXB promotes the survival of DA neurons in a concentration-dependent manner. Survival is assessed after 10 days in vitro (DIV). Survival promotion by OXB is compared to that provided by GDNF, a highly potent trophic peptide for DA neurons (Lin et al, Science, 1993). Based on these observations, we tested whether a treatment with the alkaloid NIC could act in synergy with OXB. As shown in Figure 2, NIC had no effect by itself but OXB (3 nM) was able to reveal NIC (1 μM)-mediated protection so that the level of protection provided by a combined treatment with NIC and OXB exceeded that of OXB, alone, demonstrating the presence of a synergy, which is a potentiating effect, between OXB and NIC. A non-specific blocker of acetylcholine nicotinic receptors (nAChRs), mecamylamine (50μΜ) and a specific blocker of brain cc7 nAChRs cc-bungaro toxin (ΙμΜ) prevented the effect of NIC but not that of OXB, confirming the key role played by the cc7 nicotinic receptor subtype in mediating the protective action of the alkaloid (Toulorge et al, Faseb J, 2011). Interestingly, TCS-1102 (1 nM), a dual orexin receptor antagonist prevented the protective effect of OXB and also that of NIC, confirming that the action of the alkaloid relied on that provided by the peptide. Example 2: Characterization of OX receptors in DA neurons

In this example, we performed immunodetection of the two orexin receptors OX1R and OX2R in the population of midbrain DA neurons.

Materials and methods

The immunodetection of OX receptors was carried out in 6-7 DIV midbrain cultures. Briefly, midbrain cultures fixed for 12 min with 4% formaldehyde in PBS were then incubated for 48 h with primary polyclonal antibodies for OX1R (1/100 in PBS; Abeam #ab68718) and OX2R (1/100 in PBS; Abeam #abl04701). The detection of OX1R and OX2R was performed by incubating the cultures for 20 min with an Alexa Fluor-488 conjugate of an anti-rabbit antibody (1/300 in PBS; Molecular Probes). Immunodetection of DA neurons was carried out subsequently with a monoclonal anti- TH antibody (1:5000; Diasorin, Stillwater, MN, USA). Note that immunostainings of OXR were performed in the absence of Triton-X 100, owing to the preferential location of OXR proteins at the plasma membrane level.

Figure 3 shows that both receptors are widely expressed by midbrain neurons and more specifically by the subpopulation of DA neurons characterized on the basis of its content in TH.

Example 3: Treatments combining the OXB peptide and cc7 nicotinic receptor agonists

We have shown initially that OXB has the capacity to reveal the protective action of NIC for these neurons (see Example 1). The effect of NIC being mediated by cc7nAChRs, we performed complementary studies in order to determine whether OXB had also the potential to reveal protective effects of a specific cc7 nAChR agonist. The present set of data demonstrates that OXB can unmask the protective action of the cc7 nAChR agonist N-(3R)-l-azabicyclo[2.2.2]oct-3-yl-4-chlorobenzamide (PNU 282,987) (Figure 4). Moreover, the results presented on Figure 4 demonstrate the presence of a synergy, which is a potentiating effect, of OXB and PNU 282,987 for neuroprotection. Example 4: The protective effects of OXB is mediated by OX2R

Dopamine neurons (tyrosine hydroxylase+) in midbrain cultures were exposed continuously for 10 days to OXB (10 nM) in the presence or not of TCS-1102 (Ν-[1,Γ- Biphenyl] -2-yl- 1 - [2- [( 1 -methyl- 1 H-benzimidazol-2-yl)thio] acetyl-2- pyrrolidinedicarboxamide, 1 nM), a dual OXR antagonist, SB408124 (N-(6,8-Difluoro- 2-methyl-4-quinolinyl)-N'-[4-(dimethylamino)phenyl]urea, 1 μΜ), a selective OX1R antagonist or EMPA (N-Ethyl-2-[(6-methoxy-3-pyridinyl)[(2- methylphenyl)sulfonyl]amino]-N-(3-pyridinylmethyl)acetamide, 100 nM), a selective OX2R antagonist. Some cultures were also exposed to the modified OXB peptide, [Alal l, D-Leu] OXB (10 nM), a highly potent and selective OX2R agonist that displays a 400-fold selectivity over OXlRs. The effects of [Alal l, D-Leu] OXB were tested in the presence or not of EMPA.

Figure 5 shows that the protective effects of OXB on dopamine neurons are mediated by OX2R.

Example 5: OXB-mediated protection of dopamine neurons requires activation of intracellular calcium-release channels

Dopamine neurons in midbrain cultures were exposed continuously for 10 days to OXB (10 nM) in the presence or not of 2-APB (2-AminoethoxydiPhenylBorane, 10 μΜ), an IP3 receptor antagonist or dantrolene (30 μΜ), a blocker of ryanodine receptor channels. Figure 6 shows that OXB-mediated protection of dopamine neurons requires activation of intracellular calcium-release channels. Example 6: OXA provides limited protection to dopamine neurons in comparison to OXB

Dopamine neurons (tyrosine hydroxylase+) in midbrain cultures were exposed continuously for 10 days to OXB or OXA, each at 10 nM. The effect of OXA was sensitive to the OX2R antagonist EMPA.

Figure 7 shows that OXA provides limited protection to dopamine neurons in comparison to OXB.