JP2008516961A - Carbamate compounds for use in treating neurodegenerative disorders - Google Patents

Abstract

［式中、
フェニルはフッ素、塩素、臭素およびヨウ素よりなる群から選択される１〜５個のハロゲン原子でＸで置換され；そして
Ｒ_１、Ｒ_２、Ｒ_３、Ｒ_４、Ｒ_５およびＲ_６は水素およびＣ_１〜Ｃ_４アルキルよりなる群から独立して選択され；ここで、Ｃ_１〜Ｃ_４アルキルは場合によりフェニルで置換されていてもよい（ここで、フェニルは場合によりハロゲン、Ｃ_１〜Ｃ_４アルキル、Ｃ_１〜Ｃ_４アルコキシ、アミノ、ニトロおよびシアノよりなる群から独立して選択される置換基で置換されていてもよい）］
よりなる群から選択される化合物またはその製薬学的に許容しうる塩もしくはエステルの治療的に有効な量を神経保護を必要とする被験体に投与することを含んでなる神経保護を与える方法に関する。The present invention relates to formula (I) and formula (II):
[Chemical 1]

[Where:
Phenyl is substituted with X with 1 to 5 halogen atoms selected from the group consisting of fluorine, chlorine, bromine and iodine; and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrogen and Independently selected from the group consisting of C ₁ -C ₄ alkyl; wherein C ₁ -C ₄ alkyl is optionally substituted with phenyl, where phenyl is optionally halogen, C ₁ -C _And optionally substituted with a substituent independently selected from the group consisting of ₄ alkyl, C ₁ -C ₄ alkoxy, amino, nitro and cyano)]
A method of providing neuroprotection comprising administering to a subject in need of neuroprotection a therapeutically effective amount of a compound selected from the group consisting of or a pharmaceutically acceptable salt or ester thereof. .

Description

[Cross-reference to related applications]
This application claims the benefit of US Provisional Application No. 60 / 619,402 filed October 15, 2004 and US Provisional Application No. 60 / 698,403 filed July 12, 2005. These two provisional applications are incorporated herein by reference.

[Field of the Invention]
The present invention relates generally to the fields of pharmacology, neurology and psychiatry, and to methods for protecting cells of the mammalian central nervous system from injury or injury. More particularly, the present invention provides methods for the use of certain carbamate compounds for neuroprotection.

[Description of related technology]
Various types of damage or trauma to the central nervous system (CNS) or peripheral nervous system (PNS) can cause serious and long-term neurological and / or psychiatric symptoms and disorders. One form this may take is the progressive death of neurons or other cells of the central nervous system (CNS), ie neurodegeneration or neuronal degeneration. For example, neurons as a result of Alzheimer's disease, multiple sclerosis, cerebrovascular disorder (CVA) attacks, traumatic brain injury, spinal cord injury, optic nerve degeneration, such as ischemic optic neuropathy or retinal degeneration and other central nervous system disorders Degeneration is a major medical and public health problem due to both its high incidence and the frequency of long-term sequelae. Animal studies and clinical trials have shown that amino acid transmitters (particularly glutamate), oxidative stress and inflammatory responses contribute strongly to cell death in these conditions.

  Upon injury or upon ischemic injury, damaged neurons release large amounts of the neurotransmitter glutamate, which is excitotoxic to surrounding neurons (Non-Patent Document 1; Non-Patent Document 2; Non-patent document 3; Non-patent document 4; Non-patent document 5). Glutamate is a negatively charged amino acid that is an excitatory synaptic transmitter in the mammalian nervous system. The concentration of glutamate can reach the millimolar range at the nerve ending, but its extracellular concentration is kept at a low level to prevent neurotoxicity. It has been recognized that glutamate can be toxic to neurons when given at high concentrations. The term “excitotoxicity” is used to describe the cytotoxic effects that glutamate (and other such excitatory amino acids) can have on neurons when applied at high doses.

  Physiologically, excessive release, inhibition of uptake, or both can lead to high levels of glutamate. Usually, low concentrations of extracellular glutamate are maintained by both neurons and astrocytes. Neurons store glutamate in intracellular stores and regulate its release. See Non-Patent Document 6. Astrocytes take up extracellular glutamate by a specific transporter and convert glutamate to glutamine, which is then released for neuronal uptake. See Non-Patent Document 7. In the process of excitotoxicity, glutamate is released in a self-perpetuating manner by neurons, resulting in excessive or sustained activation of glutamate receptors.

Such excessive glutamate-stimulated conjugation to energy-depleted neurons performed in conjunction with the impaired ability of neurosupportive astrocytes to sequester toxic levels of extracellular glutamate is necrotic. And leads to neuronal death by apoptosis. Various interventions are currently being investigated to reduce neuronal death associated with central nervous system damage and disease. See Non-Patent Document 8. Such therapies include glutamate release inhibitors, glutamate receptor antagonists, Cs2 + channel blockers, GABA receptor agonists, gangliosides, neurotrophic factors, calpain inhibitors, caspase inhibitors, free radical scavengers, immune and cellular metabolism modulators. Is done.

  For example, 1) Huntington's disease (HD) (Non-patent document 9); 2) Alzheimer's disease (AD) (Non-patent document 10); 3) Acupuncture (Non-patent document 11); 4) Latirism (Non-patent document 12); 5) Neurons in the pathophysiology of amyotrophic lateral sclerosis (ALS) and Parkinson's dementia on Guam (Non-Patent Document 13) and associated with stroke, ischemia and reperfusion (see Non-Patent Document 14) Several studies have shown the involvement of glutamate in pathology.

  Thus, damage to neurons can be caused by over-stimulation of receptors with excitatory amino acids including glutamate and aspartate (see Non-Patent Document 15). Indeed, the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors has many important roles in normal brain function, including synaptic transmission, learning and memory, and neuronal development (For example, see Non-Patent Document 15; Non-Patent Document 16). However, overstimulation of the glutamate receptor NMDA subtype results in increased free radical production and neuronal cell death, which can be regulated by antioxidants (see Non-Patent Document 17; Non-Patent Document 18).

  In addition, inflammation and oxidative stress are important components of pathology in many chronic neurodegenerative conditions. These symptoms include Alzheimer's disease (AD). Alzheimer's disease (AD) is characterized by neurofibrillary tangles and senile plaque accumulation, and extensive progressive degeneration of neurons in the brain. Senile plaques are rich in amyloid precursor protein (APP) encoded by the APP gene located on chromosome 21. The generally accepted hypothesis underlying the development of AD is excessive extracellular accumulation of beta-amyloid (Aβ) peptide, where abnormal proteolytic cleavage of APP has been shown to be toxic to neurons. (See Non-Patent Document 19; Non-Patent Document 20; Non-Patent Document 21; Non-Patent Document 22).

  Parkinson's disease (PD) is a progressive neurodegenerative injury characterized by motor dysfunction consisting of inability to move, stiffness, tremor and postural abnormalities. The disease involves substantial loss of dopaminergic neurons in the substantia nigra (SNpc) (see Non-Patent Document 23) and content in the striatum, reduction of dopamine synapses and vesicular transporters. (See, for example, Non-Patent Document 24), which is associated with a loss of integrity and functionality of nigrostriatal dopaminergic neurons.

  Neurons in the mammalian central (CNS) or peripheral (PNS) nervous system, including humans as a result of trauma, numerous types of injury, ischemia, metabolic abnormalities such as diabetes, hypoxia, toxins or surgical interventions Supporter cell death causes both acute and chronic and progressive loss and impairment of function. Accordingly, there is a need for the development of methods and compounds that can protect cells of the mammalian nervous system from this degeneration, ie, protect the nerves.

Choi et al. , (1988), Neuron 1: 623-634. Rothman et al. , (1984), J. Am. Neurosci. 4: 1884-1891 Choi end Rothman, (1990), Ann. Rev. Neurosci. 13: 171-182 David et al. , (1988), Exp. Eye Res. 46: 657-662 Drejer et al. , (1985), J. Am. Neurosci. 45: 145-151 Regan, R.A. F. Excitotoxicity and Central Nervous System Trauma, The Neurobiology of Central Nervous Trauma, New York, Oxford University Press, 94. 173-181 (Salzman SK, Faden Al. Eds) Robinson, M .; B. & Dow LA, Adv Pharmacol, 1997; 37: 69-115. Kermer et al. , Cell Tissue Res 298: 383-395, 1999. Coyle and Schwartz, (1976), Nature 263: 244-246. Maragos et al, (1987), TINS 10: 65-68. Nadler et al, (1978), Nature 271: 676-677. Spencer et al, (1986), Lancet 239: 1066-1067. Caine et al, (1986), Lancet 2: 1067-1070. Dykens et al, (1987), J. Am. Neurochem. 49: 1222-1228 Lipton et al. (1994) New Engl. J. et al. Med. 330: 613-621 Meldrum et al. (1990) Trends Pharm. Sci. 11: 379-387 Herin et al. (2001) J. Org. Neurochem. 78: 1307-1314 Rossato et al. (2002) Neurosci. Lett. 318: 137-140 Selkoe et al. , (1996), J. et al. Biol. Chem. 271: 487-498 Quinn et al. , (2001), Exp. Neurol. 168: 203-212 Mattson et al. (1997), Alzheimer's Dis. Rev. 12: 1-14 Fakuyama et al. , (1994), Brain Res. 667: 269-272 Pakenbergberg et al. (1991) J. MoI. Neurol. Neurosurg. Psychiat. 54: 30-33 Guttnan et al. (1997) Neurology 48: 1578-1583.

[Summary of Invention]
The present invention relates generally to neuroprotective methods, and more particularly to methods and compounds for the prevention of damage to mammalian central and peripheral nervous system cells resulting from injury, trauma, surgery or acute or chronic disease processes.

  The present invention provides, in part, that the administration of one or more members of a group of carbamate compounds, alone or in combination with one or more other neuroprotective agents, has a neuroprotective effect on the mammalian nervous system. Based on the discovery of giving.

  Neuroprotection provided by the present invention includes protection from damage caused by neurotoxicity, including neurotoxicity or excitotoxicity. Thus, the neuroprotection provided by the present invention includes seizure / ischemia, surgical trauma, traumatic brain injury (TBI), blunt, closed or penetrating head trauma, epilepsy, Huntington's disease, muscle atrophic side It is useful in the treatment of acute and chronic neurodegenerative disorders that can be associated with excitotoxicity, such as glutamate excitotoxicity, including cordiosclerosis (ALS), diabetic neuropathy and hypoglycemic encephalopathy.

  The neuroprotection provided by the present invention can be provided in a prophylactic manner on damaged or diseased tissue or during or before an event that is expected to result in nerve injury.

  The present invention provides an effective amount of a compound of the invention, or a pharmaceutically acceptable salt or ester thereof, in combination with a pharmaceutically acceptable excipient, alone or in combination with another agent. by administration to a subject, to provide neuroprotection in a subject in need of neuroprotection; to inhibit cell degeneration or cell death; to treat or prevent neurodegenerative diseases; or a compound (eg, A prophylactic amino acid such as glutamate; a toxin; or a prophylactic or therapeutic compound that provides cytotoxic side effects). In various embodiments, the methods of the invention include protection from excitotoxicity, eg, glutamate excitotoxicity.

  In various embodiments, a subject, eg, a human, can suffer from nerve injury or injury; or drug abuse, trauma, stroke, ischemia, Huntington's disease, Alzheimer's disease, Parkinson's disease, prion disease, mutant Creutzfeldt May be suffering from a symptom selected from Jacob disease, muscular atrophy or hypoglycemic encephalopathy; or may have undergone surgery or other intervention. The subject may have pre-existing symptoms that benefit from neuroprotection, or the patient will reduce the detrimental effects of concurrent or subsequent nerve damage, such as may occur during surgery or other intervention Can be treated.

  Accordingly, the present invention provides Formula 1 or Formula 2:

[Wherein R ₁ , R ₂ , R ₃ and R ₄ are independently hydrogen or C ₁ -C ₄ alkyl; and X ₁ , X ₂ , X ₃ , X ₄ and X ₅ are independently hydrogen. , Fluorine, chlorine, bromine or iodine]
A method of providing neuroprotection comprising administering to a subject in need of neuroprotection a therapeutically effective amount of a composition comprising at least one compound having: The C ₁ -C ₄ alkyl group of formula 1 or formula 2 can be substituted or unsubstituted. In one aspect of the present invention, C ₁ -C ₄ alkyl group is substituted with a phenyl group. The phenyl group can be unsubstituted or substituted. In some embodiments, the phenyl group or halogen is _{unsubstituted,} C 1 -C _{4 alkyl,} C 1 -C ₄ alkoxy, amino, nitro or cyano.

In the present invention, X ₁ , X ₂ , X ₃ , X ₄ and X ₅ can be hydrogen, fluorine, chlorine, bromine or iodine. In some embodiments, X ₁ , X ₂ , X ₃ , X ₄ and X ₅ are independently hydrogen or chlorine. In a preferred embodiment of the present invention, X ₁ is fluorine, chlorine, bromine or iodine. In one embodiment, X ₁ is chlorine and X ₂ , X ₃ , X ₄ and X ₅ are independently hydrogen. In another preferred embodiment, R ₁ , R ₂ , R ₃ and R ₄ are independently hydrogen.

  The present invention provides an enantiomer of Formula 1 or Formula 2 for providing neuroprotection in a subject. In certain embodiments, the compound of Formula 1 or Formula 2 is in the form of its single enantiomer. In another embodiment, the compound of Formula 1 or Formula 2 is in the form of an enantiomeric mixture in which one enantiomer is dominant over the other. In one embodiment, the enantiomer predominates to the extent of 90% or greater or to the extent of 98% or greater.

The invention also provides Formula 1 or Formula 2 wherein R ₁ , R ₂ , R ₃ and R ₄ are independently hydrogen or C ₁ -C ₄ alkyl; and X ₁ , X ₂ , X ₃ , A method comprising administering to a subject a neuroprotective amount of a composition comprising at least one compound having X ₄ and X ₅ independently are hydrogen, fluorine, chlorine, bromine or iodine) provide. In one embodiment, prior to administration of the composition to a subject, a determination is made as to whether the subject is suffering from any form of acute or chronic neurodegeneration or nervous system damage.

  The invention also identifies a patient at risk of developing acute or chronic neurodegeneration or nervous system damage or a patient in need of treatment with a neuroprotective drug (NPD), as defined below, and Also provided is a method comprising administering to a subject a composition comprising at least one compound having Formula 1 or Formula 2.

  In certain embodiments of the invention, the therapeutically effective amount of a compound having Formula 1 or Formula 2 to confer neuroprotection is from about 1.0 mg / Kg / dose to about 150 mg / Kg / dose. In a 70 kg human, this corresponds to a daily dose of about 70 mg / day to about 10,500 mg / day.

  In certain embodiments, to provide neuroprotection comprising one or more of the enantiomers of the invention or a pharmaceutically acceptable salt or ester thereof and a pharmaceutically acceptable carrier or excipient. A therapeutically effective amount of the pharmaceutical composition is administered to a subject or patient in need of treatment with a neuroprotective drug or NPD.

  A pharmaceutical composition comprising at least one compound having Formula 1 or Formula 2 is administered to a subject in need thereof. In certain embodiments, the subject or patient in need of treatment with a neuroprotective drug or NPD is experiencing some form of acute trauma or damage to cells of the central or peripheral nerve, or acute or chronic neurodegeneration It can have some form of failure. In one embodiment, the subject or patient is determined to be a patient at risk of developing an acute or chronic neurodegenerative disorder at the time of administration, i.e. requiring treatment with a neuroprotective agent. In another embodiment, a subject in need thereof has acute damage or trauma to cells of their nervous system at the time of administration.

[Detailed Description of the Invention]
Carbamate Compounds of the Invention The present invention provides a method of using certain 2-phenyl-1,2-ethanediol monocarbamates and dicarbamates to provide neuroprotection to a mammal in need thereof. .

  Suitable methods for synthesizing and purifying carbamate compounds, including carbamate enantiomers, used in the methods of the present invention are well known to those skilled in the art. For example, pure enantiomers and enantiomeric mixtures of 2-phenyl-1,2-ethanediol monocarbamate and dicarbamate are described in US Pat. Nos. 5,854,283, 5,698,588 and 6 No. 103,759, the disclosures of which are hereby incorporated by reference in their entirety.

  Representative carbamate compounds of the invention include Formula 1 or Formula 2:

Wherein R ₁ , R ₂ , R ₃ and R ₄ are independently hydrogen or C ₁ -C ₄ alkyl, and X ₁ , X ₂ , X ₃ , X ₄ and X ₅ are independently hydrogen , Fluorine, chlorine, bromine or iodine]
Are included.

“C ₁ -C ₄ alkyl” as used herein refers to a substituted or unsubstituted aliphatic hydrocarbon having from 1 to 4 carbon atoms. Specifically included within the definition of “alkyl” are those aliphatic hydrocarbons that are optionally substituted. In a preferred embodiment of the present invention, C ₁ -C ₄ alkyl is substituted with one or phenyl is unsubstituted.

The term “phenyl”, whether used alone or as part of another group, as used herein, as a substituted or unsubstituted aromatic hydrocarbon ring group having 6 carbon atoms. Defined. Specifically included within the definition of “phenyl” are those phenyl groups that are optionally substituted. For example, in a preferred embodiment of the invention, the “phenyl” group is unsubstituted or substituted with halogen, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, amino, nitro or cyano.

In a preferred embodiment of the invention, X ₁ is fluorine, chlorine, bromine or iodine and X ₂ , X ₃ , X ₄ and X ₅ are hydrogen.

In another preferred embodiment of the present invention, X ₁ , X ₂ , X ₃ , X ₄ and X ₅ are independently chlorine or hydrogen.

In another preferred embodiment of the invention, R ₁ , R ₂ , R ₃ and R ₄ are all hydrogen.

  The substituents and substitution patterns on the compounds of the present invention are intended to provide compounds that are chemically stable and can be readily synthesized by techniques known in the art and methods provided herein. It will be understood that these can be selected by those skilled in the art.

  Representative 2-phenyl-1,2-ethanediol monocarbamates and dicarbamates include, for example, the following compounds:

  The present invention includes the use of isolated enantiomers of Formula 1 or Formula 2. In one preferred embodiment, a pharmaceutical composition comprising an isolated S-enantiomer of Formula 1 is used to provide neuroprotection in a subject. In another preferred embodiment, a pharmaceutical composition comprising an isolated R-enantiomer of Formula 2 is used to provide neuroprotection in a subject. In another embodiment, a pharmaceutical composition comprising an isolated S-enantiomer of formula 1 and an isolated R-enantiomer of formula 2 is used to provide neuroprotection in a subject. can do.

  The invention also encompasses the use of a mixture of enantiomers of Formula 1 or Formula 2. In one embodiment of the invention, one enantiomer is dominant. An enantiomer that is predominant in a mixture is one that is present in the mixture in an amount greater than any of the other enantiomers present in the mixture, eg, greater than 50%. In one embodiment, one enantiomer predominates to the extent of 90% or to the extent of 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% or more. In one preferred embodiment, the predominant enantiomer in a composition comprising a compound of formula 1 is the S-enantiomer of formula 1. In another preferred embodiment, the predominant enantiomer in a composition comprising a compound of formula 2 is the R-enantiomer of formula 2.

In a preferred embodiment of the invention, the enantiomer present as the sole enantiomer or as the predominant enantiomer in the composition of the invention is represented by formula 3 or formula 5 wherein X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , R ₁ , R ₂ , R ₃ and R ₄ are defined as above) or are represented by formula 7 or formula 8.

  The present invention provides methods of using enantiomers and enantiomeric mixtures of the compounds represented by Formula 1 and Formula 2. The carbamate enantiomer of Formula 1 or Formula 2 contains an asymmetric chiral carbon at the benzyl position, which is an aliphatic carbon adjacent to the phenyl ring.

  An isolated enantiomer is one that is substantially free of the corresponding enantiomer. Thus, an isolated enantiomer refers to a compound that is separated by separation techniques or prepared without the corresponding enantiomer. The term “substantially free” as used herein means that the compound is made up of a significantly larger proportion of one enantiomer. In a preferred embodiment, the compound includes at least about 90% by weight of the preferred enantiomer. In another embodiment of the invention, the compound includes at least about 99% by weight of a preferred enantiomer. Preferred enantiomers can be isolated from racemic mixtures by any method known to those skilled in the art, including high performance liquid chromatography (HPLC) and chiral salt formation and crystallization, or preferred enantiomers Can be produced by the methods described herein.

Methods for the preparation of preferred enantiomers are known to those skilled in the art and are described, for example, in Jacques, et al. , Enantiomers, Racemates and Solutions (Wiley Interscience, New York, 1981); Wilen, S .; H. , Et al. Tetrahedron 33: 2725 (1977); Eliel, E .; L. Stereochemistry of Carbon
Compounds (McGraw-Hill, NY, 1962); and Wilen, S .; H. Tables of Resolving Agents and Optical Resolutions p. 268 (EL Liel, Ed., Univ. Of Notre Name Press, Notre Name, IN 1972).

  In addition, the compounds of the present invention are disclosed in US Pat. No. 3,265,728, the disclosure of which is incorporated herein by reference in its entirety and for all purposes, US Pat. No. 3,313,692. (The disclosure of which is incorporated herein by reference in its entirety and for all purposes) and previously referenced US Pat. Nos. 5,854,283, 5,698,588 and No. 6,103,759 (the disclosures of which are incorporated herein by reference in their entirety and for all purposes).

Neuroprotective properties Patients with any type of injury or damage to the central (CNS) or peripheral (PNS) nervous system, including the retina, can benefit from these neuroprotective methods. This nervous system damage includes, but is not limited to, acute damage that results in neuronal cell death or damage, hypoxic ischemia or a combination thereof. It can take the form of injury or acute injury. Acute injuries include traumatic brain injury (TBI), including obstructive, blunt or penetrating head trauma, focal brain injury, diffuse brain injury, spinal cord injury, intracranial or intraspinal injury (bruise, penetration) Including, but not limited to, shear, compression or laceration), including but not limited to spinal cord injury or whiplash babbling syndrome.

  In addition, a lack of oxygen or blood supply generally results in cerebrovascular failure, cerebral ischemia or cerebral infarction (including cerebral ischemia or infarction resulting from embolic obstruction and thrombosis), retinal ischemia (diabetic or not ), Glaucoma, retinal degeneration, multiple sclerosis, toxic and ischemic optic neuropathy, reperfusion after acute ischemia, perinatal hypoxic ischemic injury, cardiac arrest or any type of intracranial hemorrhage (hard Such as in hypoxia and / or ischemia, including but not limited to epidural, subdural, subarachnoid or intracerebral hemorrhage). Can cause acute damage.

  Trauma or damage to nervous system tissues can also include Alzheimer's disease, Pick's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson syndrome), multisystem degeneration (Shy-Drager syndrome), neurodegeneration Related chronic epilepsy, motor neuron disease (amyotrophic lateral sclerosis), multiple sclerosis, degenerative ataxia, corticobasal degeneration, ALS-Parkinson dementia syndrome in Guam, sub Acute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathies (including multiple system atrophy), primary progressive aphasia, striatal nigra degeneration, Machado-Joseph disease or spinocerebellar ataxia 3 Type and olive bridge cerebellar degeneration, bulbar and pseudobulbar paralysis, spinal and bulbospinal amyotrophy (Kennedy's disease), primary lateral sclerosis Familial spastic paraplegia, Weldnich-Hoffmann disease, Kugelberg-Welander disease, Tay-Sachs disease, Sandhoff disease, familial convulsive disease, Wohlfart-Kügelberg-Welander disease, convulsive insufficiency paraplegia, progressive multifocal disease Including leukoencephalopathy, familial autonomic neuropathy (Riley-Day syndrome) or prion disease (Kreuzfeld-Jakob disease, Gerstmann-Streisler-Scheinker disease, Kourou disease or lethal familial insomnia) Of chronic and progressive neurodegenerative disorders such as those associated with progressive neuronal cell death or disorder over a period of time, including but not limited to It can also take forms.

  In addition, trauma and progressive injury to the nervous system can be bipolar disorder or schizophrenic emotional disorder or schizophrenia, impulse control disorder, obsessive compulsive disorder (OCD), behavioral changes in temporal lobe epilepsy, and progression of personality disorder It can occur in a variety of psychiatric disorders, including but not limited to sexually impaired forms.

  In one preferred embodiment, the compounds of the invention are used to provide neuroprotection in disorders involving trauma and progressive damage to the nervous system in various psychiatric disorders. These disorders are selected from the group consisting of schizophrenic emotional disorder, schizophrenia, impulse control disorder, obsessive compulsive disorder (OCD) and personality disorder.

  In addition, trauma and injury include age-related dementia, vascular dementia, diffuse white matter disease (Binswanger disease), endocrine or metabolic dementia, dementia of head trauma and diffuse brain injury, fist fighting Associated with obvious and severe memory loss, including but not limited to neurodegenerative disorders associated with frontal dementia, including but not limited to home dementia or Pick's disease Can take the form of obstacles.

Other disorders associated with neuronal damage include chemical, toxic, infectious and radiation damage to the nervous system, including the retina, injury during fetal development, prematurity at birth, anoxic ischemia, liver, blood sugar, Uremia, electrolyte and endocrine injuries, psychiatric injuries (including but not limited to psychopathology, depression or anxiety), peripheral diseases and plexus disorders (including plexus palsy) ) Injury or neuropathy (multifocal, sensory, motor, sensory-motor, autonomy, sensory-autonomous or demyelinating neuropathy (Guillain-Barre syndrome or chronic inflammatory demyelinating polyradiculoneuropathy) Neuropathies or infections selected from, including but not limited to, inflammation, immune injury, drug abuse, pharmacological treatment, toxins, trauma (compression, contusion, laceration or segment) Segmentation trauma (including but not limited to), metabolic disorders (including but not limited to endocrine or paraneoplastic), Charcot-Marie-Tooth disease (Including but not limited to 1a, 1b, 2, 4a or 1-X linkage), Friedreich ataxia, metachromatic leukotrophy, refsum disease, adrenal spinal neuropathy, capillary Injuries from vasodilatory ataxia, dejerin-sotta (including but not limited to type A or B), neuropathy from Lambert-Eaton syndrome or cranial nerve disorders But not limited to these.

  Thus, the term “neuroprotection” as used herein inhibits the severity of dysfunction, degeneration or death of neurons, axons or their supporting cells in the central or peripheral nervous system of mammals, including humans. Means to prevent, prevent, improve or reduce. This includes treatment or prevention of neurodegenerative diseases in patients in need thereof; protection from excitotoxicity or compounds (eg excitatory amino acids such as glutamate; toxins; or immediate or delayed induction of apoptosis) Including, but not limited to, improving the cytotoxic effects of prophylactic or therapeutic compounds that provide immediate or delayed cytotoxic side effects.

  Thus, the term “patient in need of treatment with a neuroprotective drug (NPD)” as used herein refers to any of the above syndromes or disorders, or any neurological or psychiatric disorder. Any who currently has or can develop any disorder that can benefit the patient's current clinical symptoms or prognosis from providing neuroprotection to prevent onset, progression, worsening or increased resistance to treatment Point to the patient.

  The term “antiepileptic drug (AED)” is used interchangeably with the term “anticonvulsant drug” and, as used herein, both terms are used when the drug is administered to a subject or patient. An agent that can suppress (eg, prevent, slow, stop, or improve) seizure activity or seizure motility.

  The terms “treating” or “treatment”, as used herein, reduce; remission; reduce or reduce symptoms, or make a lesion or symptom more tolerable for a patient; slow down the rate of degeneration or decline To make the final point of degeneration less debilitating; or any damage or lesion or symptom, including any objective or subjective parameters such as improving the physical or mental health of the patient Any sign of success in prevention or improvement. The treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of physical examination, neurological examination and / or psychological examination. Accordingly, the term “treating” or “treatment” includes administration of a compound or agent of the invention to confer neuroprotection. In certain cases, treatment with a compound of the invention may be combined with other neuroprotective compounds or AEDs to prevent, inhibit or stop neuronal death or injury or progression of brain dysfunction or brain hyperexcitability. Done.

  The term “therapeutic effect” as used herein refers to the effective provision of a neuroprotective effect to prevent or minimize the death or damage or dysfunction of cells in the central or peripheral nervous system of a patient. .

  The term “therapeutically effective amount” as used herein refers to the present invention for providing a therapeutic effect as defined above in a subject or patient in need of such neuroprotective treatment. A sufficient amount of one or more of the following compounds:

  The terms “subject” or “patient” are used interchangeably herein and, as used herein, include human patients or subjects to whom the compositions of the invention can be administered. It means any mammal, including but not limited to. The term mammal includes human patients and non-human primates, as well as laboratory animals such as rabbits, rats and mice, and other animals.

In certain embodiments, the methods of the invention do not suffer from or are known to suffer from symptoms known in the art to be effectively treated with carbamate compounds or currently known neuroprotective compounds or AEDs. Conveniently used to treat non-patients. In these cases, the decision to use the methods and compounds of the invention is whether the patient is a “patient in need of treatment with a neuroprotective drug (NPD)” (as the term is defined above). It is based on determining whether.

  In certain embodiments, the present invention provides a method of neuroprotection. In certain embodiments, these methods have not yet developed overt clinical signs or symptoms of injury or damage to cells of the nervous system, but are due to injury or trauma to the nervous system or biochemical or genetic. Carbamates of the present invention for patients who may enter the high risk group of developing neuronal damage due to the discovery of any known predisposition or one or more confirmed biomarkers of these disorders Administering a therapeutically effective amount of the compound.

  Accordingly, in certain embodiments, the methods and compositions of the invention are directed against neuroprotection in a subject at risk of developing neuronal damage but not yet developing clinical evidence. This patient is simply exposed to a “greater risk” as determined by the recognition of any factor in subjects or their families, medical history, physical examination or testing that present a greater than average risk for developing neuronal damage. May have been. Thus, this determination that the patient may be at “greater risk” by any available means is to determine whether the patient should be treated with the method of the present invention. Can be used.

  Thus, in an exemplary embodiment, subjects who can benefit from treatment with the methods and compounds of the present invention can be identified using generally accepted screening methods to determine risk factors for neuronal damage. it can. For example, these screening methods include, for example, either closed or penetrating head trauma, bacterial or viral CNS infection, stroke, brain tumor, brain edema, cystosis, porphyria, metabolic encephalopathy Cerebrovascular diseases, hypnotic sedatives or alcohol withdrawal symptoms, including but not limited to, drug withdrawal symptoms, hypoxia at birth or any type of delivery trauma Abnormal perinatal history, including cerebral palsy, learning disabilities, hyperactivity, history of febrile convulsions in childhood, history of intussusception, family history of epilepsy or any seizure related disorder, lupus ( inflammatory diseases of the brain, including lupus), drug addiction, either directly or by placenta passage, including but not limited to cocaine addiction, parent Consanguinity, and treatment with drugs that are toxic to psychotropic drugs include nervous system encompasses conventional test to determine the risk factors are not limited thereto, and the like.

  The determination of which patients can benefit from treatment with NPD in patients without clinical signs or symptoms can be based on various “surrogate markers” or “biomarkers”.

  As used herein, the terms “surrogate marker” and “biomarker” are used interchangeably and can be any anatomical that can be reliably correlated with the current presence or future onset of neuronal damage, A biochemical, structural, electrical, genetic or chemical indicator or marker. In some cases, brain imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI) or positron emission tomography (PET) to determine whether a subject is at risk of neuronal damage Can be used.

Suitable biomarkers for the methods of the present invention include MRI of sclerosis, atrophy or volume loss in the hippocampus or overt medial temporal lobe sclerosis (MTS) or similar associated anatomical lesions,
Determination by CT or other imaging techniques; detection of molecular species such as proteins or other biochemical biomarkers in the patient's blood, serum or tissue, eg elevated levels of ciliary neurotrophic factor (CNTF) or Elevated serum levels of neuronal degradation products; or other evidence from surrogate markers or biomarkers that the patient requires treatment with a neuroprotective agent are included, but are not limited to.

  Many more such biomarkers utilizing a wide variety of detection techniques are expected to be developed in the future. Any such marker or indicator of the presence or possible future onset of neuronal damage can be used to determine the need for treatment with the compounds and methods of the invention when the latter term is used herein. It can be used in the method of the invention.

Determining that a subject may have or be at risk of developing neuronal damage also includes medical evaluation, including, for example, a detailed medical history, physical examination, and a series of related blood tests. It can also include an electroencephalogram (EEG), CT, MRI or PET scan. Determination of an increased risk of developing neuronal damage or injury can also be done using genetic testing, including gene expression profiling or proteomic techniques (Schmidt, D. Rogawski, MA Epilepsy).
Research 50; 71-78 (2002) and Loscher, W. et al. Schmidt D.C. Epilepsy Research 50; see 3-16 (2002)).

  For psychiatric disorders that can be stabilized or ameliorated by neuroprotective drugs, such as bipolar disorder, schizophrenic emotional disorder, schizophrenia, impulsive dysregulation, etc., the above tests are also Detailed history of the patient's symptoms, such as mood disorders and psychotic symptoms related to the examination and over time and other treatments that the patient may be undergoing over time, such as a life chart Can also be included. These and other specialized and routine methods allow the clinician to select patients in need of treatment using the methods and formulations of the present invention.

  In certain embodiments of the present invention, carbamate compounds suitable for use in the practice of the present invention are alone or in combination with at least one or more other compounds or therapeutic agents, such as other neuroprotective or antiepileptic drugs, It is given at the same time as a convulsant. In these embodiments, the present invention provides a method of treating or preventing neuronal damage in a patient. The method includes one or more other compounds or therapeutic agents that have the ability to confer neuroprotection or to treat or prevent seizures or epilepsy or to increase the neuroprotective effects of the compounds of the invention. Administering an effective amount of one of the carbamate compounds disclosed herein to a patient in need of treatment in combination with an effective amount of.

  As used herein, the term “simultaneous administration” or “combination administration” of a compound of the invention and a compound, therapeutic agent or known agent refers to a time period during which both the known agent and the compound have a therapeutic effect. Means administration of the drug and one or more compounds. In some cases, this therapeutic effect is synergistic. Such co-administration can include simultaneous (ie, simultaneous), prior or subsequent administration of the agents relative to the administration of the compound of the invention. Those skilled in the art have no difficulty in determining the appropriate timing, sequence and dosage of administration of a particular drug and the compounds of the present invention.

The one or more other compounds or therapeutic agents have the following properties: antioxidant activity; NMDA receptor antagonist activity; increased endogenous GABA inhibition; NO synthase inhibitor activity; iron binding capacity, eg, iron chelator; Calcium binding capacity, eg Ca (II)
A chelating agent; a zinc-binding ability, such as a Zn (II) chelator; one or more of the ability to effectively block sodium or calcium ion channels or open potassium or chloride ion channels in the patient's CNS It can be selected from compounds.

  In certain preferred embodiments, one or more other compounds or therapeutic agents neutralize the NMDA receptor by binding to the NMDA receptor (eg, by binding to the glycine binding site of the NMDA receptor). And / or the agent increases GABA inhibition by decreasing GABA uptake of glia.

  Further, the one or more other compounds or therapeutic agents can be any agent known to inhibit seizure activity even if the compound is not known to confer neuroprotection. Such agents include, but are not limited to, any effective AED known to those skilled in the art or discovered in the future, for example, suitable agents include carbamazepine, clobazam, clonazepam , Etosuximide, ferbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, phenobarbital, phenytoin, pregabalin, primidone, retigabine, talampanel, thiagabine, topiramate, valproate, vigabatrin, benzoxamide, sleep Drugs are included, but are not limited to these.

Further, in certain embodiments, the compounds of the present invention can be used alone or in combination with each other or as described above to produce a medicament for the purpose of providing neuroprotection to a patient or subject in need of neuroprotection. Used in combination with one or more other therapeutic agents, or salts or esters thereof.
Carbamate compounds as drugs:
The present invention provides enantiomeric mixtures of Formula 1 and / or Formula 2 and isolated enantiomers as drugs. These carbamate compounds are formulated as drugs to provide neuroprotection in the subject.

  In general, the carbamate compounds of the present invention include therapeutic agents including oral, buccal, topical, systemic (eg, transdermal, intranasal, or suppository) or parenteral (eg, intramuscular, subcutaneous or intravenous injection). It can be administered as a pharmaceutical composition by any method known in the art for administration. Direct compound administration to the nervous system includes, for example, intracranial, intraventricular, intraventricular, intrathecal, of administration by delivery via intracranial or intraspinal needles or catheters with or without a pump device Administration to the internal, intracapsular, intraspinal or perispinal routes can be included.

  In addition, eye diseases including but not limited to retinal ischemia (diabetic or otherwise), glaucoma, retinal degeneration, macular degeneration, multiple sclerosis, toxic and ischemic optic neuropathy Alternatively, in the case of a disorder, the compounds of the invention, including combinations of compounds, can be applied directly to the eye, ie directly to the sclera or not, such as with eye drops or ocular implantation or vitreous humor It can be administered by other slow delivery devices, including microspheres, including by direct injection into.

The composition may take the form of tablets, pills, capsules, semi-solids, powders, sustained release formulations, solutions, suspensions, emulsions, syrups, elixirs, aerosols or any other suitable composition. Possible; and comprises at least one compound of the invention in combination with at least one pharmaceutically acceptable excipient. Suitable excipients are well known to those skilled in the art, and methods for formulating them and compositions are described in Alfonso AR: Remington's.
Pharmaceutical Sciences , 17th ed. , Mack Publishing Company, Easton PA, 1985, the disclosure of which is incorporated herein by reference in its entirety and for all purposes. Liquid carriers particularly suitable for injectable solutions include water, saline solution, dextrose solution and glycol.

  The carbamate compound can be provided as an aqueous suspension. The aqueous suspensions of the present invention can contain carbamate compounds mixed with excipients suitable for the manufacture of aqueous suspensions. Such excipients include, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, suspending agents such as gum tragacanth and gum arabic, and naturally occurring phospholipids (eg lecithin), Condensation products of fatty acids and alkylene oxides (for example, polyoxyethylene stearate), condensation products of long-chain aliphatic alcohols and ethylene oxide (for example, heptadecaethyleneoxycetanol), partial esters obtained from fatty acids and hexitol and ethylene oxide Condensation products (eg, polyoxyethylene sorbitol monooleate) or partial esters of fatty acids and anhydrous hexitol with ethylene oxide For example a dispersing or wetting agents such as polyoxyethylene sorbitan monooleate) can be included.

  Aqueous suspensions also contain one or more preservatives, such as ethyl p-hydroxybenzoate or n-propyl, one or more colorants, one or more perfumes, and sucrose, One or more sweeteners such as aspartame or saccharin may also be included. The formulation can be adjusted for osmolarity.

  Oily suspensions for use in the method of the present invention comprise suspending the carbamate compound in a vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil, or a mineral oil such as liquid paraffin; or a mixture thereof. Can be prepared. Oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweeteners such as glycerol, sorbitol or sucrose can be added to provide a palatable oral preparation. These formulations can be preserved by the addition of an anti-oxidant such as ascorbic acid. As examples of injectable oil excipients, see Minto, J. et al. Pharmacol. Exp. Ther. 281: 93-102, 1997. The pharmaceutical formulations of the present invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable or mineral oil as described above, or a mixture of these.

  Suitable emulsifiers include naturally occurring gums such as gum arabic and tragacanth, naturally occurring phospholipids such as soy lecithin, esters or partial esters derived from fatty acids and anhydrous hexitol, such as sorbitan monooleate. And condensation products of ethylene oxide with these partial esters, such as polyoxyethylene sorbitan monooleate. Emulsions can also contain sweetening and flavoring agents, as in syrup and elixir formulations. Such formulations can also contain a demulcent, a preservative, or a coloring agent.

  Selected compounds, alone or in combination with other suitable ingredients, can be in aerosol formulations (ie, they can be “nebulized”) to be administered by inhalation. The aerosol formulation can be placed in a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen and the like.

For example, formulations of the present invention suitable for parenteral administration, such as by intra-articular (in the joint), intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes, include antioxidants, buffers, bacteriostatic agents, and Aqueous and non-aqueous isotonic sterile infusion solutions that can contain solutes that are isotonic with the blood of the recipient intended for the formulation, as well as suspending agents, solubilizers, thickeners, stabilizers and preservatives Aqueous and non-aqueous sterile suspensions can be included. Among the acceptable excipients and solvents that can be employed are water and Ringer's solution, isotonic sodium chloride. In addition, sterile, fixed oils can be conveniently employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the manufacture of injectables. These solutions are sterile and generally free of undesirable matter.

  If the compounds are sufficiently soluble, they can be dissolved directly in physiological saline with or without the use of a suitable organic solvent such as propylene glycol or polyethylene glycol. Finely divided compound dispersants may be made up in aqueous starch or sodium carboxymethylcellulose solution, or in a suitable oil such as peanut oil. These preparations can be sterilized by ordinary well-known sterilization techniques. The formulation may be pharmaceutically acceptable as necessary to approach physiological conditions such as pH adjusters and buffers, toxicity modifiers such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. An adjuvant can be contained.

  The concentration of the carbamate compound in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, weight, etc., according to the particular method of administration selected and the needs of the patient. For IV administration, the formulation can be a sterile injectable formulation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. . Recommended formulations can be given in unit-dose or multi-dose sealed containers such as ampoules and vials. Infusion solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

  Carbamate compounds suitable for use in the practice of the present invention can and are preferably administered orally. The amount of the compound of the invention in the composition can vary widely depending on the type of composition, the size of the unit dosage, the type of excipient and other factors well known to those skilled in the art. Generally, the final composition can comprise, for example, 0.000001 weight percent (% w) to 10% w carbamate compound, preferably 0.00001% w to 1% w, with the remainder being excipients Or a plurality of excipients.

  Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art at dosages suitable for oral administration. Such carriers include unit dosage forms such as tablets, pills, powders, dragees, capsules, liquids, troches, gels, syrups, slurries, suspensions, etc., suitable for consumption by the patient. Allows to formulate in the object.

  Formulations suitable for oral administration include: (a) a liquid solution such as an effective amount of a pharmaceutical formulation suspended in a diluent such as water, saline or PEG400; (b) as a liquid, solid, granule or gelatin Capsules, sachets or tablets each containing a predetermined amount of the active ingredient; (c) a suspension in a suitable liquid; and (d) a suitable emulsion.

Pharmaceutical formulations for oral use are obtained by combination of solid excipients and compounds of the invention, optionally milling the resulting mixture and adding appropriate additional compounds as desired Later, the mixture of granules can be processed to obtain tablets or dragee cores. Suitable solid excipients are carbohydrates or protein extenders and sugars including lactose, sucrose, mannitol or sorbitol; starch from corn, wheat, rice, potato or other plants; methylcellulose, hydroxymethylcellulose, Cellulose such as hydroxypropylmethylcellulose or sodium carboxymethylcellulose; and gums including gum arabic and tragacanth; and proteins such as gelatin and collagen are included, but are not limited to these. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof such as sodium alginate. Tablet forms include lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid and other excipients, colorants, One or more of bulking agents, binders, diluents, buffers, wetting agents, preservatives, perfumes, dyes, disintegrants and pharmaceutically compatible carriers may be included. The lozenge forms are flavors such as gelatin and glycerin or sucrose and acacia emulsions, gels and the like containing active ingredients in sucrose and carriers known in the art in addition to the active ingredients An aromatic tablet comprising an active ingredient on an inert base can be included.

  The compounds of the present invention can also be administered in the form of suppositories for rectal administration of the drug. These formulations can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ambient temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycol.

  The compounds of the present invention can also be administered by intrarectal routes including nasal, intraocular, intravaginal, and suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see Rohatagi, J. et al. Clin. Pharmacol.35: 1187-1193, 1995; Tjwa, Ann.Allergy Asthma Immunol.75: 107-111, 1995).

  The compounds of the invention can be formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pasta, jelly, coatings, powders and aerosols and delivered transdermally via the topical route. it can.

  Encapsulating materials can also be used with the compounds of the invention, and the term “composition” includes the active ingredient in combination with the encapsulating material as a formulation, with or without other carriers. be able to. For example, the compounds of the present invention can also be delivered as microspheres for sustained release in the body. In one embodiment, the microspheres are obtained by intradermal injection of microspheres containing a drug that slowly releases subcutaneously (eg, mifepristone) (eg, Rao, J. Biomaterial Sci. Polym. Ed. 7: 623-645, 1995): as a biodegradable and injectable gel formulation (see, eg, Gao, Pharm. Res. 12: 857-863, 1995); or as a microsphere for oral administration (eg, Eyles, J. Pharm). Pharmacol.49: 669-674, 1997). Both transdermal and intradermal routes provide constant delivery for weeks or months. Cachets can also be used in delivering the compounds of the present invention.

In another embodiment, the compounds of the present invention are produced by the use of liposomes that fuse or are taken up by the cell membrane, i.e., by using a ligand bound to the liposome that binds to the surface membrane protein receptor of the cell resulting in endocytosis. Can be delivered. By using liposomes, the delivery of carbamate compounds is focused on target cells in vivo, especially when the liposome surface carries a ligand specific for the target cell or is otherwise preferentially directed to a specific organ. Can be matched. (For example, Al-Muhammed, J. Microencapsul. 13: 293-306, 1996; Chon, Curr. Opin. Biotechnol. 6: 698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46: 1576-1587. , 1989).

  The pharmaceutical formulations of the present invention can be provided as salts and include a number of acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Can be formed. Salts are more soluble in aqueous or other protic solvents than the corresponding free base form. In another case, a preferred formulation is in the pH range of 4.5 to 5.5, combined with the buffer prior to use, such as: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, It can be a lyophilized powder that can contain any or all of 2% to 7% mannitol.

  Pharmaceutically acceptable salts and esters refer to salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the compound can react with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals such as sodium and potassium, magnesium, calcium and aluminum. Suitable organic salts include amine bases such as those formed with organic bases such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. Pharmaceutically acceptable salts also include inorganic acids (eg, hydrochloric acid and hydrobromic acid) and organic acids (eg, acetic acid, citric acid, maleic acid and alkanes such as methanesulfonic acid and benzenesulfonic acid) It may also include acid addition salts formed from the reaction of arenesulfonic acid) with the amine moiety in the parent compound. Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy and phosphonoxy groups present in the compound. Where there are two acidic groups present, the pharmaceutically acceptable salt or ester can be a mono-acid-mono salt or ester or a di-salt or ester; and more than two present as well If there are acidic groups, some or all of such groups can be salified or esterified.

  The compounds named in the present invention can exist in non-chlorinated or non-esterified forms, or in chlorinated and / or esterified forms, and the nomenclature of such compounds includes the original (non-chlorinated and non-esterified forms). Both esterified compounds and their pharmaceutically acceptable salts and esters are intended to be included. The present invention includes pharmaceutically acceptable salt and ester forms of Formula 1 and Formula 2. There can be more than one crystalline form of the enantiomer of Formula 1 or Formula 2, and as such are encompassed by the present invention.

  The pharmaceutical composition of the present invention may optionally contain, in addition to the carbamate compound, at least one other therapeutic agent useful in the treatment of a disease or condition associated with providing neuroprotection.

Methods for formulating pharmaceutical compositions are described in Pharmaceutical Dosage Forms: Tables . 2nd edition, revisions and augmentations, Volumes 1-3, edited by Lieberman et al; Pharmaceutical Dosage Forms : Parental Medicines. Volumes 1-2, A
vis et al editing; and Pharmaceutical Dosage Forms: Disperse Systems . Numerous publications such as Volumes 1-2, edited by Lieberman et al; published by Marcel Dekker, Inc. (the disclosures of which are incorporated herein by reference in their entirety and for all purposes) It is described in the thing.

  Pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration.

Dosage regimen The present invention provides a method of providing neuroprotection in a mammal, including a human subject or patient, using a carbamate compound or composition of the present invention. The amount of carbamate compound required to confer neuroprotection is defined as a therapeutically or pharmaceutically effective dose. The effective dosing schedule and amount for this application, i.e., dosing or dosing regimen, will depend on various factors including stage, patient physical condition, age and the like. In calculating the patient's dosage regimen, the method of administration is also taken into account. In order to accomplish this goal, the compounds or compositions of the invention must be used in the exact therapeutically effective amount or dose as described below.

  The effective dosing schedule and amount for this application, ie the dosing or dosing regimen, will depend on various factors including the exact nature of the disease or injury, the patient's physical condition, weight, age, etc. In calculating the patient's dosage regimen, the method of administration is also taken into account.

  Neuroprotective effects in humans in severe and acute clinical situations similar to the lithium-pilocarpine rat model in Examples 1 and 2 below and the transient cerebral ischemia middle cerebral artery occlusion (MCAO) rat model in Example 4 The range of doses that are expected to be effective in bringing about is determined by comparing known effective doses and blood levels in rats and humans.

Pharmacokinetics In humans, one of the compounds of the present invention, referred to herein as test compound (TC), ie the pharmacokinetics of formula 7, should be linear after single and repeated oral administration in healthy adults Is known (see Example 5).

Blood levels in humans In human toxicology studies, oral administration of test compound (TC) at various doses for 7 days resulted in the following Cmax and AUC (0-24):
1) 100 mg. b. i. d. (200 mg at 24 hours or 2.85 mg / kg / day in a 70 kg human), Cmax is 3.6 micrograms / mL, and AUC is 42.2 micrograms-hour / mL;
2) 250 mg. b. i. d. (7. 14 mg / kg / day in 500 mg or 70 kg human in 24 hours), Cmax is 8.2 micrograms / mL, and AUC is 102.3 micrograms-hour / mL;
3) 500 mg. b. i. d. (14.28 mg / kg / day in 1000 mg or 70 kg human in 24 hours), Cmax is 17.2 microgram / mL, and AUC is 204.1 microgram-hour / mL;
4) 750 mg. b. i. d. (21.4 mg / kg / day in 1500 mg or 70 kg human in 24 hours), Cmax was 28.2 micrograms / mL and AUC was 322.7 micrograms-hour / mL.

Blood levels in rats ;
In a toxicological study in rats, 8-day oral administration of test compound (TC) resulted in the following Cmax and AUC:
1) At 30 mg / kg / day, Cmax is 9.33 microgram / mL and AUC is 97.32 microgram-hour / mL;
2) At 100 mg / kg / day, Cmax is 20.63 microgram / mL and AUC is 230.33 microgram-hour / mL;
3) At 300 mg / kg / day, the Cmax was 70.34 microgram / mL and the AUC was 525.95 microgram-hour / mL.

  The dose tested in rats for antidepressant development and neuroprotective effect in Example 2 was between 30 mg / kg / day and 120 mg / kg / day. The lowest dose tested in this example, ie 30 mg / kg, provided some measurable protective effect, while the lowest dose tested in Example 1 was 10 mg / kg / day and the least protection Effected (see Examples 1 and 2 below). However, in the transient cerebral ischemic middle cerebral artery occlusion (MCAO) rat model of Example 4, the 10 mg / kg dose in rats showed a moderate but significant reduction in infarct size.

  In rats, a 30 mg / kg / day dose of test compound (TC) is expected to result in a blood level of 9.33 microgram / mL Cmax and 97.32 microgram-hour / mL AUC. In humans, these blood levels are expected from a dose of about 7.1 to about 8.6 mg / kg / day in a human of about 500 mg / day to about 600 mg / day or 70 kg.

  However, in Examples 1 and 2, relatively high doses and blood levels were required due to the acute and very severe animal model used and the need to provide a demonstrated neuroprotective anti-epileptic effect. In this severe acute animal model, the compound was also given after a traumatic event or injury, i.e., after induction of an intussusception by administration of Li-pilocarpine. In the transient cerebral ischemic middle cerebral artery occlusion (MCAO) rat model in Example 4, the compound was also administered 1 hour after occlusion of the middle cerebral artery. This type of post-injury model appears to correlate with similarly acute and severe clinical situations in human patients who do not begin medication until after severe CNS injury has already occurred. In such situations, the dosage required for neuroprotective and anti-epileptic effects is needed in lower acute or severe situations or in chronic situations and especially when the drug is used prophylactically Expected to be higher than expected.

In situations where drugs are used prophylactically in a primary prevention or pre-treatment paradigm, the required dose and blood levels required to produce clinically significant neuroprotective effects are 10 mg / kg used in Example 4. It is expected to be somewhat lower than the human equivalent of 1 mg and significantly lower than the 30 mg / kg / day dose found to be effective in Example 2. As such, in most cases, the dose expected to be therapeutically effective in clinical practice in humans is less than that identified in these severe animal models. Test compounds to prevent seizures in rats have an ED50 of about 4 mg / kg to about 30 mg / kg (depending on time and experimental type), and therefore a minimum effective dose of 10 mg / kg in a neuroprotective rat model is not unexpected . Based on this data, the expected effective neuroprotective human dose is similar to the minimum dose required for anticonvulsant efficacy in humans. In a primary prevention paradigm where administration takes place well before any injury or pathological process is initiated, the effective dose and blood level in humans is the transient cerebral ischemic middle cerebral artery occlusion (MCAO in Example 4). 10) found to be minimally effective in the rat model
It is expected to be somewhat lower than the mg / kg human equivalent.

  In a primary prevention paradigm where medication therapy begins in a human patient prior to any injury or damage to the human nervous system, the lower limit of the neuroprotective effective dose is from about 100 mg / day to about 200 mg / day or about 1. 43 mg / kg / day to about 2.85 mg / kg / day, resulting in an expected Cmax of about 3.6 microgram / mL (at 200 mg / day) and an AUC of about 42.2 microgram-hour / mL is expected.

  In situations where medication is initiated after injury has persisted, the dose range is somewhat higher, for example from about 2.85 to about 5.71 mg / kg / day in about 200 mg / day to about 400 mg / day or 70 kg human. Expected to be.

  The compounds and compositions of the present invention do not have a theoretical upper limit in their clinically effective dose range. Therefore, the upper limit of the therapeutically effective range is determined by the maximum amount that can be tolerated by the patient. However, the highest dose tested in rats that showed very significant neuroprotective and anti-epileptic effects, i.e., 120 mg / kg, was based on the above data, a dose of 750 mg twice a day (1500 mg / day or about 21.4 mg / kg / day) is expected to have Cmax and AUC similar to or lower than those provided in humans. A twice daily dose of 750 mg (1500 mg total daily dose) is used in humans and has been found to be easily tolerated. Based on this, the maximum tolerated dose is expected to be significantly higher for many patients. Probably 2500 mg / day to 3000 mg / day or about 35.7 mg / kg / day to about 42.9 mg / kg / day in a 70 kg human.

  Thus, for the purpose of providing neuroprotection to a human subject or patient, the pharmaceutical compounds and compositions of the present invention are about 1.4 mg / kg / day to about 43.0 mg / kg / day (in a 70 kg human). 100 mg / day to 3000 mg / day), preferably about 2.9 mg / kg / day to about 35.7 mg / kg / day (200 mg / day to 2500 mg / day in a 70 kg human), more preferably about 3.6 mg / day. kg / day to about 28.6 mg / kg / day (250 mg / day to 2000 mg / day in a 70 kg human) or even more preferably from about 4.3 mg / kg / day to about 21.4 mg / kg / day (70 kg 300 mg / day to 1500 mg / day in humans or most preferably from about 5.0 mg / kg / day to about 17.1 mg / kg / day (70 kg human) There may be administered at a dosage of 350 mg / day ～1200Mg / day) with. However, these dosages can vary depending on the individual characteristics and tolerability of the subject and the exact nature of the condition being treated.

Based on this disclosure, those skilled in the art will be able to consider the skill without undue experimentation,
A therapeutically effective dose or amount of a particular substituted carbamate compound of the present invention for treating epilepsy and for providing a clinically significant neuroprotective effect can be determined. (See, e.g., Lieberman, Pharmaceutical Dosage Forms (Vols. 1-3, 1992); Lloyd, 1999, Theart, Science and Technology of Pharmaceutical Compounding; and Pickar, 1999, Puls.

A therapeutically effective dose is also one in which the therapeutically beneficial effect outweighs any toxic or harmful side effects of the active ingredient in the clinical sense. For each particular subject, the particular dosing regimen should be evaluated and adjusted over time according to individual needs and the professional judgment of the person administering or instructing the administration of the compound. Should be more careful. It is also anticipated that the compositions of the present invention can be initiated at low or moderate doses and then increased to a sufficiently therapeutically effective dose and blood level over a period of time.

  For treatment purposes, the compositions or compounds disclosed herein may be administered in a single bolus delivery, by continuous delivery over a long period of time, or in a repeated dose protocol (eg, hourly, daily or weekly). , By repeated dose protocols). The pharmaceutical formulations of the invention can be administered, for example, 1, 2 or more times daily, 3 times a week, or weekly. In one embodiment of the invention, the pharmaceutical formulation of the invention is orally administered once or twice daily.

  In certain embodiments, a treatment regimen with a compound of the invention can be initiated after the subject has suffered from other initial injury, such as, for example, an injury or stroke that damages the brain. In yet another embodiment, a regimen of treatment with a compound of the present invention begins before any damage or injury to the nervous system occurs, but at the same time such damage or injury can be expected or expected can do. For example, such a treatment regimen is likely to result in a subject undergoing neurosurgical treatment or having other forms of head or brain trauma, such as combat, intense sports or competition, recurrent seizures, Can be started before TIA etc.

  In certain embodiments, the carbamate compounds of the invention can be administered daily for a set period of time (weeks, months, years) following the occurrence of brain damage or initial injury. The attending physician knows how to determine that the carbamate compound has reached a therapeutically effective level (eg, by patient clinical trials or by measuring drug levels in blood or cerebrospinal fluid). One skilled in the art can determine the maximum tolerated dose using physical examination to determine the presence and severity of side effects such as ambiguous speech, coma or impaired coordination.

  In this regard, therapeutically effective dosages of the biologically active agent (s) provide a long-term treatment regimen that provides clinically significant results to provide a neuroprotective effect. Of which repeated doses can be included. The determination of effective dosage in this context is typically based on animal model studies followed by human clinical trials and effective dosages that significantly reduce the occurrence or severity of targeted exposure symptoms or symptoms in a subject. Guided by determining amount and administration protocol. Suitable models in this regard include, for example, mice, rats, pigs, cats, non-human primates, and other generally accepted animal model subjects known in the art.

  Alternatively, effective dosages can be determined using in vitro models (eg, immunological and histopathological assays). With such a model, a therapeutically effective amount (eg, to elicit the desired response) of the biologically active agent (s) typically requiring only routine calculations and adjustments Determine the appropriate concentration and dose to administer nasally effective, transdermally effective, intravenously effective or intramuscularly effective amount).

  In an exemplary embodiment of the invention, the unit dosage form of the compound is manufactured for a standard dosage regimen. In this way, the composition can be easily subdivided into smaller doses as directed by the physician. For example, unit dosages can be made up in packed powders, vials or ampoules, and preferably in capsule or tablet form.

The active compound present in these unit dosage forms of the composition may be administered in an amount of, for example, from about 25 mg to about 800 mg, or preferably the activity of the invention for single or multiple daily administration, according to the patient's specific needs One or more of the carbamate compounds can be present in unit dosages of about 50, 100, 200, 250, 400, 450, 500 and 600 mg.

  The methods of the invention also provide kits for use in providing neuroprotection. After formulating a pharmaceutical composition comprising one or more carbamate compounds of the invention in a suitable carrier, optionally with one or more other compounds having therapeutic benefit, Can be placed in a suitable container and labeled for providing neuroprotection. In addition, another container comprising at least one other therapeutic agent useful in providing neuroprotection, sputum development, treatment of sputum or another disorder or condition associated with neuronal damage is also placed in the container, and Labels can be provided for treatment of the indicated diseases. Such labeling can include, for example, instructions regarding the amount, frequency and method of administration of each drug.

Although the foregoing invention has been described in detail by way of example for purposes of clarity of understanding, certain changes and modifications are encompassed by the disclosure and presented for purposes of illustration and not limitation. It will be apparent to those skilled in the art that the present invention can be performed without undue experimentation within the scope of the term. The following examples are provided to illustrate certain embodiments of the invention and are not intended to be limiting.
[Example]

Experimental Examples The activity of compounds of formula (I) and formula (II) for use in providing neuroprotection was evaluated in the following experimental examples. An isolated S-enantiomer of formula 1, referred to herein as “Test Compound” or “TC” or “test compound” (eg, Formula 7 shown above) Activity in the rats of Examples 1 and 2 in the model of temporal lobe epilepsy induced by lithium and pilocarpine and in other animal models useful for predicting neuroprotective effects and in the treatment of epilepsy The following experiments were evaluated to determine the efficacy of the compounds. These examples are meant to illustrate various aspects of the present invention, but are not intended to limit the invention in any way.

Lipo-Pilocarpine Model of Temporal Lobe The model induced in rats by lithium-associated pilocarpine (Li-Pilo) reproduces most of the clinical and neurophysiological features of human temporal lobe (Turski et al., 1989, Synapse
3: 154-171; Cavalheiro, 1995, Ital J Neurol
Sci 16: 33-37). In adult rats, systemic administration of pilocarpine results in a status epilepticus (SE). The fatality rate reaches 30-50% during the first day. In surviving animals, neuronal damage is prominent in the hippocampus, piriform and entorhinal cortex, thalamus, amygdala, neocortex and substantia nigra. This acute seizure period is followed by a “silent” seizure period that lasts for an average period of 14-25 days, after which all animals show spontaneous recurrent seizures with a normal frequency of 2-5 times per week ( Turski et al., 1989, Synapse 3: 154-171; Cavalheiro, 1995, Ital J Neuro Sci 16: 33-37; Dube et al., 2001, Exp Neurol 167: 227-241).

Treatment with Lithium-Pilocarpine and Test Compound Provided by Janvier Breeding Center (Le Genest-St-Iste, France), male Wistar rats weighing 225-250 g under controlled standard conditions (light-dark cycle, 7. 00a.m. to 7.00pm., With lights on), food and water were optionally available. All animal experiments were performed in accordance with the rules of the European Community Council Directive (86/609 / EEC) of November 24, 1986 and the French Ministry of Agriculture (License N ° 67-97). For electrode implantation, i.e. 2.5 mg / kg diazepam (DZP, Valium, Roche, France) and 1 mg / kg ketamine chlorhydrate (Imalgene 1000, Rhone Merrieux, France). p. Rats were anesthetized by injection. Four single contact recording electrodes, two on each side, were placed on the parietal cortex and on the skull.

癲癇 Intussusception induction:
Treatment with test compound and development of spontaneous recurrent seizures (SRS) All rats receive lithium chloride (3 meq / kg, ip, Sigma, St Louis, MO, USA); After 20 h, animals were placed in a Plexiglas box to record baseline cortical EEG. Methyl scopolamine bromide (1 mg / kg, sc, Sigma) was administered to limit the peripheral effects of convulsants. SE was induced by injecting pilocarpine hydrochloride (25 mg / kg, sc, Sigma) 30 minutes after methyl scopolamine. Bilateral EEG cortical activity was recorded during the entire period of SE and behavioral changes were noted.

  The effect of increasing doses of test compound was studied in 3 groups of rats. Group 1 animals were given 10 mg / kg of test compound 1 h after the onset of SE (ip) (pilo-TC10), whereas animals in groups 2 and 3 were 30 and 60 mg / kg, respectively. Test compounds were given (pilo-TC30 and pilo-TC60).

  Another group was infused with 2 mg / kg diazepam (DZP, im) 1 h after the onset of SE, which is our standard treatment to improve animal survival after SE (pilo-DZP). ). The control group was given saline instead of pilocarpine and the test compound (saline-saline). The pilo-test compound rats that survive SE are then subjected to a second i.d. of the same dose of test compound approximately 10 h after the first test compound injection. p. Injections were infused and maintained under twice daily treatment with test compounds for an additional 6 days. Pilo-DZP was given a second infusion of 1 mg / kg DZP on the day of SE approximately 10 h after the first. Thereafter, Pilo-DZP and saline-saline rats were given an equal volume of saline twice daily.

  The effect of the test compound on the EEG and on the latency to the onset of SRS was examined by daily video recording of animals over 10 h per day and recording of electrographic activity twice a week for 8 h.

Quantification of cell density Quantification of cell density was performed with SE in 8 pilo-DZP, 8 pilo-TC10, 7 pilo-TC30, 7 pilo-TC60 and 6 saline-saline rats. 6 days later. In 14 days after SE, pentobarbital animals ^{1.8g / kg (Dolethal (R)} , Ventoquinol, Lure, France) were deeply anesthetized with. The brain was then removed and frozen. Serial 20 μm sections were cut in a cryostat and allowed to air dry for several days prior to thionine staining.

Quantification of cell density was performed on a coronal section according to the stereotaxic coordinates of a rat brain map with a 10 × 10 box 1 cm ² microscope grid (Paxinos G, Watson C (1986) The Rat Brain in Stereotaxic Coordinator.
es, 2nd ed. (See Academic Press, San Diego).
Cell counts were performed twice in a blinded manner and were the average of at least 3 values from 2 adjacent sections in each individual animal. Counts included only cells larger than 10 μm and smaller were considered glial cells.

Two months after the onset of Tim staining spontaneous recurrent seizures, mossy fiber sprouting was examined on rats in the chronic period exposed to test compound or DZP and in 3 saline-saline rats. The animals were deeply anesthetized and saline followed by 1.15% (w / v) Na ₂ S in 100 ml 0.1 M phosphate buffer and 4% (v / v) in 100 ml 0.1 M phosphate buffer. ) Formaldehyde was transcardially perfused. The brain was removed from the skull, postfixed in 4% formaldehyde for 3-5 h, and 40 μm sections were cut on a sliding vibratome and sampled on a gelatin-coated glass slide.

  The next day, sections were cut with 50% (w / v) gum arabic (160 ml), sodium citrate buffer (30 ml), 5.7% (w / v) hydroquinone (80 ml) and 10% (w / v) silver nitrate. Color was developed in a dark place in a solution (2.5 ml) of 26 ° C. for 40-45 minutes. The sections were then rinsed with tap water for at least 45 minutes at 40 ° C., quickly rinsed with distilled water, and dried. They were dehydrated in ethanol and covered with a coverslip.

  Mossy fiber sprouting was evaluated according to previously described criteria in the dorsal hippocampus (Cavazos et al., 1991, J Neurosci 11: 2795-803), which is: 0-DG tips and ridges No granules between; 1- sparse granules in the supragranular region distributed patchy between the tips and edges of the DG; 2- more granules continuously distributed between the tips and edges of the DG 3-with occasional patchy fusogenic granules between tip and ridge, with a continuous pattern of prominent granules between tip and ridge; 4- dense thin lamina with fusibility between tip and ridge; A dense, thin lamellar band of prominent granules forming the band and a granule spreading over the 5-inner molecular layer.

Data Analysis Unpaired Student t-test was used for comparison of SE properties in pilo-saline and pilo-test compound animals. Comparison of the number of rats seizing in both groups was performed using the chi-square test. For neuronal injury, statistical analysis between groups was performed using ANOVA, followed by Statview software (Fisher RA, 1946a, Statistical Methods for Research Workers (10th edition) Oliver & Boyd, RAF 46; , The Design of Experiments (4th edition) Oliver & Boyd, Edinburgh).

The total number of Sprague-Dawley rats weighing lithium-pilocarpine intussusception and EEG traits weighing 250-330 g were subjected to SE induced by L-pilo. The behavioral characteristics of SE were the same in both pilo-saline and pilo-test compound groups. Within 5 minutes after pilocarpine infusion, rats developed diarrhea, napped and other signs of cholinergic stimulation. During the following 15-20 minutes, the rats exhibited head up and down movements, scratching, biting and exploratory behavior. Recurrent seizures began approximately 15-20 minutes after pilocarpine administration. These seizures that correlate episodes of head and bilateral forelimb myoclonus with rising and falling are as previously described (Turski et al., 1983, Behav Brain Res 9: 315-335), about 35 to 35 after pilocarpine. Progressed to SE in 40 minutes.

EEG pattern in SE During the first hour of SE, in the absence of pharmacological treatment, the EEG amplitude gradually increased while the frequency decreased. Within 5 minutes after pilocarpine injection, normal background EEG activity was replaced by low potential fast waves in the cortex, while theta waves (5-7 Hz) appeared in the hippocampus. By 15-20 minutes, high potential fast waves and isolated high potential spikes on the hippocampal theta waves were recorded only in the hippocampus, while cortical activity did not change substantially.

  By 35-40 minutes after pilocarpine injection, the animal develops a typical electrogram seizure with high potential fast waves present in both the hippocampus and cortex, which first occurs as an outbreak of activity prior to the seizure, and thereafter Followed by a continuous series of high-potential and polyspinous waves that continued until administration of DZP or test compound. With an SE of about 3-4 h, hippocampal EEG was characterized by periodic electrogram discharge (PED, about 1 time / second) in pilo-DZP and in pilo-10 group in both hippocampus and cortex. The amplitude of EEG background activity was low in pilo-TC60 animals. By 6-7 h SE, spinal wave activity is still present in the cortex and hippocampus in DZP and TC10 treated rats, while the amplitude of EEG decreases in the hippocampus of TC30 rats and in the structure of both TC60 treated rats, and Returned to baseline level. There was no difference between the TC10, TC30 and TC60 groups. By 9 h SE, isolated spike waves were still recorded in the hippocampus of test compound-treated rats and occasionally in the cortex. In both structures, background activity was of very low amplitude at that time.

Mortality induced by SE During the first 48 h after SE, the extent of mortality was pilo-DZP rats (23%, 5/22), pilo-TC10 rats (26%, 6/23) and pilo- Similar in TC30 rats (20%, 5/25). Mortality was greatly reduced in pilo-TC60 rats, where it only reached 4% (1/23). This difference was statistically significant (p <0.01).

EEG characteristics during the silent phase and the development of spontaneous recurrent seizures The EEG pattern during the silent period was similar in pilo-DZP and pilo-TC10, 30 or 60 rats. At 24 and 48 h after SE, the baseline EEG was still characterized by the occurrence of PED on which large or spike waves could be superimposed. There was no change in pilo-DZP or pilo-TC10 groups between 1-8 h after test compound injection or vehicle injection. In TC30 and TC60 rats, the frequency and amplitude of PED decreased as soon as 10 minutes post-injection and were replaced by high amplitude spikes in the TC30 group and low amplitude spikes in the TC60 group. By 4 h after injection, EEG had returned to baseline levels in the two latter groups. By 6 days after SE, EEG was still of lower amplitude than before pilocarpine injection, and spikes could still be recorded in occasional pilo-DZP, -TC10 and -TC30 rats in most groups . In pilo-TC60 rats, the frequency of large amplitude spikes was higher than in all other groups.

  After test compound or vehicle injection, EEG recordings were not affected by injection in the pilo-DZP and pilo-TC10 groups. In pilo-TC30 rats, injection induced the development of slow waves on both hippocampal and cortical EEG as well as the reduced frequency of spikes in pilo-TC60 rats.

All rats exposed to DZP, TC10, and TC30 and examined until the chronic phase developed spontaneous recurrent seizures (SRS) with similar latency. Latency times were 18.2 ± 6.9 days (n = 9) in pilo-DZP rats, 15.4 ± 5.1 days (n = 7) in pilo-TC10 rats, and 18.9 in pilo-TC30 rats. It was ± 9.0 days (n = 10). In the group of rats subjected to TC60, the sub-group of rats became addicted with a latency similar to that of the other groups, ie 17.6 ± 8.7 days (n = 7), while The group of mice became pupae with a much longer delay between 109-191 days after SE (149.8 ± 36.0 days, n = 4), and one rat was delayed 9 months after SE Did not become a habit. The difference in latency to SRS between the first subgroup of pilo-DZP, pilo-TC10, pilo-TC30 and pilo-TPM60 rats was not statistically significant. None of the saline-saline rats (n = 5) developed SRS.

  To calculate the frequency of spontaneous recurrent seizures (SRS) in rats exposed to pilocarpine, seizure severity and significant stage III (facial muscle and forelimb chronic seizures) and stage IV-V seizures (rising and (Falling) was measured. The frequency of stage III SRS per week in pilo-DZP and pilo-test compound rats varied between groups. It was low and constant in the pilo-DZP and pilo-TC60 (with early SRS onset) group during the first 3 weeks and disappeared during the 4th week in the pilo-DZP group. The frequency of stage III SRS was higher in the pilo-TC10 group, where it was significantly increased above the pilo-DZP value during weeks 3 and 4. The frequency of more severe IV-V SRS was pi60-TC30 and TC60 with late-onset seizures (where the SRS frequency was IV for all 4 weeks in the TC30 group and no seizures were recorded after the second week) It was highest during the first week in most groups, except in the pilo-TC60 group with late SRS onset without ~ V-phase seizures, which was constant over the first two weeks). The frequency of stage IV-V SRS was observed during the first week in the TC10, TC30 and TC60 (with early SRS onset) group (2 per week) compared to the pilo-DZP group (11.3 SRS per week). .3 to 6.1 SRS) was significantly reduced. Has early SRS onset with a significantly reduced seizure frequency from 0.6 to 0.9 seizures per week compared to the pilo-DZP group with SRS frequency between 3.3 and 5.8 Except in the pilo-TC60 group, between 2 and 4 weeks, the frequency of IV-V SRS was reduced in all groups compared to the first week reach of 2-6 seizures per week. .

Cell density in the hippocampus, thalamus and cortex In pilo-DZP rats compared to saline-saline rats, the number of cells is greatly reduced in the CA1 region of the hippocampus (70% cell loss in the pyramidal cell layer), On the other hand, the CAS region was not significantly damaged (54% cell loss in CA3a and 31% in CA3b). In the dentate gyrus, pilo-DZP rats experienced extensive cell loss in the portal (73%), while the granule cell layer showed no visible damage. Similar damage was observed in the ventral hippocampus, but cell counting was not performed in this area. Large scale damage was also recorded in the lateral thalamic nucleus (91% cell loss), while the dorsolateral nucleus of the thalamus was more moderately damaged (56%). In piriform cortex, in pilo-DZP rats, cell loss was all in the III-IV layer, which was no longer visible and reached 53% in the II layer. In the dorsal entorhinal cortex, layers II and III-IV were slightly damaged (9 and 15%, respectively). The layer II of the ventral entorhinal cortex was fully maintained, while layers III-IV suffered 44% cell loss.

In the hippocampus of pilo-test compound animals, cell loss is reduced in the CA1 pyramidal layer compared to pilo-DZP rats, where cell loss is 75% in pilo-DZP and pilo-TC30 or pilo-TC60. Reached 35 and 16% in animals, respectively. This difference was statistically significant at the two test compound doses. In the CAS cone layer, the test compound does not provide any protection in the CA3a region, while 6
A test compound dose of 0 mg / kg significantly protected the nerve in CA3b. In the dentate gyrus, cell loss in the portal was similar in pilo-test compounds (69-72%) and pilo-DZP animals (73%). In the two thalamic nuclei, the 60 mg / kg dose also protected in reducing 65 and 42% neuronal damage in the lateral and dorsal medial nuclei, respectively. In the cerebral cortex, treatment with the test compound provided neuronal protection compared to DZP only at the highest dose, 60 mg / kg. At the two lowest doses, 10 and 30 mg / kg, the total cell loss and tissue disruption observed in the III-IV layers of the piriform cortex are identical in pilo-DZP and pilo-test compound rats, and these Did not allow counting in any of the groups. In layers II and III-IV of the piriform cortex, TC60 treatment reduced the neuronal damage recorded in pilo-DZP rats by 41 and 44%, respectively. In the ventral entorhinal cortex, neuroprotection was induced by TC60 administration in layers III-IV and reached 31% compared to pilo-DZP rats. In the entorhinal cortex, pilo compared to pilo-DZP rats in the dorsal entorhinal cortex III-IV (more than 28% damage) and ventral entorhinal cortex III-IV (more than 35% damage). -There was a slight exacerbation of cell loss in TC10 rats. At other doses of test compound, cell loss in the entorhinal cortex was similar to that recorded in pilo-DZP rats.

All rats showing SRS in the mossy fiber sprouting pilo-DZP and pilo-TPM groups in the hippocampus showed similar intensity of Tim staining in the inner molecular layer of the dentate gyrus (score 2-4). Tim staining was present both on the upper and lower blades of the dentate gyrus. The mean value of the Tim score in the upper blade is 2.8 ± 0.8 in pilo-DZP rats (n = 9), 1.5 ± 0.6 in pilo-TC10 rats (n = 7), pilo-TC30 rats It reached 2.6 ± 1.0 at (n = 10) and 1.5 ± 0.7 in all groups of pilo-TC60 rats (n = 11). When the 60 mg / kg pilo-test compound group was further divided according to the latency to SRS, the subgroup with early SRS development showed a Tim score of 1.8 ± 0.6 (n = 6) and the SRS A subgroup of rats with late onset or absence had a Tim score of 1.2 ± 0.6 (n = 5). The values recorded in pilo-DZP rats are statistically significant with those in pilo-TC10 (p = 0.032) and the pilo-TC60 subgroup with or without late seizures (p = 0.016) Different.

Explanation and Conclusions The results of this study show that brain areas in which 7 days of treatment with test compounds beginning 1 h after the onset of SE have resulted from neuronal damage (eg, pyramidal cell layers of CA1 and CA3b areas, thalamic dorsum, pear (In the II and II MV layers of the cortex and the III-IV layers of the ventral entorhinal cortex), but only the highest dose of the test compound, ie 60 mg / kg, shows protection. The latter dose of test compound was also culled with at least a subgroup of animals (they had a mean delay of about 9 times longer than in other groups of animals, and one animal was delayed 9 months after SE The occurrence of SRS can also be delayed.

  These results indicate that certain compounds with anti-seizure properties, which are the classic properties of most anti-epileptic drugs, can also delay the occurrence of epilepsy, ie, are anti-epileptic. The data in this study also show that test compound treatment reduced the number of IV-V seizures, primarily during the first week of development and during the entire 4-week observation period at 60 mg / kg test compound treatment. So it also shows that whatever the dose used, it reduces the severity of sputum. In addition, there is a shift in the TC10 group to an increased incidence of less severe stage III seizures, which is greater than in the pilo-DZP group.

  The purpose of this extension of the study is reported in Example 1 above for the potential neuroprotective and anti-epileptic properties of the same test compound (TC) in the Lithium-Pilocarpine (Li-Pilo) model of temporal lobe It was to continue the research. In the first study, TC was shown to be able to protect the hippocampal CA1 and CA3 regions, piriform and ventral entorhinal cortex from neuronal damage induced by Li-Pilo epilepticus (SE). Most of these neuroprotective properties occurred at the highest dose examined, 60 mg / kg, and the treatment was able to delay the development of spontaneous seizures in 36% of rats (4 of 11). In this example, the results of treatment with higher doses of TC to neuronal damage and epilepsy development are examined.

  The model of epilepsy induced in rats by pilocarpine associated with lithium (Li-Pilo) reproduces most of the clinical and neurophysiological features of human temporal lobe epilepsy (Turski L, Ikonomidou C, Turski WA, Bortolotto ZA, Cavalheiro EA (1989) Review: Cholinergic mechanisms and epileptogenesis.The seizures induced by pilocarpine: a novel experimental model of intractable epilepsy, Synapse 3: 154-171; Cavalheiro EA (1995) The pilocarpine model o f epilepsy.Ital J Neuro Sci 16: 33-37).

In adult rats, systemic administration of pilocarpine results in an SE that can last up to 24 h. The fatality rate reaches 30-50% during the first day. In surviving animals, neuronal damage is prominent in the hippocampus, piriform and entorhinal cortex, thalamus, amygdala, neocortex and substantia nigra. This acute seizure period is followed by a “silent” seizure period that lasts for an average period of 14-25 days, after which all animals show spontaneous recurrent seizures with a normal frequency of 2-5 times per week ( Turski L, Ikonomidou C, Turski WA, Bortolotto ZA, Cavalheiro EA (1989) Review: Cholinergic mechanisms and epileptogenesis, The seizures induced by pilocarpine: a novel experimental model of intractable epilepsy Synapse 3: 154-171; Cavalheiro EA (1995) The pilocarpine model of epile Psy.Ital J Neuro Sci 16: 33-37; Dube C, Boyet S, Marescuux C, Nehlig A (2001) Relationship.
between neuronal loss and interactive glucose metabolism, the chronic phase of the lithium-pilocarpine model of epilepsy in the immunity and the imprint. Exp
Neurol 167: 227-241).

  Current antiepileptic drugs (AEDs) do not prevent epilepsy and are only temporarily effective against recurrent seizures.

In our previous study we studied the potential neuroprotective and anti-epileptic effects of increasing doses of test compound (TC) given in monotherapy and given primarily to prevent high mortality Compared to standard diazepam (DZP) treatment. These data indicate that 7 days treatment with 10, 30 or 60 mg / kg TC starting 1 h after the onset of SE can protect certain brain regions from neuronal damage. This effect is seen in the pyramidal cell layer of the CA1 and CA3b regions, dorsal thalamus, layers II and III-IV of the piriform cortex and layers III-IV of the ventral entorhinal cortex, but the highest dose of TC, ie 60 mg Only at / kg is statistically significant. In addition, the latter dose of TC was also culled with at least a subgroup of animals (they had a mean delay of about 9 times longer than in other groups of animals, and one animal was 9 months after SE It appears to be the only one that can delay the onset of spontaneous recurrent seizures (SRS).

  In this study, the effects of different doses of test compound (TC), namely 30, 60, 90 and 120 mg / kg (TC30, TC60, TC90 and TC120) were tested using the same design as in the previous study. Treatment started 1 hour after the onset of SE and the animals were treated with a second infusion of the same dose of drug. This early treatment of SE was followed by 6 days of TC treatment. This report relates to the effect of four different doses of TC on neuronal damage assessed in the hippocampus, parahippocampal cortex, thalamus and tonsils 14 days after SE and on the latency and frequency to spontaneous epileptic seizures.

Method Animals Adult male Sprague-Dawley rats provided by Janvier Breeding Center (Le Genest-St-Isle, France) were controlled at 20-22 ° C. under non-congested standard conditions (light-dark cycle, 7.00 a. m. to 7.00 pm (lights up) and food and water were freely available. All animal experiments were performed in accordance with the rules of the European Community Council Directive (86/609 / EEC) of November 24, 1986 and the French Ministry of Agriculture (License N ° 67-97).

Intussusception induction, test compound (TC) treatment and development of SRS All rats were given lithium chloride (3 meq / kg, ip, Sigma, St Louis, Mo, USA) And about 20 h later, all animals were also given methylscopolamine bromide (1 mg / kg, sc, Sigma) administered to limit the peripheral effects of convulsants. SE was induced by injecting pilocarpine hydrochloride (25 mg / kg, sc, Sigma) 30 minutes after methyl scopolamine. The effect of increasing doses of TC was investigated in 5 groups of rats. The animals received 2.5 mg / kg DZP 1 h after the onset of SE, i. m. Or 30, 60, 90 or 120 mg / kg TC (TC30, TC60, TC90, TC120), i. p. Gave one of them. The control group was given vehicle instead of pilocarpine and TC, then about 10 h after the first TC injection to rats surviving SE, the DZP group had 1.25 mg / kg DZP or in the morning A second i. Of the same dose of TC. p. Injections were infused and maintained under TC treatment (sc) twice daily for an additional 6 days, while DZP rats were given vehicle infusion.

  The effect of 4 doses of DZP and TC on epileptic development was examined by daily video recording of animals for 10 hours per day. Video recording takes place for 4 weeks, during which time the first seizure occurs as well as the total number of seizures over the entire period. The animals were then removed from the video recording system and kept in our animal facility for an additional 4 weeks, after which they were killed after a total 8 week drought period. Rats that did not show seizures were killed after 5 months of video recording.

Quantification of cell density Quantification of cell density was performed at two time points after SE: the first group was examined 14 days after SE and 7 DZP, 8 TC30, 11 TC60, 10 Consists of TC90, 8 TC120 and 8 control rats not subjected to SE. The second group used to study latency to SRS was killed 8 weeks after the first SRS or 5 months if SRS could not be observed in that delay, and 14 DZP, 8 It consisted of 1 TC30, 10 TC60, 11 TC90, 9 TC120 rats. Currently, neuronal counts are still ongoing in a second group of animals studying epilepsy, and long-term counts and data relating to that part of the study are not included in this report.

For neuronal counting, pentobarbital animals ^{1.8g / kg (Dolethal (R)} , Vetoquinol, Lure, France) were deeply anesthetized with. The brain was then removed and frozen. Serial 20 μm sections were cut in a cryostat and allowed to air dry for several days prior to thionine staining. Quantification of cell density was performed on a coronal section according to the stereotaxic coordinates of a rat brain map with a 10 × 10 box 1 cm ² microscope grid (Paxinos G, Watson C (1986) The Rat Brain in Stereotaxic.
Coordinates, 2nd ed. (See Academic Press, San Diego). Count grids were placed on well-defined areas of the brain structure of interest, and counts were performed with a 200 or 400-fold microscope magnification specified for each single brain structure. Cell counts were performed twice on each side of three adjacent sections for each region by a single observer unaware of the animal's treatment. The number of cells obtained in 12 counted fields in each brain structure was averaged. This method was used to minimize potential errors that could be attributed to double counting leading to overestimation of cell number. Neurons touching the bottom and right edge of the grid were not counted. Counts included only neurons with cell bodies larger than 10 μm. Cells with small cell bodies were considered glial cells and were not counted.

Data analysis For neuronal damage and wrinkle development, statistical analysis between groups was performed using one-way analysis of variance followed by Statistica software using post hoc Dunnett or Fisher test.

Results Behavioral Characteristics of Lithium-Pilocarpine Suspension Sustained State A total of 143 Sprague-Dawley rats weighing 250-330 g were subjected to SE induced by lithium-pilocarpine (L-pilo). In this number, 10 animals did not develop SE, while 133 rats developed fully characteristic Li-pilo SE. The behavioral characteristics of SE were the same in both li-pilo-DZP and li-pilo-TC groups. Within 5 minutes after pilocarpine infusion, rats developed diarrhea, napped and other signs of cholinergic stimulation. During the following 15-20 minutes, the rats exhibited head up and down movements, scratching, biting and exploratory behavior. Recurrent seizures began approximately 15-20 minutes after pilocarpine administration. These seizures that correlate episodes of head and bilateral forelimb myoclonus with rise and fall are as previously described (Turski L, Ikonomidou C, Turki WA, Bautrotto ZA, Cavallero EA (1989) Review: Choline: Choline epithelogenesis. The seizures induced by pilocarpine: a novel experimental model of intactable epilepile epsilon. Cnap3: 154-171; loss and interictal glucose metabolism during the chronic phase of the lithium-pilocarpine model of epilepsy in the immature and adult rat.Exp Neurol 167: 227-241; Andre V, Rigoulot MA, Koning E, Ferran
don A, Nehlig A (2003) Long-term pregabalin
treatment protections basal cortices and delays the occurrence of spontaneous strains in the lithium-pilocarpine model in
the rat. Epilesia 44: 893-903), progressed to SE about 35-40 minutes after pilocarpine. The control group not subjected to SE and given lithium and saline consisted of 20 rats.

  In a group of 57 animals subjected to cell counts 14 days after SE, a total of 13 rats died over the first 48 h after SE. The degree of mortality varies from treatment to treatment: 36% (4/11) in DZP rats, 33% (4/12) in TC30 rats, 8% (1/12) in TC60 rats, 0% (0/0) in TC90 rats. 10) and 33% (4/12) of TC120 rats died. In the DZP group, 4 rats died within the first 24 h after SE. In the group of TC30 rats, one died on the day of SE, one rat died by 24h after SE, and two rats died by 48h. In the group of TC60 rats, one rat died 48 h after SE. In the group of TC120 rats, 2 rats died by 24 h after SE and 2 died by 48 h.

  In the group of 55 animals subjected to latency to SRS and late cell count studies, the degree of mortality over the first 48 h after SE was as follows: 7% (1/14) of DZP rats 27% (3/11) of TC30 rats, 0% (0/10) of TC60 rats, 0% (0/11) of TC90 rats and 0% (0/9) of TC120 rats died. In the group of DZP rats, one rat died during the first 24 h after SE. In the TC30 group, two rats died by 24h after SE and one by 48h.

Cell density in the hippocampus and cortex at an early stage (14 days after SE) In DZP rats compared to control rats, the number of neurons is greatly reduced in the CA1 region of the hippocampus (85% loss in the pyramidal cell layer), whereas The CA3 region was not significantly damaged (40% loss) (Table 1 and FIG. 1). In the dentate gyrus, DZP rats experienced massive neuronal loss in the portal (65%), while the granule cell layer showed no obvious damage. The same distribution of injury was observed in the ventral hippocampus, but cell counting was not performed in this area.

  In the thalamus, neuronal loss is moderate in the dorsal medial and lateral, dorsolateral medial dorsal and in the medial central nucleus (18, 24, 40, and 34% dropout, respectively) and the dorsal medial nucleus In the ventral lateral part of the dorsolateral nucleus (90%) (Table 1 and Figure 2). In tonsils, neuronal loss was moderate in the medial ventral posterior nucleus (38%) and more prominent in the basolateral and medial dorsal pronuclei (73 and 53% dropout, respectively). There was no neuronal damage in the central nucleus (Table 1 and FIG. 3).

  In piriform cortex, neuronal loss is almost all in layer III (94%), which is no longer actually visible, and dorsal and ventral layer II in DZP rats compared to control saline-treated rats. Reached 66 and 89% in each. In the dorsal entorhinal cortex, layers II and III-IV were slightly damaged (18 and 124%, respectively), and in the ventral II and III / IV layers, the damage reached 22 and 74%, respectively ( Table 1 below and FIG. 4).

In the hippocampus of TC-treated animals, cell loss was significantly reduced in the CA1 pyramidal cell layer compared to DZP rats. This reduction was significant in TC30, 60 or 90 rats (36-47% cell loss) and significant in the TC120 group (12% cell loss). These differences were statistically significant at all TC doses (Table 1 and FIG. 1). In the CA3 pyramidal layer, only a dose of 120 mg / kg tended to have a slight neuroprotection induced by the test compound, but the difference from the DZP group was not significant. In the dentate gyrus,
Cell loss in the portal is similar in DZP and TC30, 60 and 90 groups (61-66% loss) and reduced in TC120 group (53% neuronal loss) compared to DZP animals (66% loss) There was a slight tendency to damage. None of these differences were statistically significant.

  In the thalamus, neuronal loss was similar in DZP and TC30 and TC60 rats. The difference did not reach significance in the dorsal medial and medial central nuclei of TC90 rats, but TC was at a dose of 60 mg / kg in the dorsolateral medial dorsal nucleus and the two highest doses in all thalamic nuclei, Significant protection was obtained at 90 and 120 mg / kg. In TC120 rats, neuronal loss was significantly reduced compared to DZP rats. It was between 4-19% and the number of neurons was no longer significantly different from the control animals except in the dorsolateral medial dorsal nucleus (Table 1 and FIG. 2). In the tonsils, TC also significantly protected in the basolateral nucleus at a dose of 30 mg / kg and also in the medial dorsal pronucleus at a dose of 60 mg. At the highest dose, TC generally protected nerves; the number of neurons was no longer significantly different from the control level and reached 86-99% of the control level in all amygdala (Table 1 and FIG. 3).

  In the cerebral cortex, treatment with TC did not significantly protect any cortical area compared to DZP treatment at a dose of 30 mg / kg. At 60 mg / kg, TC significantly reduced neuronal loss only in layer II of the dorsal piriform cortex (25% loss compared to 66% in the DZP group). At 90 and 120 mg / kg, TC significantly protected all three areas of the piriform cortex compared to DZP treatment, and at the highest dose of TC, 120 mg / kg, neuron density was the neuronal population in the DZP group. Even in the piriform cortex, dorsal layer II and layer III, which were almost completely depleted, 78-96% of the control level was reached. In all layers of the dorsal and ventral entorhinal cortex, the two lowest doses of TC, 30 and 60 mg / kg, did not give any neuroprotection. The 90 mg / kg dose of TC significantly protected the II and III / IV layers of the ventral entorhinal cortex (in the dorsal II and II / IV layers compared to 19 and 73% in the DZP group and 4 and 17% injury remaining in layer II of ventral part). At the highest dose of TC, 120 mg / kg, all parts of both the dorsal and ventral entorhinal cortex were protected, and the number of neurons in these areas was no longer significantly different from levels in controls ( 85-94% neurons surviving compared to 27-81% in the DZP group).

  Latency to spontaneous seizures reached a mean value of 15.5 ± 2.3 days in the DZP group (14 rats) and was similar in the TC30 group (8 rats) (11.6 ± 2.5 days). At higher concentrations of TC, the animals could be further divided into subgroups with short and long latency times. A short incubation time was considered as any period shorter than 40 days after SE. One rat showed latency to the first spontaneous seizure similar to that recorded in the DZP and TC groups, but the number of rats showing this short latency value gradually increased with increasing TC concentration. Diminished. Thus, at 30 mg / kg, 70% (7/10) of the rats have a short latency to seizure, whereas at 90 and 120 mg / kg, this percentage is 36% (4/11) and 11%, respectively. (1/9) was reached (Table 2 below and FIG. 5).

  In the TC60, 90 and 120 groups, the mean values for rats with long incubation times were similar and between 52 and 85 days. Finally, with the two highest doses of TC, it was possible to identify a certain percentage of rats that did not develop any seizures over a period of 150 days after SE. The percentage of non-fertile rats reached 45% at both doses of TC.

  The frequency of spontaneous seizures was similar across the 4 week record. It showed a higher trend in the DZP and TC30 groups, while it was lower in the TC60, 90 and 120 groups (FIG. 6). These differences did not reach a statistical advantage at the level of each individual weekly frequency, but reached an advantage over the total or average number of seizures over 4 weeks.

  The number of seizures was also plotted according to the duration of latency to the first spontaneous seizure. Animals with a short incubation time tended to display 2-3 times more seizures over the 4-week recording than rats with a long incubation period. Statistical analysis could not be performed because ANOVA did not show any significance, probably because there was only one animal in the short latency subgroup of TC120 animals (FIG. 7). However, when all latency values were plotted against the number of seizures, there was a significant inverse correlation that resulted in a straight line with a correlation coefficient of −0.4 (FIG. 8).

  To summarize this analysis, two more measurements are made. The first was video-recorded to investigate the potential correlation between the extent and location of brain damage and the occurrence and / or latency to date of spontaneous seizures and 2 after the first spontaneous seizure. Cell counts in animals followed for months or killed at 5 months. The second is that we conduct a 1-year follow-up of seizures in a group of rats to see if animals that are “non-fertile” at 5 months are still seizures.

The results of this study show that treatment with TC starting 1 h after the onset of Li-pilo induced SE in the hippocampal CA1 pyramidal cell layer and all of the ventral and dorsal piriform and entorhinal cortex Indicates that it has neuroprotective properties in the layer. TC also protects the thalamus and amygdala. However, TC does not protect at a dose of 30 mg / kg except in CA1, one thalamic nucleus and one amygdala. At a dose of 60 mg / kg, dorsal piriform cortex layer II and the second amygdala are also protected. At 90 and 120 mg / kg, the drug protects most of the brain regions studied except for hippocampal CA3 and dentate gyrus. In addition to the latter two structures, the dorsolateral ventral dorsal thalamic nucleus is the only area where the number of neurons remains significantly different from the control at a dose of 120 mg / kg TC. From these data, the very strong neuroprotective properties of TC are clearly displayed. The molecule appears to prevent neuronal death in most areas belonging to the circuit of the limbic fistula induced by Li-pilo, namely the hippocampus, thalamus, tonsils and hippocampal cortex. These are all areas where we detect MRI signals during the course of epilepsy in Li-pilo-treated rats (Roch C, Leroy C, Nehlig A, Namer IJ (2002a) Tribulation of magnetic resonance imaging to the test. of the lithium-pilocarpine model of temporal lobe epilepsy in adult
rats. Epilesia 43: 325-335). The only two regions that are not efficiently protected by TC are the CA3 pyramidal cell layer and the dentate gyrus. The latter area, rapid and received a large amount of cell damage (Andre V, Marescaux C, Nehlig A, Fritschy JM (2001) Alterations of the hippocampal GABAergic system contribute to the development of spontaneous recurrent seizures in the lithium-pilocarpine model of temporal lobe epilepsy.Hippocampus 11: 452-468.; Roch C, Leroy C, Nehlig A, Namer IJ (2002a) Contribution of magnetic resonance tomato. of the lithium-pilocarpine model of temporal lobe
epilepsy in adult rats. Epilesia 43: 325-335), and none of the neuroprotection we used in previous studies has been able to protect this structure. We have also identified this structure as an important region in the initiation and maintenance of epileptic seizures in the Li-pilo model based on previous studies (Dube C, Marescuux C, Nehrig A (2000) A metabolic and neuropathological approach). to the
understanding of plastic changes occurring in the immediate and adult brain braining lithium-pilocarpine induced epitogenesis. Epilesia 41 (Suppl 6): S36-S43).

  Obviously, the data show that wrinkling can be prevented even if the damage remains quite noticeable in this area. Long-term cell counts in the group of animals recording video can indicate whether the extent of damage in this area is important for wrinkle development in this model.

Treatment did not affect the latency to first spontaneous attack at a dose of 30 mg / kg. At the three higher doses, a certain percentage of animals developed epilepsy as early as DZP or TC30 rats, but the relative importance of this subgroup was inversely related to the dose of TC used. Another subgroup of constant size (2-4 animals per group) developed epilepsy after a 4-6 fold longer incubation time, while at the two highest doses of the drug, 4-5 Rats were not trapped after 5 months (ie, about 10 times the duration of the short incubation period and 2-3 times that of the long incubation period). This delay in wrinkle development may correlate with the number of neurons protected in the animal's basal cortex. This assumption is based on the fact that we noticed some heterogeneity in the degree of neuroprotection in the basal cortex of animals that were subjected to short-term neuronal counts 14 days after SE. However, currently we do not perform neuron counting in the animals used for the study of epilepsy, and therefore there is a potential relationship between the number of neurons surviving in the basal cortex and the rate or even the occurrence of epilepsy. No conclusion can be drawn.

The data obtained in this study is consistent with previous studies from this group reporting that a 60 mg / kg dose of test compound (TC) protected the hippocampus and basal cortex from neuronal damage and delayed the occurrence of recurrent seizures. Match (see Example 1). They support that protection of the basal cortex can be an important factor in inducing disease modifying effects in the lithium-pilocarpine model of epilepsy. The critical role of the basal cortex as an acupuncture process initiator was previously shown by our group in the lithium-pilocarpine model (Andre V, Rigulot MA, Konning E, Ferrandon
A, Nehrig A (2003) Long-term pregabalin treatment protections basal cortices and delays the occultence of the stenoids in the intiminepile. Epissia 44: 893-903; Roch C, Leroy C, Nehlig A, Name IJ (2002a) Contribution of magnetic resorpment of the energy of the symposium. Epilepsia 43: 325-335; Roch C, Leroy C, Nehlig A, Namer IJ (2002b) Predictive value of cortical injury for the development of temporal lobe epilepsy in P21-day-old rats: a MRI approach using the lithium-pilocarpine model . Epilepsia 43: 1129-1136).

References Andre V of Example 2, Marescaux C, Nehlig A, Fritschy JM (2001) Alterations of the hippocampal GABAergic system contribute to the development of spontaneous recurrent seizures in the lithium-pilocarpine model of temporal lobe epilepsy. Hippocampus 11: 452-468.
・ Andre V, Rigolot MA, Koning E, Ferrandon A, Nehrig A (2003) Long-term pregabalin treatment protections basal cortices and delays theorenous therapies
in the lithium-pilocarpine model in the
rat. Epilesia 44: 893-903.
Cavaleiro EA (1995) The pilocarpine model of epilepsy. Ital J Neuro Sci 16: 33-37
.
・ Dube C, Marescuux C, Nehlig A (2000) A metabolic and neuropathological to the unbalanced urine in circulatory and circulatory circulatory in circulatory and circulatory in circulatory and circulatory in circulatory and circulatory. Epilesia 41 (Suppl 6): S36-S43.
・ Dube C, Boyet S, Marescux C, Nehrig A (2001) Relationship between neuron loss and
interactive glucose metabolism of the chronic phase of the lithium-pilocarpine model of epilepsy in the image and
adult rat. Exp Neurol 167: 227-241.
Paxinos G, Watson C (1986) The Rat Brain in Stereocoordinates, 2nd ed. Academic Press, San Diego.
· Roch C, Leroy C, Nehlig A, Name IJ (2002a) Conviction of magnetic resilience imaging to the of the thorium in the shape of the scale. Epilesia 43: 325-335.
・ Roch C, Leroy C, Nehlig A, Name IJ (2002b) Predictive value of correlatively the development of the temporary lobe in the shape of the role of the epilepsy. Epilesia 43: 1129-1136.
・ Turski L, Ikonomidou C, Turki WA, Bolotlotto ZA, Cavaleiro EA (1989) Review: Cholinerchanisms and epiptogenesis. The seizures induced by pilotcarpine: a novel experimental model of intractable epilepsy. Synapse 3: 154-171.

PC12 cell serum depletion model serum depletion is a cytotoxic environmental challenge that results in cell death in cultured cell lines as well as in primary cells from a variety of tissues, including neurons. In particular, pheochromocytoma (PC) 12 cells are widely used as an in vitro neuronal cell model for a wide variety of neurodegenerative and cell death related disorders (Muriel, et al, Mitochondrial).
free calcium levels (Rhod-2 fluorescens) and ultrastructural alterations in newly differentiated PC12 cells during ceramide-dependent cell death. Comp. Neurol. , 2000, 426 (2), 297-315; Dermitzaki, et al, Opioids transient prevent activation.
of apoptotic mechanisms following shorting
periods of serum withdrawal, J. et al. Neurochem. , 2000, 74 (3), 960-969; Carlele, et al, Redu.
ced apoptosis after nerve growth factor and serum withdrawal: conversion of tetrameric glyceraldehyde-3, phosphate dehydrogenaseol dimermer. 2000, 57 (1), 2-12). PC12 cells were cultured in sterile medium (RPMI 1640) supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum (FBS). The culture medium also contained penicillin-streptomycin-neomycin antibiotics (50.mu.g, 50.mu.g, 100.mu.g, respectively). The medium was changed every other day and the cells were passaged in log phase near the fusion.

  Control cells were cultured in normal media without any treatment. The enantiomer of formula 7 or formula 8 (10.mu.M) was mixed well in the medium and then applied to the cells. In the 2-day assay, the enantiomer of formula 7 or formula 8 (10.mu.M) was only applied to the cells once at the time of serum deprivation. In the 7 day assay, the enantiomer of formula 7 or formula 8 (10.mu.M) is applied to the cells at the time of serum depletion and every 48 hr thereafter when the cells are replaced with fresh fresh serum-free medium. did. In the serum-depleted group, cells were cultured in serum-free medium without the additional enantiomer of Formula 7 or Formula 8. Cell survival was observed at 3- or 7-days after serum deprivation by 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxy-methoxyphenyl) -2- (4-sulfophenyl) -2H- Determined by tetrazolium inner salt (MTS) assay.

  At the end of the experiment, cells were washed with fresh media and incubated with MTS solution for 1.5 hr in a humidified 37 ° C. incubator with 5% CO 2. After the incubation period, the cells were analyzed immediately using the Softmax program (Molecular Devices). The MTS assay is a calorimetric method for determining the number of viable cells in a given experimental setting. The assay is based on the intracellular conversion of tetrazolium salt, MTS to formazan, which is soluble in tissue culture medium and measured directly at 490 nm in a 96-well assay plate. Absorbance is directly proportional to the number of living cells in culture. Any absorbance reading in control cells is expressed as 100% viability.

  Table 3 lists data showing the effect on cell viability of orally administered enantiomers of Formula 7 and Formula 8 in the PC12 cell serum depletion model.

Transient cerebral ischemic rat model Formula 7 enantiomer (test compound) at 10 and 100 mg / kg (iv) in the transient cerebral ischemic middle cerebral artery occlusion (MCAO) rat model (Nagazawa H. and Kogure K., Stroke, 1989
, 20, 1037; and Zea Longa E .; , Weinstein P .; R. , Carlson S. and Cummins R.M. , Stroke, 1989, 20, 84). MK801 (dizocilpine maleate; CAS registry number 77086-22-7, commercially available neuroprotective compound) was used as a positive control (3 mg / kg, ip).

  Rats (n = 12) were randomly assigned to one of four experimental groups and anesthetized. Blood flow from the internal carotid artery, the anterior cerebral artery and the posterior cerebral artery to the middle cerebral artery was blocked by this method. At 1 hour after block, animals are given vehicle (administered iv over 1 hour period), control (administered as a single ip dose at the beginning of the 1 hour period). And 2 doses of the enantiomer of Formula 7 (administered iv over a 1 hour period). Reperfusion was performed 2 hours after blocking. The animals were sacrificed and 20 mm thick coronal sections of each brain were prepared. The extent of brain lesions was quantified using one for every 40 sections of the pre-occipital cortex (ie every 800 nM). Glass slides were prepared using sections stained with cresyl violet (according to the Nissl method) and examined under a light microscope.

  The local ischemic surface area in coronal sections of individual rats was determined according to the presence of cells with morphological changes. The area of neuronal injury or infarct was measured and then added. Cortical and striatum volumes were calculated for each animal (total ischemic surface area x 0.8 mm (thickness)).

MCAO model analysis The mean volume (. +-. SEM) of each animal randomly assigned to the four experimental groups is one-way ANOVA (one-way ANOVA is 3 or more matches) This is a statistical method of comparing non-groups) followed by comparison using Dunnett's t-test (both methods are incorporated into Statview 512+ software, BarinPower, Calabasas, Calif., USA).

As shown in Table 4 below, the results were considered statistically significant when the p-value was <0.05 compared to the vehicle group ( ¹ p <0.01; ² p <0.05).

  The test compound (TC) referred to in the following examples is the same compound as the compound of formula 7 and the other examples 1 and 2 above.

  The purpose of this study was to evaluate the pharmacokinetics (PK) of test compound (TC) after single and repeated oral administration in healthy adults at clinically relevant doses.

Method:
Two single center, placebo-controlled, double-blind, incremental dose studies were performed in > 18 and < 45 year old healthy individuals. In Study 1 (N = 70), subjects were randomly assigned to a single dose of test compound (TC) or placebo. Increasing doses were given as 100, 250, 400, 750, 1000, 1250 and 1500 mg. PK parameters were estimated from plasma and urine samples collected up to 3 days after dosing. Study 2 (N = 53) evaluated the repeated dose PK of the test compound (TC) in four dose groups (100, 250, 500 or 750 mg). Within each group, 12 subjects were assigned q12h treatment with 1 wk of drug or placebo and crossed after a 14 day washout period. PK parameters were estimated from plasma and urine samples on days 1 and 7.

result:
Single dose: Test compound (TC) was rapidly absorbed after oral administration. C _max and AUC _0-∞ increased proportionally over the range of 100-1500 mg. The average t _max was between 1.3 and 2.7 h. Mean t _1/2 (11.5 to 13.9 h), CL / F (2.87 to 3.67 L / h) and Vd / F (52.1 to 66.3 L / h) values for all 7 The same was true for the administration group.

Repeated administration: The plasma concentration of the test compound (TC) reached steady state after 3-4 days as predicted by its single dose half-life. Mean t _max occurred 1.3-1.8 h after dosing. Mean t _1/2 (11.9 to 12.8 h) and CL / F (3.40 to 3.78 L / h) values at steady state were observed on day 1 and after single dose administration in Study 1. Comparable to PK parameters. Steady state C _max and AUC _0-12 increased in proportion to dose.

As expected, there was moderate test compound (TC) accumulation; C _max and AUC _0-12 were about twice as high on day 7 as compared to day 1 (P <0.001). The average CL _R approximate test compound (TC) is <5% of the average oral clearance, suggesting non renal clearance as the primary mechanism of the test compound (TC) elimination.

Conclusion:
The test compound (TC) showed linear PK after single (100-1500 mg) and repeated (100-750 mg bid) administration. It is rapidly absorbed and has an average elimination half-life of 11.5 to 13.9 h, allowing bid dosing. After q12h administration, the test compound (TC) accumulated twice and was largely removed by non-renal route.

All references cited herein are specifically and individually indicated when each individual publication or patent or patent application is incorporated by reference in its entirety for all purposes. To the same extent, all of them are incorporated herein by reference for all purposes.

  The discussion of references herein is merely a summary of the claims made by their authors, and no admission is made that any reference constitutes prior art. Applicant reserves the right to challenge the accuracy and appropriateness of the cited references.

The present invention is not limited with respect to the particular embodiments described herein, which are intended as an illustration of individual embodiments of the invention. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. In addition to those enumerated herein, functionally equivalent methods and apparatus within the scope of the invention will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications and variations are intended to fall within the scope of the appended claims. The invention is limited only with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

FIG. 5 is a graph showing the effect of increasing doses of TC on the number of neurons in different regions of the hippocampus counted 14 days after li-pilo SE. Values are the number of neuronal cell bodies in each region of interest ± S. E. M.M. Represented as: FIG. 5 is a graph showing the effect of increasing doses of TC on the number of neurons in different nuclei of tonsils counted 14 days after li-pilo SE. Values are the number of neuronal cell bodies in each region of interest ± S. E. M.M. Represented as: FIG. 6 is a graph showing the effect of increasing doses of TC on the number of neurons in different nuclei of the thalamus counted 14 days after li-pilo SE. Values are the number of neuronal cell bodies in each region of interest ± S. E. M.M. Represented as: FIG. 6 is a graph showing the effect of increasing doses of TC on the number of neurons in different areas of the cortex counted 14 days after li-pilo SE. Values are the number of neuronal cell bodies in each region of interest ± S. E. M.M. Represented as: FIG. 3 is a graph showing the effect of increasing doses of TC on latency to first spontaneous seizure. Values are the daily mean latency of each group ± S. E. M.M. Represented as: FIG. 4 is a graph showing the effect of increasing doses of TC on the frequency of spontaneous seizures recorded video over a 4 week period. Values are mean number of seizures ± S. E. M.M. Represented as: The total represents the total number of seizures observed during the 4 week video recording, and the average represents the average number of seizures per week. The Anova test showed the effect of treatment on the total number of seizures (p = 0.045) and the average number of seizures per week (p = 0.045). The total number of seizures recorded video over 4 weeks plotted according to latency to first spontaneous seizure is shown (SL = short latency, LL = long latency). Values are mean number of seizures in each subgroup ± SEM E. M.M. Represented as: The ANOVA test did not show any significant effect of treatment. Figure 2 shows the correlation between the latency to first spontaneous seizure and the total number of seizures observed during the subsequent 4 weeks.

Claims (46)

Formula (I) and Formula (II):

[Where:
Phenyl is substituted at X with 1 to 5 halogen atoms selected from the group consisting of fluorine, chlorine, bromine and iodine; and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrogen and Independently selected from the group consisting of C ₁ -C ₄ alkyl; wherein C ₁ -C ₄ alkyl is optionally substituted with phenyl, where phenyl is optionally halogen, C ₁ -C _And optionally substituted with a substituent independently selected from the group consisting of ₄ alkyl, C ₁ -C ₄ alkoxy, amino, nitro and cyano)]
Administering to a patient in need of treatment with a neuroprotective drug (NPD) a therapeutically effective amount of a compound selected from the group consisting of or a pharmaceutically acceptable salt or ester thereof. How to give neuroprotection.   The method of claim 1, wherein X is chlorine.   The method of claim 1, wherein X is substituted at the ortho position of the phenyl ring. The method of claim 1, wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are selected from hydrogen. Formula (I) and Formula (II):

[Where:
Phenyl is substituted at X with 1 to 5 halogen atoms selected from the group consisting of fluorine, chlorine, bromine and iodine; and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrogen and Independently selected from the group consisting of C ₁ -C ₄ alkyl; wherein C ₁ -C ₄ alkyl is optionally substituted with phenyl, where phenyl is optionally halogen, C ₁ -C _And optionally substituted with a substituent independently selected from the group consisting of ₄ alkyl, C ₁ -C ₄ alkoxy, amino, nitro and cyano)]
Enantiomers selected from the group consisting of or pharmaceutically acceptable salts or esters thereof or one enantiomer selected from the group consisting of formulas (I) and (II) A method of providing neuroprotection comprising administering to a patient in need of treatment with a neuroprotective drug (NPD) a therapeutically effective amount of a body mixture.   The method of claim 5, wherein X is chlorine.   6. The method of claim 5, wherein X is substituted at the ortho position of the phenyl ring. _{R 1, R 2, R 3} , R 4, The method of claim _{5 wherein R 5} and _{R 6} are selected from hydrogen.   6. The method of claim 5, wherein one enantiomer selected from the group consisting of formula (I) and formula (II) is predominant to the extent of about 90% or more.   6. The method of claim 5, wherein one enantiomer selected from the group consisting of formula (I) and formula (II) predominates to a degree of about 98% or more. Enantiomers selected from the group consisting of Formula (I) and Formula (II) are Formula (Ia) and Formula (IIa):

[Where:
Phenyl is substituted at X with 1 to 5 halogen atoms selected from the group consisting of fluorine, chlorine, bromine and iodine; and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrogen and Independently selected from the group consisting of C ₁ -C ₄ alkyl; wherein C ₁ -C ₄ alkyl is optionally substituted with phenyl, where phenyl is optionally halogen, C ₁ -C _And optionally substituted with a substituent independently selected from the group consisting of ₄ alkyl, C ₁ -C ₄ alkoxy, amino, nitro and cyano)]
6. The method of claim 5, which is an enantiomer selected from the group consisting of:   The method of claim 11 wherein X is chlorine.   12. The method of claim 11, wherein X is substituted at the ortho position of the phenyl ring. _{R 1, R 2, R 3} , R 4, The method of claim _{11, wherein R 5} and _{R 6} are selected from hydrogen.   12. The method of claim 11, wherein one enantiomer selected from the group consisting of formula (Ia) and formula (IIa) predominates to the extent of about 90% or more.   12. The method of claim 11, wherein one enantiomer selected from the group consisting of formula (Ia) and formula (IIa) predominates to the extent of about 98% or more. Enantiomers selected from the group consisting of Formula (I) and Formula (II) are Formula (Ib) and Formula (IIb):

6. The method of claim 5, which is an enantiomer selected from the group consisting of:   18. The method of claim 17, wherein one enantiomer selected from the group consisting of formula (Ib) and formula (IIb) predominates to the extent of about 90% or more.   18. The method of claim 17, wherein one enantiomer selected from the group consisting of formula (Ib) and formula (IIb) predominates to the extent of about 98% or more.   Any possible cause (s) of neuronal damage that makes the patient in need of neuroprotection: any to CNS or PNS, including traumatic brain injury (TBI), blunt and penetrating head trauma Types of injury or trauma; CNS infection; hypoxia; seizures (CVA); autoimmune diseases affecting CNS, eg lupus; labor trauma, eg perinatal asphyxia; cardiac arrest; therapeutic or diagnostic vascular surgical procedures Carotid endarterectomy or cerebral angiography; spinal cord injury; hypotension; damage to the CNS from embolism, hyperperfusion or hypoperfusion; metabolic disorders such as diabetes, hypoxia; responsive to NPD Known genetic predisposition to disorders known to be; spatially-occupying lesions of the CNS; brain tumors such as glioblastoma; bleeding in or around the CNS (bleedin) ) Or hemorrhage, such as intracerebral hemorrhage or subdural hematoma; brain edema; thermal convulsions; hyperthermia; Felt-Jakob disease, amyotrophic lateral sclerosis (ALS), diabetic neuropathy, olive bridge cerebellar atrophy, epilepsy, stroke, hypoglycemia, surgery or other intervention, retinal ischemia (diabetic or not ), Glaucoma, retinal degeneration, multiple sclerosis, toxic and ischemic optic neuropathy, macular degeneration, exposure of CNS or PNS to toxic or toxic drugs; drug addiction or withdrawal symptoms such as cocaine or alcohol; nerves Family history of degenerative disorder or related symptoms, history of status epilepticus; patients require treatment with neuroprotective drugs (NPD) Evidence from surrogate markers or biomarkers such as MRI scans showing structural or functional lesions, elevated serum levels of neuronal degradation products, elevated levels of ciliary neurotrophic factor (CNTF) The method according to claim 1 or 5.   Predisposition (s) that make a patient in need of neuroprotection are: traumatic brain injury (TBI), blunt, obstructive and penetrating head trauma; surgery, stroke or other cerebrovascular disorders ( 21. The method of claim 20, wherein the method is selected from the group consisting of: CVA); Scum status as well as CNS space-occupying lesions.   24. The method of claim 21, wherein the predisposition (s) is traumatic brain injury (TBI), including blunt, obstructive or penetrating head trauma and surgical intervention.   24. The method of claim 21, wherein the predisposition (s) is seizures or other cerebrovascular disorders (CVA).   24. The method of claim 23, wherein the predisposition is a neurodegenerative disease.   6. The method of claim 1 or 5, wherein said compound (or enantiomer) or pharmaceutically acceptable salt or ester thereof is administered in combination with one or more other compounds or therapeutic agents.   The one or more other compounds or therapeutic agents have the following properties so that a neuroprotective effect is conferred on the patient: antioxidant activity; NMDA receptor antagonism; ability to increase endogenous GABA inhibition; NO Synthase inhibitor activity; iron binding capacity, eg iron chelator; calcium binding capacity, eg Ca (II) chelator; zinc binding capacity, eg Zn (II) chelator; ability to block sodium or calcium ion channels; potassium or chloride 26. The method of claim 25, selected from the group consisting of compounds having one or more of the ability to open physical ion channels.   27. The method of claim 26, wherein the one or more compounds can be further selected from the group consisting of an antidepressant (AED).   The antidepressant (AED) is: carbamazepine, clobazam, clonazepam, ethosuximide, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, phenobarbital, phenytoin, pregabalin, primidone, retigabine, talgampan 28. The method of claim 27, selected from the group consisting of:, topiramate, valproate, vigabatrin, zonisamide, benzodiazepine, barbiturate or a hypnotic sedative. Formula (I) and Formula (II):

[Where:
Phenyl is substituted at X with 1 to 5 halogen atoms selected from the group consisting of fluorine, chlorine, bromine and iodine; and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are hydrogen and Independently selected from the group consisting of C ₁ -C ₄ alkyl; wherein C ₁ -C ₄ alkyl is optionally substituted with phenyl, where phenyl is optionally halogen, C ₁ -C _And optionally substituted with a substituent independently selected from the group consisting of ₄ alkyl, C ₁ -C ₄ alkoxy, amino, nitro and cyano)]
Enantiomers selected from the group consisting of or pharmaceutically acceptable salts or esters thereof or one enantiomer selected from the group consisting of formulas (I) and (II) A pharmaceutical composition for providing neuroprotection comprising a pharmaceutically effective amount of a body mixture and a pharmaceutically acceptable carrier or excipient.   30. Therapeutic of the pharmaceutical composition of claim 29 in a suitable package or container, along with information or instructions for its proper use, to provide neuroprotection to a patient in need thereof. A kit comprising an effective dosage form.   6. The method of claim 1 or 5, wherein the therapeutically effective amount is from about 1.0 mg / Kg / day to about 150 mg / Kg / day.   6. The method according to claim 1 or 5, wherein the patient has not developed clinical signs or symptoms of neuronal damage or dysfunction at the time of administration.   6. The method according to claim 1 or 5, wherein the patient is at risk of developing neuronal damage or dysfunction at the time of administration.   6. The method of claim 1 or 5, wherein the patient has developed clinical evidence of neurodegenerative disorder or neuronal damage at the time of administration.   6. The method of claim 1 or 5, wherein the therapeutically effective amount is from about 1.4 mg / Kg / day to about 43.0 mg / Kg / day.   6. The method of claim 1 or 5, wherein the therapeutically effective amount is from about 2.9 mg / Kg / day to about 35.7 mg / Kg / day.   6. The method of claim 1 or 5, wherein the therapeutically effective amount is from about 3.6 mg / Kg / day to about 28.6 mg / Kg / day.   6. The method of claim 1 or 5, wherein the therapeutically effective amount is from about 4.3 mg / Kg / day to about 21.4 mg / Kg / day.   6. The method of claim 1 or 5, wherein the therapeutically effective amount is from about 5.0 mg / Kg / day to about 17.1 mg / Kg / day.   6. The method of claim 1 or 5, wherein the therapeutically effective amount is from about 100 mg / day to about 3000 mg / day.   6. The method of claim 1 or 5, wherein the therapeutically effective amount is from about 200 mg / day to about 2500 mg / day.   6. The method of claim 1 or 5, wherein the therapeutically effective amount is from about 250 mg / day to about 2000 mg / day.   6. The method of claim 1 or 5, wherein the therapeutically effective amount is from about 300 mg / day to about 1500 mg / day.   6. The method of claim 1 or 5, wherein the therapeutically effective amount is from about 350 mg / day to about 1200 mg / day.   6. A method according to claim 1 or 5, wherein the therapeutic amount is gradually reduced over time.   The amount of the one or more other compounds or therapeutics administered in combination with the compound (or enantiomer) or pharmaceutically acceptable salt or ester thereof is gradually reduced over time. The method according to 25, 26, 27 or 28.

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