WO2009022345A1 - Phenyl carbamates for the treatment of multiple sclerosis - Google Patents

Phenyl carbamates for the treatment of multiple sclerosis Download PDF

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Publication number
WO2009022345A1
WO2009022345A1 PCT/IL2008/001126 IL2008001126W WO2009022345A1 WO 2009022345 A1 WO2009022345 A1 WO 2009022345A1 IL 2008001126 W IL2008001126 W IL 2008001126W WO 2009022345 A1 WO2009022345 A1 WO 2009022345A1
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subject
multiple sclerosis
rivastigmine
compound
pharmaceutically acceptable
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PCT/IL2008/001126
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French (fr)
Inventor
Marta Weinstock-Rosin
Talma Brenner
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Yissum Research Development Company Of The Hebrew University Of Jerusalem
Hadasit Medical Research Services And Development Ltd.
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Publication of WO2009022345A1 publication Critical patent/WO2009022345A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/21Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/27Esters, e.g. nitroglycerine, selenocyanates of carbamic or thiocarbamic acids, meprobamate, carbachol, neostigmine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the invention relates to the use of phenyl carbamates, including rivastigmine, for the treatment of multiple sclerosis.
  • MS Multiple sclerosis
  • CNS central nervous system
  • MBP myelin basic protein
  • corticosteroid treatment may be undesirable as these drugs are often poorly tolerated in patients and also inhibit endogenous immunosuppressive mechanisms.
  • Other current treatments of MS include beta interferons, mitoxantrone, natalizumab and glatiramer acetate (GA, Copaxone).
  • G glatiramer acetate
  • Such therapies are typically accompanied by adverse effects, including flu-like symptoms, depression, cytopenias, skin reactions and local pain (as consequences of the subcutaneous administration).
  • Current therapies used for symptomatic treatment of multiple sclerosis are discussed in Henze et al., 2006.
  • WO 2006/130726 is directed to methods for the treatment of a form of multiple sclerosis comprising administering an amount of R(+)-6-(N-methyl, N- ethyl-carbamoyl oxy)-N'-propargyl-l-aminoindan or a pharmaceutically acceptable salt thereof.
  • Ri is hydrogen, lower alkyl, cyclohexyl, allyl or benzyl
  • R 2 is hydrogen, methyl, ethyl or propyl, or
  • Ri and R 2 together with the nitrogen to which they are attached form a morpholino or piperidino radical
  • R 3 is hydrogen or lower alkyl
  • Rj and R 5 are the same or different and each is a lower alkyl
  • the dialkylaminoalkyl group is in the meta, ortho or para position.
  • Rivastigmine is a cholinesterase inhibitor (ChEI) classified as an intermediate- acting or pseudo-irreversible agent and is primarily indicated for the treatment of
  • Rivastigmine is (S)-N-ethyl-N-methyl-3-
  • Rivastigmine Tartrate is represented in Formula (II) below:
  • rivastigmine is a well known drug for preventing cognitive decline and loss of cholinergic function in certain types of dementia.
  • Controlled release oral compositions comprising rivastigmine are described in US 6,565,883.
  • US 6,335,031 discloses formulations of rivastigmine containing an antioxidant suitable for transdermal delivery.
  • EAE allergic encephalomyelitis
  • Parry et al. (2003) reported that administration of rivastigmine to MS patients 150 minutes prior to performing a cognitive task was associated with a relative normalization of the abnormal task-associated brain activation.
  • D'Intino et al. (2005) examined cognitive dysfunction in EAE rats that had recovered their motor function.
  • Daily oral administration of rivastigmine was found to restore cognitive performance, choline acetyltransferase activity in the basal forebrain but not in the cerebral cortex and hippocampus, and nerve growth factor mRNA expression in the cerebral cortex.
  • An anti-inflammatory effect of rivastigmine or an effect of the drug on the motor functions of EAE rats were neither taught nor suggested in these studies.
  • U.S. Patent Application Publication No. 2003/0225031 is directed to pharmaceutical compositions for nasal administration comprising ChE inhibitors, including, inter alia, rivastigmine, and methods of using same for the treatment of Alzheimer's disease and other neurological conditions associated with cognitive impairment in a mammal. According to the '031 application, these conditions include, among others, cognitive disorder associated with MS.
  • cholinergic up-regulation may result in anti-inflammatory effects. It was shown that immune system cells possess various subtypes of muscarinic cholinergic receptors and nicotinic receptors. In addition, lymphocytes synthesize acetylcholine (ACh), which is degraded by acetylcholinesterase (AChE)
  • nicotinic acetylcholine receptor ⁇ 7 subunit is required for inhibition of macrophage TNF release by ACh or nicotine.
  • Saeed et al. (2004) reported that human microvascular endothelial cells express ⁇ 7 nicotinic acetylcholine receptor ( ⁇ 7 nAChR), and that nicotine, a cholinergic agonist thereof, and vagus nerve stimulation inhibit endothelial cell activation, thereby inhibiting leukocyte recruitment in the carrageenan air pouch model of local inflammation.
  • Nizri et al. disclose bi-functional compounds consisting of the non-steroidal anti-inflammatory drug ibuprofen and pyridostigmine, a cholinesterase inhibitor that acts as a cholinergic up-regulator. These bi-functional compounds exhibited improved efficiency in an EAE mouse model compared to ibuprofen alone. Although treatment of mice by pyridostigmine alone resulted in reduced lymphocyte proliferation, such treatment did not change disease severity. Another such bi-functional compound, namely IBU- Octyl-Cytisine, containing ibuprofen and Cytisine as the nicotinic agonist, has been described by Nizri et al. (2007a), which further report that each moiety separately failed to reproduce the effect of this compound.
  • the disclosure further reports that administration of an antisense oligodeoxynucleotide targeted to AChE mRNA, reduced the clinical severity and CNS inflammation intensity in an EAE model. It has been suggested that inhibition of antibody-forming cell response by ChE inhibitors requires their presence in the CNS, based on experiments performed with physostigmine, physostigmine bromide and edrophonium (Langley et al., 2004).
  • U.S. Patent Application Publication No. 2005/0222123 is directed to a method of treating a subject with a cytokine-mediated inflammatory disorder comprising administering to the subject a cholinesterase inhibitor other than galantamine.
  • the '123 application lists a wide range of structurally-unrelated compounds, including nicotinic and muscarinic AChE inhibitors and butyrylcholinesterase inhibitors, as potential inhibitors for treating a variety of disorders.
  • the specification of '123 exemplifies the use of galantamine, tacrine, huperzine, neostigmine and physostigmine in acute inflammatory models of sepsis and endotoxemia in mice.
  • the present invention provides pharmaceutical compositions comprising phenyl carbamates including rivastigmine for treating multiple sclerosis and inhibiting symptoms associated therewith.
  • the invention is based, in part, on the unexpected discovery that rivastigmine can reduce the clinical symptoms associated with inflammation in experimental autoimmune encephalitis (EAE), a well-known model system of multiple sclerosis (MS).
  • EAE experimental autoimmune encephalitis
  • MS model system of multiple sclerosis
  • Rivastigmine was surprisingly found to reduce loss of motor functions when administered chronically to mice upon induction of EAE.
  • rivastigmine was in fact able to reduce demyelination and microglia activation and to preserve axons in treated mice.
  • Rivastigmine also decreased reactivity of encephalitogenic T-cells and production of pro-inflammatory cytokines (TNF- ⁇ , IFN- ⁇ and IL-17).
  • the present invention provides compositions comprising rivastigmine and methods thereof for inhibiting the clinical symptoms associated with inflammation in a subject afflicted with MS, for improving motor function and for inhibiting neurodegeneration and disease progression in MS patients.
  • the present invention is directed to treatment of a subject not otherwise in need of treatment with rivastigmine or other phenyl carbamates.
  • a method of treating multiple sclerosis in a subject in need thereof comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I): O
  • Ri is hydrogen, lower alkyl, cyclohexyl, allyl or benzyl
  • R 2 is hydrogen, methyl, ethyl or propyl, or
  • Ri and R 2 together with the nitrogen to which they are attached form a morpholino or piperidino radical
  • R 3 is hydrogen or lower alkyl
  • R 4 and R 5 are the same or different and each is a lower alkyl, and the dialkylaminoalkyl group is in the meta, ortho or para position,
  • the active ingredient is N-ethyl-N-methyl- 3-[l-(dimethylamino)ethyl]-phenyl carbamate or a pharmaceutically acceptable salt, hydrate or solvate thereof.
  • the active ingredient is (S)-N- ethyl-N-methyl-3-[l-(dimethylamino)ethyl]-phenyl carbamate (rivastigmine) or a pharmaceutically acceptable salt, hydrate or solvate thereof.
  • the active ingredient is rivastigmine tartrate.
  • the invention provides a method of improving motor function in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides a method of reducing demyelination in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides a method of reducing neurodegeneration associated with demyelination in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • a method of inhibiting inflammation in a subject afflicted with multiple sclerosis comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides a method of inhibiting a clinical symptom associated with inflammation in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides a method of inhibiting the progression of multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • a method of treating a multiple sclerosis-related disease selected from the group consisting of: Neuromyelitis Optica (Devic's Disease), Diffuse Sclerosis, Transitional Sclerosis, Acute Disseminated Encephalomyelitis, and Optic Neuritis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the multiple sclerosis-related disease is a virus-, bacteria- or parasite- related demyelinating brain disease.
  • the compound utilized in the methods of the invention is rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof, preferably rivastigmine tartrate.
  • the subject is human. In other embodiments, the subject is a non-human mammal.
  • the compounds of the invention are useful for decreasing the frequency or duration of acute exacerbations (or relapses) of the disease in a subject afflicted with MS.
  • the compounds are administered to a subject that is in remission of the disease, to prevent, delay or reduce recurrence of exacerbations.
  • the subject is afflicted with relapsing- remitting MS.
  • the active ingredient may be administered enterally, parenterally or topically.
  • the active ingredient is administered in a manner selected from the group consisting of: orally, subcutaneously and intramuscularly.
  • the active ingredient is formulated in the form of extended release, sustained release or controlled-release formulations.
  • oral sustained release and controlled release formulations are contemplated.
  • the active ingredient is formulated in the form of a transdermal formulation, e.g. as a transdermal delivery device.
  • SI Stimulation Index
  • Figure 8 Amelioration of EAE by treatment with rivastigmine.
  • Figure 8A daily treatment with rivastigmine (0.75 mg/kg, s.c.) ameliorated clinical score of EAE. This effect was reversed in animals treated with the nicotinic antagonist mecamylamine (10 mg/kg, s.c).
  • Figure 8B treatment of EAE induced animals with rivastigmine-loaded mini- osmotic pumps (4.8 mg/kg/day, s.c.) also ameliorated clinical score, even to a larger extent.
  • FIG. 9 Rivastigmine treatment reduced neuropathological parameters of inflammation, demyelination and neurodegeneration. Histological analysis from control- EAE spinal cord tissue taken at peak of disease showed marked inflammatory infiltration and demyelination (A), microglia activation (B) and axonal damage (C). Rivastigmine treatment ameliorated all these parameters (D, E, F). Figure 10. Inhibitory effect of rivastigmine on lymphocyte reactivity ex vivo. Figure 1OA, Treatment with rivastigmine (0.75 mg/kg, s.c.) reduced T-cell proliferation toward the encephalitogenic MOG peptide.
  • Figure 1OB Rivastigmine treatment reduced pro-inflammatory (TNF- ⁇ and IFN- ⁇ ) cytokine production in response to MOG (24 hrs) and also down-regulated IL- 17 mRNA expression (48 hrs, Figure 10C), as assayed by real-time PCR.
  • Figure 1OD IL-10 levels, an anti-inflammatory cytokine, were unaltered (96 hrs).
  • Figure 11 Effects of rivastigmine are dependent on the ⁇ 7 nAChR nicotinic receptor.
  • Figure HA inhibition of T-cell proliferation exerted by rivastigmine (l ⁇ M) is reversed when cells are incubated with ⁇ -bungarotoxin, a nicotinic muscle and neuronal- type blocker.
  • Figure HB T-cells were incubated with anti-sense to the ⁇ 5 nAChR or to ⁇ 7 nAChR (AS- ⁇ 5, AS- ⁇ 7, respectively) for 24 hrs, and then levels of ⁇ 7 nAChR mRNA were determined in the indicated treatments.
  • Rivastigmine reversed the decline in spatial learning performance seen in EAE animals in the Morris water maze. Performance of naive animals in first trial was compared to EAE-induced animals and to animals induced for EAE and treated with Rivastigmine (4.8 mg/kg, mini-pumps).
  • FIG. 14 Antigen presentation ability is affected by rivastigmine treatment. Splenocytes from untreated and treated mice were irradiated and incubated with MOG- specific T-cells in the presence of MOG. Proliferation of T-cells in response to antigen was normalized to the untreated group.
  • FIG. 15 Treatment with rivastigmine suppressed T-cell reactivity during various stages of EAE course.
  • Figure 15 A T-cell proliferation towards the encephalitogenic peptide (MOG 3S-55 ) was assessed at the indicated time points after disease induction.
  • Figure 15B-D Production of pro-inflammatory cytokines was suppressed by rivastigmine at the same time points ( Figure 15B - TNF- ⁇ ; Figure 15C - IFN- ⁇ ; Figure 15D - IL-17).
  • the treatment protocol was similar to that described for assessment of clinical score and reactivity of T-cells on day 9 post immunization.
  • EAE is associated with inflammatory infiltrates in lepto-meninges near the hippocampus.
  • the present invention is directed to the use of rivastigmine, related phenyl carbamates and salts thereof for preparing a medicament for the treatment of multiple sclerosis (MS) and related diseases.
  • the present invention provides compositions comprising these compounds and methods thereof for treating MS and inhibiting clinical symptoms associated therewith.
  • compositions comprising a compound of the general Formula (I):
  • Ri is hydrogen, lower alkyl, cyclohexyl, allyl or benzyl
  • R 2 is hydrogen, methyl, ethyl or propyl, or
  • Ri and R 2 together with the nitrogen to which they are attached form a morpholino or piperidino radical, R 3 is hydrogen or lower alkyl,
  • R 4 and R 5 are the same or different and each is a lower alkyl, and the dialkylaminoalkyl group is in the meta, ortho or para position.
  • the compound is N-ethyl-N-methyl-3-[l- (dimethylamino)ethyl] -phenyl carbamate or a pharmaceutically acceptable salt, hydrate or solvate thereof.
  • the compound is (S)-N-ethyl-N-methyl- 3-[l-(dimethylamino)ethyl]-phenyl carbamate (rivastigmine) or a pharmaceutically acceptable salt, hydrate or solvate thereof.
  • the compound is rivastigmine tartrate.
  • MS Multiple sclerosis
  • CNS central nervous system
  • MS is characterized by chronic inflammation, demyelination and gliosis.
  • Pathologically, MS is characterized by well-demarcated, macroscopic lesions, called plaques, in the brain white matter and, less frequently, gray matter.
  • Acute lesions are characterized by perivenular cuffing and infiltration of T lymphocytes and macrophages, along with a few B cells and plasma cells.
  • MS is reportedly an autoimmune disorder, likely triggered by environmental exposure in a genetically susceptible host.
  • a pharmaceutical composition comprising a compound of the general Formula (I) described above can be administered to patients having multiple sclerosis, including e.g. multiple sclerosis variants such as Neuromyelitis Optica (Devic's Disease), Diffuse Sclerosis, Transitional Sclerosis, Acute Disseminated Encephalomyelitis, and Optic Neuritis.
  • the patients are afflicted with a MS-related disorder selected from the group consisting of virus-, bacteria- or parasite-related demyelinating degenerative brain disease.
  • a MS-related disorder selected from the group consisting of virus-, bacteria- or parasite-related demyelinating degenerative brain disease.
  • Symptoms of MS which are prevented or ameliorated or treated may include: weakness and/or numbness in one or more limbs; tingling of the extremities and tight band-like sensations around the trunk or limbs; dragging or poor control of one or both legs to spastic or ataxic paraparesis; hyperactive tendon reflexes; disappearance of abdominal reflexes; Lhermitte's sign; retrobulbar or optic neuritis; unsteadiness in walking; increased muscle fatiguability; brain stem symptoms (diplopia, vertigo, vomiting); disorders of micturition; hemiplegia; trigeminal neuralgia; other pain syndromes; nystagmus and ataxia; cerebellar-type ataxia; Charcot's triad; diplopia; bilateral internuclear ophthalmoplegia; myokymia or paralysis of facial muscles; deafness; tinnitus; unformed auditory hallucinations (because of involvement cochlear connections
  • cognitive impairment as used herein relates to a disorder in memory, problem solving, abstract reasoning and orientation that weakens an individual's ability to maintain an independent lifestyle. A hallmark is memory impairment with resulting confusion in the conduct of daily affairs.
  • the invention is directed to the treatment of a subject afflicted with relapsing-remitting MS.
  • EAE Experimental autoimmune encephalomyelitis
  • CNS inflammation with macrophage and lymphocytic infiltrates and varying degrees of demyelination.
  • the disease manifests clinically with paralysis, beginning at the tail and spreading rostrally to the hindlimbs and forelimbs, and in advanced stages affects breathing and causes death.
  • a method of treating multiple sclerosis in a subject in need thereof comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • treating refers to administering a therapy in amount, manner, and/or mode effective to improve a condition, symptom, or parameter associated with a disorder or to prevent progression of a disorder, to either a statistically significant degree or to a degree detectable to one skilled in the art.
  • An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject.
  • the term “treating” includes reducing, inhibiting, alleviating and/or ameliorating the non-cognitive clinical manifestations of MS.
  • “treating” may include blocking or reducing the physiological and pathogenic deterioration associated with MS.
  • the treatment may reduce or inhibit the inflammatory response in the brain and other regions of the nervous system, breakdown or disruption of the blood-brain barrier, appearance of lesions in the brain, tissue destruction, demyelination, autoimmune inflammatory response, chronic inflammatory response, neuronal death, and/or neuroglia death.
  • the term further includes inhibition, alleviation and/or amelioration of one or more of the clinical symptoms associated with MS that were listed above, an increase in frequency and duration of MS symptom-free periods, and improvement or inhibition of deterioration in motor performance.
  • Treatment of cognitive impairment associated with MS is explicitly excluded from the scope of the present invention.
  • a method of treating multiple sclerosis in a subject in need thereof comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • a method of inhibiting inflammation in a subject afflicted with multiple sclerosis comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • a method of inhibiting inflammation in a subject afflicted with multiple sclerosis comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides a method of improving motor function in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides a method of improving motor function in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides a method of reducing demyelination in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides a method of reducing demyelination in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides methods of inhibiting neurodegeneration and axonal damage associated with demyelination in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides methods of inhibiting neurodegeneration and axonal damage associated with demyelination in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides a method of inhibiting a clinical symptom associated with inflammation in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides a method of inhibiting a clinical symptom associated with inflammation in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • inhibiting a clinical symptom it is meant reduction in severity and/or frequency of one or more MS associated symptoms, elimination of symptom(s) and prevention of the occurrence of symptom(s).
  • the invention provides a method of inhibiting the progression of multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the invention provides a method of inhibiting the progression of multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • the compounds of Formula (I) as defined herein are known as being useful for treating a subject suffering from senile dementia, Alzheimer's disease, Huntingdon's chorea, tardive dyskinesias, hyperkinesia, mania, acute confusion disorders, Friedrich's ataxia or Down's syndrome.
  • the treatment of a subject afflicted with any of these diseases is excluded from the scope of the present invention.
  • the MS-afflicted subject treated by the methods of the present invention should
  • the compounds of the invention are useful for decreasing the frequency or duration of acute exacerbations (or relapses) of the disease in a subject afflicted with MS.
  • the subject is in a state of remission.
  • the compounds of Formula (I) as defined herein may be used in the form of the free base or a pharmaceutically acceptable salt thereof.
  • a pharmaceutically acceptable salt thereof preferably the hydrogen tartrate (hta) is used.
  • Other pharmacologically acceptable salts of these compounds include, but are not limited to, the acetate, salicylate, fumarate, phosphate, sulphate, maleate, succinate, citrate, tartrate, propionate and butyrate salts thereof.
  • the compounds of Formula (I) as defined herein or physiologically acceptable salt(s) thereof may be compounded with one or more physiologically acceptable vehicles, carriers, excipients, binders, preservatives, stabilizers, flavors, etc., in a unit dosage form as called for by accepted pharmaceutical practice.
  • the amount of active substance in these compositions or preparations is such that a suitable dosage is obtained.
  • pharmaceutically acceptable or “physiologically acceptable” is meant herein a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
  • “Pharmacologically active” is meant herein a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
  • a pharmaceutically active derivative or metabolite refers to a derivative or metabolite having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
  • pharmaceutically acceptable or “physiologically acceptable” is used to refer to a derivative (e.g., a salt) of an active agent, it is to be understood that the compound is pharmacologically active as well, i.e., therapeutically effective for the treatment of multiple sclerosis.
  • Carriers or “vehicles” as used herein refer to conventional pharmaceutically acceptable carrier materials suitable for drug administration, and include any such materials known in the art that are nontoxic and do not interact with other components of a pharmaceutical composition or drug delivery system in a deleterious manner.
  • a "pharmaceutically acceptable carrier, excipient or diluent” may refer to a single auxiliary material or to various mixtures and combinations of such non-active ingredients.
  • Illustrative of the adjuvants which may be incorporated in tablets, capsules and the like are the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; a flavoring agent such as peppermint, oil of wintergreen or cherry.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • an excipient such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin
  • a flavoring agent such as peppermint
  • tablets may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and a flavoring such as cherry or orange flavor.
  • suitable pharmaceutical adjuvants and techniques for formulation as well as administration of drugs are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
  • rivastigmine tartrate may be formulated as capsules for oral administration, containing as inactive ingredients hydroxypropyl methylcellulose, magnesium stearate, microcrystalline cellulose, and silicon dioxide.
  • Each hard-gelatin capsule may contain, for example, gelatin, titanium dioxide and dyes (e.g. red and/or yellow iron oxides; Exelon ® capsules).
  • rivastigmine tartrate may be formulated as an oral solution (comprising e.g. a rivastigmine tartrate concentration equivalent to 2 mg/mL of rivastigmine base), comprising as inactive ingredients citric acid, a dye (e.g. D&C yellow #10), purified water, sodium benzoate and sodium citrate (Exelon ® oral solution).
  • an oral solution comprising e.g. a rivastigmine tartrate concentration equivalent to 2 mg/mL of rivastigmine base
  • inactive ingredients citric acid, a dye (e.g. D&C yellow #10), purified water, sodium benzoate and sodium citrate (Exelon ® oral solution).
  • Sterile compositions for injection can be formulated according to conventional pharmaceutical practice by dissolving or suspending the active substance in a vehicle such as water for injection. Buffers, preservatives, antioxidants and the like can be incorporated as required.
  • Preferred antioxidants for use with the compounds of the present invention include sodium metabisulphite and ascorbic acid.
  • the pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.
  • the invention is directed to the use of extended release, sustained release or controlled release formulations of the compounds of Formula (I) as defined herein or salts thereof, including, but not limited to those disclosed by U.S. Patent No. 6,565,883.
  • the composition may comprise a core coated with two films, the first inner film being a semi-permeable to water or body fluids film applied directly on said core and comprising cellulose acetate, the second outer film being a permeable to water or body fluids film comprising ethylcellulose.
  • controlled release is intended to refer to any drug-containing formulation in which release of the drug is not immediate, i.e., with a “controlled release” formulation, oral administration does not result in immediate release of the drug into an absorption pool.
  • controlled release is used interchangeably with "nonimmediate release” as defined in Remington: The Science and Practice of Pharmacy, Nineteenth Ed. (Easton, Pa.: Mack Publishing Company, 1995). As discussed therein, immediate and nonimmediate release can be defined.
  • the drug level achieved is the outcome of rate constants for (1) release of the drug from the formulation, (2) absorption, and (3) elimination, respectively.
  • rate constant for drug release is far greater than the absorption rate constant.
  • controlled release formulations the opposite is true, such that the rate of release of drug from the dosage form is the rate-limiting step in the delivery of the drug to the target area.
  • controlled release as used herein is intended to include any nonimmediate release formulation, including but not limited to sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of drug over an extended time period.
  • delayed release is used in its conventional sense to refer to a drug formulation in which there is a time delay provided between oral administration of a drug dosage form and the release of the drug therefrom. "Delayed release” may or may not involve gradual release of drug over an extended period of time, and thus may or may not be “sustained release”.
  • a controlled release formulation may comprise, for example, controlled release beads comprising the drug.
  • a common type of controlled release beads comprises an inert core, such as a sugar sphere, coated with an inner drug-containing layer and an outer membrane layer controlling drug release from the inner layer.
  • an example of such controlled release beads is described in U.S. Pat. No. 5,783,215 where each bead comprises (i) a core unit of a soluble or insoluble inert material, (ii) a first layer on the core unit comprising an active ingredient dispersed in a hydrophilic polymer, (iii) an optional second layer of hydrophilic polymer covering the first layer, and (iv) an outermost membrane layer effective for controlled release of the active ingredient.
  • a "sealcoat” in the form of a small amount (e.g. 1-3%) of a water-soluble polymer, such as hydroxypropylmethyl cellulose (HPMC) or polyvinylpyrrolidone (PVP), between the inert core and the layer containing the active ingredient.
  • HPMC hydroxypropylmethyl cellulose
  • PVP polyvinylpyrrolidone
  • the purpose thereof is generally to isolate the drug from the core surface in the event that a drug-core chemical interaction is possible, and/or to smooth the surface of the inert core such that the surface area is more consistent from lot to lot to thereby improve the coating quality when the drug layer and the controlled release membrane layers are applied.
  • the cores are typically of a water-soluble or swellable material, and may be any such material that is conventionally used as cores or any other pharmaceutically acceptable water-soluble or water-swellable material made into beads or pellets.
  • the beads are spheres of sucrose/starch (Sugar Spheres NF), sucrose crystals, or extruded and dried spheres typically comprised of excipients such as microcrystalline cellulose and lactose.
  • the substantially water-insoluble material in the first, or sealcoat layer is generally a "GI insoluble” or "GI partially insoluble” film forming polymer (latex or dissolved in a solvent).
  • GI insoluble or "GI partially insoluble” film forming polymer (latex or dissolved in a solvent).
  • ethyl cellulose, cellulose acetate, cellulose acetate butyrate polymethacrylates such as ethyl acrylate/methyl methacrylate copolymer (Eudragit NE-30-D) and ammonio methacrylate copolymer types A and B (Eudragit RL30D and RS30D), and silicone elastomers.
  • a plasticizer is used together with the polymer.
  • plasticizers include: dibutylsebacate, propylene glycol, triethylcitrate, tributylcitrate, castor oil, acetylated monoglycerides, acetyl triethylcitrate, acetyl butylcitrate, diethyl phthalate, dibutyl phthalate, triacetin, fractionated coconut oil (medium-chain triglycerides).
  • the second layer containing the active ingredient may be comprised of the active ingredient (drug) with or without a polymer as a binder.
  • the binder when used, is usually hydrophilic but may be water-soluble or water-insoluble.
  • Exemplary polymers to be used in the second layer containing the active drug are hydrophilic polymers such as polyvinylpyrrolidone (PVP), polyalkylene glycol such as polyethylene glycol, gelatine, polyvinyl alcohol, starch and derivatives thereof, cellulose derivatives, such as hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, carboxyethyl cellulose, carboxymethylhydroxyethyl cellulose, acrylic acid polymers, polymethacrylates, or any other pharmaceutically acceptable polymer.
  • PVP polyvinylpyrrolidone
  • HPMC hydroxypropylmethyl cellulose
  • HPMC hydroxyprop
  • transdermal pharmaceutical compositions comprising the compounds of Formula (I) as defined herein may be used in accordance with the methods of the present invention. These formulations may optionally be administered using a transdermal device, e.g. a transdermal patch.
  • a transdermal device e.g. a transdermal patch.
  • Exemplary transdermal formulations comprising inter alia rivastigmine and transdermal devices thereof are disclosed in U.S. Patent No. 6,335,031.
  • the compounds according to the invention may be administered by any conventional route, in particular enterally, preferably orally, e.g. in the form of tablets or capsules, or parenterally, e.g. in the form of injectable solutions or suspensions (e.g. for subcutaneous or intramuscular administration).
  • the exact amounts of the compound of Formula (I) as defined herein to be administered may depend on a number of factors, e.g. the drug release characteristics of the compositions, the drug penetration rate observed in vitro and in vivo tests, the duration of action required, the form of the compound of Formula (I), and for transdermal compositions the size of the skin contact area, and the part of the body to which the unit is fixed.
  • the amount of and, e.g. area of the transdermal composition etc. may be determined by routine bioavailability tests comparing the blood levels of active agents after administration of the compound of Formula (I) as defined herein in a composition according to the invention to intact skin and blood levels of the compound of Formula (I) observed after oral administration of a therapeutically effective dose of the compound.
  • rivastigmine is well tolerated in humans at an initial dose of 1.5 mg twice a day orally and the dose may be stepped up to 3 mg twice daily in week 2. Higher dosages are possible, for example 4.5 mg twice daily and even 6 mg twice daily. Tolerability was reported to be even better for a transdermal device, wherein, for example, 24 mg may be absorbed in 24 hours.
  • oral formulations of rivastigmine tartrate as described above may be administered to a subject in need thereof so that a dose equivalent to 1.5 mg rivastigmine is administered twice daily, and this dose may be gradually increased to a maximal daily dose equivalent to between 9-12 mg rivastigmine.
  • Intramuscular and subcutaneous rivastigmine administration can be e.g. from 0.75 mg twice daily to a maximal daily dose equivalent to 5-6 mg rivastigmine.
  • compositions of the invention can be administered alone or in conjunction with other therapeutic modalities. It is appropriate to administer the pharmaceutical compositions of the invention as part of a treatment regimen involving other therapies, such as drug therapy, which comprises e.g. immunosuppressive drugs (e.g. methotrexate, azathioprine, cyclophosphamide, cladribine) and/or other agents used for the treatment of MS such as interferon (IFN)- ⁇ and copolymer-1.
  • immunosuppressive drugs e.g. methotrexate, azathioprine, cyclophosphamide, cladribine
  • MS interferon
  • a method of inhibiting inflammation in a subject afflicted with multiple sclerosis or a multiple sclerosis-related disease comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein, or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
  • said subject is afflicted with multiple sclerosis.
  • the method is used for improving motor function in said subject.
  • the method is used for reducing demyelination in said subject.
  • the method is used for inhibiting neurodegeneration associated with demyelination in said subject.
  • the method is used for inhibiting a clinical symptom associated with inflammation in said subject. In another embodiment, the method is used for inhibiting the progression of multiple sclerosis in said subject. In another embodiment, the method is used for treating a multiple sclerosis-related disease selected from the group consisting of: Neuromyelitis Optica (Devic's Disease), Diffuse Sclerosis, Transitional Sclerosis, Acute Disseminated Encephalomyelitis, and Optic Neuritis, wherein each possibility represents a separate embodiment of the present invention. In another embodiment, the method is used for treating a multiple sclerosis- related disease selected from the group consisting of virus-, bacteria- and parasite-related demyelinating degenerative brain disease wherein each possibility represents a separate embodiment of the present invention.
  • Neuromyelitis Optica Devic's Disease
  • Diffuse Sclerosis Diffuse Sclerosis
  • Transitional Sclerosis Transitional Sclerosis
  • Acute Disseminated Encephalomyelitis and Optic Neuritis
  • the invention provides a method of treating multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically- effective amount of a pharmaceutical composition comprising a compound of the general Formula (I) as defined herein, and pharmaceutically acceptable salts, hydrates or solvates thereof, and a pharmaceutically acceptable carrier, excipient or diluent, whereby the treatment inhibits neurological deterioration or improves non-cognitive functions in said subject.
  • the subject is not otherwise in need of treatment with rivastigmine or other phenyl carbamates.
  • the invention provides use of a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof for the preparation of a medicament useful for treating multiple sclerosis and inhibiting clinical symptoms associated therewith.
  • the medicament is useful for inhibiting inflammation in a subject afflicted with multiple sclerosis or a multiple sclerosis- related disease, for improving motor function in a subject afflicted with multiple sclerosis, for reducing demyelination in a subject afflicted with multiple sclerosis, for inhibiting neurodegeneration associated with demyelination in a subject afflicted with multiple sclerosis, for inhibiting a clinical symptom associated with inflammation in a subject afflicted with multiple sclerosis or for inhibiting the progression of multiple sclerosis.
  • the medicament is useful for treating multiple sclerosis, whereby said medicament inhibits neurological deterioration or improves non-cognitive functions.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula (I) as defined herein, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for treating multiple sclerosis, whereby the composition inhibits neurological deterioration or improves non-cognitive functions.
  • a pharmaceutical composition comprising a compound of the general Formula (I) as defined herein, and pharmaceutically acceptable salts, hydrates or solvates thereof, for inhibiting inflammation in a subject afflicted with multiple sclerosis or a multiple sclerosis-related disease, for improving motor function in a subject afflicted with multiple sclerosis, for reducing demyelination in a subject afflicted with multiple sclerosis, for inhibiting neurodegeneration associated with demyelination in a subject afflicted with multiple sclerosis, for inhibiting a clinical symptom associated with inflammation in a subject afflicted with multiple sclerosis or for inhibiting the progression of multiple sclerosis.
  • a pharmaceutical composition comprising a compound of the general Formula (I) as defined herein, and pharmaceutically acceptable salts, hydrates or solvates thereof, for inhibiting inflammation in a subject afflicted with multiple sclerosis or a multiple sclerosis-related disease, for improving motor function in a subject afflicted with multiple sclerosis
  • the composition is useful for treating a multiple sclerosis-related disease selected from: Neuromyelitis Optica (Devic's Disease), Diffuse Sclerosis, Transitional Sclerosis, Acute Disseminated Encephalomyelitis, and Optic Neuritis, or a multiple sclerosis-related disease selected from virus-, bacteria- and parasite-related demyelinating degenerative brain disease wherein each possibility represents a separate embodiment of the present invention.
  • a multiple sclerosis-related disease selected from: Neuromyelitis Optica (Devic's Disease), Diffuse Sclerosis, Transitional Sclerosis, Acute Disseminated Encephalomyelitis, and Optic Neuritis
  • a multiple sclerosis-related disease selected from virus-, bacteria- and parasite-related demyelinating degenerative brain disease wherein each possibility represents a separate embodiment of the present invention.
  • the compound is N-ethyl-N-methyl-3-[l- (dimethylamino)ethyl] -phenyl carbamate or a pharmaceutically acceptable salt, hydrate or solvate thereof.
  • the compound is (S)-N-ethyl-N-methyl-3-[l- (dimethylamino)ethyl] -phenyl carbamate (rivastigmine) or a pharmaceutically acceptable salt, hydrate or solvate thereof.
  • the compound is rivastigmine tartrate.
  • the subject is selected from the group consisting of humans and non-human mammals.
  • the compound is administered in a manner selected from the group consisting of: orally, subcutaneously and intramuscularly.
  • said compound is formulated in a form selected from the group consisting of extended release, sustained release and controlled-release formulations.
  • said compound is formulated in the form of a transdermal delivery device.
  • the subject is in a state of remission.
  • composition is useful for the preparation of a medicament for treating relapsing-remitting multiple sclerosis.
  • mice C57BL/6JOlaHsd female mice (Harlan) were housed under specific pathogen-free conditions in the Animal Facility at the Hebrew University Medical School, in accordance with NIH guidelines for the care and use of laboratory animals.
  • EAE Experimental Autoimmune Encephalomyelitis
  • mice An additional injection of MOG35-55 peptide in CFA was delivered 7 days later into the right para-lumbar region. All animals were examined daily and evaluated for clinical signs of disease. The clinical status of the mice was scored according to the following scale: 0, without clinical disease; 1, tail weakness; 2, hind limb weakness sufficient to impair righting; 3, one limb plagic; 4, paraplegia with forelimb weakness; 5, quadriplegia; 6, death. Evaluation of central nervous system (CNS) pathology. The spinal cords and brains were removed, fixed in 4% buffered formalin, sectioned, and routinely processed for paraffin embedding. Sections prepared from the same level were stained with hematoxylin and eosin.
  • CNS central nervous system
  • Inflammatory foci containing at least 20 perivascular mononuclear cells were counted in each section. The tissues were sampled on day 32 after disease induction. EAE treatment. Female C57BL were injected daily with rivastigmine from the day of disease induction with 0.75 mg/kg/day, administered either intraperitoneally (i.p.) or subcutaneously (s.c). Control animals received the same volume of vehicle (normal saline or phosphate buffered saline, PBS).
  • vehicle normal saline or phosphate buffered saline, PBS.
  • LNCs lymph node cells
  • cytokine production The levels (pg/ml) of the inflammatory cytokines, tumor necrosis factor-a (TNF- ⁇ ) and interferon- ⁇ (INF- ⁇ ), and of the anti inflammatory cytokine interlukine-10 (IL-IO), were measured in supernatants of lymphocyte cultures by a solid phase ELISA (R&D, Minneapolis, MN, USA). This assay employs a quantitative "sandwich” enzyme immunoassay technique (Barak et al., 2002).
  • Cytotoxicity assay Lymphocyte viability was determined by trypan blue (0.4%, Sigma) exclusion. All cells were viable under the experimental conditions.
  • Statistical analysis Group differences were analyzed by One Way Repeated Measures Analysis of Variance (One Way ANOVA), according to Dunnet's method. Differences in disease severity were analyzed using the Mann - Whitney Rank sum test. A difference between the groups of P ⁇ 0.05 was considered significant.
  • the data are expressed as the mean score ⁇ SE, in 9 control-PBS treated mice, 6 mice receiving rivastigmine.
  • mice were injected i.p. with rivastigmine 0.75mg/kg/day in treated groups and with vehicle in the control group.
  • the data are expressed as the mean score ⁇ SE, in 18 control-PBS treated mice, 16 mice receiving rivastigmine. # Cumulative score: number of days animals were sick x clinical score
  • mice were injected s.c. with rivastigmine 0.75mg/kg/day in treated groups and with vehicle in the control group.
  • the inflammatory process in EAE involves activation of CNS immunocompetent cells, i.e. astrocytes and microglia, and extravasation of activated T cells and macrophages through vessel walls and their subsequent entry and accumulation in inflammatory sites.
  • CNS immunocompetent cells i.e. astrocytes and microglia
  • extravasation of activated T cells and macrophages through vessel walls and their subsequent entry and accumulation in inflammatory sites.
  • the inhibitory effect of rivastigmine on the production of inflammatory mediators by CNS glial cells and on T cell reactivity was tested.
  • Example 1.2 Ex-vivo reactivity of lymphocytes is inhibited by rivastigmine
  • T cell reactivity towards the encephalitogenic MOG peptide was inhibited by daily injection of rivastigmine as shown by the reduction in the stimulation index from 14.4 to 4.9 (Figure 4A).
  • the T-cell reactivity towards the mitogens ConA and LPS was also significantly reduced (Figure 4B).
  • Example 1.3 Effect on CNS inflammation in vivo
  • Brains and spinal cords of PBS and rivastigmine treated mice were fixed and processed for pathological examination of tissue sections.
  • the data are expressed as the mean ⁇ SE number of inflammatory foci (containing at least 20 perivascular mononuclear cells) in each section of tissue meninges and parenchyma.
  • Mini osmotic pumps (Alzet, 2004), containing rivastigmine that was released at the rate of 0.082mg/24hours or PBS as placebo treatment for 28 days, were implanted subcutaneously, one day before disease induction.
  • EAE induction and disease severity assessment were performed as described above. As can be seen in Figure 6, treatment with rivastigmine clearly reduced the clinical symptoms of EAE. As summarized in Table 4, controlled release of rivastigmine 0.082mg rivastigmine/24hours/mouse caused a significant reduction in disease severity and cumulative score, and a significant increase in survival of the mice.
  • Example 1.5 amelioration of EAE with a control drug
  • FIG. 7 and table 5 hereinbelow summarize the course of EAE under daily treatment with s.c. 2mg/day/mouse of the known drug glatiramer acetate (GA, also known as Copaxone) as compared with placebo group. EAE was induced and evaluated as described above.
  • GA glatiramer acetate
  • the brains (minus cerebellum) and tibialis muscles were rapidly removed and homogenized in 0.25mM phosphate containing 1% Triton X-100.
  • Cholinesterase activity was measured in 25 ⁇ l aliquots of the supernatant using acetylthiocholine as a substrate by the spectrophotometic method of EUman et al (Ellman et al.
  • EAE induction and treatment EAE was induced in 8-week-old female C57BL/6 mice by injecting s.c. into the left para-lumbar region 125 ⁇ g of myelin oligodendrocyte glycoprotein 35-55 peptide (MOG 35 - 55 ; synthesized by Sigma Laboratories, Israel) emulsified in complete Freund's adjuvant (CFA) containing 5 mg/ml heat-killed Mycobacterium tuberculosis. Immediately thereafter, and, again, at 48 h, the mice were inoculated i.p. with 0.5 ml of pertussis toxin (350 ng).
  • CFA complete Freund's adjuvant
  • mice were implanted s.c with ALZET ⁇ (DURECT, Cupertino, CA, USA) mini-osmotic pumps, according to the manufacturer's instructions. Pumps were loaded with 3 mg rivastigmine in 220 ⁇ l saline each, corresponding to a daily dose of 4.8 mg/kg (0.8 x 6, as rivastigmine half life in serum is 4 hrs.) for 28 days. Control animals were implanted with pumps releasing the same volume of saline.
  • ALZET ⁇ DURECT, Cupertino, CA, USA
  • the spinal cords were removed 21 days after disease induction (at disease peak) after transcardial perfusion as described previously (Irony-Tur-Sinai et ah, 2006).
  • Longitudinal sections were cut to include the majority of the length of the spinal cord including both gray and white matter, and stained with hematoxylin and eosin. Inflammatory foci containing at least 20 perivascular mononuclear cells were counted in each section.
  • Sections were stained with luxol-fast- blue for myelin visualization and analyzed by light microscopy.
  • immunohistochemistry sections were incubated with primary antibodies anti- neuro filament and anti CD68 (1:300 dilution) and processed according to standard procedures (Das Sarma et ah, 2000). Quantification of the pathologic parameters was based on the methods previously described for the pathologic evaluation of EAE.
  • inflammation none (0), a few inflammatory cells (1), organization of perivascular infiltrates (2), increasing severity of perivascular cuffing with extension into the adjacent tissue (3). Inflammatory foci containing at least 20 perivascular mononuclear cells were counted in each section.
  • the degree of myelin loss, and axonal damage was graded as absent (0), weak (1), moderate (2) and severe (3). For each animal 3 section were studied. The grade of the pathologic parameter was added and the final score was divided by the number of mice in that group.
  • LNCs Mouse lymphocyte proliferation assay: Pooled lymph node cells (LNCs) were prepared from inguinal, axilliary and mesenteric lymph nodes of mice that had been inoculated 9 days earlier with MOG 35-55 peptide in CFA with or without treatment.
  • T-cell reactivity was tested during the various stages of disease (days 14- disease onset, 21- disease peak and day 28 after immunization- chronic phase), T-cells were obtained from spleens and tested as above. The results are expressed as Stimulation
  • SI Setimal cpm of the stimulated cells/mean cpm of the unstimulated cells.
  • Cell treatment with anti-sense Cells were incubated for 24 hrs with anti-sense (AS) to the ⁇ 7 nAChR or ⁇ 5 nAChR as a negative control. After the incubation, cells were treated with rivastigmine as indicated.
  • AS anti-sense
  • the AS used was comprised of phosphorothioate- bond nucleotides (IDT Technologies, Rehovot, Israel) and the sequences used were:
  • RNA and cDNA were prepared using an SV total RNA kit (Promega, Madison, WI, USA). For determination of IL- 17 mRNA level, RNA was prepared from spleens of mice on day 9 after induction of EAE as described above. cDNA was prepared from 200 ⁇ g of total RNA, using MuLV reverse transcriptase (Applied Biosystems, Warrington, UK) and random hexamers according to the manufacturer's instructions for first-strand cDNA synthesis.
  • PCR Quantitative Real-Time Polymerase Chain Reaction
  • HPRT reverse CGAGAGGTCCTTTTCACCAGC (SEQ ID NO:4).
  • ⁇ 7 nAChR forward GACTGTTCGTTTCCCAGATGG (SEQ ID NO:5); ⁇ 7 nAChR reverse: ACGAAGTTGGGAGCCGACATCA (SEQ ID NO:6).
  • 18S forward TCGAGGCCCTGTAATTGGAA (SEQ ID NO:7); 18S reverse: CCCTCCAATGGATCCTCGTT (SEQ ID NO:8).
  • IL- 17 forward CCGCAATGAAGACCCTGATAGA (SEQ ID NO:9);
  • IL-17 reverse TCATGTGGTGGTCCAGCTTTC (SEQ ID NO:10)
  • MOG 35-55 using a commercially available ELISA kit (Biolegend, San Diego, CA, USA). The lymphocytes were collected from the mice 9 days after inoculation with MOG 35 . 55 with or without rivastigmine treatment (0.75 mg/kg, s.c).
  • Flow cytometry analysis Surface markers of leukocytes from pooled spleen cells obtained from control and treated mice (as described in the "Mouse lymphocyte proliferation assay") were analyzed. Cell suspensions were prepared as described previously (Irony-Tur-Sinai et ah, 2006). Stained cells were counted in a fluorescence- activated cell sorter (FACScan, Becton Dickinson, San Jose, CA USA).
  • anti-CD4-FITC (clone GK 1.5, Pharmingen, USA), anti-CD8-FITC (clone 53-6.7, Pharmingen, USA), anti-CD 1 Ib-FITC (clone Ml/70, Pharmingen, USA), anti-MHC class II-FITC (clone 25-5-16S, Serotec, Kidlington, UK).
  • FITC-conjugated rat IgG 2bK (clone A 95-1, Pharmingen, USA)
  • FITC-conjugated rat IgG 2a ⁇ (clone R35-95, Pharmingen, USA)
  • FITC-conjugated mouse IgM (cat.no. X 0934, DakoCytomation, Denmark).
  • mice were placed into the water, facing the maze wall, from one of four start positions evenly spaced around the pool (N, S, E & W). Start positions were chosen randomly at the beginning of each day for all mice. If the mouse failed to find the escape platform within 120 s it was placed on it for 20 sec and then removed from the pool. The mouse was given two trials a day for 5 days between 15:00 and 19:00 hr with an inter-trial interval of 15 min (Wang et al, 2000).
  • MOG- induced EAE mice were treated from the day of induction with 0.75 mg/kg (s.c). This dose of drug inhibited brain cholinesterase by 56.1 ⁇ 2.0% and muscle cholinesterase by 42.4 ⁇ 2.8%, 45 min after injection. Treatment with rivastigmine reduced disease severity and cumulative score by 50- 54% (Fig. 8A and Table 6), and delayed disease onset by 3.5 days (p ⁇ 0.001).
  • a nicotinic receptor blocker, mecamylamine was tested.
  • mice were inoculated daily s.c. with 0.75 mg/kg rivastigmine and with saline in the control group.
  • mice were implanted s.c. with miniosmotic pumps delivering rivastigmine at 4.8 mg/kg/day (see materials and methods) and with saline in the control group.
  • Example 2.2 Attenuation of CNS inflammation, demyelination and neuronal damage by rivastigmine treatment
  • Example 2.3 Rivastigmine treatment reduced MOG-specific T-cell proliferation and pro-inflammatory cytokine production
  • T-cell reactivity was further tested at 14, 21 and 28 days after immunization, corresponding to disease onset, peak and chronic phase, respectively.
  • Figure 15A T-cell proliferation was reduced by 40-80% following rivastigmine treatment as well as the production of pro inflammatory cytokine (40-80%) ( Figure 15B-D).
  • Example 2.4 Effect of rivastigmine on T-cells is ⁇ 7 nAChR dependent
  • the effect of rivastigmine was abolished by ⁇ -bungarotoxin ( ⁇ -btx) a nicotinic antagonist of neuronal and muscle nicotinic receptors. Since the muscle-type nAChR is not expressed on T-cells, ⁇ -btx blockade can be attributed to the ⁇ 7 nAChR. This was confirmed by pre-incubation of T-cells with anti-sense (AS) to this receptor and to the ⁇ 5 nAChR.
  • AS anti-sense
  • ⁇ 7 mRNA was down-regulated by this protocol
  • cells were incubated with rivastigmine and tested for its ability to suppress mitogen-induced proliferation. It was found that T-cells treated with AS- ⁇ 7 were less sensitive to the effects of rivastigmine, while those treated with AS- ⁇ 5 were still responsive (Fig. HC).
  • Example 2.5 Treatment with rivastigmine reduced number of MHC-II + cells
  • Splenocytes from animals treated with the AChEI were analyzed for several cell markers associated with the inflammatory response and antigen presentation.
  • the number of MHC-II + cells was reduced by 30% (from 47% to 32%) (Fig. 12). There was no change in the intensity of the signal, indicating that the number of MHC-II + cells was reduced, and not the number of MHC-II molecules expressed per cell. This was accompanied by a reduction in CDl Ib + (macrophages) (from 28% to 19%), whereas there was no change in CDl Ic + (dendritic cells) or CD19 + (B-cells). Taken together these data support that rivastigmine treatment affected macrophage antigen presentation ability.
  • Example 2.6 Improvement of spatial memory function upon rivastigmine treatment
  • Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 405: 458—462.
  • Kawashima, K., Fujii, T. 2003 a The lymphocytic cholinergic system and its contribution to the regulation of immune activity. Life Sci.74 675-696.
  • Nizri E., Irony-Tur-Sinai, M., Lavon, I., Meshulam, H., Amitai, G., Brenner, T., 2007a.
  • IBU-Octyl-Cytisine a novel bifunctional molecule eliciting anti-inflammatory and cholinergic activity, ameliorates CNS inflammation by inhibition of T-cell activity.
  • Nizri, E., et al., 2007b The role of cholinergic balance perturbation in neurological diseases. Drug News Perspect. 20, 421-9.
  • Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature. 421:384-388.

Abstract

The present invention is directed to the use of rivastigmine, related phenyl carbamates and salts thereof for modulation of multiple sclerosis (MS) and related disorders. The present invention provides compositions comprising rivastigmine and methods thereof for inhibiting the clinical symptoms associated with inflammation in a subject afflicted with MS, for improving motor function and for inhibiting neurodegeneration and disease progression in MS patients.

Description

PHENYL CARBAMATES FOR THE TREATMENT OF MULTIPLE SCLEROSIS
FIELD OF THE INVENTION The invention relates to the use of phenyl carbamates, including rivastigmine, for the treatment of multiple sclerosis.
BACKGROUND OF THE INVENTION
Multiple sclerosis (MS) is a severe chronic disabling disease with characteristic demyelination in the central nervous system (CNS) triggered by presumed autoimmune mechanisms in a genetically susceptible population. According to prevailing hypotheses, an environmental factor which most probably acts in childhood, activates a specific population of T-cells (which have the potential of attacking myelin-associated antigens such as myelin basic protein, MBP) which normally is controlled by suppressor cells. In many cases of MS, non-specific stress results in disease exacerbations with opening of the blood brain barrier (BBB), edema, migration and activation of T-cells and macrophages and subsequent destruction of oligodendroglia-associated myelin, followed by failing attempts at remyelination and finally a glial scar (relapsing-remitting form of MS). These exacerbations which can be visualized by magnetic resonance imaging (MRI) are associated, depending on location, with severe functional disabilities. Furthermore, with an increasing number of relapses, the disease becomes more generalized and progresses with less clear-cut intervals (progressive form of MS, secondary or primary) and increasing and persisting disability which causes, in this mostly young population, impairment of quality of life, with loss of employment and independent life, hospitalization and eventually death. For a review on the neurobiology of MS, see Hauser et al. (2006). Acute exacerbations resulting in functional impairment (e.g., loss of vision, strength, or coordination) are treated with brief courses of corticosteroids (prednisone, methylprednisolone). However, corticosteroids have not been demonstrated to affect long- term outcome of the disease. Furthermore, corticosteroid treatment may be undesirable as these drugs are often poorly tolerated in patients and also inhibit endogenous immunosuppressive mechanisms. Other current treatments of MS include beta interferons, mitoxantrone, natalizumab and glatiramer acetate (GA, Copaxone). However, such therapies are typically accompanied by adverse effects, including flu-like symptoms, depression, cytopenias, skin reactions and local pain (as consequences of the subcutaneous administration). Current therapies used for symptomatic treatment of multiple sclerosis are discussed in Henze et al., 2006.
Recent research efforts have been focused on an understanding of the immunology of MS which, in combination with computational methods and emerging high-throughput technologies that characterize variations in DNA, RNA, proteins, and metabolites, are used to define subsets of patients with different forms of the demyelinating disease. These efforts may allow tailoring immunotherapy to the underlying pathophysiologic processes of individual patients using current therapies, emerging treatments, and combinations thereof (De Jager et al, 2007).
WO 2006/130726 is directed to methods for the treatment of a form of multiple sclerosis comprising administering an amount of R(+)-6-(N-methyl, N- ethyl-carbamoyl oxy)-N'-propargyl-l-aminoindan or a pharmaceutically acceptable salt thereof.
Rivastigmine
US Patent 4,948,807 is directed to a novel group of phenyl carbamates of the general formula (I):
Figure imgf000003_0001
wherein
Ri is hydrogen, lower alkyl, cyclohexyl, allyl or benzyl, R2 is hydrogen, methyl, ethyl or propyl, or
Ri and R2 together with the nitrogen to which they are attached form a morpholino or piperidino radical, R3 is hydrogen or lower alkyl, Rj and R5 are the same or different and each is a lower alkyl, and the dialkylaminoalkyl group is in the meta, ortho or para position.
Included within the scope of Formula I, and specifically claimed in US 4,948,807, is the compound N-ethyl-N-methyl-3-[l-(dimethylamino)ethyl] phenyl carbamate. Rivastigmine is the trivial chemical name of the isolated (S) enantiomer of this compound. US 4,948,807 further discloses and claims use of rivastigmine and related phenyl carbamates for alleviation of symptoms associated with decline in cholinergic function as is known to occur in certain neurodegenerative diseases and dementias.
Rivastigmine is a cholinesterase inhibitor (ChEI) classified as an intermediate- acting or pseudo-irreversible agent and is primarily indicated for the treatment of
Alzheimer's disease. The full chemical name of Rivastigmine is (S)-N-ethyl-N-methyl-3-
[l-(dimethylamino)ethyl]-phenyl carbamate which is specifically disclosed and claimed in
US 5,602,176. The tartrate salt of Rivastigmine (Rivastigmine Tartrate), chemically named
(S)-N-ethyl-N-methyl-3-[l -(dimethylamino)ethyl] -phenyl carbamate hydrogen-(2R,3R)- tartrate, is currently marketed under the trade name EXELON . The chemical structure of
Rivastigmine Tartrate is represented in Formula (II) below:
Figure imgf000004_0001
(H) Thus, rivastigmine is a well known drug for preventing cognitive decline and loss of cholinergic function in certain types of dementia. Controlled release oral compositions comprising rivastigmine are described in US 6,565,883. US 6,335,031 discloses formulations of rivastigmine containing an antioxidant suitable for transdermal delivery.
The known effects of rivastigmine on cognitive improvement were also examined in MS patients and in experimental allergic encephalomyelitis (EAE) which is an experimental animal model system resembling some aspects of MS. Parry et al. (2003) reported that administration of rivastigmine to MS patients 150 minutes prior to performing a cognitive task was associated with a relative normalization of the abnormal task-associated brain activation. D'Intino et al. (2005) examined cognitive dysfunction in EAE rats that had recovered their motor function. Daily oral administration of rivastigmine was found to restore cognitive performance, choline acetyltransferase activity in the basal forebrain but not in the cerebral cortex and hippocampus, and nerve growth factor mRNA expression in the cerebral cortex. An anti-inflammatory effect of rivastigmine or an effect of the drug on the motor functions of EAE rats were neither taught nor suggested in these studies.
U.S. Patent Application Publication No. 2003/0225031 is directed to pharmaceutical compositions for nasal administration comprising ChE inhibitors, including, inter alia, rivastigmine, and methods of using same for the treatment of Alzheimer's disease and other neurological conditions associated with cognitive impairment in a mammal. According to the '031 application, these conditions include, among others, cognitive disorder associated with MS.
Several recent studies indirectly suggest that cholinergic up-regulation may result in anti-inflammatory effects. It was shown that immune system cells possess various subtypes of muscarinic cholinergic receptors and nicotinic receptors. In addition, lymphocytes synthesize acetylcholine (ACh), which is degraded by acetylcholinesterase (AChE)
(Kawashima et al., 2003a; Kawashima et al., 2003b).
Borovikova et al. (2000) demonstrated that administration of acetylcholine or its agonist nicotine in vitro attenuated the release of the cytokine tumor necrosis factor (TNF), interleukin (IL)- lβ, IL-6 and IL-18, but not the anti-inflammatory cytokine IL-10, in lipopolysaccharide (LPS)-stimulated human macrophage cultures. Direct electrical stimulation of the peripheral vagus nerve in vivo during lethal endotoxaemia in rats inhibited TNF synthesis and peak serum levels, and prevented the development of endotoxic shock. Wang et al. (2003) further showed that the nicotinic acetylcholine receptor α7 subunit is required for inhibition of macrophage TNF release by ACh or nicotine. Saeed et al. (2004) reported that human microvascular endothelial cells express α7 nicotinic acetylcholine receptor (α7 nAChR), and that nicotine, a cholinergic agonist thereof, and vagus nerve stimulation inhibit endothelial cell activation, thereby inhibiting leukocyte recruitment in the carrageenan air pouch model of local inflammation. Other studies performed in animal models disclose that nicotine suppresses the progression of experimental ulcerative colitis (Sykes et al., 2000; Eliakim et al., 2003) and cutaneous inflammation (Sopori et al., 1998) and improves survival during endotoxemia and sepsis (Wang et al., 2004). Nicotine was also reported to impair antigen presentation, and α7 nAChR has also been disclosed to be expressed on microglia and astrocytes. In addition, the use of nicotine patches for the treatment of human ulcerative colitis was examined in clinical trials (Sandborn et al., 1997). However, the use of nicotine as a therapeutic agent is limited by its toxicity.
Nizri et al. (2005) disclose bi-functional compounds consisting of the non-steroidal anti-inflammatory drug ibuprofen and pyridostigmine, a cholinesterase inhibitor that acts as a cholinergic up-regulator. These bi-functional compounds exhibited improved efficiency in an EAE mouse model compared to ibuprofen alone. Although treatment of mice by pyridostigmine alone resulted in reduced lymphocyte proliferation, such treatment did not change disease severity. Another such bi-functional compound, namely IBU- Octyl-Cytisine, containing ibuprofen and Cytisine as the nicotinic agonist, has been described by Nizri et al. (2007a), which further report that each moiety separately failed to reproduce the effect of this compound.
Nizri et al. (2006) report suppressed lymphocyte proliferation and pro- inflammatory cytokine production induced by the ChE inhibitors pyridostigmine and edrophonium administered to the cells in vitro. The disclosure further reports that administration of an antisense oligodeoxynucleotide targeted to AChE mRNA, reduced the clinical severity and CNS inflammation intensity in an EAE model. It has been suggested that inhibition of antibody-forming cell response by ChE inhibitors requires their presence in the CNS, based on experiments performed with physostigmine, physostigmine bromide and edrophonium (Langley et al., 2004).
Reale et al. (2004, 2006) disclose that donepezil treatment down-regulates IL-I, IL-6 and TNF, and up-regulates the expression and production of IL-4 and MCP-I in peripheral blood mononuclear cells (PBMC) in Alzheimer's disease (AD) patients.
For general discussion on the role of cholinergic balance perturbation in neurological diseases, including neuroinflammatory diseases such as multiple sclerosis, see Nizri et al. (2007b), and references cited therein. Pollak et al. (2005) report that peripheral administration of certain cholinesterase inhibitors including rivastigmine to mice attenuates the production of interleukin-lbeta in the hippocampus and blood, concomitantly with the reduction in acetylcholinesterase activity.
Tyagi et al. (2007) studied the effect of tacrine, rivastigmine and donepezil in mice that received intraperitoneal administration of LPS, and found that LPS-induced increased levels of IL-2 were attenuated, independently from a reduction of brain AChE activity.
U.S. Patent Application Publication No. 2005/0222123 is directed to a method of treating a subject with a cytokine-mediated inflammatory disorder comprising administering to the subject a cholinesterase inhibitor other than galantamine. The '123 application lists a wide range of structurally-unrelated compounds, including nicotinic and muscarinic AChE inhibitors and butyrylcholinesterase inhibitors, as potential inhibitors for treating a variety of disorders. The specification of '123 exemplifies the use of galantamine, tacrine, huperzine, neostigmine and physostigmine in acute inflammatory models of sepsis and endotoxemia in mice. None of the prior art discloses that rivastigmine or related phenyl carbamates are useful for the treatment and amelioration of neurodegeneration and loss of motor function in multiple sclerosis. There remains an unmet medical need for safe and effective therapeutic modalities for treating multiple sclerosis and for interventions that address their neurodegenerative component. SUMMARY OF THE INVENTION
The present invention provides pharmaceutical compositions comprising phenyl carbamates including rivastigmine for treating multiple sclerosis and inhibiting symptoms associated therewith. The invention is based, in part, on the unexpected discovery that rivastigmine can reduce the clinical symptoms associated with inflammation in experimental autoimmune encephalitis (EAE), a well-known model system of multiple sclerosis (MS). Rivastigmine was surprisingly found to reduce loss of motor functions when administered chronically to mice upon induction of EAE. Unexpectedly, it is now disclosed that rivastigmine was in fact able to reduce demyelination and microglia activation and to preserve axons in treated mice. Rivastigmine also decreased reactivity of encephalitogenic T-cells and production of pro-inflammatory cytokines (TNF-α, IFN-γ and IL-17).
Thus, the present invention provides compositions comprising rivastigmine and methods thereof for inhibiting the clinical symptoms associated with inflammation in a subject afflicted with MS, for improving motor function and for inhibiting neurodegeneration and disease progression in MS patients. In other words, the present invention is directed to treatment of a subject not otherwise in need of treatment with rivastigmine or other phenyl carbamates.
According to a first aspect of the present invention, there is provided a method of treating multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I): O
Figure imgf000008_0001
( I ) wherein
Ri is hydrogen, lower alkyl, cyclohexyl, allyl or benzyl,
R2 is hydrogen, methyl, ethyl or propyl, or
Ri and R2 together with the nitrogen to which they are attached form a morpholino or piperidino radical,
R3 is hydrogen or lower alkyl,
R4 and R5 are the same or different and each is a lower alkyl, and the dialkylaminoalkyl group is in the meta, ortho or para position,
or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent, whereby the treatment inhibits neurological deterioration or improves non-cognitive functions in said subject.
In one currently preferred embodiment, the active ingredient is N-ethyl-N-methyl- 3-[l-(dimethylamino)ethyl]-phenyl carbamate or a pharmaceutically acceptable salt, hydrate or solvate thereof. In another preferred embodiment, the active ingredient is (S)-N- ethyl-N-methyl-3-[l-(dimethylamino)ethyl]-phenyl carbamate (rivastigmine) or a pharmaceutically acceptable salt, hydrate or solvate thereof. In yet another particularly preferred embodiment, the active ingredient is rivastigmine tartrate.
In another aspect, the invention provides a method of improving motor function in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In yet another aspect, the invention provides a method of reducing demyelination in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In another aspect, the invention provides a method of reducing neurodegeneration associated with demyelination in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
According to one aspect of the present invention, there is provided a method of inhibiting inflammation in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In another aspect, the invention provides a method of inhibiting a clinical symptom associated with inflammation in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent. In another aspect, the invention provides a method of inhibiting the progression of multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent. According to another aspect of the present invention, there is provided a method of treating a multiple sclerosis-related disease selected from the group consisting of: Neuromyelitis Optica (Devic's Disease), Diffuse Sclerosis, Transitional Sclerosis, Acute Disseminated Encephalomyelitis, and Optic Neuritis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent. In another embodiment, the multiple sclerosis-related disease is a virus-, bacteria- or parasite- related demyelinating brain disease.
In a preferable embodiment, the compound utilized in the methods of the invention is rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof, preferably rivastigmine tartrate. In another embodiment, the subject is human. In other embodiments, the subject is a non-human mammal.
In another embodiment, the compounds of the invention are useful for decreasing the frequency or duration of acute exacerbations (or relapses) of the disease in a subject afflicted with MS. In a particular embodiment, the compounds are administered to a subject that is in remission of the disease, to prevent, delay or reduce recurrence of exacerbations.
In yet another particular embodiment, the subject is afflicted with relapsing- remitting MS. In other embodiments, the active ingredient may be administered enterally, parenterally or topically. According to currently preferred embodiments, the active ingredient is administered in a manner selected from the group consisting of: orally, subcutaneously and intramuscularly.
In another embodiment, the active ingredient is formulated in the form of extended release, sustained release or controlled-release formulations. In certain particular embodiments, oral sustained release and controlled release formulations are contemplated.
In other embodiments, the active ingredient is formulated in the form of a transdermal formulation, e.g. as a transdermal delivery device.
Other objects, features and advantages of the present invention will become clear from the following description and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Daily treatment with Rivastigmine ameliorates EAE. Rivastigmine (0.75mg/kg) was daily injected i.p from the day of EAE induction (n=6). Animals from the control-EAE group were administered vehicle (n=9). The results are expressed as the mean daily clinical score.
Figure 2. Daily treatment with Rivastigmine ameliorates EAE. Rivastigmine (0.75mg/kg) was daily injected s.c from the day of EAE induction (n=18). Animals from the control EAE group were administered vehicle (n=16). The results are expressed as the mean daily clinical score.
Figure 3. Summary of three individual experiments
Figure 4. Rivastigmine inhibits lymphocyte reactivity towards the specific encephalitogenic peptide MOG (Figure 4A) and mitogens (Figure 4B). Lymphocytes were obtained from the lymph nodes of the treated animals (n=6 for each group) and tested in vitro for the proliferative ability by a standard thymidine proliferation assay. The results are expressed as Stimulation Index (SI), according to the formula: SI=Mean cpm of the stimulated cells/mean cpm of the unstimulated cells. *p<0.05 vs. control EAE group.
Figure 5. Rivastigmine inhibits ex- vivo secretion of IFN-γ (Figure 5A) and TNF-α
(Figure 5B). Lymphocytes from MOG-induced EAE mice, PBS and rivastigmine treated (0.75mg/kg/day) were analyzed for INFγ secretion (Figure 5A) and TNF α secretion (Figure 5B) in the presence of the MOG encephalitogenic peptide 35-55 (100 μg/ml). The data are expressed as the mean ±SE of INFγ secreted after 24 and 48 hours of incubation, and TNF α after 4 and 24 hours. * p<0.05
Figure 6. Rivastigmine administered via a miniosmotic pump ameliorates EAE. The results are expressed as the mean daily clinical score.
Figure 7. Amelioration of EAE with glatiramer acetate (GA, Copaxone).
Figure 8. Amelioration of EAE by treatment with rivastigmine. Figure 8A, daily treatment with rivastigmine (0.75 mg/kg, s.c.) ameliorated clinical score of EAE. This effect was reversed in animals treated with the nicotinic antagonist mecamylamine (10 mg/kg, s.c). Figure 8B, treatment of EAE induced animals with rivastigmine-loaded mini- osmotic pumps (4.8 mg/kg/day, s.c.) also ameliorated clinical score, even to a larger extent.
Figure 9. Rivastigmine treatment reduced neuropathological parameters of inflammation, demyelination and neurodegeneration. Histological analysis from control- EAE spinal cord tissue taken at peak of disease showed marked inflammatory infiltration and demyelination (A), microglia activation (B) and axonal damage (C). Rivastigmine treatment ameliorated all these parameters (D, E, F). Figure 10. Inhibitory effect of rivastigmine on lymphocyte reactivity ex vivo. Figure 1OA, Treatment with rivastigmine (0.75 mg/kg, s.c.) reduced T-cell proliferation toward the encephalitogenic MOG peptide. Figure 1OB, Rivastigmine treatment reduced pro-inflammatory (TNF-α and IFN-γ) cytokine production in response to MOG (24 hrs) and also down-regulated IL- 17 mRNA expression (48 hrs, Figure 10C), as assayed by real-time PCR. Figure 1OD, IL-10 levels, an anti-inflammatory cytokine, were unaltered (96 hrs).
Figure 11. Effects of rivastigmine are dependent on the α7 nAChR nicotinic receptor. Figure HA, inhibition of T-cell proliferation exerted by rivastigmine (lμM) is reversed when cells are incubated with α-bungarotoxin, a nicotinic muscle and neuronal- type blocker. Figure HB, T-cells were incubated with anti-sense to the α5 nAChR or to α7 nAChR (AS- α5, AS- α7, respectively) for 24 hrs, and then levels of α7 nAChR mRNA were determined in the indicated treatments. Figure HC, T-cells treated as above were tested for rivastigmine (lμM) inhibition in response to ConA. Treatment with AS- α7 abolished the effects of rivastigmine, whereas treatment with AS- α5 had no effect.
Figure 12. Rivastigmine reduced the number of MHC-II+ and CDl Ib+ cells. Splenocytes obtained from treated animals were analyzed by flow cytometry.
Figure 13. Rivastigmine reversed the decline in spatial learning performance seen in EAE animals in the Morris water maze. Performance of naive animals in first trial was compared to EAE-induced animals and to animals induced for EAE and treated with Rivastigmine (4.8 mg/kg, mini-pumps).
Figure 14. Antigen presentation ability is affected by rivastigmine treatment. Splenocytes from untreated and treated mice were irradiated and incubated with MOG- specific T-cells in the presence of MOG. Proliferation of T-cells in response to antigen was normalized to the untreated group.
Figure 15. Treatment with rivastigmine suppressed T-cell reactivity during various stages of EAE course. Figure 15 A, T-cell proliferation towards the encephalitogenic peptide (MOG 3S-55) was assessed at the indicated time points after disease induction.
Figure 15B-D, Production of pro-inflammatory cytokines was suppressed by rivastigmine at the same time points (Figure 15B - TNF-α; Figure 15C - IFN-γ; Figure 15D - IL-17). The treatment protocol was similar to that described for assessment of clinical score and reactivity of T-cells on day 9 post immunization.
Figure 16. EAE is associated with inflammatory infiltrates in lepto-meninges near the hippocampus. Representative sections from two placebo-treated mice with inflammatory infiltrates stained near the hippocampus. Hematoxylin and eosin staining, magnification x40.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to the use of rivastigmine, related phenyl carbamates and salts thereof for preparing a medicament for the treatment of multiple sclerosis (MS) and related diseases. The present invention provides compositions comprising these compounds and methods thereof for treating MS and inhibiting clinical symptoms associated therewith.
The present invention provides novel therapeutic uses for compositions comprising a compound of the general Formula (I):
O
Figure imgf000014_0001
wherein
Ri is hydrogen, lower alkyl, cyclohexyl, allyl or benzyl,
R2 is hydrogen, methyl, ethyl or propyl, or
Ri and R2 together with the nitrogen to which they are attached form a morpholino or piperidino radical, R3 is hydrogen or lower alkyl,
R4 and R5 are the same or different and each is a lower alkyl, and the dialkylaminoalkyl group is in the meta, ortho or para position.
In one preferable embodiment, the compound is N-ethyl-N-methyl-3-[l- (dimethylamino)ethyl] -phenyl carbamate or a pharmaceutically acceptable salt, hydrate or solvate thereof. In another preferable embodiment, the compound is (S)-N-ethyl-N-methyl- 3-[l-(dimethylamino)ethyl]-phenyl carbamate (rivastigmine) or a pharmaceutically acceptable salt, hydrate or solvate thereof. In yet another particularly preferable embodiment, the compound is rivastigmine tartrate. The compounds of the invention, methods of preparation thereof, and uses thereof for preventing cognitive decline and loss of cholinergic function in certain types of dementia, were disclosed by U.S. Patent Nos. 4,948,807 and 5,602,176. The present invention unexpectedly discloses novel uses of these known compounds for the treatment of multiple sclerosis. Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system (CNS) characterized by chronic inflammation, demyelination and gliosis. Pathologically, MS is characterized by well-demarcated, macroscopic lesions, called plaques, in the brain white matter and, less frequently, gray matter. Acute lesions are characterized by perivenular cuffing and infiltration of T lymphocytes and macrophages, along with a few B cells and plasma cells. MS is reportedly an autoimmune disorder, likely triggered by environmental exposure in a genetically susceptible host.
According to the present invention, a pharmaceutical composition comprising a compound of the general Formula (I) described above can be administered to patients having multiple sclerosis, including e.g. multiple sclerosis variants such as Neuromyelitis Optica (Devic's Disease), Diffuse Sclerosis, Transitional Sclerosis, Acute Disseminated Encephalomyelitis, and Optic Neuritis. In other embodiments, the patients are afflicted with a MS-related disorder selected from the group consisting of virus-, bacteria- or parasite-related demyelinating degenerative brain disease. Each possibility represents a separate embodiment of the present invention. Symptoms of MS which are prevented or ameliorated or treated may include: weakness and/or numbness in one or more limbs; tingling of the extremities and tight band-like sensations around the trunk or limbs; dragging or poor control of one or both legs to spastic or ataxic paraparesis; hyperactive tendon reflexes; disappearance of abdominal reflexes; Lhermitte's sign; retrobulbar or optic neuritis; unsteadiness in walking; increased muscle fatiguability; brain stem symptoms (diplopia, vertigo, vomiting); disorders of micturition; hemiplegia; trigeminal neuralgia; other pain syndromes; nystagmus and ataxia; cerebellar-type ataxia; Charcot's triad; diplopia; bilateral internuclear ophthalmoplegia; myokymia or paralysis of facial muscles; deafness; tinnitus; unformed auditory hallucinations (because of involvement cochlear connections); vertigo and vomiting (vestibular connections); transient facial anesthesia or of trigeminal neuralgia; bladder dysfunction; fatigue; dull, aching pain in the low back; sharp, burning, poorly localized pains in a limb or both legs and girdle pains; abrupt attacks of neurologic deficit; dysarthria and ataxia; paroxysmal pain and dysesthesia in a limb; flashing lights; paroxysmal itching; and/or tonic seizures, taking the form of flexion (dystonic) spasm of the hand, wrist, and elbow with extension of the lower limb. A patient having MS may have one or more of these associated with MS and one or more can be ameliorated by administrating of a pharmaceutical composition according to the present invention.
It should be noted that treatment of cognitive symptoms that may be associated with MS, particularly with progressive subtypes of the disease, such as dementia and other forms of cognitive impairment, is explicitly excluded from the methods of the present invention. The term "cognitive impairment" as used herein relates to a disorder in memory, problem solving, abstract reasoning and orientation that weakens an individual's ability to maintain an independent lifestyle. A hallmark is memory impairment with resulting confusion in the conduct of daily affairs. In a particular embodiment, the invention is directed to the treatment of a subject afflicted with relapsing-remitting MS.
Experimental autoimmune encephalomyelitis (EAE) is a T cell mediated autoimmune disease of the central nervous system that serves as an experimental model for multiple sclerosis. Some models of EAE are relapsing inflammatory disorders that can be induced in certain strains of rodents by injection of purified myelin basic protein or crude spinal cord homogenate together with adjuvant. Histopathologically, EAE is characterized by CNS inflammation with macrophage and lymphocytic infiltrates and varying degrees of demyelination. The disease manifests clinically with paralysis, beginning at the tail and spreading rostrally to the hindlimbs and forelimbs, and in advanced stages affects breathing and causes death. According to one aspect of the present invention, there is provided a method of treating multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
The term "treating" refers to administering a therapy in amount, manner, and/or mode effective to improve a condition, symptom, or parameter associated with a disorder or to prevent progression of a disorder, to either a statistically significant degree or to a degree detectable to one skilled in the art. An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject. In the case of multiple sclerosis, the term "treating" includes reducing, inhibiting, alleviating and/or ameliorating the non-cognitive clinical manifestations of MS. For example, "treating" may include blocking or reducing the physiological and pathogenic deterioration associated with MS. Without wishing to be bound by any theory or mechanism of action, the treatment may reduce or inhibit the inflammatory response in the brain and other regions of the nervous system, breakdown or disruption of the blood-brain barrier, appearance of lesions in the brain, tissue destruction, demyelination, autoimmune inflammatory response, chronic inflammatory response, neuronal death, and/or neuroglia death. The term further includes inhibition, alleviation and/or amelioration of one or more of the clinical symptoms associated with MS that were listed above, an increase in frequency and duration of MS symptom-free periods, and improvement or inhibition of deterioration in motor performance. Treatment of cognitive impairment associated with MS is explicitly excluded from the scope of the present invention.
According to another aspect of the present invention, there is provided a method of treating multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
According to another aspect, there is provided a method of inhibiting inflammation in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
According to another aspect, there is provided a method of inhibiting inflammation in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In another aspect, the invention provides a method of improving motor function in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In another aspect, the invention provides a method of improving motor function in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In yet another aspect, the invention provides a method of reducing demyelination in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In yet another aspect, the invention provides a method of reducing demyelination in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In another aspect, the invention provides methods of inhibiting neurodegeneration and axonal damage associated with demyelination in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In another aspect, the invention provides methods of inhibiting neurodegeneration and axonal damage associated with demyelination in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In another aspect, the invention provides a method of inhibiting a clinical symptom associated with inflammation in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In another aspect, the invention provides a method of inhibiting a clinical symptom associated with inflammation in a subject afflicted with multiple sclerosis, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
By "inhibiting a clinical symptom" it is meant reduction in severity and/or frequency of one or more MS associated symptoms, elimination of symptom(s) and prevention of the occurrence of symptom(s).
In another aspect, the invention provides a method of inhibiting the progression of multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
In another aspect, the invention provides a method of inhibiting the progression of multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising rivastigmine or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent. It is to be understood, that the compounds of Formula (I) as defined herein are known as being useful for treating a subject suffering from senile dementia, Alzheimer's disease, Huntingdon's chorea, tardive dyskinesias, hyperkinesia, mania, acute confusion disorders, Friedrich's ataxia or Down's syndrome. The treatment of a subject afflicted with any of these diseases is excluded from the scope of the present invention. Preferably, the MS-afflicted subject treated by the methods of the present invention should not suffer simultaneously from any of said diseases or from dementia or cognitive impairment associated with MS.
In a particular embodiment, the compounds of the invention are useful for decreasing the frequency or duration of acute exacerbations (or relapses) of the disease in a subject afflicted with MS. In another particular embodiment, the subject is in a state of remission.
According to various embodiments of the present invention, the compounds of Formula (I) as defined herein may be used in the form of the free base or a pharmaceutically acceptable salt thereof. For rivastigmine formulations, preferably the hydrogen tartrate (hta) is used. Other pharmacologically acceptable salts of these compounds include, but are not limited to, the acetate, salicylate, fumarate, phosphate, sulphate, maleate, succinate, citrate, tartrate, propionate and butyrate salts thereof.
The compounds of Formula (I) as defined herein or physiologically acceptable salt(s) thereof may be compounded with one or more physiologically acceptable vehicles, carriers, excipients, binders, preservatives, stabilizers, flavors, etc., in a unit dosage form as called for by accepted pharmaceutical practice. The amount of active substance in these compositions or preparations is such that a suitable dosage is obtained.
By "pharmaceutically acceptable", or "physiologically acceptable", is meant herein a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. "Pharmacologically active"
(or simply "active"), as in a "pharmacologically active" derivative or metabolite, refers to a derivative or metabolite having the same type of pharmacological activity as the parent compound and approximately equivalent in degree. When the term "pharmaceutically acceptable" or "physiologically acceptable" is used to refer to a derivative (e.g., a salt) of an active agent, it is to be understood that the compound is pharmacologically active as well, i.e., therapeutically effective for the treatment of multiple sclerosis.
"Carriers" or "vehicles" as used herein refer to conventional pharmaceutically acceptable carrier materials suitable for drug administration, and include any such materials known in the art that are nontoxic and do not interact with other components of a pharmaceutical composition or drug delivery system in a deleterious manner. As used herein, a "pharmaceutically acceptable carrier, excipient or diluent" may refer to a single auxiliary material or to various mixtures and combinations of such non-active ingredients.
Illustrative of the adjuvants which may be incorporated in tablets, capsules and the like are the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; a flavoring agent such as peppermint, oil of wintergreen or cherry. When the dosage unit form is a capsule, it may contain in addition to materials of the above type a liquid carrier such as a fatty oil. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and a flavoring such as cherry or orange flavor. Other examples of suitable pharmaceutical adjuvants and techniques for formulation as well as administration of drugs are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
By means of a non-limitative example, rivastigmine tartrate may be formulated as capsules for oral administration, containing as inactive ingredients hydroxypropyl methylcellulose, magnesium stearate, microcrystalline cellulose, and silicon dioxide. Each hard-gelatin capsule may contain, for example, gelatin, titanium dioxide and dyes (e.g. red and/or yellow iron oxides; Exelon® capsules).
In another non-limitative example, rivastigmine tartrate may be formulated as an oral solution (comprising e.g. a rivastigmine tartrate concentration equivalent to 2 mg/mL of rivastigmine base), comprising as inactive ingredients citric acid, a dye (e.g. D&C yellow #10), purified water, sodium benzoate and sodium citrate (Exelon® oral solution).
Sterile compositions for injection can be formulated according to conventional pharmaceutical practice by dissolving or suspending the active substance in a vehicle such as water for injection. Buffers, preservatives, antioxidants and the like can be incorporated as required.
Preferred antioxidants for use with the compounds of the present invention include sodium metabisulphite and ascorbic acid. The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.
In other embodiments, the invention is directed to the use of extended release, sustained release or controlled release formulations of the compounds of Formula (I) as defined herein or salts thereof, including, but not limited to those disclosed by U.S. Patent No. 6,565,883. For example, the composition may comprise a core coated with two films, the first inner film being a semi-permeable to water or body fluids film applied directly on said core and comprising cellulose acetate, the second outer film being a permeable to water or body fluids film comprising ethylcellulose. The term "controlled release" is intended to refer to any drug-containing formulation in which release of the drug is not immediate, i.e., with a "controlled release" formulation, oral administration does not result in immediate release of the drug into an absorption pool. The term is used interchangeably with "nonimmediate release" as defined in Remington: The Science and Practice of Pharmacy, Nineteenth Ed. (Easton, Pa.: Mack Publishing Company, 1995). As discussed therein, immediate and nonimmediate release can be defined.
For a formulation of the drug administered at a particular absorption site, the drug level achieved is the outcome of rate constants for (1) release of the drug from the formulation, (2) absorption, and (3) elimination, respectively. For immediate release dosage forms, the rate constant for drug release is far greater than the absorption rate constant. For the controlled release formulations, the opposite is true, such that the rate of release of drug from the dosage form is the rate-limiting step in the delivery of the drug to the target area. The term "controlled release" as used herein is intended to include any nonimmediate release formulation, including but not limited to sustained release, delayed release and pulsatile release formulations. The term "sustained release" is used in its conventional sense to refer to a drug formulation that provides for gradual release of drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of drug over an extended time period. The term "delayed release" is used in its conventional sense to refer to a drug formulation in which there is a time delay provided between oral administration of a drug dosage form and the release of the drug therefrom. "Delayed release" may or may not involve gradual release of drug over an extended period of time, and thus may or may not be "sustained release". A controlled release formulation may comprise, for example, controlled release beads comprising the drug. A common type of controlled release beads comprises an inert core, such as a sugar sphere, coated with an inner drug-containing layer and an outer membrane layer controlling drug release from the inner layer. An example of such controlled release beads is described in U.S. Pat. No. 5,783,215 where each bead comprises (i) a core unit of a soluble or insoluble inert material, (ii) a first layer on the core unit comprising an active ingredient dispersed in a hydrophilic polymer, (iii) an optional second layer of hydrophilic polymer covering the first layer, and (iv) an outermost membrane layer effective for controlled release of the active ingredient. In the above and similar controlled release beads it is not uncommon to apply a "sealcoat" in the form of a small amount (e.g. 1-3%) of a water-soluble polymer, such as hydroxypropylmethyl cellulose (HPMC) or polyvinylpyrrolidone (PVP), between the inert core and the layer containing the active ingredient. The purpose thereof is generally to isolate the drug from the core surface in the event that a drug-core chemical interaction is possible, and/or to smooth the surface of the inert core such that the surface area is more consistent from lot to lot to thereby improve the coating quality when the drug layer and the controlled release membrane layers are applied.
The cores are typically of a water-soluble or swellable material, and may be any such material that is conventionally used as cores or any other pharmaceutically acceptable water-soluble or water-swellable material made into beads or pellets. Especially, the beads are spheres of sucrose/starch (Sugar Spheres NF), sucrose crystals, or extruded and dried spheres typically comprised of excipients such as microcrystalline cellulose and lactose.
The substantially water-insoluble material in the first, or sealcoat layer is generally a "GI insoluble" or "GI partially insoluble" film forming polymer (latex or dissolved in a solvent). As examples may be mentioned ethyl cellulose, cellulose acetate, cellulose acetate butyrate, polymethacrylates such as ethyl acrylate/methyl methacrylate copolymer (Eudragit NE-30-D) and ammonio methacrylate copolymer types A and B (Eudragit RL30D and RS30D), and silicone elastomers. Usually, a plasticizer is used together with the polymer. Exemplary plasticizers include: dibutylsebacate, propylene glycol, triethylcitrate, tributylcitrate, castor oil, acetylated monoglycerides, acetyl triethylcitrate, acetyl butylcitrate, diethyl phthalate, dibutyl phthalate, triacetin, fractionated coconut oil (medium-chain triglycerides).
The second layer containing the active ingredient may be comprised of the active ingredient (drug) with or without a polymer as a binder. The binder, when used, is usually hydrophilic but may be water-soluble or water-insoluble. Exemplary polymers to be used in the second layer containing the active drug are hydrophilic polymers such as polyvinylpyrrolidone (PVP), polyalkylene glycol such as polyethylene glycol, gelatine, polyvinyl alcohol, starch and derivatives thereof, cellulose derivatives, such as hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, carboxyethyl cellulose, carboxymethylhydroxyethyl cellulose, acrylic acid polymers, polymethacrylates, or any other pharmaceutically acceptable polymer.
In further alternate embodiments, transdermal pharmaceutical compositions comprising the compounds of Formula (I) as defined herein may be used in accordance with the methods of the present invention. These formulations may optionally be administered using a transdermal device, e.g. a transdermal patch. Exemplary transdermal formulations comprising inter alia rivastigmine and transdermal devices thereof are disclosed in U.S. Patent No. 6,335,031. The compounds according to the invention may be administered by any conventional route, in particular enterally, preferably orally, e.g. in the form of tablets or capsules, or parenterally, e.g. in the form of injectable solutions or suspensions (e.g. for subcutaneous or intramuscular administration).
The exact amounts of the compound of Formula (I) as defined herein to be administered may depend on a number of factors, e.g. the drug release characteristics of the compositions, the drug penetration rate observed in vitro and in vivo tests, the duration of action required, the form of the compound of Formula (I), and for transdermal compositions the size of the skin contact area, and the part of the body to which the unit is fixed. The amount of and, e.g. area of the transdermal composition etc. may be determined by routine bioavailability tests comparing the blood levels of active agents after administration of the compound of Formula (I) as defined herein in a composition according to the invention to intact skin and blood levels of the compound of Formula (I) observed after oral administration of a therapeutically effective dose of the compound.
Orally, rivastigmine is well tolerated in humans at an initial dose of 1.5 mg twice a day orally and the dose may be stepped up to 3 mg twice daily in week 2. Higher dosages are possible, for example 4.5 mg twice daily and even 6 mg twice daily. Tolerability was reported to be even better for a transdermal device, wherein, for example, 24 mg may be absorbed in 24 hours.
For example, oral formulations of rivastigmine tartrate as described above may be administered to a subject in need thereof so that a dose equivalent to 1.5 mg rivastigmine is administered twice daily, and this dose may be gradually increased to a maximal daily dose equivalent to between 9-12 mg rivastigmine. Intramuscular and subcutaneous rivastigmine administration can be e.g. from 0.75 mg twice daily to a maximal daily dose equivalent to 5-6 mg rivastigmine.
The compositions of the invention can be administered alone or in conjunction with other therapeutic modalities. It is appropriate to administer the pharmaceutical compositions of the invention as part of a treatment regimen involving other therapies, such as drug therapy, which comprises e.g. immunosuppressive drugs (e.g. methotrexate, azathioprine, cyclophosphamide, cladribine) and/or other agents used for the treatment of MS such as interferon (IFN)-β and copolymer-1.
In another aspect, there is provided a method of inhibiting inflammation in a subject afflicted with multiple sclerosis or a multiple sclerosis-related disease, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I) as defined herein, or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent. In another embodiment, said subject is afflicted with multiple sclerosis. In another embodiment, the method is used for improving motor function in said subject. In another embodiment, the method is used for reducing demyelination in said subject. In another embodiment, the method is used for inhibiting neurodegeneration associated with demyelination in said subject. In another embodiment, the method is used for inhibiting a clinical symptom associated with inflammation in said subject. In another embodiment, the method is used for inhibiting the progression of multiple sclerosis in said subject. In another embodiment, the method is used for treating a multiple sclerosis-related disease selected from the group consisting of: Neuromyelitis Optica (Devic's Disease), Diffuse Sclerosis, Transitional Sclerosis, Acute Disseminated Encephalomyelitis, and Optic Neuritis, wherein each possibility represents a separate embodiment of the present invention. In another embodiment, the method is used for treating a multiple sclerosis- related disease selected from the group consisting of virus-, bacteria- and parasite-related demyelinating degenerative brain disease wherein each possibility represents a separate embodiment of the present invention.
In another aspect, the invention provides a method of treating multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically- effective amount of a pharmaceutical composition comprising a compound of the general Formula (I) as defined herein, and pharmaceutically acceptable salts, hydrates or solvates thereof, and a pharmaceutically acceptable carrier, excipient or diluent, whereby the treatment inhibits neurological deterioration or improves non-cognitive functions in said subject. In another embodiment, the subject is not otherwise in need of treatment with rivastigmine or other phenyl carbamates.
In another aspect, the invention provides use of a compound of Formula (I) as defined herein or a pharmaceutically acceptable salt, hydrate or solvate thereof for the preparation of a medicament useful for treating multiple sclerosis and inhibiting clinical symptoms associated therewith. In various embodiments, the medicament is useful for inhibiting inflammation in a subject afflicted with multiple sclerosis or a multiple sclerosis- related disease, for improving motor function in a subject afflicted with multiple sclerosis, for reducing demyelination in a subject afflicted with multiple sclerosis, for inhibiting neurodegeneration associated with demyelination in a subject afflicted with multiple sclerosis, for inhibiting a clinical symptom associated with inflammation in a subject afflicted with multiple sclerosis or for inhibiting the progression of multiple sclerosis. Each possibility represents a separate embodiment of the present invention. In another embodiment, the medicament is useful for treating multiple sclerosis, whereby said medicament inhibits neurological deterioration or improves non-cognitive functions.
In another aspect, the invention provides a pharmaceutical composition comprising a compound of Formula (I) as defined herein, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for treating multiple sclerosis, whereby the composition inhibits neurological deterioration or improves non-cognitive functions.
In another aspect, there is provided a pharmaceutical composition comprising a compound of the general Formula (I) as defined herein, and pharmaceutically acceptable salts, hydrates or solvates thereof, for inhibiting inflammation in a subject afflicted with multiple sclerosis or a multiple sclerosis-related disease, for improving motor function in a subject afflicted with multiple sclerosis, for reducing demyelination in a subject afflicted with multiple sclerosis, for inhibiting neurodegeneration associated with demyelination in a subject afflicted with multiple sclerosis, for inhibiting a clinical symptom associated with inflammation in a subject afflicted with multiple sclerosis or for inhibiting the progression of multiple sclerosis. Each possibility represents a separate embodiment of the present invention.
In another embodiment, the composition is useful for treating a multiple sclerosis- related disease selected from: Neuromyelitis Optica (Devic's Disease), Diffuse Sclerosis, Transitional Sclerosis, Acute Disseminated Encephalomyelitis, and Optic Neuritis, or a multiple sclerosis-related disease selected from virus-, bacteria- and parasite-related demyelinating degenerative brain disease wherein each possibility represents a separate embodiment of the present invention.
In another embodiment the compound is N-ethyl-N-methyl-3-[l- (dimethylamino)ethyl] -phenyl carbamate or a pharmaceutically acceptable salt, hydrate or solvate thereof.
In another embodiment, the compound is (S)-N-ethyl-N-methyl-3-[l- (dimethylamino)ethyl] -phenyl carbamate (rivastigmine) or a pharmaceutically acceptable salt, hydrate or solvate thereof.
In another embodiment, the compound is rivastigmine tartrate. In another embodiment, the subject is selected from the group consisting of humans and non-human mammals. In another embodiment the compound is administered in a manner selected from the group consisting of: orally, subcutaneously and intramuscularly.
In another embodiment said compound is formulated in a form selected from the group consisting of extended release, sustained release and controlled-release formulations. In another embodiment, said compound is formulated in the form of a transdermal delivery device.
In another embodiment, the subject is in a state of remission.
In another embodiment the composition is useful for the preparation of a medicament for treating relapsing-remitting multiple sclerosis.
The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, however, in no way be construed as limiting the broad scope of the invention.
EXAMPLES
Immunomodulation of Multiple Sclerosis by Rivastigmine Example 1
Materials: Rivastigmine hydrogen tartrate.
Experimental animals. C57BL/6JOlaHsd female mice (Harlan) were housed under specific pathogen-free conditions in the Animal Facility at the Hebrew University Medical School, in accordance with NIH guidelines for the care and use of laboratory animals.
Experimental Autoimmune Encephalomyelitis (EAE) induction and evaluation. EAE was induced in 8-week-old female C57BL/6 mice by injecting 125mg of myelin oligodendrocyte glycoprotein 35-55 peptide (MOG 35-55; synthesized by Sigma Laboratories, Israel) emulsified in complete Freund's adjuvant (CFA) containing 5 mg/ml heat-killed Mycobacterium tuberculosis (Mt), subcutaneously (s.c.) into the left para- lumbar region. Immediately thereafter, and again at 48h, the mice were inoculated i.p. with 0.5 ml of pertussis toxin (250 ng). An additional injection of MOG35-55 peptide in CFA was delivered 7 days later into the right para-lumbar region. All animals were examined daily and evaluated for clinical signs of disease. The clinical status of the mice was scored according to the following scale: 0, without clinical disease; 1, tail weakness; 2, hind limb weakness sufficient to impair righting; 3, one limb plagic; 4, paraplegia with forelimb weakness; 5, quadriplegia; 6, death. Evaluation of central nervous system (CNS) pathology. The spinal cords and brains were removed, fixed in 4% buffered formalin, sectioned, and routinely processed for paraffin embedding. Sections prepared from the same level were stained with hematoxylin and eosin. Inflammatory foci containing at least 20 perivascular mononuclear cells were counted in each section. The tissues were sampled on day 32 after disease induction. EAE treatment. Female C57BL were injected daily with rivastigmine from the day of disease induction with 0.75 mg/kg/day, administered either intraperitoneally (i.p.) or subcutaneously (s.c). Control animals received the same volume of vehicle (normal saline or phosphate buffered saline, PBS).
Lymphocyte proliferation assay. Pooled lymph node cells (LNCs) were prepared from inguinal, axillary and mesenteric lymph nodes from naive mice or from mice that had been inoculated s.c. 9 days earlier with MOG 35-55 peptide in CFA with or without treatment with rivastigmine. The in vitro response of the LNCs was assayed in triplicate wells of 96-well flat-bottom microtiter plates. A total of 2x105 LNCs, suspended in 0.2 ml RPMI supplemented with 5% fetal calf serum (FCS), was added to each well with or without lμg/ml concanavalin A (Con A) (Sigma, USA) or lOμg/ml of phytohemaglutinin (PHA) or 50μg/ml of lipopolysaccharide (LPS) or lOOμg/ml of MOG 35-55 peptide. At 48 hrs after seeding, lμCi 3(H)Thymidine (Amersham, UK) was added to each well and the plates were incubated for an additional 18 hrs. They were then harvested with a semiautomatic harvester onto a glass fiber filter, and the radioactivity was determined by liquid scintillation.
Determination of cytokine production The levels (pg/ml) of the inflammatory cytokines, tumor necrosis factor-a (TNF-α) and interferon-γ (INF-γ), and of the anti inflammatory cytokine interlukine-10 (IL-IO), were measured in supernatants of lymphocyte cultures by a solid phase ELISA (R&D, Minneapolis, MN, USA). This assay employs a quantitative "sandwich" enzyme immunoassay technique (Barak et al., 2002).
Cytotoxicity assay. Lymphocyte viability was determined by trypan blue (0.4%, Sigma) exclusion. All cells were viable under the experimental conditions. Statistical analysis. Group differences were analyzed by One Way Repeated Measures Analysis of Variance (One Way ANOVA), according to Dunnet's method. Differences in disease severity were analyzed using the Mann - Whitney Rank sum test. A difference between the groups of P < 0.05 was considered significant.
Example 1.1; Clinical amelioration of EAE by Rivastigmine
Treatment with rivastigmine from the day of disease induction clearly reduced the clinical symptoms of EAE. The reduction in clinical symptoms was achieved when rivastigmine was injected either intraperitoneally (Figure 1) or subcutaneously (Figure 2). A significant reduction in disease severity was observed in three independent experiments (Figure 3).
As summarized in Table 1, intraperitoneal treatment with rivastigmine 0.75 mg/kg/day caused a 55% reduction in disease severity, 57% reduction in the cumulative score and a significant delay in disease onset.
Table 1. Amelioration of EAE clinical parameters in mice by i.p. rivastigmine treatment.
Treatment Mean disease Cumulative score# Disease onset day severity
EAE+ placebo 3.5 ± 0.3 59.7 ± 7.8 16 3 ± 1.2
EAE+ rivastigmine 1.6* ± 0.3 25.4* ± 4. 6 20 7* ± 1.0
The data are expressed as the mean score ± SE, in 9 control-PBS treated mice, 6 mice receiving rivastigmine.
# Cumulative score: mean number of days that the animals were sick x clinical score
* Significant difference between control and treated groups. The mice were injected i.p. with rivastigmine 0.75mg/kg/day in treated groups and with vehicle in the control group.
When rivastigmine was injected subcutaneously at the same daily dose disease severity and cumulative score were reduced by about 40% as summarized in Table 2. Table 2 Amelioration of EAE clinical parameters in mice by s.c. Rivastigmine treatment.
Figure imgf000031_0001
The data are expressed as the mean score ± SE, in 18 control-PBS treated mice, 16 mice receiving rivastigmine. # Cumulative score: number of days animals were sick x clinical score
* # Cumulative score: mean number of days that the animals were sick x clinical score
* Significant difference between control and treated groups. The mice were injected s.c. with rivastigmine 0.75mg/kg/day in treated groups and with vehicle in the control group.
The inflammatory process in EAE involves activation of CNS immunocompetent cells, i.e. astrocytes and microglia, and extravasation of activated T cells and macrophages through vessel walls and their subsequent entry and accumulation in inflammatory sites. In order to determine how specific cell types are affected by the treatment, the inhibitory effect of rivastigmine on the production of inflammatory mediators by CNS glial cells and on T cell reactivity was tested.
Example 1.2: Ex-vivo reactivity of lymphocytes is inhibited by rivastigmine
T cell reactivity towards the encephalitogenic MOG peptide was inhibited by daily injection of rivastigmine as shown by the reduction in the stimulation index from 14.4 to 4.9 (Figure 4A). The T-cell reactivity towards the mitogens ConA and LPS was also significantly reduced (Figure 4B).
In addition, ex vivo secretion of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) was also markedly decreased by rivastigmine treatment (Figures 5A and 5B). Lymphocytes from MOG-induced EAE mice, PBS and rivastigmine treated (0.75mg/kg/day) were analyzed for INFγ secretion (A) and TNF α secretion (B) in the presence of MOG encephalitogenic peptide 35-55 (100 μg/ml). The figures show suppressive effect of rivastigmine on the secretion of the pro-inflammatory cytokines.
Example 1.3: Effect on CNS inflammation in vivo
Daily treatment with rivastigmine 0.75mg/kg from the day of induction ameliorated clinical severity. Clinical amelioration was accompanied by a reduction in CNS inflammatory cellular infiltrates (Table 3).
Table 3 Amelioration of CNS inflammatory infiltrates by rivastigmine treatment
Figure imgf000032_0001
Brains and spinal cords of PBS and rivastigmine treated mice were fixed and processed for pathological examination of tissue sections. The data are expressed as the mean ± SE number of inflammatory foci (containing at least 20 perivascular mononuclear cells) in each section of tissue meninges and parenchyma.
Example 1.4; Clinical amelioration of EAE by Rivastigmine controlled release administration
Mini osmotic pumps (Alzet, 2004), containing rivastigmine that was released at the rate of 0.082mg/24hours or PBS as placebo treatment for 28 days, were implanted subcutaneously, one day before disease induction.
EAE induction and disease severity assessment were performed as described above. As can be seen in Figure 6, treatment with rivastigmine clearly reduced the clinical symptoms of EAE. As summarized in Table 4, controlled release of rivastigmine 0.082mg rivastigmine/24hours/mouse caused a significant reduction in disease severity and cumulative score, and a significant increase in survival of the mice.
Table 4 Amelioration of EAE clinical parameters in mice by Rivastigmine treatment via a miniosmotic pump.
Figure imgf000033_0001
Example 1.5: amelioration of EAE with a control drug
Figure 7 and table 5 hereinbelow summarize the course of EAE under daily treatment with s.c. 2mg/day/mouse of the known drug glatiramer acetate (GA, also known as Copaxone) as compared with placebo group. EAE was induced and evaluated as described above.
Table 5 Amelioration of EAE clinical parameters in mice by GA treatment
Figure imgf000033_0002
Example 2 Materials: Rivastigmine hydrogen tartrate.
AChE inhibition in the CNS and in the periphery by rivastigmine: C57BL/6 female mice 8-week-old were injected s.c. with saline (n=5) or rivastigmine 0.75 mg/kg (n=8) and sacrificed 45 min later. The brains (minus cerebellum) and tibialis muscles were rapidly removed and homogenized in 0.25mM phosphate containing 1% Triton X-100. Cholinesterase activity was measured in 25 μl aliquots of the supernatant using acetylthiocholine as a substrate by the spectrophotometic method of EUman et al (Ellman et al. , A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961;7:88-95). Percent inhibition of cholinesterase in brain and muscle by rivastigmine treatment was calculated by comparison with the value obtained in saline- injected controls.
EAE induction and treatment: EAE was induced in 8-week-old female C57BL/6 mice by injecting s.c. into the left para-lumbar region 125 μg of myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55; synthesized by Sigma Laboratories, Israel) emulsified in complete Freund's adjuvant (CFA) containing 5 mg/ml heat-killed Mycobacterium tuberculosis. Immediately thereafter, and, again, at 48 h, the mice were inoculated i.p. with 0.5 ml of pertussis toxin (350 ng). An additional injection of MOG35- 55 peptide in CFA was delivered 7 days later into the right para-lumbar region, as described previously (Irony-Tur-Sinai et ah, 2006). All animals were examined daily and evaluated for clinical signs of disease according to the common scale. Mice were injected daily s.c. with rivastigmine 0.75 mg/kg in a volume of 0.2 ml/mouse from the day of induction of EAE. Mecamylamine (Sigma, USA) (10 mg/kg) was injected s.c. 30 min. before rivastigmine or alone. Control animals received the same volume of normal saline, 0.2 ml/mouse/day. To provide continuous AChE inhibition, mice were implanted s.c with ALZET© (DURECT, Cupertino, CA, USA) mini-osmotic pumps, according to the manufacturer's instructions. Pumps were loaded with 3 mg rivastigmine in 220 μl saline each, corresponding to a daily dose of 4.8 mg/kg (0.8 x 6, as rivastigmine half life in serum is 4 hrs.) for 28 days. Control animals were implanted with pumps releasing the same volume of saline.
Evaluation of CNS pathology: Histological studies were performed on separate groups of animals treated for 21 days (n=6, n=7 in the control and rivastigmine groups, respectively) according to the above protocol. The spinal cords were removed 21 days after disease induction (at disease peak) after transcardial perfusion as described previously (Irony-Tur-Sinai et ah, 2006). Longitudinal sections were cut to include the majority of the length of the spinal cord including both gray and white matter, and stained with hematoxylin and eosin. Inflammatory foci containing at least 20 perivascular mononuclear cells were counted in each section. Sections were stained with luxol-fast- blue for myelin visualization and analyzed by light microscopy. For immunohistochemistry sections were incubated with primary antibodies anti- neuro filament and anti CD68 (1:300 dilution) and processed according to standard procedures (Das Sarma et ah, 2000). Quantification of the pathologic parameters was based on the methods previously described for the pathologic evaluation of EAE. For inflammation: none (0), a few inflammatory cells (1), organization of perivascular infiltrates (2), increasing severity of perivascular cuffing with extension into the adjacent tissue (3). Inflammatory foci containing at least 20 perivascular mononuclear cells were counted in each section. The degree of myelin loss, and axonal damage was graded as absent (0), weak (1), moderate (2) and severe (3). For each animal 3 section were studied. The grade of the pathologic parameter was added and the final score was divided by the number of mice in that group.
Mouse lymphocyte proliferation assay: Pooled lymph node cells (LNCs) were prepared from inguinal, axilliary and mesenteric lymph nodes of mice that had been inoculated 9 days earlier with MOG 35-55 peptide in CFA with or without treatment.
Spleens from same animals were analyzed by flow cytometry (see below). The ex vivo response of the LNCs was assayed as described previously (Irony-Tur-Sinai et al., 2006).
In addition, T-cell reactivity was tested during the various stages of disease (days 14- disease onset, 21- disease peak and day 28 after immunization- chronic phase), T-cells were obtained from spleens and tested as above. The results are expressed as Stimulation
Index (SI) according to the equation: SI=Mean cpm of the stimulated cells/mean cpm of the unstimulated cells.
Cell treatment with anti-sense: Cells were incubated for 24 hrs with anti-sense (AS) to the α7 nAChR or α5 nAChR as a negative control. After the incubation, cells were treated with rivastigmine as indicated. The AS used was comprised of phosphorothioate- bond nucleotides (IDT Technologies, Rehovot, Israel) and the sequences used were:
AS- α7: GTCTTTCTCGGCTTCTTCTCT (SEQ ID NO:1) AS- α5: TCACCTCACTCTCATCCTGGC (SEQ ID NO:2) Preparation of RNA and cDNA: Total RNA was prepared using an SV total RNA kit (Promega, Madison, WI, USA). For determination of IL- 17 mRNA level, RNA was prepared from spleens of mice on day 9 after induction of EAE as described above. cDNA was prepared from 200 μg of total RNA, using MuLV reverse transcriptase (Applied Biosystems, Warrington, UK) and random hexamers according to the manufacturer's instructions for first-strand cDNA synthesis.
Quantitative Real-Time Polymerase Chain Reaction (PCR): Semi-quantitative real-time PCR reaction was performed as previously described (Nizri et al., 2006). The results for gene expression were normalized according to the HPRT gene for α7 nAChR or 18S for IL- 17. The primers used were: HPRT forward: CAGCCCAAAATGGTTAAGGT (SEQ ID NO:3);
HPRT reverse: CGAGAGGTCCTTTTCACCAGC (SEQ ID NO:4). α7 nAChR forward: GACTGTTCGTTTCCCAGATGG (SEQ ID NO:5); α7 nAChR reverse: ACGAAGTTGGGAGCCGACATCA (SEQ ID NO:6). 18S forward: TCGAGGCCCTGTAATTGGAA (SEQ ID NO:7); 18S reverse: CCCTCCAATGGATCCTCGTT (SEQ ID NO:8). IL- 17 forward: CCGCAATGAAGACCCTGATAGA (SEQ ID NO:9);
IL-17 reverse: TCATGTGGTGGTCCAGCTTTC (SEQ ID NO:10)
Analysis of cytokine production: The presence of IFN-γ, TNF-α, and IL-IO was determined in the culture media of the lymph node cells incubated in the presence of
MOG 35-55 using a commercially available ELISA kit (Biolegend, San Diego, CA, USA). The lymphocytes were collected from the mice 9 days after inoculation with MOG 35.55 with or without rivastigmine treatment (0.75 mg/kg, s.c).
Flow cytometry analysis: Surface markers of leukocytes from pooled spleen cells obtained from control and treated mice (as described in the "Mouse lymphocyte proliferation assay") were analyzed. Cell suspensions were prepared as described previously (Irony-Tur-Sinai et ah, 2006). Stained cells were counted in a fluorescence- activated cell sorter (FACScan, Becton Dickinson, San Jose, CA USA). The following antibodies were used: anti-CD4-FITC (clone GK 1.5, Pharmingen, USA), anti-CD8-FITC (clone 53-6.7, Pharmingen, USA), anti-CD 1 Ib-FITC (clone Ml/70, Pharmingen, USA), anti-MHC class II-FITC (clone 25-5-16S, Serotec, Kidlington, UK). The following isotype controls were used: FITC-conjugated rat IgG2bK (clone A 95-1, Pharmingen, USA), FITC-conjugated rat IgG2aκ (clone R35-95, Pharmingen, USA), FITC-conjugated mouse IgM (cat.no. X 0934, DakoCytomation, Denmark).
Spatial memory in water maze: Rivastigmine is used in clinical practice for the treatment of cognitive dysfunction. After appearance of the clinical signs of EAE it is impossible to determine whether any decrement in spatial memory is due to cognitive dysfunction or to neurological motor impairment caused by the disease. Hence, animals were tested for spatial memory in the Morris water maze (MWM) before appearance of EAE clinical signs (7-14 days post induction). The MWM consisted of a circular pool (150 cm in diameter, 60 cm high) filled to depth of 20 cm with water at a temperature of 22 ± I0C. A transparent glass escape platform (12 cm in diameter) was placed 1 cm below the surface, midway between the centre and rim of the pool in one quadrant where it remained for all the acquisition trials. To begin each trial the mouse was placed into the water, facing the maze wall, from one of four start positions evenly spaced around the pool (N, S, E & W). Start positions were chosen randomly at the beginning of each day for all mice. If the mouse failed to find the escape platform within 120 s it was placed on it for 20 sec and then removed from the pool. The mouse was given two trials a day for 5 days between 15:00 and 19:00 hr with an inter-trial interval of 15 min (Wang et al, 2000).
Statistical analysis: Group differences were analyzed by t-test for two groups or by One Way Analysis of Variance (One Way ANOVA) for several groups, according to the Holm-Sidak method. Differences in disease severity were analyzed using the Mann - Whitney Rank sum test. For analysis of real-time PCR ΔCt was calculated for each sample according to the formula: ΔCt = Ct target- Ct endogenous and data was analyzed using ΔCt. The Morris water maze data was analyzed by One Way Repeated Measures ANOVA according to Holm-Sidak method. P < 0.05 was considered significant.
Example 2.1; Amelioration of EAE by treatment with rivastigmine
In order to assess the influence of rivastigmine on CNS inflammation, MOG- induced EAE mice were treated from the day of induction with 0.75 mg/kg (s.c). This dose of drug inhibited brain cholinesterase by 56.1 ± 2.0% and muscle cholinesterase by 42.4 ± 2.8%, 45 min after injection. Treatment with rivastigmine reduced disease severity and cumulative score by 50- 54% (Fig. 8A and Table 6), and delayed disease onset by 3.5 days (p<0.001). In order to verify previous suggestion that the anti-inflammatory properties of AChEI depend on the presence of nAChR, the ability of a nicotinic receptor blocker, mecamylamine, to reverse the effect of rivastigmine, was tested. Daily treatment with mecamylamine significantly reduced the effects of rivastigmine (Fig. 8A and Table 6). Treatment with mecamylamine alone did not change the course of the disease or its severity (n=7, p=0.15). Treatment with rivastigmine after the appearance of clinical signs did not change disease severity.
In Fig. 8A, results for control and rivastigmine groups are presented as a summary of 3 different experiments (n=27, n=22, respectively). For mecamylamine group n=10. In Fig. 8B, the results are a summary of 2 different experiments n=10 for each group. Table 6. Amelioration of EAE by rivastigmine according to clinical and pathological parameters.
Figure imgf000038_0001
The data are expressed as the mean ± S.E. Treated mice were inoculated daily s.c. with 0.75 mg/kg rivastigmine and with saline in the control group.
# Cumulative score: number of days animal was sick x clinical score ** pO.Ol, *** pO.OOl vs. control.
When rivastigmine was delivered by mini-osmotic pumps to ensure more uniform and continuous AChE inhibition, disease severity and cumulative score were reduced by 73% and 72%, respectively (Fig. 8B and Table 7).
Table 7. Amelioration of EAE by continuous treatment with rivastigmine (mini- osmotic pumps).
Figure imgf000038_0002
The data are expressed as the mean ± S.E. Treated mice were implanted s.c. with miniosmotic pumps delivering rivastigmine at 4.8 mg/kg/day (see materials and methods) and with saline in the control group.
# Cumulative score: number of days animal was sick x clinical score ** p=0.006, *** p<0.001.
Example 2.2: Attenuation of CNS inflammation, demyelination and neuronal damage by rivastigmine treatment
The marked differences in clinical scores in rivastigmine-treated and saline treated mice were consistent with the histological analysis of spinal cord tissues removed at the peak of disease. In the placebo (saline treated) group, pronounced perivascular infiltration was observed, with marked demyelination, microglia activation and axonal damage (Fig. 9 A, B, C, respectively). In the rivastigmine-treated group only minimal inflammation was found. A comparison of the number of inflammatory infiltrates showed a 75% reduction in the treated group (from 42.4 ± 3.4 to 10.6 ± 7, p=0.002, for control and rivastigmine groups, respectively). Demyelination, degree of activation and number of microglia were also reduced (Fig. 9 D, E). Altogether, these reduced parameters of inflammation culminated in decreased axonal loss and damage as manifested by neurofilament staining (Fig. 9 F). A grade of the various pathological parameters is presented in Table 8. Treatment with rivastigmine decreased consistently and significantly by 50-60% all pathological parameters evaluated.
Table 8. Grading of various pathological parameters and assessment of treatment effects.
Figure imgf000039_0001
The various pathological parameters were graded as described in the Materials and Methods section. Animals (n=6, n=7 for control and rivastigmine, respectively) were treated for 21 days with same protocol used in Figure 8 A. Spinal cord tissue was obtained after transcardial perfusion and stained as described in Materials and Methods. * p<0.05,
Example 2.3: Rivastigmine treatment reduced MOG-specific T-cell proliferation and pro-inflammatory cytokine production
Treatment with rivastigmine markedly decreased the proliferation of MOG specific encephalitogenic T-cells (Fig. 10A). Pooled lymph node cells from treated and non-treated animals were tested in vitro for their reactivity by a standard thymidine proliferation assay. The stimulation index dropped from 8.6 in the control group to 3.8 in the rivastigmine- treated group (p=0.015). Upon recognition of their cognate antigen, encephalitogenic T-cells secrete proinflammatory cytokines, which contribute to pathogenesis of EAE. Hence, cytokine production upon MOG stimulation was tested. Rivastigmine treatment was associated with reduced production of TNF-α and IFN-γ (Fig. 10B). Expression of IL- 17 mRNA was also inhibited upon treatment (Fig. 10C). On the other hand, production of the antiinflammatory cytokine IL-IO was not changed (Fig. 10D).
In Fig. 1OA, the results summarize 3 different experiments (n=12 for each group) and are expressed as Stimulation Index (SI) according to the equation: SI=Mean cpm of the stimulated cells/mean cpm of the unstimulated cells. *p=0.015 vs. control. In Fig. lOB-D, the results summarize 3 different experiments (n=12 for each group), *p=0.01, **p=0.0032 vs. control. For IL- 17 mRNA expression the results summarize 2 different experiment (n=8 for each group).
T-cell reactivity was further tested at 14, 21 and 28 days after immunization, corresponding to disease onset, peak and chronic phase, respectively. As can be seen, (Figure 15A) T-cell proliferation was reduced by 40-80% following rivastigmine treatment as well as the production of pro inflammatory cytokine (40-80%) (Figure 15B-D).
In Figure 15, for rivastigmine treatment n=3 for each time point, for control, n=3 for day 14 and n=4 each for days 21, 28. *p<0.05; **p<0.01; ***p<0.001.
Example 2.4: Effect of rivastigmine on T-cells is α7 nAChR dependent
Rivastigmine reduced Concanvalin-A (ConA)-induced proliferation by 70% (Fig. HA). The effect of rivastigmine was abolished by α-bungarotoxin (α-btx) a nicotinic antagonist of neuronal and muscle nicotinic receptors. Since the muscle-type nAChR is not expressed on T-cells, α-btx blockade can be attributed to the α7 nAChR. This was confirmed by pre-incubation of T-cells with anti-sense (AS) to this receptor and to the α5 nAChR. Fig HB demonstrates that AS to α7 nAChR specifically and efficiently reduced the α7 mRNA level by 88% (p=0.005), while level of α5 mRNA was unaffected. After levels of α7 mRNA were down-regulated by this protocol, cells were incubated with rivastigmine and tested for its ability to suppress mitogen-induced proliferation. It was found that T-cells treated with AS-α7 were less sensitive to the effects of rivastigmine, while those treated with AS- α5 were still responsive (Fig. HC). In Fig. 1 IA, the results are expressed as fraction of stimulated cells and summarize 3 different experiments, where cells were incubated in triplicates. **p=0.001 vs. Concanavalin A (ConA)-treated cells. In Fig. HB, the results are the summary of 3 different experiments. **p=0.005. In Fig. HC, the results are expressed as fraction of stimulated cells (for each treatment) and summarize 3 different experiments where cells were incubated in triplicates. ***p<0.001.
Example 2.5: Treatment with rivastigmine reduced number of MHC-II+ cells
Splenocytes from animals treated with the AChEI were analyzed for several cell markers associated with the inflammatory response and antigen presentation. The number of MHC-II+ cells was reduced by 30% (from 47% to 32%) (Fig. 12). There was no change in the intensity of the signal, indicating that the number of MHC-II+ cells was reduced, and not the number of MHC-II molecules expressed per cell. This was accompanied by a reduction in CDl Ib+ (macrophages) (from 28% to 19%), whereas there was no change in CDl Ic+ (dendritic cells) or CD19+ (B-cells). Taken together these data support that rivastigmine treatment affected macrophage antigen presentation ability.
In addition, as seen in Figure 14, after 9 days of treatment with rivastigmine (0.75 mg/kg), the ability of APC to induce MOG-specific T-cell proliferation was reduced by 25% (pO.OOl). In Fig. 12, presented are results of one representative experiment out of 3 (n=12 for each group). In Fig. 13, the results are the mean of 2 different experiments (n=6 for each group). ***p<0.001
Example 2.6: Improvement of spatial memory function upon rivastigmine treatment
Induction of EAE was associated with impairment in spatial memory in the MWM, compared to that in naϊve mice (Fig. 13). This was characterized by a failure to improve escape latencies over time and was indicated by the difference from those of controls on days 4 and 5 (p<0.025, p<0.005, respectively). Treatment with rivastigmine restored spatial memory to the level of that in control mice. To ascertain that the difference in latency between the groups is not due to subtle motor deficit, the time to reach a visible platform was compared. No significant difference was found under these conditions (10.1 ± 1.4 sec, 11.7 ± 1.6 sec, 10.4 ± 1.3 sec, for naive, EAE and EAE + rivastigmine groups, respectively).
In Fig. 13, *p=0.02, **p=0.003 vs. EAE-+Rivastigmine and control groups. The results are a summary of two different experiments, n=16 for each group.
Inflammatory infiltrates were found near the hippocampus in several mice (Figure 16).
References
Borovikova, L.V., S. Ivanova, M. Zhang, H. Yang, G.I. Botchkina, L.R. Watkins, H. Wang, N. Abumrad, J. W. Eaton, K.J. Tracey 2000. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 405: 458—462.
D'Intino et al., 2005. Cognitive deficit associated with cholinergic and nerve growth factor down-regulation in experimental allergic encephalomyelitis in rats. Proc Natl Acad Sci U S A. 102(8):3070-5.
Das Sarma J, Fu L, Tsai JC et al. 2000 Demyelination determinants map to the spike glycoprotein gene of coronavirus mouse hepatitis virus. J Virol.;74:9206-9213.
De Jager PL, Hafler DA. New therapeutic approaches for multiple sclerosis. Annu Rev Med. 2007;58:417-432.
Eliakim, R., and F. Karmeli. 2003. Divergent effects of nicotine administration on cytokine levels in rat small bowel mucosa, colonic mucosa, and blood. Isr. Med. Assoc. J. 5:178-180.
Hauser SL, Oksenberg JR. 2006. The neurobiology of multiple sclerosis: genes, inflammation, and neurodegeneration. Neuron;52:61-76.
Henze, T., et al., 2006. Symptomatic treatment of multiple sclerosis. Multiple Sclerosis Therapy Consensus Group (MSTCG) of the German Multiple Sclerosis Society. Eur Neurol. 56, 78-105. Irony-Tur-Sinai M, Grigoriadis N, Lourbopoulos A et al. 2006. Amelioration of autoimmune neuroinflammation by recombinant human alpha-fetoprotein. Exp Neurol.;198:136-144.
Kawashima, K., Fujii, T. 2003 a. The lymphocytic cholinergic system and its contribution to the regulation of immune activity. Life Sci.74 675-696.
Kawashima K, Fujii T. 2003b. The lymphocytic cholinergic system and its biological function. Life Sci.;72:2101-2109.
Langley et al., 2004. Central but not the peripheral action of cholinergic compounds suppresses the immune system. J Neuroimmunol. 148(l-2):140-5. Nizri E, Adani R, Meshulam H, Amitai G, Brenner T. 2005. Bifunctional compounds eliciting both anti-inflammatory and cholinergic activity as potential drugs for neuroinflammatory impairments. Neurosci Lett. 376(l):46-50.
Nizri E, Hamra-Amitay Y, Sicsic C, Lavon I, Brenner T. 2006. Anti-inflammatory properties of cholinergic up-regulation: A new role for acetylcholinesterase inhibitors. Neuropharmacology 50(5):540-7.
Nizri, E., Irony-Tur-Sinai, M., Lavon, I., Meshulam, H., Amitai, G., Brenner, T., 2007a. IBU-Octyl-Cytisine, a novel bifunctional molecule eliciting anti-inflammatory and cholinergic activity, ameliorates CNS inflammation by inhibition of T-cell activity. International Immunopharmacology. 7, 1129-1139. Nizri, E., et al., 2007b. The role of cholinergic balance perturbation in neurological diseases. Drug News Perspect. 20, 421-9.
Reale M, Iarlori C, Gambi F et al. 2004. Treatment with an acetylcholinesterase inhibitor in Alzheimer patients modulates the expression and production of the pro- inflammatory and anti-inflammatory cytokines. J Neuroimmunol.148: 162-171. Reale M, Iarlori C, Gambi F, Feliciani C, Isabella L, Gambi D. 2006. The acetylcholinesterase inhibitor, Donepezil, regulates a Th2 bias in Alzheimer's disease patients. Neuropharmacology. Apr;50(5):606-13. Epub 2006 Jan 30.
Saeed et al., 2005. Cholinergic stimulation blocks endothelial cell activation and leukocyte recruitment during inflammation. J Exp Med. 201(7): 1113-23. Sandborn, W.J., WJ. Tremaine, K.P. Offord, G.M. Lawson, B.T. Petersen, K.P. Batts, LT. Croghan, L.C. Dale, D.R. Schroeder, and R.D. Hurt. 1997. Transdermal nicotine for mildly to moderately active ulcerative colitis. A randomized, double-blind, placebo- controlled trial. Ann. Intern. Med. 126:364-371 Sopori, M.L., W. Kozak, S.M. Savage, Y. Geng, MJ. Kluger. 1998. Nicotine- induced modulation of T Cell function. Implications for inflammation and infection. Adv. Exp. Med Biol. 437:279-289.
Sykes, A.P., C. Brampton, S. Klee, CL. Chander, C. Whelan, M.E. Parsons. 2000. An investigation into the effect and mechanisms of action of nicotine in inflammatory bowel disease. Inflamm. Res. 49:311-319.
Tyagi, E., Agrawal, R., Nath C, Shukla R. 2007. Effect of anti-dementia drugs on LPS induced neuroinflammation in mice. Life Sci.;80: 1977-1983.
Wang, R.,H., Bejar, C, Weinstock, M. 2000. Gender differences in the effect of rivastigmine on brain cholinesterase activity and cognitive function in rats. Neuropharmacology. 39: 497-506.
Wang, H., H. Liao, M. Ochani, M. Justiniani, X. Lin, L. Yang, Y. Al Abed, H. Wang, C. Metz, EJ. Miller, et al. 2004. Cholinergic agonists inhibit HMGBl release and improve survival in experimental sepsis. Nat. Med. 10:1216-1221.
Wang, H., M. Yu, M. Ochani, CA. Amelia, M. Tanovic, S. Susarla, J.H. Li, H. Yang, L. Ulloa, Y. Al-Abed, et al. 2003. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature. 421:384-388.

Claims

1. A method of inhibiting inflammation in a subject afflicted with multiple sclerosis or a multiple sclerosis-related disease, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of Formula (I):
Figure imgf000045_0001
wherein
Ri is hydrogen, lower alkyl, cyclohexyl, allyl or benzyl,
R2 is hydrogen, methyl, ethyl or propyl, or
Ri and R2 together with the nitrogen to which they are attached form a morpholino or piperidino radical,
R3 is hydrogen or lower alkyl,
R4 and R5 are the same or different and each is a lower alkyl, and the dialkylaminoalkyl group is in the meta, ortho or para position, or a pharmaceutically acceptable salt, hydrate or solvate thereof and a pharmaceutically acceptable carrier, excipient or diluent.
2. The method of claim 1, wherein said subject is afflicted with multiple sclerosis.
3. A method according to claim 2 for improving motor function in said subject.
4. A method according to claim 2 for reducing demyelination in said subject.
5. A method according to claim 2 for inhibiting neurodegeneration associated with demyelination in said subject.
6. A method according to claim 2 for inhibiting a clinical symptom associated with inflammation in said subject.
7. A method according to claim 2 for inhibiting the progression of multiple sclerosis in said subject.
8. A method according to claim 1 for treating a multiple sclerosis-related disease selected from the group consisting of: Neuromyelitis Optica (Devic's Disease), Diffuse Sclerosis, Transitional Sclerosis, Acute Disseminated Encephalomyelitis, and Optic Neuritis.
9. A method according to claim 1 for treating a multiple sclerosis-related disease selected from the group consisting of virus-, bacteria- and parasite-related demyelinating degenerative brain disease.
10. The method of claim 1, wherein the compound is N-ethyl-N-methyl-3-[l- (dimethylamino)ethyl]-phenyl carbamate or a pharmaceutically acceptable salt, hydrate or solvate thereof.
11. The method of claim 1, wherein the compound is (S)-N-ethyl-N-methyl-3-[l-
(dimethylamino)ethyl] -phenyl carbamate (rivastigmine) or a pharmaceutically acceptable salt, hydrate or solvate thereof.
12. The method of claim 1, wherein the compound is rivastigmine tartrate.
13. The method of claim 1, wherein the subject is selected from the group consisting of humans and non-human mammals.
14. The method of claim 1, wherein the compound is administered in a manner selected from the group consisting of: orally, subcutaneously and intramuscularly.
15. The method of claim 1, wherein said compound is formulated in a form selected from the group consisting of extended release, sustained release and controlled-release formulations.
16. The method of claim 1, wherein said compound is formulated in the form of a transdermal delivery device.
17. The method of claim 1, wherein the subject is in a state of remission.
18. The method of claim 1, wherein the subject is afflicted with relapsing-remitting multiple sclerosis.
19. A method of treating multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically-effective amount of a pharmaceutical composition comprising a compound of the general Formula (I) as defined in claim 1, and pharmaceutically acceptable salts, hydrates or solvates thereof, and a pharmaceutically acceptable carrier, excipient or diluent, whereby the treatment inhibits neurological deterioration or improves non- cognitive functions in said subject.
20. The method of claims 19, wherein the compound is N-ethyl-N-methyl-3-[l-
(dimethylamino)ethyl]-phenyl carbamate or a pharmaceutically acceptable salt, hydrate or solvate thereof.
21. The method of claim 20, wherein the compound is (S)-N-ethyl-N-methyl-3-[l- (dimethylamino)ethyl] -phenyl carbamate (rivastigmine) or a pharmaceutically acceptable salt, hydrate or solvate thereof.
22. The method of claim 21, wherein the pharmaceutical composition comprises rivastigmine tartrate.
23. The method of claim 19, wherein said compound is formulated in a form selected from the group consisting of extended release, sustained release and controlled-release formulations.
24. The method of claim 19, wherein the subject is in a state of remission.
25. The method of claim 19, wherein the subject is afflicted with relapsing- remitting multiple sclerosis.
26. Use of a pharmaceutical composition comprising a compound of the general Formula (I):
Figure imgf000048_0001
wherein
Ri is hydrogen, lower alkyl, cyclohexyl, allyl or benzyl,
R2 is hydrogen, methyl, ethyl or propyl, or
Ri and R2 together with the nitrogen to which they are attached form a morpholino or piperidino radical,
R3 is hydrogen or lower alkyl,
R4 and R5 are the same or different and each is a lower alkyl, and the dialkylaminoalkyl group is in the meta, ortho or para position, and pharmaceutically acceptable salts, hydrates or solvates thereof, for the preparation of a medicament for inhibiting inflammation in a subject afflicted with multiple sclerosis or a multiple sclerosis-related disease, for improving motor function in a subject afflicted with multiple sclerosis, for reducing demyelination in a subject afflicted with multiple sclerosis, for inhibiting neurodegeneration associated with demyelination in a subject afflicted with multiple sclerosis, for inhibiting a clinical symptom associated with inflammation in a subject afflicted with multiple sclerosis or for inhibiting the progression of multiple sclerosis.
27. The use of claim 26 for treating a multiple sclerosis-related disease selected from: Neuromyelitis Optica (Devic's Disease), Diffuse Sclerosis, Transitional Sclerosis, Acute Disseminated Encephalomyelitis, and Optic Neuritis, or a multiple sclerosis-related disease selected from virus-, bacteria- and parasite- related demyelinating degenerative brain disease.
28. Use of a pharmaceutical composition comprising a compound of Formula (I) as defined in claim 26, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for the preparation of a medicament for treating multiple sclerosis, whereby the medicament inhibits neurological deterioration or improves non- cognitive functions.
29. The use of any one of claims 26-28, wherein the compound is N-ethyl-N- methyl-3-[l-(dimethylamino)ethyl]-phenyl carbamate or a pharmaceutically acceptable salt, hydrate or solvate thereof.
30. The use of any one of claims 26-28, wherein the compound is (S)-N-ethyl-N- methyl-3-[l-(dimethylamino)ethyl]-phenyl carbamate (rivastigmine) or a pharmaceutically acceptable salt, hydrate or solvate thereof.
31. The use of any one of claims 26-28, wherein the compound is rivastigmine tartrate.
32. The use of any one of claims 26-28, wherein the subject is selected from the group consisting of humans and non-human mammals.
33. The use of any one of claims 26-28, wherein the compound is administered in a manner selected from the group consisting of: orally, subcutaneously and intramuscularly.
34. The use of any one of claims 26-28, wherein said compound is formulated in a form selected from the group consisting of extended release, sustained release and controlled-release formulations.
35. The use of any one of claims 26-28, wherein said compound is formulated in the form of a transdermal delivery device.
36. The use of any one of claims 26-28, wherein the subject is in a state of remission.
37. The use of any one of claims 26-28, for the preparation of a medicament for treating relapsing-remitting multiple sclerosis.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102846612A (en) * 2011-06-30 2013-01-02 复旦大学 Application of Huperzine A in preparation of medicines preventing and treating multiple sclerosis disease
US20130184338A1 (en) * 2011-12-27 2013-07-18 Bio-Pharm Solutions Co., Ltd. Phenylpropyl carbamate derivatives for use in preventing or treating multiple sclerosis
WO2013118126A1 (en) 2012-02-12 2013-08-15 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Ladostigil therapy for immunomodulation

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999034782A1 (en) * 1998-01-12 1999-07-15 Novartis Ag Tts containing an antioxidant
US20030039692A1 (en) * 1998-10-01 2003-02-27 Jorg Ogorka Sustained release oral formulations
US20030119832A1 (en) * 1999-10-19 2003-06-26 David Goldblum Treatment of ocular disorders
US20060183733A1 (en) * 2005-02-11 2006-08-17 Stephen Wills Treating microvasculature diseases with acetyl cholinesterase inhibitors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999034782A1 (en) * 1998-01-12 1999-07-15 Novartis Ag Tts containing an antioxidant
US20030039692A1 (en) * 1998-10-01 2003-02-27 Jorg Ogorka Sustained release oral formulations
US20030119832A1 (en) * 1999-10-19 2003-06-26 David Goldblum Treatment of ocular disorders
US20060183733A1 (en) * 2005-02-11 2006-08-17 Stephen Wills Treating microvasculature diseases with acetyl cholinesterase inhibitors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NIZRI ET AL: "Anti-inflammatory properties of cholinergic up-regulation: A new role for acetylcholinesterase inhibitors", NEUROPHARMACOLOGY, vol. 50, no. 5, 1 April 2006 (2006-04-01), PERGAMON PRESS, OXFORD, GB, pages 540 - 547, XP005353225, ISSN: 0028-3908 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102846612A (en) * 2011-06-30 2013-01-02 复旦大学 Application of Huperzine A in preparation of medicines preventing and treating multiple sclerosis disease
US20130184338A1 (en) * 2011-12-27 2013-07-18 Bio-Pharm Solutions Co., Ltd. Phenylpropyl carbamate derivatives for use in preventing or treating multiple sclerosis
US9624164B2 (en) 2011-12-27 2017-04-18 Bio-Pharm Solutions Co., Ltd. Phenyl carbamate compounds for use in preventing or treating epilepsy
WO2013118126A1 (en) 2012-02-12 2013-08-15 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Ladostigil therapy for immunomodulation
US9867798B2 (en) 2012-02-12 2018-01-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Ladostigil therapy for immunomodulation

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