WO2007038610A2 - Use of natural products for treatment of neurological disorders - Google Patents

Use of natural products for treatment of neurological disorders Download PDF

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Publication number
WO2007038610A2
WO2007038610A2 PCT/US2006/037692 US2006037692W WO2007038610A2 WO 2007038610 A2 WO2007038610 A2 WO 2007038610A2 US 2006037692 W US2006037692 W US 2006037692W WO 2007038610 A2 WO2007038610 A2 WO 2007038610A2
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compound
extract
compounds
subject
seizure
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PCT/US2006/037692
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French (fr)
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WO2007038610A3 (en
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Steven C. Schachter
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President & Fellows Of Harvard College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/539—Scutellaria (skullcap)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A61K36/232—Angelica
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/898—Orchidaceae (Orchid family)
    • A61K36/8988—Gastrodia

Definitions

  • Epilepsy is one of the most prevalent neurological disorders, affecting approximately 2.3 million Americans, or about 1% of the population.
  • the Center for Disease Control and Prevention (CDC) estimates that about 181,000 new cases of epilepsy are diagnosed each year. While epilepsy can be idiopathic, about half of diagnosed epilepsy cases are linked to neurological insults, such as status epilepticus (SE), stroke, and traumatic brain injury (TBI).
  • SE status epilepticus
  • TBI traumatic brain injury
  • the present invention is based on the discovery that an isolated compound or a combination of compounds (e.g., an isolated extract) of a natural product such as a botanical or herbal source is useful to alleviate or prevent seizures such as those associated with epilepsy and/or other neurological disorders associated with N-methyl-D-aspartate (NMDA) receptor pathology.
  • An isolated compound of a botanical source is one that occurs naturally in the botanical source but has been purified from one or more other components with which it naturally occurs or is synthetically made (e.g., chemically synthesized). The isolated compound demonstrates at least 10% greater activity in reduction of neurological seizures compared to the level of activity of the botanical source from which it is derived or in which is naturally occurs.
  • the isolated compound possesses at least 25%, 50%, 75%, 100%, 3-fold, 5-fold, 10- fold or more activity compared to the naturally-occurring botanical source.
  • isolated compounds include, for example, rhynchophylline, isorhynchophylline, hyperin, baicalin, wogonin, oroxyloside, gastrodin, peoniflorin, albiflorin, vanillin, pentagalloylglucose, imperatorin, oxypeucedaninhydrate, decursinol, decursin, wogonoside, ganhuangenin, baicalein, aporphine type alkaloids, protoberberine type I alkaloids, protoberberine type II alkaloids, and protopine-type alkaloids as well as analogs and combinations thereof.
  • imperatorin and oxypeucedanin hydrate are isolated from Angelica dahurica, paoniflorin is isolated from Paeonia lcatiflora, gastrodin is isolated from Gastrodia elata; and decursinol and decursin are isolated from Angelica gigas.
  • the isolated compounds and extracts described herein exhibit neuroprotective and anticonvulsive properties and are administered in a substantially purified form, either alone or in combination, in an amount sufficient to treat, prevent, or delay the onset of neurological disorders.
  • An extract contains at least two but less than all of the compounds present in the naturally-occurring botanical source. For example, an extract contains 2, 5, 10, 25, 50, 100, 500, 1000, 2000 or 5000 compounds and the extract is separated from other compounds that are present in the naturally-occurring botanical source.
  • the isolated compounds Upon administration, the isolated compounds reduce one or more of the mental or cognitive effects such as age-associated cognitive or memory decline, mental decline, and likelihood of age related brain, and cognitive disorders.
  • the isolated compound or functional analog thereof is administered such that it will penetrate the blood-brain barrier and interact with a target receptor in the central nervous system of the subject. The interaction with the target receptor modifies the target receptor in a way to treat or prevent seizures or other neurological disorders in the subject.
  • the therapeutic compositions of the invention are administered with any pharmaceutical excipient known in the art.
  • the dosage that is administered is one that reduces or prevents a disorder.
  • the isolated compounds, mixtures of isolated compounds, or extracts are formulated in a sustained release composition.
  • Such slow release formulations deliver the compounds over a time period of greater than 4 hours, e.g., 5, 6, 7, 8, 10, 12, 18, 24 hours or over a period of days (1, 2, 3, 4, 6, 6, 7 days).
  • the compounds are in the form of a slowly degrading capsule or tablet, a dermal patch, or an solid, semi-permeable, or permeable implant that is placed into a mammal.
  • the implant is placed into or adjacent to a bodily tissue or in a vascularized location such that the compound gains access to the vasculature and is then delivered via the vasculature to the target tissue.
  • the implant is biodegradable.
  • the isolated compounds are administered in combination with other therapeutic agent including the isolated compounds described herein.
  • the isolated compound is provided in the form of a dietary supplement.
  • the invention also provides methods of isolating the active compounds present within herbal extracts.
  • Methods for separation and isolation of the active compounds include standard techniques known in the art, including thin layer chromatography (using silica-coated plates, for example), column chromatography, and high pressure liquid chromatography (HPLC). Active compounds are identified by re-testing of individual bands or fractions (separated by thin layer chromatography, column chromatography and/or HPLC) using specific assay tests as described herein.
  • Sufficient isolation of active ingredients contained within individual bands and/or fractions is tested using, for example, scanning electron microscopes equipped with energy dispersive x-ray analyzer to detect and spatially map elements present in each sample, elemental analysis by combustion to determine the relative amount (e.g., percentage) of carbon, hydrogen and nitrogen, high resolution mass spectroscopy to determine molecular weight and elemental composition, fourier transform infrared spectroscopy to determine functional groups and make comparisons to the spectra of known compounds, differential scanning calorimetry to determine melting point, atomic absorption, gel chromatography, high performance liquid chromatography, proton and 13 C nuclear magnetic resonance spectroscopy for material characterization and to provide information regarding the position of atoms relative to each other, and UVVVIS spectroscopy.
  • scanning electron microscopes equipped with energy dispersive x-ray analyzer to detect and spatially map elements present in each sample
  • elemental analysis by combustion to determine the relative amount (e.g., percentage) of carbon, hydrogen and nitrogen
  • the invention provides methods and compositions for treating or preventing neurological disorders using compounds purified or isolated from natural products such as botanical or herbal extracts.
  • Such compounds include, for example, rhynchophylline, isorhynchophylline, hyperin, baicalin, wogonin, oroxyloside, gastrodm, peom ' fiorin, albiflorin, vanillin, pentagalloylglucose, imperatorin, oxypeucedaninhydrate, decursinol, decursin, wogonoside, ganhuangenin, baicalein, aporphine type alkaloids, protoberberine type I alkaloids, protoberberine type II alkaloids, and protopine-type alkaloids as well as analogs, derivatives, homologs, positional isomers, stereoisomers and mixtures of steroisomers in optically active or racemic form, salts, and hydrates thereof.
  • the terms “isolated”, “purified” and “substantially purified,” are used interchangeably and refer to a compound that is at least 30%, 40%, or preferably 50%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. More preferably, the preparation is at least 60%, more preferably 75%, more preferably 90%, and most preferably at least 99%, by weight, chemical compound, e.g., natural product compound.
  • a purified compound may be obtained by any method known in the art or described herein including, for example, high pressure liquid chromatography, thin layer chromatography, or by synthesis.
  • analog refers to a chemical compound that is structurally similar to a parent compound and has chemical properties or pharmaceutical activity in common with the parent compound.
  • Analogs include, but are not limited to, homologs, i.e., where the analog differs from the parent compound by one or more carbon atoms in series; positional isomers; compounds that differ by interchange of one or more atoms by a different atom, for example, replacement of a carbon atom with an oxygen, sulfur, or nitrogen atom; and compounds that differ in the identity one or more functional groups, for example, the parent compound differs from its analog by the presence or absence of one or more suitable substituents.
  • Suitable substituents include, but not limited to, (Ci-C 8 )alkyl; (Ci ⁇ C 8 )alkenyl; (Ci-C 8 )alkynyl: aryl; (C 2 -C 5 )heteroaryl; (Ci- C 6 )heterocycloaklyl; (C 3 -C 7 )cycloalkyl; O(Ci ⁇ -C 8 )alkyl; O(C 1 -C 8 )alkenyl; O(C 1 -C 8 )alkynyl; Oaryl; CN; OH; oxo; halo, C(O)OH; COhalo; O(CO)halo; CF 3 ; N 3 ; NO 2 , NH 2 ; NH((Ci- QOalkyl); N((C,-C 8 )alkyl) 2 ; NH(aryl); N(aryl) 2 N((C 1 -C 8 )
  • alkyl means a saturated, monovalent, unbranched or branched hydrocarbon chain.
  • alkyl groups include, but are not limited to, (Ci-C 8 )alkyl groups, such as methyl, ethyl, propyl, isopropyl, 2-methyl-l -propyl, 2-methyl-2-propyl, 2 -methyl- 1 -butyl, 3- methyl-1 -butyl, 2-me thyl-3-butyl, 2,2-dimethyl-l -propyl, 2-methyl-l -pentyl, 3 -methyl- 1-pentyl, 4-methyl-l-pentyl, 2-methyl-2-pentyl, 3- methyl-2 -pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l- butyl, 3, 3 -dimethyl- 1 -butyl, 2-ethyl-l-buty 1, butyl, isobutyl, t-butyl,
  • alkenyl means a monovalent, unbranched or branched hydrocarbon chain having one or more double bonds therein.
  • the double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group.
  • Suitable alkenyl groups include, but are not limited to (C 2 -C 8 )alkenyl groups, such as vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl,2-propyl-2-butenyl,4-(2-methyl-3-butene)- pentenyl.
  • An alkenyl group can be unsubstituted or substituted with one or two suitable substituents.
  • alkynyl means monovalent, unbranched or branched hydrocarbon chain having one or more triple bonds therein.
  • the triple bond of an alkynyl group can be unconjugated or conjugated to another unsaturated group.
  • Suitable alkynyl groups include, but are not limited to, (C 2 -C 8 )alkynyl groups, such as ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-l-butynyl,4-propyl-2-pentynyl, and 4-butyl-2-hexynyl.
  • An alkynyl group can be unsubstituted or substituted with one or two suitable substituents.
  • aryl means a monocyclic or polycyclic-aromatic group comprising carbon and hydrogen atoms.
  • suitable aryl groups include, but are not limited to, phenyl, tolyl, anthacenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl.
  • An aryl group can be unsubstituted or substituted.
  • the aryl group is a monocyclic ring, wherein the ring comprises 6 carbon atoms, referred to herein as "(C 6 )aryl".
  • heteroaryl means a monocyclic- or poly cyclic aromatic ring comprising carbon atoms, hydrogen atoms, and one or more heteroatoms, preferably, 1 to 3 heteroatoms, independently selected from nitrogen, oxygen, and sulfur.
  • heteroaryl rings have less aromatic character than their all-carbon counter parts.
  • a heteroaryl group need only have some degree of aromatic character.
  • heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidyl, pyrazyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3,)- and (1,2,4)- triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, phenyl, isoxazolyl, and oxazolyl.
  • a heteroaryl group can be unsubstituted or substituted with one or two suitable substituents.
  • a heteroaryl group is a monocyclic ring, wherein the ring comprises 2 to 5 carbon atoms and 1 to 3 heteroatoms, referred to herein as "(C 2 -C 5 )heteroaryl".
  • cycloalkyl means a non-aromatic, monocyclic or polycyclic ring comprising carbon and hydrogen atoms.
  • a cycloalkyl group can have one or more carbon-carbon double bonds in the ring so long as the ring is not rendered aromatic by their presence.
  • cycloalkyl groups include, but are not limited to, (C 3 -C 7 )cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, and saturated cyclic and bicyclic terpenes and (C 3 -C 7 )cycloalkenyl groups, such as cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl, and unsaturated cyclic and bicyclic terpenes.
  • a cycloalkyl group can be unsubstituted or substituted by one or two suitable substituents.
  • the cycloalkyl group is a monocyclic ring or bicyclic ring.
  • heterocycloalkyl means a non-aromatic monocyclic or polycyclic ring comprising carbon and hydrogen atoms and at least one heteroatom, preferably, 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur.
  • a heterocycloalkyl group can have one or more carbon-carbon double bonds or carbon-heteroatom double bonds in the ring as long as the ring is not rendered aromatic by their presence.
  • heterocycloalkyl groups include aziridinyl, pyrrolidinyl, pyrrolidino, piperidinyl, piperidino, piperazinyl, piperazino, morpholinyl, morpholino, thiomorpholinyl, thiomorpholino, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, and pyranyl.
  • a heterocycloalkyl group can be unsubstituted or substituted with one or two suitable substituents.
  • the heterocycloalkyl group is a monocyclic or bicyclic ring, more preferably, a monocyclic ring, wherein the ring comprises from 2 to 6 carbon atoms and form 1 to 3 heteroatoms, referred to herein as (Q-C ⁇ heterocycloalkyl.
  • halogen means fluorine, chlorine, bromine, or iodine.
  • halo means fluoro, chloro, bromo, and iodo.
  • derivative refers to an analog, as defined above, that can be obtained in one or more chemical reactions from its parent compound.
  • phrases "pharmaceutically acceptable salt(s),” as used herein includes, but is not limited to, salts of acidic or basic groups that may be present in the natural product compounds, and hydrates thereof. Natural product compounds, and hydrates thereof that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids.
  • acids that may be used to prepare pharmaceutically acceptable salts of such basic compounds are those that form salts comprising pharmacologically acceptable anions including, but not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, edetate, camsylate, carbonate, bromide, chloride, iodide, citrate, dihydrochloride, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydroxynaphthoate, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, muscate, napsylate, nitrate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, succinate
  • Natural product compounds, and hydrates thereof that include an amino moiety can also form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above.
  • Natural product compounds, and hydrates thereof that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.
  • hydrate means an natural product compound, or a pharmaceutically acceptable salt thereof that further includes a stoichiometric or non-stoichiometric amount of water bound to it by non-covalent intermolecular forces.
  • the compound represents a certain isomer for convenience in some cases, but the present invention includes all isomers such as geometrical isomer, optical isomer based on an asymmetrical carbon, stereoisomer, tautomer and the like which occur structurally and an isomer mixture and is not limited to the chemical structure for convenience, and may be any one of isomer or a mixture. Therefore, an asymmetrical carbon atom may be present in the molecule and an optically active compound and a racemic compound may be present in the present compound, but the present invention is not limited to them and includes any one.
  • a crystal polymorphism may be present but is not limiting, but any crystal form may be single or a crystal form mixture, or an anhydride or hydrate. Further, so-called metabolite which is produced by degradation of the present compound in vivo is included in the scope of the present invention.
  • the structure of some of the compounds of the invention include asymmetric (chiral) carbon atoms e.g., decursinol. It is to be understood accordingly that the isomers arising from such asymmetry are included within the scope of the invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. The compounds of this invention may exist in stereoisomeric form, therefore can be produced as individual stereoisomers or as mixtures. "Isomerism” means compounds that have identical molecular formulae but that differ in the nature or the sequence of bonding of their atoms or in the arrangement of their atoms in space.
  • stereoisomers that differ in the arrangement of their atoms in space are termed “stereoisomers”.
  • stereoisomers that are not mirror images of one another are termed “diastereoisomers”, and stereoisomers that are non-superimposable mirror images are termed “enantiomers”, or sometimes optical isomers.
  • enantiomers or sometimes optical isomers.
  • a carbon atom bonded to four non-identical substituents is termed a "chiral center”.
  • Chiral isomer means a compound with at least one chiral center. It has two enantiomeric forms of opposite chirality and may exist either as an individual enantiomer or as a mixture of enantiomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a "racemic mixture”. A compound that has more than one chiral center has 2" "1 enantiomeric pairs, where n is the number of chiral centers. Compounds with more than one chiral center may exist as either an individual diastereomer or as a mixture of diastereomers, termed a "diastereomeric mixture”.
  • a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center.
  • Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center.
  • the substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et al, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem. Educ. 1964, 41, 116).
  • “Geometric Isomers” means the diastereomers that owe their existence to hindered rotation about double bonds. These configurations are differentiated in their names by the prefixes cis and trans, or Z and E, which indicate that the groups are on the same or opposite side of the double bond in the molecule according to the Cahn-Ingold-Prelog rules.
  • atropic isomers are a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropic isomers owe their existence to a restricted rotation caused by hindrance of rotation of large groups about a central bond. Such atropic isomers typically exist as a mixture, however as a result of recent advances in chromatography techniques, it has been possible to separate mixtures of two atropic isomers in select cases.
  • subject as used herein is intended to include a living organism in which alleviation of symptoms or inhibition of a neurological disorder is sought. Preferred subjects are mammals. Examples of subjects include but are not limited to, humans, monkeys, dogs, cats, mice, rats, cows, horses, pigs, goats and sheep.
  • neurodegenerative disorder or a "neurological disorder * ' as used herein refers to a disorder which causes morphological and/or functional abnormality of a neural cell or a population of neural cells.
  • the neurodegenerative disorder can result in an impairment or absence of a normal neurological function or presence of an abnormal neurological function in a subject.
  • neurodegenerative disorders can be the result of disease, injury, and/or aging.
  • Non-limiting examples of morphological and functional abnormalities include physical deterioration and/or death of neural cells, abnormal growth patterns of neural cells, abnormalities in the physical connection between neural cells, under- or over production of a substance or substances, e.g., a neurotransmitter, by neural cells, failure of neural cells to produce a substance or substances which it normally produces, production of substances, e.g., neurotransmitters, and/or transmission of electrical impulses in abnormal patterns or at abnormal times.
  • Neurodegeneration can occur in any area of the brain of a subject and is seen with many disorders including, for example, epilepsy, head trauma, stroke, ALS, multiple sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease.
  • the terms "neurodegenerative disorder” and "neurological disorder” are intended to cover memory disorders.
  • memory disorder refers to a diminished mental registration, retention or recall of past experiences, knowledge, ideas, sensations, thoughts or impressions.
  • Memory disorder may affect short and/or long-term information retention, facility with spatial relationships, memory (rehearsal) strategies, and verbal retrieval and production.
  • the term memory disorder is intended to include dementia, slow learning and the inability to concentrate. Common causes of a memory disorder are age, severe head trauma, brain anoxia or ischemia, alcoholic-nutritional diseases, drug intoxications, and neurodegenerative diseases.
  • a memory disorder is a common feature of neurodegenerative diseases, such as epilepsy or Alzheimer's disease (i.e. Alzheimer-type dementia).
  • Memory disorders also occur with other kinds of dementia such as AIDS Dementia; Wernicke-Korsakoff s related dementia (alcohol induced dementia); age related dementia, multi-infarct dementia, a senile dementia caused by cerebrovascular deficiency, and the Lewy-body variant of Alzheimer's disease with or without association with Parkinson's disease.
  • Creutzfeldt- Jakob disease a spongiform encephalopathy caused by the prion protein, is a rare dementia with which memory disorder is associated. Loss of memory is also a common feature of brain-damaged patients.
  • Non-limiting examples of causes of brain damage which may result in a memory disorder include stroke, seizure, an anaesthetic accident, epilepsy, ischemia, anoxia, hypoxia, cerebral edema, arteriosclerosis, hematoma or epilepsy; spinal cord cell loss; and peripheral neuropathy, head trauma, hypoglycemia, carbon monoxide poisoning, lithium intoxication, vitamin (Bl, thiamine and B 12) deficiency, or excessive alcohol use.
  • Korsakoff s amnesic psychosis is a rare disorder characterized by profound memory loss and confabulation, whereby the patient invents stories to conceal his or her memory loss. It is frequently associated with excessive alcohol intake. Memory disorder may furthermore be age-associated; the ability to recall information such as names, places and words seems to decrease with increasing age. Transient memory loss may also occur in patients, suffering from a major depressive disorder, after electro-convulsive therapy.
  • seizure refers to a change in behavior, or spasms or convulsions that arise naturally in a subject as a result of a natural chemical imbalance or lack of homeostasis in a subject. Such natural convulsions may arise due to a disease or disorder (e.g., epilepsy), age, or the occurrence of an event (e.g., stroke).
  • a disease or disorder e.g., epilepsy
  • age e.g., age
  • an event e.g., stroke
  • seizure also refers to seizures that are chemically induced, for example those brought on by intake, uptake, or ingestion of chemicals such as organophosphates.
  • treatment refers to a decrease in the symptoms associated with the disorder or an amelioration of the recurrence of the symptoms of the disorder, prophylaxis, or reversal of a disease or disorder, or at least one discernible symptom thereof.
  • treatment refers to inhibiting or slowing the progression of a disease or disorder, e.g., epilepsy, physically, e.g., stabilization of a discernible symptom, such as seizures.
  • treatment or “treating” refers to delaying the onset of a disease or disorder, e.g., seizures.
  • prevention or “preventing” refers to delaying the onset of the symptoms of the disorder.
  • At least one natural product compound is administered as a preventative measure to a subject having a genetic or non-genetic predisposition to a neurological disorder such as epilepsy.
  • at least one natural product compound is administered as a preventative measure for a subject at risk of developing seizures as a result of another medical event. For example, patients who have a suffered a stroke are often at risk of developing seizures. In these instances, at least one natural product compound can be administered after the stroke as a preventative measure against seizures.
  • Isolated Compounds and Analogs of Natural Products The invention provides methods and compositions for treating or preventing neurological disorders using a compound that is isolated from a natural product.
  • the invention includes analogs of compounds isolated from natural products where the compound is selected from chromen-one derivatives, carbohydrate-derivatized compounds, and alkaloid compounds.
  • Substantially purified compounds are prepared from herbal extracts.
  • the dry powder of the herbal plant (3 kg) is extracted in 80% aqueous MeOH (5 L) at room temperature overnight. Then, the filtrates are evaporated in vacuum e.g., under 40 0 C, and the concentrates are fractionated into three fractions such as n-EtOAc, n-BuOH, and aqueous.
  • Each solvent extracts flow through the column chromatography (SiO 2 , ODS, Sephadex etc.) and are eluted with several mixed solvents. Several obtained fractions are repeatedly chromatographed while varying packing materials or eluting solutions. Purity of the isolated compounds is assessed by TLC or HPLC.
  • the molecular structure and weight are determined by: 1) hypothesizing chemical and physical characteristics of compounds; 2) determining functional groups in the compound molecule through IR spectra; 3) reading molecular weight and molecular formula from Mass data; 4) identifying chemical environment and integration value of proton, and neighboring protons using 1 H-NMR data; 5) measuring chemical environment and numbers of carbon from 13 C-NMR data; 6) multiplicity of carbon by reading DEPT NMR data; 7) investigating the correlation between neighboring proton signals from 1 H- 1 H 2D COSY NMR;
  • Rhvnchophylline and Isorhynchophylline are indole alkaloids that are isolated from gou teng (Ramulus Uncariae cum Uncis).
  • Botanical names for gou teng include Ourouparia rhynchophylla Matsum, Uncaria rlynchophylla (Miq.) Jacks, Uncaria macrophylla Wall, Uncaria hirsute Haviland, Uncaria sinensis Haviland, Uncaria sessilifructus Roxb, and Uncaria scandens (Smith) Hutch.
  • Rhynchophylline and isorhynchophylline are isolated from the hook of Uncaria rhynchophylla, Uncaria sinensis Haviland Uncaria, macrophylla Wallich (Rubiaceae) (Chung Kuo Yao Li Hsueh Pao 1986;7(5)426).
  • Isorhynchophylline is isolated from M. rubrostipulata (Seaton, J. C, Tondeur, R. and Marion, L. Canad. J. Chem., 36, 1031 (1958)).
  • Uncaria rhynchophyllina has about 0.2 % alkaloid content, in which rhynchophylline (Rhy) is 28%-50 %, isorhynchophylline (Isorhy) is 15 %.
  • Other trace components include hirsutine, hirsuteine, corynantheine, dihydrocorynantheine, isocorynoxeine, akuammigine, geissoschijine, and methylethe (Drug Dicimasia Institute of Tianjing. Extract and pharmacological research of effective components in Uncar ia rhynchophylla. Chin Herb Med 1974; 4: 212-7; Phillipson JD, Hemingway SR.
  • the invention includes compounds that are derivatives of alkaloids.
  • the compound is a derivative of rhynchophylline and isorhynchophylline.
  • Compounds of the invention include compounds of Formula I:
  • Ri, R 2 , R 3 , R4, R 5 , and R 6 are selected from hydrogen, alkyl, alkenyl, alkoxy, carboxyalkyl, acylamino, and halogen.
  • R 7 , R 8 , R 9 , and Ri 0 are selected from hydrogen, hydroxyl, alkoxy, substituted alkoxy, alkyl, alkenyl, CF 3 , amino, carboxy, and halogen.
  • Ri or R 2 is alkyl and the other is hydrogen.
  • One OfR 1 or R 2 is methyl and the other is hydrogen.
  • One of Ri or R 2 is ethyl and the other is hydrogen.
  • R 5 is carboxyalkyl.
  • R 3 is alkoxy.
  • R 3 is methoxy.
  • R 4 is hydrogen.
  • R 7 , R 8 , R 9 , and Rio are each hydrogen Alkaloids isolated from Corydalis tuber Four classes of alkaloids (shown below) are isolated from Corydalis tuber (Rhizoma),
  • Corydalis a root found in the Zhejiang province of China.
  • the genus of Corydalis consists of about 300 species of rhizomatous or tuberous plants, which are native to Asian and European woodlands. Currently, only about 25 species are commonly known.
  • the tubers contain approximately 6% of alkaloids, which are often bound to malic or fumaric acid.
  • aporphine type e.g., bulbocapnine, corydine, magnoflorine and jatrorrhicine iodide
  • protoberberine type I e.g., berberine, coptisine, corysamine, palmatine
  • protoberberine type II e.g., canadine, corybulbine, corydaline, corypalmine, scoulerine, stylopine, tetrahydropalmatine
  • protopine-type e.g., (protopine, allocryptopine, corycavamine
  • Analogs and derivatives of aporphine type, protoberberine type I, protoberberine type II, and protopine-type alkaloids are useful for the treatment of neurological disorders as described herein.
  • One of skill in the art can readily identify analogs and derivatives of the isolated alkaloid compounds suitable for use with the present invention by obtaining compounds with at least one of the core structures shown below and testing those compounds for neuroprotective activity as set forth in the examples below.
  • R in the formulae above depicts the various side chains. In one embodiment, the side chains are selected from the group consisting of H and CH 3 .
  • the molecular structures of CTEB-3-5, CTEB-11-6, CTB-7, and CTEB-7-5 are shown below:
  • the invention also includes derivatives of alkaloids isolated from Corydalis tuber, including multi-cyclic ring compounds e.g., tri- and tetra-cyclic compounds.
  • Compounds of the invention include compounds of Formula II:
  • Ri, R 2 , R 3 , and R 4 are selected from alkyl, alkenyl, hydrogen, carbonylalkyl or Ri and R 2 together form a 5- or 6-membered ring.
  • the 5- or 6-membered ring is substituted or unsubstituted.
  • the 5-membered heterocycle ring is dioxole.
  • the 6-membered heterocycle ring is dioxine or dihydrodioxine.
  • At least one of Ri, R 2 , R 3 , or R 4 is alkyl.
  • At least two of R], R 2 , R 3 , or R 4 is alkyl.
  • At least three of Ri, R 2 , R 3 , or R 4 is alkyl.
  • R 1 and R 4 are methyl.
  • X is alkyl or halogen.
  • X is methyl.
  • X is ethyl.
  • Compounds of the invention also include compounds of Formula III: (III) where, Y is alkyl, halogen, or absent,
  • Ri, R 2 , R 3 , and Rio are selected from hydrogen or alkyl, or together R 1 and R 2 form a carbonyl, or one of Ri or R 2 and Y form a covalent bond, or one of Rj or R 2 and R 3 form a covalent bond, or Y and R 8 form a covalent bond.
  • R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are selected from alkyl, alkenyl, hydrogen, carbonylalkyl, or together R 4 and R 5 form a 5- or 6-membered ring, or together R 4 and R 9 form a 5- or 6- membered ring, or together R 6 and R 7 form a 5- or 6-membered ring, or together R 7 and R 8 form a 5- or 6-membered ring.
  • the 5- or 6-membered ring is substituted or unsubstituted.
  • the 5- membered heterocycle ring is dioxole.
  • the 6-membered heterocycle ring is dioxine or dihydrodioxine.
  • At least one of is R 4 , R 5 , R 6 , R 7 , R 8 , or R9 alkyl. At least two OfR 4 , R 5 , R 6 , R 7 , R 8 , or R 9 is alkyl. At least three OfR 4 , R 5 , R 6 , R 7 , R 8 , or R 9 is alkyl.
  • R 4 and R 5 form a 5- membered ring.
  • R 7 and R 8 form a 5-membered ring.
  • the 5-membered ring is dioxine.
  • R 4 and R 5 are methyl.
  • R 7 is methyl.
  • R 6 and R 7 are methyl.
  • Y is methyl.
  • Y is halogen, where halogen is Cl. Y is absent. Together Ri and R 2 form a carbonyl.
  • Gastrodin Gastrodin (Ci 3 Hi 8 O 7 , molecular weight 286, soluble in MeOH, DMSO, EtOH, H 2 O) is isolated from the rhizome of Gastrodia elata. A preferred method describing its isolation is described.
  • the dried powder (5 kg) of Gastrodia elata was extracted with 80% aqueous MeOH (4L x 2) at room temperature overnight and filtered through N02 Whatmann filter paper. The filtrate was evaporated under reduced pressure to give rise to MeOH extracts (187 g).
  • the 15O g of the extracts was poured into H 2 O (1600 mL) and extracted with EtOAc (1500 mL x 2) and n- BuOH (1000 ml x 2), successively. Each solution was evaporated under vacuum to afford EtOAc (32 g), ft-BuOH (86 g) and aqueous (32 g) fractions, respectively.
  • the molecular structure of gastrodin is below:
  • Peoniflorin (C 23 H 28 O n , molecular weight 480, soluble in MeOH, DMSO, EtOH, H 2 O) is isolated from the root of Paeonia lactiflora. Methods of purification of paeoniflorin (5b-
  • the molecular structure of paeoniflorin is below:
  • Albiflorin (9-((benzoyloxy)methyl)- 1 -(beta-D-glucopyranosyloxy)-4- hydroxy)-6- methyl-7-oxatricyclononan-8-one) is a water-soluble compound that is isolated from the root of Paeonia lactifloria and Paeonia sinjiangensis. (See, for example, Song, Z.H., Zhongguo Zhong Yao Za ZhL 2004 Aug;29(8):748-51; S. Shibata, Chem. Pharm. Bull. 11 (1963) 372; K Yamasaki, Tetrahedon Lett. AA (1976) 3965). The molecular structures of albiflorin is below:
  • Pentagalloylglucose (beta-Penta-O-galloyl-glucose)
  • Pentagalloylglucose (C 41 H 32 O 26 , molecular weight 940.68) is a polyphenol that is isolated from leaves of Euphorbia hirta L. (Chen, L. Zhongguo Zhong Yao Za Zhi. 1991 Jan;16(l):38-9, 64) and from Peonia lactiflora (peony) root. The molecular structure of pentagalloylglucose is shown below:
  • the invention also includes analogs of compounds derivatized with one or more carbohydrates.
  • the carbohydrate is ⁇ glycoside i.e., the anomeric carbon is blocked by acetal formation e.g., methyl ⁇ -D-glucopyranoside is a glycoside, wherein the anomeric carbon is blocked or derivatized with a methoxy group (OCH 3 ).
  • the glycoside includes a sugar that is naturally occurring.
  • the glycoside includes a sugar that is non-naturally occurring.
  • the glycoside includes the sugar glucose.
  • the anomeric carbon of the carbohydrate is derivatized with aryl or substituted aryl.
  • the aryl group is phenyl.
  • the aryl group is substituted with hydroxyalkyl.
  • the aryl group is substituted with -CH 2 OH.
  • the anomeric carbon of the carbohydrate is derivatized with a compound containing a bicyclic ring system.
  • Bicyclic ring systems are characterized by two carbon atoms, the bridgehead carbons, being shared by two rings.
  • Bridgehead carbon Bicyclic ring systems include compounds which contain the chemical structures of e.g., cubane, tetrahedrane, and dodecahedrane.
  • Examples of bicyclic structures include 5b-((benzoyloxy)methyl)tetrahydro-5-hydroxy-2-methyl-2,5,-methano- 1 H- 3 ,4-dioxacyclobuta(cd)pentalen- 1 a(2H)-yl-beta-D-glycopyranoside and 9-((benzoyloxy)methyl)- 4-hydroxy)-6-methyl-7-oxatricyclononan-8-one.
  • Bicyclic ring systems can be optionally substituted with one or more substituents selected from e.g., alkyl, cycloalkyl, cycloalkenyl, aryl, heterocycle, alkoxyaryl, alkaryl, alkoxy, halogen, hydroxy, amine, cyano, nitro, alkylthio, phenoxy, ether, trifluoromethyl and the like.
  • substituents selected from e.g., alkyl, cycloalkyl, cycloalkenyl, aryl, heterocycle, alkoxyaryl, alkaryl, alkoxy, halogen, hydroxy, amine, cyano, nitro, alkylthio, phenoxy, ether, trifluoromethyl and the like.
  • the anomeric carbon of the carbohydrate includes an acyl or substituted acyl group.
  • “Acyl” includes compounds and moieties that contain the acyl radical (CH 3 CO-) or a carbonyl group.
  • “Substituted acyl” includes acyl groups where one or more of the hydrogen atoms are replaced by for example, alkyl, alkynyl, halogen, or hydroxyl.
  • the carbohydrate includes a benzoyl or substituted benzoyl group.
  • the carbohydrate includes a benzoyl or substituted benzoyl group (i.e., hydroxyl groups on the carbohydrate in addition to the anomeric carbon are derivatized).
  • Benzoyl includes compounds and moieties that contain the benzoyl radical (PhCO-).
  • Substituted benzoyl includes benzoyl groups where the phenyl group is substituted. The benzoyl group is substituted with one or more hydroxyl groups. The benzoyl group is substituted with one hydroxyl group. The benzoyl group is substituted with two hydroxyl groups. The benzoyl group is substituted with three hydroxyl groups.
  • Imperatorin (Ci 6 Hi 4 O 4 , molecular weight 270, soluble in CHCl 3 , EtOH, MeOH, DMSO) and oxypeucedaninhydrate (Ci 6 Hi 6 O 6 , molecular weight 304, soluble in EtOH, MeOH, DMSO) are isolated from the root of Angelica dahurica.
  • One preferred method of isolation is described. Coarsely powdered plant material (lkg) was extracted with 80% aqueous MeOH (4Lx2) at room temperature overnight. The extract were evaporated under reduced pressure and partitioned between EtOAc (700 mL x 2) and H 2 O (500 mL).
  • Decursin (Ci 9 H 20 O 5 , molecular weight 328, soluble in CHCl 3 , DMSO, EtOH) and decursinol (C 14 H 14 O 4 , molecular weight 246, soluble in CHCl 3 , DMSO, EtOH, MeOH) are isolated from the root of Angelica gigas. A preferred method of isolation is described. The dried underground part (2 kg) of Angelica gigas was extracted with MeOH and upon removal of solvent in vacuo, the MeOH extract yielded 15O g of residue. This was then suspended in H 2 O (500 ml) and partitioned successively with CH 2 Cl 2 (500 ml x 2).
  • the compounds of the present invention act through different receptors, or bind to different regions of the same receptor.
  • the invention provides combinations of the compounds that act synergistically to improve therapeutic effects.
  • the combination can include two or more of the compounds of the present invention.
  • the combination of compounds also means that lower dosages of the individual compounds are used to achieve the same therapeutic effect as each compound used alone. This is important when a desired therapeutic effect occurs at a high dose of a natural product compound, but which also leads to adverse side effects. In such an instance, a lower dosage is combined with a dosage of a synergistic compound to provide the desired therapeutic effect, but without the adverse side effects.
  • Hyperin Hyperin (C 2 iH 20 O 12 , molecular weight 464.38, 2-(3,4-dihydroxyphenyl)-3-( ⁇ -D- galactopyranosyloxy)-5,7-dihydroxy-4H-l-benzopyran-4-one) is isolated from gou teng
  • Hyperin can be isolated from the hook of Uncaria rhynchophylla, Uncaria sinensis, as well as from other plants, such as Acanthopanax chiisanensis roots and Drosera burmannii Vahl, Taraxacum mongolicum Hand.-Mazz, Canarium album and Euphorbia nematocypha, and Onobrychis angustifolia (Lee, S. Arch Pharm Res. 2004 Jun;27(6):628-32, Wang, O. Zhong Yao Cai. 1998 Aug;21(8):401-3, Ling, Y.
  • hyperin is intended to include isoforms, derivatives and functional analogs of hyperin.
  • hyperin is intended to cover all synonyms including, but not limited to, hyperoside, hyperasid, hyperozide, quercetin 3-0-beta-D-galactopyranosid, Quercetin 3-beta-D-galactopyranoside, Quercetin-3-O-galactoside, Quercetin-3-galactoside and isoforms thereof.
  • hyperin can be isolated from Uncaria sinensis. One method of isolation is described below.
  • Uncaria sinensis is extracted with boiling water three times. Each extract is combined and lyophilized to give a brown mass.
  • the water extract is chromatographed on polus polymer gel (Diaion HP-20, 2 L), eluted with water, [H 2 ]O-methanol (1:1) and then with methanol.
  • the [H 2 ]O -methanol (1:1) eluate is purified by Sephadex LH-20 column chromatography, and eluted with [H 2 ]O -ethanol (1 : 1) to obtain hyperin.
  • the molecular structure of hyperin is shown below:
  • Baicalin, 7-D-glucuronic acid-5,6-dihydroxyflavone, (C 2 IH 18 On, beta-D- Glucopyranosiduronic acid, 5,6-dihydroxy-4-oxo-2-phenyl-4H-l-benzopyran-7-yl) is isolated from plants, such as Scutellariae baicalensis, Scutellariae Radix, Shuang ⁇ Huang-Lian % and Scutellaria rehderiana (Wu, S. J Chromatogr A. 2005 Feb 25;1066(l-2):243-7, Zhang, L. J Ethnopharmacol. 2006 Jan 3;103(l):120-5. Epub 2005 Sep 12, Feng, N.
  • baicalin is intended to include isoforms, derivatives and functional analogs of baicalin.
  • a preferred method of isolation is described. Baicalin can be separated with a one-step separation from crude sample from Scutellaria baicalensis, Georgi using ethyl acetate-methanol- 1% acetic acid water (5:0.5:5, v/v) as the two-phase solvent system.
  • baicalin The upper phase of ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) can be used as the stationary phase of highspeed counter-current chromatography (HSCCC).
  • HSCCC highspeed counter-current chromatography
  • Baicalein 5,6,7-trihydroxy-2-phenyl-4H-l-benzopyran-4-one (C15H10O5, molecular weight 270.24) is isolated from the roots of Scutellaria baicalensis (Bargellini, Gazz. Chim. Ital. 49, II, 47 (1919); Shibata et al. Acta Phytochim. 1, 109 (1923)).
  • the term baicalein is intended to include isoforms, derivatives, and functional analogs of baicalein.
  • Baicalein is soluable in alcohol, methanol, ether, acetone, ethyl acetate, and hot glacial acetic acid. The molecular structure of baicalein is shown below:
  • Wogonin 5,7-dihydroxy-8-methoxyflavone (Ci 6 Hi 2 Os, molecular weight 284.27, 4H-1- Benzopyran-4-one, 5,7-dihydroxy-8-methoxy-2-phenyl-) is isolated from plants such as Scutellariae baicalensis, Scutellariae Radix, Shuang-Huang-Lian, and Scutellaria rehderiana (Wu, S. J Chromatogr A. 2005 Feb 25;1066(l-2):243-7).
  • the term wogonin is intended to include isoforms, derivatives and functional analogs of wogonin. One method to isolate wogonin is described.
  • Wogonin is separated with a one-step separation from a crude sample from Scutellaria baicalensis, Georgi using ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) as the two- phase solvent system.
  • the upper phase of ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) can be used as the stationary phase of high-speed counter-current chromatography (HSCCC).
  • HSCCC high-speed counter-current chromatography
  • Wogonoside (Wu et al, 2005, J. Chromatogr. A., 1055(l-2):243-247), a flavonoid from the plant Radix Scutellariae, is isolated according to established methods, e.g., Tang et al., 2004, Phytomedicine 11(4):277-284.
  • the molecular structure of wogonoside is shown below:
  • Ganhuangenin Ganhuangenin (Liu et al, 1984, Yao Sue Sue Bao 19(11):830-835), a flavonoid from the root of Scutellaria gehderiana, is isolated as described by Su et al., 2004, Zhongguo Zhong Yao Za Zhi 29(9): 863-864).
  • the molecular structure of ganhuangenin is shown below:
  • Oroxyloside (oroxylin A-glucuronide), (Ci 6 Hi 2 O 5 , 4H-l-benzopyran-4-one, 5,7- dihydroxy-6-methoxy-2-phenyl-) is isolated from plants such as Scutellariae baicalensis, Scutellariae Radix, Shuang-Huang-Lian, and Scutellaria rehderiana (Wu, S. J Chromatogr A. 2005 Feb 25;1066(l-2):243-7, Li, H. J Chromatogr A. 2005 May 13;1074(l-2): 107-10).
  • a method of isolating oroxyloside is described.
  • Oroxyloside is separated with a one-step separation from a crude sample from Scutellaria baicalensis, Georgi using high-speed counter-current chromatography (HSCCC) with n-hexane- ethyl acetate-n-butanol-water (1:1:8:10, v/v/v/v) as the two-phase solvent system and increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml min(-l) after 4 h.
  • HSCCC high-speed counter-current chromatography
  • the purified compounds can be analyzed by high-performance liquid chromatography.
  • oroxyloside is intended to include isoforms, derivatives and functional analogs of oroxyloside.
  • derivatives of oroxyloside see Suresh, B., BioorgMed Chem Lett. 2005 Sep l;15(17):3953-6.
  • the molecular structure of oroxyloside is shown below:
  • the invention includes compounds that are chromen-one derivatives.
  • the compound is a derivative of 2H-chromen-2-one.
  • Compounds of the invention include compounds of Formula IV:
  • Ri and R 2 are hydrogen, unsubstituted aryl, substituted aryl, heterocycle, substituted heterocycle, carbohydrate, alkyl, alkenyl, halogen, or hydroxyl.
  • Aryl or heterocycle are substituted with hydrogen, hydroxyl, alkoxy, substituted alkoxy, alkyl, alkenyl, CF 3 , amino, carboxy, or halogen.
  • Aryl or heterocycle are mono-, di-, or tri-substituted.
  • the term "carbohydrate” refers to a monosaccharide, disaccharide, trisaccharide, oligomeric, and polymeric form of a sugar.
  • a sugar is a naturally occurring sugar or a non-naturally occurring sugar.
  • the compound is substituted with one or more monosaccharides.
  • the monosaccharide is the sugar galactose.
  • the monosaccharide is the sugar glucose.
  • R 3 , R 4 , R 5 , and R 6 are hydrogen, hydroxy, alkoxy, alkyl, alkenyl, CF 3 , amino, carboxy, halogen or together R 3 and R 4 or R 4 and R 5 or R 5 and R 6 form a 5- or 6-membered ring.
  • the 5- or 6-membered ring is unsubstituted or substituted.
  • R] is hydrogen.
  • R 2 is hydrogen.
  • Rj and R 2 are both hydrogen.
  • R 4 and R 5 form a 5- membered ring, where the ring is a heterocycle or cycloalkyl.
  • the 5-membered heterocycle ring is selected from tetrahydrofuran, furan, pyrrolidine, pyrrole, tetrahydrothiophene, thiophene, and dioxole.
  • the 5-membered heterocycle is furan.
  • the 5-membered cycloalkyl ring is cyclopentane.
  • R 4 and R 5 form a 6-membered ring, where the ring is a heterocycle or cycloalkyl.
  • the 6- membered heterocycle ring is selected from piperazine, piperidine, tetrahydrothiopyran, thiomorpholine, or tetrahydropyran.
  • the 6-membered heterocycle ring is tetrahydrofuran.
  • the heterocycle ring is substituted.
  • the heterocycle ring is mono, di-, or tri-substituted.
  • the heterocycle ring is substituted with alkyl, e.g., methyl, hydroxyl, alkoxy, alkoxycarbonyl, or alkenoxycarbonyl.
  • the 6-membered cycloalkyl ring is cyclohexane.
  • R 3 is alkoxy.
  • R 6 is alkoxy.
  • R 6 Is OCH 2 CH(OH)C(CH 3 ) 2 OH.
  • the invention includes compounds that are chromen-one derivatives.
  • the compound is a derivative of 4H-chromen-4-one.
  • Compounds of the invention include compounds of Formula V:
  • Ri and R 2 are hydrogen, unsubstituted aryl, substituted aryl, heterocycle, substituted heterocycle, carbohydrate, alkyl, alkenyl, halogen, or hydroxyl.
  • Aryl or heterocycle are substituted with hydrogen, hydroxyl, alkoxy, substituted alkoxy, alkyl, alkenyl, CF 3 , amino, carboxy, or halogen.
  • Aryl or heteroaryl are mono, di-, or tri-substituted.
  • the carbohydrate is a monosaccharide.
  • the monosaccharide is the sugar glucose.
  • the monosaccharide is the sugar galactose.
  • R 3 , R 4 , R 5 , and R 6 are hydrogen, hydroxy, alkoxy, alkyl, alkenyl, CF 3 , amino, carboxy, halogen or together R 3 and R 4 or R 4 and R 5 or R 5 and R 6 form a 5- or 6-membered ring.
  • the 5- or 6-membered ring is unsubstituted or substituted.
  • Ri is carbohydrate or hydrogen.
  • R 2 is substituted or unsubstituted aryl.
  • Aryl is mono- or di-substituted. Aryl is substituted with hydroxyl.
  • At least one of R 3 , R 4 , or R 5 is not hydrogen. At least two of R 3 , R 4 , or R 5 is not hydrogen.
  • R 3 , R 4 , or R 5 is not hydrogen.
  • R 3 , R 4 , and R 5 are selected from hydrogen, hydroxyl, carbohydrate, and alkoxy.
  • R 3 and R 5 are methoxy.
  • R 5 is carbohydrate.
  • R 5 is monosaccharide.
  • R 5 is glucopyranosiduronic acid.
  • R 3 and R 5 are each hydroxyl.
  • R 3 and R 4 are each hydroxyl.
  • R 4 is alkoxy.
  • R 6 is alkoxy.
  • R 4 and R 5 form a 5-membered ring, where the ring is a heterocycle or cycloalkyl.
  • the 5- membered heterocycle ring is selected from tetrahydrofuran, furan, pyrrolidine, pyrrole, tetrahydrothiophene, thiophene, and dioxole.
  • the 5-membered heterocycle is furan.
  • the 5- membered cycloalkyl ring is cyclopentane.
  • R 4 and R 5 form a 6-membered ring, where the ring is a heterocycle or cycloalkyl.
  • the 6- membered heterocycle ring is selected from piperazine, piperidine, tetrahydrothiopyran, thiomorpholine, or tetrahydropyran.
  • the 6-membered heterocycle ring is tetrahydrofuran.
  • the heterocycle ring is substituted.
  • the heterocycle ring is mono, di-, or tri-substiruted.
  • the heterocycle ring is substituted with alkyl, e.g., methyl, hydroxyl, alkoxy, alkoxycarbonyl, or alkenoxycarbonyl.
  • the 6-membered cycloalkyl ring is cyclohexane. Vanillin
  • Vanillin (CsH 8 O 3 , molecular weight 152) (4-hydroxy-3-methoxybenzaldehyde), is an aromatic compound that can be isolated from vanilla beans and a variety of plants, such as Buddlejapurdomii (Gao, Y. Zhong Yao Cai. 2004 May;27(5):339-41.). Vanillin occurs widely in plants in nature, usually as a glycoside bound to sugar or as a percusor to vanillin bound to the large lignin molecule abundant in wood. While a majority of vanillin is produced from guaiacol, it can also be made from lignin, a by-product from the wood-pulp industry and synthetically.
  • Isoeugenol can be converted into vanillin from crude enzyme extracted from soybean (Li, YH, Appl Biochem Biotechnol. 2005 Apr;125(l):l-10). Vanillin can also be obtained by treatment of spruce wood with alkali in the presence of nitrobenzene (J. Am. Chem. Soc. 63, 312 (1941)). Vanillin is a white crystalline material. Vanillin has a characteristic pleasant smell and taste of vanilla which accounts for its widespread use. Vanillin is soluble in water and its solubility increases with increasing temperature. Vanillin is converted to L-dopa and related compounds. In addition, vanillin is the starting material for chemical synthesis of Aldomet. The molecular structure of vanillin is shown below:
  • Neurological disorders are associated with neuronal loss or dysfunction, including, but not limited to, Parkinson's Disease, Alzheimer's Disease, Huntington's Disease, ALS, stroke, epilepsy, ADD, and neuropsychiatric syndromes.
  • the neurological disorder is a neurodegenerative disorder.
  • the neurological disorder is a seizure-associated disorder such as a stroke, brain ischemic condition, and epilepsy.
  • Compounds of the present invention are administered therapeutically (including prophylactically): (1) in diseases, disorders, or conditions involving neuronal loss or dysfunction, including, but not limited to, Parkinson's Disease, Alzheimer's Disease, Huntington's Disease, ALS, stroke, ADD, and neuropsychiatric syndromes; or (2) in diseases, disorders, or conditions wherein in vitro (or in vivo) assays indicate the utility of the compounds of the present invention.
  • a memory disorder is associated with a diminished mental registration, retention or recall of past experiences, knowledge, ideas, sensations, thoughts or impressions.
  • Memory disorders affect short and/or long-term information retention, facility with spatial relationships, memory (rehearsal) strategies, and verbal retrieval and production.
  • Memory disorders include, for example, dementia, slow learning and the inability to concentrate.
  • Common causes of a memory disorder are age, severe head trauma, brain anoxia or ischemia, alcoholic-nutritional diseases, drug intoxications and neurodegenerative diseases.
  • a memory disorder is a common feature of neurodegenerative diseases, such as Alzheimer's disease (i.e. Alzheimer-type dementia).
  • AIDS Dementia Wernicke- Korsakoff s related dementia (alcohol induced dementia); age related dementia, multi-infarct dementia, a senile dementia caused by cerebrovascular deficiency, and the Lewy-body variant of Alzheimer's disease with or without association with Parkinson's disease.
  • Loss of memory is also a common feature of brain-damaged patients.
  • Non-limiting examples of causes of brain damage which may result in a memory disorder include stroke, seizure, an anaesthetic accident, ischemia, anoxia, hypoxia, cerebral edema, arteriosclerosis, hematoma or epilepsy; spinal cord cell loss; and peripheral neuropathy, head trauma, hypoglycemia, carbon monoxide poisoning, lithium intoxication, vitamin (Bl, thiamine and B 12) deficiency, or excessive alcohol use.
  • the purified compounds or functional analogs thereof are antagonists of NMDA receptors and are therefore useful in the treatment of disorders associated with the NMDA receptor function, e.g., seizures, epilepsy, epileptogenesis, and other neurological disorders.
  • Glutamate is recognized as the predominant excitatory neurotransmitter (messenger molecule) in the mammalian central nervous system (CNS); for a review, see the chapter by Olney entitled "Glutamate” in The Encyclopedia of Neuroscience, edited by Adelman (either the 1987 or the 1995 edition). Glutamate is involved in transmitting messages from one nerve cell (neuron) to another in many different circuits within the CNS, and therefore serves many important functions.
  • NMDA receptors There are several different subtypes of receptors through which glutamate transmits messages.
  • a particularly important receptor through which glutamate mediates a wide range of functions is the NMDA receptor.
  • Other major classes of glutamate receptors are kainic acid receptors and Quis/AMPA receptors; these two classes are collectively referred to as non-NMDA receptors. Both NMDA and non-NMDA receptors are normally activated by glutamate.
  • Antagonist drugs that block glutamate receptors, such as the NMDA receptor are classified in two broad classes of compounds.
  • NMDA antagonists bind at the NMDA/glutamate binding site (such drugs include CPP, DCPP-ene, CGP 40116, CGP 37849, CGS 19755, NPC 12626, NPC 17742, D-AP5, D-AP7, CGP 39551, CGP-43487, MDL-100,452, LY-274614, LY-233536, and LY233053).
  • NMDA antagonists Another class is referred to as non-competitive NMDA antagonists; these agents bind at other sites in the NMDA receptor complex (such drugs include phencyclidine, dizocilpine, ketamine, tiletamine, CNS 1102, dextromethorphan, memantine, kynurenic acid, CNQX, DNQX, 6,7-DCQX, 6,7- DCHQC, R(+)-HA-966, 7-chloro-kynurenic acid, 5,7-DCKA, 5-iodo-7-chloro-kynurenic acid, MDL-28,469, MDL-100,748, MDL-29,951, L-689,560, L-687,414, ACPC, ACPCM, ACPCE, arcaine, diethylenetriamine, 1,10-diaminodecane, 1,12-diaminododecane, ifenprodil, and SL- 82.0715).
  • NMDA receptor antagonists to ameliorate, reduce or treat the effects of a disorder associated with the NMDA receptor.
  • a combination of purified compounds and functional analogs are used to antagonize NMDA receptors.
  • Seizures and Epilepsy The invention provides methods for treating or preventing seizures as well as methods for delaying the onset of seizures (epileptogenesis). There are many different types of seizures. Seizures are typically divided into generalized seizures (absence, atonic, tonic-clonic, myoclonic) and partial (simple and complex) seizures. Generalized Seizures Generalized seizures are thought to affect both cerebral hemispheres (sides of the brain) from the beginning of the seizure. They produce loss of consciousness, either briefly or for a longer period of time, and are sub-categorized into several major types: generalized tonic clonic; myoclonic; absence; and atonic.
  • Absence seizures also called petit mal seizures
  • Some absence seizures are accompanied by brief myoclonic jerking of the eyelids or facial muscles, or by variable loss of muscle tone. More prolonged attacks may be accompanied by automatisms, which may lead them to be confused with complex partial seizures.
  • complex partial seizures last longer, may be preceded by an aura, and are usually marked by some type of confusion following the seizure.
  • Myoclonic seizures are rapid, brief contractions of bodily muscles, which usually occur at the same time on both sides of the body. Occasionally, they involve one arm or a foot.
  • Partial Seizures In partial seizures the onset of the electrical disturbance is limited to a specific area of one cerebral hemisphere (side of the brain). Partial seizures are subdivided into simple partial seizures (in which consciousness is retained); and complex partial seizures (in which consciousness is impaired or lost). Partial seizures may spread to cause a generalized seizure, in which case the classification category is partial seizures secondarily generalized.
  • Partial seizures are the most common type of seizure experienced by people with epilepsy. Virtually any movement, sensory, or emotional symptom can occur as part of a partial seizure, including complex visual or auditory hallucinations. There are two types of partial seizure, simple partial seizures and complex partial seizures.
  • Simple partial seizures include autonomic and mental symptoms and sensory symptoms such as olfaction, audition, or vision, sometimes concomitant with symptoms of experiences such as deja-vu and Zealand-vu.
  • Complex partial seizures affect a larger area of the brain than simple partial seizures and they affect consciousness.
  • a person cannot interact normally with other people, is not in control of his/her movements, speech, or actions.
  • a person may even be able to speak, but the words are unlikely to make sense and he or she will not be able to respond to others in an appropriate way.
  • complex partial seizures can affect any area of the brain, they often take place in one of the brain's two temporal lobes. Because of this, the condition is sometimes called "temporal lobe epilepsy.”
  • Epileptic seizures are the outward manifestation of excessive and/or hypersynchronous abnormal activity of neurons in the cerebral cortex. Many types of seizures occur, as described above.
  • the neuromechanism responsible for seizures includes the amygdala, the hippocampus, the hypothalamus, the parolfactory cortex, the frontal and temporal lobes, and the involvement of the substantia nigra, a particular portion of the brain considered to be part of neural circuitry referred to as the basal ganglia (See e.g., Depaulis, et al. (1994) Prog. Neurobiology, 42: 33-52).
  • the neural connections that make up. the basal ganglia are important in epilepsy. These connections are reviewed by Alexander et al. (Alexander, et al. Prog. Brain Res. 85: 119-146).
  • the substantia nigra receives input from the subthalamic nucleus (STN) which is excitatory and involves glutamate as the neurotransmitter conveying information at the synapse.
  • STN subthalamic nucleus
  • glutamate glutamate as the neurotransmitter conveying information at the synapse.
  • a lesion of the subthalamic nucleus will reduce the inhibitory output of the internal segment of the globus pallidus and substantia nigra reticulata (SN) (Bergman, et al. (1990), Science, 249: 1436-1438).
  • the subthalamic nucleus receives input from the external segment of the globus pallidus (GPe).
  • This input is inhibitory using GABA as a transmitter substance.
  • GABA GABA
  • the methods and compositions of the invention are used to inhibit, reduce, or treat seizures that include, but are not limited to, tonic seizures, tonic-clonic seizures, atypical absence seizures, atonic seizures, myoclonic seizures, clonic seizures, simple partial seizures, complex partial seizures, and secondary generalized seizures.
  • seizures and epilepsy There are various in vivo models of seizures and epilepsy that can be used to test compounds against specific forms of seizures and epilepsy.
  • the kainate model is an epileptic model in which kainic acid, one of the excitatory amino acids found in the brain, is injected to nuclei (amygdala, hippocampus, etc.) in the limbic system in an microamount to induce focal epilepsy.
  • the kainate model serves as a model for an epileptic seizure; more particularly, as a model for status epilepticus induced from the limbic system in an acute phase, and as a model for evolution of a spontaneous limbic seizure to a secondary generalized seizure in a chronic phase.
  • the kainate model may also be used as a cortex epilepsy model through injection of kainic acid to the cortex (sensory motor field).
  • seizures and epilepsy include, but are not limited to the Maximal Electroshock (MES) model, which was used to investigate the efficacy of compounds against grand mal seizures. Maximal seizures were induced by applying an electrical current to the brain of the animal via corneal electrodes. The electrical stimulus was applied at about 50mA in a pulse of 60Hz for 200ms. After the onset of seizures, the compound was administered and the inhibition of spasms recorded.
  • MES Maximal Electroshock
  • Another suitable model is the Subcutaneous Metrazole (ScMET) Model for epilepsy, and in particular was useful for investigating petite mal seizures.
  • a metrazole dose of 85mg/kg was administered subcutaneously to the animal (e.g., mouse) to induce seizures. The compound was then be administered and the animals observed.
  • the Toxicity Model (TOX) using a rotorod was employed.
  • the animals e.g., mice
  • the trained animal was given the compound at various doses and the effect of compound on their motor skills, was determined.
  • the dose at which the animals fell off the rotorod is the toxic dose.
  • Epileptogenesis is the process by which a normal brain becomes chronically prone to seizures. Many brain insults (stroke, trauma, neurodegenerative disease etc) can induce epileptogenesis, yet no therapies exist to disrupt this process. Although reorganization of specific neuronal circuits and alterations in individual synapses are associated with epileptogenesis, the functional consequences and relative importance of these changes to epileptogenesis and seizure genesis are unknown, as are many of the molecular and cellular mechanisms underlying these alterations.
  • Seizures and epilepsy are common sequelae of acute brain insults such as stroke, traumatic brain injury, and central nervous system infections.
  • Early, or acute symptomatic, seizures occur at the time of the brain insult and may be a marker of severity of injury.
  • a cascade of morphologic and biologic changes in the injured area over months to years leads to hyperexcitability and epileptogenesis.
  • After a variable latency period late unprovoked seizures and epilepsy occur.
  • the drugs that presently used in the treatment of epilepsy treat the symptom, seizures, but do not modify the epileptogenic process.
  • purified compounds and functional analogs are administered during the latent period for the prevention of epileptogenesis and the development of unprovoked seizures and epilepsy.
  • substantially purified compounds and functional analogs are used as a neuroprotectant and an antiepileptogenic agent.
  • Seizure kindling models are characterized by giving a sub- seizure eliciting electrical or chemical stimulus (i.e., sub-threshold) over a period of time (Goddard et al., (1969) Exp. Neurol. 25: 295-330).
  • the kindling phenomenon has been proposed to underlie the development of disorders such as certain types of epilepsy syndromes.
  • Several kindling models of seizure development have been characterized. For a review of animal models used for epilepsy and seizures, see for example, Sarkisian (2001) Epilepsy and Behavior, 2: 201-216).
  • Purified compounds or functional analogs that delay or block acquisitions of seizures in these kindling models are used for effective therapy following cerebral insults including, but not limited to, ischemia, haemorrhagic stroke, trauma, and infection, that can lead to an elevated incidence of seizure disorders (The Epilepsies: Etiologies and Prevention, 1999, Eds. Kotagal and Luders; Ballenger, et al, (1978) Br. J. Psychiatry 133: 1-14).
  • a substantially purified natural product such as an NMDA receptor antagonist is used to ameliorate, reduce, prevent or treat the effects of a disorder associated with NMDA receptor function.
  • a combination of substantially purified natural product compounds are used together (administered simultaneously or sequentially) as NMDA receptor antagonists.
  • Compounds identified from botanical sources are purified from the natural source or synthetically made de novo.
  • compounds are formulated as mixtures of 2, 3, 4, 5, 8, 10 or more compounds.
  • the compound is formulated as a prodrug.
  • the prodrug is inactive until it is ingested, processed by the body (digested), or comes in contact with a target cell
  • the prodrug chemical structure undergoes conversion to an active drug within a biological system, e.g., metabolism or contact with a component of a target cell such as an enzyme or cell surface structure.
  • Some prodrugs include the active compound to which a chemical moiety has been linked, i.e., the compound has been derivatized When the prodrug is metabolized, the chemical moiety is removed thereby activating the compound.
  • targeted prodrug formulations include antibody-directed enzyme prodrugs, gene- directed enzyme prodrugs, and peptide transporter-associated prodrugs.
  • the purified compounds, extracts, and functional analogs of the present invention are administered by virtually any mode and are administered simultaneously or serially. When administered serially, the purified compounds and functional analogs are administered sufficiently close in time so as to provide the desired effect, for example within 1-3 hours of each other. In some examples, the purified compounds and functional analogs are administered topically, transdermally via a transdermal patch.
  • the purified compounds and functional analogs are administered therapeutically to treat, prevent, or slow the rate of onset of neuronal dysfunctions, such as epilepsy and seizures, or prophylactically to either protect against further seizures associated with epilepsy or to avoid or forestall the onset of seizures associated with other disorders.
  • the purified compounds and functional analogs are administered prophylactically to slow or halt the progression of seizures and epilepsy in a patient who has had a stroke and has a risk of developing seizures as a result of the stroke.
  • the purified compounds and functional analogs are administered to a subject, using a wide variety of routes or modes of administration.
  • Suitable routes of administration for particular compositions include, but are not limited to, oral inhalation; nasal inhalation; transdermal; oral; rectal; transmucosal; intestinal; and parenteral administration, including intramuscular, subcutaneous, and intravenous injections.
  • the purified compounds are administered via the same or via a different mode of administration.
  • an isolated compound with a pharmaceutically acceptable salt or hydrate is administered orally or via a transdermal patch, an aerolized formulation, by nasal inhalation, or via nano- or microencapsulated formulations.
  • the purified compounds or functional analogs are administered by intrathecal and intraventricular modes of administration.
  • the compounds can be administered in a combination with other therapeutic agents.
  • therapeutic agents that are co-administered with the composition of the invention will depend, in part, on the condition being treated.
  • the compounds of the invention are administered in cocktails comprising other agents used to treat symptoms and associated with epilepsy or seizures. In the latter case, the combination therapy approach may permit a lower dose of the agents, thereby reducing undesired side effects.
  • the compounds can be formulated either as single compounds per se or as mixtures of compounds of the same type (e.g., two different analogs), as well as mixtures of compounds.
  • Such compositions will generally comprise at least one purified compound or functional analogs formulated as a pharmaceutically acceptable salt or hydrate.
  • compositions for use in accordance with the present invention are formulated in conventional manner using one or more physiologically acceptable carriers, excipients, diluents or auxiliaries that further facilitate processing of the substantially purified natural product compounds.
  • the choice of formulation is dependent upon the selected administration route.
  • the compounds are formulated in the form of an ointment, paste, spray, patch, cream, gel, sponge, or foam.
  • the formulations are administered by implantation or transcutaneous delivery (for example subcutaneously or intramuscularly), intramuscular injection, or transdermally.
  • the compounds are formulated with suitable polymeric or hydrophobic materials (such as an emulsion in an acceptable oil) or ion exchange resins.
  • suitable polymeric or hydrophobic materials such as an emulsion in an acceptable oil
  • ion exchange resins such as an emulsion in an acceptable oil
  • Formulations suitable for transdermal administration of compounds are described in U.S. Pat. Nos. 5,725,876; 5,716,635; 5,633,008; 5,603,947; 5,411,739; 5,364,630; 5,230,896; 5,004,610; 4,943,435; 4,908,213; and 4,839,174, which patents are hereby incorporated herein by reference.
  • pharmaceutically acceptable salts or hydrates are readily absorbed and cross cell membranes and the blood-brain barrier. Any of these formulations are routinely adapted for transderma
  • the purified compounds or functional analogs are formulated in physiologically compatible aqueous solutions, such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible aqueous solutions such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the purified compounds or functional analogs are formulated with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated for oral administration as tablets, pills, gums dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like.
  • the compounds are formulated into candies, cookies, or other edible foodstuffs.
  • Pharmaceutical preparations for oral use are obtained by mixing the compounds of the invention with a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after. adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients include, but are not limited to, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • disintegrating agents can be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Concentrated sugar solutions are used that can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments are added to the tablets or coatings for identification or to characterize different combinations of active compound doses.
  • compositions that are used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules contain the compounds of the invention in an admixture with filler, such as lactose; binders, such as starches; or lubricants, such as talc or magnesium stearate; or stabilizers.
  • filler such as lactose
  • binders such as starches
  • lubricants such as talc or magnesium stearate
  • stabilizers are added to the soft-capsule formulation. All formulations for oral administration are in dosages suitable for such administration.
  • compositions takes the form of oral sprays, tablets, gums, or lozenges formulated by well-known methods.
  • a candy formulation suitable for oral or buccal administration of therapeutic compounds, pharmaceutically acceptable salts and hydrates is described in U.S. Pat. No. 6,083,962, which is hereby incorporated herein by reference. Additional formulations suitable for oral or buccal administration of therapeutic compounds, are described in U.S. Pat. Nos.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the compositions are. administered by the oral or nasal respiratory route for local or systemic effect.
  • the compounds of the invention are conveniently delivered in the form of an aerosol spray delivered via pressurized packs or a nebulizer, with a suitable propellant, e.g., carbon dioxide or other suitable gas.
  • a suitable propellant e.g., carbon dioxide or other suitable gas.
  • the dosage unit is controlled by a dose-metered valve.
  • gelatin for use in an inhaler or insufflator are formulated as a powder mix of the compounds if the invention and a suitable powder base, such as lactose or starch.
  • a suitable powder base such as lactose or starch.
  • Formulations suitable for nasal inhalation are well known in the art.
  • a nasal aerosol spray contains compound, a water soluble diluent such as an organic acid, and a thickening agent such as a natural or synthetic polymer or an oil substance comprising the oil phase of an emulsion.
  • the compounds of the invention are also administered in a vaporizer that delivers a volume of vapor containing compounds.
  • the vaporizer is battery operated and designed to deliver a dosage of compound effective to inhibit seizures.
  • the compounds of the invention in a sterile pharmaceutically acceptable solvent, are nebulized by use of inert gases. Nebulized solutions are breathed directly from the nebulizing device or the nebulizing device is attached to a face mask, tent or intermittent positive pressure breathing machine.
  • an aerosol spray containing substantially purified natural product compounds is used to treat or prevent seizure clustering. Some patients with epilepsy are prone to having consecutive seizures after the initial seizure. An aerosol formulation of compounds is used as a spray mist in such patients after the first seizure as a preventative measure against subsequent seizures.
  • the aerosol formulation is administered in a spray mist in a subject that has been chemically induced to have seizures or is at the risk of developing seizures, such as those at risk of bioterror attacks.
  • the aerosolized substantially purified natural product compounds are provided in the form of a portable kit or package and used prior to, or immediately after, exposure to the seizure inducing chemical, e.g., organophosphate.
  • the compounds of the invention are formulated with a surface-active agent (or wetting agent or surfactant) or in the form of an emulsion (as a water- in-oil or oil-in-water, emulsion).
  • Suitable surface-active agents include, but are not limited to, non-ionic agents, such as polyoxyethylenesorbitans (e.g. TweenTM 20, 40, 60, 80 or 85) and other sorbitans (e.g. SpanTM 20, 40, 60, 80 or 85).
  • Compositions with a surface-active agent comprise between 0.05 and 5% surface-active agent, and preferably between 0.1 and 2.5%. It will be appreciated that other ingredients are added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.
  • Suitable emulsions are prepared using commercially available fat emulsions, such as Intralipid , Liposyn , Infonutrol , Lipofundin and Lipiphysan .
  • the active ingredient is either dissolved in a pre-mixed emulsion composition or alternatively it is dissolved in an oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil. corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water.
  • an oil e.g. soybean oil, safflower oil, cottonseed oil, sesame oil. corn oil or almond oil
  • a phospholipid e.g. egg phospholipids, soybean phospholipids or soybean lecithin
  • other ingredients are added, for example gylcerol or glucose, to adjust the tonicity of the emulsion.
  • Suitable emulsions typically contain up to 20% oil, for example, between 5 and 20%.
  • the fat emulsion preferably comprises fat droplets between 0.1 and 1.0 ⁇ m, particularly 0.1 and 0.5 ⁇ m, and have a pH in the range of 5.5 to 8.0.
  • An injectable formulation containing compound is used to treat or prevent seizure clustering. Some patients with epilepsy are prone to having consecutive seizures after the initial seizure.
  • the injectable formulation of compound is after the first seizure as a preventative measure against subsequent seizures.
  • the injectable formulation is administered to a subject that has been chemically induced to have seizures or is at the risk of developing seizures, such as those at risk of bioterror attacks.
  • the injectable formulation of compounds are provided in the form of a portable kit or package and used prior to, or immediately after, exposure to the seizure inducing chemical, e.g., organophosphate.
  • the purified compound is formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection are presented in unit- dosage form, e.g., in ampules or in multi-dose containers, optionally with an added preservative.
  • the compositions take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain formulatory agents, such as suspending, stabilizing, or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the compounds of the invention in water-soluble form. Additionally, suspensions of the compounds of the invention are prepared as appropriate oily-injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic-fatty-acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous-injection suspensions contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension contains suitable stabilizers or agents that increase the solubility of the compounds of the invention to allow for the preparation of highly concentrated solutions. Alternatively, the compounds are in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds are also be formulated in rectal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases, such as cocoa butter or other glycerides.
  • rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases, such as cocoa butter or other glycerides.
  • the pharmaceutical compositions also comprise suitable solid- or gel-phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers, such as polyethylene glycols.
  • the appropriate dose of the pharmaceutical composition is that amount effective to prevent occurrence of the symptoms of the disorder or to treat some symptoms of the disorder from which the patient suffers.
  • effective amount By “effective amount”, “therapeutic amount” or “effective dose” is meant that amount sufficient to elicit the desired pharmacological or therapeutic effects, thus resulting in effective prevention or treatment of the disorder.
  • the effective dose varies, depending upon factors such as the condition of the patient, the severity of the symptoms of the disorder, and the manner in which the pharmaceutical composition is administered.
  • the effective dose of the composition differs from patient to patient but in general includes amounts starting where desired therapeutic effects occur, but below that amount where significant undesirable side effects are observed.
  • an effective amount of composition is an amount sufficient to pass across the blood-brain barrier of the subject and to interact with relevant receptor sites in the brain of the subject and alter the actions of neurotransmitters on those receptors, thus resulting in effective prevention or treatment of the disorder.
  • compositions suitable for use with the present invention include compositions wherein the purified compounds or functional analogs are present in effective amounts, i.e., in amounts effective to achieve the intended purpose, for example, treating, preventing, or reducing seizures, or orthostatic hypotension.
  • effective amounts i.e., in amounts effective to achieve the intended purpose, for example, treating, preventing, or reducing seizures, or orthostatic hypotension.
  • the actual amounts of the compounds effective for a particular application depends upon a variety of factors including, inter alia, the type of disorder being treated, and the age and weight of the subject.
  • When administered to treat or prevent seizures such compositions contain amounts of compound effective to achieve these results. Determination of effective amounts is well within the capabilities of those skilled in the art.
  • the compounds can be administered in any manner that achieves the requisite therapeutic or prophylactic effect.
  • Therapeutically or prophylactically effective doses of the compounds of the invention can be determined from animal or human data for analogous compounds that are known to exhibit similar pharmacological activities.
  • the applied doses are adjusted based on the relative bioavailability, potency and in vivo half-life of the administered compounds as compared with these other agents.
  • the dosage range of natural product compounds administered subcutaneously is about 0.1mg/kg/day to 0.4mg/kg/day.
  • the dosage range of natural product compounds capsules for oral administration is in the range of about lOO ⁇ g/day to 2000 ⁇ g/day (See e.g., Ye et al (2000) Acta Pharmacol Sin: 21, 65; Ma et al. (1998) Ann NY Acad Sd.
  • the dosage range of compounds administered subcutaneously is about 3mg/kg/day to 12mg/kg/day.
  • the dosage range of substantially purified natural product compounds capsules for oral administration is in the range of about 3mg/day to 60mg/day. It will be appreciated that the duration of action, and the therapeutic effect of the compounds is increased by combining two or more purified compounds or functional analogs of the present invention without causing adverse side effects. Furthermore, the dosage of each purified compound is reduced to achieve the same therapeutic effect.
  • the dosage of that natural product of the present invention may be reduced, for example to lOOO ⁇ g/day, when used in combination with a different natural product of the present invention, for example, at a dosage of about 30mg/day.
  • This combination provides the same therapeutic effect but without the adverse side effects.
  • the duration of therapeutic effect may also increase by a using a combination compounds.
  • the effective dosage amount can be manipulated to achieve the desired therapeutic effect.
  • the compounds of the invention are administered to achieve either a therapeutic or a prophylactic effect.
  • the purified compound is prophylactically administered to a subject who has not yet suffered a seizure, but one who may be prone to, or at risk of seizures, for example as a result of a stroke, thereby protecting the subject against seizures.
  • the compounds of the invention are administered to subjects who suffer from epilepsy. Regardless of the condition of the subject, the compounds of the invention are typically be administered as part of a daily r regimen.
  • disorders that are also treated include, but are not limited to, cognitive impairment; severe neurodegenerative disorders, such as Alzheimer's disease; and neuronal dysfunction associated with loss of motor skills, such as Parkinson's disease and amyotrophic lateral sclerosis.
  • the compounds of the invention also treat, prevent, or reverse neuronal dysfunction resulting from CNS injury, such as stroke, spinal-cord injury, and peripheral- nerve injury.
  • the ratio between toxicity and therapeutic effect for a particular compound is its therapeutic index and is expressed as the ratio between LD 50 (the amount of compound lethal in 50% of the population) and ED 50 (the amount of compound effective in 50% of the population).
  • LD 50 the amount of compound lethal in 50% of the population
  • ED 50 the amount of compound effective in 50% of the population.
  • the compounds of the invention that exhibit high therapeutic indices are preferred.
  • Therapeutic index data is obtained from animal studies and used in formulating a range of dosages for use in humans.
  • the dosage of such compounds preferably lies within a range of plasma concentrations that include the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.
  • the invention is illustrated by way of working examples that demonstrate the anticonvulsive effect of the substantially purified natural product compounds of the invention in established in vitro and in vivo models of epilepsy.
  • Herbal extracts were evaluated in the Maximal Electroshock (MES) and Subcutaneous Metrazol (pentylenetetrazol; SCMET) models of generalized tonic-clonic and myoclonic seizures, respectively in mice and rats.
  • the herbal extracts were evaluated in a 6 Hz "psychomotor" seizure stimulation, a proven useful model of therapy-resistant limbic seizures (See, for example, Barton ME, Klein BD, WoIfHH, White HS., Epilepsy Res. 2001 Dec;47(3):217-27).
  • MES Maximal Electroshock
  • the maximal electroshock (MES) model is an art recognized test, useful to investigate the efficacy of therapeutic agents against grand mal seizures.
  • Maximal seizures were induced by the application of electrical current to the brain via corneal electrodes.
  • the stimulus parameters for mice were 5OmA in a pulse of 60Hz for 200ms.
  • the animals were given the herbal extract dissolved in methyl cellulose and spasm inhibition was recorded as a measure of anticonvulsant activity.
  • the Subcutaneous Metrazole (SCMET) model is also an art-recognized test for therapeutic agents for epilepsy, and in particular, is useful for investigating petite mal seizures.
  • a metrazole dose of 85mg/kg was administered subcutaneously to induce seizures.
  • the herbal extracts were then administered and the animals observed,
  • Toxicity Model (TOX) The neurotoxicity of the herbal extracts was tested in the rotorod test.
  • the mice were trained to stand on an accelerating rotorod rotating at 10 rev min-1 with a diameter of 3.2cm.
  • the trained animal were given the herbal extracts at various doses and the effect of the herbal extracts on their motor skills was determined.
  • the dose at which the animals fell off the rotorod was the toxic dose.
  • the herbal extracts were tested for their ability to block 6-Hz seizures after intraperitoneal (i.p.) administration to the mice.
  • the "psychomotor" seizures characterized by stun, forelimb clonus, twitching of the vibrissae, and Straub tail, were induced according to a previously described procedure (See, for example, Barton ME, Klein BD, WoIfHH, et al. Pharmacological characterization of the 6-Hz psychomotor seizure model of parial epilepsy.
  • the herbal extracts were administered in an i.p. form, soluble form (SOL) or as a suspension (SUS).
  • SOL soluble form
  • SUS suspension
  • the herbal extract can be ground to a powder using a mortar and pestle, and the powder mixed with methyl cellulose. This mixture was then sonicated to produce a suspension that was subsequently administered to mice or rats.
  • Various time points were analyzed at each concentration of herbal extract with each animal model and the results were recorded.
  • Gou teng extract is active in MES, ScMET and 6-Hz models.
  • ED50 is 119 mg/kg (95% Cl: 69-183 mg/kg); 5/8 animals were protected at 200 mg/kg; and all eight animals were protected at 400 mg/kg with minimal motor impairment at 1 hr.
  • the ED50 > 400 mg/kg.
  • the ED50 is between 200 and 400 mg/kg.
  • TD50 is >500 mg/kg.
  • the tuber of Gastrodia elata plant contains the compound gastrodin.
  • Tian Ma is an extract of the plant tissue or tuber.
  • the Tian ma extract is active in ScMET and 6-Hz model.
  • the ED50 is 122 mg/kg (95% Cl: 76-178 mg/kg), 7/8 animals were protected at 250 mg/kg, and all eight animals (100%) were protected at 500 mg/kg.
  • the mouse-i.p. toxicity model up to 1000 mg/kg is non-toxic.
  • the lowest anti- convulsant dose is 75 mg/kg.
  • Gastrodin is also useful in reducing frequency and severity of seizures. Effect of TJ-960 extract in Mouse
  • TJ-960 (mixture of extracts of Bupleuri Radix, Paeoniae Radix, Pinelliae Tuber, Cinnamomi Cortex, Zizyphi Fructus, Scutellariae Radix, Ginseng Radix, Glycyrrhizae Radix, Zingiberis Rhizoma) was found to be active in the mouse-i.p. 6 Hz model.
  • the TJ-960 extract was active in the 6-Hz model with an ED50 of 36.4 mg/kg.
  • 300 mg/kg is non-toxic.
  • the lowest anti-convulsant dose is 15 mg/kg in the mouse-i.p. model and 30 mg/kg in the rat-i.p. model.
  • TJ-IO extract in mouse and rat and effect of TJ-IO extract in rat oral TJ-IO (mixture of extracts of Bupleurum falcatum (thorowax) root, 19%; Peonia lacttflora (peony) root, 19%; Pinellia Ternata (ban xia) rhizome, 15%; Zizyphus jujuba (jubube) fruit, 12%; Panax ginseng (Asian ginseng) root, 9%; Scutellaria baicalensis (Skullcap) root, 9%; Zingiber officinale (ginger) rhizome, 6%; Cinnamomum cassia (cassia) bark, 6%; Glycyrrhiza uralensis (licorice, gan cao) rhizome, 5%) was active in ScMET rat-i.p.
  • TJ- 12 extract was found to be active in the MES rat-i.p. model. Effect of Brahmi rasavan extract in mouse and rat Brahmi rasayan extract was active in the mouse-i.p. 6 Hz model using stimulations of 32,
  • the peoniflorin compound was active in the ScMET model and 6-Hz model using 22 mA stimulation. No toxicity is observed at doses up to 300 mg/kg. Effect of Oxypeucedaninhiydrate in mouse The oxypeucedaninhiydrate compound was active in the MES mouse-i.p. and in the
  • the imperatorin compound was active in the MES rat-i.p. model. At 50 mg/kg, protection from MES seizures was observed. Effect of Decursinol in mouse and rat
  • the decursinol compound was active in the MES and ScMET mouse-i.p. models at doses of 300 mg/kg. Effect of Baicalein, Baicalin, Wogonoside, and Ganhuangenin
  • baicalein baicalin
  • baicalin showed a neuroprotective effect against glutatmate-mediated cell death.
  • Whole-cell patch-clamp recording of ligand- and voltage-gated currents is carried out in cultured rodent cortical and hippocampal neurons to evaluate the activity of purified compoundsn and enriched compositions from botanical sources (extracts).
  • the extracts and substantially purified natural product compounds inhibit, evoke or modulate currents mediated by AMPA ( ⁇ -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), NMDA (N-methyl-D- aspartate) or GABAA ( ⁇ -amino-butyric acid A) receptors or block or enhance voltage-dependent sodium, potassium, or calcium ion channels.
  • AMPA ⁇ -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
  • NMDA N-methyl-D- aspartate
  • GABAA ⁇ -amino-butyric acid A
  • the whole-cell configuration of the patch-clamp technique is used to study voltage- and ligand-gated currents in cultured rodent cortical neurons as described previously by Sucher et al. (Sun X, et al. NeuroSignals, 2003;12:31-8; Sucher NJ, et al. Neuroscience 1991;43:135-50). Specific currents are evoked by application of ligands (ligand-gated conductances) or voltage steps (voltage-gated conductances) in the presence or absence of extracts or single compounds while other currents are blocked by the addition of specific antagonists to the extra- and/or intracellular solutions.
  • I/ V Current/voltage relationships are constructed by performing voltage ramps in the presence of buffer alone, extract or single compound alone, and specific agonist and extract or single compound together. Each trial consists of three 4 seconds ramps from -80 to +40 mV. Ramps are performed before, during, and after drug (extracts or single compounds) application, and each set is averaged. The net I/V plot is constructed by subtracting the averages of the trials before and after drug application from the average during drug application. Should these experiments reveal any acute blocking effect of the drugs, complete dose/response curves are performed in order to determine the IC 5O for each antagonist.
  • Patch-clamp recording Patch-clamp recordings are performed using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) as used previously by Sucher et al. (Sun X, et al. NeuroSignals, 2003;12:31-8; Sucher NJ, et al. Neuroscience 1991;43:135-50). Patch pipettes (2- 5 M ⁇ ) are pulled from borosilicate glass tubing using a computer controlled horizontal puller (Sutter Instrument).
  • Pipettes are fire-polished before use. Extra- and intracellular solutions for the isolation of specific conductances are prepared. Extracts and compounds are applied by the use of a linear array of seven plastic pipes connected to solution reservoirs. Solution flow through each pipe is individually controlled in the pClampex 7 menu. Neurons are continuously bathed in a stream of solution from the flow pipe (0.5 ml per min); only one barrel is used for perfusion at a given time. Data analysis: The amplitudes of the whole-cell currents recorded in the presence and absence of extracts or compounds are measured and compared. The data is analyzed by ANOVA and Tukey's post test.
  • NMDARs Functional neuronal NMDA receptors
  • NRl NMDARl
  • NR2A- D NMDAR2A-D subunits
  • NR3 additional NR3 subunit.
  • both the location (extrasynaptic vs. synaptic) and subunit composition (NR1/NR2B vs. NR1/NR2A) of NMDARs may determine whether or not their stimulation will lead to excitotoxic injury of neurons.
  • HEK293 cells do not express endogenous NMDAR mRNAs or protein and they are easily manipulated and transfected with expression vectors containing recombinant NMDAR subunits.
  • the electrophysiological and biophysical characteristics of various NMDAR subunit combinations expressed in HEK293 cells have been well characterized (Sucher NJ, Awobuluyi M, Choi Y-B, Lipton SA. NMDA receptors: from genes to channels.
  • HEK 293 cells are transfected with rat NMDA receptor subunit cDNAs using calcium phosphate precipitation.
  • Mixed plasmids (1 ⁇ g NRl-GFP and 2 ⁇ g NR2A/B/C or D) are added to the dish and incubated for 24 hours.
  • the medium is removed, and cells are rinsed twice with culture medium and further incubated in the presence of 20 ⁇ M 5,7-dichlorokynurenic acid, which is added in order to protect the transfected cells from NMDAR-mediated cell death.
  • Cells are used for recording 40-50 hours after transfection.
  • the NRl subunit is cloned in frame with green fluorescent protein (GFP).
  • NMDAR-induced cytotoxicity leads to fulminant cell death, necrosis, or a delayed form of cell death (apoptosis). While necrosis leads to immediate cell lysis, apoptotic cell death develops over time and is distinguished by a number of criteria such as characteristic morphological changes and DNA fragmentation.
  • the colorimetric lactate dehydrogenase (LDH) assay is used to assess primary necrosis as well as secondary, necrotic cell death subsequent to apoptosis.
  • Cytotoxicity assays are conducted in primary cultures of mouse cortical neurons after 10 days in vitro as described previously (Sun X, Chan LN, Gong X, Sucher NJ. NeuroSignals, 2003; 12:31-8.). Cultured neurons are exposed to NMDA (200 ⁇ M; 5 minutes) and co-agonist glycine (10 ⁇ M) in the nominal absence of magnesium and the presence of increasing concentrations of extract or single compound. Measurements are performed for each drug and dose in triplicate in at least three independent experiments. The herbal-derived extracts and compounds are added to the culture in three different ways: 1) before, concomitant with and after NMDA, 2) concomitant with and after NMDA and 3) after NMDA. Twenty- four hours after exposure to NMDA cell death is determined by the LDH assay.
  • Extracts and/or substantially purified natural product compounds derived from herbal extracts also reduce NMDA-induced nitric oxide (NO) production in neurons.
  • NMDA receptors are coupled to nNOS through their interaction with postsynaptic density protein (PSD) 95 via PDZ domains. Either deletion of nNOS or PSD-95 was previously reported to suppress NMDA-induced nitric oxide (NO) production in neurons.
  • PSD postsynaptic density protein
  • NMDAR-mediated toxicity the disruption of the interaction between nNOS and PSD-95 suffices to abrogate NMDAR-mediated excitotoxicity and represents a potential therapeutic approach aimed at abating NMDAR-mediated neurotoxicity.
  • mice hippocampal neurons utilize primary cultures of mouse hippocampal neurons.
  • the cultures are virtually purely neuronal (containing less than 1% astrocytes) and express high levels of nNOS.
  • Cultured hippocampal neurons are exposed to NMDA (200 ⁇ M; 5 minutes) and co-agonist glycine (10 ⁇ M) in the nominal absence of magnesium and the presence of increasing concentrations of each extract and compound.
  • the production of NO using the Griess reaction is measured for each extract and/or compound in triplicate in at least three independent experiments.
  • the extract or compound is added to the culture in three different ways: 1) before, concomitant with and after NMDA, 2) concomitant with and after NMDA and 3) after NMDA.
  • Nitrite (NO 2 " ) in culture superaatants is measured to assess NO production in primary neurons using the Griess reagent system' (Promega, Madison, WI) as described previously (Jiang X, et al. Exp Neurol 2004; 190:224-32.). Sample aliquots (50 ⁇ l) are mixed with 100 ⁇ l of Griess reagent (1% sulfanilamide/0.1% naphthylethylene diamine dihydrochloride/5% phosphoric acid) in 96-well plates and incubated at 25 °C for 10 min. The absorbance at 540 nm is measured on a microplate reader. NaNO 2 is used as standard to calculate NO 2 " concentrations.
  • a reduction in NMDA-induced NO production by one or more extracts or substantially purified compounds indicates that the compound or extract 1) directly or indirectly inhibits the NMDA receptor, 2) displaces nNOS from PSD-95, or 3) directly or indirectly inhibits nNOS.
  • Substantially purified natural product compounds are tested in the screening epilepsy animal models for the amygdala kindling and lamotrigine-resistant animal models.
  • the extracts are also tested, in addition to the compounds, because extracts may contain active compounds that are too low in concentration to be isolated and identified.
  • Anticonvulsant profiles are evaluated in a battery of well-established mouse and rat seizure and epilepsy models as shown in the diagram below.
  • Initial identification studies are conducted in CF#1 mice (The Jackson Laboratories, Bar Harbor, ME; Charles River Laboratories, Wilmington, MA) using the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazol (sc PTZ) tests following intraperitoneal administration (White HS, et al. Discovery and preclinical development of anticonvulsants. In: Levy R, Mattson R, Meldrum B, Perucca E, eds. Antiepileptic Drugs, Fifth Edition. New York: Raven Press; 2002:36-48.).
  • MES maximal electroshock seizure
  • sc PTZ subcutaneous pentylenetetrazol
  • levetiracetam With the exception of levetiracetam, all of the approved anti-epileptic drugs (AEDs) currently on the market have demonstrated activity in one or both of these models that are routinely performed. Subsequent investigations demonstrated that levetiracetam was active in pathologically abnormal models of partial and primary generalized seizures (e.g., kindling, audiogenic seizures, and 6-Hz psychomotor seizures). In this regard levetiracetam appears unique among the established and newer AEDs and demonstrates the need for flexibility when screening for efficacy and the need to incorporate levetiracetam-sensitive models into the early evaluation process.
  • AEDs anti-epileptic drugs
  • extracts and substantially purified natural product compounds found ineffective in the initial MES and sc PTZ screens are subsequently evaluated in the levetiracetam-sensitive 6-Hz psychomotor seizure test.
  • Activity in one or more of these three identification tests can be quantified at the time-to-peak effect (TPE) and the median effective dose (ED 50 ) determined by probit analysis of data obtained from quantal dose-response curves.
  • TPE time-to-peak effect
  • ED 50 median effective dose
  • the lamotrigine (LTG)-resistant kindled rat results when rats are kindled in the presence of a low-dose of LTG (Postma T Krupp E, Li XL, Post RM, Weiss SR. Lamotrigine treatment during amygdala-kindled seizure development fails to inhibit seizures and diminishes subsequent anticonvulsant efficacy. Epilepsia 2002;41 : 1514-21.). Studies in the White laboratory have substantiated Postma' s original findings and extended them to include phenytoin and carbamazepine (Srivastava AK, Woodhead, JH, White HS.
  • a complete dose- response study is conducted to quantitate its efficacy.
  • a similar study is conducted in the lamotrigine-resistant kindled rat in an effort to differentiate it from lamotrigine, phenytoin, carbamazepine, and topiramate (all inactive in this model).
  • the substantially purified natural product compounds are also evaluated for their ability to prevent the development of kindling.
  • rats implanted with a bipolar stimulating/recording electrode receive a dose previously determined to be effective in one or more of the acute seizure tests (i.e., MES, scPTZ, or 6 Hz).
  • mice behavioral impairment is determined by the rotarod test; behavioral impairment in rats is estimated from a battery of behavioral assessments including: gait, stance, placing response, muscle tone, etc.
  • the dose producing behavioral impairment in 50% of the population i.e., TD 50
  • the dose producing behavioral impairment in 50% of the population is calculated from probit analysis of quantal dose-response data as described by White et al (White HS, Woodhead JH, Wilcox KS, Stables JP, Kupferberg HJ, WoIf HH. Discovery and preclinical development of anticonvulsants. In: Levy R, Mattson R, Meldrum B, Perucca E, eds. Antiepileptic Drugs, Fifth Edition. New York: Raven Press; 2002:36-48).
  • the protective index (TDso/EDso) is calculated.
  • MMI minimal motor impairment
  • Previous studies have found that non-selective NMDA antagonists display more behavioral impairment in kindled vs. non-kindled rats and that this shift to the left in the toxicity dose-response curve correlates with increased toxicity in the epilepsy patient population.
  • the tolerability profile for those compounds and extracts found to possess a favorable anticonvulsant profile is established in both kindled and non-kindled rats and those compounds and extracts for which the TD 50 is similar regardless of whether animals have been kindled or not are chosen for further investigation.

Abstract

The invention is directed to methods and compositions for using extracts, compounds isolated from natural products and functional analogs thereof for the prevention and/or treatment of neurological seizures such as those associated with epilepsy. The invention is also directed to methods and compositions for using extracts, compounds isolated from natural products and functional analogs thereof for the prevention and/or treatment of neurological disorders.

Description

USE OF NATURAL PRODUCTS FOR TREATMENT OF NEUROLOGICAL DISORDERS
BACKGROUND OF THE INVENTION
Epilepsy is one of the most prevalent neurological disorders, affecting approximately 2.3 million Americans, or about 1% of the population. The Center for Disease Control and Prevention (CDC) estimates that about 181,000 new cases of epilepsy are diagnosed each year. While epilepsy can be idiopathic, about half of diagnosed epilepsy cases are linked to neurological insults, such as status epilepticus (SE), stroke, and traumatic brain injury (TBI).
Many different mechanisms have been associated with the development of epilepsy. For example, repetitive firing of individual neurons caused by overactive voltage-dependent sodium or calcium channels, increases in sodium-dependent action potentials, increases in neuronal calcium uptake, decreased inhibitory events mediated by γ-aminobutyric acid (GABA), and increase in extracellular glutamate concentration that causes increased intracellular neuronal calcium.
To date, treatments of neurological disorders have met with limited success due to side effects and toxicities associated with these therapies. Accordingly, a need exists for better approaches to neuroprotection and for improved therapies for epilepsy, and other neurological disorders.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that an isolated compound or a combination of compounds (e.g., an isolated extract) of a natural product such as a botanical or herbal source is useful to alleviate or prevent seizures such as those associated with epilepsy and/or other neurological disorders associated with N-methyl-D-aspartate (NMDA) receptor pathology. An isolated compound of a botanical source is one that occurs naturally in the botanical source but has been purified from one or more other components with which it naturally occurs or is synthetically made (e.g., chemically synthesized). The isolated compound demonstrates at least 10% greater activity in reduction of neurological seizures compared to the level of activity of the botanical source from which it is derived or in which is naturally occurs. Preferably, the isolated compound possesses at least 25%, 50%, 75%, 100%, 3-fold, 5-fold, 10- fold or more activity compared to the naturally-occurring botanical source. Such isolated compounds include, for example, rhynchophylline, isorhynchophylline, hyperin, baicalin, wogonin, oroxyloside, gastrodin, peoniflorin, albiflorin, vanillin, pentagalloylglucose, imperatorin, oxypeucedaninhydrate, decursinol, decursin, wogonoside, ganhuangenin, baicalein, aporphine type alkaloids, protoberberine type I alkaloids, protoberberine type II alkaloids, and protopine-type alkaloids as well as analogs and combinations thereof. These compounds are separated, extracted or isolated from portions of plants, such as tian ma, gou teng (Ramulus Uncariae cum Uncis), Angelica gigas, Paeonia lactiflora, Gastodia elata, Angelica gigas, Bupleuri Radix, Paeoniae Radix, Pinelliae Tuber, Cinnamomi Cortex, Zizyphi Fructus, Scutellariae Radix, Ginseng Radix, Glycyrrhizae Radix, Zingiberis Rhizoma, Bupleurum falcatum (thorowax) root, Peonia lactiflora (peony) root, Pinellia Ternata (ban xia) rhizome, Zizyphus jujuba (jubube) fruit, Panax ginseng (Asian ginseng) root, Scutellaria baicalensis (Skullcap) root, Zingiber officinale (ginger) rhizome, Cinnamomum cassia (cassia) bark, Glycyrrhiza uralensis (licorice, gan cao) rhizome, Shuang-Huang-Lian, Scutellaria rehderiana, Corydalis tuber, Acanthopanax chiisanensis roots, Drosera burmannii Vahl, Taraxacum mongolicum Hand.-Mazz, Canarium album, Euphorbia nematocypha, Brahmi rasayan, and Onobrychis angustifolia. For example, imperatorin and oxypeucedanin hydrate are isolated from Angelica dahurica, paoniflorin is isolated from Paeonia lcatiflora, gastrodin is isolated from Gastrodia elata; and decursinol and decursin are isolated from Angelica gigas.
The isolated compounds and extracts described herein exhibit neuroprotective and anticonvulsive properties and are administered in a substantially purified form, either alone or in combination, in an amount sufficient to treat, prevent, or delay the onset of neurological disorders. An extract contains at least two but less than all of the compounds present in the naturally-occurring botanical source. For example, an extract contains 2, 5, 10, 25, 50, 100, 500, 1000, 2000 or 5000 compounds and the extract is separated from other compounds that are present in the naturally-occurring botanical source.
Upon administration, the isolated compounds reduce one or more of the mental or cognitive effects such as age-associated cognitive or memory decline, mental decline, and likelihood of age related brain, and cognitive disorders. The isolated compound or functional analog thereof is administered such that it will penetrate the blood-brain barrier and interact with a target receptor in the central nervous system of the subject. The interaction with the target receptor modifies the target receptor in a way to treat or prevent seizures or other neurological disorders in the subject. The therapeutic compositions of the invention are administered with any pharmaceutical excipient known in the art. The dosage that is administered is one that reduces or prevents a disorder. For example, the isolated compounds, mixtures of isolated compounds, or extracts are formulated in a sustained release composition. Such slow release formulations deliver the compounds over a time period of greater than 4 hours, e.g., 5, 6, 7, 8, 10, 12, 18, 24 hours or over a period of days (1, 2, 3, 4, 6, 6, 7 days). The compounds are in the form of a slowly degrading capsule or tablet, a dermal patch, or an solid, semi-permeable, or permeable implant that is placed into a mammal. For example, the implant is placed into or adjacent to a bodily tissue or in a vascularized location such that the compound gains access to the vasculature and is then delivered via the vasculature to the target tissue. Optionally, the implant is biodegradable. If desired, the isolated compounds are administered in combination with other therapeutic agent including the isolated compounds described herein. Optionally, the isolated compound is provided in the form of a dietary supplement.
The invention also provides methods of isolating the active compounds present within herbal extracts. Methods for separation and isolation of the active compounds include standard techniques known in the art, including thin layer chromatography (using silica-coated plates, for example), column chromatography, and high pressure liquid chromatography (HPLC). Active compounds are identified by re-testing of individual bands or fractions (separated by thin layer chromatography, column chromatography and/or HPLC) using specific assay tests as described herein. Sufficient isolation of active ingredients contained within individual bands and/or fractions is tested using, for example, scanning electron microscopes equipped with energy dispersive x-ray analyzer to detect and spatially map elements present in each sample, elemental analysis by combustion to determine the relative amount (e.g., percentage) of carbon, hydrogen and nitrogen, high resolution mass spectroscopy to determine molecular weight and elemental composition, fourier transform infrared spectroscopy to determine functional groups and make comparisons to the spectra of known compounds, differential scanning calorimetry to determine melting point, atomic absorption, gel chromatography, high performance liquid chromatography, proton and 13C nuclear magnetic resonance spectroscopy for material characterization and to provide information regarding the position of atoms relative to each other, and UVVVIS spectroscopy.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. All references cited herein are hereby incorporated by reference
DETAILED DESCRIPTION OF THE INVENTION
The invention provides methods and compositions for treating or preventing neurological disorders using compounds purified or isolated from natural products such as botanical or herbal extracts. Such compounds include, for example, rhynchophylline, isorhynchophylline, hyperin, baicalin, wogonin, oroxyloside, gastrodm, peom'fiorin, albiflorin, vanillin, pentagalloylglucose, imperatorin, oxypeucedaninhydrate, decursinol, decursin, wogonoside, ganhuangenin, baicalein, aporphine type alkaloids, protoberberine type I alkaloids, protoberberine type II alkaloids, and protopine-type alkaloids as well as analogs, derivatives, homologs, positional isomers, stereoisomers and mixtures of steroisomers in optically active or racemic form, salts, and hydrates thereof. As used herein, the terms "isolated", "purified" and "substantially purified," are used interchangeably and refer to a compound that is at least 30%, 40%, or preferably 50%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. More preferably, the preparation is at least 60%, more preferably 75%, more preferably 90%, and most preferably at least 99%, by weight, chemical compound, e.g., natural product compound. A purified compound may be obtained by any method known in the art or described herein including, for example, high pressure liquid chromatography, thin layer chromatography, or by synthesis.
The term "analog" refers to a chemical compound that is structurally similar to a parent compound and has chemical properties or pharmaceutical activity in common with the parent compound. Analogs include, but are not limited to, homologs, i.e., where the analog differs from the parent compound by one or more carbon atoms in series; positional isomers; compounds that differ by interchange of one or more atoms by a different atom, for example, replacement of a carbon atom with an oxygen, sulfur, or nitrogen atom; and compounds that differ in the identity one or more functional groups, for example, the parent compound differs from its analog by the presence or absence of one or more suitable substituents. Suitable substituents include, but not limited to, (Ci-C8)alkyl; (Ci~C8)alkenyl; (Ci-C8)alkynyl: aryl; (C2-C5)heteroaryl; (Ci- C6)heterocycloaklyl; (C3-C7)cycloalkyl; O(Ci~-C8)alkyl; O(C1-C8)alkenyl; O(C1-C8)alkynyl; Oaryl; CN; OH; oxo; halo, C(O)OH; COhalo; O(CO)halo; CF3; N3; NO2, NH2 ; NH((Ci- QOalkyl); N((C,-C8)alkyl)2; NH(aryl); N(aryl)2 N((C1-C8)alkyl)(aryl); (CO)NH2; (C0)NH((d- C8)alkyl); (CO)N((C,-C8)alkyl)2; (CO)NH(aryl); (CO)N(aryl)2; 0(CO)NH2; NHOH; NOH((Ci- C8)alkyl); NOH(aryl); O(CO)NH((Ci-C8)alkyl); O(CO)N((C1-C8)alkyl)2; O(CO)NH(aryl); O(CO)N(aryl)2 ; CHO; CO((C1-C8)alkyl); CO(aryl); C(O)O((Ci-C8)alkyl); C(O)O(aryl); OtCOXtQ-QOalkyl); O(CO)(aryl); O(CO)O((C,-C8)alkyl); O(CO)O(aryl); S-(C1-C8)^yI; S(C,-C8)alkenyl; S(C1-C8)alkynyl; Saryl; S(O)(C1-C8)alkyl; S(O)(C ,-C8)alkenyl; S(O)(Cj- C8)alkynyl; and S(O)aryl; S(O)2(C ,-C8)alkyl; S(O)2(C ,-C8)alkenyl; S(O)2(C i-C8)alkynyl; and S(O)2-aryl. One of skill in art can readily choose a suitable substituent based on the stability and pharmacological activity of the compound.
The term "alkyl" means a saturated, monovalent, unbranched or branched hydrocarbon chain. Examples of alkyl groups include, but are not limited to, (Ci-C8)alkyl groups, such as methyl, ethyl, propyl, isopropyl, 2-methyl-l -propyl, 2-methyl-2-propyl, 2 -methyl- 1 -butyl, 3- methyl-1 -butyl, 2-me thyl-3-butyl, 2,2-dimethyl-l -propyl, 2-methyl-l -pentyl, 3 -methyl- 1-pentyl, 4-methyl-l-pentyl, 2-methyl-2-pentyl, 3- methyl-2 -pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l- butyl, 3, 3 -dimethyl- 1 -butyl, 2-ethyl-l-buty 1, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl. An alkyl group can be unsubstituted or substituted with one or two suitable substituents.
The term "alkenyl" means a monovalent, unbranched or branched hydrocarbon chain having one or more double bonds therein. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. Suitable alkenyl groups include, but are not limited to (C2-C8)alkenyl groups, such as vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl,2-propyl-2-butenyl,4-(2-methyl-3-butene)- pentenyl. An alkenyl group can be unsubstituted or substituted with one or two suitable substituents.
The term "alkynyl" means monovalent, unbranched or branched hydrocarbon chain having one or more triple bonds therein. The triple bond of an alkynyl group can be unconjugated or conjugated to another unsaturated group. Suitable alkynyl groups include, but are not limited to, (C2-C8)alkynyl groups, such as ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-l-butynyl,4-propyl-2-pentynyl, and 4-butyl-2-hexynyl. An alkynyl group can be unsubstituted or substituted with one or two suitable substituents.
The term "aryl" means a monocyclic or polycyclic-aromatic group comprising carbon and hydrogen atoms. Examples of suitable aryl groups include, but are not limited to, phenyl, tolyl, anthacenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. An aryl group can be unsubstituted or substituted. Preferably, the aryl group is a monocyclic ring, wherein the ring comprises 6 carbon atoms, referred to herein as "(C6)aryl". The term "heteroaryl" means a monocyclic- or poly cyclic aromatic ring comprising carbon atoms, hydrogen atoms, and one or more heteroatoms, preferably, 1 to 3 heteroatoms, independently selected from nitrogen, oxygen, and sulfur. As well known to those skilled in the art, heteroaryl rings have less aromatic character than their all-carbon counter parts. Thus, for the purposes of the invention, a heteroaryl group need only have some degree of aromatic character. Illustrative examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidyl, pyrazyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3,)- and (1,2,4)- triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, phenyl, isoxazolyl, and oxazolyl. A heteroaryl group can be unsubstituted or substituted with one or two suitable substituents. Preferably, a heteroaryl group is a monocyclic ring, wherein the ring comprises 2 to 5 carbon atoms and 1 to 3 heteroatoms, referred to herein as "(C2-C5)heteroaryl". The term "cycloalkyl" means a non-aromatic, monocyclic or polycyclic ring comprising carbon and hydrogen atoms. A cycloalkyl group can have one or more carbon-carbon double bonds in the ring so long as the ring is not rendered aromatic by their presence. Examples of cycloalkyl groups include, but are not limited to, (C3-C7)cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, and saturated cyclic and bicyclic terpenes and (C3-C7)cycloalkenyl groups, such as cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl, and unsaturated cyclic and bicyclic terpenes. A cycloalkyl group can be unsubstituted or substituted by one or two suitable substituents. Preferably, the cycloalkyl group is a monocyclic ring or bicyclic ring. The term "heterocycloalkyl" means a non-aromatic monocyclic or polycyclic ring comprising carbon and hydrogen atoms and at least one heteroatom, preferably, 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur. A heterocycloalkyl group can have one or more carbon-carbon double bonds or carbon-heteroatom double bonds in the ring as long as the ring is not rendered aromatic by their presence. Examples of heterocycloalkyl groups include aziridinyl, pyrrolidinyl, pyrrolidino, piperidinyl, piperidino, piperazinyl, piperazino, morpholinyl, morpholino, thiomorpholinyl, thiomorpholino, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, and pyranyl. A heterocycloalkyl group can be unsubstituted or substituted with one or two suitable substituents. Preferably, the heterocycloalkyl group is a monocyclic or bicyclic ring, more preferably, a monocyclic ring, wherein the ring comprises from 2 to 6 carbon atoms and form 1 to 3 heteroatoms, referred to herein as (Q-C^heterocycloalkyl.
The term "halogen" means fluorine, chlorine, bromine, or iodine. Correspondingly, the term "halo" means fluoro, chloro, bromo, and iodo.
The term "derivative" refers to an analog, as defined above, that can be obtained in one or more chemical reactions from its parent compound.
The phrase "pharmaceutically acceptable salt(s)," as used herein includes, but is not limited to, salts of acidic or basic groups that may be present in the natural product compounds, and hydrates thereof. Natural product compounds, and hydrates thereof that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable salts of such basic compounds are those that form salts comprising pharmacologically acceptable anions including, but not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, edetate, camsylate, carbonate, bromide, chloride, iodide, citrate, dihydrochloride, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydroxynaphthoate, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, muscate, napsylate, nitrate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, succinate, sulfate, tannate, tartrate, teoclate, triethiodide, and pamoate (i.e., l,r-methylene-bis-(2-hydroxy-3-naphthoate)). Natural product compounds, and hydrates thereof that include an amino moiety can also form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Natural product compounds, and hydrates thereof that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts. . The term "hydrate" means an natural product compound, or a pharmaceutically acceptable salt thereof that further includes a stoichiometric or non-stoichiometric amount of water bound to it by non-covalent intermolecular forces.
In the present specification, the compound represents a certain isomer for convenience in some cases, but the present invention includes all isomers such as geometrical isomer, optical isomer based on an asymmetrical carbon, stereoisomer, tautomer and the like which occur structurally and an isomer mixture and is not limited to the chemical structure for convenience, and may be any one of isomer or a mixture. Therefore, an asymmetrical carbon atom may be present in the molecule and an optically active compound and a racemic compound may be present in the present compound, but the present invention is not limited to them and includes any one. In addition, a crystal polymorphism may be present but is not limiting, but any crystal form may be single or a crystal form mixture, or an anhydride or hydrate. Further, so-called metabolite which is produced by degradation of the present compound in vivo is included in the scope of the present invention.
It will be noted that the structure of some of the compounds of the invention include asymmetric (chiral) carbon atoms e.g., decursinol. It is to be understood accordingly that the isomers arising from such asymmetry are included within the scope of the invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. The compounds of this invention may exist in stereoisomeric form, therefore can be produced as individual stereoisomers or as mixtures. "Isomerism" means compounds that have identical molecular formulae but that differ in the nature or the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of one another are termed "diastereoisomers", and stereoisomers that are non-superimposable mirror images are termed "enantiomers", or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is termed a "chiral center".
"Chiral isomer" means a compound with at least one chiral center. It has two enantiomeric forms of opposite chirality and may exist either as an individual enantiomer or as a mixture of enantiomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a "racemic mixture". A compound that has more than one chiral center has 2""1 enantiomeric pairs, where n is the number of chiral centers. Compounds with more than one chiral center may exist as either an individual diastereomer or as a mixture of diastereomers, termed a "diastereomeric mixture". When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et al, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem. Educ. 1964, 41, 116).
"Geometric Isomers" means the diastereomers that owe their existence to hindered rotation about double bonds. These configurations are differentiated in their names by the prefixes cis and trans, or Z and E, which indicate that the groups are on the same or opposite side of the double bond in the molecule according to the Cahn-Ingold-Prelog rules.
Further, the structures and other compounds discussed in this application include all atropic isomers thereof. "Atropic isomers" are a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropic isomers owe their existence to a restricted rotation caused by hindrance of rotation of large groups about a central bond. Such atropic isomers typically exist as a mixture, however as a result of recent advances in chromatography techniques, it has been possible to separate mixtures of two atropic isomers in select cases. The term "subject" as used herein is intended to include a living organism in which alleviation of symptoms or inhibition of a neurological disorder is sought. Preferred subjects are mammals. Examples of subjects include but are not limited to, humans, monkeys, dogs, cats, mice, rats, cows, horses, pigs, goats and sheep.
The terms "neurodegenerative disorder" or a "neurological disorder*' as used herein refers to a disorder which causes morphological and/or functional abnormality of a neural cell or a population of neural cells. The neurodegenerative disorder can result in an impairment or absence of a normal neurological function or presence of an abnormal neurological function in a subject. For example, neurodegenerative disorders can be the result of disease, injury, and/or aging. Non-limiting examples of morphological and functional abnormalities include physical deterioration and/or death of neural cells, abnormal growth patterns of neural cells, abnormalities in the physical connection between neural cells, under- or over production of a substance or substances, e.g., a neurotransmitter, by neural cells, failure of neural cells to produce a substance or substances which it normally produces, production of substances, e.g., neurotransmitters, and/or transmission of electrical impulses in abnormal patterns or at abnormal times. Neurodegeneration can occur in any area of the brain of a subject and is seen with many disorders including, for example, epilepsy, head trauma, stroke, ALS, multiple sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease. The terms "neurodegenerative disorder" and "neurological disorder" are intended to cover memory disorders.
The term "memory disorder," as used herein, refers to a diminished mental registration, retention or recall of past experiences, knowledge, ideas, sensations, thoughts or impressions. Memory disorder may affect short and/or long-term information retention, facility with spatial relationships, memory (rehearsal) strategies, and verbal retrieval and production. The term memory disorder is intended to include dementia, slow learning and the inability to concentrate. Common causes of a memory disorder are age, severe head trauma, brain anoxia or ischemia, alcoholic-nutritional diseases, drug intoxications, and neurodegenerative diseases. For example, a memory disorder is a common feature of neurodegenerative diseases, such as epilepsy or Alzheimer's disease (i.e. Alzheimer-type dementia). Memory disorders also occur with other kinds of dementia such as AIDS Dementia; Wernicke-Korsakoff s related dementia (alcohol induced dementia); age related dementia, multi-infarct dementia, a senile dementia caused by cerebrovascular deficiency, and the Lewy-body variant of Alzheimer's disease with or without association with Parkinson's disease. Creutzfeldt- Jakob disease, a spongiform encephalopathy caused by the prion protein, is a rare dementia with which memory disorder is associated. Loss of memory is also a common feature of brain-damaged patients. Non-limiting examples of causes of brain damage which may result in a memory disorder include stroke, seizure, an anaesthetic accident, epilepsy, ischemia, anoxia, hypoxia, cerebral edema, arteriosclerosis, hematoma or epilepsy; spinal cord cell loss; and peripheral neuropathy, head trauma, hypoglycemia, carbon monoxide poisoning, lithium intoxication, vitamin (Bl, thiamine and B 12) deficiency, or excessive alcohol use. Korsakoff s amnesic psychosis is a rare disorder characterized by profound memory loss and confabulation, whereby the patient invents stories to conceal his or her memory loss. It is frequently associated with excessive alcohol intake. Memory disorder may furthermore be age-associated; the ability to recall information such as names, places and words seems to decrease with increasing age. Transient memory loss may also occur in patients, suffering from a major depressive disorder, after electro-convulsive therapy.
The term "seizure" as used herein refers to a change in behavior, or spasms or convulsions that arise naturally in a subject as a result of a natural chemical imbalance or lack of homeostasis in a subject. Such natural convulsions may arise due to a disease or disorder (e.g., epilepsy), age, or the occurrence of an event (e.g., stroke). The term "seizure" also refers to seizures that are chemically induced, for example those brought on by intake, uptake, or ingestion of chemicals such as organophosphates.
The term "treatment" or "treating" refers to a decrease in the symptoms associated with the disorder or an amelioration of the recurrence of the symptoms of the disorder, prophylaxis, or reversal of a disease or disorder, or at least one discernible symptom thereof. The term "treatment" or "treating" refers to inhibiting or slowing the progression of a disease or disorder, e.g., epilepsy, physically, e.g., stabilization of a discernible symptom, such as seizures. The term "treatment" or "treating" refers to delaying the onset of a disease or disorder, e.g., seizures. The term "prevention" or "preventing" refers to delaying the onset of the symptoms of the disorder. In one embodiment, at least one natural product compound is administered as a preventative measure to a subject having a genetic or non-genetic predisposition to a neurological disorder such as epilepsy. In another embodiment, at least one natural product compound is administered as a preventative measure for a subject at risk of developing seizures as a result of another medical event. For example, patients who have a suffered a stroke are often at risk of developing seizures. In these instances, at least one natural product compound can be administered after the stroke as a preventative measure against seizures. Isolated Compounds and Analogs of Natural Products The invention provides methods and compositions for treating or preventing neurological disorders using a compound that is isolated from a natural product. The invention includes analogs of compounds isolated from natural products where the compound is selected from chromen-one derivatives, carbohydrate-derivatized compounds, and alkaloid compounds.
Compounds purified from, or otherwise based upon, natural products such as botanical or herbal extracts are used to modulate neurological disorders. Purification or isolation of compounds from natural products is performed by methods known in the art, including those described below.
Substantially purified compounds are prepared from herbal extracts. For example, the dry powder of the herbal plant (3 kg) is extracted in 80% aqueous MeOH (5 L) at room temperature overnight. Then, the filtrates are evaporated in vacuum e.g., under 40 0C, and the concentrates are fractionated into three fractions such as n-EtOAc, n-BuOH, and aqueous. Each solvent extracts flow through the column chromatography (SiO2, ODS, Sephadex etc.) and are eluted with several mixed solvents. Several obtained fractions are repeatedly chromatographed while varying packing materials or eluting solutions. Purity of the isolated compounds is assessed by TLC or HPLC. The molecular structure and weight are determined by: 1) hypothesizing chemical and physical characteristics of compounds; 2) determining functional groups in the compound molecule through IR spectra; 3) reading molecular weight and molecular formula from Mass data; 4) identifying chemical environment and integration value of proton, and neighboring protons using 1H-NMR data; 5) measuring chemical environment and numbers of carbon from 13C-NMR data; 6) multiplicity of carbon by reading DEPT NMR data; 7) investigating the correlation between neighboring proton signals from 1H-1H 2D COSY NMR;
8) studying the correlation between carbon and neighboring proton signals using HSQC 2D NMR; 9) reading the long range correlations between carbon and proton signals from HMBC 2D NMR; 10) stereochemistry of chiral carbons from NOESY 2D NMR; 11) stereochemistry of molecular structure by reading Polaimeter and CD; and 12) adaptation of molecular modification as needed (eg, acetylation, methylation, methyl esterification, acid hydrolysis, alkaline hydrolysis). Alkaloid Compounds
Rhvnchophylline and Isorhynchophylline Rhynchophylline and isorhynchophylline are indole alkaloids that are isolated from gou teng (Ramulus Uncariae cum Uncis). Botanical names for gou teng include Ourouparia rhynchophylla Matsum, Uncaria rlynchophylla (Miq.) Jacks, Uncaria macrophylla Wall, Uncaria hirsute Haviland, Uncaria sinensis Haviland, Uncaria sessilifructus Roxb, and Uncaria scandens (Smith) Hutch. For example, Rhynchophylline and isorhynchophylline are isolated from the hook of Uncaria rhynchophylla, Uncaria sinensis Haviland Uncaria, macrophylla Wallich (Rubiaceae) (Chung Kuo Yao Li Hsueh Pao 1986;7(5)426). Isorhynchophylline is isolated from M. rubrostipulata (Seaton, J. C, Tondeur, R. and Marion, L. Canad. J. Chem., 36, 1031 (1958)). Uncaria rhynchophyllina has about 0.2 % alkaloid content, in which rhynchophylline (Rhy) is 28%-50 %, isorhynchophylline (Isorhy) is 15 %. Other trace components include hirsutine, hirsuteine, corynantheine, dihydrocorynantheine, isocorynoxeine, akuammigine, geissoschijine, and methylethe (Drug Dicimasia Institute of Tianjing. Extract and pharmacological research of effective components in Uncar ia rhynchophylla. Chin Herb Med 1974; 4: 212-7; Phillipson JD, Hemingway SR. Chromatographic and spectroscopic methods for the identification of alkaloids from herbarium samples of the genus Uncaria. J Chromatogr 1975; 105: 163-78). The molecular structures of rhynchophylline and isorhynchophylline are below:
Figure imgf000015_0001
Rhynchophylhne
Figure imgf000015_0002
The invention includes compounds that are derivatives of alkaloids. In one aspect of the invention, the compound is a derivative of rhynchophylline and isorhynchophylline. Compounds of the invention include compounds of Formula I:
Figure imgf000015_0003
(I) where,
Ri, R2, R3, R4, R5, and R6 are selected from hydrogen, alkyl, alkenyl, alkoxy, carboxyalkyl, acylamino, and halogen. R7, R8, R9, and Ri0 are selected from hydrogen, hydroxyl, alkoxy, substituted alkoxy, alkyl, alkenyl, CF3, amino, carboxy, and halogen.
One of Ri or R2 is alkyl and the other is hydrogen. One OfR1 or R2 is methyl and the other is hydrogen. One of Ri or R2 is ethyl and the other is hydrogen. R5 is carboxyalkyl. R3 is alkoxy. R3 is methoxy. R4 is hydrogen. R7, R8, R9, and Rio are each hydrogen Alkaloids isolated from Corydalis tuber Four classes of alkaloids (shown below) are isolated from Corydalis tuber (Rhizoma
Corydalis, Yan Hu Suo) a root found in the Zhejiang province of China. The genus of Corydalis consists of about 300 species of rhizomatous or tuberous plants, which are native to Asian and European woodlands. Currently, only about 25 species are commonly known. The tubers contain approximately 6% of alkaloids, which are often bound to malic or fumaric acid. Four groups of alkaloids have been identified: (1) aporphine type (e.g., bulbocapnine, corydine, magnoflorine and jatrorrhicine iodide); (2) protoberberine type I (e.g., berberine, coptisine, corysamine, palmatine) (3) protoberberine type II (e.g., canadine, corybulbine, corydaline, corypalmine, scoulerine, stylopine, tetrahydropalmatine); and (4) protopine-type (e.g., (protopine, allocryptopine, corycavamine).
Figure imgf000016_0001
1) Aporphine type
Figure imgf000016_0002
Protoberberine type I
Figure imgf000016_0003
3)Protoberberine type II
Figure imgf000016_0004
Protopine-type
Analogs and derivatives of aporphine type, protoberberine type I, protoberberine type II, and protopine-type alkaloids are useful for the treatment of neurological disorders as described herein. One of skill in the art can readily identify analogs and derivatives of the isolated alkaloid compounds suitable for use with the present invention by obtaining compounds with at least one of the core structures shown below and testing those compounds for neuroprotective activity as set forth in the examples below. R in the formulae above depicts the various side chains. In one embodiment, the side chains are selected from the group consisting of H and CH3. The molecular structures of CTEB-3-5, CTEB-11-6, CTB-7, and CTEB-7-5 are shown below:
Figure imgf000017_0001
Figure imgf000017_0002
The invention also includes derivatives of alkaloids isolated from Corydalis tuber, including multi-cyclic ring compounds e.g., tri- and tetra-cyclic compounds. Compounds of the invention include compounds of Formula II:
Figure imgf000017_0003
(II), where Ri, R2, R3, and R4 are selected from alkyl, alkenyl, hydrogen, carbonylalkyl or Ri and R2 together form a 5- or 6-membered ring. The 5- or 6-membered ring is substituted or unsubstituted. The 5-membered heterocycle ring is dioxole. The 6-membered heterocycle ring is dioxine or dihydrodioxine. At least one of Ri, R2, R3, or R4 is alkyl. At least two of R], R2, R3, or R4 is alkyl. At least three of Ri, R2, R3, or R4 is alkyl. R1 and R4 are methyl. X is alkyl or halogen. X is methyl. X is ethyl.
Compounds of the invention also include compounds of Formula III:
Figure imgf000018_0001
(III) where, Y is alkyl, halogen, or absent,
Ri, R2, R3, and Rio are selected from hydrogen or alkyl, or together R1 and R2 form a carbonyl, or one of Ri or R2 and Y form a covalent bond, or one of Rj or R2 and R3 form a covalent bond, or Y and R8 form a covalent bond.
R4, R5, R6, R7, R8, and R9 are selected from alkyl, alkenyl, hydrogen, carbonylalkyl, or together R4 and R5 form a 5- or 6-membered ring, or together R4 and R9 form a 5- or 6- membered ring, or together R6 and R7 form a 5- or 6-membered ring, or together R7 and R8 form a 5- or 6-membered ring. The 5- or 6-membered ring is substituted or unsubstituted. The 5- membered heterocycle ring is dioxole. The 6-membered heterocycle ring is dioxine or dihydrodioxine. At least one of is R4, R5, R6, R7, R8, or R9 alkyl. At least two OfR4, R5, R6, R7, R8, or R9 is alkyl. At least three OfR4, R5, R6, R7, R8, or R9 is alkyl. R4 and R5 form a 5- membered ring. R7 and R8 form a 5-membered ring. The 5-membered ring is dioxine. R4 and R5 are methyl. R7 is methyl. R6 and R7 are methyl. Y is methyl. Y is halogen, where halogen is Cl. Y is absent. Together Ri and R2 form a carbonyl.
Carbohydrate-derivatized Compounds Gastrodin Gastrodin (Ci3Hi8O7, molecular weight 286, soluble in MeOH, DMSO, EtOH, H2O) is isolated from the rhizome of Gastrodia elata. A preferred method describing its isolation is described. The dried powder (5 kg) of Gastrodia elata was extracted with 80% aqueous MeOH (4L x 2) at room temperature overnight and filtered through N02 Whatmann filter paper. The filtrate was evaporated under reduced pressure to give rise to MeOH extracts (187 g). The 15O g of the extracts was poured into H2O (1600 mL) and extracted with EtOAc (1500 mL x 2) and n- BuOH (1000 ml x 2), successively. Each solution was evaporated under vacuum to afford EtOAc (32 g), ft-BuOH (86 g) and aqueous (32 g) fractions, respectively. The n-BuOH fraction (30 g) was applied over silica gel (200 g) column eluting with CHCl3-MeOH- H2O (65:35:10 (lower phase) -> 6:4: 1) to afford 8 fractions monitored by TLC. The fourth fraction was chromatographed on the silica gel column (70 g, CHCl3-Me0H=3: 1) to yield pure gastrodin (232 mg). The molecular structure of gastrodin is below:
Figure imgf000019_0001
Peoniflorin
Peoniflorin (C23H28On, molecular weight 480, soluble in MeOH, DMSO, EtOH, H2O) is isolated from the root of Paeonia lactiflora. Methods of purification of paeoniflorin (5b-
((benzoyloxy)methyl)tetrahydro-5-hydroxy-2-methyl-2,5-methano-lH-3,4-dioxacyclobuta(cd) pentalen-la(2H)-yl-beta-D-glucopyranoside) are known in the art, (see for example, J Chromatogr A. 2004 Jun 25;1040(2):205-8). A preferred method of isolation is described. The dried powder (5 kg) of Paeonia lactiflora was extracted with 80% aqueous MeOH (1OL x 2) at room temperature overnight and filtered through NO2 Whatmann filter paper. The filtrate was evaporated under reduced pressure to give rise to MeOH extracts. The extracts were poured into H2O (3000 mL) and extracted with EtOAc (3000 mL x 2) and Λ-BUOH (2500 ml x 2), successively. Each solution was evaporated under vacuum to afford EtOAc (52 g), o-BuOH (140 g) and aqueous (1250 g) fractions, respectively. The «-BuOH fraction (60 g) was applied over silica gel (300 g) column eluting with CHCl3-MeOH- H2O (10:1 -=> 7:3:1 -» 65:35:10 -» 6:4:1 -> MeOH) to afford 6 fractions monitored by TLC. The second fraction (14 g) was chromatographed on a silica gel column (150 g, CHCl3-MeOH- H2O= 7:3:1) to yield pure paeoniflorin (2.5 g). The molecular structure of paeoniflorin is below:
Figure imgf000019_0002
Albiflorin
Albiflorin (9-((benzoyloxy)methyl)- 1 -(beta-D-glucopyranosyloxy)-4- hydroxy)-6- methyl-7-oxatricyclononan-8-one) is a water-soluble compound that is isolated from the root of Paeonia lactifloria and Paeonia sinjiangensis. (See, for example, Song, Z.H., Zhongguo Zhong Yao Za ZhL 2004 Aug;29(8):748-51; S. Shibata, Chem. Pharm. Bull. 11 (1963) 372; K Yamasaki, Tetrahedon Lett. AA (1976) 3965). The molecular structures of albiflorin is below:
Figure imgf000020_0001
Albiflorin
Pentagalloylglucose (beta-Penta-O-galloyl-glucose)
Pentagalloylglucose (C41H32O26, molecular weight 940.68) is a polyphenol that is isolated from leaves of Euphorbia hirta L. (Chen, L. Zhongguo Zhong Yao Za Zhi. 1991 Jan;16(l):38-9, 64) and from Peonia lactiflora (peony) root. The molecular structure of pentagalloylglucose is shown below:
Figure imgf000020_0002
Pentagalloylglucose
The invention also includes analogs of compounds derivatized with one or more carbohydrates. The carbohydrate is α glycoside i.e., the anomeric carbon is blocked by acetal formation e.g., methyl α-D-glucopyranoside
Figure imgf000021_0001
is a glycoside, wherein the anomeric carbon is blocked or derivatized with a methoxy group (OCH3). The glycoside includes a sugar that is naturally occurring. The glycoside includes a sugar that is non-naturally occurring. The glycoside includes the sugar glucose. The anomeric carbon of the carbohydrate is derivatized with aryl or substituted aryl. For example, the aryl group is phenyl. The aryl group is substituted with hydroxyalkyl. The aryl group is substituted with -CH2OH. The anomeric carbon of the carbohydrate is derivatized with a compound containing a bicyclic ring system. Bicyclic ring systems are characterized by two carbon atoms, the bridgehead carbons, being shared by two rings.
Figure imgf000021_0002
Bridgehead carbon Bicyclic ring systems include compounds which contain the chemical structures of e.g., cubane, tetrahedrane, and dodecahedrane. Examples of bicyclic structures include 5b-((benzoyloxy)methyl)tetrahydro-5-hydroxy-2-methyl-2,5,-methano- 1 H- 3 ,4-dioxacyclobuta(cd)pentalen- 1 a(2H)-yl-beta-D-glycopyranoside and 9-((benzoyloxy)methyl)- 4-hydroxy)-6-methyl-7-oxatricyclononan-8-one. Bicyclic ring systems can be optionally substituted with one or more substituents selected from e.g., alkyl, cycloalkyl, cycloalkenyl, aryl, heterocycle, alkoxyaryl, alkaryl, alkoxy, halogen, hydroxy, amine, cyano, nitro, alkylthio, phenoxy, ether, trifluoromethyl and the like.
The anomeric carbon of the carbohydrate includes an acyl or substituted acyl group. "Acyl" includes compounds and moieties that contain the acyl radical (CH3CO-) or a carbonyl group. "Substituted acyl" includes acyl groups where one or more of the hydrogen atoms are replaced by for example, alkyl, alkynyl, halogen, or hydroxyl. The carbohydrate includes a benzoyl or substituted benzoyl group. The carbohydrate includes a benzoyl or substituted benzoyl group (i.e., hydroxyl groups on the carbohydrate in addition to the anomeric carbon are derivatized). "Benzoyl" includes compounds and moieties that contain the benzoyl radical (PhCO-). "Substituted benzoyl" includes benzoyl groups where the phenyl group is substituted. The benzoyl group is substituted with one or more hydroxyl groups. The benzoyl group is substituted with one hydroxyl group. The benzoyl group is substituted with two hydroxyl groups. The benzoyl group is substituted with three hydroxyl groups. Chromen-one Compounds
Imperatorin and Oxypeucedaninhydrate
Imperatorin (Ci6Hi4O4, molecular weight 270, soluble in CHCl3, EtOH, MeOH, DMSO) and oxypeucedaninhydrate (Ci6Hi6O6, molecular weight 304, soluble in EtOH, MeOH, DMSO) are isolated from the root of Angelica dahurica. One preferred method of isolation is described. Coarsely powdered plant material (lkg) was extracted with 80% aqueous MeOH (4Lx2) at room temperature overnight. The extract were evaporated under reduced pressure and partitioned between EtOAc (700 mL x 2) and H2O (500 mL). The EtOAc extracts (36 g) obtained by removal of the solvent were chromatographed on a silica gel column. Gradient elution with n- hexane-EtOAc with increasing proportions of the EtOAc gave eleven subfractions (ADE-I - ADE-11). Silica gel column chromatography of subfraction ADE-5 (1.89g) eluting with n- hexane-CHCl3-Et0H (20:20:1) afforded isoimperatorin (628 mg). ADE-11 was applied on silica gel column eluting with n-hexane-EtO Ac-acetone (4:6:1) to give oxypeucedanin hydrate (517 mg). Molecular structures of imperatorin and oxypeucedaninhydrate hydrate are below:
'- f -.
'. Imperatorin f cXJ. °X ° Oxypeucedaninhydrate hydrate
Decursin and Decursinol
Decursin (Ci9H20O5, molecular weight 328, soluble in CHCl3, DMSO, EtOH) and decursinol (C14H14O4, molecular weight 246, soluble in CHCl3, DMSO, EtOH, MeOH) are isolated from the root of Angelica gigas. A preferred method of isolation is described. The dried underground part (2 kg) of Angelica gigas was extracted with MeOH and upon removal of solvent in vacuo, the MeOH extract yielded 15O g of residue. This was then suspended in H2O (500 ml) and partitioned successively with CH2Cl2 (500 ml x 2). Silica gel column chromatography of the CH2Cl2 fraction (90 g) with a mixture of n-hexane-CHCl3-Me0H as eluent afforded seven fractions (F1-F7). A part of F2 (4 g in 78 g) was subjected to silica gel column chromatography with π-hexane-EtOAc-(5:l) mixture and yielded decurin (2.4 g). F5 fraction (3.1 g) was subjected to silica gel column chromatography with n-hexane-EtOAc-(l:l) mixture and yielded decursinol (l.lg). Molecular structures of decursin and decursinol are below:
Figure imgf000023_0001
Decursin Decursinol
The compounds of the present invention act through different receptors, or bind to different regions of the same receptor. In one aspect, the invention provides combinations of the compounds that act synergistically to improve therapeutic effects. The combination can include two or more of the compounds of the present invention. The combination of compounds also means that lower dosages of the individual compounds are used to achieve the same therapeutic effect as each compound used alone. This is important when a desired therapeutic effect occurs at a high dose of a natural product compound, but which also leads to adverse side effects. In such an instance, a lower dosage is combined with a dosage of a synergistic compound to provide the desired therapeutic effect, but without the adverse side effects. Hyperin Hyperin (C2iH20O12 , molecular weight 464.38, 2-(3,4-dihydroxyphenyl)-3-(β-D- galactopyranosyloxy)-5,7-dihydroxy-4H-l-benzopyran-4-one) is isolated from gou teng
(Ramulus Uncariae cum Uncis). For example, hyperin can be isolated from the hook of Uncaria rhynchophylla, Uncaria sinensis, as well as from other plants, such as Acanthopanax chiisanensis roots and Drosera burmannii Vahl, Taraxacum mongolicum Hand.-Mazz, Canarium album and Euphorbia nematocypha, and Onobrychis angustifolia (Lee, S. Arch Pharm Res. 2004 Jun;27(6):628-32, Wang, O. Zhong Yao Cai. 1998 Aug;21(8):401-3, Ling, Y. Zhongguo Zhong Yao Za ZhI 1999 Apr;24(4):225-6, 256, Ito, M. Chem Pharm Bull (Tokyo). 1990 Aug;38(8):2201-3). The term hyperin is intended to include isoforms, derivatives and functional analogs of hyperin. The term hyperin is intended to cover all synonyms including, but not limited to, hyperoside, hyperasid, hyperozide, quercetin 3-0-beta-D-galactopyranosid, Quercetin 3-beta-D-galactopyranoside, Quercetin-3-O-galactoside, Quercetin-3-galactoside and isoforms thereof. In some embodiment, hyperin can be isolated from Uncaria sinensis. One method of isolation is described below.
Uncaria sinensis is extracted with boiling water three times. Each extract is combined and lyophilized to give a brown mass. The water extract is chromatographed on polus polymer gel (Diaion HP-20, 2 L), eluted with water, [H2]O-methanol (1:1) and then with methanol. The [H2]O -methanol (1:1) eluate is purified by Sephadex LH-20 column chromatography, and eluted with [H2]O -ethanol (1 : 1) to obtain hyperin. The molecular structure of hyperin is shown below:
Figure imgf000024_0001
Hyperin
Baicalin
Baicalin, 7-D-glucuronic acid-5,6-dihydroxyflavone, (C2IH18On, beta-D- Glucopyranosiduronic acid, 5,6-dihydroxy-4-oxo-2-phenyl-4H-l-benzopyran-7-yl) is isolated from plants, such as Scutellariae baicalensis, Scutellariae Radix, Shuang~Huang-Lian% and Scutellaria rehderiana (Wu, S. J Chromatogr A. 2005 Feb 25;1066(l-2):243-7, Zhang, L. J Ethnopharmacol. 2006 Jan 3;103(l):120-5. Epub 2005 Sep 12, Feng, N. Chem Pharm Bull (Tokyo). 2005 Aug;53(8):978-83, Su, Y. Zhongguo Zhong Yao Za ZhL 2004 Sep;29(9):863-4). The term baicalin is intended to include isoforms, derivatives and functional analogs of baicalin. A preferred method of isolation is described. Baicalin can be separated with a one-step separation from crude sample from Scutellaria baicalensis, Georgi using ethyl acetate-methanol- 1% acetic acid water (5:0.5:5, v/v) as the two-phase solvent system. The upper phase of ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) can be used as the stationary phase of highspeed counter-current chromatography (HSCCC). The molecular structure of baicalin is shown below:
Figure imgf000025_0001
Baicalin
Baicalein
Baicalein, 5,6,7-trihydroxy-2-phenyl-4H-l-benzopyran-4-one (C15H10O5, molecular weight 270.24) is isolated from the roots of Scutellaria baicalensis (Bargellini, Gazz. Chim. Ital. 49, II, 47 (1919); Shibata et al. Acta Phytochim. 1, 109 (1923)). The term baicalein is intended to include isoforms, derivatives, and functional analogs of baicalein. Baicalein is soluable in alcohol, methanol, ether, acetone, ethyl acetate, and hot glacial acetic acid. The molecular structure of baicalein is shown below:
Figure imgf000025_0002
Baicalein
Baicalein isolated using known procedures, e.g., that described in Zhang et al., J. Chromatogr. A., 2005, 1129(2):304-7.
Wogonin
Wogonin, 5,7-dihydroxy-8-methoxyflavone (Ci6Hi2Os, molecular weight 284.27, 4H-1- Benzopyran-4-one, 5,7-dihydroxy-8-methoxy-2-phenyl-) is isolated from plants such as Scutellariae baicalensis, Scutellariae Radix, Shuang-Huang-Lian, and Scutellaria rehderiana (Wu, S. J Chromatogr A. 2005 Feb 25;1066(l-2):243-7). The term wogonin is intended to include isoforms, derivatives and functional analogs of wogonin. One method to isolate wogonin is described.
Wogonin is separated with a one-step separation from a crude sample from Scutellaria baicalensis, Georgi using ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) as the two- phase solvent system. The upper phase of ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) can be used as the stationary phase of high-speed counter-current chromatography (HSCCC). The molecular structure of wogonin is shown below:
Figure imgf000026_0001
OH 0 Wogonin
Wogonoside
Wogonoside (Wu et al, 2005, J. Chromatogr. A., 1055(l-2):243-247), a flavonoid from the plant Radix Scutellariae, is isolated according to established methods, e.g., Tang et al., 2004, Phytomedicine 11(4):277-284. The molecular structure of wogonoside is shown below:
Figure imgf000026_0002
Ganhuangenin Ganhuangenin (Liu et al, 1984, Yao Sue Sue Bao 19(11):830-835), a flavonoid from the root of Scutellaria gehderiana, is isolated as described by Su et al., 2004, Zhongguo Zhong Yao Za Zhi 29(9): 863-864). The molecular structure of ganhuangenin is shown below:
Figure imgf000026_0003
Ganhuangenin
Oroxyloside
Oroxyloside (oroxylin A-glucuronide), (Ci6Hi2O5, 4H-l-benzopyran-4-one, 5,7- dihydroxy-6-methoxy-2-phenyl-) is isolated from plants such as Scutellariae baicalensis, Scutellariae Radix, Shuang-Huang-Lian, and Scutellaria rehderiana (Wu, S. J Chromatogr A. 2005 Feb 25;1066(l-2):243-7, Li, H. J Chromatogr A. 2005 May 13;1074(l-2): 107-10). A method of isolating oroxyloside is described.
Oroxyloside is separated with a one-step separation from a crude sample from Scutellaria baicalensis, Georgi using high-speed counter-current chromatography (HSCCC) with n-hexane- ethyl acetate-n-butanol-water (1:1:8:10, v/v/v/v) as the two-phase solvent system and increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml min(-l) after 4 h. The purified compounds can be analyzed by high-performance liquid chromatography. The upper phase of ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) is used as the stationary phase of highspeed counter-current chromatography (HSCCC). Alternative solvent systems or protocols are used. In addition, the term oroxyloside is intended to include isoforms, derivatives and functional analogs of oroxyloside. For non- limiting examples of derivatives of oroxyloside, see Suresh, B., BioorgMed Chem Lett. 2005 Sep l;15(17):3953-6. The molecular structure of oroxyloside is shown below:
Figure imgf000027_0001
The invention includes compounds that are chromen-one derivatives. The compound is a derivative of 2H-chromen-2-one. Compounds of the invention include compounds of Formula IV:
Figure imgf000027_0002
(IV) where: Ri and R2 are hydrogen, unsubstituted aryl, substituted aryl, heterocycle, substituted heterocycle, carbohydrate, alkyl, alkenyl, halogen, or hydroxyl. Aryl or heterocycle are substituted with hydrogen, hydroxyl, alkoxy, substituted alkoxy, alkyl, alkenyl, CF3, amino, carboxy, or halogen. Aryl or heterocycle are mono-, di-, or tri-substituted. The term "carbohydrate" refers to a monosaccharide, disaccharide, trisaccharide, oligomeric, and polymeric form of a sugar. A sugar is a naturally occurring sugar or a non-naturally occurring sugar. The compound is substituted with one or more monosaccharides. The monosaccharide is the sugar galactose. The monosaccharide is the sugar glucose.
R3, R4, R5, and R6 are hydrogen, hydroxy, alkoxy, alkyl, alkenyl, CF3, amino, carboxy, halogen or together R3 and R4 or R4 and R5 or R5 and R6 form a 5- or 6-membered ring. The 5- or 6-membered ring is unsubstituted or substituted.
R] is hydrogen. R2 is hydrogen. Rj and R2 are both hydrogen. R4 and R5 form a 5- membered ring, where the ring is a heterocycle or cycloalkyl. The 5-membered heterocycle ring is selected from tetrahydrofuran, furan, pyrrolidine, pyrrole, tetrahydrothiophene, thiophene, and dioxole. The 5-membered heterocycle is furan. The 5-membered cycloalkyl ring is cyclopentane.
R4 and R5 form a 6-membered ring, where the ring is a heterocycle or cycloalkyl. The 6- membered heterocycle ring is selected from piperazine, piperidine, tetrahydrothiopyran, thiomorpholine, or tetrahydropyran. The 6-membered heterocycle ring is tetrahydrofuran. The heterocycle ring is substituted. The heterocycle ring is mono, di-, or tri-substituted. The heterocycle ring is substituted with alkyl, e.g., methyl, hydroxyl, alkoxy, alkoxycarbonyl, or alkenoxycarbonyl. The 6-membered cycloalkyl ring is cyclohexane.
R3 is alkoxy. R3 is -OCH2CH=CH(CH3)2. R6 is alkoxy. R6 Is OCH2CH(OH)C(CH3)2OH.
The invention includes compounds that are chromen-one derivatives. The compound is a derivative of 4H-chromen-4-one. Compounds of the invention include compounds of Formula V:
Figure imgf000028_0001
(V) where:
Ri and R2 are hydrogen, unsubstituted aryl, substituted aryl, heterocycle, substituted heterocycle, carbohydrate, alkyl, alkenyl, halogen, or hydroxyl. Aryl or heterocycle are substituted with hydrogen, hydroxyl, alkoxy, substituted alkoxy, alkyl, alkenyl, CF3, amino, carboxy, or halogen. Aryl or heteroaryl are mono, di-, or tri-substituted. The carbohydrate is a monosaccharide. The monosaccharide is the sugar glucose. The monosaccharide is the sugar galactose.
R3, R4, R5, and R6 are hydrogen, hydroxy, alkoxy, alkyl, alkenyl, CF3, amino, carboxy, halogen or together R3 and R4 or R4 and R5 or R5 and R6 form a 5- or 6-membered ring. The 5- or 6-membered ring is unsubstituted or substituted. Ri is carbohydrate or hydrogen. R2 is substituted or unsubstituted aryl. Aryl is mono- or di-substituted. Aryl is substituted with hydroxyl. At least one of R3, R4, or R5 is not hydrogen. At least two of R3, R4, or R5 is not hydrogen. At least three of R3, R4, or R5 is not hydrogen. R3, R4, and R5 are selected from hydrogen, hydroxyl, carbohydrate, and alkoxy. R3 and R5 are methoxy. R5 is carbohydrate. R5 is monosaccharide. R5 is glucopyranosiduronic acid. R3 and R5 are each hydroxyl. R3 and R4 are each hydroxyl. R4 is alkoxy. R6 is alkoxy.
R4 and R5 form a 5-membered ring, where the ring is a heterocycle or cycloalkyl. The 5- membered heterocycle ring is selected from tetrahydrofuran, furan, pyrrolidine, pyrrole, tetrahydrothiophene, thiophene, and dioxole. The 5-membered heterocycle is furan. The 5- membered cycloalkyl ring is cyclopentane. R4 and R5 form a 6-membered ring, where the ring is a heterocycle or cycloalkyl. The 6- membered heterocycle ring is selected from piperazine, piperidine, tetrahydrothiopyran, thiomorpholine, or tetrahydropyran. The 6-membered heterocycle ring is tetrahydrofuran. The heterocycle ring is substituted. The heterocycle ring is mono, di-, or tri-substiruted. The heterocycle ring is substituted with alkyl, e.g., methyl, hydroxyl, alkoxy, alkoxycarbonyl, or alkenoxycarbonyl. The 6-membered cycloalkyl ring is cyclohexane. Vanillin
Vanillin (CsH8O3, molecular weight 152) (4-hydroxy-3-methoxybenzaldehyde), is an aromatic compound that can be isolated from vanilla beans and a variety of plants, such as Buddlejapurdomii (Gao, Y. Zhong Yao Cai. 2004 May;27(5):339-41.). Vanillin occurs widely in plants in nature, usually as a glycoside bound to sugar or as a percusor to vanillin bound to the large lignin molecule abundant in wood. While a majority of vanillin is produced from guaiacol, it can also be made from lignin, a by-product from the wood-pulp industry and synthetically. Isoeugenol can be converted into vanillin from crude enzyme extracted from soybean (Li, YH, Appl Biochem Biotechnol. 2005 Apr;125(l):l-10). Vanillin can also be obtained by treatment of spruce wood with alkali in the presence of nitrobenzene (J. Am. Chem. Soc. 63, 312 (1941)). Vanillin is a white crystalline material. Vanillin has a characteristic pleasant smell and taste of vanilla which accounts for its widespread use. Vanillin is soluble in water and its solubility increases with increasing temperature. Vanillin is converted to L-dopa and related compounds. In addition, vanillin is the starting material for chemical synthesis of Aldomet. The molecular structure of vanillin is shown below:
Figure imgf000030_0001
Vanillin
Neurological Disorders The purified compounds and functional analogs thereof that are described herein are useful for the therapeutic and prophylactic treatment of neurological disorders. Neurological disorders are associated with neuronal loss or dysfunction, including, but not limited to, Parkinson's Disease, Alzheimer's Disease, Huntington's Disease, ALS, stroke, epilepsy, ADD, and neuropsychiatric syndromes. In one embodiment, the neurological disorder is a neurodegenerative disorder. Desirably, the neurological disorder is a seizure-associated disorder such as a stroke, brain ischemic condition, and epilepsy.
Compounds of the present invention are administered therapeutically (including prophylactically): (1) in diseases, disorders, or conditions involving neuronal loss or dysfunction, including, but not limited to, Parkinson's Disease, Alzheimer's Disease, Huntington's Disease, ALS, stroke, ADD, and neuropsychiatric syndromes; or (2) in diseases, disorders, or conditions wherein in vitro (or in vivo) assays indicate the utility of the compounds of the present invention.
Purified compounds and analogs are also useful for the therapeutic and prophylactic treatment of memory disorders or to improve learning and cognition. A memory disorder is associated with a diminished mental registration, retention or recall of past experiences, knowledge, ideas, sensations, thoughts or impressions. Memory disorders affect short and/or long-term information retention, facility with spatial relationships, memory (rehearsal) strategies, and verbal retrieval and production. Memory disorders include, for example, dementia, slow learning and the inability to concentrate. Common causes of a memory disorder are age, severe head trauma, brain anoxia or ischemia, alcoholic-nutritional diseases, drug intoxications and neurodegenerative diseases. For example, a memory disorder is a common feature of neurodegenerative diseases, such as Alzheimer's disease (i.e. Alzheimer-type dementia). Memory disorders also occur with other kinds of dementia such as AIDS Dementia; Wernicke- Korsakoff s related dementia (alcohol induced dementia); age related dementia, multi-infarct dementia, a senile dementia caused by cerebrovascular deficiency, and the Lewy-body variant of Alzheimer's disease with or without association with Parkinson's disease. Loss of memory is also a common feature of brain-damaged patients. Non-limiting examples of causes of brain damage which may result in a memory disorder include stroke, seizure, an anaesthetic accident, ischemia, anoxia, hypoxia, cerebral edema, arteriosclerosis, hematoma or epilepsy; spinal cord cell loss; and peripheral neuropathy, head trauma, hypoglycemia, carbon monoxide poisoning, lithium intoxication, vitamin (Bl, thiamine and B 12) deficiency, or excessive alcohol use. Glutamate Neurotransmitter System and NMDA Subtypes of Glutamate Receptors
The purified compounds or functional analogs thereof are antagonists of NMDA receptors and are therefore useful in the treatment of disorders associated with the NMDA receptor function, e.g., seizures, epilepsy, epileptogenesis, and other neurological disorders. Glutamate is recognized as the predominant excitatory neurotransmitter (messenger molecule) in the mammalian central nervous system (CNS); for a review, see the chapter by Olney entitled "Glutamate" in The Encyclopedia of Neuroscience, edited by Adelman (either the 1987 or the 1995 edition). Glutamate is involved in transmitting messages from one nerve cell (neuron) to another in many different circuits within the CNS, and therefore serves many important functions.
There are several different subtypes of receptors through which glutamate transmits messages. A particularly important receptor through which glutamate mediates a wide range of functions is the NMDA receptor. Other major classes of glutamate receptors are kainic acid receptors and Quis/AMPA receptors; these two classes are collectively referred to as non-NMDA receptors. Both NMDA and non-NMDA receptors are normally activated by glutamate. Antagonist drugs that block glutamate receptors, such as the NMDA receptor, are classified in two broad classes of compounds. One class is referred to as competitive NMDA antagonists; these agents bind at the NMDA/glutamate binding site (such drugs include CPP, DCPP-ene, CGP 40116, CGP 37849, CGS 19755, NPC 12626, NPC 17742, D-AP5, D-AP7, CGP 39551, CGP-43487, MDL-100,452, LY-274614, LY-233536, and LY233053). Another class is referred to as non-competitive NMDA antagonists; these agents bind at other sites in the NMDA receptor complex (such drugs include phencyclidine, dizocilpine, ketamine, tiletamine, CNS 1102, dextromethorphan, memantine, kynurenic acid, CNQX, DNQX, 6,7-DCQX, 6,7- DCHQC, R(+)-HA-966, 7-chloro-kynurenic acid, 5,7-DCKA, 5-iodo-7-chloro-kynurenic acid, MDL-28,469, MDL-100,748, MDL-29,951, L-689,560, L-687,414, ACPC, ACPCM, ACPCE, arcaine, diethylenetriamine, 1,10-diaminodecane, 1,12-diaminododecane, ifenprodil, and SL- 82.0715). The invention relates to the use of isolated compounds, or functional analogs thereof, as
NMDA receptor antagonists to ameliorate, reduce or treat the effects of a disorder associated with the NMDA receptor. Optionally, a combination of purified compounds and functional analogs are used to antagonize NMDA receptors. Seizures and Epilepsy , The invention provides methods for treating or preventing seizures as well as methods for delaying the onset of seizures (epileptogenesis). There are many different types of seizures. Seizures are typically divided into generalized seizures (absence, atonic, tonic-clonic, myoclonic) and partial (simple and complex) seizures. Generalized Seizures Generalized seizures are thought to affect both cerebral hemispheres (sides of the brain) from the beginning of the seizure. They produce loss of consciousness, either briefly or for a longer period of time, and are sub-categorized into several major types: generalized tonic clonic; myoclonic; absence; and atonic.
Absence seizures (also called petit mal seizures) are lapses of awareness, sometimes with staring, that begin and end abruptly, lasting only a few seconds. There is no warning and no after-effect. Some absence seizures are accompanied by brief myoclonic jerking of the eyelids or facial muscles, or by variable loss of muscle tone. More prolonged attacks may be accompanied by automatisms, which may lead them to be confused with complex partial seizures. However, complex partial seizures last longer, may be preceded by an aura, and are usually marked by some type of confusion following the seizure. Myoclonic seizures are rapid, brief contractions of bodily muscles, which usually occur at the same time on both sides of the body. Occasionally, they involve one arm or a foot. People usually think of them as sudden jerks or clumsiness. A variant of the experience, common to many people who do not have epilepsy, is the sudden jerk of a foot or limb during sleep. Atonic seizures produce an abrupt loss of muscle tone. Other names for this type of seizure include drop attacks, astatic or akinetic seizures. They produce head drops, loss of posture, or sudden collapse. Because they are so abrupt, without any warning, and because the people who experience them fall with force, atonic seizures can result in injuries, such as to the head and face. Generalized tonic clonic seizures (grand mal seizures) are the most common and best known type of generalized seizure. They begin with stiffening of the limbs (the tonic phase), followed by jerking of the limbs and face (the clonic phase). During the tonic phase, breathing may decrease or cease altogether, producing cyanosis (blueing) of the lips, nail beds, and face. Breathing typically returns during the clonic (jerking) phase, but it may be irregular. This clonic phase usually lasts less than a minute. Some people experience only the tonic, or stiffening phase of the seizure; others exhibit only the clonic or jerking movements; still others may have a tonic-clonic-tonic pattern. Partial Seizures In partial seizures the onset of the electrical disturbance is limited to a specific area of one cerebral hemisphere (side of the brain). Partial seizures are subdivided into simple partial seizures (in which consciousness is retained); and complex partial seizures (in which consciousness is impaired or lost). Partial seizures may spread to cause a generalized seizure, in which case the classification category is partial seizures secondarily generalized.
Partial seizures are the most common type of seizure experienced by people with epilepsy. Virtually any movement, sensory, or emotional symptom can occur as part of a partial seizure, including complex visual or auditory hallucinations. There are two types of partial seizure, simple partial seizures and complex partial seizures.
People who have simple partial seizures do not lose consciousness during the seizure. However, some people, although fully aware of what's going on, find they cannot speak or move until the seizure is over. Simple partial seizures include autonomic and mental symptoms and sensory symptoms such as olfaction, audition, or vision, sometimes concomitant with symptoms of experiences such as deja-vu and jamais-vu.
Complex partial seizures affect a larger area of the brain than simple partial seizures and they affect consciousness. During a complex partial seizure, a person cannot interact normally with other people, is not in control of his/her movements, speech, or actions. A person may even be able to speak, but the words are unlikely to make sense and he or she will not be able to respond to others in an appropriate way. Although complex partial seizures can affect any area of the brain, they often take place in one of the brain's two temporal lobes. Because of this, the condition is sometimes called "temporal lobe epilepsy." Epileptic Seizures
Epileptic seizures are the outward manifestation of excessive and/or hypersynchronous abnormal activity of neurons in the cerebral cortex. Many types of seizures occur, as described above. The neuromechanism responsible for seizures includes the amygdala, the hippocampus, the hypothalamus, the parolfactory cortex, the frontal and temporal lobes, and the involvement of the substantia nigra, a particular portion of the brain considered to be part of neural circuitry referred to as the basal ganglia (See e.g., Depaulis, et al. (1994) Prog. Neurobiology, 42: 33-52). The neural connections that make up. the basal ganglia are important in epilepsy. These connections are reviewed by Alexander et al. (Alexander, et al. Prog. Brain Res. 85: 119-146).
The substantia nigra receives input from the subthalamic nucleus (STN) which is excitatory and involves glutamate as the neurotransmitter conveying information at the synapse. A lesion of the subthalamic nucleus will reduce the inhibitory output of the internal segment of the globus pallidus and substantia nigra reticulata (SN) (Bergman, et al. (1990), Science, 249: 1436-1438).
The subthalamic nucleus receives input from the external segment of the globus pallidus (GPe).
This input is inhibitory using GABA as a transmitter substance. Hence, increased activity of the neurons in GPe will increase inhibition of neurons in the subthalamic nucleus which will reduce the excitation of neurons in the substantia nigra.
The methods and compositions of the invention are used to inhibit, reduce, or treat seizures that include, but are not limited to, tonic seizures, tonic-clonic seizures, atypical absence seizures, atonic seizures, myoclonic seizures, clonic seizures, simple partial seizures, complex partial seizures, and secondary generalized seizures. There are various in vivo models of seizures and epilepsy that can be used to test compounds against specific forms of seizures and epilepsy. For example, the kainate model is an epileptic model in which kainic acid, one of the excitatory amino acids found in the brain, is injected to nuclei (amygdala, hippocampus, etc.) in the limbic system in an microamount to induce focal epilepsy. The kainate model serves as a model for an epileptic seizure; more particularly, as a model for status epilepticus induced from the limbic system in an acute phase, and as a model for evolution of a spontaneous limbic seizure to a secondary generalized seizure in a chronic phase. The kainate model may also be used as a cortex epilepsy model through injection of kainic acid to the cortex (sensory motor field). For a review of animal models used for epilepsy and seizures, see for example, Sarkisian (2001) Epilepsy and Behavior, 2: 201τ216).
Other examples of seizures and epilepsy include, but are not limited to the Maximal Electroshock (MES) model, which was used to investigate the efficacy of compounds against grand mal seizures. Maximal seizures were induced by applying an electrical current to the brain of the animal via corneal electrodes. The electrical stimulus was applied at about 50mA in a pulse of 60Hz for 200ms. After the onset of seizures, the compound was administered and the inhibition of spasms recorded.
Another suitable model is the Subcutaneous Metrazole (ScMET) Model for epilepsy, and in particular was useful for investigating petite mal seizures. In this model, a metrazole dose of 85mg/kg was administered subcutaneously to the animal (e.g., mouse) to induce seizures. The compound was then be administered and the animals observed.
To investigate the neurotoxicity levels of the compounds, the Toxicity Model (TOX) using a rotorod was employed. The animals (e.g., mice) were trained to stand on an accelerating rotorod rotating at 10 rev min"1 with a diameter of 3.2cm. The trained animal was given the compound at various doses and the effect of compound on their motor skills, was determined. The dose at which the animals fell off the rotorod is the toxic dose.
Epileptogenesis
Epileptogenesis is the process by which a normal brain becomes chronically prone to seizures. Many brain insults (stroke, trauma, neurodegenerative disease etc) can induce epileptogenesis, yet no therapies exist to disrupt this process. Although reorganization of specific neuronal circuits and alterations in individual synapses are associated with epileptogenesis, the functional consequences and relative importance of these changes to epileptogenesis and seizure genesis are unknown, as are many of the molecular and cellular mechanisms underlying these alterations.
Seizures and epilepsy are common sequelae of acute brain insults such as stroke, traumatic brain injury, and central nervous system infections. Early, or acute symptomatic, seizures occur at the time of the brain insult and may be a marker of severity of injury. A cascade of morphologic and biologic changes in the injured area over months to years leads to hyperexcitability and epileptogenesis. After a variable latency period, late unprovoked seizures and epilepsy occur. The drugs that presently used in the treatment of epilepsy, treat the symptom, seizures, but do not modify the epileptogenic process. In one embodiment, purified compounds and functional analogs are administered during the latent period for the prevention of epileptogenesis and the development of unprovoked seizures and epilepsy. In this regards, substantially purified compounds and functional analogs are used as a neuroprotectant and an antiepileptogenic agent.
There are several recognized models that are used to test the effect neuroprotectant and an anti-epileptogenic of therapeutic agents, for example by using a kindling model (Wada, (1974) Epilepsia 19: 217-227; Sato et al, (1990) Epilepsy Research 5: 117-124); Silver et al, (1991) Ann. Neurol. 29: 356-363). Seizure kindling models are characterized by giving a sub- seizure eliciting electrical or chemical stimulus (i.e., sub-threshold) over a period of time (Goddard et al., (1969) Exp. Neurol. 25: 295-330). The majority of initially non-convulsive animals that are exposed to such stimuli over a number of days, eventually exhibit seizure activity to these stimuli, have a permanently lowered threshold, exhibit altered manifestations of normal behavior and, therefore, are considered "kindled." The kindling phenomenon has been proposed to underlie the development of disorders such as certain types of epilepsy syndromes. Several kindling models of seizure development have been characterized. For a review of animal models used for epilepsy and seizures, see for example, Sarkisian (2001) Epilepsy and Behavior, 2: 201-216).
Purified compounds or functional analogs that delay or block acquisitions of seizures in these kindling models are used for effective therapy following cerebral insults including, but not limited to, ischemia, haemorrhagic stroke, trauma, and infection, that can lead to an elevated incidence of seizure disorders (The Epilepsies: Etiologies and Prevention, 1999, Eds. Kotagal and Luders; Ballenger, et al, (1978) Br. J. Psychiatry 133: 1-14).
A substantially purified natural product (compounds, extracts) such as an NMDA receptor antagonist is used to ameliorate, reduce, prevent or treat the effects of a disorder associated with NMDA receptor function. Optionally, a combination of substantially purified natural product compounds are used together (administered simultaneously or sequentially) as NMDA receptor antagonists. Pharmaceutical Compositions
Compounds identified from botanical sources are purified from the natural source or synthetically made de novo. Optionally, compounds are formulated as mixtures of 2, 3, 4, 5, 8, 10 or more compounds. In some cases the compound is formulated as a prodrug. The prodrug is inactive until it is ingested, processed by the body (digested), or comes in contact with a target cell The prodrug chemical structure undergoes conversion to an active drug within a biological system, e.g., metabolism or contact with a component of a target cell such as an enzyme or cell surface structure. Some prodrugs include the active compound to which a chemical moiety has been linked, i.e., the compound has been derivatized When the prodrug is metabolized, the chemical moiety is removed thereby activating the compound. Examples of targeted prodrug formulations include antibody-directed enzyme prodrugs, gene- directed enzyme prodrugs, and peptide transporter-associated prodrugs. The purified compounds, extracts, and functional analogs of the present invention are administered by virtually any mode and are administered simultaneously or serially. When administered serially, the purified compounds and functional analogs are administered sufficiently close in time so as to provide the desired effect, for example within 1-3 hours of each other. In some examples, the purified compounds and functional analogs are administered topically, transdermally via a transdermal patch.
The purified compounds and functional analogs are administered therapeutically to treat, prevent, or slow the rate of onset of neuronal dysfunctions, such as epilepsy and seizures, or prophylactically to either protect against further seizures associated with epilepsy or to avoid or forestall the onset of seizures associated with other disorders. For example, the purified compounds and functional analogs are administered prophylactically to slow or halt the progression of seizures and epilepsy in a patient who has had a stroke and has a risk of developing seizures as a result of the stroke. The purified compounds and functional analogs are administered to a subject, using a wide variety of routes or modes of administration. Suitable routes of administration for particular compositions include, but are not limited to, oral inhalation; nasal inhalation; transdermal; oral; rectal; transmucosal; intestinal; and parenteral administration, including intramuscular, subcutaneous, and intravenous injections. The purified compounds are administered via the same or via a different mode of administration. For example, an isolated compound with a pharmaceutically acceptable salt or hydrate is administered orally or via a transdermal patch, an aerolized formulation, by nasal inhalation, or via nano- or microencapsulated formulations. The purified compounds or functional analogs are administered by intrathecal and intraventricular modes of administration.
Various combinations of the purified compounds or functional analogs are administered. In addition, the compounds can be administered in a combination with other therapeutic agents. The choice of therapeutic agents that are co-administered with the composition of the invention will depend, in part, on the condition being treated. For example, the compounds of the invention are administered in cocktails comprising other agents used to treat symptoms and associated with epilepsy or seizures. In the latter case, the combination therapy approach may permit a lower dose of the agents, thereby reducing undesired side effects.
The compounds can be formulated either as single compounds per se or as mixtures of compounds of the same type (e.g., two different analogs), as well as mixtures of compounds. Such compositions will generally comprise at least one purified compound or functional analogs formulated as a pharmaceutically acceptable salt or hydrate.
Pharmaceutical compositions for use in accordance with the present invention are formulated in conventional manner using one or more physiologically acceptable carriers, excipients, diluents or auxiliaries that further facilitate processing of the substantially purified natural product compounds. The choice of formulation is dependent upon the selected administration route. For example, the compounds are formulated in the form of an ointment, paste, spray, patch, cream, gel, sponge, or foam.
The formulations are administered by implantation or transcutaneous delivery (for example subcutaneously or intramuscularly), intramuscular injection, or transdermally. Thus, for example, the compounds are formulated with suitable polymeric or hydrophobic materials (such as an emulsion in an acceptable oil) or ion exchange resins. Formulations suitable for transdermal administration of compounds are described in U.S. Pat. Nos. 5,725,876; 5,716,635; 5,633,008; 5,603,947; 5,411,739; 5,364,630; 5,230,896; 5,004,610; 4,943,435; 4,908,213; and 4,839,174, which patents are hereby incorporated herein by reference. As purified compounds or functional analogs, pharmaceutically acceptable salts or hydrates are readily absorbed and cross cell membranes and the blood-brain barrier. Any of these formulations are routinely adapted for transdermal administration.
For injection, the purified compounds or functional analogs are formulated in physiologically compatible aqueous solutions, such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the purified compounds or functional analogs are formulated with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated for oral administration as tablets, pills, gums dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. Alternatively, the compounds are formulated into candies, cookies, or other edible foodstuffs. Pharmaceutical preparations for oral use are obtained by mixing the compounds of the invention with a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after. adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
Concentrated sugar solutions are used that can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments are added to the tablets or coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations that are used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules contain the compounds of the invention in an admixture with filler, such as lactose; binders, such as starches; or lubricants, such as talc or magnesium stearate; or stabilizers. In soft capsules, the compounds of the invention are dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers are added to the soft-capsule formulation. All formulations for oral administration are in dosages suitable for such administration.
For buccal administration, the compositions takes the form of oral sprays, tablets, gums, or lozenges formulated by well-known methods. A candy formulation suitable for oral or buccal administration of therapeutic compounds, pharmaceutically acceptable salts and hydrates is described in U.S. Pat. No. 6,083,962, which is hereby incorporated herein by reference. Additional formulations suitable for oral or buccal administration of therapeutic compounds, are described in U.S. Pat. Nos. 5,939,100; 5,799,633; 5,662,920; 5,603,947; 5,549,906; D358,683; 5,326,563; 5,293,883; 5,147,654; 5,035,252; 4,967,773; 4,907,606; 4,848,376; and 4,776,353, which are hereby incorporated herein by reference. All of these formulations are routinely adapted for administration of the purified compounds or functional analogs thereof, pharmaceutically acceptable salts and hydrates.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. Preferably the compositions are. administered by the oral or nasal respiratory route for local or systemic effect. For administration by oral or nasal inhalation, the compounds of the invention are conveniently delivered in the form of an aerosol spray delivered via pressurized packs or a nebulizer, with a suitable propellant, e.g., carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit is controlled by a dose-metered valve. Capsules and cartridges, e.g. gelatin, for use in an inhaler or insufflator are formulated as a powder mix of the compounds if the invention and a suitable powder base, such as lactose or starch. Formulations suitable for nasal inhalation are well known in the art. For example, a nasal aerosol spray contains compound, a water soluble diluent such as an organic acid, and a thickening agent such as a natural or synthetic polymer or an oil substance comprising the oil phase of an emulsion. The compounds of the invention are also administered in a vaporizer that delivers a volume of vapor containing compounds. The vaporizer is battery operated and designed to deliver a dosage of compound effective to inhibit seizures. The compounds of the invention, in a sterile pharmaceutically acceptable solvent, are nebulized by use of inert gases. Nebulized solutions are breathed directly from the nebulizing device or the nebulizing device is attached to a face mask, tent or intermittent positive pressure breathing machine. In one embodiment, an aerosol spray containing substantially purified natural product compounds is used to treat or prevent seizure clustering. Some patients with epilepsy are prone to having consecutive seizures after the initial seizure. An aerosol formulation of compounds is used as a spray mist in such patients after the first seizure as a preventative measure against subsequent seizures. In another embodiment, the aerosol formulation is administered in a spray mist in a subject that has been chemically induced to have seizures or is at the risk of developing seizures, such as those at risk of bioterror attacks. In such instances, the aerosolized substantially purified natural product compounds are provided in the form of a portable kit or package and used prior to, or immediately after, exposure to the seizure inducing chemical, e.g., organophosphate.
For administration by injection, the compounds of the invention are formulated with a surface-active agent (or wetting agent or surfactant) or in the form of an emulsion (as a water- in-oil or oil-in-water, emulsion). Suitable surface-active agents include, but are not limited to, non-ionic agents, such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with a surface-active agent comprise between 0.05 and 5% surface-active agent, and preferably between 0.1 and 2.5%. It will be appreciated that other ingredients are added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.
Suitable emulsions are prepared using commercially available fat emulsions, such as Intralipid , Liposyn , Infonutrol , Lipofundin and Lipiphysan . The active ingredient is either dissolved in a pre-mixed emulsion composition or alternatively it is dissolved in an oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil. corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients are added, for example gylcerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to 20% oil, for example, between 5 and 20%. The fat emulsion preferably comprises fat droplets between 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH in the range of 5.5 to 8.0.
An injectable formulation containing compound is used to treat or prevent seizure clustering. Some patients with epilepsy are prone to having consecutive seizures after the initial seizure. The injectable formulation of compoundis after the first seizure as a preventative measure against subsequent seizures. In another embodiment, the injectable formulation is administered to a subject that has been chemically induced to have seizures or is at the risk of developing seizures, such as those at risk of bioterror attacks. In such instances, the injectable formulation of compounds are provided in the form of a portable kit or package and used prior to, or immediately after, exposure to the seizure inducing chemical, e.g., organophosphate. The purified compound is formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection are presented in unit- dosage form, e.g., in ampules or in multi-dose containers, optionally with an added preservative. The compositions take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain formulatory agents, such as suspending, stabilizing, or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the compounds of the invention in water-soluble form. Additionally, suspensions of the compounds of the invention are prepared as appropriate oily-injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic-fatty-acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous-injection suspensions contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension contains suitable stabilizers or agents that increase the solubility of the compounds of the invention to allow for the preparation of highly concentrated solutions. Alternatively, the compounds are in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds are also be formulated in rectal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases, such as cocoa butter or other glycerides. The pharmaceutical compositions also comprise suitable solid- or gel-phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers, such as polyethylene glycols.
The appropriate dose of the pharmaceutical composition is that amount effective to prevent occurrence of the symptoms of the disorder or to treat some symptoms of the disorder from which the patient suffers. By "effective amount", "therapeutic amount" or "effective dose" is meant that amount sufficient to elicit the desired pharmacological or therapeutic effects, thus resulting in effective prevention or treatment of the disorder. The effective dose varies, depending upon factors such as the condition of the patient, the severity of the symptoms of the disorder, and the manner in which the pharmaceutical composition is administered. The effective dose of the composition differs from patient to patient but in general includes amounts starting where desired therapeutic effects occur, but below that amount where significant undesirable side effects are observed. Thus, when treating a seizures and epilepsy, an effective amount of composition is an amount sufficient to pass across the blood-brain barrier of the subject and to interact with relevant receptor sites in the brain of the subject and alter the actions of neurotransmitters on those receptors, thus resulting in effective prevention or treatment of the disorder. Administration and Dosages
Pharmaceutical preparations suitable for use with the present invention include compositions wherein the purified compounds or functional analogs are present in effective amounts, i.e., in amounts effective to achieve the intended purpose, for example, treating, preventing, or reducing seizures, or orthostatic hypotension. Of course, the actual amounts of the compounds effective for a particular application depends upon a variety of factors including, inter alia, the type of disorder being treated, and the age and weight of the subject. When administered to treat or prevent seizures, such compositions contain amounts of compound effective to achieve these results. Determination of effective amounts is well within the capabilities of those skilled in the art. The compounds can be administered in any manner that achieves the requisite therapeutic or prophylactic effect. Therapeutically or prophylactically effective doses of the compounds of the invention can be determined from animal or human data for analogous compounds that are known to exhibit similar pharmacological activities. The applied doses are adjusted based on the relative bioavailability, potency and in vivo half-life of the administered compounds as compared with these other agents.
Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods that are well-known is well within the capabilities of the ordinarily skilled artisan.
Typically, the dosage range of natural product compounds administered subcutaneously, is about 0.1mg/kg/day to 0.4mg/kg/day. The dosage range of natural product compounds capsules for oral administration is in the range of about lOOμg/day to 2000μg/day (See e.g., Ye et al (2000) Acta Pharmacol Sin: 21, 65; Ma et al. (1998) Ann NY Acad Sd.
Al 854:506-7; and Ma et al. (1998) N-S Arch Pharmacol 358:suppl 1, P53194). Typically, the dosage range of compounds administered subcutaneously, is about 3mg/kg/day to 12mg/kg/day. The dosage range of substantially purified natural product compounds capsules for oral administration is in the range of about 3mg/day to 60mg/day. It will be appreciated that the duration of action, and the therapeutic effect of the compounds is increased by combining two or more purified compounds or functional analogs of the present invention without causing adverse side effects. Furthermore, the dosage of each purified compound is reduced to achieve the same therapeutic effect. For the sake of illustration only, if a dosage of 2000μg/day of one compound of the present invention is effective at treating seizures, but causes some undesired side effects, then the dosage of that natural product of the present invention may be reduced, for example to lOOOμg/day, when used in combination with a different natural product of the present invention, for example, at a dosage of about 30mg/day. This combination provides the same therapeutic effect but without the adverse side effects. Furthermore, the duration of therapeutic effect may also increase by a using a combination compounds. As will be obvious to the skilled practitioner, the effective dosage amount can be manipulated to achieve the desired therapeutic effect.
Depending upon the neuroprotective effect desired, the compounds of the invention are administered to achieve either a therapeutic or a prophylactic effect. For example, the purified compound is prophylactically administered to a subject who has not yet suffered a seizure, but one who may be prone to, or at risk of seizures, for example as a result of a stroke, thereby protecting the subject against seizures. Alternatively, the compounds of the invention are administered to subjects who suffer from epilepsy. Regardless of the condition of the subject, the compounds of the invention are typically be administered as part of a daily r regimen. Other disorders that are also treated include, but are not limited to, cognitive impairment; severe neurodegenerative disorders, such as Alzheimer's disease; and neuronal dysfunction associated with loss of motor skills, such as Parkinson's disease and amyotrophic lateral sclerosis. The compounds of the invention also treat, prevent, or reverse neuronal dysfunction resulting from CNS injury, such as stroke, spinal-cord injury, and peripheral- nerve injury.
The ratio between toxicity and therapeutic effect for a particular compound is its therapeutic index and is expressed as the ratio between LD50 (the amount of compound lethal in 50% of the population) and ED50 (the amount of compound effective in 50% of the population). The compounds of the invention that exhibit high therapeutic indices are preferred. Therapeutic index data is obtained from animal studies and used in formulating a range of dosages for use in humans. The dosage of such compounds preferably lies within a range of plasma concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.
Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All U.S. Patents, scientific journal articles, and other references noted herein for whatever reason are specifically incorporated by reference. The specification and examples should be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.
The invention is illustrated by way of working examples that demonstrate the anticonvulsive effect of the substantially purified natural product compounds of the invention in established in vitro and in vivo models of epilepsy.
In vivo Neuroprotection against Seizures Using Herbal Extracts Containing Natural Products.
Herbal extracts were evaluated in the Maximal Electroshock (MES) and Subcutaneous Metrazol (pentylenetetrazol; SCMET) models of generalized tonic-clonic and myoclonic seizures, respectively in mice and rats. In addition, the herbal extracts were evaluated in a 6 Hz "psychomotor" seizure stimulation, a proven useful model of therapy-resistant limbic seizures (See, for example, Barton ME, Klein BD, WoIfHH, White HS., Epilepsy Res. 2001 Dec;47(3):217-27). (i) Maximal Electroshock (MES) model The maximal electroshock (MES) model is an art recognized test, useful to investigate the efficacy of therapeutic agents against grand mal seizures. Maximal seizures were induced by the application of electrical current to the brain via corneal electrodes. The stimulus parameters for mice were 5OmA in a pulse of 60Hz for 200ms. The animals were given the herbal extract dissolved in methyl cellulose and spasm inhibition was recorded as a measure of anticonvulsant activity.
(ii) Subcutaneous Metrazole (SCMET) Model
The Subcutaneous Metrazole (SCMET) model is also an art-recognized test for therapeutic agents for epilepsy, and in particular, is useful for investigating petite mal seizures. A metrazole dose of 85mg/kg was administered subcutaneously to induce seizures. The herbal extracts were then administered and the animals observed, (iii) Toxicity Model (TOX) The neurotoxicity of the herbal extracts was tested in the rotorod test. The mice were trained to stand on an accelerating rotorod rotating at 10 rev min-1 with a diameter of 3.2cm. The trained animal were given the herbal extracts at various doses and the effect of the herbal extracts on their motor skills was determined. The dose at which the animals fell off the rotorod was the toxic dose. (iv) 6-Hz "Psychomotor" Seizures
The herbal extracts were tested for their ability to block 6-Hz seizures after intraperitoneal (i.p.) administration to the mice. The "psychomotor" seizures characterized by stun, forelimb clonus, twitching of the vibrissae, and Straub tail, were induced according to a previously described procedure (See, for example, Barton ME, Klein BD, WoIfHH, et al. Pharmacological characterization of the 6-Hz psychomotor seizure model of parial epilepsy.
Epilepsy Res. 2001; 47; 217-227; Brown WC et al. Comparative assay of antiepileptic drugs by "psychomotor" seizures and minimal electroshock threshold test. J Pharmacol Exp Ther 1953: 107: 273-83). A current of 22 niA, 32 mA, or 44 mA at 6 Hz for 3 sec was delivered through corneal electrodes. The median effective does (ED50) was determined at these three different current intensities. Animals in which none of these characteristics of the "psychomotor" seizures were observed were considered protected. Effect of Herbal Extracts in Mice and Rats
The herbal extracts were administered in an i.p. form, soluble form (SOL) or as a suspension (SUS). To produce a suspension, the herbal extract can be ground to a powder using a mortar and pestle, and the powder mixed with methyl cellulose. This mixture was then sonicated to produce a suspension that was subsequently administered to mice or rats. Various time points were analyzed at each concentration of herbal extract with each animal model and the results were recorded.
Studies were also conducted to determine the time course of herbal extracts in mice and rats following i.p. or oral administration of the extracts. The herbal extracts were prepared as described above at various concentrations. The effects were tested in the MES, ScMET, TOX and 6-Hz animal models.
Effect of Gou teng Extract in Rat and Mouse
Gou teng extract is active in MES, ScMET and 6-Hz models. In the mouse-i.p. 6-Hz; 22 mA model, ED50 is 119 mg/kg (95% Cl: 69-183 mg/kg); 5/8 animals were protected at 200 mg/kg; and all eight animals were protected at 400 mg/kg with minimal motor impairment at 1 hr. In the rat-i.p. MES model, the ED50 > 400 mg/kg. In the rat-i.p. ScMET model, the ED50 is between 200 and 400 mg/kg. In the rat-i.p. tox model, TD50 is >500 mg/kg. Effect of Gastrodia elata compounds and extract in Rat and Mouse The tuber of Gastrodia elata plant contains the compound gastrodin. Tian Ma is an extract of the plant tissue or tuber. The Tian ma extract is active in ScMET and 6-Hz model. In the mouse-i.p. 6 Hz model, the ED50 is 122 mg/kg (95% Cl: 76-178 mg/kg), 7/8 animals were protected at 250 mg/kg, and all eight animals (100%) were protected at 500 mg/kg. In the mouse-i.p. toxicity model, up to 1000 mg/kg is non-toxic. In the rat-i.p. model, the lowest anti- convulsant dose is 75 mg/kg. Gastrodin is also useful in reducing frequency and severity of seizures. Effect of TJ-960 extract in Mouse
TJ-960 (mixture of extracts of Bupleuri Radix, Paeoniae Radix, Pinelliae Tuber, Cinnamomi Cortex, Zizyphi Fructus, Scutellariae Radix, Ginseng Radix, Glycyrrhizae Radix, Zingiberis Rhizoma) was found to be active in the mouse-i.p. 6 Hz model. The TJ-960 extract was active in the 6-Hz model with an ED50 of 36.4 mg/kg. In the mouse-i.p. toxicity model, 300 mg/kg is non-toxic. The lowest anti-convulsant dose is 15 mg/kg in the mouse-i.p. model and 30 mg/kg in the rat-i.p. model. Effect of TJ-IO extract in mouse and rat and effect of TJ-IO extract in rat oral TJ-IO (mixture of extracts of Bupleurum falcatum (thorowax) root, 19%; Peonia lacttflora (peony) root, 19%; Pinellia Ternata (ban xia) rhizome, 15%; Zizyphus jujuba (jubube) fruit, 12%; Panax ginseng (Asian ginseng) root, 9%; Scutellaria baicalensis (Skullcap) root, 9%; Zingiber officinale (ginger) rhizome, 6%; Cinnamomum cassia (cassia) bark, 6%; Glycyrrhiza uralensis (licorice, gan cao) rhizome, 5%) was active in ScMET rat-i.p. and rat-oral models and 6-Hz mouse-i.p. The lowest anticonvulsant dose was 100 mg/kg in the mouse-i.p. 6-Hz model and doses up to 300 mg/kg are non-toxic, including a 250 mg/kg dose given qd x 3 days. TJ- 12 extract in Rat and Mouse
TJ- 12 extract was found to be active in the MES rat-i.p. model. Effect of Brahmi rasavan extract in mouse and rat Brahmi rasayan extract was active in the mouse-i.p. 6 Hz model using stimulations of 32,
22, and 44 mA. In the mouse i.p. 6 Hz model, the ED50 is 21.76 mg/kg (95%C1: 5.08-69.55 mg/kg)(0.5hr). Brahmi rasayan (Shukla et al, 1987, J. Ethnopharmacol 21(1)65-74) contains Kateri, Gokhru, Shalparni, Punarnva, AmIa, and Shatavar. Brahma Rasayanam extract tested was prepared according to the formula from the text "Ashtanga Hrudayam". This commercially available product was obtained from The Arya Vaidya Pharmacy (Coimbatore) Limited. Effect of Peoniflorin in mouse
The peoniflorin compound was active in the ScMET model and 6-Hz model using 22 mA stimulation. No toxicity is observed at doses up to 300 mg/kg. Effect of Oxypeucedaninhiydrate in mouse The oxypeucedaninhiydrate compound was active in the MES mouse-i.p. and in the
ScMET mouse-i.p. and rat-oral models. Effect of Imperatorin in mouse
The imperatorin compound was active in the MES rat-i.p. model. At 50 mg/kg, protection from MES seizures was observed. Effect of Decursinol in mouse and rat
The decursinol compound was active in the MES and ScMET mouse-i.p. models at doses of 300 mg/kg. Effect of Baicalein, Baicalin, Wogonoside, and Ganhuangenin
Each of these compounds, baicalein, baicalin, wogonoside, and ganhuangenin were found to be active in the in vitro NMDA receptor excitotoxicity model using primary neuronal cultures from mouse cortex. Baicalin showed a neuroprotective effect against glutatmate-mediated cell death. These data indicate an inhibitory effect on NMDA receptors. Effect of Wogonin in mouse.
This compound was active in the ScMET mouse-i.p. model at a dose of 300 mg/kg. Isolation and Purification of Natural Product Compounds from Herbal Extracts Substantially purified natural product compounds or "the compounds" of the present invention were prepared from herbal extracts. For example, the dry powder of the herbal plant (3 kg) were extracted in 80% aqueous MeOH (5 L) at room temperature overnight. Then, the filtrates were evaporated in vacuum under 400C, and the concentrates were fractionized into three fractions (n-EtOAc, n-BuOH, and aqueous). Each solvent extract was flowed through column chromatography (SiO2, ODS, Sephadex etc.) and eluted with several mixed solvents. Several fractions were repeatedly chromatographed while varying the packing materials or eluting solutions. Purity of the compounds was assessed by TLC or HPLC. The molecular structure and weight were determined using one or more of the following techniques: 1) hypothesizing chemical and physical characteristics of compounds; 2) determining functional groups in the compound through IR spectra; 3) reading molecular weight and molecular formula from Mass data; 4) identifying chemical environment and integration value of proton, and neighboring protons using IH-NMR data; 5) measuring chemical environment and numbers of carbon from 13C-NMR data; 6) multiplicity of carbon by reading DEPT NMR data; 7) investigating the correlation between neighboring proton signals from 1H-1H 2D COSY NMR; 8) studying the correlation between carbon and neighboring proton signals using HSQC 2D NMR; 9) reading the long range correlations between carbon and proton signals from HMBC 2D NMR; 10) determining the stereochemistry of chiral carbons from NOESY 2D NMR; 11) determining the stereochemistry of a molecular structure by reading Polarimeter and CD; and 12) adaptating molecular modifications as needed (e.g., acetylation, methylation, methyl esterification, acid hydrolysis, alkaline hydrolysis).
Screening of Substantially Purified Natural Product Compounds and Their Parent Extracts Using in vitro Assays related to Currents Mediated by AMPA, NMDA and GABAA Receptors and Voltage-dependent Sodium, Potassium and Calcium Ion Channels.
Whole-cell patch-clamp recording of ligand- and voltage-gated currents is carried out in cultured rodent cortical and hippocampal neurons to evaluate the activity of purified compoundsn and enriched compositions from botanical sources (extracts). The extracts and substantially purified natural product compounds inhibit, evoke or modulate currents mediated by AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), NMDA (N-methyl-D- aspartate) or GABAA (γ-amino-butyric acid A) receptors or block or enhance voltage-dependent sodium, potassium, or calcium ion channels.
The whole-cell configuration of the patch-clamp technique is used to study voltage- and ligand-gated currents in cultured rodent cortical neurons as described previously by Sucher et al. (Sun X, et al. NeuroSignals, 2003;12:31-8; Sucher NJ, et al. Neuroscience 1991;43:135-50). Specific currents are evoked by application of ligands (ligand-gated conductances) or voltage steps (voltage-gated conductances) in the presence or absence of extracts or single compounds while other currents are blocked by the addition of specific antagonists to the extra- and/or intracellular solutions. Current/voltage (I/ V) relationships are constructed by performing voltage ramps in the presence of buffer alone, extract or single compound alone, and specific agonist and extract or single compound together. Each trial consists of three 4 seconds ramps from -80 to +40 mV. Ramps are performed before, during, and after drug (extracts or single compounds) application, and each set is averaged. The net I/V plot is constructed by subtracting the averages of the trials before and after drug application from the average during drug application. Should these experiments reveal any acute blocking effect of the drugs, complete dose/response curves are performed in order to determine the IC5O for each antagonist.
Cortical neurons from mouse embryos at day 16 are isolated and cultured as previously described (Sun X, et al. NeuroSignals, 2003;12:31-8; Chan SF, et al. JNeurosci 2001;21:7985-92.). Patch-clamp recording: Patch-clamp recordings are performed using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) as used previously by Sucher et al. (Sun X, et al. NeuroSignals, 2003;12:31-8; Sucher NJ, et al. Neuroscience 1991;43:135-50). Patch pipettes (2- 5 MΩ) are pulled from borosilicate glass tubing using a computer controlled horizontal puller (Sutter Instrument). Pipettes are fire-polished before use. Extra- and intracellular solutions for the isolation of specific conductances are prepared. Extracts and compounds are applied by the use of a linear array of seven plastic pipes connected to solution reservoirs. Solution flow through each pipe is individually controlled in the pClampex 7 menu. Neurons are continuously bathed in a stream of solution from the flow pipe (0.5 ml per min); only one barrel is used for perfusion at a given time. Data analysis: The amplitudes of the whole-cell currents recorded in the presence and absence of extracts or compounds are measured and compared. The data is analyzed by ANOVA and Tukey's post test.
Analysis of results: A reduction of the current amplitude in the presence of extract or compound indicates antagonist activity. An increase in the current amplitude or induction of a whole-cell current by the extract or compound alone indicates an agonist-like activity. Whole-cell patch-clamp recording of recombinant NMDA receptors in HEK293 cells.
Functional neuronal NMDA receptors (NMDARs) are thought to be heteromultimers that are composed of at least one NMDARl (NRl) with one or more of the NMDAR2A-D (NR2A- D) subunits and in some cases an additional NR3 subunit. Recent evidence suggests that both the location (extrasynaptic vs. synaptic) and subunit composition (NR1/NR2B vs. NR1/NR2A) of NMDARs may determine whether or not their stimulation will lead to excitotoxic injury of neurons.
Experimental design: These experiments are performed using the whole-cell variant of the patch-clamp technique in human embryonic kidney (HEK) 293 cells that are transiently transfected with the NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D subunit combinations. HEK293 cells do not express endogenous NMDAR mRNAs or protein and they are easily manipulated and transfected with expression vectors containing recombinant NMDAR subunits. The electrophysiological and biophysical characteristics of various NMDAR subunit combinations expressed in HEK293 cells have been well characterized (Sucher NJ, Awobuluyi M, Choi Y-B, Lipton SA. NMDA receptors: from genes to channels. TiPS, 1996;17:348-55). The Sucher group has established the conditions for transfection and subsequent patch-clamp recording of HEK293 cells with recombinant NMDAR subunits. To facilitate recognition of transfected cells, NRl constructs that have been fused to a green fluorescent protein (GFP) tag are used. Methods: HEK 293 cell culture and cDNA transfection: HEK 293 cells (American Type
Culture Collection, Rockville MD) are cultured as previously described by Sucher' s group (Chan SF, Sucher NJ. JNeurosci 2001;21:7985-92 ; Sucher NJ, Akbarian S, Chi CL, Leclerc CL, Awobuluyi M, Deitcher DL, Wu MK, Yuan JP, Jones EG, Lipton SA JNeurosci 1995;10:6509- 20). HEK 293 cells are transfected with rat NMDA receptor subunit cDNAs using calcium phosphate precipitation. Mixed plasmids (1 μg NRl-GFP and 2 μg NR2A/B/C or D) are added to the dish and incubated for 24 hours. The medium is removed, and cells are rinsed twice with culture medium and further incubated in the presence of 20 μM 5,7-dichlorokynurenic acid, which is added in order to protect the transfected cells from NMDAR-mediated cell death. Cells are used for recording 40-50 hours after transfection. In order to facilitate identification of transfected cells, the NRl subunit is cloned in frame with green fluorescent protein (GFP).
Data analysis: The amplitudes of the whole-cell currents recorded in the presence and absence of extracts or compounds are measured, normalized and compared in HEK293 cells that have been transfected with either the NR1/NR2A or the NR1/NR2B subunit combinations. The data is analyzed by ANOVA and Tukey's post test. Analysis of results: A greater reduction of the NMDA-induced current amplitude by an extract or compound in one particular subunit combination (e.g. NR1/NR2B > NR1/NR2A) indicates a subunit preferring activity.
Characterization of NMDAR-mediated excitotoxicity in primary neuronal cultures from mouse cortex. Application of the extracts and/or substantially purified natural product compounds prevents NMDAR-mediated excitotoxicity in primary neuronal cultures from mouse cortex in vitro.
Experiments are performed in vitro so that the experimental conditions are precisely controlled. In particular, detailed dose/response curves are obtained to determine the IC50 of the tested extracts and compounds (applied together with and for the duration of application of NMDA). NMDAR-induced cytotoxicity leads to fulminant cell death, necrosis, or a delayed form of cell death (apoptosis). While necrosis leads to immediate cell lysis, apoptotic cell death develops over time and is distinguished by a number of criteria such as characteristic morphological changes and DNA fragmentation. The colorimetric lactate dehydrogenase (LDH) assay is used to assess primary necrosis as well as secondary, necrotic cell death subsequent to apoptosis.
Methods: Cytotoxicity assays are conducted in primary cultures of mouse cortical neurons after 10 days in vitro as described previously (Sun X, Chan LN, Gong X, Sucher NJ. NeuroSignals, 2003; 12:31-8.). Cultured neurons are exposed to NMDA (200 μM; 5 minutes) and co-agonist glycine (10 μM) in the nominal absence of magnesium and the presence of increasing concentrations of extract or single compound. Measurements are performed for each drug and dose in triplicate in at least three independent experiments. The herbal-derived extracts and compounds are added to the culture in three different ways: 1) before, concomitant with and after NMDA, 2) concomitant with and after NMDA and 3) after NMDA. Twenty- four hours after exposure to NMDA cell death is determined by the LDH assay.
Data analysis: Means and standard deviation are calculated and further analyzed by ANOVA and Tukey's post test.
Analysis of results: Three outcomes are possible: 1) Some or all of the drugs abrogate toxicity, 2) they do not abrogate toxicity or 3) the drugs are toxic. Determination of NMDA-induced nitric oxide (NO) production in cultured primary neurons from mouse hippocampus.
Extracts and/or substantially purified natural product compounds derived from herbal extracts also reduce NMDA-induced nitric oxide (NO) production in neurons. NMDA receptors are coupled to nNOS through their interaction with postsynaptic density protein (PSD) 95 via PDZ domains. Either deletion of nNOS or PSD-95 was previously reported to suppress
NMDAR-mediated toxicity. Thus, the disruption of the interaction between nNOS and PSD-95 suffices to abrogate NMDAR-mediated excitotoxicity and represents a potential therapeutic approach aimed at abating NMDAR-mediated neurotoxicity.
Experiments utilize primary cultures of mouse hippocampal neurons. The cultures are virtually purely neuronal (containing less than 1% astrocytes) and express high levels of nNOS. Cultured hippocampal neurons are exposed to NMDA (200 μM; 5 minutes) and co-agonist glycine (10 μM) in the nominal absence of magnesium and the presence of increasing concentrations of each extract and compound. The production of NO using the Griess reaction is measured for each extract and/or compound in triplicate in at least three independent experiments. The extract or compound is added to the culture in three different ways: 1) before, concomitant with and after NMDA, 2) concomitant with and after NMDA and 3) after NMDA. Methods: Nitrite (NO2 ") in culture superaatants is measured to assess NO production in primary neurons using the Griess reagent system' (Promega, Madison, WI) as described previously (Jiang X, et al. Exp Neurol 2004; 190:224-32.). Sample aliquots (50 μl) are mixed with 100 μl of Griess reagent (1% sulfanilamide/0.1% naphthylethylene diamine dihydrochloride/5% phosphoric acid) in 96-well plates and incubated at 25 °C for 10 min. The absorbance at 540 nm is measured on a microplate reader. NaNO2 is used as standard to calculate NO2 " concentrations.
Data analysis: Means and standard deviation are calculated and further analyzed by ANOVA and Tukey's post test.
Analysis of results: A reduction in NMDA-induced NO production by one or more extracts or substantially purified compounds indicates that the compound or extract 1) directly or indirectly inhibits the NMDA receptor, 2) displaces nNOS from PSD-95, or 3) directly or indirectly inhibits nNOS. Analysis of Substantially Purified Natural Product Compounds and Their Parent Herbal Extracts using in vivo Animal Models of Focal and Generalized Epilepsy
Substantially purified natural product compounds are tested in the screening epilepsy animal models for the amygdala kindling and lamotrigine-resistant animal models. The extracts are also tested, in addition to the compounds, because extracts may contain active compounds that are too low in concentration to be isolated and identified.
Anticonvulsant profiles are evaluated in a battery of well-established mouse and rat seizure and epilepsy models as shown in the diagram below. Initial identification studies are conducted in CF#1 mice (The Jackson Laboratories, Bar Harbor, ME; Charles River Laboratories, Wilmington, MA) using the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazol (sc PTZ) tests following intraperitoneal administration (White HS, et al. Discovery and preclinical development of anticonvulsants. In: Levy R, Mattson R, Meldrum B, Perucca E, eds. Antiepileptic Drugs, Fifth Edition. New York: Raven Press; 2002:36-48.). These two tests are highly predictive of efficacy against human generalized tonic- clonic (MES) and generalized myoclonic (sc PTZ) seizures (White HS, et al. The early identification of anticonvulsant activity: Role of the maximal electroshock and subcutaneous pentylenetetrazol seizure models. ItalJ Neurol Sd 1995; 16:73-7).
With the exception of levetiracetam, all of the approved anti-epileptic drugs (AEDs) currently on the market have demonstrated activity in one or both of these models that are routinely performed. Subsequent investigations demonstrated that levetiracetam was active in pathologically abnormal models of partial and primary generalized seizures (e.g., kindling, audiogenic seizures, and 6-Hz psychomotor seizures). In this regard levetiracetam appears unique among the established and newer AEDs and demonstrates the need for flexibility when screening for efficacy and the need to incorporate levetiracetam-sensitive models into the early evaluation process. To this end, extracts and substantially purified natural product compounds found ineffective in the initial MES and sc PTZ screens are subsequently evaluated in the levetiracetam-sensitive 6-Hz psychomotor seizure test. Activity in one or more of these three identification tests can be quantified at the time-to-peak effect (TPE) and the median effective dose (ED50) determined by probit analysis of data obtained from quantal dose-response curves. Subsequent differentiation studies evaluate the efficacy of active herbs in the amygdala kindled rat model of partial epilepsy and the lamotrigine-resistant model. Those herbal medicine-derived extracts and substantially purified natural product compounds that are found to be inactive in the initial identification tests (i.e., MES, sc PTZ, and 6-Hz) need not be further tested.
The lamotrigine (LTG)-resistant kindled rat results when rats are kindled in the presence of a low-dose of LTG (Postma T Krupp E, Li XL, Post RM, Weiss SR. Lamotrigine treatment during amygdala-kindled seizure development fails to inhibit seizures and diminishes subsequent anticonvulsant efficacy. Epilepsia 2002;41 : 1514-21.). Studies in the White laboratory have substantiated Postma' s original findings and extended them to include phenytoin and carbamazepine (Srivastava AK, Woodhead, JH, White HS. Effect of lamotrigine, carbamazepine, and sodium valproate on lamotrigine-resistant kindled rats. Epilepsia 2003;44:42.). Thus, this particular model displays resistance to three commonly employed AEDs and is proposed to represent a model of therapy resistant epilepsy. For these studies, fully kindled rats are treated with an effective dose (determined from the acute seizure tests) of the substantially purified natural product compound or extract by the i.p. route. At the time of peak effect, animals are challenged with the same electrical stimulus employed in the kindling procedure. Their behavior and afterdischarge duration is recorded. When a compound or extract is found to reduce either the seizure severity (as estimated by a change in the Racine seizure score), a complete dose- response study is conducted to quantitate its efficacy. A similar study is conducted in the lamotrigine-resistant kindled rat in an effort to differentiate it from lamotrigine, phenytoin, carbamazepine, and topiramate (all inactive in this model). The substantially purified natural product compounds are also evaluated for their ability to prevent the development of kindling. For these studies, rats implanted with a bipolar stimulating/recording electrode receive a dose previously determined to be effective in one or more of the acute seizure tests (i.e., MES, scPTZ, or 6 Hz). At the time to peak effect, animals are challenged with an electrical stimulus sufficient to elicit an afterdischarge in vehicle-treated rats. Their behavior and duration of afterdischarge duration is recorded and this procedure repeated on a daily basis until animals in the control group have become fully kindled (i.e., five consecutive Stage 5 generalized seizures). Based on previous investigations with NMDA receptor antagonists, a delay in kindling acquisition is observed for those substantially purified natural product compounds and extracts shown in in vitro assays to be NMDAR antagonists. In addition to the efficacy studies described above, additional studies are undertaken for herbal medicine-derived extracts and substantially purified natural product compounds with activity in one or more of the animal models to determine their propensity for causing behavioral impairment. For mice, behavioral impairment is determined by the rotarod test; behavioral impairment in rats is estimated from a battery of behavioral assessments including: gait, stance, placing response, muscle tone, etc. The dose producing behavioral impairment in 50% of the population (i.e., TD50) is calculated from probit analysis of quantal dose-response data as described by White et al (White HS, Woodhead JH, Wilcox KS, Stables JP, Kupferberg HJ, WoIf HH. Discovery and preclinical development of anticonvulsants. In: Levy R, Mattson R, Meldrum B, Perucca E, eds. Antiepileptic Drugs, Fifth Edition. New York: Raven Press; 2002:36-48). The protective index (TDso/EDso) is calculated. In rats, minimal motor impairment (MMI) is determined by overt evidence of ataxia, abnormal gait and stance. Previous studies have found that non-selective NMDA antagonists display more behavioral impairment in kindled vs. non-kindled rats and that this shift to the left in the toxicity dose-response curve correlates with increased toxicity in the epilepsy patient population. Thus, the tolerability profile for those compounds and extracts found to possess a favorable anticonvulsant profile is established in both kindled and non-kindled rats and those compounds and extracts for which the TD50 is similar regardless of whether animals have been kindled or not are chosen for further investigation.
At the conclusion of this analysis, active substantially purified natural product compounds and/or herbal extracts are evaluated following oral administration in at least one of the above models to assess the extent of its oral absorption. Collectively, the results from these studies provide an anticonvulsant profile for the substantially purified natural product compounds.

Claims

CLAIMS:
1. A method of reducing the severity, frequency, or duration of a neurological seizure in a subject comprising administering an isolated compound of Scutellaria baicalensis or an extract of said Scutellaria baicalensis.
2. The method of claim 1, wherein said compound is baicalein, ganhuangen, wogonin, wogonoside, baicalin, oroxyloside, or baicalin.
3. A method of reducing the severity, frequency, or duration of a neurological seizure in a subject comprising administering an isolated compound of Gastrodia elata or an extract of said Gastrodia elata.
4. The method of claim 3, wherein said compound is gastrodin or tian ma.
5. A method of reducing the severity, frequency, or duration of a neurological seizure in a subject comprising administering an isolated compound of Angelica gigas or an extract of said Angelica gigas.
6. The method of claim 5, wherein said compound is decursin or decursinol.
7. A method of reducing the severity, frequency, or duration of a neurological seizure in a subject comprising administering an isolated compound of Angelica dahurica or an extract of said Angelica dahurica.
8. The method of claim 7, wherein said compound is imperatorin or oxypeucedanin.
9. A method of reducing the severity, frequency, or duration of a neurological seizure in a subject comprising administering an isolated compound Paeonia lactiflora or an extract of said Paeonia lactiflora.
10. The method of claim 9, wherein said compound is peoniflorin or albiflorin.
11. A method of reducing the severity, frequency, or duration of a neurological seizure in a subject comprising administering an isolated compound ofBrahmi rasayan or an extract of said Brami rasayan.
12. A method of reducing the severity, frequency, or duration of a neurological seizure in a subject comprising administering an isolated compound of TJ-960 (a mixture of extracts of Bupleurumfalcatum (thorowax) root, 19%; Peonia lactiflora (peony) root, 19%; Pinellia
Ternata (ban xia) rhizome, 15%; Zizyphus jujuba (jubube) fruit, 12%; Panax ginseng (Asian ginseng) root, 9%; Scutellaria baicalensis (Skullcap) root, 9%; Zingiber officinale (ginger) rhizome, 6%; Cinnamomum cassia (cassia) bark, 6%; Glycyrrhiza uralensis (licorice, gan cao) rhizome, 5%) or an extract of TJ-960.
13. A method of reducing, the severity, frequency, or duration of a neurological seizure in a subject comprising administering an isolated compound of TJ-12 or an extract of TJ-12.
14. A method of reducing the severity, frequency, or duration of a neurological seizure in a subject comprising administering an isolated compound of TJ-IO (mixture of extracts of
Bupleurumfalcatum (thorowax) root, 19%; Peonia lactiflora (peony) root, 19%; Pinellia Ternata (ban xia) rhizome, 15%; Zizyphus jujuba (jubube) fruit, 12%; Panax ginseng (Asian ginseng) root, 9%; Scutellaria baicalensis (Skullcap) root, 9%; Zingiber officinale (ginger) rhizome, 6%; Cinnamomum cassia (cassia) bark, 6%; Glycyrrhiza uralensis (licorice, gan cao) rhizome, 5%) or an extract of TJ-IO.
15. A method of reducing the severity, frequency, or duration of a neurological seizure in a subject comprising administering an isolated compound of tian ma or an extract of said tian ma.
16. A method of reducing the severity, frequency, or duration of a neurological seizure in a subject comprising administering an isolated compound of gou teng or an extract of said gou teng.
17. The method of claim 16, wherein said compound is rhynchophylline, isorhynchophylline, or hyperin.
18. A method of reducing the severity, frequency, or duration of a neurological seizure in a subject comprising administering a combination of an isolated compound of tian ma and an isolated compound of gou teng or a combination of an extract of said tian ma and an extract of gou teng.
19. The method of claims 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 or 17, wherein said compound or extract is administered to said subject in a sustained release delivery vehicle.
20. The method of claim 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 or 17 wherein said compound or extract is formulated as dermal patch.
21. The method of claim 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 or 17, wherein said compound or extract is formulated as an implant, said implant being biodegradable or erodible.
22. The method of claim 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 or 17, wherein said compound or extract is formulated into a semipermeable membrane, said membrane controlling the delivery rate.
23. The method of claim 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 or 17, wherein compound or extract is formulated in a vehicle comprises a plurality of particles, each of said particles comprising a different rate of dissolution.
24. The method of claim 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 or 17, wherein said compound or extract is continuously infused into said subject.
25. The method of claim 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 or 17, wherein said compound or extract is administered in the form of an ointment, paste, spray, patch, cream, gel, sponge, foam, or subcutaneous depo formulation.
26. The method of claim 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 or 17, wherein said compound or extract is administered before or at the onset of a seizure.
27. The method of claim 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 or 17, wherein the compound or extract is administered after the occurrence of a seizure.
28. The method of claim 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 or 17, wherein said neurological seizure is epilepsy.
29. The method of claim 1, 3, 5, 7, 9, 11, 12, 13, 14, 15, 16 or 17, wherein said subject is diagnosed with epilepsy and has a score of less than 27 on a Mini Mental State Examination.
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