WO2008062296A2

WO2008062296A2 - Nop receptor agonists for the treatment of l-dopa induced dyskinesias

Info

Publication number: WO2008062296A2
Application number: PCT/IB2007/003597
Authority: WO
Inventors: Michele Morari; Matteo Marti
Original assignee: Universita' Degli Studi Di Ferrara
Priority date: 2006-11-23
Filing date: 2007-11-23
Publication date: 2008-05-29
Also published as: WO2008062296A3; ITFE20060036A1

Abstract

The present invention relates to the chronic treatment with L,3-4-dihydroxyphenylalanine (L-DOPA). Chronic L-DOPA administration still represents the most effective pharmacological therapy of Parkinson's disease (PD) although it is invariably associated with the appearance (within 5-10 years after start of the therapy, in about 80 % of patients) of motor complications that limit its clinical effectiveness and greatly reduce the quality of life of patients (Obeso et al., 2000). These motor complications encompass motor fluctuations (e.g. wearing off and 'on-off fluctuations) and abnormal involuntary movements or dyskinesias (peak dose and diphasic dyskinesias, dystonia). We report for the first time that in an animal model of dyskinesias, administration of agonists at nociceptin/orphanin FQ receptors, termed NOP receptors, dramatically reduces abnormal involuntary movements induced by L-DOPA administration. NOP receptor agonists thus represent a novel class of drugs useful for the treatment of L-DOPA induced dyskinesias.

Description

NOP RECEPTOR AGONISTS FOR THE TREATMENT OF L-DOPA INDUCED DYSKINESIAS

STATE OF THE ART Idiopathic Parkinson's disease (PD) is a progressive neurodegenerative disorder, clinically characterized by hypo/akinesias, rigidity, gait disturbance and resting tremor. It is also often associated with mood changes (namely depression), dementia, dysautonomias and other non motor symptoms. PD is typically a senile disease (it affects about 1 % of population over 60) although juvenile onset has been described. In addition to reducing the quality of life of patients, particularly in the more advanced stages of the disease, PD causes heavy social burdens. Indeed, a recent study (Keranen et al., 2003) has estimated that direct social costs (essentially those due to drugs and hospitalization) amount to 4,900 euros/year per patient, while indirect costs due to loss of productivity (e.g. absence from work and early retirement) and informal home care amount to 6,700 euros/year per patient. Pharmacological therapy of PD still relies on L-DOPA (L,3-4- dihydrohydroxyphenylalanine) which, more than 30 years after its discovery, still represents the gold standard of symptomatic PD therapy (see for a recent review Obeso et al., 2000). Indeed, administration of the biosynthetic precursor of catecholamines (namely L-DOPA) and its subsequent decarboxylation to DA, has been proven the most effective strategy to normalize the loss of endogenous dopamine (DA) signal (caused by progressive degeneration of mesencephalic DA neurons) and to control typical PD symptoms. L- DOPA is decarboxylated by L-DOPA decarboxylases both in the central nrevous system and periphery. To avoid decarboxylation of L-DOPA to DA in the periphery, an event responsible not only for reduction of drug bioavailability but also unwanted side effects, L-DOPA is administered in combination with L-DOPA decarboxylase inhibitors that do not cross the blood brain barrier (carbidopa and benserazide). Chronic L- DOPA administration is invariably associated with the appearance (within 5-10 years in about 80 % of patients) of motor complications that limit its clinical effectiveness and greatly reduce the quality of life of patients (Obeso et al., 2000). These motor complications encompass motor fluctuations (e.g. wearing off and "on-off fluctuations) and abnormal involuntary movements or dyskinesias (peak dose and diphasic dyskinesias, dystonia). The pathogenic mechanisms underlying such a phenomenon have not yet been fully identified. Nevertheless, it has been shown that progressive degeneration of DA structures together with pulsatile stimulation of DA receptors induce changes of gene expression and firing in non DA neurons, thereby leading to development of abnormal involuntary movements (AIMs) during L-DOPA treatments. Therefore, one of the strategies followed by clinicians to prevent development of dyskinesias in de novo patients is to delay as much as possible the introduction of L-DOPA or to combine it with other antiparkinsonian drugs (e.g. D2 receptor agonists) to reduce L-DOPA dosage. Monotherapy with DA agonists, despite less dyskinesiogenic, is less effective than monotherapy with L-DOPA in control of motor symptoms. The management of patients with advanced PD who have already developed dyskinesias also appears problematic. By its nature, dyskinesias represents an irreversible modification of nervous structures (a pathological form of synaptic plasticity; Picconi et al., 2003), particularly in the basal ganglia, where these phenomena are thought to originate. In the advanced stage of the disease, clinical response to L-DOPA becomes strictly dependent on drug blood levels. Thus, strategies have been developed to improve bioavailability. Among these, the improvement of absorption through oral route (strongly dependent on gastric emptying and food protein content), stabilization of blood levels via intake of slow release L-DOPA preparations, or the combination of L-DOPA with an inhibitor of catechol-0-methyl-transferase (COMT), which are enzymes that degrade L-DOPA in the periphery, have been attempted. Finally, in order to provide a long lasting stimulation of DA receptors, L-DOPA can be paired with DA agonists administered through oral, or in the most severe cases, parenteral route (e.g. apomorphine). Symptomatic drugs able to treat L- DOPA-induced dyskinesias are still lacking. The only marketed drug able to exert modest antidyskinetic activity is amantadine (Goetz et al., 2002). However, recent studies have shown that its clinical efficacy is short-lasting since its action tends to disappear during chronic treatment. This indicates that amantadine cannot be considered a long lasting therapy of dyskinesias. In the mid '90s, a new peptide, termed nociceptin/orphanin FQ (N/OFQ; Meunier et al., Nature 377, 532-535, 1995; Reinscheid et al., Science 270, 792-794, 1995) was identified. N/OFQ belongs to the opioid family, but interacts with a G-protein-coupled receptor which is different from the classical mu, kappa and delta opioid receptors (now re-named MOP, KOP and DOP according to recent IUPHAR recommendations Cox et al., 2000). This receptor, termed NOP, shows high affinity for N/OFQ but no affinity for classical opioid ligands, and is widely expressed in the central and peripheral nervous system, as well as in other systems (e.g. cardiovascular and genitourinary) where it can also be expressed by non neuronal cells. Studies focusing on the biological activity of NOP receptor agonists (CaIo et al., 2000; Mogil and Pasternak, 2001) have demonstrated that exogenous N/OFQ: i) induces hyperalgesia (i.e. reduces pain threshold) or analgesia, when administered supraspinally or spinally, respectively; ii) induces anxiolysis (mimicked by nonpeptide NOP receptor agonists administered systemically); iii) reduces locomotion, iv) increases food intake, v) reduces self-administration of drugs of abuse. Moreover, N/OFQ affects vi) the renal system (increases the diuresis when given i.v. or i.c.v.), vii) the cardiovascular system (hypotension and bradycardia after i.v. administration, vii) the gastrointestinal system (relaxation and contraction, depending on the segment affected). The pharmacology of the N/OFQ-NOP receptor system can now take advantage of selective peptide antagonists such as [Nphe']N/OFQ(l-13)NH₂ (Calό et al., 2000) and [Nphe¹, Arg ¹⁴, Lys ¹⁵]N/OFQ(1-13)NH₂ (UFP-101; Calό et al., 2002), and nonpeptide antagonists such as J-113397 (1-[3R, 4R)-l-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-l,3- dihydro-2H-benzimidazol-2-one; Kawamoto et al., 1999; Ozaki et al., 2000), JTC-801 (N-(4-amino-2- methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamide hydrochloride; Shinkai et al., 2000; Yamada et al., 2002) and SB-612111 ((-)-cis-l-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-l-yl]methyl]-6,7,8,9- tetrahydro-5H-benzocyclohepten-5-ol; Zaratin et al., 2004). These compounds have been proven effective in counteracting the effects of NOP receptor agonists in several experimental in vitro and in vivo models. In rodents in vivo, they also displayed primary effects opposite to those of the natural ligand N/OFQ, suggesting the existence of an endogenous N/OFQergic tone on some biological functions, such as food intake, where [Nphe']N/OFQ(l-13)NH2 induced anorexia (Polidori et al., 2000), pain threshold, where i.c.v. administration of [Nphe']N/OFQ(l-13)NH2 and UFP-101 induced analgesia (CaIo et al., 2000), and motor activity where UFP-101 and J-113397 improved physiologically-stimulated locomotion (Marti et al., 2004). Moreover, systemic administration of J-113397 exerts antiparkinsonian effects (Marti et al., 2005) whereas i.c.v. administration of [Nphe']N/OFQ(l-13)NH2 and UFP-101 induced antidepressant-like effects (Redrobe et al., 2002; Gavioli et al., 2003). In addition to selective antagonists, peptide and non peptide selective agonists are also available. Among N/OFQ analogues, [Arg^l4,Lys¹⁵]N/OFQ (Okada et al., 2000), UFP-102 ([(pF)Phe⁴, Arg¹⁴,Lys¹⁵]N/OFQ-NH₂; Carra et al., 2004; Guerrini et al., 2005) and UFP-112 ([(pF)Phe⁴, Aib⁷, Arg¹⁴, Lys¹⁵]N/OFQ-NH₂; Rizzi et al., 2006; PCT/EP2006/050958) have emerged with higher potency than the natural peptide. Among non peptide compounds, two compounds must be mentioned, Ro 64-6198 ([(lS,3aS)-8-(2,3_;3a,4,5,6-hexahydro-lH-phenalen-l-yl)-l-phenyl-l,3,8-triaza-spiro[4. 5]decan-4-one];

Rover et al., 2000) and 65-6570 (8-acenaphthen-l-yl-l-phenyl-l,3,8-triaza-spiro[4,5]decan-4-one; Wichmann et al., 1999). Both exert marked in vivo anxiolytic activity and hypolocomotion (Jenck et al., 2000; Higgins et al., 2001). Also the piperidin-4-yl-l,3dihydroindol-2-one derivatives endowed with agonist activity (Zaveri et al., 2004) have been reported to be selective for NOP receptors depending on the substitute at the piperidinic nitrogen. Besides full agonists, partial agonists are available that are known to prevent the actions of the endogenous agonist while maintaining a certain degree of receptor activation. Among these, the pseudopeptides [Phe'ψ(CH₂-NH)GIy²]N/OFQ(l-13)NH, (Guerrini et al., 1998), UFP-103 ([Phe'ψ(CH₂- NH)GIy², (pF)Phe⁴, Arg¹⁴, Lys^l3]N/OFQ-NH₂; Guerrini et al. 2005 ), UFP-113 ([Phe'ΨGly², (pF)Phe⁴, Aib⁷, Arg¹⁴, Lys¹⁵]N/OFQ-NH₂; PCT/EP2006/050958), the peptides Ac-RYYRWK-NH₂, Ac-RYYRIK-NH₂ (Dooley et al., 1997) and their derivatives Ac-RYYRJK-OH (Gunduz et al., 2006) as well as ZP 120 (Kapusta et al., 2005; Rizzi et al., 2002).

DESCRIPTION

The invention provides the use of one or more compounds able to stimulate the

nociceptin/orphanin FQ receptor (NOP receptor) for the preparation of medications to treat

L-DOP A-induced dyskinesias. Such compounds are preferentially full or partial agonists,

selected among peptide, pseudopeptide, peptide-mimetic, peptide-derivative and non-

peptide molecules. Among compounds of peptide and pseudopeptide nature, are preferred

those-having the--following~structures;-where-(pF-)Phe-stands-for-4-fluoride~phenylalanine,

Aib stands for amminoisobutiric acid, and Ψ(CH₂NH) indicates substitution of the CO-

NH peptidic bond with a CH₂-NH bond:

H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln-OH (SEQ ID NO: 1);

H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-NH₂ (SEQ ID NO: 2);

H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-Asn-Gln-OH

(SEQ ID NO: 3);

H-Phe-Gly-Gly-(pF)Phe-Thr-Gly-Aib-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-Asn-Gln- NH₂ (SEQ ID NO: 4) ;

H-Phe-Gly-Gly-(pF)Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-Asn-Gln-

NH₂ (SEQ ID NO: 5); H-Phe-Gly-Gly-(pF)Phe-Thr-Gly-Aib-Arg-Lys-Ser-Ala-Arg-Lys-NH₂ (SEQ ID NO:

6);

H-PheΨ(CH₂NH)Gly-Gly-(pF)Phe-Thr-Gly-Aib-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-

Asn-Gln-NH₂ (SEQ ID NO: 7); H-PheΨ(CH₂NH)Gly-Gly-(pF)Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-

Asn-Gln-NH₂ (SEQ ID NO: 8);

H-PheΨ(CH₂NH)Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-NH₂ (SEQ ID

NO: 9);

Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH₂ (SEQ ID NO: 10); Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH₂ (SEQ ID NO: 11) ;

Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-OH (SEQ ID NO: 12);

Ac-Arg-Tyr-Tyr-Arg-Tφ-Lys-Lys-Lys- Lys-Lys-Lys-Lys-NH₂ (SEQ ID NO: 13).

Among non peptide compounds bearing the above mentioned biological activity, four preferred subgroups can be distinguished, each representing a specific embodiment of the

invention.

According to the first of them, the compounds have general structure 1 :

1 wherein X and Z are, independent of each other, carbon (CH) or nitrogen (N)

Y is carbon (CH₂) or nitrogen (NH)

R] is an aromatic or heteroaromatic ring, with a number of ring members between 5 and

12.

R₂ is an aromatic or aliphatic cyclic or polycyclic substituent, optionally substituted

R₃ is hydrogen (H), or an aliphatic chain with a number of carbons between 1 and 10, substituted with one or more alcohol, ester, acid or amine groups.

Preferably in formula 1, X and Z are nitrogen (N), Y is carbon (CH₂), R₁ is a benzene ring, R₂ is selected among one of the following groups:

R₃ is a hydrogen (H), or an aliphatic chain with a number of carbons between 1 and 10, substituted with one or more ester groups.

According to a second embodiment, non peptide molecules covered by the invention have general structure 2:

wherein the hatched line represents a single or double bond, R₁ is a hydrogen (H) or a hydroxymethyl group (-CH₂-OH), R₂ is an aromatic or aliphatic cyclic or polycyclic substituent, optionally substituted R₃ is an aromatic or heteroaromatic substituent, optionally substituted.

Preferably in formula 2, the hatched line indicates a single bond, R₁ is a hydrogen (H) or a hydroxymethyl group (-CH₂-OH), R₂ is selected among the following groups:

wherein A and B are, independent of each other, a hydrogen (H), halogen (F, Cl, Br, or I), methyl (CH₃), or hydroxymethyl (CH₂OH), R₃ is selected among the following groups:

wherein R₄ is an aliphatic substituent with a number of carbon atoms between 1 and 13 optionally bearing ester, acid (COOH) or amine (NH₂) groups , R₅ and R₆ are, independent of each other, halogen (F, Cl, Br or I), methyl (CH₃) or methoxyl (OCH₃). According to a third embodiment, non peptide molecules covered by the invention have general formula 3:

wherein X is carbon (CH₂), oxygen (O), sulphur (S), nitrogen (NH), and R is an aromatic or aliphatic cyclic or polycyclic substituent, optionally substituted. Preferably in formula 3, R is selected among the following groups:

According to a fourth embodiment, non peptide molecules covered by the invention have general formula 4:

wherein X₅Y and Z are, independent of each other, carbon (CH) or nitrogen (N), Rj is an aromatic or aliphatic substituent, optionally substituted, R₂ is a hydrogen (H) or a linear or cyclic aliphatic chain substituted with an amine and guanidine group, R₃ is a hydrogen (H), methyl (CH₃) or methoxyl (OCH₃), R₄ is a hydrogen (H) or an amine group (NH₂). Preferably in formula 4, R₁ is selected among the following groups:

R₂ is a hydrogen (H) or a linear or cyclic aliphatic chain^{^}substituted^~with^~an^~amine-and guanidine group, R₃ is a hydrogen (H), methyl (CH₃) or methoxyl (OCH₃), R₄ is a hydrogen (H) or an amine group (NH₂). Even more specifically, the above mentioned use extends to specific compounds detailed below:

7-[4-(2,6-Dιchloro-phenyl)-pιpeπdιn-1- , ,

ylmethyl]-1-methyl-6,7,8,9-tetrahydro- W-(4-Ammo-2-meth_y -_;qu,nol₁n-6-yl)-2-(4-eth dihydro-benzo.mriazol^-one SH-benzocyclohepten-5-ol yl-phenoxymethyl)-benzaπn,de

[1 -Cyclooctylmethyl-4-

8-Acenaphthen-1-yl-1-phenyl- 8-(2,3,3a,4,5,6-Hexahydro-1H-phenalen-1-yl)- (2,6-dιmethyl-phenyl)- 1 ,3,8-tπaza-spιro[4 5]decan-4-one 1 -phenyl-1 ,3,8-tπaza-spιro[4 5]decaπ-4-one pipeπdιn-3-yl]-methanol

[i-CyclooctylmethyM-ta.e-dimethyl-phenyl)- 1-Benzyl-pyrrolιdιne-2-carboxylιc acid 1 ,2,5,6-tetrahydro-pyrιdιn-3-yl]-πnethanol {3-[4-(2,6-dιchloro-phenyl)- pιpeπdιn-1 -yl]-propyl}-amιde

Λ/-Methyl-2-{3-[1-(1-methyl-2-naphthalen-1-yl-ethyl) (8-Naphthalen-1-ylmethyl-4-oxo-1-

-piperιdιn-4-yl]-2-oxo-2,3-dιhydro- phenyl-1 ,3,8-triaza-spiro[4.5]dec-3-yl)- benzoιmidazol-1-yl}-acetamide acetic acid methyl ester

According to the invention, the compounds able to activate the NOP receptor for nociceptin/orphanin FQ can be administered to each patient requiring L-DOPA treatment. The patient might have already received L- DOPA, be under treatment with L-DOPA, or plan to undergo treatment with L-DOPA; such a patient might already suffer from dyskinesias or be at risk to develop it. These compounds can be administered for a therapeutic or prophylactic purpose. Such a treatment might outlast L-DOPA therapy, either for therapeutic or prophylactic aims In the case of combination therapy, the above mentioned compounds and L-DOPA might be administered separately, i.e. through different dosage forms, or be combined in the same dosage form, with the advantage of a single administration.

The invention covers the use of a combination of L-DOPA with one or more compounds as previously defined, or their pharmaceutically acceptable salts, for the preparation of a medication to treat L-DOPA induced dyskinesias. Such a medication can be formulated as a dosage form containing both active principles: such a preparation, which represents a further object of the invention, can be administered to each patient that needs L-DOPA treatment and has the advantage of being less dyskinesiogenic or not dyskinesiogenic at all.

The invention also includes all the above mentioned compounds, able to activate the NOP receptor for nociceptin/orphanin FQ, for treatment of L-DOP A-induced dyskinesias. The invention also covers a method to treat or prevent L-DOP A-induced dyskinesias, which is based on the administration of an effective dose of one or more compounds able to activate the NOP receptor for nociceptin/orphanin FQ, as previously defined.

To the best of the inventors' knowledge, this use has not been previously reported.

Indeed, we observed for the first time that intracerebro ventricular (i.c.v.) injection, trough an infusion cannula, of N/OFQ and its analog UFP-112, as well as systemic administration of Ro 65-6570, prevents AIM appearance in a model of L-DOP A-induced dyskinesias, i.e. the rat unilaterally lesioned with-6-hydroxydopamine^~(6=ΘHDA) and-chronically-treated with low doses of L-DOPA (6 mg/Kg; Cenci et al., 1998).

EXPERIMENTAL SECTION

METHODS Dyskinetic rat model

This model has been described for the first time by Cenci and collaborators (Cenci et al., 1998). Male Sprague-Dawley rats weighing 15O g are anesthetized with isoflurane and placed on an stereotaxic apparatus. After drilling a hole into parietal skull, 4 μl of saline solution containing 8 μg of 6-0HDA in 0.2 mg/ml of ascorbic acid are injected at the 1 μl/min rate, according to the following stereotaxic coordinates from the bregma: AP = -4.4 mm; ML = -1.2 mm; VD = -7.8 mm (Paxinos and Watson, 1982). 6-OHDA is a toxin that damages DA neurons projecting to the striatum and reduces dramatically (>95%) the levels of DA in that nucleus. After surgery rats are housed under standard conditions. Two weeks after lesion, the degree of DA depletion is assessed through two different behavioral tests:

A) Evaluation of the number of ipsilateral rotations (i.e. in the same direction of the lesion) induced by intraperitoneal administration of amphetamine (5 mg/kg);

B) Rotarod test.

Four weeks after lesion, the animals selected, i.e. those with a >95% lesion of the DA system, are subcutaneously injected with L-DOPA (6 mg/kg) + benserazide (15 mg/kg), once daily for 21 days.

Every other day, animals are evaluated by an operator (1 min observation every 20 min, from 20 to 180 min after L-DOPA administration), and dyskinesias is scored using four parameters related to various motor and postural conditions (Winkler et al, 2002):

-Locomotive: the rat begins rotating around its axis, contralateral to the lesion side;

-Limb: the rat repetitively moves the contralateral forepaw;

-Axial: the rat turns the trunk and neck in direction contralateral to the lesion side; - Orolingual: the rat shows uncontrolled movements of the facial muscles, especially of the mouth and tongue. A typical movement is the actual protrusion of the tongue outside the mouth.

Each of these four categories is assigned a score which allow the classification of them on a duration scale. Moreover, to better quantify the degree of dyskinesias of the contralateral forepaw and body axis, abnormal involuntary movements are also scored on a severity scale. Duration:

1= the disorder is present for a period less than half of the observation time (less than 30 sec); 2= the disorder is present for more than half of the observation time (more than 30 sec); 3= the disorder is always present throughout the period of observation, but can be suppressed with a stress manoeuvre (snap of the fingers); 4= the disorder is always present throughout the period of observation and cannot be suppressed. Severity: Axial AIM:

1= deviation of the neck and head by an angle of approximately 30° ; 2= deviation of the neck and head by an angle between 30° and 60°: 3= lateral deviation and/or twisting of the neck, head and upper torso by an angle between 60° and 90° 4= torsion of the head, neck and trunk by an angle exceeding 90°, resulting in the loss of balance of the animal. AIM limb: 1= subtle tremors of the proximal and distal part of the leg around a fixed position;

2= small movements that cause a clear translocation of the proximal and distal part of the leg;

3= translocation of the entire limb with visible contraction of the muscles of the shoulder;

4= intense movements of maximal amplitude of the leg and shoulder that can have ballistic nature.

At the end of treatment, the animals that develop dyskinesias undergo surgery. The rat is placed on a stereotaxic apparatus and a stainless steel infusion cannula (15 mm long, 24 gauge) is positioned over the lateral ventricle (stereotaxic coordinates: AP = -0.9 mm; ML = -1.4 mm; VD = -3.2 mm) in accordance with the procedures described above. A stainless steel speculum is inserted into the cannula to avoid its occlusion by external material. The cannula is then fixed to the skull with methacrylic cement and anaesthesia is interrupted. The animal is housed individually in a polycarbonate cage with free access to food and water. In the following days, the animal is treated every other day with maintenance doses of L-DOPA (6 mg/kg + benserazide 15 mg/kg) and trained during daily sessions to stay on a rotating cylinder (rotarod test; Rozas et al., 1997) until its motor performance becomes constant (Marti et al., 2004). The experiment is performed seven days after surgery. The animal is first tested on the rotarod (control session). After 30 min, the speculum occluding the cannula is removed and 0.5 microliters of saline (control rats) or saline containing N/OFQ (0.1 nmol) or UFP-112 (0.001 nmol) are injected into the lateral ventricle through a stainless steel injector. Alternatively, systemic administration of saline or Ro 65-6570 (0.1 mg/kg, i.p.) is performed. Five minutes after the injection of saline or N/OFQ, and 30 min after the administration of UFP-1 12 or Ro 65- 6570, a dyskinetogenic dose of L-DOPA (6 mg/kg + benserazide 15 mg/Kg) is administered subcutaneously. The appearance of AIM is evaluated every 20 min (1 min observation every 20 min, starting from 20 min after L-DOPA administration) according to the scales previously described. To verify that the treatment selectively reduces dyskinesias without depressing animal motor activity, the rotarod test is performed (according to the protocol described by Marti et al., 2004) 60 min after L-DOPA administration, i.e. when dyskinesias is maximal. EXPERIMENTAL RESULTS Effect of acute treatment with L-DOPA and benserazide in dyskinetic rats.

As previously described (Cenci et al., 1998), repetitive administration of L-DOPA (6 mg/kg i.p.) combined with benserazide (15 mg/kg i.p.) in hemiparkinsonian rats for 21 days induces the occurrence of abnormal involuntary movements (dyskinesias). Once established, the dyskinetic condition remains for an extended duration even in the absence of treatment with L-DOPA, and dyskinesias appear with constant intensity each time the animal is acutely treated with L-DOPA. Dyskinesias are already evident 20 min after L-DOPA administration, reaches their maximum after 60 min, and then tend to gradually decline within 120 min (Table 1). During the whole observation period (180 min) dyskinetic animals show abnormal movements affecting locomotion (Lo), or body axis, limb and orolingual muscles (ALO) quantifiable in 10.3 ± 0.9 and 74.4 ± 4.8 arbitrary units, respectively (Cenci et al., 1998).

Since dyskinesias affects rat mobility in proportion to their intensity, the rotarod test is used to quantify the degree of overall motor impairment caused by a dyskinetogenic dose of L-DOPA. As shown in Table 2, 60 min after L-DOPA administration (i.e. when dyskinesias is maximal; Tab 1), rat motor ability on the rotarod is reduced by approximately 85 %. Effect ofN/OFQ on dyskinesias induced by acute treatment with L-DOPA. Dyskinesias test.

To test the hypothesis that stimulation of NOP receptors is effective in reducing L-DOP A-induced dyskinesias, we first examined if N/OFQ, the endogenous agonist of NOP receptors, prevents the effect of L- DOPA. The intracerebroventricular (i.c.v.) microinjection of saline (0.5 μl) or N/OFQ (0.03-1 nmol/0.5 μl) does not induce per se abnormal movements in dyskinetic rats off L-DOPA (data not shown). N/OFQ, administered i.c.v. 5 min before L-DOPA, attenuates the appearance of dyskinesias in a dose-dependent fashion (Tab. 3). The antidyskinetic effect is already present at the dose of 0.03 nmol N/OFQ which reduces by about 50 % both Lo and ALO abnormal movements. Rotarod test. Microinjection of 0.03-0.1 nmol N/OFQ does not affect the rotarod performance of dyskinetic rats off L- DOPA while depressing it at higher doses (1 nmol; Tab. 4). When co-administered with L-DOPA, 0.1 nmol N/OFQ, which reduces dyskinesias by about 60% (Tab. 3), produces full motor recovery on the rotarod (Tab. 4). Conversely, 1 nmol N/OFQ, despite showing a better antidyskinetic effect (Tab. 3), causes minor motor recovery on the rotarod, probably because of its primary inhibitory effect on motor activity (Tab. 4). Receptor antagonism

To investigate whether the antidyskinetic action of N/OFQ is due to NOP receptor selective stimulation, the NOP receptor peptide antagonist, UFP-101 (CaIo et al., 2002) and the non peptide compound J-113397 (Kawamoto et al., 1999) were administerd i.c.v. and i.p, respectively. Administration of J-113397 (3 mg/kg, i.p.) or UFP-101 (10 nmol, i.c.v.) does not induce per se abnormal movements in dyskinetic rats off L-DOPA (data not shown). When administered together with L-DOPA, J-113397 does not change the dyskinetogenic action of L-DOPA (Tab. 5) while UFP-101 increases, albeit slightly, ALO but not Lo abnormal movements (Tab. 5). Administration of J-113397 (1 mg/kg i.p.; 15 min before N/OFQ) or UFP-101 (10 nmol i.c.v.; co- injected with N/OFQ) prevents the antidyskinetic effect of 0.1 nmol N/OFQ (Tab. 6). This pharmacological antagonism is also evident on the rotarod since in the presence of J-113397 or UFP-101, N/OFQ loses its antidyskinetic action (Tab. 6). It should be stressed that, in agreement with previous studies (Marti et al., 2004, 2005), blockade of NOP receptors with UFP-101 (10 nmol) and J-113397 (3 mg/kg) in dyskinetic rats off L-DOPA facilitates motor performance on the rotarod (Tab. 7). Overall, these results suggest that the antidyskinetic action of N/OFQ is selectively mediated by NOP receptor stimulation. We therefore claim the use of medications based on N/OFQ (peptide structures, formula 1) for treatment of L-DOP A-induced dyskinesias.

Effect of UFP-112 on dyskinesias induced by acute treatment with L-DOPA Dyskinesias test. To further demonstrate the potential efficacy of NOP receptor agonists in reducing L-DOP A-induced dyskinesias, we used UFP-112, a NOP receptor peptide agonist 100-fold more potent than N/OFQ (Rizzi et al., 2006). I.c.v. microinjection of UFP-112 (0.01-10 pmol) does not induce per se abnormal movements in dyskinetic rats off L-DOPA (data not shown). Given 30 min before L-DOPA, UFP-112 prevents in a dose- dependent manner the appearance of dyskinesias (Tab. 9). The antidyskinetic effect is already present at a dose of 0.01 pmol, which reduces both Lo and ALO abnormal movements by about 50 %. Rotarod test.

Microinjection of UFP-112 does not affect the motor performance of dyskinetic rat off L-DOPA at 0.01 pmol while depressing it at higher doses (0.1-1 pmol; Tab. 10). At 0.01 pmol (effective in reducing dyskinesias by about 50 %; Tab. 9), UFP-112 induces recovery of motor activity on the rotarod (Tab. 10). The same effect is observed at higher doses even though they exert primary inhibitory effects on motor performance (Tab. 10). Receptor antagonism

To investigate whether the antidyskinetic effect of UFP-112 is due to NOP receptor selective stimulation, UFP-112 has been tested in the presence of J-113397 or UFP-101. Administration of J-113397 (1 mg/kg i.p.; given 15 min before UFP-112) and UFP-101 (10 nmol i.c.v.; co-injected with UFP-112) prevents the antidyskinetic effect of 1 pmol UFP-112. These data show that not only N/OFQ but also its analogs like UFP-112 reduce L-DOPA-induced dyskinesiass through stimulation of NOP receptors. We therefore claim the use of medications based on analogs of the peptide N/OFQ (peptide structures, formulas 2-13) for treatment of L-DOPA-induced dyskinesias. Effect of Ro 65-6570 on dyskinesias induced by acute treatment with L-DOPA. Dyskinesias test

To demonstrate the potential efficacy of the systemic use of NOP receptor agonists in reducing L-DOPA- induced dyskinesias we used Ro 65-6570, a non peptide NOP receptor agonist (Rover et al., 2000). Systemic administration of Ro 65-6570 (0.01-1 mg/kg i.p.) does not induce per se abnormal movements in dyskinetic rats off L-DOPA (data not shown). Given 30 minutes before L-DOPA, Ro 65-6570 prevents in a dose- dependent manner the appearance of dyskinesias (Tab. 13). The antidyskinetic effect is already present at a dose of 0.01 mg/kg which reduces both Lo and ALO abnormal movements by about 70 %. Rotarod test.

Administration of Ro 65-6570 does not affect the motor performance of dyskinetic rat off L-DOPA at 0.01 and 0.1 mg/kg while reducing it at 1 mg/kg (Tab. 14). At 0.01 and 0.1 mg/kg, Ro 65-6570 attenuates motor impairment induced by dyskinesias while allowing full recovery at 1 mg/Kg (Tab. 14). Receptor antagonism

As shown for the other NOP receptor agonists such as N/OFQ and UFP-112, administration of J-113397 (1 mg/kg i.p., 15 min before Ro 65-6570) and UFP-101 (10 nmol i.c.v.; injected 5 min before Ro 65-6570) prevents the antidyskinetic effect of Ro 65-6570 (0.1 mg/Kg; Tab. 15) and motor recovery on the rotarod (Tab. 16). Overall, the data show that also Ro 65-6570 attenuates the L-DOPA-induced dyskinesias through stimulation of NOP receptors. We therefore claim the use of medications based on non peptide agonists (non- peptide structures, general formulas 1-4) of NOP receptor for the treatment of L-DOPA-induced dyskinesias. Effect of amantadine on dyskinesias induced by acute treatment with L-DOPA. Dyskinesias test. Amantadine is currently the only compound used in clinics to treat dyskinesias (Goetz et al., 2002). Therefore, we used this drug as a reference compound to assess the antidyskinetic efficacy of NOP receptor agonists. Subcutaneous administration of amantadine at the reference dose of 40 mg/Kg (Lundblad et al., 2002) does not induce per se abnormal movements in dyskinetic rats off L-DOPA (data not shown). Given 40 min before L-DOPA, amantadine reduces both Lo and ALO abnormal movements by about 80 % (Tab. 17). Rotarod test. At a dose of 40 mg/Kg, amantadine induces full motor recovery on the rotarod (Tab. 18).

TABLES

Table 1. Time-course of the expression of locomotive (Lo), or body axis (axial), limb and orolingual (overall ALO) dyskinesias. Data are expressed as arbitrary units and represent the mean ± SEM of at least 47 determinations made every 20 min after the injection of L-DOPA (6 mg/kg + benserazide 15 mg/kg). In the far right column of the table the total value of dyskinesias (calculated in 180 min) is reported. The table clearly indicates that dyskinesias shows an intensity peak at 60-80 min after L-DOPA injection.

Table 2. Rotarod performance of dyskinetic rats after saline treatment (o/fL-DOPA) or during dyskinesias induced by injection of L-DOPA (6 mg/Kg + benserazide 15 mg/Kg). The rotarod test is performed 60 min after saline or L-DOPA administration. Data are expressed as percent of pre-treatment values (control section) and represent the mean ± SEM of at least 47 determinations. The table shows that L-DOPA administration as well as the appearance of dyskinesias (Tab. 1) causes an impairment of the animal's ability to^^"eTfoττnπ;o^"ordinated^"and^~cornplex^{^}motor^"actions-(performance-on-the-rotarodrcalculated-60-min-after-L DOPA administration, i.e. when dyskinesias is maximal, vd Tab. 1). Conversely, saline administration is ineffective.

** p<0.01 significantly different from saline.

Table 3. Effect of i.c.v. injection of N/OFQ (0.03-1 nmol) or saline on locomotive (Lo), or axial, limb and orolingual (overall ALO) dyskinesias induced by L-DOPA. Data are expressed as percent of L-DOPA effect and represent the mean ± SEM of 10 determinations. N/OFQ or saline were administered 5 min before L- DOPA. The table clearly shows that N/OFQ attenuates the severity of dyskinesias in a dose-related fashion (lower values correspond to stronger antidyskinetic effect) while saline is ineffective. o^op<0.01 significantly different from L-DOP A+ saline

Table 4. Effect of i.e. v. injection of N/OFQ (0.03-1 nmol) or saline on the rotarod performance of dyskinetic rats in the absence and presence of dyskinesias (off and on L-DOPA, respectively). Data are expressed as percent of pretreatment values (control session) and represent the mean ± SEM of 10 determinations. N/OFQ was administered 5 min before L-DOPA. The table clearly shows that in the absence of dyskinesias (off L- DOPA) N/OFQ inhibits (but only at high doses) rotarod performance. However, if one considers the animal with dyskinesias (on L-DOPA), where the motor activity is severely compromised, N/OFQ, due to its antidyskinetic effect, actually improves rotarod activity (higher values correspond to stronger effect). The reason of the weaker effect of 1 nmol compared to 0.1 nmol N/OFQ is due to the hypolocomotion induced by high doses of N/OFQ (see effects OTJL-DOPA). **p<0.01 significantly different from saline °°p<0.01 significantly different from L-DOP A+ saline

Table 5. Effect of administration of J-1 13397 (3 mg/kg ip) and UFP-101 (10 nmol icv) on locomotive (Lo), or axial, limb and orolingual (overall ALO) dyskinesias induced by L-DOPA. Data are expressed as percent of L-DOPA effect and represent the mean ± SEM of 10 determinations. J-113397 and UFP-101 were given 15 and 5 min before L-DOPA, respectively. The table clearly shows that the NOP receptor antagonists of N/OFQ, administered systemically or i.c.v., do not attenuate (UFP-101 slightly worsens) L-DOPA induced dyskinesias, while saline is ineffective.

° p <0.05, significantly different from L-DOPA + saline

Table 6. Blockade of the antidyskinetic effect induced by N/OFQ 0.1 nmol by J-113397 (1 mg/kg i.p.) and UFP-101 (10 nmol i.c.v.). Data are expressed as percent of L-DOPA effect and represent the mean ± SEM of 10 determinations. N/OFQ was injected 5 min before L-DOPA. J-113397 was administered 15 min before N/OFQ while UFP-101 was co-injected with N/OFQ. The table shows that N/OFQ reduces the severity of locomotive (Lo) or axial, forepaw and orolingual (overall ALO) dyskinesias induced by L-DOPA (lower values correspond to stronger antidyskinetic effect) while NOP receptor antagonists pharmacologically block the action of N/OFQ. In the presence of J-113397 and N/OFQ there was even a slight worsening of ALO dyskinesias.

°p <0.05; ^oop <0.01 significantly different from L-DOP A+ saline ^p <0.01 significantly different from L-DOP A+ N/OFQ

Table 7. Effect of J-113397 (3 mg/kg i.p.) and UFP-101 (10 nmol i.c.v.) administration on the rotarod performance of dyskinetic rats in the absence and presence of dyskinesias (offana on L-DOPA, respectively). Data are expressed as percent of pretreatment values (control session) and represent the mean ± SEM of 10 determinations. J-113397 and UFP-101 were given 15 and 5 min before saline or L-DOPA, respectively. The table clearly shows that in the absence of dyskinesias (off L-DOPA), NOP antagonists improve motor performance on the rotarod while in the presence of dyskinesias (on L-DOPA) are ineffective. **P<0.01 significantly different from saline

Table 8. Blockade of the antidyskinetic action of 0.1 nmol N/OFQ on the rotarod by J-113397 (1 mg/kg i.p.) and UFP-101 (10 nmol i.c.v.) administration. Data are expressed as percent of pre-treatment values (control session) and represent the mean ± SEM of 10 determinations. N/OFQ was injected 5 min before L-DOPA. J- 1 13397 was administered 15 min before N/OFQ while UFP-101 was co-injected with N/OFQ. The table clearly shows that L-DOPA inhibits motor performance on the rotarod (due to induction of dyskinesias). This effect was blocked by N/OFQ acting through NOP receptors. Indeed, NOP receptor antagonists counteract the beneficial effect of N/OFQ eliciting the negative effect of L-DOPA. °°p <0.01 significantly different from L-DOPA + saline; §^§p <0.01 significantly different from L-DOPA + N/OFQ

Table 9. Effect of i.c.v. injection of UFP-112 (0.01-10 pmol) on locomotive (Lo), or axial, limb and orolingual (overall ALO) dyskinesias induced by L-DOPA. Data are expressed as percent of L-DOPA effect and represent the mean ± SEM of 10 determinations. UFP-112 was administered 30 min before L-DOPA. The table clearly shows that UFP-112 attenuates dyskinesias in a dose-related fashion (lower values correspond to stronger antidyskinetic effect) while saline is ineffective. °°p<0.01 significantly different from L-DOP A+ saline

Table 10. Effect of i.e. v. injection of UFP-112 (0.01-10 pmol) on rotarod performance of dyskinetic rats in the absence or presence of dyskinesias {off and on L-DOPA, respectively). Data are expressed as percent of pre-treatment values (control session) and represent the mean ± SEM of 10 determinations. UFP-112 was administered 30 min before L-DOPA. The table clearly shows that in the absence of dyskinesias {off L- DOPA) UFP-112 impairs the performance on the rotarod. However, if one considers the animal with dyskinesias {on L-DOPA), where the motor activity is severely compromised, UFP-112, due to its antidyskinetic effect, actually improves rotarod activity (higher values correspond to stronger effect). It is worth noting that the improvement of performance in animals on L-DOPA is also seen for doses that, in the absence of dyskinesias, worsen motor activity (see effect off L-DOPA). **p<0.01 significantly different from saline °°p<0.01 significantly different from L-DOP A+ saline

Table 11. Blockade of the antidyskinetic action of 1 pmol UFP-112 by J-113397 (1 πig/kg i.p.) and UFP-101 (10 nmol i.c.v.) administration. Data are expressed as percent of L-DOPA effect and represent the mean ± SEM of 10 determinations. UFP-112 was injected 30 min before L-DOPA. J-113397 was administered 15 min before UFP-112 while UFP-101 was co-injected with UFP-112. The table shows that UFP-112 reduces locomotive (Lo), or axial, limb and orolingual (overall ALO) dyskinesias induced by L-DOPA (lower values correspond to stronger antidyskinetic effect) while NOP receptor antagonists block the action of UFP-112. In the presence of J-1 13397 and UFP-101 there was a slight worsening of ALO dyskinesias. o^op <0.01 significantly different from L-DOPA + saline §^§p <0.01 significantly different from L-DOPA + UFP-112

Table 12. Blockade of the antidyskinetic action of 1 pmol UFP-112 on the rotarod by J-113397 (1 mg/kg i.p.) and UFP-101 (10 nmol i.c.v.) administration. Data are expressed as percent of pre-treatment values (control session) and represent the mean ± SEM of 10 determinations. UFP-1 12 was injected 30 min before L-DOPA. J-113397 was administered 15 min before UFP-1 12 while UFP-101 was co-injected with UFP- 112. The table clearly shows that L-DOPA inhibits the rotarod performance (due to induction of dyskinesias). This effect was blocked by UFP-112 acting through NOP receptors. Indeed, NOP receptor antagonists counteracted the beneficial effect of UFP-112, eliciting the negative effect of L-DOPA. o^op <0.01 significantly different from L-DOPA + saline §^§p <0.01 significantly different from L-DOP A+ UFP-1 12

Table 13. Effect of systemic administration of Ro 65-6570 (0.01-1 mg/Kg) on locomotive (Lo), or axial, limb and orolingual dyskinesias (overall ALO) induced by L-DOPA. Data are expressed as percent of L- DOPA effect and represent the mean ± SEM of 10 determinations. Ro 65-6570 was administered 30 min before L-DOPA. The table clearly shows that Ro 65-6570 attenuates dyskinesias in a dose-related fashion (lower values correspond to stronger antidyskinetic effect) while saline was ineffective. o^op<0.01 significantly different from L-DOP A+ saline

Table 14. Effect of systemic administration of Ro 65-6570 (0.01-1 ing/Kg) on rotarod performance of dyskinetic rats in the absence or presence of dyskinesias (off and on L-DOPA, respectively). Data are expressed as percent of pre-treatment values (control session) and represent the mean ± SEM of 10 determinations. Ro 65-6570 was administered 30 min before L-DOPA. The table clearly shows that in the absence of dyskinesias (off L-DOPA) Ro 65-6570 (but only at the highest doses) impairs rotarod performance. However, if one considers the animal with dyskinesias (on L-DOPA), where the motor activity is severely compromised, Ro 65-6570, due to its antidyskinetic effect, actually improves rotarod activity (higher values correspond to stronger effect). It is worth noting that the improvement of performance in animals on L-DOPA is also seen for doses that, in the absence of dyskinesias, worsen motor activity (see effect off L-DOPA).

**p<0.01 significantly different from saline o^op<0.01 significantly different from L-DOP A+ saline

Table 15. Blockade of the antidyskinetic action of 0.1 mg/Kg Ro 65-6570 by J-113397 (1 mg/kg i.p.) and

UFP-101 (10 nmol i.c.v.) administration. Data are expressed as percent of L-DOPA effect and represent the mean ± SEM of 10 determinations. Ro 65-6570 was injected 30 min before L-DOPA. J-113397 was administered 15 min before Ro 65-6570 while UFP-101 was injected 5 min before Ro 65-6570. The table shows that Ro 65-6570 reduces locomotive (Lo), or axial, limb and orolingual (overall ALO) dyskinesias induced by L-DOPA (lower values correspond to stronger antidyskinetic effect) while NOP receptor antagonists block the action of UFP-112. o^op <0.01 significantly different from L-DOPA + saline

^§§p <0.01 significantly different from L-DOPA + Ro 65-6570

Table 16. Blockade of the antidyskinetic action of Ro 65-6570 (0.1 mg/Kg) on the rotarod by J-113397 (1 mg/kg i.p.) and UFP-101 (10 nmol i.c.v.) administration. Data are expressed as percent of pre-treatment values (control session) and represent the mean ± SEM of 10 deteπninations. Ro 65-6570 was injected 30 min before L-DOPA. J-113397 was administered 15 min before Ro 65-6570 while UFP-101 was injected 5 min before Ro 65-6570. The table clearly shows that L-DOPA inhibits motor performance on the rotarod (due to induction of dyskinesiass). This effect was blocked by Ro 65-6570 acting through NOP receptors. Indeed, NOP receptor antagonists counteract the beneficial effect of Ro 65- 6570, disclosing the negative effect of L-DOPA. ^oop <0.01 significantly different from L-DOPA + saline §^§p <0.01 significantly different from L-DOPA + Ro 65-6570

Table 17. Effect of systemic administration of amantadine (40 mgKg s.c.) on locomotive (Lo), and axial, limb and orolingual dyskinesias (overall ALO) induced by L-DOPA. Data are expressed as percent of L- DOPA effect and represent the mean ± SEM of 6 determinations. Amantadine was administered 40 min before L-DOPA. The table clearly shows that amantadine attenuates the dyskinesias (lower values correspond to stronger antidyskinetic effect) while saline was ineffective. °°p<0.01 significantly different from L-DOP A+saline

Dyskinesias test: effect of amantadine treatment Lo ALO

(% of L-DOPA) (% ofL-DOPA)

L-DOPA + saline 104.4±6.1 107.4±4.9

L-DOPA + amantadine 20.3±8.2°° 43.5±5.4°°

10

ft

Table 18. Effect of systemic administration of amantadine (40 mgKg s.c.) on the rotarod performance in the presence of dyskinesias (on L-DOPA). Data are expressed as percent of pre-treatment values (control 0 session) and represent the mean ± SEM of 6 determinations. Amantadine was administered 40 min before L- DOPA. The effect of amantadine in the absence of dyskinesias {off L-DOPA) was not tested (nt). The table shows that amantadine, due to its antidyskinetic effect, ameliorates rotarod performance impaired by L- DOPA (higher values correspond to stronger effect) while saline was ineffective. °°p<0.01 significantly different from L-DOP A+ saline 5 nt= not tested

An example list of full/partial agonists for NOP receptors is provided below:

1. peptide agonists:

Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln (Peptidg sequence of nociceptin is abbreviated as N/OFQ)

[Arg¹⁴,Lys¹⁵]N/OFQ (Okada et al., 2000);

[(pF)Phe⁴, Arg¹⁴, Lys¹⁵]N/OFQ-NH₂ (UFP102; Carra' et al., 2005; Guerrini et al., 2005); [(pF)Phe⁴, Aib⁷, Arg¹⁴, Lys¹⁵]N/OFQ-NH₂ (UFP-112; Rizzi et al., 2006; PCT/EP2006/050958). 2. peptide partial agonists:

[Phe^(CH₂-NH)Gly²]N/OFQ(l-13)NH₂ ([F/G];Guerrini et. al. 1998)

[PlIe¹T(CH₂-NH)GIy², pFPhe⁴, Arg¹⁴, Lys¹⁵]N/OFQ-NH₂ (UFP-103 ;Guerrini et. al. 2005); [PlIe¹T(CH₂-NH)GIy², pFPhe⁴, Aib⁷, Arg¹⁴, Lys¹⁵]N/OFQ-NH₂ (UFP-113; PCT/EP2006/050958); Ac-RYYRIK-NH₂ e Ac-RYYRWK-NH₂ (Dooley et al., 1997) Ac-RYYRIK-ol (Gunduz et al, 2006); ZP 120, (Ac-RYYRWKKKKKKK-NH,) ( Zealand Pharma WO 01/98324; Kapusta et al., 2005. Rizzi et al.,

2002);

3. non peptide agonists:

Ro 64-6198 ((lS,3aS)-8-(2,3,3a,4,5, 6-hexahydro-lH-phenalen-l-yl)-l-phenyl-l,3,8-triazaspiro[4. 5]decan- 4-one; Hoffmann La Roche EP 921125; Wichmann et al., 1999; Rover et al., 2000) e hexahydrospiro- [piperidine-4,l'[3,4-c]pyrroles] (Kolczewski et al., 2003); piperidin-4-il-l,3-dihydroindol-2one ad attivita agonista in dipendenza del sostituente sull'azoto piperidinico (Zaveri et.al., 2004);

Ro 65-6570, (ΛS)-8-Acenaphten-l-yl-l -phenyl- 1, 3, 8-triazaspiro[4.5]-decan-4-one. (Hoffmann La Roche EP 856514; Rover et al.,2000); NNC 63-0532 (S-naphthalen-l-ylmethyM-oxo-l-phenyl-l^^-triaza-spiro^.Sl-deco-yO-acetic acid methyl ester ( Novo Nordisk WO 9929696; Watson et al., 1999; Thomsen and Hohlweg, 2000) W-212393 (2- {3-[ 1 -(( 1 R)-acenaphthen- 1 -yl)piperidin-4-yl]-2,3-dihydro-2-oxo-benzimidazol- 1 -yl} -N- methylacetamide) (Teshima et al., 2005).

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Claims

1. The use of one or more compounds able to activate the nociceptin/orphanin FQ NOP receptor for the preparation of a medication to treat L-DOPA induced dyskinesias.

2. The use according to claim 1, where such compounds are full or partial agonists at NOP receptors selected among peptides, pseudopeptides, peptide-mimetics, peptide-derivatives or non peptide molecules.

3. The use according to claims 1-2, where such compounds are selected among

peptide or pseudopeptide molecules having the following structures, where (pF)Phe stands

for 4-fluoride phenylalanine, Aib stands for amminoisobutiric acid, and Ψ(CH₂NH)

indicates substitution of the CO-NH peptidic bond with a CH₂-NH bond:

H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-NH₂ (SEQ ID NO: 2);

H-Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-Asn-Gln-OH

(SEQ ID NO: 3);

H-Phe-Gly-Gly-(pF)Phe-Thr-Gly-Aib-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-Asn-Gln- NH2_(SEQJD NOL4) ;

H-Phe-Gly-Gly-(pF)Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-Asn-Gln-

NH₂ (SEQ ID NO: 5);

H-Phe-Gly-Gly-(pF)Phe-Thr-Gly-Aib-Arg-Lys-Ser-Ala-Arg-Lys-NH₂ (SEQ ID NO:

6); H-PheΨ(CH₂NH)Gly-Gly-(pF)Phe-Thr-Gly-Aib-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-

Asn-Gln-NH₂ (SEQ ID NO: 7);

H-PheΨ(CH₂NH)Gly-Gly-(pF)Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Arg-Lys-

Asn-Gln-NH₂ (SEQ ID NO: 8);

H-PheΨ(CH₂NH)Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-NH₂ (SEQ ID NO:^" 9); ^{" '}

Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-NH₂ (SEQ ID NO: 10);

Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH₂ (SEQ ID NO: 11) ; Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-OH (SEQ ID NO: 12); Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys- Lys-Lys-Lys-Lys-NH₂ (SEQ ID NO: 13).

4. The use according to calims 1-2, where such compounds have general formula 1 :

1

wherein X and Z are, independent of each other, carbon (CH) or nitrogen (N)

Y is carbon (CH₂) or nitrogen (NH) R₁ is an aromatic or heteroaromatic ring, with a number of ring members between 5 and 12 R₂ is an aromatic or aliphatic cyclic or polycyclic substituent, optionally substituted

5. The use according to claim 4, where: X and Z are nitrogen (N)

Y is carbon (CH₂) R₁ is a benzene ring

R₂ is selected among one of the following groups:

R₃ is a hydrogen (H)₅ or an aliphatic chain with a number of carbons between 1 and 10, substituted with one or more ester groups.

6. The use according to claims 1-2, where such compounds have general formula 2:

wherein the hatched line represents a single or double bond

R₁ is a hydrogen (H) or a hydroxymethylic group (-CH₂-OH)

R₃ is an aromatic or heteroaromatic substituent, optionally substituted

7. The use according to claim 6, wherein: the hatched line represents a single bond

R₁ is a hydrogen (H) or a hydroxymethylic group (-CH₂-OH)

R₂ is selected among one of the following groups:

wherein A- and B are, independent of each other,- a hygrogen (H), halogen (F, Gl₅-Br, or-I),— methyl (CH₃), or hydroxymethyl (CH₂OH),

R₃ is selected among one of the following groups:

wherein R₄ is an aliphatic substituent with a number of carbon atoms between 1 and 13, optionally bearing ester, acid (COOH) or amine (NH₂) groups, R₅ and R₆ are independent of each other halogen (F, Cl, Br or I), methyl (CH₃) or methoxyl (OCH₃).

8. The use according to claims 1-2, where such compounds have general formula 3:

wherein X is carbon (CH₂), oxygen (O), sulphur (S) o nitrogen (NH)

R is an aromatic or aliphatic cyclic or polycyclic substituent, optionally substituted.

9. The use according to claim 8, where R is selected among one of the following groups:

10. The ^"use according to claims 1-2^", where such compounds have general formula 4:

wherein X, Y and Z are independent of each other carbon (CH) or nitrogen (N)

R₁ is an aromatic or aliphatic substituent, optionally substituted

R₂ is a hydrogen (H) or a linear or cyclic aliphatic chain substituted with an amine and guanidine group

R₃ is a hydrogen (H), methyl (CH₃) or methoxyl (OCH₃)

R₄ is a hydrogen (H) or an amine group (NH₂).

11. The use according to claim 10, where:

R₁ is selected among one of the following groups:

R₃ is a hydrogen (H), methyl (CH₃) or methoxyl (OCH₃)

R₄ is a hydrogen (H) or an amine group (NH₂).

12. The use according to claims 1-11, where the compounds is selected among the following structures:

1 -( 1 -Cyclooctylmethy l-3-hydroxy 7-[4-(2,6-Dichloro-phenyl)-pipeπdin-1- „ , , . . ,_ . . , „ „ , methyl-pipeιϊdin-4-yl)-3-ethyl-1,3- ylmethyl]-1-methyl-6,7,8,9-tetrahydro- W-(4-Am,no-2-methyl-quιnolιn^yl)-2-(4-eth dihydro-benzoimidazol-2-oπe 5H-benzocydohepten-5-ol yl-phenoxymethyl)-benzamιde

[i-Cyclooctylmethyl-4-

8-Acenaphthen-1 -yl-1 -phenyl- 8-(2,3,3a,4,5,6-Hexahydro-1H-phenalen-1-yl)- (2,6-dimethyl-phenyl)- 1 ,3,8-triaza-spiro[4.5]decan-4-one 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one piperidin-3-yl]-methanol

[_1^Cyclooctylmethylr4r(2,6rdimethylzphenyl)- 1 -Benzyl-pyrrolidine-2-carboxy lie acid 1 ,2,5,6-tetrahydro-pyridin-3-yl]-methanol {3-[4-(2,6-dichloro-phenyl)- piperidin-1-yl]-propyl}-amide

Λ/-Methyl-2-{3-[1 -(1-methyl-2-naphthalen-1-yl-ethyl) (8-Naphthaien-1-ylmethyl-4-oxo-1-

-piperidin-4-yl]-2-oxo-2,3-dihydro- phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl)- benzoimidazol^ylX-acetamide acetic acid jTiethyi _ester

13. The use of a combination including one or more compounds as defined in claims 1- 12, or their pharmaceutically acceptable salts, and L-DOPA, to prepare a medication for the treatment of L-DOP A- induced dyskinesias.

14. Pharmaceutical composition including one or more compounds as defined in claims 1-12 or their pharmaceutically acceptable salts, and L-DOPA.

15. A compound as defined in claims 1-12 or a pharmaceutical composition according to claim 13, for use in the treatment of L-DOP A-induced dyskinesias.