WO2008064296A1 - Procédé de traitement de troubles neurologiques avec des inhibiteurs d'anhydrase carbonique - Google Patents

Procédé de traitement de troubles neurologiques avec des inhibiteurs d'anhydrase carbonique Download PDF

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Publication number
WO2008064296A1
WO2008064296A1 PCT/US2007/085358 US2007085358W WO2008064296A1 WO 2008064296 A1 WO2008064296 A1 WO 2008064296A1 US 2007085358 W US2007085358 W US 2007085358W WO 2008064296 A1 WO2008064296 A1 WO 2008064296A1
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Prior art keywords
carbonic anhydrase
flies
disease
dosage form
anhydrase inhibitor
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PCT/US2007/085358
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English (en)
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Carol M. Singh
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Envivo Pharmaceuticals, Inc.
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Publication of WO2008064296A1 publication Critical patent/WO2008064296A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/421—1,3-Oxazoles, e.g. pemoline, trimethadione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • HD is a progressive hereditary disorder of the neurodegenerative type involving the basal ganglia (cerebral areas in charge of controlling involuntary movement), can cause highly debilitating motor and psychiatric symptoms.
  • onset of Huntington's disease occurs between ages 35 and 40, with an incidence of one case in 10,000 and a mean duration of the disease of about 17 years.
  • the onset is insidious and is characterized by abnormalities of coordination, movement, and behavior. Movement abnormalities include restlessness, mild postural abnormalities, and quick jerking movements of the fingers, limbs, and trunk. The movement abnormalities may be accompanied by substantial weight loss. Depression is common, and cognitive abnormalities and inappropriate behavior may develop.
  • juvenile patients may exhibit rigidity, tremor, and dystonia. In the course of eight to 15 years, the disorder progresses to complete incapacitation, with most patients dying of aspiration pneumonia or inanition.
  • Huntington's disease was the first major inherited disorder with an unidentified basic defect to be linked with a DNA marker.
  • the genetic defect responsible for the disease consists of an expansion of the CAG triplet encoding glutamine (polyQ expansion) at the amino-terminal end of the protein known as huntingtin.
  • polyQ expansion glutamine residues at the amino terminus.
  • those affected by HD there is an increase in these repetitions ranging from about 38 to about 120 units.
  • Described herein are methods for treating a subject, such as an animal or human, having a neurological disorder or at risk for developing a neurological disorder (e.g., a patient that has a genetic defect associated with a neurological disorder, but does not appear to suffer from any symptoms commonly associated with the neurological disorder) by administering to the subject having the neurological disorder an effective amount of a carbonic anhydrase inhibitor.
  • the method comprises administering a carbonic anhydrase inhibitor to the subject at risk for developing a neurological disorder.
  • Described herein is a method for treating a subject having a neurological disorder, the method comprising administering to the subject an effective amount of a carbonic anhydrase inhibitor.
  • the neurological disorder is selected from the group consisting of Huntington's disease, Alzheimer's disease, and a neurological disorder arising from a polyglutamine expansion.
  • the carbonic anhydrase inhibitor is selected from the group consisting of dichlorphenamide, dorzolamide, methazolamide, topiramate or acetazolamide.
  • the carbonic anhydrase inhibitor is selected from the group consisting of benzthiazide, chlorothiazide, hydroflumethiazide, diazoxide, celecoxib, metolazone and althiazide. In some cases the carbonic anhydrase inhibitor is selected from the group consisting of bendroflumethiazide, hydrochlorthiazide, trichlomethiazide, benzolamide and furosemide.
  • the method comprises of administering a carbonic anhydrase inhibitor to a patient, either as the sole active agent or in combination with one or more additional active agents.
  • the carbonic anhydrase inhibitor can be administered as a pharmaceutical composition that further contains a pharmaceutically acceptable excipient, carrier or diluent.
  • the pharmaceutical composition can be administered in a variety of solid and liquid dosage forms, and through various routes of administration, including oral delivery or continuous injection via a shunt.
  • the neurological disorder can be a neurodegenerative disorder, a disorder of movement, an extrapyrimidal disorder, a cerebellar disorder or a hyperkinetic movement disorder.
  • Neurological disorders that can be treated include, but are not limited to, neurological diseases arising from a polyglutamine expansion, such as Huntington's disease.
  • Pharmaceutical compositions for treating neurological disorders containing an effective amount of carbonic anhydrase inhibitor or a derivative are described in the disclosure.
  • Figure 1 depicts the results of a study analyzing the age-dependent decline in speed of locomotion between the wild type flies treated with vehicle (DMSO) and HD model flies treated with vehicle, HD model flies treated with the positive control compounds Trichostatin A (TSA) and different concentrations of dorzolamide (DRZ). From the top, the lines depict: wt-DMSO; HD-TSA 250 micromolar; HD-DRZ 300 micromolar; HD-DRZ 200 micromolar; HD-DRZ 100 micromolar; and HD-DMSO.
  • TSA Trichostatin A
  • DRZ dorzolamide
  • Figure 2 depicts the results of a study analyzing the age-dependent decline in speed of locomotion between the wild type flies treated with vehicle (DMSO) and HD model flies treated with vehicle, HD model flies treated with the positive control compounds Trichostatin A (TSA) and different concentrations of dichlorphenamide (DCP).
  • DMSO vehicle
  • TSA Trichostatin A
  • DCP dichlorphenamide
  • Figure 3 is a table that shows the effect sizes of compound-treated vs. vehicle-treated HD Drosophila model flies in locomotor assay.
  • Figure 4 depicts the result of an experiment analyzing the effect of dichlorphenamide on survival in the HD fly model. Dichlorphenamide treatment results are shown in the upper line. The control (DMSO) results are shown in the lower line.
  • Figure 5 is a table that shows the effect sizes of compound-treated vs. vehicle treated AD Drosophila model flies in a locomotor assay.
  • compositions and methods for treating a subject such as a human or an animal that has a neurological disorder by administering a carbonic anhydrase inhibitor or a derivative, analog, or a salt thereof are described as are pharmaceutical compositions and methods for treating a patient having a neurological disorder, comprising administering to the patient an effective amount of a carbonic anhydrase inhibitor or a pharmaceutically acceptable salt thereof.
  • small molecule refers to compounds having a molecular mass of less than 3000 Daltons.
  • a “therapeutic agent” includes a carbonic anhydrase inhibitor that ameliorates one or more of the symptoms of a neurological disorder.
  • a therapeutic agent can reduce one or more symptoms of the disorder, delay onset of one or more symptoms, or prevent or cure the disease.
  • an “additional active agent” refers to an agent that ameliorates one or more of the symptoms of a neurological disorder, including a neurodegenerative disorder.
  • An “additional active agent” can reduce one or more symptoms of the disorder, delay onset of one or more symptoms, or prevent or cure the disease.
  • An “additional active agent” can mean a second (different) therapeutic agent (i.e., a different carbonic anhydrase inhibitor), or a structurally distinct active agent for treating a neurological disorder.
  • an "effective amount” as referred to herein relates to the amount of the carbonic anhydrase inhibitor that is capable of rendering a beneficial clinical outcome of the condition being treated with a derivative or analog thereof compared with the absence of such treatment.
  • the effective amount of the therapeutic agent administered will depend on the degree, severity, and type of the disease or condition, the amount of therapy desired, and the release characteristics of the pharmaceutical formulation. It will also depend on the subject's health, size, weight, age, sex and tolerance to specific compounds, which are determinable pharmaceutical parameters to those skilled in the field. Generally, treatment is considered “effective” if one or more symptoms of the disease or disorder improves (e.g., at least 10% relative to pre-treatment) during the course of treatment.
  • the compounds described herein can also be given to prevent or delay the onset of symptoms in an individual predisposed to such disorder, e.g. Huntington's disease. A delay or absence of the onset of one or more symptoms relative to the time one would expect such symptoms to arise in a similar individual not treated with the drug would indicate efficacy.
  • the neurological disorder treated in the disclosure can be, but is not limited to, a disorder of movement, an extrapyramidal disorder, a cerebellar disorder excluding episodic ataxia-2, or a hyperkinetic movement disorder.
  • the neurological disorder also can be, but is not limited to, Huntington's disease, age-related memory impairment, amyotrophic lateral sclerosis, ataxia-telangiectasia, Biswanger's disease, cerebral amyloid angiopathies,
  • Creutzfeldt- Jacob disease including variant form, corticobasal degeneration, multi infarct dementia, subcortical dementia, dementia with Lewy Bodies, dementia due to human immunodeficiency virus (HIV), Friedreich ataxia, fronto-temporal dementia linked to chromosome 17 (FTDP- 17), frontotemporal lobar degeneration, frontal lobe dementia, Kennedy disease, Korsakoff s syndrome, mild cognitive impairment, neurological manifestations of HIV, neurological conditions arising from polyglutamine expansions excluding polyglutamine expansion in the CACNAlA gene affected in SCA-6, Pick's disease, prion diseases, Kuru disease, fatal familial insomnia, Gerstmann-Straussler- Scheinker disease, prion protein cerebral amyloid angiopathy, postencephalitic Parkinsonism, progressive supernuclear palsy, spinal muscular atrophy, transmissable spongiform encephalopathies or vascular dementia.
  • HIV human immunodeficiency virus
  • FTDP- 17
  • the disclosure also can provide therapeutic benefit to diseases or conditions including, but is not limited to, agyrophilic grain dementia, Parkinsonism-dementia complex of Guam, auto-immune conditions such as Guillain-Barre syndrome or Lupus, brain and spinal tumors (including neurofibromatosis), cerebral amyloid angiopathies, cerebral palsy, chronic fatigue syndrome, corticobasal degeneration, conditions due to developmental dysfunction of the CNS parenchyma, conditions due to developmental dysfunction of the cerebro vasculature, dementia lacking distinct histology, Dementia Pugilistica, diffuse neurofibrillary tangles with calcification, diseases of the eye, ear and vestibular systems involving neurodegeneration (including macular degeneration and glaucoma), Down's syndrome, dyskinesias (Paroxysmal), dystonias, essential tremor, Fahr's syndrome, hepatic encephalopathy, hereditary spastic paraplegia, hydrocephalus, pseudo
  • the neurological disease is a neurological condition arising from a polyglutamine expansion, excluding polyglutamine expansion in the CACNAlA gene.
  • the polyglutamine expansion can be of at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, or at least about 100 or more residues.
  • the polyglutamine expansion is typically between 21 and 33 residues in length.
  • the subject that the method of treatment is administered to can be an animal, such as Drosophila or a mammal.
  • the mammal can be, but is not limited to, a mouse, a rat, a cat, a dog, a primate, or a human.
  • mice transgenic for Huntington's disease see, e.g., Mangiarini et al, 1996, Cell 87: 493-506, Lin et al., 2001, Hum. MoI. Genet. 10: 137-144) and can be used to determine the efficacy of a given compound in the treatment or prevention of neurodegenerative disease.
  • Alzheimer's disease Hsiao, 1998, Exp. Gerontol.
  • Animal models are not limited to mammalian models.
  • Drosophila strains provide accepted models for a number of neurodegenerative disorders (reviewed in Fortini & Bonini, 2000, Trends Genet. 16: 161-167; Zoghbi & Botas, 2002, Trends Genet. 18: 463-471).
  • These models include not only flies bearing mutated fly genes, but also flies bearing human transgenes, optionally with targeted mutations.
  • Drosophila models available are, for example, Huntington's disease (Kazemi-Esfarjani & Benzer, 2000, Science 287: 1837-1840), Parkinson's disease (Feany et al, 2000, Nature 404: 394-398; Auluck et al., 2002, Science 295: 809-810), age-dependent neurodegeneration (Palladino et al., 2002, Genetics 161 : 1197-1208), Alzheimer's disease (Selkoe et al., 1998, Trends Cell Biol. 8: 447-453; Ye et al., 1999, J. Cell Biol. 146: 1351-1364), amyotrophic lateral sclerosis (Parkes et al., 1998, Nature Genet. 19: 171-174), and adrenoleukodystrophy.
  • Huntington's disease Korean azemi-Esfarjani & Benzer, 2000, Science 287: 1837-18
  • Drosophila as a model organism has proven to be an important tool in the elucidation of human neurodegenerative pathways, as the Drosophila genome contains many relevant human orthologs that are extremely well conserved in function (Rubin, G.M., et al., Science 287: 2204-2215 (2000)).
  • Drosophila melanogaster carries a gene that is homologous to human APP which is involved in nervous system function.
  • the gene, APP- like (APPL) is approximately 40% identical to APP695, the neuronal isoform (Rosen et al., Proc. Natl. Acad. Sci. U.S.A.
  • the transgenic flies exhibit progressive neurodegeneration which can lead to a variety of altered phenotypes including locomotor phenotypes, behavioral phenotypes (e.g., appetite, mating behavior, and/or life span), and morphological phenotypes (e.g., shape, size, or location of a cell, organ, or appendage; or size, shape, or growth rate of the fly).
  • Test animals such as transgenic flies, are administered carbonic anhydrase inhibitor and evaluated for symptoms relative to animals not administered the compound. A change in the severity of symptoms (e.g., a 1%, 2%, 5%, 10%, or greater improvement in one or more symptoms), or a delay in the onset of symptoms, in treated versus untreated animals can be indicative of therapeutic efficacy.
  • dichlorphenamide has shown reproducible positive effects in a Drosophila model of HD.
  • the flies performed better than carrier controls using two metrics: early speed (days 1-7) and late speed (days 8-10).
  • the pharmaceutical composition for treating a subject having or at risk of developing a neurological disorder can comprise a therapeutically effective amount of a carbonic anhydrase inhibitor, a derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, carrier or diluent.
  • the pharmaceutical composition can comprise carbonic anhydrase inhibitor, or a derivative, analog, or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition of the disclosure can be administered in a variety of dosage forms including, but not limited to, a solid dosage form or in a liquid dosage form, an oral dosage form, a parenteral dosage form, an intranasal dosage form, a suppository, a lozenge, a troche, buccal, a controlled release dosage form, a pulsed release dosage form, an immediate release dosage form, an intravenous solution, a suspension or combinations thereof.
  • the dosage can be an oral dosage form that is a controlled release dosage form.
  • the oral dosage form can be a tablet or a caplet.
  • the compounds employed can be administered, for example, by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
  • the compounds or pharmaceutical compositions comprising the compounds are delivered to a desired site, such as the brain, by continuous injection via a shunt.
  • the carbonic anhydrase inhibitor can be administered parenterally, such as intravenous (IV) administration.
  • the formulations for administration will commonly comprise a solution of the carbonic anhydrase inhibitor (e.g., Dichlorphenamide) dissolved in a pharmaceutically acceptable carrier.
  • acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride.
  • sterile fixed oils can conventionally be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter.
  • formulations may be sterilized by conventional, well known sterilization techniques.
  • the formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of compound of the carbonic anhydrase inhibitor in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs.
  • the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
  • This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3- butanediol.
  • the carbonic anhydrase inhibitor of the disclosure can be administered by introduction into the central nervous system of the subject, e.g., into the cerbrospinal fluid of the subject.
  • the formulations for administration will commonly comprise a solution of the compound of the carbonic anhydrase inhibitor (e.g Dichlorphenamide) dissolved in a pharmaceutically acceptable carrier.
  • the carbonic anhydrase inhibitor is introduced intrathecally, e.g., into a cerebral ventricle, the lumbar area, or the cisterna magna.
  • the carbonic anhydrase inhibitor is introduced intraocullarly, to thereby contact retinal ganglion cells.
  • the pharmaceutically acceptable formulations can easily be suspended in aqueous vehicles and introduced through conventional hypodermic needles or using infusion pumps. Prior to introduction, the formulations can be sterilized with , gamma radiation or electron beam sterilization, described in U.S. Pat. No. 436,742 the contents of which are incorporated herein by reference.
  • the pharmaceutical composition comprising a compound of carbonic anhydrase inhibitor is administered into a subject intrathecally.
  • intrathecal administration is intended to include delivering a pharmaceutical composition comprising carbonic anhydrase inhibitor directly into the cerebrospinal fluid of a subject, by techniques including lateral cerebro ventricular injection through a burrhole or cisternal or lumbar puncture or the like (described in Lazorthes et al. Advances in Drug Delivery Systems and Applications in Neurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1 : 169-179, the contents of which are incorporated herein by reference).
  • lumbar region is intended to include the area between the third and fourth lumbar (lower back) vertebrae.
  • ceisterna magna is intended to include the area where the skull ends and the spinal cord begins at the back of the head.
  • cervical ventricle is intended to include the cavities in the brain that are continuous with the central canal of the spinal cord.
  • Administration of a carbonic anhydrase inhibitor to any of the above mentioned sites can be achieved by direct injection of the pharmaceutical composition comprising the carbonic anhydrase inhibitor or by the use of infusion pumps.
  • the pharmaceutical compositions can be formulated in liquid solutions, for example physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • compositions may be formulated in solid form and re-dissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • the injection can be, for example, in the form of a bolus injection or continuous infusion (e.g., using infusion pumps) of pharmaceutical composition.
  • the pharmaceutical composition comprising a carbonic anhydrase inhibitor is administered by lateral cerebro ventricular injection into the brain of a subject.
  • the injection can be made, for example, through a burr hole made in the subject's skull.
  • said encapsulated therapeutic agent is administered through a surgically inserted shunt into the cerebral ventricle of a subject.
  • the injection can be made into the lateral ventricles, which are larger, even though injection into the third and fourth smaller ventricles can also be made.
  • the pharmaceutical composition is administered by injection into the cisterna magna, or lumbar area of a subject.
  • the compounds that are useful will generally be provided in unit dosage forms of a tablet, pill, dragee, lozenge or capsule; as a powder or granules; or as an aqueous solution, suspension, liquid, gels, syrup, slurry, etc. suitable for ingestion by the patient.
  • Tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives.
  • suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents.
  • Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
  • compositions for oral use can be obtained through combination of a carbonic anhydrase inhibitor with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
  • Suitable solid excipients in addition to those previously mentioned are carbohydrate or protein fillers that include, but are not limited to, sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredients is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • the compounds will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity.
  • Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride.
  • Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin.
  • suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth
  • a wetting agent such as lecithin.
  • Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
  • the suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperatures and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperatures and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient are cocoa butter and polyethylene glycols.
  • the compounds of the disclosure can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, or aerosols.
  • the compounds useful may also be presented as aqueous or liposome formulations.
  • Aqueous suspensions contain a carbonic anhydrase inhibitor in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene
  • the aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin.
  • preservatives such as ethyl or n-propyl p-hydroxybenzoate
  • coloring agents such as a coloring agent
  • flavoring agents such as aqueous suspension
  • sweetening agents such as sucrose, aspartame or saccharin.
  • Formulations can be adjusted for osmolality.
  • Oil suspensions can be formulated by suspending a carbonic anhydrase inhibitor in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these.
  • the oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose.
  • These formulations can be preserved by the addition of an antioxidant such as ascorbic acid.
  • an injectable oil vehicle see Minto, J. Pharmacol. Exp. Ther. 281 :93-102, 1997.
  • the pharmaceutical formulations can also be in the form of oil-in- water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these.
  • Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate.
  • the emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation or transcutaneous delivery (e.g., subcutaneously or intramuscularly), intramuscular injection or a transdermal patch.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the pharmaceutical compositions also may 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 compounds for use are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable dose will be in the range of 0.01 to 1000 mg per kilogram body weight of the recipient per day.
  • the desired dose is can be presented once daily, but may be dosed as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day.
  • the compounds can be administered as the sole active agent, or in combination with other known therapeutics to be beneficial in the treatment of neurological disorders.
  • the administering physician can provide a method of treatment that is prophylactic or therapeutic by adjusting the amount and timing of drug administration on the basis of observations of one or more symptoms (e.g., motor or cognitive function as measured by standard clinical scales or assessments) of the disorder being treated.
  • Therapeutically effective amounts of a carbonic anhydrase inhibitor may range from about 0.01 to about 1000 milligrams per kilogram (mg/kg).
  • mg/kg milligrams per kilogram
  • a pharmaceutical composition of the disclosure After a pharmaceutical composition of the disclosure has been formulated in an acceptable carrier, it can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include, e.g., instructions concerning the amount, frequency and method of administration.
  • a kit for inhibiting or reversing AP-induced weight gain in a human which includes a carbonic anhydrase inhibitor and instructional material teaching the indications, dosage and schedule of administration of the carbonic anhydrase inhibitor is provided.
  • the ratio between toxicity and therapeutic effect for a particular compound is its therapeutic index and can be 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).
  • LD50 the amount of compound lethal in 50% of the population
  • ED50 the amount of compound effective in 50% of the population.
  • Therapeutic index data obtained from cell culture assays and/or animal studies can be used in formulating a range of dosages for use in humans.
  • the dosage of such compounds can lie within a range of plasma concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. See, e.g. Fingl el al., In. The Pharmacological Basis of Therapeutics, Ch.1, p.l, 1975.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition and the particular method in which the compound is used.
  • a library of compounds was screened to identify compounds that reduce the polyglutamine-induced neurodegeneration and behavior in a Drosophila HD model.
  • the Drosophila HD model used to screen compounds can be generated using standard genetic engineering technology, such as those described below.
  • tissue-specific expression control sequence refers to expression control sequences that drive expression in one tissue or a subset of tissues, while being essentially inactive in at least one other tissue. "Essentially inactive” means that the expression of a sequence operatively linked to a tissue-specific expression control sequence is less than 5% of the level of expression of that sequence in that tissue where the expression control sequence is most active.
  • tissue-specific expression control sequences include those that are specific for organs such as the eye, wing, notum, brain, as well as tissues of the central and peripheral nervous systems. Tissue-specific expression control sequences can either be used as a "driver" in the UAS-GAL4 system, or alternatively can be linked to a transgene to control its expression in a cis acting manner.
  • tissue specific control sequences include but are not limited to: promoters/enhancers important in eye development, such as sevenless (Bowtell et al., Genes Dev. 2:620-34 (1988)), eyeless (Bowtell et al., Proc. Natl. Acad. Sci. U.S.A.
  • promoters/enhancers derived from any of the rhodopsin genes, that are useful for expression in the eye; enhancers/promoters derived from the dpp or vestigial genes useful for expression in the wing (Staehling-Hampton et al., Cell Growth Differ. 5:585-93 (1994), Kim et al., Nature 382:133-8 (1996)); promoters/enhancers specific for nerve, e.g., elav (Yao and White, J. Neurochem.
  • expression control sequences include, but are not limited to the heat shock promoters/enhancers from the hsp70 and hsp83 genes, useful for temperature induced expression; and promoters/enhancers derived from ubiquitously expressed genes, such as tubulin, actin, or ubiquitin.
  • Transgenic flies that have incorporated into their genome a DNA sequence that encodes exons 1-4 of huntingtin can be utilized.
  • the transgenic flies for screening compounds for treating neurological diseases can be generated by any means known to those skilled in the art. Methods for production and analysis of transgenic Drosophila strains are well established and described in Brand et al., Methods in Cell Biology 44:635-654 (1994); Hay et al., Proc. Natl. Acad. Sci. USA 94(10):5195-200 (1997); and in Drosophila: A Practical Approach (ed. D. B. Roberts), ppl75-197, IRL Press, Oxford, UK (1986), herein incorporated by reference in their entireties.
  • a transgene of interest is stably incorporated into a fly genome.
  • Any fly can be used, such as a member of the Drosophilidae family.
  • An exemplary fly is Drosophila melanogaster.
  • a variety of transformation vectors are useful for the generation of transgenic flies, and include, but are not limited to, vectors that contain transposon sequences, which mediate random integration of a transgene into the genome, as well as vectors that use homologous recombination (Rong and Golic, Science 288:2013-2018 (2000)).
  • One vector is pUAST (Brand and Perrimon, Development 118:401-415 (1993)) which contains sequences from the transposable P-element which mediate insertion of a transgene of interest into the fly genome.
  • Another vector is PdL, which is able to yield doxycycline-dependent overexpression (Nandis, Bhole and Tower, Genome Biology 4 (R8):l-14, (2003)).
  • Yet another vector is pExP-UAS because of its ease of cloning and mapping genomic location.
  • P-element transposon mediated transformation is a commonly used technology for the generation of transgenic flies and is described in detail in Spradling, P element mediated transformation, in Drosophila: A Practical Approach (ed. D. B. Roberts), pp 175- 197, IRL Press, Oxford, UK (1986), herein incorporated by reference.
  • Other transformation vectors based on transposable elements include, for example, the hobo element (Blackman et al., Embo J.
  • the mariner element Lidholm et al., Genetics 134:859-68 (1993)), the hermes element (O'Brochta et al., Genetics 142:907-14 (1996)), the Minos element (Loukeris et al., Proc. Natl. Acad. Sci. USA 92:9485-9 (1995)), or the piggyBac element (Handler et al., Proc. Natl. Acad. Sci. USA 95:7520-5 (1998)).
  • the terminal repeat sequences of the transposon that are required for transposition are incorporated into a transformation vector and arranged such that the terminal repeat sequences flank the transgene of interest.
  • the transformation vector contains a marker gene used to identify transgenic animals.
  • marker genes affect the eye color of Drosophila, such as derivatives of the Drosophila white gene (Pirrotta V., & C. Brockl, EMBO J. 3(3):563-8 (1984)) or the Drosophila rosy gene (Doyle W. et al, Eur. J Biochem. 239(3):782-95 (1996)) genes. Any gene that results in a reliable and easily measured phenotypic change in transgenic animals can be used as a marker. Examples of other marker genes used for transformation include the yellow gene (Wittkopp P. et al., Curr Biol.
  • coli the neomycin 1 gene from the E.coli transposon Tn5; and the green fluorescent protein (GFP; Handler and Harrell, Insect Molecular Biology 8:449-457 (1999)), which can be under the control of different promoter/enhancer elements, e.g. eyes, antenna, wing and leg specific promoter/enhancers, or the poly-ubiquitin promoter/enhancer elements.
  • promoter/enhancer elements e.g. eyes, antenna, wing and leg specific promoter/enhancers, or the poly-ubiquitin promoter/enhancer elements.
  • Plasmid constructs for introduction of the desired transgene are coinjected into Drosophila embryos having an appropriate genetic background, along with a helper plasmid that expresses the specific transposase needed to incorporate the transgene into the genomic DNA.
  • Animals arising from the injected embryos (GO adults) are selected, or screened manually, for transgenic mosaic animals based on expression of the marker gene phenotype and are subsequently crossed to generate fully transgenic animals (Gl and subsequent generations) that will stably carry one or more copies of the transgene of interest.
  • transgenic Drosophila termed “target” lines
  • the gene of interest e.g., huntingtin
  • an appropriate promoter e.g., hsp70 TATA box, see Brand and Perrimon, Development 118:401-15 (1993)
  • driver lines are generated where the GAL4 coding region is operatively linked to promoters/enhancers that direct the expression of the GAL4 activator protein in specific tissues, such as the eye, antenna, wing, or nervous system or by induction, such as by heat shock.
  • the gene of interest is not expressed in the "target" lines for lack of a transcriptional activator to "drive” transcription from the promoter joined to the gene of interest.
  • the gene of interest is induced.
  • the resultant progeny display a specific pattern of expression that is characteristic for the GAL4 line.
  • Driver strains for use include, for example, apterous -GdXA for expression in wings, brain, and interneurons; elav- GAL4 for pan-neuronal expression in post-mitotic neurons; scabrous-GALA for pan-neuronal expression in the developing nervous system from neuroblasts to neurons; sevenless-GdXA, eyeless-Gd ⁇ A, and GMR-GaH for expression in eyes; Nervana 2-Gal4 for expression in the central nervous system; Cha- (choline acetyltransferase) Gal4 for expression in cholinergic neurons, TH- (tyrosine hydroxylase) for expression in dopaminergic neurons; CaMKII- (calmodulin dependent kinase II) for expression in the central nervous system of embryos and larvae as well as the brain, throacic ganglion, and gut of adults; P-GaW for expression in pharangeal sensory neurons; and gcm-GdXA for expression in glial cells.
  • transgenic Drosophila are produced using the UAS/GAL4 control system. Briefly, to generate a transgenic fly that expresses a mutant form of huntingtin containing a 128Q repeat in exon 1, a DNA sequence encoding httl28Q is cloned into a vector such that the sequence is operatively linked to the GAL4 responsive element UAS.
  • Vectors containing UAS elements are readily available in the art, such as the pUAST vector (Brand and Perrimon, Development 118:401-415 (1993)), which places the UAS sequence element upstream of the transcribed region.
  • the DNA is cloned using standard methods (Sambrook et al., Molecular Biology: A laboratory Approach, Cold Spring Harbor, N.Y. (1989); Ausubel, et al., Current protocols in Molecular Biology, Greene Publishing, Y, (1995)) and is described in more detail under the Molecular Techniques section of the present disclosure.
  • appropriate vector such as pUAST
  • the vector is injected into Drosophila embryos (e.g. yw embryos) by standard procedures (Brand et al, Methods in Cell Biology 44:635-654 (1994); Hay et al., Proc. Natl. Acad. Sci. USA 94:5195- 200 (1997)) to generate transgenic Drosophila.
  • the transgenic progeny can be crossed with Drosophila driver strains to assess the presence of an altered phenotype caused by expression of the transgene.
  • a desirable Drosophila comprises the eye specific driver strain GMR-GALA, which enables identification and classification of transgenic flies based on the severity of a eye phenotype.
  • locomotor and behavioral phenotypes e.g., climbing assay
  • an elav- GAL4 driver strain is used in the cross.
  • Ectopic overexpression of httl28Q in Drosophila central nervous system (CNS) results in locomotor deficits, such as impaired movement, climbing and flying.
  • proteins such as huntingtin in transgenic flies is confirmed by standard techniques, such as Western blot analysis or by immunostaining of fly tissue cross-sections, both of which are described below.
  • Locomotor phenotypes can be assessed, for example, as described in U.S. Application Nos. 2004/0076583, 2004/0076318, and 2004/0076999, each of which is hereby incorporated by reference in its entirety.
  • locomotor ability can be assessed in a climbing assay by placing flies in a vial, knocking them to the bottom of the vial, then counting the number of flies that climb past a given mark on the vial during a defined period of time.
  • 100% locomotor activity of control flies is represented by the number of flies that climb past the given mark, while flies with an altered locomotor activity can have 80%, 70%, 60%, 50%, less than 50%, or less than 30% of the activity observed in a control fly population.
  • the traits are measured by detecting and serially analyzing the movement of a population of flies in containers, e.g., vials. Movement of the flies can be monitored by a recording instrument, such as a CCD-video camera, the resultant images can be digitized, analyzed using processor-assisted algorithms as described herein, and the analysis data stored in a computer-accessible manner.
  • a recording instrument such as a CCD-video camera
  • the resultant images can be digitized, analyzed using processor-assisted algorithms as described herein, and the analysis data stored in a computer-accessible manner.
  • the trajectory of each animal may be monitored by calculation of one or more variables (e.g., speed, vertical only speed, vertical distance, turning frequency, frequency of small movements, etc.) for the animal. Values of such a variable are then averaged for population of animals in the vial and a global value is obtained describing the trait for each population (e.g., parental stock flies and transgenic flies).
  • Movement trait data refers to the measurements made of one or more movement traits. Examples of “movement trait data” measurements include, but are not limited to X-pos, X-speed, speed, turning, stumbling, size, T-count, P-count, T-length,
  • Examples of such "movement traits” include, but are not limited to: a) total distance (average total distance traveled over a defined period of time); b) X only distance (average distance traveled in X direction over a defined period of time; c) Y only distance (average distance traveled in Y direction over a defined period of time); d) average speed (average total distance moved per time unit); e) average X-only speed (distance moved in X direction per time unit); f) average Y-only speed (distance moved in Y direction per time unit); g) acceleration (the rate of change of velocity with respect to time); h) turning; i) stumbling; j) spatial position of one fly to a particular defined area or point (examples of spatial position traits include (1) average time spent within a zone of interest (e.g., time spent in bottom, center, or top of a container; number of visits to a defined zone within container); (2) average distance between a fly and a point of interest (e.g., the
  • Movement traits can be quantified, for example, using the following parameters:
  • the X-Pos score is calculated by concatenating the lists of x-positions for all trajectories and then computing the average of all values in the concatenated list
  • the X-Speed score is calculated by first computing the lengths of the x- components of the speed vectors by taking the absolute difference in x-positions for subsequent frames. The resulting lists of x-speeds for all trajectories are then concatenated and the average x-speed for the concatenated list is computed.
  • Turning The Turning score is calculated in the same way as the Speed score, but instead of using the length of the speed vector, the absolute angle between the current speed vector and the previous one is used, giving a value between 0 and 90 degrees.
  • Stumbling The Stumbling score is calculated in the same way as the Speed score, but instead of using the length of the speed vector, the absolute angle between the current speed vector and the direction of body orientation is used, giving a value between 0 and 90 degrees.
  • Size The Size score is calculated in the same way as the Speed score, but instead of using the length of the speed vector, the size of the detected fly is used.
  • T-Count The T-Count score is the number of trajectories detected in the movie.
  • P -Count The P-Count score is the total number of points in the movie (i.e., the number of points in each trajectory, summed over all trajectories in the movie).
  • T-Length The T-Length score is the sum of the lengths of all speed vectors in the movie, giving the total length all flies in the movie have walked.
  • F -Count The F-Count score counts the number of detected flies in each individual frame, and then takes the maximum of these values over all frames. It thereby measures the maximum number of flies that were simultaneously visible in any single frame during the movie.
  • X refers to the vertical direction (typically along the long axis of the container in which the flies are kept) and "Y” refers to movement in the horizontal direction (e.g., along the surface of the vial).
  • Neuronal degeneration in the central nervous system will give rise to alterations in behavioral phenotype, including but not limited to locomotor deficits, that can be assayed and quantified in both larvae and adult Drosophila.
  • behavioral phenotype including but not limited to locomotor deficits, that can be assayed and quantified in both larvae and adult Drosophila.
  • failure of Drosophila adult animals to climb in a standard climbing assay see, e.g. Ganetzky and Flannagan, J. Exp. Gerontology 13:189-196 (1978); LeBourg and Lints, J. Gerontology 28:59-64 (1992)
  • Neurodegenerative phenotypes include, but are not limited to, progressive loss of neuromuscular control, e.g.
  • fly behavior that can be assayed include but are not limited to circadian behavioral rhythms, feeding behaviors, courtship behaviors, inhabituation to external stimuli, and odorant conditioning. All of these phenotypes are measured by one skilled in the art by standard visual observation of the fly.
  • Another neural degeneration phenotype is a reduced life span, for example, the Drosophila life span can be reduced by 10-80%, e.g., approximately, 30%, 40%, 50%, 60%, or 70%.
  • the flies are then placed at a "choice point," where the odors come from opposite directions, and expected to decide which odor to avoid.
  • learning is defined as the fly's performance when testing occurs immediately after training.
  • a single training trial produces strong learning: a typical response is that >90% of the flies avoid the CS+.
  • Performance of wild-type flies from this single-cycle training decays over a roughly 24-hour period until flies once again distribute evenly between the two odors.
  • Flies can also form long-lasting associative olfactory memories, but normally this requires repetitive training regimens.
  • a common feature of many neurodegenerative diseases is the presence of protein aggregation in the brain.
  • Examples of neurodegenerative diseases characterized by protein aggregation include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Pick's disease, prion diseases, and other spongiform encephalopathies.
  • Female “activator” or “driver” flies were engineered to contain the neuronal specific drivers elav-GAL4 or nervana-GAL4 in a heat shock-/zz ⁇ i Y (phs-hidY or HS-hid+ on Y chomosome) background (Starz-Gaiano et al., 2001).
  • the hid gene is a pro-apoptotic gene, which is expressed in this line by a heat inducible promoter.
  • These flies were heat shocked in early development to kill male larvae and embryos and thereby produce massive numbers of elav-GAL4 or nervana-GAL4 virgin females.
  • Fl generation Their progeny, the Fl generation, were the "assay flies" (e.g., elav-GAL4 I UAS-httl28Q[F27B]). These assay flies were robotically distributed into drugged assay vials.
  • assay flies e.g., elav-GAL4 I UAS-httl28Q[F27B]
  • the HD Drosophila model expresses exons 1 to 4 of the human huntingtin gene with 128Q repeats in exon 1.
  • the construct was cloned into the Drosophila transformation vector, pUAST. Flies from a resulting transgenic line were then crossed to GAL4 driver lines that direct expression either in the eye (GMR-GAL4 promoter) or in neurons (elav-GAL4 promoter).
  • GAL4 driver lines that direct expression either in the eye (GMR-GAL4 promoter) or in neurons (elav-GAL4 promoter).
  • the model shows degeneration of photoreceptors when the transgene was expressed in the eye. Progressive degeneration of the photoreceptors was observed after several days of adult life.
  • HD128Q flies were consistent with those reported by others for two other strains in which exon- 1 -containing expanded repeats was expressed in the eye (Jackson et al, 1998; Steffan et al, 2001 -US20040142859).
  • HD128Q lines were examined in which the transgene was expressed in all neurons. Motor function was assayed by tapping flies in a vial to the bottom and measuring the number of flies that are able to climb to a specified height within a specific length of time. When tapped to the bottom of a vial, wild-type flies rapidly climb to the top, where most of them remain.
  • the HD128Q transgenic lines showed normal behavior early in life. Starting at 10-12 days of life, however, the transgenic flies were observed to make short abortive climbs and fall back to the bottom. The decline in motor performance was progressive, leading to early death. Examination of the HD128Q flies under the microscope revealed that their motor dysfunction was the result of spontaneous and uncoordinated movement of the limbs, which inhibited normal locomotion. Examination of the neurons in these flies showed progressive degeneration and loss of particular neurons, accompanied by the formation of nuclear inclusions that stain positively for molecular chaperones, ubiquitin, and components of the proteasome.
  • the HD128Q model thus has a functional deficit that in its phenotypic and neuropathological characteristics appears to be related to that seen in the human disease. Moreover, as the deficit is quantifiable and reproducible, it is suitable for automated high- throughput screening. [0090]
  • Example 2 Locomotor Activity Assay Using the Transgenic Drosophila HD Model
  • HDAC histone deacetylase
  • the trajectories consisted of a list of x- and y- coordinates mapping out the positions of all the flies in the video, as well as measurements of size, orientation, length and width of the flies, with one list entry for every frame from when the fly started moving in one frame until it stopped in another. These data were then used to provide different scoring metrics for the test flies' behavior.
  • the definitions of 11 metrics developed are summarized above.
  • Average speed of the Drosophila flies were measured by a robot at both early and late speed, and early and late speed summary metrics were created. Early speed was the average speed of eight replicate vials, ten flies/vial, four repeat videos per trial day, averaged over trial days 1-7. Late speed was determined in the same manner but measured and averaged over days 8-10. Effect sizes were the differences between average mean speeds of the summary metrics (i.e., differences between DMSO-carrier control and test drug) divided by the pooled standard deviation in the whole assay.
  • Carbonic anhydrase(s) are a collection of at least 14 Zinc containing metalloenzymes that are separated in three groups designated ⁇ , ⁇ , and ⁇ .
  • the enzymes catalyze the reversible dehydration of bicarbonate (HC(V) which is characterized by Equation 1.
  • the enzymes are also known to catalyze other chemical and biochemical reactions such as the hydrolysis of esters and lipids.
  • a carbonic anhydrase inhibitor is a compound that reduces the ability of a carbonic anhydrase to catalyze the conversion depicted in Equation 1 and/or one or more additional chemical or biochemical reactions catalyzed by a carbonic anhydrase.
  • the reaction may be inhibited from progressing in any one or both directions.
  • Examples of carbonic anhydrase inhibitors can be found in the following patents and applications US 5,095,026, US 5,157,044, US 6,214,381, US 6,683,066, US 6,242,442, US 2006/0088886A1, US 6,620,819, hereby incorporated by reference. The examples found in these documents are not meant to be limiting. Further examples of carbonic anhydrase inhibitors are depicted below. Other examples of carbonic anhydrase inhibitors include: brinzolamide and ethoxbenzolamide.
  • Ocufen sodium 2-(3-fluoro-4-phenyl-phenyl)propanoate dehydrate
  • Furbiprofen - ((2R)-2-(3-fluoro-4-phenyl-phenyl)propanoic acid)
  • homosulfamine (4-(aminomethyl)benzenesulfonamide hydrochloride
  • probenecid (4- (dipropylsulfamoyl)benzoic acid)
  • mafenide (4-(aminomethyl)benzenesulfonamide).
  • Other carbonic anhydrase inhibitors are known to those of ordinary skill in the art.
  • One of ordinary skill in the art will recognize that certain carbonic anhydrase inhibitors can exist in multiple tautomeric forms. ,4- mide,5-dichl
  • Figure 1 shows a comparison of the age-dependent decline in speed of locomotion between the wild type flies treated with vehicle (DMSO) and HD model flies treated with vehicle, HD model flies treated with the positive control compounds Trichostatin A (TSA) and different concentrations of dorzolamide (DRZ).
  • DMSO vehicle
  • TSA Trichostatin A
  • DTZ dorzolamide
  • Figure 2 shows a comparison of the age-dependent decline in speed of locomotion between the wild type flies treated with vehicle (DMSO) and HD model flies treated with vehicle, HD model flies treated with the positive control compounds Trichostatin A (TSA) and different concentrations of dichlorphenamide (DCP).
  • TSA Trichostatin A
  • DCP dichlorphenamide
  • the phenotype measured is the progressive decline in motor coordination and reduction in average walking speed following stimulation (tapping of the vial).
  • the HD model flies also had a reduced lifespan (data not shown).
  • the calculated effect sizes were weighted averages across experiments, using sample size as weight. Not all campaigns had the same number of replicates. The more replicates then a greater weight is attributed.
  • the effect sizes shown in green are the calculated effect size averaged over at least three different assays with a total n ⁇ 21. Data shown in the white boxes are effect sizes in at least one assay with n greater than or equal to 8.
  • Female “activator” or “driver” flies were engineered to contain the neuronal specific drivers elav-GAL4 or Cha-GAL4 in a heat shock-hid Y (phs-hidY or HS-hid+ on Y chomosome) background (Starz-Gaiano et al., 2001).
  • the hid gene is a pro-apoptotic gene, which is expressed in this line by a heat inducible promoter.
  • These flies were heat shocked in early development to kill male larvae and embryos and thereby produce massive numbers of elav-GAL4 or Cha-GAL4 virgin females.
  • the AD Drosophila model contains the coding sequence of the carboxy-terminal portion of the human amyloid precursor protein gene (APP-CT), often referred to as the "C99" or "ClOO" fragment linked to a human APP signal sequence.
  • APP-CT human amyloid precursor protein gene
  • the construct was cloned into the Drosophila transformation vector, pExP-UAS.
  • the human wild- type tau coding sequence was cloned into the pUAST Drosophila transformation vector. Both constructs were then incorporated into the fly to generate the AD disease model transgenic line.
  • GAL4 driver lines that direct expression either in the eye (GMR-GAL4 promoter) or in cholinergic neurons (Cha-GAL4 promoter).
  • the model shows degeneration of photoreceptors when the transgenes were expressed in the eye.
  • APP-CT tau lines were examined in which the transgenes were expressed in cholinergic neurons. Motor function was assayed by tapping flies in a vial to the bottom and measuring the number of flies that are able to climb to a specified height within a specific length of time.
  • Example 4 Locomotor Activity Assay Using the Transgenic Drosophila AD Model
  • HDAC histone deacetylase
  • TSA Trichostatin-A
  • tacrine the histone deacetylase inhibitor
  • the trajectories consisted of a list of x- and y- coordinates mapping out the positions of all the flies in the video, as well as measurements of size, orientation, length and width of the flies, with one list entry for every frame from when the fly started moving in one frame until it stopped in another. These data were then used to provide different scoring metrics for the test flies' behavior.
  • the definitions of 11 metrics developed are summarized above.
  • the percent of trajectories in the top of the vial for last 4.5 seconds of the movie was measured by dividing the number of trajectories in the top three centimeters of the vial for A- 7.5 seconds in each movie.
  • the summary metric percoftraj days 1-7 was calculated similarly as the metrics above by averaging over eight replicate vials, ten flies/vial, four repeat videos/trial day for trial days 1-7.
  • the mean of all detected fly speeds was calculated as the distance moved per frame for all pairs of detected points in all fly trajectories in the movie or movie segment.
  • WeightedSec4Speed was the mean of the detected fly speeds weighted by the time the detected speed occurred.
  • the summary metric for days 1-10 was calculated similarly to the metrics above.
  • Figure 5 is a table presenting the effect sizes of AD model flies treated with carbonic anhydrase inhibitors at various concentrations.

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Abstract

La présente invention concerne des procédés de traitement d'un sujet, tel qu'un animal ou un humain, atteint d'un trouble neurologique ou à risque de développer un trouble neurologique (par exemple, un patient qui a un défaut génétique associé à un trouble neurologique, mais ne présente aucun symptôme normalement associé au trouble neurologique) par l'administration au sujet souffrant du trouble neurologique d'une quantité efficace d'un inhibiteur d'anhydrase carbonique.
PCT/US2007/085358 2006-11-22 2007-11-21 Procédé de traitement de troubles neurologiques avec des inhibiteurs d'anhydrase carbonique WO2008064296A1 (fr)

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CN102716124B (zh) * 2012-05-25 2014-07-23 中国人民解放军军事医学科学院卫生学环境医学研究所 一种碳酸酐酶抑制剂的用途
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