EA040772B1 - COMBINATIONS CONTAINING ORTHOSTERIC AGONISTS OF METABOTROPIC GLUTAMATERGIC RECEPTOR 2 SUBTYPES AND THEIR USE - Google Patents

Description

Field of invention

The invention relates to combinations containing an orthosteric agonist of the metabotropic glutamatergic receptor subtype 2, or a pharmaceutically acceptable salt or solvate thereof, and a synaptic vesicle 2A (SV2A) protein ligand.

Background of the invention

The term epilepsy describes a condition in which an individual has recurrent epileptic seizures caused by a chronic process in the deep structures of the brain. Epilepsy refers to a clinical symptom rather than a separate nosological form, since there are a large number of forms and causes of epilepsy. Using the definition of epilepsy as two or more unprovoked epileptic seizures, the incidence of epilepsy is estimated to be approximately 0.3-0.5% in various populations worldwide, with an estimated prevalence of epilepsy of 5-10 people per 1000.

An important step in the evaluation and management of a patient with an epileptic seizure is to determine the type of epileptic seizure that has occurred. The main characteristic that distinguishes the various categories of epileptic seizures is whether the seizure activity is partial (synonymous with focal) or generalized.

Partial seizures are those seizures in which seizure activity is limited to certain areas of the cerebral cortex. If consciousness is fully preserved during an epileptic seizure, then the clinical manifestations are considered relatively simple, and the epileptic seizure is called a simple partial seizure. If consciousness is disturbed, then an epileptic seizure is called a complex partial seizure. An important additional subgroup includes epileptic seizures that begin as a partial seizure and then spread diffusely throughout the cerebral cortex, which are known as secondary generalized partial seizures.

Generalized epileptic seizures simultaneously involve diffuse areas of the brain in a bilaterally symmetrical manner. Minor epileptic seizures or a mild form of epileptic seizures are characterized by sudden short-term lapses in consciousness without loss of control over body position. Atypical petit mal seizures usually include a longer duration in the blackout, less violent onset and cessation, and more pronounced motor signs, which may include focal or lateralizing signs. Generalized tonic-clonic or grand mal seizures, which are the main type of generalized epileptic seizures, are characterized by a violent onset without precursors. In the initial phase of an epileptic seizure, there is usually a tonic muscle spasm, impaired breathing, a significant increase in sympathetic tone, leading to an increase in heart rate, blood pressure and pupil size. After 10-20 seconds, the tonic phase of an epileptic seizure usually passes into the clonic phase, which consists of alternating periods of muscle relaxation and tonic muscle contraction. The periods of relaxation gradually increase until the end of the ictal phase, which usually lasts no more than 1 min. The postictal phase is characterized by unresponsiveness, muscle relaxation, and increased salivation, which can cause wheezing and partial airway obstruction. Atonic seizures are characterized by a sudden loss of postural muscle tone lasting 1–2 s. Consciousness is briefly disturbed, but usually not accompanied by post-seizure confusion. Myoclonic seizures are characterized by sudden and brief muscle contractions that may involve part of the body or the entire body.

Synaptic vesicle protein 2A (SV2A) has been identified as a broad spectrum anticonvulsant target in models of partial and generalized epilepsy. The results of studies performed in animal models and human tissues suggest that changes in SV2A expression result in the development of epilepsy (for a review, see, for example: (a) Mendoza-Torreblanca et al., Synaptic vesicle protein 2A: basic facts and role in synaptic function European Journal of Neuroscience 2013, pp. 1-11 (b) Kaminski R.M. et al., Targeting SV2A for Discovery of Antiepileptic Drugs B: Noebels J.L., Avoli M., Rogawski M.A. et al., editors Jasper's Basic Mechanisms of the Epilepsies [Internet] 4th edition Bethesda (MD): National Center for Biotechnology Information (US) 2012 Available at http://www.ncbi.nlm.nih.gov/books/NBK98183/ ).

The exact role of SV2A remains unclear, but research suggests that changes in SV2A expression affect synaptic function (Nowack et al., Levetiracetam reverses synaptic deficits produced by overexpression of SV2A PLoS One 2011, Volume 6 (12), e29560). It has also been suggested that SV2A is a key player in exocytosis and is involved in nerve impulse transmission (Crowder et al., Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A) Proc. Nat. Acad. Sci. USA 1999, 96, pp 152 68-15273), and studies in knockout mice suggest that a lack of SV2A results in an imbalance between glutamatergic and GABAergic neurons (Venkatesan et al., Altered balance between excitatory and inhibitory inputs onto CA pyramidal neurons from SV2A-deficient but not SV2B-deficient mice J.

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neurosci. Res. 2012, 90, pp. 2317-2327). Decreased expression of SV2A may be the cause of seizure activity and may be involved in the progression of epilepsy 433; Feng et al., Down-regulation of synaptic vesicle protein 2A in the anterior temporal neocortex of patients with intractable epilepsy J. Mol. Neurosci. 2009, 39, pp. 354-359; Toering et al., Expression patterns of synaptic vesicle protein 2A in focal cortical dysplasia and TSC-cortical tubers Epilepsia 2009, 50, pp. 1409-1418) and epileptogenesis in patients with brain tumors (de Groot et al., Expression of synaptic vesicle protein 2A in epilepsy-associated brain tumors and in the peritumoral cortex Neuro-Oncology 2010, 12, pp. 265-273).

SV2A ligands include levetiracetam (Lynch et al., The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam Proc. Natl. Acad. Sci. USA 2004, Vol. 101, pp. 9861-9866), brivaracetam, and seletracetam ( Kaminski R.M. et al., Targeting SV2A for Discovery of Antiepileptic Drugs B: Noebels J.L., Avoli M., Rogawski M.A. et al., editors Jasper's Basic Mechanisms of the Epilepsies [Internet] 4th edition Bethesda (MD): National Center for Biotechnology Information (US), 2012. Available at http://www.ncbi.nlm.nih.gov/books/NBK98183/ Nowack et al., Levetiracetam reverses synaptics deficits produced by overexpression of SV2A PLoSone December 2011, Vol 6(12), e29560).

Levetiracetam, (-)-(S)-α-ethyl-2-oxo-1-pyrrolidineacetamide or (S)-2-(2-oxopyrrolidin-1yl)butanamide

ΝΗ ₂ is an antiepileptic drug. Levetiracetam has not been shown to be active in traditional models of acute forms (maximum shock and pentylenetetrazole tests for epileptic seizures), but has been found to be effective in models of chronic epilepsy and in genetic models of generalized epilepsy. A high safety margin has been shown compared to other antiepileptic drugs (Klitgaard Levetiracetam: the preclinical profile of a new class of antiepileptic drugs Epilepsia 2001, 42 (Supplement 4), pp. 13-18). It is marketed under the brand name Keppra® and is available as tablets, as an oral solution, and as a concentrate for infusion. Keppra® has been approved in Europe as monotherapy in patients aged 16 years and older with newly diagnosed epilepsy, in the treatment of partial seizures (convulsions) with or without secondary generalization and as adjunctive therapy for use with other antiepileptic drugs in the treatment of partial seizures with generalization or without it in patients aged from 1 month; myoclonic seizures in patients over 12 years of age with juvenile myoclonic epilepsy; and primary generalized tonic-clonic seizures in patients aged 12 years and older with idiopathic generalized epilepsy (www.ema.europa.eu). Keppra® has also been approved in the US as an add-on therapy for the treatment of partial seizures in patients aged 1 month or older; myoclonic seizures in patients aged 12 years and older with juvenile myoclonic epilepsy; and primary generalized tonic-clonic seizures in patients aged 6 years and older with idiopathic generalized epilepsy. Keppra XR®, available as sustained release tablets, has been approved in the US for the adjunctive treatment of partial seizures in patients 16 years of age and older with epilepsy (http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index .cfm) .

Brivaracetam, 4-p-propyl analog of levetiracetam, (2S)-2-[(4R)-oxo-4-pronylpyrrolidin-1yl]butanamide

is undergoing clinical trials and is being investigated as monotherapy for partial seizures and postherpetic neuralgia, as well as as adjunctive therapy for refractory partial seizures, Unferricht-Lundborg disease in adolescents and adults, and photogenic epilepsy (www.clinicaltrials.gov).

Seletracetam, (2S)-2-[(4S)-4-(2,2,-difluorovinyl)-2-oxopyrrolidin-1-yl]butanamide

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has been tested in clinical trials.

Methods for preparing these three compounds are known from the literature. For example, processes for the production of levetiracetam are disclosed in, for example, EP 0162036 and GB 2225322. A process for the production of brivaracetam is disclosed in, for example, WO 01/62726. A process for the production of seletracetam is known, for example, from WO 2005/121082. Alternative methods for preparing these three compounds are disclosed in EP 1806339.

Landmarck Antiepileptic drugs in non-epilepsy disorders. Relations between mechanisms of action and clinical efficacy of CNS have been established. Drugs 2008, Vol.22(1), pp.27-47 Calabresi et al., Antiepileptic drugs in migraine: from clinical aspects to cellular mechanisms Trends in Pharmacological Sciences 2007, Vol.28(4), pp.188-195 ; Rogawski and Loscher The neurobiology of antiepileptic drugs for the treatment of nonepileptic conditions Nat. Med. 2004, Vol. 10, pp. 685-692).

Levetiracetam has been found to be effective or potentially effective in a wide range of neuropsychiatric disorders, including mood disorders (Muralidharan and Bhagwagar Potential of levetiracetam in mood disorders: a preliminary review CNS Drugs 2006, Vol. 20, pp. 969-979; Mula et al. , The role of anticonvulsant drugs in anxiety disorders: a critical review of the evidence J. Clin. Pshycopharmacol. 2007, Vol. 27, pp. 263-272) disorders J Clin Psychiatry 2007 Vol 68 pp 1010-1013 Zhang et al Levetiracetam in social phobia: a placebo controlled pilot study J Psychopharmacol 2005 Vol 19 pp 551-553 Kinrys et al., Levetiracetam for treatment-refractory posttraumatic stress disorder J. Clin. Psychiatry 2006, Vol. 67, pp. 211-214, pain (Enggaard et al., Specific effect of levetiracetam in experimental human pain models Eur. J. Pain 2006, Vol 10, pp 193-198; Dunteman Levetiracetam as an adjunctive analgesic in neoplastic plexopathies: case series and commentary J. Pain Palliative Care Pharmacother. 2005 Vol. 19, pp. 3543; Price Levetiracetam in the treatment of neuropathic pain: three case studies Clin. J. Pain 2004, Vol. 20, pp. 33-36), movement disorders (Bushara et al., The effect of levetiracetam on essential tremor Neurology 2005, Vol. 64, pp. 1078-1080; McGavin et al., Levetiracetam as a treatment for tardive dyskinesia: a case report Neurology 2003, Vol. 61, pp. 419; Woods et al., Effects of levetiracetam on tardive dyskinesia: a randomized, double-blind, placebo-controlled study J. Clin. Psychiatry 2008, Vol. 69, pp. 546-554; Zivkovic et al., Treatment of tardive dyskinesia with levetiracetam in a transplant patient Acta Neurol Scand 2008, Vol. 117, pp. 351-353; Neurol. Neurosurg. Psychiatry 2007, Vol. 78, pp. 438-439) and is believed to have potentially positive effects on cognitive function (Piazzini et al., Levetiracetam: An improvement of attention and of oral fluency in patients with partial epilepsy Epilepsy Research 2006, Vol. 68, pp. 181-188 de Groot et al., Levetiracetam improv es verbal memory in high-grade glioma patients Neuro-oncology 2013, Vol. 15(2), pp. 216-223; Bakker et al., Reduction of hippocampal hyperactivity improves cognition in amnestic mild impairment cognitive Neuron 2012, Vol. 74, pp. 467-474; for review: Eddy et al., The cognitive impact of antiepileptic drugs Ther. Adv. Neurol. Discord. 2011 Vol. 4(6), pp. 385-407 and references cited herein; Wheless Levetiracetam in the treatment of childhood epilepsy Neuropsychiatric Disease and Treatment 2007, Vol. 3(4), pp. 409-421) and behavioral symptoms in dementia (Dolder and Nealy The efficacy and safety of newer anticonvulsants in patients with dementia Drugs Aging 2012, Vol. 29(8), pp. 627637). Animal data and some results from preliminary clinical trials suggest that levetiracetam may have the potential to control post-traumatic epilepsy such as status epilepticus, traumatic brain injury, and ischemic stroke, and appear to have neuroprotective effects. The potential of levetiracetam to attenuate epileptogenesis or cognitive dysfunction remains to be elucidated through definitive clinical and animal studies (for review: Loscher and Brandt Prevention or modification of epileptogenesis after brain insults: experimental approaches and translational research Pharmacol. Rev. 2010, Vol. 62, 668 -700; Shetty Prospects of levetiracetam as a neuroprotective drug against status epilepticus, traumatic brain injury and stroke Front. Neur.2013, 4:172. Doi: 10.3389/fneur.2013.00172) because it showed antiepileptogenic activity in a kindling model mice and rats. It has also been suggested that levetiracetam inhibits glutamate release (Lee et al., Levetiracetam inhibits glutamate transmission through presynaptic P/Q-type calcium channels on the granule cells of the dentate gyrus British Journal of Pharmacology 2009, Vol. 158, pp.

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1753-1762).

Seletracetam and brivaracetam have been found to reduce the severity of dystonia in a dt ^sz mutant hamster model and may be beneficial in some patients suffering from dyskinetic and dystonic movement disorder (Hamann et al., Brivaracetam and seletracetam, two new SV2A ligands , improve paroxysmal dystonia in the dt ^sz mutant hamster European Journal of Pharmacology 2008, Vol.601, pp.99-102).

Positive mGluR2 allosteric modulators have recently been discovered as promising new therapeutic approaches for the treatment of a number of CNS diseases, including epilepsy, and some mGluR2 PAMs are currently in clinical trials for the treatment of schizophrenia and depression associated with feelings of inferiority (www.clinicaltrials.gov, see for example: JNJ-40411813/ADX71149 by Addex Therapeutics and Janssen Pharmaceuticals, Inc.). The initial suggestion that drugs that attenuate glutamatergic nerve impulse transmission might be effective for the treatment of epilepsy was based on the results of non-clinical direct-effect studies with mixed mGlu2/3 receptor agonists (Moldrich et al., Glutamate metabotropic receptors as targets for drug therapy in epilepsy Eur J Pharmacol 2003 Vol 476 pp 3-16). LY379268 and LY389795, two mGlu2/3 receptor agonists, were found to be ineffective in blocking the MES of epileptic seizures up to doses that induce motor failure, but were found to be effective in a dose-dependent 6 Hz stimulation model (Barton et al., Comparison of the effect of glutamate receptor modulators in the 6 Hz and maximal electroshock seizure models (Epilepsy Research 2003, Vol. 56, pp. 17-26). Chronic administration of an mGlu2/3 agonist paradoxically induced seizure activity during long-term toxicological studies (Dunayevich et al., Efficacy and tolerability of an mGlu2/3 agonist in the treatment of generalized anxiety disorder Neuropsychopharmacology. 2008, Vol. 33(7), pp. 1603-10). This paradoxical effect may be associated with agonist-induced changes in the sensitivity of the receptor system (tachyphylaxis), but, nevertheless, it was not noted in preclinical studies of epilepsy in model subjects. On the contrary, positive allosteric modulators modulate the continuous transmission of the nerve impulse, but do not have a direct stimulatory effect, thereby reducing the risk of tachyphylaxis.

Before seizure activity, an increase in extracellular glutamate levels in the human hippocampus is measured, and this increase is maintained during epileptogenic activity (During and Spencer Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain Lancet 1993, Vol. 341(8861), pp. 1607-10), thus providing support for the idea that lowering glutamate levels may have a beneficial effect in the treatment of epilepsy. In fact, during seizure activity, glutamate levels rise to potentially neurotoxic levels. Seizure activity results in progressive structural damage to the human brain, inducing further abnormalities in glutamate metabolism (Petroff et al., Glutamate-glutamine cycling in the epileptic human hippocampus Epilepsia 2002, Vol. 43(7), pp. 703-10 ). Thus, it is hypothesized that a positive allosteric mGluR2 modulator or an orthosteric mGluR2 agonist may protect against seizure-induced neuronal damage.

Patent documents WO 2009/033704 and WO 2010/130424 disclose mGluR2 positive allosteric modulators, their uses and methods for synthesizing these compounds. Patent documents WO 1997/18199 and WO 2003/104217 disclose motor amino acid receptor modulator compounds that have later been shown to have orthosteric mGlu2/3 agonist activity (see e.g. Rorick-Kehn et al. (2007) The Journal of Pharmacology and Experimental therapeutics Vol. 321, No. 1, pp. 308-317), further examples of compounds having orthosteric mGlu2/3 agonist activity are disclosed in the scientific and patent literature, and patent document WO 2008/150233 discloses compounds with allosteric activating activity for mGluR2.

Currently available antiepileptic drugs do not exclusively affect glutamatergic transmission of the nerve impulse. Their mechanism of action is usually explained by a change in the balance between excitatory (glutamate-mediated) and inhibitory (GABA-mediated) nerve impulse transmission (Johannessen Landmark Antiepileptic drugs in non-epilepsy disorders: relations between mechanisms of action and clinical efficacy CNS Drugs 2008, Vol. 22(1 ), pp. 27-47).

A significant limiting factor in the use of SV2A ligands is tolerability and side effect profile. For example, the effective dose of levetiracetam for partial seizures is 1000 mg, 2000 mg and 3000 mg given twice daily. Side effects noted for levetiracetam include aggressive or restless behavior, anxiety, personality change, fever, cough or hoarseness, crying, depersonalization, diarrhea, xerostomia, euphoria, fever, general discomfort or soreness, headache, hyperventilation, irregular heartbeat, irritability, joint pain, decreased appetite, back or side pain, depression, muscle aches and pains, nausea, painful or difficult urination, steam

- 4 040772 anxiety, overreaction or excessive emotionality, rapidly changing moods, restlessness, shaking paralysis, tremors, shortness of breath, drowsiness or unusual lethargy, sore throat, stuffy nose or runny nose, sweating, sleep disturbance, unusual fatigue or weakness and vomiting . Thus, there is still a need to provide effective treatment with a low effective dose of levetiracetam and a more favorable side effect profile for the treatment of epilepsy and related disorders, not only for adults, but also for pediatric patients.

Brief description of graphic materials

Fig. 1 - studies of combinations for the reference Comm. No. 25-a with levetiracetam (LEV) in a 6 Hz (44 mA) stimulation assay. At a dose of 10 mg/kg, sc, reference comp. No. 25-a improves the efficiency of the LEV, resulting in an approximately 70-fold shift in ED ₅₀ . This indicates a positive pharmacodynamic relationship;

fig. 2 - studies of combinations for the reference Comm. No. 2-a with levetiracetam (LEV) in a 6 Hz (44 mA) stimulation assay. At a dose of 10 mg/kg, sc, reference comp. No. 2-a improves the efficiency of the LEV, resulting in an approximately 35-fold shift in ED ₅₀ . This indicates a positive pharmacodynamic relationship;

fig. 3 - Combination studies for LY-404039 with levetiracetam (LEV) in a 6 Hz (44 mA) stimulation assay. At a dose of 5 mg/kg, sc, LY-404039 increased the effectiveness of LEV, resulting in an approximately 27-fold shift in ED ₅₀ . This indicates a positive pharmacodynamic relationship.

Description of the invention

The invention relates to a combination for the treatment or prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, and bipolar and related disorders, comprising:

(a) a synaptic vesicle 2A (SV2A) protein ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and (b) an orthosteric subtype 2 metabotropic glutamatergic receptor agonist selected from

1) LY-404039 he

or its salt or solvate; And

2) LY-2140023

or its salt or solvate.

Preferably, the SV2A ligand is levetiracetam or brivaracetam and the metabotropic glutamatergic receptor subtype 2 orthosteric agonist is LY404039.

In another embodiment, the metabotropic glutamatergic receptor subtype 2 orthosteric agonist is LY-2140023.

Also, the metabotropic glutamatergic receptor subtype 2 orthosteric agonist may be LY-2140023 monohydrate.

The invention is also directed to a pharmaceutical composition containing the above combination and a pharmaceutically acceptable carrier.

In addition, the invention relates to a method for preparing a pharmaceutical composition according to the invention, where the above combination is intimately mixed with a pharmaceutical carrier.

Further, the invention includes a drug containing a combination of an SV2A ligand and an orthosteric agonist of the metabotropic glutamatergic receptor subtype 2, for simultaneous

- 5 040772 single, separate or sequential use in the treatment or prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, bipolar and related disorders.

In this case, the SV2A ligand and the orthosteric agonist of the metabotropic glutamatergic receptor subtype 2 can be administered separately.

In addition, the invention includes the use of the combination of the invention for the preparation of a medicament for the treatment of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, bipolar and related disorders.

Also, the combination according to the invention can be used for the preparation of a medicament for the prevention of epileptogenesis.

In addition, the invention includes the use of the combination of the invention for the preparation of a medicament for the prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, and bipolar and related disorders.

Preferably, the drug is for the treatment of epilepsy and related disorders, where epilepsy and related disorders are selected from focal seizures with or without generalization, myoclonic seizures, primary generalized tonic-clonic seizures, and treatment-resistant or refractory epilepsy. Here, the refractory epilepsy may be refractory partial epilepsy or refractory generalized epilepsy.

In one embodiment, primary generalized tonic-clonic seizures occur in patients with idiopathic generalized epilepsy.

In one embodiment, the invention relates to the use of the above combination as a neuroprotector.

In addition, the invention includes the use of the pharmaceutical composition according to the invention as a drug for the treatment of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, bipolar and related disorders, or the use of this pharmaceutical composition in the prevention of epileptogenesis.

Additionally, the invention relates to the use of said pharmaceutical composition in the prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, and bipolar and related disorders.

Preferably, the invention includes the use of said pharmaceutical composition in the treatment or prevention of epilepsy and related disorders, or as a neuroprotective agent.

In addition, the invention includes a method of treating a patient with epilepsy or related disorders, neuropathic pain, migraine or persistent headache, or bipolar and related disorders, comprising administering an orthosteric agonist of the metabotropic glutamatergic receptor subtype 2 and an SV2A ligand selected from the group, consisting of levetiracetam, brivaracetam and seletracetam, wherein the metabotropic glutamatergic receptor subtype 2 orthosteric agonist is selected from:

1) LY-404039 or a salt or solvate thereof; and 2) LY-2140023

or its salt or solvate.

Said SV2A ligand may be levetiracetam or brivaracetam.

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According to the method according to the invention, said disorder is epilepsy, in particular treatment-resistant epilepsy, wherein the epilepsy and its associated disorder are focal seizures.

In one preferred embodiment of the method, the orthosteric agonist of the mGluR2 compound and the SV2A ligand are administered simultaneously.

In a particular embodiment of this method, the orthosteric agonist of the mGluR2 compound and the SV2A ligand are administered simultaneously via separate dosage forms.

According to another embodiment of this method, the orthosteric agonist of the mGluR2 compound and the SV2A ligand are administered separately, at different time points during the course of therapy.

Components (b) of the combination of the present invention are generally referred to herein as mGluR2 compounds, or mGluR2/mGluR2 agonist compounds, or mGluR2 mortosteric agonist compound, which means that the compounds mainly have activity against the metabotropic glutamatergic receptor 2 subtype and in particular , are selected from orthosteric agonists of the metabotropic glutamatergic receptor subtype 2. One of skill in the art will be aware of the high homology between mGluR2 and mGluR3, whereby some mGluR2 orthosteric agonists also exhibit similar activity to mGluR3 orthosteric agonists. This is the case, for example, with (-)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]-hexane-4,6dicarboxylic acid (also known as LY-404,039 [CAS 635318 -11-5]) with Ki=149 nM (mGlu2 receptor) and Ki=92 nM (mGlu3 receptor), which has a 100-fold selectivity for mGlu2 and mGlu3 compared to mGlu4a, -6, -7a and -8a, and it lacks mGlula and mGlu5a activity (Rorick-Kehn et al. (2007) The Journal of Pharmacology and Experimental Therapeutics Vol. 321, No. 1, pp. 308-317). Thus, the term mGluR2 compound or mGluR2 agonist compound or mGluR2 orthosteric agonist compound does not exclude compounds exhibiting some additional secondary activity in vitro or in vivo.

PAM mGluR2 reference compounds are disclosed in WO 2009/033704. Specific reference compounds are

or a pharmaceutically acceptable salt or solvate thereof.

Known orthosteric mGluR2/mGluR2/3 agonists include, for example,

LY-404039; LY-2969822; LY-2934747; LY-379268; DCG-IV; LY-354740; LY-314582; LY-544344; LY2140023; LY-181837; LY-389795; LY-446433; LY-450477; LY-395756; LY-566332; LY-541850; LY2300559; LY-404040; LY-281223; LY-2979165; thalaglumethad; MGS008; MGS0022; MGS0028; MGS0039;

(-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate;

(+)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid;

(+)-2-amino-4-fluorobicyclo-[3.1.0]hexane-2,6-dicarboxylic acid;

S,2R,5S,6S-2-amino-6-fluoro-4-oxobicyclo-[3.1.0]hexane-2,6-dicarboxylic acid;

S,2R,4S,5S,6S-2-amino-6-fluoro-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylic acid;

1S,2R,3R,5S,6S-2-amino-3-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid;

S,2R,3S,5S,6S-2-amino-6-fluoro-3-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylic acid;

(+)-4-amino-2-sulfonylbicyclo-[3.1.0]hexane-4,6-dicarboxylic acid;

(+)-2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid;

1S,2R,5S,6S-2-amino-6-fluoro-4-oxobicyclo[3.1.0]hexane-2,6-dicarboxylic acid;

S,2R,4S,5S,6S-2-amino-6-fluoro-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylic acid;

1S,2R,3R,5S,6S-2-amino-3-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid; or

S,2R,3S,5S,6S-2-amino-6-fluoro-3-hydroxybicyclo-[3.1.0]hexane-2,6-dicarboxylic acid.

A specific group of mGluR2 agonists include LY-37 92 68; DCG-IV; LY-354740; LY-404039; LY2969822; LY-2934747; LY-544344 and LY-2140023.

Orthosteric agonists of the metabotropic glutamatergic receptor 2 subtype combination of the present invention are disclosed in WO 1997/18199 and WO 2003/104217, incorporated herein in their entirety. Specific compounds disclosed in these documents are (-)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]-hexane-4,6-dicarboxylic acid (also known as LY-404039)

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o or its salt or solvate and 2,2-dioxide (1R,4S,5S,6S)-4-[[(2S)-2-amino-4-(methylthio)-1-oxobutyl]amino]-2-thiabicyclo [3.1.0]hexane-4,6-dicarboxylic acid (also known as LY-2140023 [CAS 635318-55-7])

or a salt or solvate thereof, for example its monohydrate.

The names of the compounds of the present invention were compiled according to the nomenclature rules adopted by the Chemical Abstracts Service (C.A.S.) using Advanced Chemical Development, Inc. software. (Product version ACD/Name 10.01.0.14105, October 2006). In the case of tautomeric forms, the name of the represented tautomeric form of the structure was made up. However, it should be understood that another tautomeric form not shown is also included within the scope of the present invention.

As used herein, unless otherwise indicated, the term antiepileptic agent and the abbreviation AED will be used interchangeably with the term anticonvulsant, and in the context of the present invention, they refer to an agent that can treat, suppress, or prevent convulsive activity or ictogenesis when the agent is administered. subject or patient.

As used herein, unless otherwise indicated, the term synaptic vesicle protein ligand 2A and the abbreviation SV2A ligand will be used interchangeably. Examples of prior art SV2A ligands include, without limitation, compounds included in GB 1039113, GB 1309692, EP 1262036, EP 1806339, WO 2001/062726, US 2002/094787, WO 2004/087658, WO 2005/121082, WO 2005/121082, /054188, WO 2006/128692, WO 2006/128693, WO 2007/065595, WO 2008/132139 and WO 2008/132142, WO 2011/047860, WO 2012/143116 and WO 2012/143117.

As already mentioned, the SV2A ligand is selected from levetiracetam, brivaracetam and seletracetam.

In a specific embodiment, the SV2A ligand is levetiracetam.

In a particular embodiment, the SV2A ligand is brivaracetam.

Methods for obtaining the above SV2A ligands are known from the literature and are disclosed, for example, in EP 1806339; in EP 0162036 and GB 2225322 (levetiracetam); in WO 01/62726 (brivaracetam); and in WO 2005/121082 (seletracetam); such methods are hereby incorporated by reference in their entirety.

In a further embodiment, the combination according to the present invention comprises (a) a pharmaceutically effective amount of levetiracetam or brivaracetam, and (b) a pharmaceutically effective amount of LY-404039 or a pharmaceutically acceptable salt thereof, in particular its hydrochloride salt, or a solvate thereof.

In a further embodiment, the pharmaceutical composition according to the present invention comprises (a) a pharmaceutically effective amount of levetiracetam or brivaracetam, and (b) a pharmaceutically effective amount of LY-404039 or a pharmaceutically acceptable salt thereof, in particular its hydrochloride salt, or a solvate thereof.

In a further embodiment, the combination according to the present invention comprises (a) a pharmaceutically effective amount of levetiracetam or brivaracetam and (b) a pharmaceutically effective amount of LY-2140023, or a pharmaceutically acceptable salt or solvate thereof, in particular its monohydrate.

In a further embodiment, the pharmaceutical composition according to the present invention comprises (a) a pharmaceutically effective amount of levetiracetam or brivaracetam, and (b) a pharmaceutically effective amount of LY-2140023, or a pharmaceutically acceptable salt or solvate thereof, in particular its monohydrate.

The combination product of the present invention, in particular the pharmaceutical composition of the present invention, is mainly intended for the treatment of epilepsy

- 8 040772 and related disorders.

It should be understood that some mGluR2 compounds, in particular the mGluR2 agonist compounds of the present invention and their pharmaceutically acceptable addition salts and solvates thereof, may contain one or more chiral centers and exist as stereoisomeric forms.

As used herein, the expression for a compound of the present invention is understood to include the mGluR2 agonist compounds as disclosed herein and their salts and solvates.

In the context of this application, any chemical formula with bonds shown as solid lines only and not as solid wedge or dashed wedge bonds, or otherwise shown as having a specific configuration (e.g., R, S) around one or more atoms, provides each possible stereoisomer or mixture of two or more stereoisomers.

If a specific stereoisomer is indicated, this means that said stereoisomer is substantially free of other isomers, i.e. associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, in particular less than 2% and most preferably less than 1% of other isomers. Thus, if an mGluR2 compound is, for example, listed as (R), this means that the compound is substantially free of the (S) isomer.

For use in medicine, salts of the compounds of the present invention refer to non-toxic pharmaceutically acceptable salts (salts of the compounds of the present invention wherein the counterion is pharmaceutically acceptable). However, other salts may be useful in the preparation or purification of the compounds of the present invention or their pharmaceutically acceptable salts, and may include acids and bases that are not pharmaceutically acceptable. All salts, whether or not pharmaceutically acceptable, are included within the scope of the present invention.

The pharmaceutically acceptable acid and base addition salts mentioned above or below herein are intended to include the therapeutically active non-toxic acid and base addition salt forms that the compounds of the present invention can form. Suitable pharmaceutically acceptable salts of such compounds include acid addition salts, which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid, such as, for example, inorganic acids such as hydrohalic acids, for example, hydrochloric or hydrobromic acid, sulfuric, nitric , phosphoric and other acids; or organic acids such as, for example, acetic, propionic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic acid), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic , ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, paminosalicylic, pamoic and other acids. Conversely, said salt forms can be converted by treatment with an appropriate base to the free base form. In addition, when the compounds of the present invention carry an acidic moiety, suitable pharmaceutically acceptable salts may include organic and inorganic bases. Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts, such as lithium, sodium, potassium, magnesium, calcium, and the like salts, organic base salts, such as primary, secondary, and tertiary aliphatic and aromatic amines, such as methylamine, ethylamine, propylamine, isopropylamine, four isomers of butylamine, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidine, pyridine, quinoline and isoquinoline; salts of benzathine, N-methylD-glucamine, hydrabamine, as well as salts of amino acids such as, for example, arginine, lysine, and the like. Conversely, the salt form can be converted by treatment with acid to the free acid form.

The term solvate includes the solvent addition forms as well as their salts which the compounds of the invention may form. Examples of such solvent addition forms are, for example, hydrates, alcoholates, and the like.

As used herein, the expression composition is intended to cover a product containing certain ingredients in certain amounts, as well as any product that is obtained, directly or indirectly, by combining certain ingredients in certain amounts.

As used herein, the term "subject" refers to an animal, preferably a mammal, most preferably an adult, child, or infant, that is or has been the subject of treatment, examination, or experiment.

As used herein, a therapeutically effective amount means that amount of an active compound or pharmaceutical agent that produces a biological or medical effect in a tissue system in an animal or human that is desired by the researcher, veterinarian, physician, or other clinician, which includes alleviating one or more symptoms of the disease. or disorder being treated; and/or reducing the severity of one

- 9 040772 or several symptoms of the disease being treated.

The combination of (a) an SV2A ligand and (b) an orthosteric subtype 2 metabotropic glutamatergic receptor agonist, or a pharmaceutically acceptable salt or solvate thereof, whether compounds (a) and (b) are administered simultaneously, separately or sequentially, may provide more beneficial effect compared to the effect of compounds (a) or (b) administered separately. In particular, at least one beneficial effect can be observed, for example a mutual enhancement of the effect of the compounds (a) and (b), an effect that is greater than an additive, in particular a synergistic effect; additional advantageous effects include, for example, a significant reduction in the effective dose for the combination of (a) and (b); additional therapeutic effect that is not observed for either compound (a) or (b) alone, a more favorable side effect profile, or a combined therapeutic effect at an ineffective dosage of one of (a) and (b) or both the first and second connections.

While the present invention is directed to a complex or combination therapy involving the introduction of (a) a protein ligand of synaptic vesicles 2A (SV2A); and (b) an mGluR2 agonist compound as defined herein, wherein a pharmaceutically or therapeutically effective amount shall mean that amount of the combination of agents taken together such that the combined effect elicits the desired biological or medical response. For example, a therapeutically effective amount in combination therapy comprising the administration of (a) an SV2A ligand, as defined herein, and (b) an mGluR2 agonist compound, as defined herein, may be an amount of (a) an SV2A ligand, as defined in herein, and the amount (b) of an mGluR2 agonist compound that, when taken together or sequentially, produces a combined effect that is therapeutically effective. Additionally, one skilled in the art will recognize that in the case of combination therapy with a therapeutically effective amount as in the example above, the amount of the mGluR2 agonist compound and/or the amount of the appropriate SV2A ligand alone may or may not be therapeutically effective.

The present invention provides methods of prevention or treatment comprising administering to a subject in need of a complex therapy a therapeutically effective amount of an SV2A ligand and a therapeutically effective amount of an mGluR2 agonist compound as described herein. To achieve this goal, the compounds or compositions of the present invention should be used in the correct therapeutically effective amount or dose, which are described below.

Optimal dosages and regimens to be administered can be readily determined by those skilled in the art and will vary with the particular compound used, route of administration, drug potency, and progression of the disease state. In addition, factors associated with the particular patient being treated, including age, weight, diet, and time of administration, will necessitate dosage adjustments.

One skilled in the art will appreciate that a therapeutically effective dosage of the compounds of the present invention may include repeated doses as part of an extended treatment regimen that will provide clinically meaningful results.

The amounts of the mGluR2 agonist compound in the combinations of the present invention administered daily may vary from about 0.01 mg to about 2000 mg. An effective amount of the drug is typically administered at a dosage level of about 0.01 mg/kg to about 150.0 mg/kg of body weight per day, or any range within that range. Preferably the range is from about 0.1 to about 100.0 mg/kg of body weight per day, more preferably from about 0.5 mg/kg to about 50 mg/kg, more preferably from about 1.0 to about 25.0 mg/kg of body weight per day. Compounds can be administered 1, 2, 3 or 4 times a day. The amounts of SV2A ligand administered daily may vary from about 0.01 to about 7000 mg, preferably from 250 to 5000 mg, or more preferably from 500 to 3000 mg. Examples of daily amounts of SV2A ligand are 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250, 500, 750, 1000, 1500 and 3000 mg for symptomatic adjusting the dosage in the patient to be treated. An effective amount of the drug is typically administered at a dosage level of about 0.01 mg/kg to about 150.0 mg/kg of body weight per day, or any range within that range. Preferably the range is from about 0.1 to about 100.0 mg/kg of body weight per day, more preferably from about 0.5 mg/kg to about 50 mg/kg, more preferably from about 1.0 to about 25.0 mg/kg of body weight per day. Compounds can be administered 1, 2, 3 or 4 times a day. All quantities mentioned in this and the following paragraphs refer to the free form (ie non-salt form). The above values represent free-form equivalents, i.e. values corresponding to the intended introduction of the free form. When introducing salts, the quantities must be calculated

- 10 040772 depending on the ratio of molecular weights of the salt form and the free form.

The above daily doses are based on an average body weight of approximately 70 kg and should be recalculated if used in pediatrics or if used in patients with significant body weight variation.

Dosages may be presented as one, two, three or four or more dose portions administered at regular intervals throughout the day. The dosage used preferably corresponds to the daily amount of the mGluR2 agonist compound or SV2A ligand mentioned above, or a portion of its dose, such as its 1/2, 1/3, 1/4. The dosage form may contain an mGluR2 agonist compound or an SV2A ligand, or both together in an amount equal to the ranges or amounts mentioned in the previous paragraphs, for example, the dosage form may contain 10, 25, 50, 100, 150 or 200 mg of the mGluR2 agonist compound, 10 , 25, 50, 100, or 250 mg of SV2A ligand, either in single formulations or combined formulations. In one embodiment, the mGluR2 agonist compound is administered once a day (q.d.), in particular as a single dose per day, and the SV2A ligand is administered once a day or twice a day (q.d. or b.i.d.), in particular as a single dose. or two doses per day. In the case where both compounds are to be administered once a day, this can be done by administering two separate doses, one with the mGluR2 agonist compound, one with the SV2A ligand, or by administering a combined dose containing the mGluR2 agonist compound and the SV2A ligand.

The combinations of the present invention can be administered one, two, three, four or, if necessary, many times a day. In one embodiment, the combination is administered once a day. In another embodiment, the combination is administered twice a day or three times a day. Dosing may be via separate dosage forms, ie. via dosage forms containing only the mGluR2 agonist compound or only the SV2A ligand; or through combination dosage forms containing the active ingredients a mGluR2 agonist compound and an SV2A ligand. It is also possible to use a combination of the use of a combined dosage form and the use of separate dosage forms. Dosage forms that can be administered are described hereinafter, with oral dosage forms, in particular tablets or capsules, being preferred.

The active ingredients may be formulated into pharmaceutical compositions either singly or as a combined pharmaceutical composition. In the latter case, a pharmaceutical composition is prepared containing a therapeutically effective amount of an mGluR2 agonist compound, or a pharmaceutically acceptable salt thereof, and an SV2A ligand, the foregoing being as set forth herein, and a pharmaceutically acceptable carrier.

In a further aspect, the present invention relates to a method for preparing a pharmaceutical composition as described herein, which comprises intimately mixing a pharmaceutically acceptable carrier with a therapeutically effective amount of an mGluR2 agonist compound, or a pharmaceutically acceptable salt or solvate thereof, and a therapeutically effective amount of at least one SV2A ligand.

The combinations provided herein may also be formulated as a combined preparation for simultaneous, separate or sequential use in the prevention or treatment of epilepsy and related disorders; neuropathic pain; migraine or persistent headache; bipolar and related disorders; in neuroprotection; or in the prevention of epileptogenesis. In such a case, the mGluR2 agonist compound is formulated into a pharmaceutical composition containing other pharmaceutically acceptable excipients, and the SV2A ligand is formulated separately into a pharmaceutical composition containing other pharmaceutically acceptable excipients. Relatively speaking, these individual pharmaceutical compositions may be part of a kit for simultaneous, separate or sequential use.

The individual components of the combination of the present invention can be administered simultaneously or separately at different points in time during the course of therapy or in parallel in separate or single combined forms.

In view of the foregoing, the mGluR2 agonist compounds and the SV2A ligand, alone or in combination, can be formulated into various pharmaceutical compositions suitable for administration. In these compositions, a therapeutically effective amount of a particular compound, or both, is combined with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. Pharmaceutical compositions may be formulated for oral, parenteral (including subcutaneous (s.c.), intramuscular (i.m.) and intravenous (i.v.)), rectal, transdermal, buccal or intranasal administration. Pharmaceutical compositions can also be prepared for administration directly to the nervous system via routes including, but not limited to, intracerebral, intraventricular, intracerebroventricular, intrathecal, intracisternal, intraspinal, and/or perispinal routes via delivery through needles and/or catheters for intracranial or intravertebral administration with with pumping devices or without them. Suitable compositions for oral administration include powders,

- 11 040772 granulates, aggregates, tablets, compressed or coated pills, dragees, sachets, hard or gelatine capsules, syrups and suspensions. Suitable compositions for parenteral administration include aqueous or non-aqueous solutions or emulsions, while suitable compositions for rectal administration include hydrophilic or hydrophobic suppositories. For topical administration, suitable transdermal delivery systems can be used, and for nasal delivery, suitable aerosol delivery systems can be used.

For example, in the preparation of compositions for oral administration, any conventional pharmaceutical medium may be used, such as, for example, water, glycols, oils, alcohols, and the like, in the case of liquid compositions for oral administration, such as suspensions, syrups, elixirs, emulsions, and solutions. ; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrants and the like, in the case of solid compositions. In the case of compositions for parenteral administration, the carrier will generally contain sterile water, at least in large part, although other ingredients such as solubilizers, emulsifiers or additional auxiliaries may be added to the carrier. Injectable solutions may be prepared in which the carrier contains saline solution, glucose solution, or a mixture of both. Injectable suspensions can also be prepared, in which case suitable liquid carriers, suspending agents and the like can be used. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations such as powders for dissolution. In compositions suitable for subcutaneous administration, the carrier optionally comprises a skin penetration enhancer and/or wetting agent, optionally combined with suitable skin-compatible additives in minor amounts. The mGluR2 agonist compound or SV2A ligand, or combinations thereof, can also be administered by oral inhalation or insufflation of formulations suitable for this type of administration, such as a solution, suspension, or dry powder. Suitable pharmaceutical compositions for administration in the form of aerosols or sprays are, for example, suspensions of the mGluR2 agonist compound or the SV2A ligand, or both, in a pharmaceutically acceptable liquid carrier such as ethanol or water, or a mixture thereof. If necessary, the composition may also additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers, as well as a propellant. Such a formulation typically contains the active compound at a concentration of from about 0.1% to 50%, in particular from about 0.3% to 3% by weight.

Pharmaceutical compositions may contain the active ingredient, an mGluR2 agonist compound or an SV2A ligand, or both in combination, at a concentration of from about 0.1% to about 50%, or from about 1% to about 30%, or from about 3% to about 20%. , or from about 5% to about 20%, all percentages are by weight, and the total of all components in these pharmaceutical compositions does not exceed 100%. In compositions containing both the mGluR2 agonist compound and the SV2A ligand, the mGluR2 agonist compound is present at a concentration of from about 0.1% to about 50%, or from about 1% to about 30%, or from about 3% to about 20%, or from about 5% to about 20%; and the SV2A ligand is present at a concentration of about 3% to about 50%, or about 5% to about 50%, or about 10% to about 50%, or about 10% to about 40%, or about 10% up to approximately 30%, and the total of all components in these pharmaceutical compositions does not exceed 100%.

Pharmaceutical compositions may conveniently be presented in unit dosage form for ease of administration and dosage uniformity. Examples include tablets (including scored or coated tablets), capsules, pills, suppositories, powder packets, cachets, injectable solutions or suspensions, and the like, as well as multiples thereof. Of interest are solid dosage forms for oral administration, such as tablets or capsules.

Solid dosage forms in unit dose form may be packaged in any known package, with blister packaging being preferred in particular for tablets and capsules. If the mGluR2 agonist compound and SV2A ligand are separately formulated, they may be packaged in separate blister packs, but one blister may also contain unit dose forms of the mGluR2 agonist compound and SV2A ligand, for example, one row with units of the mGluR2 agonist compound and the other with the ligand SV2A. Other options may also be possible.

The combinations of the present invention can be used to treat or prevent epilepsy and related disorders; neuropathic pain; migraine or persistent headache and bipolar and related disorders; or they can be used as a neuroprotector or to prevent epileptogenesis.

As used herein, the term "treatment" is intended to refer to all methods that may involve slowing, disrupting, suppressing, or stopping the progression of a disease, or alleviating symptoms, but does not necessarily mean complete elimination.

- 12 040772 all symptoms.

In the context of this application, unless otherwise indicated, the terms epilepsy and related disorders or epilepsy or related disorder will mean any disorder in which a subject (preferably an adult, child or infant) experiences one or more epileptic seizures and/or tremor attacks. Suitable examples include, without limitation, epilepsy (including, but not limited to, localized forms of epilepsy, generalized forms of epilepsy, forms of epilepsy with both generalized and localized seizures, etc.), partial seizures with or without generalization, myoclonic seizures , primary generalized tonic-clonic seizures, especially in patients with idiopathic generalized epilepsy, seizures associated with Lennox-Gastaut syndrome, seizures as a complication of a disease or condition (such as seizures associated with encephalopathy, phenylketonuria, juvenile Gaucher disease , Lundborg's progressive myoclonic epilepsy, stroke, head injury, stress, hormonal changes, drug use or drug withdrawal, alcohol use or alcohol withdrawal, sleep deprivation, fever, infection, etc.), status epilepticus (convulsive or non-convulsive) , uh sensational tremor, restless limb syndrome, etc. The disorder is preferably selected from epilepsy (regardless of type, underlying cause or origin), essential tremor or restless limb syndrome. More preferably, the disorder is epilepsy (regardless of type, underlying cause or origin) or essential tremor. A specific example of epilepsy is refractory epilepsy, also referred to as treatment or therapy resistant epilepsy.

This term is often used when three or more antiepileptic drugs (AEDs) have been unsuccessful on patients. Refractory epilepsy also includes refractory partial epilepsy and refractory generalized epilepsy (including idiopathic or symptomatic).

As used herein, the term neuropathic pain includes pain resulting from chronic or debilitating conditions or disorders. Chronic or debilitating conditions or disorders that can lead to neuropathic pain include, without limitation, the pain form of diabetic peripheral neuropathy, postherpetic neuralgia, trigeminal neuralgia, post-stroke pain, pain associated with multiple sclerosis, pain associated with neuropathies, such as idiopathic or post-traumatic neuropathy and mononeuritis, HIV-associated neuropathic pain, cancer-associated neuropathic pain, carpal tunnel-associated neuropathic pain, spinal cord injury-associated pain, complex regional pain syndrome, fibromyalgia-associated neuropathic pain, lumbar and cervical pain, reflex sympathetic dystrophy, phantom pain syndrome and other pain syndromes associated with chronic and debilitating conditions.

As used herein, the term migraine will mean a chronic, episodic and debilitating clinical condition that is diagnosed by the presence of moderate to severe pulsating unilateral headaches lasting 4 to 72 hours, which includes migraine without aura and migraine with aura. As used herein, the term migraine without aura will refer to at least five attacks meeting the following criteria: (a) headache attack lasting 4 to 72 hours with headache having at least two of the following features: unilateral localization, throbbing nature, moderate or severe intensity with a direct effect on the activities of daily life and aggravation when climbing stairs or similar procedures; and (b) during a headache attack, at least the following occur: nausea and/or vomiting and fear of light and phonophobia. As used herein, the term migraine with aura will refer to at least two attacks accompanied by at least 3 of 4 of the following: (a) one or more fully reversible aura symptoms; (b) at least one aura symptom that develops gradually over more than four minutes, or two or more symptoms that appear sequentially; (c) no aura symptom lasting more than 60 minutes; (d) the headache occurs before, simultaneously with, or after the aura, with an indefinite interval between the aura and the headache of less than about 60 minutes.

As used herein, the term bipolar and related disorders will include bipolar I disorder (e.g., single manic episode, recent hypomanic episode, recent manic episode, recent mixed episode, recent depressive episode, and recent unspecified episode), bipolar II disorder, cyclothymic disorder and bipolar disorder without further specification (as these terms are defined by their diagnostic criteria in the Diagnostic and Statistical manual of Mental Disorders ^4th Edition, Text Revision, American Psychiatric Association, 2000 (DSM-IV-TR) or ^5th Edition, Text Revision, American Psychiatric Association, 2013 (DSM-5™) Preferably, bipolar disorder is characterized by depressive

- 13 040772 and manic (or hypomanic) phases, the phases being cyclic. Preferably, the bipolar disorder is Bipolar I or Bipolar II. As used herein, the term mania will include mania or a manic phase, not counting the underlying cause. As used herein, the term bipolar mania is intended to mean mania associated with, characteristic of, or a symptom of bipolar disorder. Thus, the methods for treating bipolar mania of the present invention are directed to methods that treat mania and/or the manic phase of bipolar disorders. As used herein, the term bipolar depression is intended to mean depression associated with, characteristic of, or a symptom of bipolar disorder. Thus, the methods for treating bipolar depression of the present invention are directed to methods that treat depression and/or the depressive phase of bipolar disorders. In the context of this document, unless otherwise indicated, the terms periodicity or bipolar periodicity will refer to the change in mood from depressive to manic phases characteristic of bipolar disorders. Thus, the present invention includes methods for stabilizing said cycling, including, without limitation, reducing the frequency of cycling and/or reducing the intensity of manic and/or depressive phases.

Thus, in one embodiment, the pharmaceutical composition of the present invention can be used for mood stabilization, in particular mood stabilization in manic depression.

As used herein, the term epileptogenesis refers to the gradual process of developing epilepsy. This process may occur after cerebral hemorrhage or a range of conditions, including neurodegenerative diseases, traumatic brain injury, stroke, brain tumor, central nervous system infections, and status epilepticus, or it may occur after gene mutations.

As used herein, the expression anxiety refers specifically to generalized anxiety disorder.

As used herein, the expression approximately has its generally accepted meaning. In particular embodiments, when associated with a numerical value, it may be interpreted as a numerical value of ±10%, or ±5%, or ±2%, or ±1%, or ±0.5%, or ±0.1 %. In other embodiments, the exact value is implied, ie. omitting the word approximately.

And/or means that each component or both or all components or characteristics from the list are possible options, especially two or more of them, as an alternative or together.

As used herein, the singular, plural, and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed as embracing both the singular and plural forms, unless otherwise stated herein or obviously not refuted by the context.

Examples

The following examples are set forth to assist in the understanding of the present invention and are not meant to be and should not be construed as limiting in any way the present invention as set forth in the claims which follow.

A) Anticonvulsant studies with mglur2 compounds (orthosteric agonist and reference compounds).

General information.

Preparation of test compounds and solutions.

Test compounds were administered using the optimum ratio of liquid form volume to body fluid volume. Test compounds were administered to mice in a volume of 0.01 ml/g body weight (White H.S. et al., General Principles: Experimental selection, quantification, and evaluation of antiepileptic drugs, in Antiepileptic Drugs, Fourth Edition, R.H. Levy, R.H. Mattson, and B.S. Meldrum, Editors 1995, Raven Press, Ltd.: New York, pp. 99-110). For subcutaneous (s.c.) administration, test compounds were injected into a fold of skin along the back of the animal. For each of the tests performed on test compounds, final concentrations of the compounds were administered as an aqueous solution in 20% Hp-e-CD. For inert groups, a 20% solution of Hp-e-CD was used.

For LY-404039, final concentrations of the compound were administered as sodium chloride solution s.c.

The final concentrations of levetiracetam were administered in an aqueous solution of 0.5% methylcellulose (MC) administered by intraperitoneal (i.p.) injection.

important reagents.

a) Inert solutions 0.5% methylcellulose (MC) 40% stock solution of hydroxypropyl-v-cyclodextrin (Hp-v-CD).

- 14 040772

b) Other solutions.

Tetracaine (a solution of 0.5 w/v%) was applied dropwise from a plastic dropper bottle to the eyes of all animals that were to subsequently receive electrical stimulation using corneal electrodes.

Animals and conditions of keeping animals.

Adult male albino mice CF #1 (26-35 g) were obtained from Charles River, Portage, Michigan. Animals were maintained on an appropriate diet (Prolab RMH 3000) and were given free access to food and water, except for a short period of time when they were removed from their cage for testing. Newly received animals in the laboratory were allowed sufficient time to correct for possible food and water restriction they were subjected to in transit before they were used in testing. All mice were kept in plastic cages in specially designed chambers with controlled humidity, air exchange and controlled lighting (12 hours on - 12 hours off). Animals were kept, fed and cared for in accordance with the recommendations of the National Council publication Guide for the Care and Use of Laboratory Animals.

Minimal motor impairment (MMI).

Acute MMI was assessed through a combination of direct observation of the animal for overt symptoms associated with neurological or muscle function in the animal. For mice, a rotating rod procedure was used to detect minimal muscle or neurological impairment. If the mouse was placed on a rod rotating at 6 rpm, the animal was able to maintain its balance for long periods of time. An animal was considered unhealthy if it fell off this rotating rod three times within a period of 1 min.

Determination of median effective and toxic doses (ED ₅₀ and TD ₅₀ ).

To determine the ED ₅₀ or TD ₅₀ for each test compound, the first dose administered was usually the same dose that was used in the successful determination of TPE. If the initial dose used was effective or toxic in more than 50% of the animals, then the next dose was half the original dose; if the initial dose was effective or toxic in less than 50% of the animals, then the next dose was twice the initial dose. The third and fourth doses were selected to obtain a dose-response line with equal intervals between points. There must be a minimum of 4 points that either include 0 and 100% or are in that range.

Definition of TPE.

Animals in groups were typically treated with four of the test compounds and each group was tested at one of five time points: 0.25, 0.5, 1, 2, or 4 hours post-treatment (White et al., 1995) . TPE was determined using the analysis with stimulation at 6 Hz (32 mA). The point in time (0.25, 0.5, 1, 2, or 4 hours after treatment) at which maximum protection was observed was considered the time of peak effect (TPE).

In TPE, defined for this study or previously defined, compounds were tested in the assay with stimulation at 6 Hz (32 and/or 44 mA) at multiple doses, including doses that elicit little or no protection to complete protection.

ED ₅₀ and 95% confidence interval (CI) were calculated using probit analysis in a computer program provided by the laboratory (Finney Probit Analysis 34d Ed 1971, London: Cambridge University Press).

Collection of blood serum for pK/pD analysis.

In various tests, animals were sacrificed after testing, and blood drained from the torso and/or brain tissue (whole brains) were collected for quantification of drug levels. Immediately after testing, the animals were decapitated and blood drained from the torso was collected into a BD Vacutainer® tube containing K2EDTA and chilled on ice prior to centrifugation. After centrifugation (13000-18000 rpm, 5-7 min), blood plasma was removed and transferred to a labeled microcentrifuge tube and stored at -80°C. To collect brain tissue, the brains were removed immediately after decapitation and flash-frozen. The frozen sample was placed in a labeled centrifuge tube and stored at -80°C.

Test with psychomotor seizures at 6 Hz stimulation in mice.

The psychomotor seizure test at 6 Hz stimulation is used as a model for drug-resistant limbic seizures. Seizure at 6 Hz stimulation reflects resistance to phenytoin, carbamazepine, lamotrigine, and topiramate (Barton et al. Pharmacological characterization of the 6 Hz psychomotor seizure model of partial epilepsy Epilepsy Research 2001, Vol. 47, pp. 217-222).

The method of conducting a test with psychomotor seizures during stimulation at 6 Hz.

Mice were induced with focal epileptic seizures by corneal stimulation (6 Hz, 0.2 ms square wave, 3 s duration; Barton et al., 2001). Mice were tested at 32 mA or 44 mA. Before stimulation, drops of 0.5% tetracaine were applied to each eye. Seizures induced by corneal stimulation in this analysis were characterized by a minimal clonic phase followed by

- 15 040772 stereotypical forms of automatic behavior, including stupor, forelimb clonus, vibrissa twitching and Straub's tail. Animals that did not exhibit these behaviors were considered protected.

Example 1

1.1. Study of combinations with levetiracetam.

First, each compound was tested individually at the dose that showed minimal activity in the 6 Hz 44 mA stimulation test at the TPE of each compound. In addition to recording efficacy and toxicity data for these compounds alone or in combination, both plasma and brain samples were taken from each group for pharmacokinetic/pharmacokinetic analysis. No pharmacokinetic interaction was observed based on compound levels in plasma and brain samples (data not shown).

1.2. Mouse model of corneal kindling.

Mice were electrically induced to seize with 3 s, 3 mA, 60 Hz stimuli twice daily via corneal electrodes until the criterion of 5 consecutive epileptic seizures at stage 5 according to Racine (Racine Modification of seizure activity by electrical stimulation II) was achieved. motor seizure Electroenceph Clin Neurophysiol 1972, 32, pp. 281-294). After the mice reached a steady state of kindling, the test compound or inert substance was administered and, at a predetermined TPE, each animal was given an electrical stimulus as described above. After stimulation, the animals were examined for the presence or absence of seizure activity on a Racine scale (0-5), where 5 indicates the highest degree of fall and rise.

The final data for the compounds tested separately are presented in table. 1.

Table 1 Summary data on anticonvulsant effects in acute seizure model with stimulation at 6 Hz at 32 mA and 44 mA for reference compounds PAM mGluR2 2-a and 25-a and LY-404039 after s.c. introductions

Comm. No. TRE (h) ED ₅₀ (95% Ci: ) mg/kg, S.C. Seizure score (dose) 32 mA 44 mA Seizure induction through corneal kindling Reference compound 25-a 1 7.7 (2.3- 18.4) 1 hour TRE: 25.9 (15.5- 33.7) 0.25 hour TPE: 29.1 (21.6- 39, 6) n.t. Reference compound 2-a 0.5 44.7 (23.4- 80.5) 20.8 (10.0- 31.7) 12.2 (8.4- 17.4) 50% protection at 100 mg/kg 21 (17.9-27.4) 4.4 (100 mg/kg) LY-404039 0.5 10.2 (3.62- 12.4) n.t. 3, 1 (100 mg/kg)

TPE means time of peak effect, CI means confidence interval, s.c. means subcutaneously, r.o. means oral, n.t. means not tested. TPE was determined in the 6 Hz, 32 mA stimulation test. Effects were generally observed for doses that did not cause a reduction in the rolling rod test. For reference compound 2-a, individual replicate values are shown. For reference compound 25a for studies with stimulation at 6 Hz (44 mA), time points of both 0.25 and 1 h were used.

Example 2 Studies with Reference Compounds 25-a and 2-a.

2.1. The study of combinations Comm. No. 25-a and levetiracetam.

Independent dose-response studies were performed in a 6 Hz, 44 mA stimulation test for both reference compounds to determine ED50 values at TPE 1 h, i.p., for levetiracetam, and for 1 h, s. With. reference compounds. ED50 value for conn. No. 25-a was 25.9 mg/kg, and for levetiracetam the value was estimated to be about 345 mg/kg. Dose-dependent effect

- 16 040772 for levetiracetam was repeated with a combined administration of 10 mg/kg Comm. No. 25-a (dose of Comp. No. 25-a, which alone did not protect in the model with stimulation at 6 Hz, 44 mA). Combined administration of 10 mg/kg Comm. No. 25-a produced an ED50 for the dose-dependent effect of levetiracetam at 4.9 mg/kg (~70-fold reduction compared to levetiracetam alone) and, importantly, provided complete protection in the epileptic seizure model when stimulated at 6 Hz, 44 mA. These results suggest a positive pharmacodynamic interaction between Comm. No. 25-a and levetiracetam in a model of an epileptic seizure with stimulation at 6 Hz.

Table 2. Determination of the time to peak effect for the reference compound. No. 25-a in the analysis with stimulation at 6 Hz (32 mA)

Dose (mg/kg, s . s . ) Time (h) Number protection of others/number tested Number of those with motor impairment on the results of the rotating rod test/number tested Average levels of connection. No. 25-a (ng/ml) in blood plasma 10 0.25 2/4 0/4 10.983 (2.477) 0.5 1/4 0/4 3.330 1 1/4 1/4 700 2 0/4 0/4 256 4 0/4 0/4 40 20 0.25 4/4 0/4 4.095 0.5 3/4 1/4 2,800 1 4/4 1/4 1.765 2 1/4 0/4 618 4 1/4 1/4 28

In this study, two doses, 10 and 20 mg/kg, were used at multiple time points (0.25-4 hours). The compound showed the greatest protection in the 6 Hz stimulation assay, from 0.25 to 1 hour, which was most pronounced at 20 mg/kg. Plasma levels of the compound were generally consistent with protection against behavioral seizure. A TPE of 0.25 h was used for the 6 Hz (32 mA) stimulation studies, while both 0.25 and 1 h s.c. time points were used for the 6 Hz (44 mA) stimulation studies. means subcutaneous.

The mean plasma level, shown in parentheses ( ), was calculated with the statistical test removed to determine outliers.

Table 3. Studies of dose-dependent effects for Comm. No. 25-a in the analysis with stimulation at 6 Hz (32 and 44 ^mAb )

Test Dose (mg/kg, s.s.) Number protected/number tested Number of people with motor impairment on the rotating rod test/number tested Average levels of connection. No. 25-a (ng/ml) in blood plasma

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6 Hz 32 mA 20 8/8 0/8 5,570 15 3/8 0/8 1.201 10 4/8 0/8 6, 113 5 4/8 0/8 2.558 1 1/8 0/8 466 ED ₅₀ (95% CI): 7.7 mg/kg (2.3 to 18.4) 6 Hz 44 mA 40 7/8 0/8 6.263 thirty 3/8 0/8 7.220 20 2/8 0/8 3.368 10 0/8 0/8 4.345 (1,526) 5 0/8 1/8 1.428 ED ₅₀ (95% CI): 29.1 mg/kg (21.6 to 39.6)

CI means confidence interval.

^a Time to peak effect in assay with stimulation at 6 Hz, 32 mA, for conn. No. 25-a was determined as corresponding to 0.25 hours (see table. 1).

^ъ Time to peak effect in the assay with stimulation at 6 Hz, 44 mA, for conn. No. 25-a was the same for 0.25 h and 1 h; the results for 1 hour were confirmed by an ED ₅₀ (95% CI) of 25.9 (15.5-33.7) (see Tables 1 and 6).

The mean plasma level, shown in parentheses ( ), was calculated with the statistical test removed to determine outliers.

Table 4 Combination Studies for Reference Comm. No. 25-a with levetiracetam (LEV) in a 6 Hz (44 mA) stimulation assay

Medicinal means Dose (mg/kg, s . s . ) Number protected/number tested Number of people with motor impairment on the rotating rod test/number tested LEV 200 2/8 0/8 400 4/9 0/9 800 10/12 0/12 ED ₅₀ (95% CI): 345.4 mg/kg (211.0 to 485.3) LEV + ref. conn. No. 25-a, 10 mg/kg 200 8/8 1/8 100 7/8 2/8 50 5/8 1/8 10 4/8 0/8 1 4/8 1/8 ED ₅₀ (95% CI): 4.9 (0.0-14.2)

Reference connection No. 25-a (s.c), 10mg/kg, tested in combination with LEV (i.p.).

Reference connection No. 25-a, 10 mg/kg, not active when administered separately.

2.2. The study of combinations of the reference Comm. No. 2-a and levetiracetam.

Dose-response studies were carried out in tests with stimulation at 6 Hz, 32 mA and 44

- 18 040772 mA, (Table 9 below) and in the test of combinations with levetiracetam (effect of compound No. 2-a on the dose-dependent effect of LEV in Table 10a and the effect of LEV on the dose-dependent effect of Compound No. 2-a in Table 10b below ) in the same way as described for studies with reference comp. No. 25-a and levetiracetam above.

Table 5. Studies of dose-response effects for the reference comp.

No. 2-a, in the analysis with stimulation at 6 Hz (32 and 44 mA; TPE 0.5 h)

Test Dose (mg/kg, s . s . ) Number protected/number tested Number of people with motor impairment on the rotating rod test/number tested 6 Hz 32 mA 40 8/8 2/8 20 6/8 3/8 10 4/8 0/8 5 0/8 0/8 2.5 0/8 1/8 ED ₅₀ (95% CI): 12.2 mg/kg (8.4 to 17.4) 6 Hz 44 mA 40 8/8 4/8 20 3/8 3/8 0/8 0/8 15 2/8 1/8 10 0/8 0/8 1/8 0/8 ED ₅₀ (95% CI): 21.0 mg/kg (17.9 to 27.4) TD ₅₀ : >40 mg/kg ^a

Time to peak effect 0.5 h. Determined in 6 Hz stimulation test, 32 mA (s.c), and used for 6 Hz stimulation studies (32 and 44 mA).

^and 40 mg/kg 6 out of a total of 16 (combined 32 mA and 44 mA) were deficient.

Dose selected for study combinations with LEV at 6 Hz (44 mA): reference comp. No. 2-a 10 mg/kg.

- 19 040772

Table 6a. Combination studies for reference comp. No. 2-a with levetiracetam (LEV) in a 6 Hz (44 mA) stimulation assay. Combination 10 mg/kg reference comp. No. 2-a with different doses of levetiracetam

Medicinal means Dose (mg/kg) Number protected/number tested Number of those with motor impairment on the results of the rotating rod test/number tested LEV 200 2/8 0/8 400 4/9 0/9 800 10/12 0/12 LEV ED ₅₀ (95% CI): 345.4 mg/kg (211.0 to 485.3) LEV + Ref. Comm. No. 2-a 10 mg/kg ^a 200 6/8 1/8 100 6/8 0/8 50 6/8 0/8 25 8/8 0/8 12.5 5/8 0/8 6.25 4/8 0/8 3, 125 3/8 1/8 1.5625 0/8 0/8 LEV ED ₅₀ (95% CI): 9.6 mg/kg (1.7-21.9)

aReference conn. No. 2-a (s.c.) 10 mg/kg, tested in combination with LEV (gr.).

Reference connection No. 2-a, 10 mg/kg, inactive when administered separately.

Additional controls for LEV (low dose) were tested at 25 and 6.25 mg/kg (1/8 and 0/6 protected, respectively).

Mice treated with inert (0.5% methylcellulose, i.p., (1 h)/20% HPBCD s.c. (0.5 h)) showed no protection (0/8 protected).

- 20 040772

Table 6b. Combination studies for reference comp. No. 2-a with levetiracetam (LEV) in a 6 Hz (44 mA) stimulation assay. Combination of 350 mg/kg levetiracetam ____________ with different doses of the reference comp. No. 2-а____________

Medicinal means Dose (mg/kg) Number protected/number tested Number of those with motor impairment on the results of the rotating rod test/number tested LEV (separately) ³ 350 3/8 0/8 LEV 350 mg/kg + ref. conn. No. 2-a ^b 20 8/8 2/8 10 7/8 1/8 5 7/8 1/8 2.5 5/8 0/8 1.25 4/8 0/8

Preliminary value ED ₅₀ for ref. conn. No. 2-a (95% CI):

21.0 mg/kg (from 17.9 to 27.4)

ED ₅₀ for combination LEV and ref. conn. No. 2-a (95% CI): 1.5 mg/kg (0.1-2.7) ~14-fold activity deviation ^a ED ₅₀ for LEV (presented separately), tentatively determined at 6 Hz stimulation (44 mA): 345 mg/kg.

^b Reference conn. No. 2-a (sc) 10 mg/kg, tested in combination with LEV (bp).

Reference connection No. 2-a, 10 mg/kg, not active when administered alone.

Additional controls for LEV (low dose) were tested at 25 and 6.25 mg/kg (1/8 and 0/6 protected, respectively).

Mice treated with inert (0.5% methylcellulose, i.p., (1 h)/20% HPBCD s.c. (0.5 h) showed no protection (0/8 protected).

At a dose of 10 mg/kg, sc, reference comp. No. 2-a increased the effectiveness of the LEV, resulting in an approximately 35-fold shift in ED ₅₀ . This indicated a positive pharmacodynamic relationship (Table 16). At a dose of 350 mg/kg, sc, LEV increased the effectiveness of the reference compound. No. 2-a, which resulted in approximately a 14-fold shift in ED ₅₀ . This indicated a positive pharmacodynamic relationship (Table 6b).

Example 3 Studies with LY404039 3.1.

Combination study with LY404039 and levetiracetam LY-404039 was tested alone and in combination with levetiracetam according to the procedures already described herein above. The results of studies conducted with the connection LY-404039 are shown in table. 7, 8.

- 21 040772

Table 7. Dose-response effects studies for LY404039 in the 6 Hz (32 and 44 mA) stimulation assay. Time to peak effect of 0.5 h was determined in the 6 Hz, 32 mA (s.c) stimulation test and used for 6 Hz (32 and 44 mA) stimulation studies

Test Dose (mg/kg, S.s.) Number protected/number tested Number of people with motor impairment on the rotating rod test/number tested 6Hz 32mA 40 8/8 1/8 20 6/8 1/8 10 5/8 0/8 5 1/16 1/16 ED ₅₀ (95% CI): 10.9 mg/kg (7.8 to 15.9) 6Hz 4 4 mA 40 7/8 2/8 20 7/8 1/8 10 3/8 1/8 5 0/16 0/16 ED ₅₀ (95% CI): 14.1 mg/kg (10.0 to 20.6) TD ₅₀ : > 40 mg/kg ^a

^and 40 mg/kg, 3 out of a total of 16 (combined 32 mA and 44 mA) were deficient.

Note: No activity was observed after administration of an inert substance at 32 or 44 mA. Dose selected for combination studies with LEV at 6 Hz (44 mA): LY404039 5 mg/kg.

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Table 8. Combination studies for LY404039 with levetiracetam (LEV) in the 6 Hz (44 mA) pacing assay

Medicinal means Dose (mg/kg) Number protection of others/number tested Number of people with motor impairment on the rotating rod test/number tested LEV ^a 200 2/8 0/8 400 4/9 0/9 800 10/12 0/12 LEV ED ₅₀ (95% CI): 345.4 mg/kg (211.0 to 485.3) LEV + LY404039, 5 mg/kg ^b 200 8/8 0/8 50 6/8 1/8 20 6/8 2/8 5 2/8 1/8 LEV ED ₅₀ (95% CI) : 12.8 mg/kg (2.5 - 25.2)

^a LEV has been shown alone previously, confirmation doses were given in combination with reference comp. No. 2-a (see the previous table above).

^b LY404039 (sc), 5 mg/kg, tested in combination with LEV (ip); LY404039, 5 mg/kg, was not active when given alone.

Additional controls for LEV (low dose) were tested at 25 and 6.25 mg/kg (1/8 and 0/6 protected, respectively).

Mice treated with an inert substance (10% sterile water - NaCl; s.c, 0.5 h TPE and 0.5% MC, i.p., 1 h TPE) showed no protection or deterioration in the assessment of the ability to hold onto a rotating rod.

At a dose of 5 mg/kg, LY404039 increased the effectiveness of LEV, resulting in an approximately 27-fold shift in ED ₅₀ . This indicates a positive pharmacodynamic relationship.

The current dataset indicates that mGlu2 agonist molecules had anticonvulsant activity in an animal model when stimulated at 6 Hz. In addition, all tested molecules showed synergistic effects with LEV. With regard to LY404039, an mGlu2/3 agonist, activity was observed in both tests with 6 Hz stimulation, and synergy was observed at a dose of 3 times less than the ED ₅₀ determined in the 44 mA stimulation model, which was inactive when tested alone.

Examples of possible applications.

A) Dominant-subordinate relationship (DSR) in rats in an in vivo assay.

The DSR analysis is divided into two models: Mania Reduction in the Dominant Behavior Model (RDBM) and Depression Reduction in the Submissive Behavior Model (RSBM). The RDBM model in which dominant animals were treated with a test compound predicts the ability of a test compound to treat mania. The RSBM model, in which subordinate animals were treated with a test compound, predicts the ability of a test compound to treat depression.

Male Sprague-Dawley rats (140 to 160 g) from Charles River Laboratories, Wilmington, Massachusetts were used in this assay. Batches of rats were received at two week intervals. Each batch was subjected to a five-day quarantine, a one-week acclimatization period, and a one-week selection process, followed by a five-week treatment of selected pairs with a drug or inert substance.

Rats were kept in cages, four individuals each. Access to food was limited to one hour per day after testing from Monday to Thursday. After testing on Friday, the rats were allowed free access to food until re-abstinence from food on Sunday. During all time periods, the rats were not deprived of water. Involved periods of deprivation

- 23 040772 food had little effect on weight gain since the average weight of rats at the end of the study would be approximately 300 g. facilities.

The main testing device consisted of two chambers connected only by a tunnel large enough to allow one rat to pass through at any given time. At the bottom, in the middle part of the tunnel, a container with sweetened milk was placed. This main device was duplicated so that a total of four pairs of rats could be recorded on a video camera at the same time. The camera could recognize rats labeled with different colors. Thus, the heads of rats were stained for the purpose of video recording, red in one cage and yellow in another. Only one animal at a time can have free access to the feeder, but both animals can drink milk daily for a five-minute period. During daily five minute periods, time spent in the feed area by each rat was recorded by video capture software and saved to a text file.

The test began with a randomized pairing of rats. Each pair of rats was placed in the opposite chamber of the test device. Recorded the time spent by each animal in the area of the feeder. During the first week (five days) of testing, the animals were accustomed to a new habitat. Dominance was assigned to the animal with the highest score during the second week of testing when three criteria were met. First, there must be a significant difference (two-tailed t-test, P<0.05) between the mean daily drinking rates of both animals. Secondly, the score of the dominant animal must be at least 25% higher than the score of the subordinate animal. As a result, there should be no change during the week of selection of pairs in which the hypothetically subordinate rat outperformed its dominant partner in isolated cases. Ideally, there will be minimal change during the week of acclimatization. Only pairs of animals that met these criteria continued to participate in the study.

Significant differences between time spent at the feeder by dominant and submissive rats were determined by ANOVA using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA) followed by a two-tailed t-test (P<0.05). Comparisons were made between treatment groups using normalized values for the level of dominance in paired animals. The level of dominance was a value that measured the social relationship between subjects in a pair. Dominance level (DL)=FTD-FTS, where FTD is time spent at the feeder by dominant rats and FTS is time spent at the feeder by subordinate rats. Normalization was carried out in accordance with the formula:

dominance level (week, n in %)=(dominance level (week, n))/(dominance level (week 2).

The statistical significance of the difference in the level of dominance between the control group (pairs of rats where both dominant and subordinate animals were treated with an inert substance) and the treatment group (subordinate rats were treated with drug and dominant rats with an inert substance) was determined using ANOVA followed by t-test . The time to onset of action at the 50% response level (AOT-50) and the minimum and maximum drug responses for decreasing dominance values were calculated using non-linear regression analysis (GraphPad Software, Inc., San Diego, CA). Normalized DL values were used for these calculations, where the DL values for weeks of treatment were normalized as a percentage of the second week (pre-treatment) of such a pair according to the above formula. Under these parameters, minimal response (DL) indicated positive drug activity, in line with efficacy, since DL values decreased if the drug response was positive. In the case of a negative reaction to the drug (increase in the severity of symptoms), the DL values increased. If the drug did not show such activity, then the maximum response did not exceed 100%. Any peak DL value significantly above the control value (approximately 100%) indicated negative drug activity.

Levetiracetam and an mGluR2 agonist compound (eg, LY-404039) were evaluated in the RDBM rat model according to the procedure described in more detail below.

Groups of dominant rats were treated po. QD with levetiracetam 10 mg/kg and mGluR2 magonist compound at different concentrations from about 0.05 mg/kg (n>3), at 0.5 mg/kg (n>3), at 2.5 mg/kg (n >3), in the amount of 5.0 mg/kg (n>3) and in the amount of 50.0 mg/kg (n>3). One control group of inert dominant rats was treated with 0.5% methylcellulose (n>3) and a second control group of dominant rats was treated with i.p. QD with sodium valproate at 30 mg/kg (n>6 after 2 studies n>3 each).

- 24 040772

All treatments were performed approximately 1 hour prior to testing. All treatments started on the Saturday after the second week of testing (week of selection). Levetiracetam and the mGluR2 agonist compound were administered orally (po).

When dominant animals were treated with levetiracetam at a dose of 10 mg/kg and the mGluR2 agonist compound, the difference between dominant and subordinate animals disappeared after the first or second week of treatment depending on the dosage. Similarly, if dominant animals were treated with sodium valproate, then the difference between dominant and subordinate rats also disappeared after the first week of treatment. An increase in tolerance could be observed in dominant rats treated with levetiracetam and a mGluR2 agonist compound or sodium valproate. Thus, treated dominant rats allowed their submissive partners to increase their time at the feeder.

To compare different drugs and dose effects, the data were normalized to the baseline values of the control week. The strongest effect of the combination of levetiracetam and the mGluR2 agonist compound was observed when there was a significant difference in dominance level (DL) values between inert and combination treated rats starting from the second week and continuing through the treatment period up to 5 weeks. In comparison, animals (30 mg/kg) treated with sodium valproate consistently showed a reduced level of dominance after the second week of treatment, with the effect increasing in subsequent weeks.

To assess time to onset (AOT), daily averages of time spent at the feeder for dominant and submissive pairs were plotted and significant differences between the two groups calculated using a two-tailed t-test.

To compare time to onset (AOT) between different treatments, time to onset was evaluated using a non-linear regression plot. A non-linear regression model produced a plot for each drug, combination, and daily DL values normalized for dose.

The effects of levetiracetam and the mGluR2 agonist compound on RDBM were potentially dose dependent.

According to this analysis, the combination of levetiracetam and an mGluR2 agonist compound potentially reduced dominant behavior, indicating that the combination is active as an antimanic agent.

C) Tablets for oral administration.

As a specific embodiment, oral formulations of 100 mg mGluR2 PAM/mGluR2 agonist compound were formulated with sufficiently finely dispersed lactose to give a total amount of 580 to 590 mg to fill an O size hard gelatin capsule.

Claims (30)

CLAIM 1. Combination for the treatment or prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, and bipolar and related disorders, containing: (a) a synaptic vesicle 2A (SV2A) protein ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and (b) an orthosteric metabotropic glutamatergic receptor subtype 2 agonist selected from: 1) LY-404039 he or its salt or solvate; and 2) LY-2140023 - 25 040772 or its salt or solvate. 2. The combination of claim 1 wherein the SV2A ligand is levetiracetam or brivaracetam. 3. A combination according to claim 1 or 2, wherein the orthosteric subtype 2 metabotropic glutamatergic receptor agonist is LY-404039. 4. A combination according to claim 1 or 2, wherein the subtype metabotropic glutamatergic receptor 2 orthosteric agonist is LY-2140023. 5. A combination according to claim 1 or 2, wherein the orthosteric subtype 2 metabotropic glutamatergic receptor agonist is LY-2140023 monohydrate. 6. A pharmaceutical composition containing a combination according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier. 7. A method for preparing a pharmaceutical composition according to claim 6, wherein the combination according to any one of claims 1 to 5 is intimately mixed with a pharmaceutical carrier. 8. A medicament containing a combination of an SV2A ligand and an orthosteric agonist of a metabotropic glutamatergic receptor subtype 2 according to any one of claims 1 to 5, for simultaneous, separate or sequential use in the treatment or prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, bipolar and related disorders. 9. The medicament of claim 8, wherein the SV2A ligand and the subtype 2 metabotropic glutamatergic receptor 2 orthosteric agonist are administered separately. 10. The use of a combination according to any one of claims 1 to 5 for the preparation of a medicament for the treatment of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, bipolar and related disorders. 11. The use of a combination according to any one of claims 1 to 5 for the preparation of a medicament for the prevention of epileptogenesis. 12. The use of a combination according to any one of claims 1 to 5 for the preparation of a medicament for the prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, and bipolar and related disorders. 13. Use according to claim 10, where the drug is for the treatment of epilepsy and related disorders. 14. Use according to claim 13, wherein the epilepsy and related disorders are selected from focal seizures with or without generalization, myoclonic seizures, primary generalized tonic-clonic seizures, and treatment-resistant or refractory epilepsy. 15. Use according to claim 14, wherein primary generalized tonic-clonic seizures occur in patients with idiopathic generalized epilepsy. 16. Use according to claim 14, wherein the refractory epilepsy is refractory partial epilepsy or refractory generalized epilepsy. 17. Use according to claim 13 as a neuroprotector. 18. The use of a pharmaceutical composition according to claim 6 as a medicament for the treatment of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, bipolar and related disorders. 19. The use of a pharmaceutical composition according to claim 6 in the prevention of epileptogenesis. 20. The use of a pharmaceutical composition according to claim 6 in the prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, and bipolar and related disorders. 21. Use according to claim 18 in the treatment or prevention of epilepsy and related disorders. 22. Use according to claim 21 as a neuroprotector. 23. A method of treating a patient with epilepsy or related disorders, neuropathic pain, migraine or persistent headache, or bipolar and related disorders, comprising administering an orthosteric agonist of the metabotropic glutamatergic receptor subtype 2 and an SV2A ligand selected from the group consisting of levetiracetam , brivaracetam and seletracetam, wherein the orthosteric agonist of the metabotropic glutamatergic receptor subtype 2 is selected from: 1) LY-404039 - 26 040772 or its salt or solvate; and 2) LY-2140023 or its salt or solvate. 24. The method of claim 23 wherein the SV2A ligand is levetiracetam. 25. The method of claim 23 wherein the SV2A ligand is brivaracetam. 26. The method according to any one of claims 23-25, wherein the disorder is epilepsy, in particular treatment-resistant epilepsy. 27. The method of any one of claims 23-25, wherein the epilepsy and its associated disorder are focal seizures. 28. The method of any one of claims 23-27, wherein the orthosteric agonist of the mGluR2 compound and the SV2A ligand are administered simultaneously. 29. The method of any one of claims 23-27, wherein the orthosteric agonist of the mGluR2 compound and the SV2A ligand are administered simultaneously via separate dosage forms. 30. The method of any one of claims 23-27, wherein the orthosteric agonist of the mGluR2 compound and the SV2A ligand are administered separately, at different time points during the course of therapy.