WO2003099299A1 - Agents et methode pour le traitement de troubles associes a la degenerescence motoneuronale - Google Patents

Agents et methode pour le traitement de troubles associes a la degenerescence motoneuronale Download PDF

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WO2003099299A1
WO2003099299A1 PCT/AU2003/000645 AU0300645W WO03099299A1 WO 2003099299 A1 WO2003099299 A1 WO 2003099299A1 AU 0300645 W AU0300645 W AU 0300645W WO 03099299 A1 WO03099299 A1 WO 03099299A1
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glur3
agent
receptor subunit
ampa receptor
subject
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PCT/AU2003/000645
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English (en)
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Surindar Singh Cheema
Steven Langford
Stephen Bruce
Bradley Turner
Rachel Scott
Nam Sang Cheung
Philip Mark Beart
Alan Rembach
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Monash University
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Priority to AU2003229380A priority Critical patent/AU2003229380A1/en
Publication of WO2003099299A1 publication Critical patent/WO2003099299A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • 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
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA

Definitions

  • the present invention relates to agents and methods for treating neurological disorders, and more particularly to peptide nucleic acids that regulate expression of glutamate receptors such as GluR3, and uses of those peptide nucleic acids.
  • ALS amyotrophic lateral sclerosis
  • RILUZOLE 2-amino-6-trifluoromethoxybenzothiazole
  • the antioxidant vitamin E has been suggested as an alternative treatment for ALS. This suggestion arose out of the observation that lipid peroxidation may effect motor neurons.
  • vitamin E has proven unable to prolong survival in ALS mouse models.
  • the present invention arises out of the discovery of elevated levels of a glutamate receptor subunit in the lumbar spinal cord of a mouse model (G93A SOD1) of ALS.
  • the specific receptor is known as GluR3.
  • This discovery suggested that up-regulation of a glutamate receptor subunit plays a key role in motor neuron degeneration, and formed the basis for studies which showed that down regulation of expression of a glutamate receptor subunit in a mouse model for ALS resulted in improved motor scores in treated mice relative to control mice.
  • amino acid glutamate acts as an excitatory neurotransmitter at many synapses in the central nervous system, lonotropic glutamate receptors are expressed by motor neurons and when glutamate binds to its specific receptor a series of rapid conformational changes occur that open an ion channel through the cell membrane and permit extracellular ions to flow down the concentration gradients.
  • the ionotropic glutamate receptors are divided into different glutamate receptor subtypes. Each subtype is named according to the agonist that binds the specific receptor, namely NMDA, oc-amino-3-hydroxy-5- methylisoxazolepropionic acid (hereafter referred to as 'AMPA'), Kainate and quisqualate.
  • NMDA oc-amino-3-hydroxy-5- methylisoxazolepropionic acid
  • Kainate oc-amino-3-hydroxy-5- methylisoxazolepropionic acid
  • AMPA receptors are pentameric in structure and consist of combinations of four subunits termed GluR1 , GluR2, GluR3 and GluR4. High levels of sequence identity exist between GluR1 and GluR2 (70%), GluR1 and GluR3 (69%), as well as between GluR2 and GluR3 (74%). Each subunit contains four transmembrane regions (TM1 through to TM4).
  • the present invention provides a method for treating a subject that is afflicted with a disorder associated with motor neuron degeneration, the method including the step of inhibiting or preventing up-regulation of an AMPA receptor subunit protein in the subject.
  • the AMPA receptor subunit is GluR3.
  • the present invention also provides a method for treating a subject that is afflicted with a neurological disorder associated with motor neuron degeneration, the method including the step of administering to the subject a therapeutically effective dose of an agent that inhibits or prevents up-regulation of an AMPA receptor subunit in the subject.
  • the present invention also provides a therapeutic agent that is suitable for administration to a subject that is afflicted with a neurological disorder associated with motor neuron degeneration, wherein the agent modifies the activity of an AMPA receptor subunit.
  • the agent is a peptide nucleic acid (PNA).
  • the present invention also provides a composition for treating a neurological disorder, the composition including an agent of the present invention, and a pharmaceutically acceptable carrier.
  • the present invention also provides a method for diagnosing a disorder associated with motor neuron degeneration in a subject, the method including the step of measuring the levels of an AMPA receptor subunit protein expressed in the subject and comparing the measured level with the levels of the AMPA receptor subunit protein in a subject that is not afflicted with a disorder associated with motor neuron degeneration.
  • the AMPA receptor subunit is GluR3.
  • FIG 1 shows the amino acid sequence differences within transmembrane domain 2 (TM2) of the AMPA receptor subunits.
  • Figure 2 shows the amino acid sequence selected for the design of the PNAs of the present invention.
  • Figure 3 shows the results of two-dimensional electrophoresis of wildtype and SOD1 G93A G1H transgenic mice protein expression.
  • Figure 4 shows the phase contrast observations of NSC-34D cells in culture.
  • A Phase contrast image of NSC-34 D cells after 24 hours in culture.
  • B Phase contrast image of NSC-34 D cells after 24-48 hours in culture.
  • C Phase contrast image of NSC-34 D cells after 72-96 hours in culture.
  • D Detection of neuronal markers by Western blot of SDS-PAGE analysis of NSC-34 D cell lysates after 72 hours in culture.
  • E Detection of AMPA receptor subunit proteins by Western blot of
  • Figure 5 shows a series of plots demonstrating the effect of L-glutamic acid on MTT reduction by NSC-34 D cells.
  • A NSC-34D cells exposed to L-glutamate.
  • Figure 6 shows a series of photomicrographs that show the fluorescent PNA distribution in NSC-34 D cells.
  • A DAPI stained cells 1 hour after treatment with fluorescent conjugated
  • B Puncate stained cells 1 hour after treatment with fluorescent conjugated
  • APNG3 12 Overlay of images from (D) and (E).
  • G Western blot of SDS-PAGE analysis of NSC-34 D cell lysates after treatment with APNG3 ⁇ 2 .
  • H Western blot of SDS-PAGE analysis of NSC-34 D cell lysates after treatment with NPNG3 ⁇ 2 .
  • I Western blot of SDS-PAGE analysis of NSC-34 D cell lysates after treatment with SPNG3 ⁇ 2 .
  • Figure 7 shows the effect of APNAG3 ⁇ 2 (A), NPNAG3 12 (B) and SPNAG3 i2 (C) treatment on MTT reduction of NSC-34 D cells after exposure to (S)-5-FW.
  • Figure 8 shows the results of tests of the locomotor performance of grouped
  • A Rotorod performance illustrating the locomotor capability of pooled animals receiving ANAPG3 12 (solid line) and NPNAG3 ⁇ 2 (dashed line).
  • B Statistical representation of the weights of pooled animals
  • NPNAG3i2 (dashed line).
  • D Western blot of SDS-PAGE analysis of lumbar spinal cord extracts after treatment with ANAPG3 12 and NPNAG3 ⁇ 2 .
  • inhibition of up-regulation need not be 100% inhibition.
  • “inhibition" of up-regulation of GluR3 refers to a decrease in up-regulation that is sufficient to provide a health benefit to the subject.
  • inhibition of up-regulation of GluR3 by at least 50% may be sufficient to provide a health benefit.
  • up-regulation of GluR3 is inhibited by at least 70% and more preferably by at least 90%.
  • the term 'subject' as used throughout the specification is to be understood to mean any multi-cellular system and includes isolated groups of cells to whole organisms.
  • the subject may be cells in tissue culture, a tissue or organ, or any mammalian single subject for which therapy is desired, including (but not limited to) humans, cattle, dogs, guinea pigs, rabbits, pigs, horses, or chickens. Most preferably, the subject is a human.
  • the term 'therapeutically effective dose' as used throughout the specification refers to that amount of an agent sufficient to result in a health benefit in the treated subject.
  • the therapeutically effective dose is 1 to 10 mg/kg. More preferably, the therapeutically effective dose is 1 to 5 mg/kg. Most preferably the therapeutically effective dose is about 2.5 mg/kg.
  • 'peptide nucleic acid' and 'PNA' as used throughout the specification are used interchangeably and are intended to refer to a class of compounds that includes ligands such as naturally occurring or synthetic DNA bases attached to a peptide backbone through a suitable link or linker. This may include polymers of two or more PNA subunits such as a PNA oligomer linked together by, for example, amide bonds.
  • transgenic mice expressing a mutant human CuZn-SOD1 gene with a gly93 ⁇ ala substitution were obtained from commercial sources (Jackson Laboratories, USA) and used for studies on the experession of AMPA receptor subunit(s).
  • This mouse carries a mutation found in human familial ALS. The disease onset and progression have many similarities with the human condition including limb paralysis and death.
  • This model is internationally accepted as a model for human ALS (see Gurney, M. E (1997). "The use of transgenic mouse models of amyotrophic lateral sclerosis in preclinical drug studies.” Journal of the Neurological Sciences vol. 152:S67-73).
  • amino acid sequence homology of all AMPA receptor subunits were obtained from the Genebank databases (GluR1 NM008165), (GluR2 NM013540), (GluR3 NM016886) and (GluR4 MN019691).
  • a unique target sequence for antisense action was identified within the signal peptide region of the GluR3 subunit (see figures 1 and 2).
  • An antisense peptide nucleic acid was designed and synthesised to down-regulate the expression of GluR3 in G93A SOD1 mice.
  • PNAs are a class of antisense compounds that are highly stable and readily penetrate the blood brain barrier.
  • the sequence of anti-GluR3 PNA is C-GTA AGA GTG CCT-N.
  • mice received intraperitoneal injections of anti-GluR3 PNA three times weekly until each animal reached its own disease endpoint. Mice treated with anti- GluR3 PNA lived significantly longer than those receiving nonsense PNA. Antisense treated mice showed improved survival of four, eight and twenty days beyond a control group. The prolonged survival of mice receiving antisense treatment correlated with the later onset of paralysis seen in the behavioural tests that were conducted.
  • GluR3 when expressed as a homomeric receptor or heteromeric receptor lacking GluR2 produces AMPA receptors that are permeable to calcium. Calcium loading through these AMPA receptors may account for the selective vulnerability of motor neurons seen in ALS. Accordingly, up-regulation of GluR3 expression may increase Ca 2+ influx to excititoxic levels.
  • the present invention provides a method for treating a subject that is afflicted with a disorder associated with motor neuron degeneration, the method including the step of inhibiting or preventing up- regulation of an AMPA receptor subunit in the subject.
  • AMPA receptors are pentameric in structure and consist of combinations of four subunits termed GluR1 , GluR2, GluR3 and GluR4.
  • the glutamate receptor subunit could be any one of GluR1 , GluR2, GluR3 and/or GluR4, although it is preferably GluR3.
  • the disorder associated with motor neuron degeneration may be any neurological disorder.
  • Non-specific X-linked mental retardation, Rasmussen's encephalitis, Parkinson's disease, Alzheimer's disease, Rett syndrome and ALS are some examples of disorders that are associated with motor neuron degeneration.
  • the subject is afflicted with ALS.
  • the step of inhibiting or preventing up-regulation of an AMPA receptor subunit in the subject may involve administering to the subject in need of treatment a therapeutically effective dose of an agent that is capable of inhibiting or preventing the up-regulation of the AMPA receptor subunit either directly or indirectly.
  • the agent may be any organic compound, amino acid, peptide, protein, peptide nucleic acid, nucleotide or combination of any two or more of these that modifies the activity of the AMPA receptor subunit and interferes with expression of the receptor subunit protein.
  • the agent may bind or otherwise interfere with the function of either AMPA receptor subunit protein or receptor.
  • the function of the AMPA receptor subunit in a subject that is afflicted with a disorder associated with motor neuron degeneration is modified.
  • the agent may inhibit or prevent up-regulation of the AMPA receptor subunit by binding to a nucleotide sequence in a gene that expresses the AMPA receptor subunit.
  • the agent preferably binds to a nucleotide sequence that codes an amino acid sequence in GluR3 that is non-homologous with the amino acid sequences of GluR1 , 2 and 4.
  • the non-homologous amino acid sequence is located within exon 1 of the GluR3 protein.
  • the non-homologous amino acid sequence is his-ser- his (HSH).
  • amino acid sequences are presented in the conventional manner with the N-terminal amino acid on the left of the sequence, and the C-terminal amino acid on the right of the sequence.
  • the agent that interferes with expression of the AMPA receptor subunit is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replaced with a polyamide backbone which is similar to that found in peptides.
  • PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind more strongly to a complementary DNA sequence than to a naturally occurring nucleic acid molecule due to the lack of charge repulsion between the PNA strand and the DNA strand.
  • the PNAs of the present invention may also include other nucleotides comprising polymer backbones, cyclic backbones, or acyclic backbones.
  • the nucleotides may comprise morpholino backbone structures.
  • the PNAs of the present invention are "nuclease resistant" when they have either been modified such that they are not susceptible to degradation by DNA and RNA nucleases or alternatively they have been placed in a delivery vehicle which in itself protects the oligonucleotide from DNA or RNA nucleases.
  • Nuclease resistant oligonucleotides include, for example, methyl phosphonates, phosphorothioates, phosphorodithioates, phosphotriesters, and morpholino oligomers.
  • Suitable delivery vehicles for conferring nuclease resistance include, for example Iiposomes.
  • the PNAs of the present invention may also contain groups, such as groups for improving the pharmacokinetic properties of nucleotides, or groups for improving the pharmacodynamic properties of nucleotides.
  • the PNAs of the present invention generally comprise from at least about 3 nucleotides or nucleotide analogues, preferably from about 3 to about 100 nucleotides or nucleotide analogues, more preferably, from about 3 to about 50 nucleotides or nucleotide analogues, most preferably from about 9 to about 12 nucleotide or nucleotide analogues.
  • GluR3 receptor subunit may be inhibited by administration a peptide nucleic acid that contains the sequence C-GTA AGA GTG-N, or a functional equivalent thereof.
  • a particularly preferred peptide nucleic acid is C- GTA AGA GTG CCT-N.
  • the peptide nucleic acid could also be a functional equivalent or active derivative of the peptide or nucleic acid of C-GTA AGA GTG CCT-N.
  • a derivative or functional equivalent of a peptide or nucleic acid is intended to include homologous peptides or nucleic acids in which conservative amino acid or nucleotide substitutions have been made, as well as to include amino acid or nucleotide substitutions that result in a peptide nucleic acid that retains its function.
  • the peptide nucleic acid may also include mutations, such as substitutions, insertions and deletions.
  • the derivative or functional equivalent shares at least 70% homology, and more preferably at least 90% homology, with the peptide nucleic acid C-GTA AGA GTG CCT-N.
  • a functional equivalent or derivative of the peptide nucleic acid C-GTA AGA GTG CCT-N is capable of inhibiting up regulation
  • the person skilled in the art can use any one or more of the procedures described herein to determine either in vitro or in vivo whether or not expression of the GluR3 receptor subunit protein has been inhibited after administration of the functional equivalent or derivative.
  • the method of the present invention may be used for prophylactic treatment and/or it may be used to maintain a condition or prevent further degeneration.
  • the present invention also provides an agent for use in the treatment of a disorder associated with motor neuron degeneration.
  • the agent is preferably the peptide nucleic acid C-GTA AGA GTG CCT-N or a functional equivalent or derivative thereof.
  • the peptide nucleic acids of the present invention may be prepared by conventional and well-known techniques.
  • the peptide nucleic acids may be prepared using solid-phase synthesis and in particular using commercially available equipment.
  • Isolation and purification of the peptide nucleic acids of the present invention can be effected, if desired, by any suitable separation or purification such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography, thick-layer chromatography, preparative low or high-pressure liquid chromatography or a combination of these procedures.
  • the agent of the present invention may be administered as a composition including the agent and a pharmaceutically acceptable carrier.
  • the agent can be formulated into compositions.
  • compositions of this invention can be administered to humans and other animals orally, rectally, parenterally (i.e. intravenously, intramuscularly, or sub-cutaneously), intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), transdermally, bucally, or as an oral or nasal spray. Multiple administration may be required.
  • compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • aqueous and nonaqueous carriers, diluents, solvents or vehicles examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, or dispersing agents.
  • adjuvants such as preservative, wetting agents, emulsifying agents, or dispersing agents.
  • an adjuvant such as Freund's (complete or incomplete), mineral gels, surface active substances such as peptides or oil emulsions.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like.
  • isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the compounds can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay
  • compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes. If desired, and for more effective distribution, the compounds can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.
  • the active compounds can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifier
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for exampled, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • suspending agents as, for exampled, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Dosage forms for topical administration of a compound of this invention include powders, sprays, ointments and inhalants.
  • the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers, or propellants which may be required.
  • compositions for treating neurological conditions may be administered at any time.
  • compositions are administered at or after the time of diagnosis.
  • formulations may conveniently be presented in unit-dose or multi-dose containers, e.g. sealed ampoules and vials.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, or an appropriate fraction of the administered ingredient.
  • the attending clinician will determine, in his or her judgement, an appropriate dosage and regimen, based on the patient's age and condition as well as the severity of the neurological condition.
  • Toxicity and therapeutic efficacy may be determined by standard pharmaceutical procedures that may involve cell cultures or experimental animals, e.g., for determining the LD 5 o (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of a peptide nucleic acid lies preferably within a range of circulating concentrations that include the ED 5 o with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration used in the method of the invention.
  • the PNA may be used in conjunction with other compounds, including antioxidants, calcium buffering agents, growth factors (such as Leukemia inhibitor factory), or with chemotherapeutic agents.
  • the present invention also provides a method for diagnosing a disorder associated with motor neuron degeneration in a subject, the method including the step of measuring the levels of an AMPA receptor subunit protein expressed in the subject and comparing the measured level with the levels of the AMPA receptor subunit in a subject that is not afflicted with a disorder associated with motor neuron degeneration.
  • the AMPA receptor subunit is GluR3.
  • the levels of the AMPA receptor subunit expressed in a subject may be determined by any of the techniques that are used for that purpose in the art, including competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sand
  • AMPA receptor subunit may be routinely detected in certain tissues or cell types (e.g., immune, neural, renal, urogenital, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from a subject.
  • tissues or cell types e.g., immune, neural, renal, urogenital, and cancerous and wounded tissues
  • bodily fluids e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid
  • AMPA receptor subunits (mus muscularis) were obtained from the Genebank databases (GluRI NM008165), (GluR2 NM013540), (GluR3 NM016886) and (GluR4 MN019691).
  • a unique target sequence for anti-sense action was identified within the signal peptide region of the GluR3 subunit (see Figures 1 and 2).
  • Previous PNA studies have shown a sequence length of three codons to be sufficient in the down regulation of target genes (Nielson et al. 1994).
  • the antisense sequence termed anti- GluR3 PNA was checked against the mouse genome for the likelihood of nonspecific gene down-regulation. A randomly generated nonsense sequence was created to act as a control.
  • the transgenic mouse line was maintained as hemizygous by mating transgenic males with B6SJL wildtype females. Wildtype is defined as any mouse derived from a transgenic founder that does not contain the transgene itself (i.e. genetically wildtype).
  • Experimentation involved SOD1-G93A transgenic and wildtype mice of either sex at postnatal days 30 (P30), P60, P90 and P120.
  • GluR2 expression comparisons were made between G93A SOD1 and wildtype animals throughout the disease time course.
  • anti-GluR3 PNA therapy was evaluated using an immunoprecipitation technique.
  • the lumbar spinal cord proteins were electrophoresed using a Tris-glycine buffered SDS-PAGE system.
  • the resolving gel was cast by mixing a 12.5% (w/v) acrylamide and bisacrylamide (37.5:1) solution, distilled water, 10% (w/v) SDS, 10% (w/v) ammonium persulfate and TEMED in a 2 M Tris-HCI buffer, pH 8.8.
  • the stacking gel was cast by mixing a 5% (w/v) acrylamide and bisacrylamide (37.5:1) solution, distilled water, 10% (w/v) SDS, 10% (w/v) ammonium persulfate and TEMED in a 500mM Tris-HCI buffer, pH 6.8.
  • the gels (1.5mm thickness) were loaded onto a Mini-PROTEAN II Cell (Bio-Rad) vertical electrophoresis unit.
  • Protein extracts (20 ⁇ g) were solubilized in 20 ⁇ l of SDS-sample buffer containing 2% (w/v) SDS, 1% (w/v) glycine, 0.005% (w/v) bromophenol blue, 10% (v/v) glycerol and 5% (v/v) ⁇ -mercaptoethanol in 500 mM Tris-HCI buffer, pH 6.8. Samples were boiled at 100°C for 5 minutes then loaded onto the SDS-gel. SeeBlue Plus2 molecular weight standards were also loaded. The anode and cathode buffer contained 1% SDS (w/v) and 6% (w/v) glycine in 250mM Tris-HCI, pH 8.8.
  • Electrophoresis was conducted at 50 V for 30 minutes then 90 V for 1 hour. Protein spotting pattern was visualised following Coomassie G250 staining. The results are shown in Figure 3. Arrowheads indicate the position of GluR3 as determined by APAF. Note when compared to (C) wildtype, in the (D) sOD1 G93A G H there is considerably darker staining suggesting an upregulation of this protein.
  • Proteins were electrophoretically transferred onto moistened polyvinyl difluoridine (PVDF) membranes using a Mini Trans-Bolt cell (Bio-Rad) unit containing 0.6% (w/v) glycine in 25mM Tris-HCI and 20% (v/v) methanol in distilled water, pH 8.35. Electroblotting was performed at 100 V for 1 hour.
  • PVDF polyvinyl difluoridine
  • PVDF membranes were blocked for 1 hour in 5% (w/v) defatted milk powder
  • IgG-horse radish peroxidase conjugate diluted 1:2500 in TBS for 1 hour and washed four times. Blots were incubated with chemiluminescence substrates for 5 minutes then exposed to hyperfilm for 1 minuted which were later developed. An immunopreciptation technique was employed to compare GluR3 protein levels between the PNA treatment groups.
  • the GluR2/3 antibody recognises a common epitope situated within the C-terminus of both GluR2 and GluR3. Using this antibody, GluR3 protein levels are masked by the presence of GluR2.
  • protein A Sepharose CL-4 beads were used to remove GluR2 from the protein samples.
  • Sepharose beads (1.5g) were pre-washed three times with 50ml distilled water, washed in 50 mM Tris-HCI, pH 7.5, and stored in 50% (w/v) tris buffer at a 4°C.
  • each lysate was pre- cleared with sepharose beads (30 ⁇ l) in RIPA lysis buffer, pH, 7.4, for 30 minutes. Samples were centrifuged at 14,000rpm for 1 minuted and the pre- cleared lysate transferred into clean eppendorf tubes.
  • Tail cuttings were taken from all experimental mice for genotyping purposes. A cell lysis was performed on all tails, followed by PCR to detect the presence of the human SOD1 transgene. PCR products were run on a 1.5% agarose gel with a 100bp standard ladder and negative control (i.e. master mix excluding template DNA). The negative control was used to determine if contamination had occurred. All mice display a 32bp PCR product representing the endogenous murine IL2 gene. Only animals carrying the human SOD1 transgene display a PCR product of 236bp. This procedure was carried out for all mice used in experimentation to ensure the correct genotype of each animal. As expected, offspring inherit the mutant SOD1 transgene at a ration of 1 :1.
  • a final anti-GluR2 concentration of 0.5 ⁇ g-1.O ⁇ g/rnl revealed an absence of immunoreactive cells in the tissue sections examined. Altered antibody concentrations and modified protocol design was unable to identify GluR2 positive staining in the spinal cord of wildtype or G93A SOD1 mice. Due to the apparent technical problems, a western blots analysis was undertaken to determine GluR2 expression. Blots revealed no difference in GluR2 levels between wildtype and SOD1 G93A mice throughout the disease time course (P30, 60, 90 or 120).
  • NSC34 D cells Neuroblastoma x spinal cord cell lines
  • NSC-34 D cells were cultured in 96 well tissue culture test plates at 1 x 10 4 cells/well for 48 hours in DMEM/F-12 with 1% (v/v) FCS.
  • cells were exposed to various concentrations of L-glutamic acid, (S)-AMPA, 5-(S)-FW and 5-(S)-FW in the presence of CNQX for 72 hours.
  • Cell viability was determined by the ability for cells to metabolize MTT (0.5 mg/ml) for 2 hours. The results are shown in Figure 5 where it can be seen that between 1 and 10mM [L-Glutamate] significant cell death was elicited (A).
  • (S)-5-AMPA elicited significant cell death with the maximal effect at a concentration of 1000 ⁇ M [(S)-AMPA] (B).
  • the AMPA receptor specific agonist, (S)-5-FW elicited significant cell death with the maximal effect at a concentration of 1000 ⁇ M [(S)-5-FW] (C).
  • this killing was antagonised by initially exposing cells to CNQX whereby the effect of (S)-5-FW was inhibited by approximately 85% in the 10 /M [CNQX] condition (D).
  • Statistically significant differences from control (zero agonist) were defined using one-way ANOVA with a Tuckey's post-hoc test (*P ⁇ 0.05, **P ⁇ 0.01 , ***P ⁇ 0.001).
  • NSC-34 D cells were treated with a 5(6)-carboxyfluorescein-succinimidyl-ester conjugated to the N- terminus of the APNG3j 2 construct.
  • the results are shown in Figure 6.
  • 100 /M [fluorescent APNG3 ⁇ 2 ] positive punctate staining was noted in the cytosol of most NSC-34 D cells (B).
  • B positive punctate staining was noted in the cytosol of most NSC-34 D cells
  • C overlay image
  • NSC-34 D cells were cultured at 1 x 10 4 cells/well for 48 hours in DMEM/F-12 with 1% (v/v) FCS and then treated with various concentrations of APNAG3 ⁇ 2 , SPNAG3i 2 and NPNAG3 ⁇ 2 for 72 hours.
  • Cells were lysed in ice-cold cell lysis buffer and 2x SDS-Sample buffer. Lysates were then electrophoresed through 12.5% SDS-gels, electroblotted onto nitrocellulose and incubated overnight with the GluR3 antiserum. Blots then normalised by re-probing for ⁇ -actin protein.
  • NSC-34 D cells were cultured at 1 x 10 4 cells/well for 48 hours in DMEM/F-12 with 1% (v/v) FCS and then treated with various concentrations of APNAG3- ⁇ 2 , NPNAG3 ⁇ 2 and SPNAG3 ⁇ 2 for 72 hours. Cells were then exposed to 500 /M [(S)-5-FW] for 72 hours. The results are shown in Figure 7.
  • Intraperitoneal injections of anti-GluR3 PNA or nonsense-GluR3 PNA into the previously described SOD1 G93A G1H mice began at P71 at a dose of 2.5mg/Kg. Injections continued three times weekly (Monday, Wednesday and Friday) until each animal reached its own disease endpoint (as described).
  • mice receiving PNA injections underwent behavioural testing to identify the possible therapeutic benefit of anti-GluR3 PNA. Two tests were developed to assess the progressive loss of hind-limb motor function.
  • Hind limb grip strength was assessed using a 'Cling Test'.
  • a strict training procedure was undertaken to familiarise animals with the testing conditions. Training To simulate the testing conditions mix were placed on a metallic grid (5mm x 5mm holes) and inverted over a catchment chamber for a maximum limit of 180 seconds. This procedure was repeated three times with three-minute rest periods between each session. Testing Two testing sessions were conducted weekly to assess changes in motor performance. Prior to the testing sessions, each mouse underwent a final training session to refamiliarise it with the procedure. After a three-minute rest period the two testing sessions were conducted. Motor performance was defined as the length of time between the initial inversion of the animal and the moment all four paws were released from the wire mesh. A hand timer was used to assess this performance. Testing was stopped after an arbitrary limit of 180 seconds. ⁇ Rotarod Analysis
  • mice were given equal training time on the Rotarod before testing began. Training was divided into three sessions. Sessions one and two were ramping test starting at 3rpm with a stepped increase in speed to 30rpm over five- minutes. During Session three mice were trained at a constant 16rpm for five- minutes. A ten-minute rest period was allowed between sessions. Testing Prior to weekly assessment of motor performance, all mice were given a three- minute training period at a constant speed of 16rpm, followed by a three minute rest period. Performance was defined as the time period each mouse remained on the rotating axle (3.6cm diameter; speed of rotation, 16rpm) without falling. A pressure sensor located under the chamber floor of the Rotarod electronically recorded the performance of a fallen mouse. The test procedure was stopped after an arbitrary limit of 180 seconds.
  • the results demonstrate a significant improvement in locomotor function of APNAG3 ⁇ 2 treated animals when compared to NPNAG3 ⁇ 2 treated animals.
  • mice received thrice I.P injections per week of APNAG3 12 or NPNAG3 ⁇ 2 for 15 weeks. Upon reaching stage IV of their disease progression, the mice were humanely euthanased and fresh lumbar spinal cord and kidneys were dissected. Proteins were extracted and quantified using BCA standards. The proteins were then electrophoresed through 12.5% SDS-gels, electroblotted onto nitrocellulose and incubated overnight with the GluR3 antiserum. Blots were then normalised by re-probing for ⁇ -actin protein. The results of this experiment illustrate that there was no significant different in levels of GluR3 in the lumbar spinal cords ( Figure 8D). However, there was a modest increase in GluR3 levels observed in the kidney extracts ( Figure 8E).
  • mice are considered at the end point of life when they develop complete paralysis in one or both of their hind-limbs (Stage IV of disease progression).
  • stage IV as an indication of animal survival, mice from both groups were compared using a Kaplan-Meier statistical analysis (GraphPad Prism Inc. San Diego, USA).
  • Mice treated with anti-GluR3 PNA lived significantly longer than those receiving the nonsense PNA (p ⁇ 0.05) both control mice died 114 days after birth.
  • Antisense treated mice showed improved survival of four, eight and twenty days beyond the control group. The prolonged survival of mice receiving antisense treatment correlated with the later onset of paralysis seen in the Cling Test and also in two out of three cases with the Rotarod apparatus.
  • antisense PNA designed to down-regulate the expression of GluR3 significantly improved the survival of G93A SOD1 mice.
  • GluR2 and GluR3 bands were unsuccessful. Protein A sepharose beads were used to immunoprecipitate GluR2 from the protein samples. In this way GluR3 protein levels could be identified directly.
  • GluR2 depleted lumbar cord protein was electrophoresed using a Tris-glycine SDS-PAGE system before being electrophoretically transferred onto a PVDF membrane. A final working concentration of 1.0 ⁇ g/ml of the GluR2/3 antibody was used for immunodetection.

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Abstract

L'invention concerne une méthode de traitement d'un sujet souffrant d'un trouble associé à la dégénérescence motoneuronale. Ladite méthode consiste à inhiber ou à prévenir la régulation à la hausse d'une sous-unité de récepteur AMAP chez le sujet. La sous-unité de récepteur AMPA est de préférence GluR3.
PCT/AU2003/000645 2002-05-27 2003-05-27 Agents et methode pour le traitement de troubles associes a la degenerescence motoneuronale WO2003099299A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
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US8173644B2 (en) 2007-01-03 2012-05-08 Les Laboratoires Servier 3-substituted-[1,2,3]-benzotriazinone compound for enhancing glutamatergic synaptic responses
WO2008085506A1 (fr) * 2007-01-03 2008-07-17 Cortex Pharmaceuticals, Inc. Composé de [1,2,3]-benzotriazinone 3-substituée destiné à améliorer les réponses synaptiques glutamatergiques
CN101616592A (zh) * 2007-01-03 2009-12-30 皮质制药公司 用于提高谷氨酸能突触响应的3-取代的-[1,2,3]-苯并三嗪酮化合物
WO2008085505A1 (fr) * 2007-01-03 2008-07-17 Cortex Pharmaceuticals, Inc. Composés [1,2,3]benzotriazinone substitués en position 3 destinés à améliorer les réponses synaptiques glutamatergiques
CN101616592B (zh) * 2007-01-03 2013-06-05 瑟维尔实验室 用于提高谷氨酸能突触响应的3-取代的-[1,2,3]-苯并三嗪酮化合物
EA017437B1 (ru) * 2007-01-03 2012-12-28 Ле Лаборатуар Сервье 3-замещённое-[1,2,3]-бензотриазиноновое соединение для увеличения глутаматергических синаптических ответов
US8013003B2 (en) 2007-05-17 2011-09-06 Cortex Pharmaceuticals, Inc. Di-substituted amides for enhancing glutamatergic synaptic responses
US8404682B2 (en) 2007-05-17 2013-03-26 Cortex Pharmaceuticals, Inc. Di-substituted amides for enhancing glutamatergic synaptic responses
US8168632B2 (en) 2007-08-10 2012-05-01 Cortex Pharmaceuticals, Inc. Bicyclic amide derivatives for the treatment of respiratory disorders
US8263591B2 (en) 2007-08-10 2012-09-11 Cortex Pharmaceuticals, Inc. Bicyclic amides for enhancing glutamatergic synaptic responses
US8119632B2 (en) 2007-08-10 2012-02-21 Cortex Pharmaceuticals, Inc. Bicyclic amide derivatives for enhancing glutamatergic synaptic responses
US8110584B2 (en) 2007-08-10 2012-02-07 Cotex Pharmaceuticals, Inc. Methods for the treatment of respiratory depression
US8507482B2 (en) 2007-08-10 2013-08-13 Cortex Pharmaceuticals, Inc. Bicyclic amide derivatives for enhancing glutamatergic synaptic responses
US9492440B2 (en) 2007-08-10 2016-11-15 Respirerx Pharmaceuticals Inc Bicyclic amides for enhancing glutamatergic synaptic responses

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