WO2014111525A2

WO2014111525A2 - New combination therapies for treating nervous system diseases

Info

Publication number: WO2014111525A2
Application number: PCT/EP2014/050907
Authority: WO
Inventors: Mireia COMA; Patrick ALOY; Albert PUJOL; José Manuel MAS; Jordi NAVAL; Caty CASAS; Xavier Navarro; Mireia HERRANDO
Original assignee: Anaxomics Biotech, Sl
Priority date: 2013-01-18
Filing date: 2014-01-17
Publication date: 2014-07-24
Also published as: WO2014111525A3

Abstract

The invention is directed to combinations of alitretinoin and pranlukast or to combinations of alitretinoin and mefloquine, to pharmaceutical compositions, kits and treatment methods for the treatment or prevention of nervous system disorders, particularly motor neuron disease, spinal cord disease and/or nervous system disease associated with glutamate excitotoxicity, including amyotrophic lateral sclerosis.

Description

NEW COMBINATION THERAPIES FOR TREATING NERVOUS SYSTEM

DISEASES

FIELD OF THE INVENTION

The present invention relates generally to the field of nervous system diseases. The invention features novel combinations useful for the treatment of nervous system diseases including motor neuron disease, spinal cord diseases and diseases associated with glutamate excitotoxicity, and particularly amyotrophic lateral sclerosis. The invention also relates to pharmaceutical compositions and methods for the treatment or prevention of nervous system diseases, and particularly amyotrophic lateral sclerosis.

BACKGROUND OF THE INVENTION

Motor neuron diseases are an etiologically heterogeneous group of disorders that are characterized by muscle weakness and/or spastic paralysis, which results from the selective degeneration of lower motor neurons and/or upper motor neurons, respectively (see Dion, P.A., Daoud, H. & Rouleau, G.A. Genetics of motor neuron disorders: new insights into pathogenic mechanisms. Nat Rev Genet 10, 769-82 (2009).

Amyotrophic lateral sclerosis is the most common form of motor neuron disease. Amyotrophic lateral sclerosis is characterized by the selective premature degeneration and death of motor neurons, which control voluntary actions such as breathing and walking. The disease starts in adult life, and the ensuing progressive paralysis is typically fatal within a few years, usually owing to failure of the respiratory system (see Pratt, A.J., Getzoff, E.D. & Perry, J.J. Amyotrophic lateral sclerosis: update and new developments. Degener Neurol Neuromuscul Dis 2012, 1 -14). The incidence of amyotrophic lateral sclerosis is around two to three cases per 100,000 general population annually, and the prevalence is around four to six per 100,000 (see Venkova-Hristova, K., Christov, A., Kamaluddin, Z., Kobalka, P. & Hensley, K. Progress in therapy development for amyotrophic lateral sclerosis. Neurol Res Int 2012, 187234).

Riluzole is the only approved treatment for amyotrophic lateral sclerosis, which prolongs survival by about two or three months (see Miller, R.G., Mitchell, J.D., Lyon, M. & Moore, D.H. Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND). Cochrane Database Syst Rev, CD001447 (2007)). New treatments that block the initial events leading to neuronal death and avoid disease progression are needed. Recently, a large number of clinical trials have failed, as per example vitamin E (see Desnuelle, C, Dib, M., Garrel, C. & Favier, A. A double-blind, placebo-controlled randomized clinical trial of alpha- tocopherol (vitamin E) in the treatment of amyotrophic lateral sclerosis. ALS riluzole-tocopherol Study Group. Amyotroph Lateral Scler Other Motor Neuron Disord 2, 9-18 (2001 )), memantine (see de Carvalho, M. et al. A randomized, placebo-controlled trial of memantine for functional disability in amyotrophic lateral sclerosis. Amyotroph Lateral Scler 1 1 , 456-60), minocycline (see Gordon, P.H. et al. Efficacy of minocycline in patients with amyotrophic lateral sclerosis: a phase III randomised trial. Lancet Neurol 6, 1045-53 (2007)) due to lack of efficacy, or thalidomide (see Stommel, E.W. et al. Efficacy of thalidomide for the treatment of amyotrophic lateral sclerosis: a phase II open label clinical trial. Amyotroph Lateral Scler 10, 393-404 (2009); Meyer, T. et al. Thalidomide causes sinus bradycardia in ALS. J Neurol 255, 587-91 (2008)) due to lack of safety.

Amyotrophic lateral sclerosis is a complex disease with diverse genetic and environmental etiologies. Motor neuron degeneration in amyotrophic lateral sclerosis involve multiple molecular events in neurons but also active (gain of function) and passive (loss of function) events to occur in non-neuronal cells, especially astrocytes and microglia. Due to the multifactorial nature of the pathology, it is more and more clear that multi-target polypharmacological research is needed to interact with different targets and modify different molecular pathways. The discovery of drug combinations and the understanding of their complex modes of action may be worth attempting in future amyotrophic lateral sclerosis animal and human studies to outline new therapies against degenerative diseases (see Venkova- Hristova, K., Christov, A., Kamaluddin, Z., Kobalka, P. & Hensley, K. Progress in therapy development for amyotrophic lateral sclerosis. Neurol Res Int 2012, 187234; Pujol, A., Mosca, R., Farres, J. & Aloy, P. Unveiling the role of network and systems biology in drug discovery. Trends Pharmacol Sci 31 , 1 15-23 (2010); Aloy, P. & Russell, R. Targeting and tinkering with interaction networks. FEBS Lett 582, 1219 (2008); Jia, J. et al. Mechanisms of drug combinations: interaction and network perspectives. Nat Rev Drug Discov 8, 1 1 1 -28 (2009)). Clinical success with multicomponents therapies and multi-targeted agents has been shown in other pathologies like asthma, hyperlipidemia, HIV-1 or cancer.

A large number of nervous system diseases are considered to be motor neuron diseases, spinal cord diseases and/or diseases associated with glutamate excitotoxicity such as amyotrophic lateral sclerosis, spinal muscular atrophy (SMA) (see US 7541371 B2; EP 1405637 B1 ; Garcera A et al. Cell Death Dis. 4:e686 (2013); Papadimitriou D et al. Neurobiol Dis. 37(3):493-502 (2010)), spinal and bulbar muscular atrophy (SBMA) (also known as bulbo-spinal atrophy, X-linked bulbospinal neuropathy (XBSN), X-linked spinal muscular atrophy type 1 (SMAX1 ), X-linked bulbo-spinal atrophy, Kennedy's disease (KD) or spinobulbar muscular atrophy) (see Tanaka F et al. Neural Plast 2012:369284 (2012); EP 0593516 B1 ), childhood spinal muscular atrophies (see Briese M et al. Bioessays, 27(9):946-57(2005)), progressive spinomuscular atrophy, infantile muscular atrophy, primary lateral sclerosis (PLS) (see US 6297254 B1 ), spinal cord injury (see Thomas CK and Zijdewind I. Muscle nerve, 33(1 ):21 -41 (2006); Mehta A et al. Eur J Pharmacol, 698(1 -3):6-18 (2013)), poliomyelitis (see EP 0593516 B1 ; Okumura H. et al. Ann N Y Acad Sci, 753:245-56 (1995); Shimada A et al. Acta Neuropathol, 97(3):317-21 (1999)), postpoliomyelitis syndrome (see EP 0593516 B1 ; Thomas CK and Zijdewind I. Muscle nerve, 33(1 ):21 -41 (2006); Okumura H. et al. Ann N Y Acad Sci, 753:245-56 (1995)), Charcot-Marie-Tooth disease (see EP 0593516 B1 ; Brownlees J et al. Hum Mol Genet, 1 1 (23):2837-44 (2002); Szigeti K and Lupski JR. Eur J Hum Genet, 17(6):703-10 (2009)), diffuse atrophic paralysis (see US 7541371 B2), pseudobulbar palsy (see US 7541371 B2), bulbar palsy (see US 7541371 B2), juvenile unilateral upper-limb muscular atrophy (see US 7541371 B2), progressive bulbar palsy (see US 7541371 B2), spinal progressive muscular atrophy (see US 7541371 B2), arthrogryposis multiplex congenita (AMC) (see EP 1405637 B1 ), Brown-Vialetto-Van Laere syndrome (see Sathasivam S. Orphanet J Rare Dis, 3:9 (2008)), Fazio-Londe disease (see EP 0593516 B1 ; McShane MA et al. Brain, 1 15(Pt 6):1889-9000 (1992)), spinal cord compression (see Xu W et al. Spinal Cord, 43(4):204-13 (2005)), central cord syndrome (see Li XF and Dai LY. Spine (Phila Pa 1976), 35(19):E955-64 (2010)), spinal cord ischemia (see Sakurai M et al. J Cereb Blood Flow Metab, 29(4):752-8 (2009)), Machado-Joseph disease (see Tan CF et al. Acta Neuropathol, 1 18(4):553-60 (2009)), frontotemporal dementia (see Ling SC et al. Neuron, 79(3):416-38 (2013)); Burrell JR et al. Brain, 134(Pt 9):2582-94 (201 1 ); Lomen-Hoerth C. J Mol Neurosci, 45(3):656-62 (201 1 )), frontotemporal dementia with amyotrophic lateral sclerosis (see Lomen-Hoerth C. J Mol Neurosci, 45(3):656-62 (201 1 )), stroke (see Hossain Ml et al. J Biol Chem, 288(14):9696-709 (2013); Mehta A et al. Eur J Pharmacol, 698(1 -3):6-18 (2013)), mitochondrial encephalomyopathies, spinocerebellar degeneration syndromes (see Bittigau P and Ikonomidou C. J Child Neurol, 12(8):471 -85 (1997), traumatic brain injury, epilepsy, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, alcohol withdrawal (see Mehta A et al. Eur J Pharmacol, 698(1 -3):6-18 (2013)), peripheral nerve injuries (see Watabe K et al. Neuropathology, 25(4):371 -80 (2005)) and motoneuron degeneration caused by infarction, exposure to a toxin, malignancy or autoimmune disease (see EP 0593516 B1 ). Particularly, excitotoxicity is the pathological process by which nerve cells are damaged and killed by excessive stimulation by neurotransmitters such as glutamate and similar substances. Such excitotoxic neuronal death has been implicated in spinal cord injury, stroke, traumatic brain injury, and in neurodegenerative diseases of the central nervous system such as stroke, epilepsy, multiple sclerosis, Alzheimer disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington disease and alcohol withdrawal (see Mehta A et al. Eur J Pharmacol, 698(1 -3):6-18 (2013)). A mechanism of slow excitotoxicity may also be involved in neuronal death in chronic neurodegenerative diseases such as the mitochondrial encephalomyopathies, Huntington's disease, spinocerebellar degeneration syndromes, and motor neuron diseases (see Bittigau P and Ikonomidou C. J Child Neurol, 12(8):471 - 85 (1997). Glutamate excitotoxicity has been suggested to play a role in other neuronal diseases such as spinal muscular atrophy (SMA) (see Miller RG et al. J Neurol Sci, 191 (1 -2):127-31 (2001 ); Greensmith L and Vrbova G. Neuromuscul Disord, 5(5):359-369 (1995)), childhood spinal muscular atrophies (see Greensmith L and Vrbova G. Neuromuscul Disord, 5(5):359-369 (1995)), spinal and bulbar muscular atrophy (SBMA) (see Kilpatrick TJ and Soilu- Hanninen M. Mol Neurobiol, 19(3):205-28 (1999)), poliomyelitis, postpoliomyelitis syndrome (see Sejvar JJ et al. J Neurovirol, 16(1 ):93-100 (2010)), Charcot-Marie-Tooth disease (see Wang Y et al. Mol Neurodegener, 6:75 (201 1 )), spinal cord compression (see Esposito E et al. BMC Neurosci, 12:31 (201 1 )), spinal cord ischemia, frontotemporal dementia (see Furukawa K et al. J Neurochem, 87(2):427-36 (2003)) and frontotemporal dementia with amyotrophic lateral sclerosis.

SUMMARY OF THE INVENTION Now, the inventors have surprisingly found that a combination of alitretinoin and pranlukast or a combination of alitretinoin and mefloquine are capable of providing neuroprotection against motor neuron diseases, spinal cord diseases and diseases associated with glutamate excitotoxicity, more particularly a synergistic degree of neuroprotection. Thus, the present invention is directed to a combination of alitretinoin and pranlukast or to a combination of alitretinoin and mefloquine, to pharmaceutical compositions, kits and treatment methods for the treatment or prevention of nervous system disorders, particularly motor neuron diseases, spinal cord diseases, and diseases associated with glutamate excitotoxicity and, more particularly, amyotrophic lateral sclerosis.

In a first aspect the present invention is directed to a combination comprising alitretinoin or its pharmaceutically acceptable salts and a second component selected from pranlukast and mefloquine or a pharmaceutically acceptable salt thereof.

In a second aspect the present invention is directed to a pharmaceutical composition comprising a combination comprising alitretinoin or its pharmaceutically acceptable salts and a second component selected from pranlukast and mefloquine or a pharmaceutically acceptable salt thereof.

In a third aspect the present invention is directed to a combination of alitretinoin or its pharmaceutically acceptable salts and pranlukast or its pharmaceutically acceptable salts or a combination of alitretinoin or its pharmaceutically acceptable salts and mefloquine or its pharmaceutically acceptable salts, or to a pharmaceutical composition comprising said combinations for use in the treatment of a nervous system disorder selected from the group consisting of a motor neuron disease, spinal cord disease and/or a nervous system disease associated with glutamate excitotoxicity.

The compositions and/or combinations of the present invention are particularly suitable for treating nervous system diseases, particularly motor neuron disease, spinal cord diseases, and diseases associated with glutamate excitotoxicity such as amyotrophic lateral sclerosis, spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), childhood spinal muscular atrophies, progressive spinomuscular atrophy, infantile muscular atrophy, primary lateral sclerosis (PLS), spinal cord injury, poliomyelitis, postpoliomyelitis syndrome, Charcot-Marie-Tooth disease, diffuse atrophic paralysis, pseudobulbar palsy, bulbar palsy, juvenile unilateral upper-limb muscular atrophy, progressive bulbar palsy, spinal progressive muscular atrophy, arthrogryposis multiplex congenita (AMC), Brown-Vialetto-Van Laere syndrome, Fazio-Londe disease, spinal cord compression, central cord syndrome, spinal cord ischemia, Machado-Joseph disease, frontotemporal dementia , frontotemporal dementia with amyotrophic lateral sclerosis, stroke, mitochondrial encephalomyopathies, spinocerebellar degeneration syndromes, traumatic brain injury, epilepsy, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, alcohol withdrawal, peripheral nerve injuries and motoneuron degeneration caused by infarction, exposure to a toxin, malignancy or autoimmune disease.

In a fourth aspect the present invention is directed to the use of a combination of alitretinoin or its pharmaceutically acceptable salts and pranlukast or its pharmaceutically acceptable salts or a combination of alitretinoin or its pharmaceutically acceptable salts and mefloquine or its pharmaceutically acceptable salts, or to a pharmaceutical composition comprising said combinations for the manufacture of a medicament for the treatment of a nervous system disorder selected from the group consisting of a motor neuron disease, spinal cord disease and/or a nervous system disease associated with glutamate excitotoxicity.

In a fifth aspect the present invention is directed to a method for treating a subject suffering a nervous system disorder selected from the group consisting of a motor neuron disease, spinal cord disease and/or a nervous system disease associated with glutamate excitotoxicity comprising the administration to said subject of a therapeutically effective amount of a combination of alitretinoin or its pharmaceutically acceptable salts and pranlukast or its pharmaceutically acceptable salts or a combination of alitretinoin or its pharmaceutically acceptable salts and mefloquine or its pharmaceutically acceptable salts, or to a pharmaceutical composition comprising said combinations. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 . Drug combinations reduce neuroinflammation. Levels of TNFa released to the culture medium were measured at 3 weeks post-excitotoxic treatment by specific ELISA assay. Percentage of TNFa released to the medium was represented referred to its control (0%) and THA (100%) cytokine production. Values are the mean±SEM of 3 independent experiments.

FIGURE 2. Drug combinations reduce neuroinflammation. Microphotographs of the spinal cord ventral horn at 3 weeks post-THA treatment, showing microglia stained with ibal antibody. Top panels show slices corresponding to controls, THA-alone or concomitantly with alitretionin-pranlukast, alitretionin-mefloquine or riluzole. Lower panels show schematic drawings of the shape of microglia for each condition. Scale bar 20 μΜ.

FIGURE 3. Drug combinations increase motorneuron survival after chronic excitotoxic insult. Number of motoneurons in the ventral horn (VH) of each spinal cord treated with vehicle, THA-alone or concomitantly with alitretionin- pranlukast, alitretionin-mefloquine or riluzole. SMI-32 positive cells were counted using the Z-stack images from confocal microscopy. Values are meaniSEM of 13 hemisections per treatment (^***p<0.001 and ^*p<0.05 by Dunnet's pot-hoc test vs THA condition). FIGURE 4. Drug combinations increase motorneuron survival after chronic excitotoxic insult. Fluorescent microphotographs representative of motorneuron distribution in the ventral horn of each spinal cord stained with SMI-32 antibody. Scale bar 50 μΜ.

DETAILED DESCRIPTION OF THE INVENTION

Nervous system disorders treated with the compositions and combinations of the invention

The compositions and combinations of the invention are particularly suitable for treating nervous system diseases, particularly motor neuron disease, spinal cord diseases, and diseases associated with glutamate excitotoxicity such as amyotrophic lateral sclerosis, spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), childhood spinal muscular atrophies, progressive spinomuscular atrophy, infantile muscular atrophy, primary lateral sclerosis (PLS), spinal cord injury, poliomyelitis, postpoliomyelitis syndrome, Charcot- Marie-Tooth disease, diffuse atrophic paralysis, pseudobulbar palsy, bulbar palsy, juvenile unilateral upper-limb muscular atrophy, progressive bulbar palsy, spinal progressive muscular atrophy, arthrogryposis multiplex congenita (AMC), Brown-Vialetto-Van Laere syndrome, Fazio-Londe disease, spinal cord compression, central cord syndrome, spinal cord ischemia, Machado-Joseph disease, frontotemporal dementia , frontotemporal dementia with amyotrophic lateral sclerosis, stroke, mitochondrial encephalomyopathies, spinocerebellar degeneration syndromes, traumatic brain injury, epilepsy, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, alcohol withdrawal, peripheral nerve injuries and motoneuron degeneration caused by infarction, exposure to a toxin, malignancy or autoimmune disease. In a particular embodiment, the compositions and combinations of the invention are suitable for treating diseases associated with glutamate excitotoxicity such as spinal cord injury, stroke, traumatic brain injury, epilepsy, multiple sclerosis, Alzheimer disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, alcohol withdrawal, mitochondrial encephalomyopathies, spinocerebellar degeneration syndromes, spinal muscular atrophy (SMA), childhood spinal muscular atrophies, spinal and bulbar muscular atrophy (SBMA), poliomyelitis, postpoliomyelitis syndrome, Charcot-Marie-Tooth disease, spinal cord compression, spinal cord ischemia, frontotemporal dementia and frontotemporal dementia with amyotrophic lateral sclerosis, more particularly amyotrophic lateral sclerosis. In a particular embodiment the compounds are used to prepare a medicine for treating a mammal in need thereof, in particular a human patient, wherein compounds are used in a dosage from 0.0037 mg to 37000 mg per m² of body weight, in particular from 0.37mg to 3700 mg per m² of body weight. In particular, the combination of alitretinoin plus pranlukast is used in a range of 0.037 mg/m²/day to 1480 mg/m²/day for alitretinoin, and 0.037 mg/m²/day to 16650 mg/m²/day for pranlukast. Preferably, alitretinoin is used in a range of 0.5 mg/m²/day to 100 mg/m²/day, more preferably 1 mg/m²/day to 50 mg/m²/day, more preferably 2 mg/m²/day to 25 mg/m²/day, more preferably 3 mg/m²/day to 20 mg/m²/day, even more preferably from 5 mg/m²/day to 16 mg/m²/day. Preferably, pranlukast is used in a range of 1 mg/m²/day to 1000 mg/m²/day, more preferably 5 mg/m²/day to 500 mg/m²/day, more preferably 50 mg/m²/day to 400 mg/m²/day, more preferably from 100 mg/m²/day to 300 mg/m²/day, even more preferably from 200 mg/m²/day to 300 mg/m²/day. A combination of alitretinoin plus pranlukast may be formulated using a 1 :1 ratio of alitretinoin:pranlukast or different w/w ratios, ranging for example, from 1 - 100:100-1 , preferably from 1 -50:50-1 , more preferably from 1 -20:20-1 , even more preferably from 1 -10:10-1 , the most preferred from 1 -5:5-1 . This range includes all intermediate ratios, such as 1 :4, 1 :3, 1 :2, 2:1 , 3:1 or 4:1 .

In particular, the combination of alitretinoin plus mefloquine is used in a range of 0.037 mg/m²/day to 1480 mg/m²/day for alitretinoin, and 0.037 mg/m²/day to 10138 nng/nn²/day for mefloquine. Preferably, alitretinoin is used in a range of 0.5 mg/m²/day to 100 mg/m²/day, more preferably 1 mg/m²/day to 50 mg/m²/day, more preferably 2 mg/m²/day to 25 mg/m²/day, more preferably 3 mg/m²/day to 20 mg/m²/day, even more preferably from 5 mg/m²/day to 16 mg/m²/day. Preferably, mefloquine is used in a range of 0.5 mg/m²/day to 500 mg/m²/day, more preferably 1 mg/m²/day to 300 mg/m²/day, more preferably from 5 mg/m²/day to 200 mg/m²/day, more preferably from 10 mg/m²/day to 150 mg/m²/day, even more preferably from 10 mg/m²/day to 50 mg/m²/day. A combination of alitretinoin plus mefloquine may be formulated using a 1 :1 ratio of alitretinoin:mefloquine or different w/w ratios, ranging for example, from 1 - 100:100-1 , preferably from 1 -50:50-1 , more preferably from 1 -20:20-1 , even more preferably from 1 -10:10-1 , the most preferred from 1 -5:5-1 . This range includes all intermediate ratios, such as 1 :4, 1 :3, 1 :2, 2:1 , 3:1 or 4:1 . Doses of active ingredients may be expressed either in mg of active ingredient per kg of body weight or in mg of active ingredient per square meter of body surface. The article from Reagan-Shaw S. "Dose translation from animal to human studies revisited". FASEB J 2007, 22:659-661 provides the standard conversion factors used to convert mg/kg to mg/m².

Dose (mg/kg) x K_m = Dose (mg/m²)

The article also explains that this conversion is the basis for converting dose in a first animal species to dose in a second animal species (allometric dose translation). Thus, animal dose (AD) in mg/kg can be converted to human equivalent dose (HED) in mg/kg using the following formula:

Animal K_n

HED (mg/kg) = AD (mg/kg) X

Human K, wherein the K_m for each species is shown in Table I (data extracted from Reagan-Shaw S. "Dose translation from animal to human studies revisited". FASEB J 2007, 22:659-661 ).

Table I. K_m factor for conversion of AD to HED

In the context of the present invention alitretinoin (also named 9-cis-retinoic acid) is used to designate the compound (2E,4E,6Z,8E)-3,7-dimethyl-9-(2,6,6- trimethyl-1 -cyclohexenyl)nona-2,4,6,8-tetraenoic acid or its pharmaceutically acceptable salts. Whenever the present specification contains references to quantities of alitretinoin salts they are meant to refer to the quantities expressed as alitretinoin in free form.

In the context of the present invention pranlukast is used to designate the compound A/-[4-oxo-2-(1 H-tetrazol-5-yl)-4H-chromen-7-yl]-4-(4- phenylbutoxy)benzamide or its pharmaceutically acceptable salts. Whenever the present specification contains references to quantities of pranlukast salts they are meant to refer to the quantities expressed as pranlukast in free form. In the context of the present invention mefloquine is used to designate the compound [(R^*,S^*)-2,8-bis(trifluoromethyl)quinolin-4-yl]-(2-piperidyl)methanol or its pharmaceutically acceptable salts. Whenever the present specification contains references to quantities of mefloquine salts they are meant to refer to the quantities expressed as mefloquine in free form. Pharmaceutical Compositions

This invention also provides pharmaceutical compositions that comprise compounds of this invention formulated together with one or more non-toxic pharmaceutically acceptable carriers. The pharmaceutical compositions may be specially formulated for any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.

In one preferred embodiment, compositions of the invention are administered orally. Other modes of administration include topical, buccal, transdermal, within/on implants, or parenteral routes. The term "parenteral" includes subcutaneous, intrathecal, intraventricular, intravenous, intraocular, intramuscular, intraperitoneal, intraarticular, subarachnoid, dorsalis venous plexis injection or infusion. Drug delivery includes conjugates, gels, liposomes, microspheres, nanoparticles, carriers, pupms, catheters, implants, controlled- release microchips, and the similar. Compositions comprising a composition of the invention can be added to a physiological fluid.

The invention particularly provides pharmaceutical compositions that comprise alitretinoin and pranlukast, formulated together with one or more pharmaceutically acceptable carriers. The invention further provides pharmaceutical compositions that comprise alitretinoin and mefloquine, formulated together with one or more pharmaceutically acceptable carriers. The pharmaceutical compositions may be specially formulated for oral administration in solid or liquid form, for parenteral injection, or for rectal administration. The term "pharmaceutically acceptable carrier" as used herein means a nontoxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. This invention provides pharmaceutical compositions which comprise compounds of the invention formulated together with one or more non-toxic pharmaceutically acceptable carriers. The pharmaceutical compositions can be formulated for oral administration in solid or liquid form, for parenteral injection or for rectal administration.

The pharmaceutical compositions of this invention can be administered to humans (patients) and other mammals orally, rectally, parenterally, intracisternally, intraperitoneally, topically (as by powders, ointments or drops both over skin or over body external mucoses, including but not limited to bucal, oral or nasal application. The term "parenterally," as used herein, refers to modes of administration which include intraocular, subcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal, intra-articular injection or infusion. Compositions comprising a composition of the invention can be added to a physiological fluid. Pharmaceutical compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservative agents, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Suspensions, in addition to the active compounds, may contain suspending agents, as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.

If desired, and for more effective distribution, the compounds of this invention can be incorporated into slow-release or targeted-delivery systems such as polymer matrices, liposomes, and microspheres. They may be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporation of sterilizing agents in the form of sterile solid compositions, which may be dissolved in sterile water or some other sterile injectable medium immediately before use.

The active compounds can also be in micro-encapsulated form, if appropriate, with one or more pharmaceutically acceptable carriers as noted above. The solid dosage forms of tablets, dragees, capsules, pills, granules and device can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of such composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as a solution in 1 ,3- butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable carrier such as sodium citrate or calcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and salicylic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

Compositions for rectal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non- irritating carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum and release the active compound.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Dosage forms for topical administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to the compounds of this invention, lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.

Compounds of this invention may also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes may be used. The present compositions in liposome form may contain, in addition to the compounds of this invention, stabilizers, preservatives, and the like. The preferred lipids are the natural and synthetic phospholipids and phosphatidylcholines (lecithins) used separately or together.

Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y., (1976), p 33 et seq.

The phrase "therapeutically effective amount" of the compound of this invention means a sufficient amount of the compound to treat neurological and/or neurodegenerative disorders, or to prevent the onset of neurological and/or neurodegenerative disorders, at a reasonable benefit/risk ratio applicable to any medical treatment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention can be varied so as to obtain an amount of the active compound(s) which is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. A "pharmaceutically-acceptable derivative" denotes any salt, ester of a compound of this invention, or any other compound which upon administration to a patient is capable of providing (directly or indirectly) a compound of this invention, or a metabolite or residue thereof. The term "pharmaceutically-acceptable salts" embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, example of which are formic, acetic, adipic, butyric, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, 4- hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, ethanedisulfonic, benzenesulfonic, pantothenic, 2- hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, camphoric, camphorsulfonic, digluconic, cyclopentanepropionic, dodecylsulfonic, glucoheptanoic, glycerophosphonic, heptanoic, hexanoic, 2- hydroxy-ethanesulfonic, nicotinic, 2-naphthalenesulfonic, oxalic, palmoic, pectinic, persulfuric, 2-phenylpropionic, picric, pivalic propionic, succinic, tartaric, thiocyanic, mesylic, undecanoic, stearic, algenic, [beta]-hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable pharmaceutically-acceptable base addition salts include metallic salts, such as salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or salts made from organic bases including primary, secondary and tertiary amines, substituted amines including cyclic amines, such as caffeine, arginine, diethylamine, N- ethyl piperidine, aistidine, glucamine, isopropylamine, lysine, morpholine, N- ethyl morpholine, piperazine, piperidine, triethylamine, trimethylamine. All of these salts may be prepared by conventional means from the corresponding compound of the invention by reacting, for example, the appropriate acid or base with the compound of the invention. When a basic group and an acid group are present in the same molecule, a compound of the invention may also form internal salts. Kits containing compositions

The invention is also directed to a method of administration of the combination. More particularly the active agents of the combination therapy are administered sequentially in either order or simultaneously. When the active agents are administered simultaneously, one skilled in the art will understand that the second agent can be administered some time after the first agent. The particular period of delay between the administration of the individual compounds in the combination can extend to days, hours, minutes or seconds. The invention also relates to a kit, wherein the individual compounds of the combination (alitretinoin and pranlukast) are disposed in separate containers.

The invention also relates to a kit, wherein the individual compounds of the combination (alitretinoin and mefloquine) are disposed in separate containers. The invention also relates to a kit where, additionally to the compounds of the combination, other devices such as injection devices, and other materials such as pharmaceutical carriers, are included. The invention also relates to a kit according to any of the foregoing, further comprising integrally thereto or as one or more separate documents, information pertaining to the contents or the kit and the use of the inhibitors.

As used in relation to the invention, the term "treating" or "treatment" and the like should be taken broadly. They should not be taken to imply that an animal is treated to total recovery. Accordingly, these terms include amelioration of the symptoms or severity of a particular condition or preventing or otherwise reducing the risk of further development of a particular condition. The term "comprising" is meant to be open ended, including the indicated component but not excluding other elements.

The phrase "therapeutically-effective" is intended to qualify the amount of each agent, which will achieve the goal of improvement in disorder severity and the frequency of incidence.

It should be appreciated that methods of the invention may be applicable to various species of subjects, preferably mammals, more preferably humans. Where the plural form is used for compounds, salts, and the like, this is taken to mean also a single compound, salt and the like.

Methods of treatment The invention thus provides a method for treating nervous system diseases, particularly motor neuron disease, spinal cord diseases, and diseases associated with glutamate excitotoxicity such as amyotrophic lateral sclerosis, spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), childhood spinal muscular atrophies, progressive spinomuscular atrophy, infantile muscular atrophy, primary lateral sclerosis (PLS), spinal cord injury, poliomyelitis, postpoliomyelitis syndrome, Charcot-Marie-Tooth disease, diffuse atrophic paralysis, pseudobulbar palsy, bulbar palsy, juvenile unilateral upper- limb muscular atrophy, progressive bulbar palsy, spinal progressive muscular atrophy, arthrogryposis multiplex congenita (AMC), Brown-Vialetto-Van Laere syndrome, Fazio-Londe disease, spinal cord compression, central cord syndrome, spinal cord ischemia, Machado-Joseph disease, frontotemporal dementia , frontotemporal dementia with amyotrophic lateral sclerosis, stroke, mitochondrial encephalomyopathies, spinocerebellar degeneration syndromes, traumatic brain injury, epilepsy, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, alcohol withdrawal, peripheral nerve injuries and motoneuron degeneration caused by infarction, exposure to a toxin, malignancy or autoimmune disease in a mammal in need thereof, particularly in a human patient, that includes the step of administering to the mammal, particularly to the human mammal, a therapeutically effective amount, particularly a synergistically effective amount of a combination comprising alitretinoin plus pranlukast or of a combination comprising alitretinoin plus mefloquine either in the form of a pharmaceutical composition comprising said combination or in the form of a kit wherein the individual compounds of each combination are disposed in separate containers.

In a particular embodiment the disease treated is a motor neuron disease, preferably amyotrophic lateral sclerosis.

In another embodiment, the present combinations may also be used with other types of therapies for treating motor neuron disease, spinal cord diseases, and diseases associated with glutamate excitotoxicity.

In another embodiment, the present combinations may also be used or administered in combination with other drugs for the treatment of motor neuron disease, spinal cord diseases, and diseases associated with glutamate excitotoxicity.

As will be appreciated, the dose of a combination of the present invention to be administered, the period of administration, and the general administration regime may differ between subjects depending on such variables as the severity of symptoms, the type of ocular disorder to be treated, the mode of administration chosen, type of composition, size of a unit dosage, kind of excipients, the age and/or general health of a subject, and other factors well known to those of ordinary skill in the art.

Administration may include a single daily dose or administration of a number of discrete divided doses as may be appropriate. An administration regime may also include administration of one or more of the active agents, or compositions comprising same, as described herein. The period of administration may be variable. It may occur for as long a period is desired.

Administration may include simultaneous administration of suitable agents or compositions or sequential administration of agents or compositions.

In a further embodiment, the compositions and methods described herein may be used prophylactically as a means to prevent the development and/or onset of nervous system disorders. The compounds and methods described herein can be used also as motor neuron protective and/or preventive treatments.

The following examples further illustrate specific embodiments of the invention; however, the following illustrative examples should not be interpreted in any way to limit the extent of the invention.

EXAMPLES EXAMPLE 1 Experimental model

The efficacy of selected drug combinations (alitretinoin plus pranlukast or alitretinoin plus mefloquine) on amyotrophic lateral sclerosis was studied in organotypic spinal cord cultures because they retain many organizational features of the host tissue, such as neuronal connectivity, relatively well- preserved cellular architecture and glial-neuronal interactions (see Guzman- Lenis M.S. et al. Neuroscience, 155:104-13 (2008); Guzman-Lenis M.S. et al. Restor Neurol Neurosci, 27:335-49 (2009)). Organotypic spinal cord cultures were prepared from lumbar spinal cords of 8-day-old Sprague-Dawley rat pups, as previously described (see Guzman-Lenis M.S. et al. Neuroscience, 155:104- 13 (2008)). Cultures were incubated at 37 °C in a 5% CO2/05% air humidified environment. Cultures were let to stabilize for 1 week, and thereafter the medium was changed twice per week. All experiments started at least 7 days after the explants settled down. A well-established model to study the neuroprotective effect of drugs in the field of amyotrophic lateral sclerosis relies on inducing chronic excitotoxicity in spinal cord cultures to cause progressive motoneuron death (see Guzman-Lenis M.S. et al. Restor Neurol Neurosci, 27:335-349 (2009); Lee S. et al. Korean J Physiol Pharmacol, 16:43-8 (2012)). Chronic glutamate excitotoxicity was induced by addition of 100μΜ THA (threohydroxyaspartate), a potent glutamate transporter inhibitor, which is known to produce a dose-dependent sustained elevation of glutamate levels causing motoneuron degeneration (see Rothstein, J.D. et al. Proc Natl Acad Sci U S A, 90:6591 -5 (1993)). Treatment The neuroprotective effect of drug combinations (alitretinoin plus pranlukast or alitretinoin plus mefloquine) was assessed by their concomitant addition in the culture with THA. Five experimental groups were defined, out of which one was treated with drug combination alitretinoin plus pranlukast, another was treated with drug combination alitretinoin plus mefloquine, another was treated with riluzole, another with THA + vehicle and the latter with vehicle only (control). CHCI3 and DMSO were used as vehicles in variable concentrations. Each experiment was performed with five replicas. Riluzole is the only approved treatment for amyotrophic lateral sclerosis, which prolongs survival by about two or three months (see Miller R.G. et al. Cochrane Database Syst Rev, CD001447 (2007)), so it was used as reference in the study. Drugs, including riluzole, were administered two weeks after THA addition and during two additional weeks as follows: 1 μΜ alitretinoin plus 1 μΜ pranlukast, 1 μΜ alitretinoin plus 1 μΜ mefloquine or 5μΜ riluzole.

Experimental results

The intensity of microglial activation is correlated with severity of motor neuron damage in human amyotrophic lateral sclerosis. In response to several types of damage stimulus, microglia changes their morphology from ramified to amoeboid form, migrate to the damaged cells, and subsequently clear the debris of the dead cells. Through such processes, microglia release reactive oxygen species, proinflammatory cytokines, complement factors, and neurotoxic molecules, leading to further neuronal dysfunction and death (see Lasiene J. and Yamanaka K. Neurol Res Int, 201 1 :718987 (201 1 )).

The abovementioned parameters were measured in order to evaluate the efficacy of tested combinations against ALS. Statistical significance was determined by one-way ANOVA followed by Dunnett's post-hoc test, with p<0.05 considered as the significance threshold. To study the effect of the combinations on the inflammatory process triggered by THA, we quantified the amount of the pro-inflammatory cytokine TNF-a released to the medium by ELISA assay (Invitrogen, Carlsbad, CA, USA). The combinations composed by alitretinoin plus pranlukast and alitretionin plus mefloquine reduce TNF-a level as riluzole (Figure 1 ).

Moreover, changes in pro-inflammatory cytokine release matched well with changes in microglia morphology related to its reactivity state, which was qualitatively assessed by visualization after anti-lbal immunostaining (Figure 2). Anti-lba1 selectively binds to microglia cells, since Iba1 is specifically expressed in them (see Ito D. et al. Brain Res Mol Brain Res, 57(1 ): 1 -9 (1998)). The combinations composed by alitretinoin plus pranlukast and alitretionin plus mefloquine presented a phenotype more ramified than the ones treated with THA alone, and more similar to the phenotype of the controls.

To determine the neuroprotective effect in motoneurons for each drug combination administered simultaneously to a chronic excitotoxicity insult, we counted the number of surviving motoneurons 4 weeks post-insult, using SMI- 32 immunostaining. SMI-32 antibody selectively labels motoneurons, so positive staining is correlated with better preservation of motoneurons (see Crow JP. et al. Ann Neurol, 58(2):258-265 (2005)). The combinations composed by alitretinoin plus pranlukast and alitretionin plus mefloquine presented a significant increase in the number of motoneurons preserved respect to the THA-alone slides (Figures 3 and 4).

Conclusions

Thus, drug-drug combinations of the invention show an unexpected neuroprotective effect for motoneurons in the THA-based in-vitro model of amyotrophic lateral sclerosis by reducing the effects of THA chronic excitotoxic insult on both microglia and motoneurons.

Claims

1 . A combination comprising alitretinoin or its pharmaceutically acceptable salts and a second component selected from pranlukast and mefloquine or a pharmaceutically acceptable salt thereof.

2. The combination according to claim 1 wherein the second component is pranlukast or a pharmaceutically salt thereof.

3. The combination according to claim 1 wherein the second component is mefloquine or a pharmaceutically salt thereof.

4. Pharmaceutical composition comprising a combination as defined in any of claims 1 to 3.

5. A combination as defined in any of claims 1 to 3 or a pharmaceutical composition as defined in claim 4 for use in the treatment of a nervous system disorder selected from the group consisting of a motor neuron disease, spinal cord disease and/or a nervous system disease associated with glutamate excitotoxicity.

6. A combination or a pharmaceutical composition for use according to claim 5 wherein the nervous system disorder is selected from the group consisting of amyotrophic lateral sclerosis, spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), childhood spinal muscular atrophies, progressive spinomuscular atrophy, infantile muscular atrophy, primary lateral sclerosis (PLS), spinal cord injury, poliomyelitis, postpoliomyelitis syndrome, Charcot-Marie-Tooth disease, diffuse atrophic paralysis, pseudobulbar palsy, bulbar palsy, juvenile unilateral upper-limb muscular atrophy, progressive bulbar palsy, spinal progressive muscular atrophy, arthrogryposis multiplex congenita (AMC), Brown-Vialetto-Van Laere syndrome, Fazio-Londe disease, spinal cord compression, central cord syndrome, spinal cord ischemia, Machado-Joseph disease, frontotemporal dementia, frontotemporal dementia with amyotrophic lateral sclerosis, stroke, mitochondrial encephalomyopathies, spinocerebellar degeneration syndromes, traumatic brain injury, epilepsy, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, alcohol withdrawal, peripheral nerve injuries and motoneuron degeneration caused by infarction, exposure to a toxin, malignancy or autoimmune disease.

7. A combination or a pharmaceutical composition for use according to claim 6 wherein the nervous system disorder is selected from the group consisting of spinal cord injury, stroke, traumatic brain injury, epilepsy, multiple sclerosis, Alzheimer disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, alcohol withdrawal, mitochondrial encephalomyopathies, spinocerebellar degeneration syndromes, spinal muscular atrophy (SMA), childhood spinal muscular atrophies, spinal and bulbar muscular atrophy (SBMA), poliomyelitis, postpoliomyelitis syndrome, Charcot- Marie-Tooth disease, spinal cord compression, spinal cord ischemia, frontotemporal dementia and frontotemporal dementia with amyotrophic lateral sclerosis.

8. A combination or a pharmaceutical composition for use according to claim 7 wherein the nervous system disorder is amyotrophic lateral sclerosis.

9. Use of a combination as defined in any of claims 1 to 3 or of a pharmaceutical composition as defined in claim 4 for the manufacture of a medicament for the treatment of a nervous system disorder selected from the group consisting of a motor neuron disease, spinal cord disease and/or a nervous system disease associated with glutamate excitotoxicity.

10. Use according to claim 9 wherein the nervous system disorder is selected from the group consisting of amyotrophic lateral sclerosis, spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), childhood spinal muscular atrophies, progressive spinomuscular atrophy, infantile muscular atrophy, primary lateral sclerosis (PLS), spinal cord injury, poliomyelitis, postpoliomyelitis syndrome, Charcot-Marie-Tooth disease, diffuse atrophic paralysis, pseudobulbar palsy, bulbar palsy, juvenile unilateral upper- limb muscular atrophy, progressive bulbar palsy, spinal progressive muscular atrophy, arthrogryposis multiplex congenita (AMC), Brown-Vialetto-Van Laere syndrome, Fazio-Londe disease, spinal cord compression, central cord syndrome, spinal cord ischemia, Machado-Joseph disease, frontotemporal dementia, frontotemporal dementia with amyotrophic lateral sclerosis, stroke, mitochondrial encephalomyopathies, spinocerebellar degeneration syndromes, traumatic brain injury, epilepsy, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, alcohol withdrawal, peripheral nerve injuries and motoneuron degeneration caused by infarction, exposure to a toxin, malignancy or autoimmune disease.

1 1 . Use according to claim 10 wherein the nervous system disorder is selected from the group consisting of spinal cord injury, stroke, traumatic brain injury, epilepsy, multiple sclerosis, Alzheimer disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, alcohol withdrawal, mitochondrial encephalomyopathies, spinocerebellar degeneration syndromes, spinal muscular atrophy (SMA), childhood spinal muscular atrophies, spinal and bulbar muscular atrophy (SBMA), poliomyelitis, postpoliomyelitis syndrome, Charcot-Marie-Tooth disease, spinal cord compression, spinal cord ischemia, frontotemporal dementia and frontotemporal dementia with amyotrophic lateral sclerosis.

12. Use according to claim 1 1 wherein the nervous system disorder is amyotrophic lateral sclerosis.

13. Method of treating a subject suffering a nervous system disorder selected from the group consisting of a motor neuron disease, spinal cord disease and/or a nervous system disease associated with glutamate excitotoxicity comprising the administration to said subject of a therapeutically effective amount of a combination as defined in any of claims 1 to 3 or of a pharmaceutical composition as defined in claim 4.

14. Method according to claim 13 wherein the nervous system disorder is selected from the group consisting of amyotrophic lateral sclerosis, spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), childhood spinal muscular atrophies, progressive spinomuscular atrophy, infantile muscular atrophy, primary lateral sclerosis (PLS), spinal cord injury, poliomyelitis, postpoliomyelitis syndrome, Charcot-Marie-Tooth disease, diffuse atrophic paralysis, pseudobulbar palsy, bulbar palsy, juvenile unilateral upper- limb muscular atrophy, progressive bulbar palsy, spinal progressive muscular atrophy, arthrogryposis multiplex congenita (AMC), Brown-Vialetto-Van Laere syndrome, Fazio-Londe disease, spinal cord compression, central cord syndrome, spinal cord ischemia, Machado-Joseph disease, frontotemporal dementia , frontotemporal dementia with amyotrophic lateral sclerosis, stroke, mitochondrial encephalomyopathies, spinocerebellar degeneration syndromes, traumatic brain injury, epilepsy, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, alcohol withdrawal, peripheral nerve injuries and motoneuron degeneration caused by infarction, exposure to a toxin, malignancy or autoimmune disease.

15. Method according to claim 14 wherein the nervous system disorder is selected from the group consisting of spinal cord injury, stroke, traumatic brain injury, epilepsy, multiple sclerosis, Alzheimer disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, alcohol withdrawal, mitochondrial encephalomyopathies, spinocerebellar degeneration syndromes, spinal muscular atrophy (SMA), childhood spinal muscular atrophies, spinal and bulbar muscular atrophy (SBMA), poliomyelitis, postpoliomyelitis syndrome, Charcot-Marie-Tooth disease, spinal cord compression, spinal cord ischemia, frontotemporal dementia and frontotemporal dementia with amyotrophic lateral sclerosis.

16. Method according to claim 15 wherein the nervous system disorder is amyotrophic lateral sclerosis.