EP1465609A2 - Modulierung von dopaminergischen neuronen - Google Patents

Modulierung von dopaminergischen neuronen

Info

Publication number
EP1465609A2
EP1465609A2 EP02770126A EP02770126A EP1465609A2 EP 1465609 A2 EP1465609 A2 EP 1465609A2 EP 02770126 A EP02770126 A EP 02770126A EP 02770126 A EP02770126 A EP 02770126A EP 1465609 A2 EP1465609 A2 EP 1465609A2
Authority
EP
European Patent Office
Prior art keywords
channel
agent
dopaminergic neuron
type channel
coupling
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02770126A
Other languages
English (en)
French (fr)
Inventor
Jakob Wolfart
Jochen Roeper
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Medical Research Council
Original Assignee
Medical Research Council
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB0126781.4A external-priority patent/GB0126781D0/en
Application filed by Medical Research Council filed Critical Medical Research Council
Publication of EP1465609A2 publication Critical patent/EP1465609A2/de
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates, inter alia, to modulation of the activity of dopaminergic neurons.
  • key aspects of the present invention relate to: Use of an agent in the manufacture of a medicament, wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode.
  • a method of treatment comprising administering to a subject in need of same an agent, wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode.
  • indirect means would be means to determine the level of Ca 2+ influx and/or K + efflux.
  • the diagnostic composition or kit may also contain an agent accoding to the present invention.
  • the present invention also includes a kit comprising the agent of the present invention and another pharmaceutically active agent, wherein said other agent is optionally capable of acting as an agent according to the present invention.
  • preferred aspects of the present invention relate to:
  • an agent in the manufacture of a medicament wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in dopamine release from said dopaminergic neuron, wherein said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • a method of treatment comprising administering to a subject in need of same an agent, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in dopamine release from said dopaminergic neuron, wherein said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • An assay method for identifying and/or improving the effect of an agent wherein said assay utilises: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in " dopamine release from said dopaminergic neuron, wherein said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • a process comprising the steps of:
  • a process comprising the steps of:
  • SK preferably SK3
  • a modulator of a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel channel for use in the preparation of a medicament for the treatment of a condition that can be alleviated with an increase in dopamine levels, preferably endogeneous dopamine levels, preferably wherein the condition is Parkinson's disease.
  • a blocker of a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel channel for use in the preparation of a medicament for the treatment of a condition that can be alleviated with an increase in dopamine levels, preferably endogeneous dopamine levels, preferably wherein the condition is Parkinson's disease.
  • nucleotide sequence of the present invention and “amino acid sequence of the present invention” refer respectively to any one or more nucleotide sequences presented or discussed herein and to any one or more of the amino acid sequences presented or discussed herein.
  • amino acid refers to peptide or protein sequence and may refer to portions thereof.
  • amino acid sequence of the present invention is synonymous with the phrase “polypeptide of the present invention”.
  • nucleotide sequence of the present invention is synonymous with the phrase “poly-nucleotide sequence of the present invention”.
  • the agent indirectly causes the dopaminergic neuron to enter bursting mode by affecting T-type channel and/or.an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • the agent is for the treatment of conditions that can be alleviated by an increase in dopamine levels.
  • the dopaminergic neuron is a mid-brain dopaminergic neuron.
  • the dopaminergic neuron is a dopaminergic Substantia nigra neuron.
  • the disorder is a neuronal disorder.
  • the disorder affects the mid-brain
  • the disorder affects the Substantia nigra.
  • the agent is for the treatment of Parkinson's disease.
  • Disorders to be treated may be genetic in origin. Thus, they may arise due to one or more mutations that result in a deleterious effect, e.g. mutations in genes or in other regions, utations may result in excessive, insufficient, or otherwise aberrant expression or activity of a gene product. Disorders to be treated may also/alternatively arise due to environmental factors.
  • the agent affects a dopaminergic neuron entering bursting mode and/or of preventing it from leaving bursting mode, wherein said agent modulates: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • Neurons are highly polarised cells whose most obvious feature is their extensive system of axons and dendrites. Neurons are cell populations that are distinct in their morphology, function and/or biochemical characteristics.
  • the term "dopaminergic neuron” relates to neurons which have cell bodies in the region of the ventral midbrain (VM) known as the substantia nigra pars compacta and project to the striatum. These neurons are distinguished biochemically by the fact that they secrete dopamine as a neurotransmitter and thus express at high levels the enzyme tyrosine hydroxylase (TH) which catalyzes the rate- limiting step in the biosynthesis of dopamine.
  • VM ventral midbrain
  • TH tyrosine hydroxylase
  • Dopaminergic neurons are of enormous clinical significance because it is these cells that progressively lose function in patients with neurodegenerative disorders such as Parkinson's disease.
  • Dopaminergic (DA) midbrain neurones play an essential role in a variety of brain functions such as voluntary movement, working memory, and reward (Goldman-Rakic, 1999; Kitai et al., 1999; Spanagel and Weiss, 1999). In addition, they are intimately involved in neuropsychiatric and neurological disorders such as schizophrenia, drug addiction, and Parkinson's disease (Dunnett and Bjorklund, 1999; Verhoeff, 1999; Svensson, 2000). Since these brain functions and diseases are associated with anatomically distinct DA neurone subpopulations, the question arises whether the functional characteristics of mesencephalic DA neurones are differentiated in accordance to their anatomical subgroups.
  • DA neurones exert their function by integrating synaptic inputs in the context of their intrinsic pacemaker to generate pattern of electrical activity that control dopamine release and their functional effects on their respective target cells.
  • potential diversity within the DA system might originate both from differences in axonal projections and synaptic connectivity as well as from diverging properties of the somato-dendritic integrator and pacemaker.
  • Dopaminergic midbrain neurones are distributed in three partially overlapping nuclei: the retrorubral area (RRA, A8), substantia nigra (SN Conduct A9), and the ventral tegmental area (VTA, A 10), which correspond to different mesotelencephalic projections (Gardner and Ashby, 2000; Joel and Weiner, 2000).
  • Substantia nigra neurones mainly target the dorsal striatum (mesostriatal projection) and are involved in motor function, whereas the neurones of the VTA project predominantly to the ventral striatum e.g.
  • nucleus accumbens (mesolimbic projection) and to prefrontal cortex (mesocortical projection) and are thus associated with limbic functions (Gardner and Ashby, 2000; Joel and Weiner, 2000).
  • Recent tracing studies have revealed a more refined concept of the topographical organisation of mesotelencephalic connections.
  • two neurochemically distinct tiers project to and receive input from different neurochemical compartments in the striatum, namely the limbic and sensori-motor regions (Maurin et al., 1999; Haber et al., 2000).
  • Ventral tier DA neurones that do not express the calcium-binding protein calbindin D 28 -K (calbindin-negative, CB-), project to patch compartments and in turn receive innervation from striatal projection neurones in the matrix. Conversely, calbindin-positive (CB+) dorsal tier DA neurones project to the matrix while receiving input from the limbic patch compartment (Gerfen, 1992; Barrot et al., 2000).
  • CB+ DA neurones are less vulnerable (Liang et al, 1996; Damier et al, 1999a; Gonzalez-Hernandez and Rodriguez, 2000; Tan et al., 2000).
  • DA neurones have a number of features suggestive of the expression of a highly conserved repertoire of ion channels namely, low-frequency pacemaker activity, broad action potentials followed by a pronounced afterhyperpolarisation, a strong sag-component mediated by hyperpolarisation activated channels (Ih channels) and a D2 autoreceptor-mediated hyperpolarisation (Sanghera et al., 1984; Grace and Onn, 1989; Lacey et al, 1989; Richards et al., 1997).
  • Ih channels hyperpolarisation activated channels
  • D2 autoreceptor-mediated hyperpolarisation Roshera et al., 1984; Grace and Onn, 1989; Lacey et al, 1989; Richards et al., 1997.
  • Neurons in general, have a single axon which is specialised for the conductance of a particular type of electric impulse called an action potential.
  • An action potential is a series of sudden changes in the electric potential across the neuron plasma membrane. The potential varies according to the physiological mode of the plasma membrane of the neuronal cell which may be resting, firing, pacemaker or bursting.
  • resting mode relates to a neuron plasma membrane which is non-stimulated- and in which any voltage-gated ion channels, such as a T-type calcium channel or an SK (preferably SK3) potassium channel, are open.
  • any voltage-gated ion channels such as a T-type calcium channel or an SK (preferably SK3) potassium channel
  • the voltage-gated ion channels are open only for a fraction of a second which propagates action potentials along the neuron plasma membrane.
  • firing mode relates to a stimulated neuron plasma membrane which is capable of generating action potentials.
  • pacemaker mode relates to a regular or maintained low level of firing action potentials.
  • bursting mode relates to a high frequency firing of action potentials.
  • T-type channels are so called because they carry a transient current, with a low voltage of activation and rapid inactivation.
  • the main factor which defines the different calcium currents is which ⁇ i subtype is included in the channel complex.
  • the subfamily of C ⁇ G, a and an subunits display the low-voltage activation characteristic of T-type channels.
  • T-type channels are located in cardiac & vascular smooth muscle; and in the nervous system.
  • Perez- Reyes et al discuss the molecular characterization of a neuronal low- voltage-activated T-type calcium channel (Nature 391, 896-900, 1998).
  • T-type channels may be blocked for example by nickel ions, Mibefradil (available from Roche), and Kurtoxin (a peptide from South African scorpion, Parabuthus transvaalicus).
  • Voltage-activated calcium channels can be distinguished based on their voltage-dependence, deactivation, and single-channel conductance.
  • Low- voltage-activated calcium channels are referred to as 'T type because their currents are both transient, owing to fast inactivation, and tiny, owing to small conductance.
  • T-type channels are thought to be involved in pacemaker activity, low-threshold calcium spikes, neuronal oscillations and resonance, and rebound burst firing.
  • the rat alpha- 1G channel When expressed in Xenopus oocytes, the rat alpha- 1G channel exhibited the properties of a low-voltage-activated T-type calcium channel.
  • Perez-Reves et al. ( “ 1998 " ) mapped the CACNA1G gene to 17q22. Using interspecific backcross analysis, they mapped the mouse Cacnalg gene to the distal portion of chromosome 11, in a region showing homology of synteny with 17q22. Native calcium channels have been classified by their electrophysiological and pharmacological properties as T, L, N, P and Q types (for views see McCleskey, E. W. et al. Curr Topics Membr (1991) 39:295-326, and Dunlap, K. et al. Trends Neurosci (1995) 18:89-98).
  • T-type (or low voltage-activated) channels describe a broad class of molecules that transiently activate at negative potentials and are highly sensitive to changes in resting potential.
  • the L, N, P and Q-type channels activate at more positive potentials (high voltage activated) and display diverse kinetics and voltage-dependent properties. There is some overlap in biophysical properties of the high voltage-activated channels, consequently pharmacological profiles are usefull to further distinguish them.
  • L-type channels are sensitive to dihydropyridine agonists and antagonists
  • N-type channels are blocked by the Conus geographus peptide toxin, .omega.-conotoxin GVIA
  • P-type channels are blocked by the peptide aagatoxm IVA from the venom of the funnel web spider, Agelenopsis aperta.
  • Q-type high voltage-activated calcium channel
  • ⁇ subunit is a nonglycosylated, hydrophilic protein with a high affinity of binding to a cytoplasmic region of the
  • a fourth subunit, .gamma. is unique to L-type calcium channels expressed in skeletal muscle T-tubules.
  • the isolation and characterization of .gamma.-subunit-encoding cDNAs is described in U.S. Pat. No. 5.386.025
  • .alpha. 1A -.alpha, n and .alpha are and correlated with the subtypes set forth above, .alpha, IA channel s are of the P/Q type; .alpha. 1B represents N; .alpha. lc , .alpha.' 1D , .alpha. ⁇ F and acs represent L; .alpha. 1E represents a novel type of calcium conductance, and .alpha. ⁇ G -.alpha.n represent members of the T-type family, reviewed in Stea, A. et al.
  • SK channels are activated in a voltage-independent manner and have a relatively small unit conductance and high sensitivity to calcium.
  • Kohler et al (Science 273 1709-1714, 1996) isolated rat and human brain cDNAs encoding a family of SK channels which they designated SKI, SK2, and SK3. All 3 proteins contain intracellular N and C termini and 6 highly conserved transmembrane segments. In situ hybridization revealed that mRNAs encoding these subunits are widely expressed in rat brain with distinct but overlapping patterns.
  • Chandy et al (Molec. Psychiat. 3: 32-37, 1998) identified cDNAs encoding the human SK3, or SKCa3, homolog. The predicted protein is 731 amino acids long.
  • the second CAG repeat was highly polymorphic in control individuals, with alleles ranging in size from 12 to 28 repeats.
  • AHP afterhyperpolarization
  • SK channels are activated in a voltage-independent manner and have a relatively small unit conductance and high sensitivity to calcium.
  • Kohler et al. (1996) isolated rat and human brain cDNAs encoding a family of SK channels which they designated SKI (KCNNl), SK2 and SK3. All 3 proteins contain intracellular N and C termini and 6 highly conserved transmembrane segments. In situ hybridization revealed that mRNAs encoding these subunits are widely expressed in rat brain with distinct but overlapping patterns.
  • Chandy et al. identified cDNAs encoding the human SK3, or SKCa3, homolog.
  • the predicted protein is 731 amino acids long.
  • the authors determined that the SK3 gene contains 2 arrays of CAG trinucleotide (polyglutamine) repeats in the N-terminal region of the protein.
  • the second CAG repeat was highly polymorphic in control individuals, with alleles ranging in size from 12 to 28 repeats.
  • Several human hereditary neurodegenerative diseases, such as Huntington disease are caused by expanded trinucleotide repeats within genes. Citing previous reports of expanded CAG arrays in patients with schizophrenia and bipolar disorder I. Chandy et al.
  • Navon et al. (1998) used FISH to localize the KCNN3 gene to lq21, noting also the alignment of an STS previously mapped to this region with the KCNN3 gene. They observed an association with the larger CAG repeat within this gene in Israeli Jewish schizophrenia patients compared to controls.
  • Bond et al. (2000) targeted the SK3 gene by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function.
  • An absence of SK3 did not present overt phenotypic consequences.
  • SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition, presumably by effects on uterine contraction. Both conditions were corrected by silencing the gene. Bond et al. (2000) concluded that their results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.
  • moduleates'V'modulating preferably mean any one or more of: adversely affecting, decreasing, removing, inhibiting, antagonising, blocking or down regulating T-type channel activity and/or SK (preferably SK3) channel activity and/or the coupling thereof.
  • SK preferably SK3
  • the agent blocks T-type channel activity and/or SK3 channel activity and/or the coupling thereof.
  • blocks'V'blocking include partial or complete blocking. In one preferred aspect, the terms preferably mean at least substantially blocks.
  • Neural transmitters are chemicals in the brain that are used to send messages from one brain cell to another. Neurotransmitters bind to special receptor proteins in the membranes of nerve cells, like a lock in a key, triggering a chemical reaction within the cell.
  • Dopamine is an example of a central nervous system (CNS) neurotransmitter which is a catecholamine belonging to a class of biogenic amine neurotransmitters, along with norepinephrine, serotonin, and histamine.
  • the catecholomines are involved in the control of movement; mood; attention; and possibly, certain endocrine, cardiovascular, and stress responses. Imbalances in neurotransmitter production have been implicated in a variety of mental and physical disorders, such as Parkinson's disease (PD). It is thus desirable to diagnose and monitor such imbalances and to monitor the effectiveness of drugs and substances that affect brain chemistry.
  • PD Parkinson's disease
  • the agent is capable of reducing calcium efflux through a calcium channel of a dopaminergic neuron and/or of reducing potassium efflux through a potassium channel of a dopaminergic neuron in the manufacture of a medicament for treating a disorder that is alleviated by increased dopamine release.
  • the calcium channel is a T-type calcium channel and/or the potassium channel is an SK (preferably SK3) potassium channel.
  • the agent of the present invention may also be capable of displaying one or more other beneficial functional properties.
  • the agent has an EC 50 value of less than 300nM, 250nM, 200nM, 150nM, preferably less than about 100 nM, preferably less than about 75 nM, preferably less than about 50 nM, preferably less than about 25 nM, preferably less than about 20 nM, preferably less than about 15 nM, preferably less than about 10 nM, preferably less than about 5 nM.
  • substituents being a cyclic group
  • a combination of substituents may form a cyclic group.
  • the hydrocarbyl group comprises more than one C then those carbons need hot necessarily be linked to each other.
  • at least two of the carbons may be linked via a suitable element or group.
  • the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
  • the agent may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups - which may be unbranched- or branched-chain.
  • An agent of the present invention may be provided in substantially pure or substantially isolated form. It may be provided in the form of a pharmaceutically acceptable composition - e,g admixed with a suitable pharmaceutically acceptable carrier, diluent, or excipient.
  • Agents that can be used to provide increased dopamine release include agents capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode.
  • T-type calcium channel blockers and/or SK (preferably SK3) channel blockers can be identified by screening, as described later.
  • T-type calcium channel blockers such as Mibefradil and/or nickel ions.
  • calcium channel blocker examples include Kurtoxin, benzothiazepine, such' as diltiazem (dilzem) and TA 3090 and phenylalkylamine, such as verapamil (isoptin), desmethoxyverapamil, methoxy verapamil (D-600, gallopamil or (-)D-888), prenylamine, fendiline, terodiline, caroverine, perhexiline.
  • Kurtoxin benzothiazepine
  • benzothiazepine such' as diltiazem (dilzem) and TA 3090
  • phenylalkylamine such as verapamil (isoptin), desmethoxyverapamil, methoxy verapamil (D-600, gallopamil or (-)D-888
  • prenylamine fendiline
  • terodiline caroverine, perhexiline.
  • 5.428.038 discloses compounds which are said to exert a neural protective and antiallergic effect. These compounds are coumarin dervatives which may include derivatives of piperazine and other six- membered heterocycles. A permitted substituent on the heterocycle is diphenylhydroxymethyl.
  • Certain compounds containing both benzhydril moieties and piperidine or piperazine are known to be calcium, channel antagonists and neuroleptic drugs.
  • the agent may be in the form of - and/or may be administered as - a pharmaceutically acceptable salt - such as an acid addition salt or a base salt - or a solvate thereof, including a hydrate thereof.
  • a pharmaceutically acceptable salt - such as an acid addition salt or a base salt - or a solvate thereof, including a hydrate thereof.
  • a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base, as appropriate.
  • the salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
  • the agent of the present invention may exist in polymorphic form.
  • Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of the agent or a suitable salt or derivative thereof.
  • An individual enantiomer of a compound of the agent may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.
  • isotopic variations of the agent and pharmaceutically acceptable salts thereof are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
  • pro-moieties for example as described in "Design of Prodrugs” by H. Bundgaard, Elsevier, 1985 (the disclosure of which is hereby incorporated by reference), may be placed on appropriate functionalities of the agents. Such prodrugs are also included within the scope of the invention.
  • the agent is selected from the group consisting of an antagonist, a partial antagonist and a competitive antagonist of a T-type channel and/or an SK (preferably SK3) channel and/or the coupling thereof.
  • An antagonist of a given moiety may inhibit one or more activities of that moiety (e.g. all activities). It may, for example, bind in a competitive or non-competitive manner to the moiety or to something with which the moiety interacts (e.g. binds).
  • the agent may be associated with or may be a binding agent.
  • binding agents may be useful as channel blockers in accordance with the present invention. For example they may block a channel by binding to a part thereof or to another moiety operatively associated with the channel.
  • binding agent is an antibody or a fragment thereof that is specific for a T- type channel and/or an SK (preferably SK3) channel.
  • a further type of binding agent that can be used in the present invention is a lectin.
  • Lectins are carbohydrate-binding proteins of non-immune (e.g. plant) origin (see e.g. the discussion of lectins by Manualr in Methods in Enzymology, Guide to Protein Purification, 182 (1990)). Different lectins can be used to select particular glycoproteins based upon the presence of particular carbohydrate moieties (e.g. sialic acid, galactose, mannose, fucose, N-acetyl glucosamine, N-acetyl galactosamine, etc). In some cases a plurality of different lectins may be used - e.g. if a glycoprotein is known to include three different sugars, then three different lectins may be used to purify it. They may be used sequentially (e.g. in sequential affinity columns).
  • binding agent is a ligand or a part thereof that binds to a polypeptide of the present invention or to a moiety with which said polypeptide interacts (e.g. binds).
  • the ligand or part thereof may be provided in immobilised or non-immobilised (soluble) form.
  • the agent of the present invention may be an antibody.
  • the target of the present invention may be an antibody.
  • the term "antibody”, unless specified to the contrary, includes but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
  • Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
  • the antibodies and fragments thereof may be humanised antibodies. Neutralizing antibodies, i.e., those which inhibit biological activity of the substance polypeptides, are especially preferred for diagnostics and therapeutics.
  • BCG Bacilli Calmette-Gueri ⁇
  • Corynebacterium parvum are potentially useful human adjuvants which may be employed if purified the substance polypeptide is administered to immunologically compromised individuals for the purpose of stimulating systemic defence.
  • Monoclonal antibodies directed against epitopes obtainable from an identifed agent and/or substance of the present invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
  • Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
  • Monoclonal antibodies to the substance and/or identified agent of the present invention may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al
  • Antibodies both monoclonal and polyclonal, which are directed against epitopes obtainable from an identifed agent and/or substance of the present invention are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy.
  • Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
  • Anti- idiotype antibodies are immunoglobulins which carry an "internal image" of the substance and/or agent against which protection is desired. Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful in therapy.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991 ; Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for the substance may also be generated.
  • fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-128 1).
  • agent of the present invention will be prepared by chemical synthesis techniques.
  • the agent of the present invention or variants, homologues, derivatives, fragments or mimetics thereof may be produced using chemical methods to synthesize the agent in whole or in part.
  • peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
  • Direct synthesis of the agent or variants, homologues, derivatives, fragments or mimetics thereof can be performed using various solid-phase techniques (Roberge JY et al (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequences comprising the agent or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant agent, such as, for example, a variant a T-type channel and/or a variant SK (preferably SK3) channel.
  • a variant agent such as, for example, a variant a T-type channel and/or a variant SK (preferably SK3) channel.
  • the coding sequence of the agent or variants, homologues, derivatives, fragments or mimetics thereof may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).
  • the term "mimetic” relates to any chemical which includes, but is not limited to, a peptide, polypeptide, antibody or other organic chemical which has the same qualitative activity or effect as a reference entity.
  • derivative or "derivatised” as used herein includes chemical modification of an agent. Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group.
  • the agent may be a chemically modified agent.
  • the chemical modification of an agent of the present invention may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, Van Der Waals interaction or dipole interaction between the agent and the target.
  • the identified agent may act as a model (for example, a template) for the development of other compounds.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the agent of the present invention and a pharmaceutically acceptable carrier, diluent or excipients (including combinations thereof).
  • the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
  • the formulation may be designed to be delivered by both routes.
  • the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
  • compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously.
  • compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
  • the agents of the present invention may also be used in combination with a cyclodextrin.
  • Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules. Formation of a drug-cyclodextrin complex may modify the solubility, dissolution rate, bioavailability and/or stability property of a drug molecule. Drug- cyclodextrin complexes are generally useful for most dosage forms and administration routes.
  • the cyclodextrin may be used as an auxiliary additive, e.g. as a carrier, diluent or solubiliser.
  • Alpha-, beta- and gamma- cyclodextrins are most commonly used and suitable examples are described in WO-A- 91/11172, WO-A-94/02518 and WO-A-98/55148.
  • compositions of the present invention may be provided in controlled release form. This can be achieved by providing a pharmaceutically active agent in association with a substance that degrades under physiological conditions in a predetermined manner. Degradation may be enzymatic or may be pH-dependent.
  • compositions may be deigned to pass across the blood brain barrier (BBB).
  • BBB blood brain barrier
  • a carrier such as a fatty acid, inositol or cholesterol may be selected that is able to penetrate the BBB.
  • the carrier may be a substance that enters the brain through a specific transport system in brain endothelial cells, such as insulin-like growth factor I or II.
  • the carrier may be coupled to the active agent or may contain/be in admixture with the active agent.
  • Liposomes can be used to cross the BBB.
  • WO-A-91/04014 describes a liposome delivery system in which an active agent can be encapsulated/embedded and in which molecules that are normally transported across the BBB (e.g. insulin or insulin-like growth factor I or II) are present on the liposome outer surface. Liposome delivery systems are also discussed in US-A-4704355.
  • Different drug delivery systems may be used to administer pharmaceutical compositions of the present invention, depending upon the desired route of acuministration.
  • Drug delivery systems are described, for example, by Langer (Science 249:1527 - 1533 (1991)) and by Ilium and Davis (Current Opinions in Biotechnology 2 254 - 259 (1991)).
  • the asents of the present invention are delivered systemically (such as orally, buccally, sublingually), more preferably orally.
  • the agent is in a form that is suitable for oral delivery.
  • the term "administered” includes delivery by viral or non-viral techniques.
  • Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectos, herpes viral vectors, refroviral vectors, lentiviral vectors, and baculoviral vectors.
  • Non- viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.
  • agents of the present invention may be administered alone but will generally be administered as a pharmaceutical composition - e.g. when the agent is in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the agent can be administered (e.g. orally or topically) in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
  • the tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
  • disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene, glycols.
  • the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying arid/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • the routes for administration include, but are not limited to, one or more of: oral (e.g. as a tablet, capsule, or as an ingestable solution), topical, mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, infravaginal, infracerebroventricular, infracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, via the pensis, vaginal, epidural, sublingual.
  • oral e.g. as a tablet, capsule, or as an ingestable solution
  • mucosal e.g. as a nasal spray or aerosol for inhalation
  • nasal parenteral (e.g. by an injectable form)
  • composition comprises more than . one active component, then those components may be administered by different routes.
  • agents of the present invention are administered parenterally, then examples of such administration include one or more of: intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the agent; and/or by using infusion techniques.
  • the agent is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well- known to those skilled in the art.
  • the agent of the present invention can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotefrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134ATM) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EATM), carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotefrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134ATM
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
  • a lubricant e.g. sorbitan trioleate.
  • Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the agent and a suitable powder base such as lactose or starch.
  • the agent of the present invention can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder.
  • the agent of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. They may also be administered by the ocular route.
  • the compounds can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.
  • a preservative such as a benzylalkonium chloride.
  • they may be formulated in an ointment such as petrolatum.
  • the agent of the present invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.
  • compositions of the present invention may be administered by direct injection.
  • nucleotide sequence is synonymous with the term “polynucleotide”.
  • the nucleotide sequence is cDNA.
  • themucleotide sequence may be the same as the naturally occurring form.
  • variant in relation to the known nucleotide sequences include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence encoding the channel has T-type channel and/or an SK (preferably SK3) channel activity, preferably having at least the same channel activity as the known sequences.
  • SK preferably SK3
  • the present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above.
  • Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.
  • hybridization shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction (PCR) technologies.
  • Nucleotide sequences of the invention capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement, will be generally at least 75%, preferably at least 85 or 90% and more preferably at least 95% or 98% homologous to the corresponding complementary nucleotide sequence presented herein over a region of at least 20, preferably at least.25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
  • Preferred nucleotide sequences of the invention will comprise regions homologous to the nucleotide sequence set out in SEQ ID No 2 of the sequence listings of the present invention preferably at least 80 or 90% and more preferably at least 95% homologous to the nucleotide sequence set out in SEQ JJD No 2 of the sequence listings of the present invention.
  • both strands of the duplex either individually or in combination, are encompassed by the present invention.
  • the nucleotide sequence is single-stranded, it is to be understood that the complementary sequence of that nucleotide sequence is also included within the scope of the present invention.
  • Nucleotide sequences which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of sources. In addition, other viral/bacterial, or cellular homologues particularly cellular homologues found in mammalian cells (e.g. rat, mouse, bovine and primate cells), may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the -sequences shown in the sequence listing herein.
  • mammalian cells e.g. rat, mouse, bovine and primate cells
  • sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of the nucleotide sequence set out in SEQ ID No 2 of the sequence listings of the present invention under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the amino acid and/or nucleotide sequences of the present invention.
  • nucleotide sequences may be obtained by site directed mutagenesis of characterised sequences, such as the nucleotide sequence set out in SEQ ID No 2 of the sequence listings of the present invention. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the nucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the activity of the T-type channel and/or an SK (preferably SK3) channel encoded by the nucleotide sequences.
  • SK preferably SK3
  • nucleotide sequences such as a DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • RNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used to clone and express a T-type channel and/or an SK (preferably SK3) channel.
  • SK preferably SK3
  • an agent of the present invention or an T-type channel and/or an SK (preferably SK3) channel may be administered directly to an individual.
  • the recombinant channel is prepared and/or delivered to a target site using a genetic vector.
  • a vector is-a tool that allows or faciliates the transfer of an entity from one environment to another.
  • some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a host and/or a target cell for the purpose of replicating the vectors comprising the nucleotide sequences of the present invention and/or expressing the proteins of the invention encoded by the nucleotide sequences of the present invention.
  • vectors used in recombinant DNA techniques include but are not limited to plasmids, chromosomes, artificial chromosomes or viruses.
  • vector includes expression vectors and/or transformation vectors.
  • expression vector means a construct capable of in vivo or in vitrolex vivo expression.
  • transformation vector means a construct capable of being transferred from one 0 species to another.
  • naked DNA refers to a plasmid comprising a nucleotide sequences 20 encoding an agent of the present invention or a channel of the present invention together with a short promoter region to control its production. It is called “naked” DNA because the plasmids are not carried in any delivery vehicle.
  • a DNA plasmid enters a host cell, such as a eukaryotic cell, the proteins it encodes (such as an agent of the present invention or a T-type channel and/or an SK (preferably SK3) channel) are transcribed and 25. translated within the cell.
  • the vectors comprising nucleotide sequences of the present invention or an 30 agent of the present invention may be introduced into suitable host cells using a variety of non-viral techniques known in the art, such as transfection, transformation, electroporation and biolistic transformation.
  • transfection refers to a process using a non-viral vector to deliver a gene to a target mammalian cell.
  • Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556), multivalent cations such as spermine, cationic lipids or polylysine, 1, 2,-bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) and combinations thereof.
  • CFAs cationic facial amphiphiles
  • DOTAP 1, 2,-bis (oleoyloxy)-3-(trimethylammonio) propane
  • DOTAP 1, 2,-bis (oleoyloxy)-3-(trimethylammonio) propane
  • DOTAP 1, 2,-bis (oleoyloxy)-3-(trimethylammoni
  • Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents.
  • transfection agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectamTM and transfectamTM).
  • cationic agents for example calcium phosphate and DEAE-dextran
  • lipofectants for example lipofectamTM and transfectamTM.
  • nucleic acid constructs are mixed with the transfection agent to produce a composition.
  • the vectors comprising an agent of the present invention or nucleotide sequences of the present invention may be introduced into suitable host cells using a variety of viral techniques which are known in the art, such as for example infection with recombinant viral vectors such as retroviruses, herpes simplex viruses and adenoviruses.
  • the vector is a recombinant viral vectors.
  • Suitable -recombinant viral vectors include but are not limited to adenovirus vectors, adeno-associated viral (AAV) vectors, herpes-virus vectors, a refroviral vector, lentiviral vectors, baculoviral vectors, pox viral vectors or parvovirus vectors (see Kestler et al 1999 Human Gene Ther 10( 10): 1619-32).
  • AAV adeno-associated viral
  • lentiviral vectors lentiviral vectors
  • baculoviral vectors pox viral vectors or parvovirus vectors (see Kestler et al 1999 Human Gene Ther 10( 10): 1619-32).
  • delivery of the nucleotide sequence encoding the channel is mediated by viral infection of a target cell.
  • targeted vector refers to a vector whose ability to infect/transfect/transduce a cell or to be expressed in a host and/or target cell is restricted to certain cell types within the host organism, usually cells having a common or similar phenotype.
  • the nucleotide sequences encoding an agent of the present invention o the channel of the present invention may be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate the nucleotide sequence in a compatible host cell.
  • the invention provides a method of making the channel of the present invention by introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into-a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell.
  • an agent of the present invention or a nucleotide sequence of present invention which is inserted into a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence, such as the coding sequence of the channel of the present invention by the host cell, i.e. the vector is an expression vector.
  • a control sequence that is capable of providing for the expression of the coding sequence, such as the coding sequence of the channel of the present invention by the host cell, i.e. the vector is an expression vector.
  • An agent of the present invention or a channel produced by a host recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing an agent of the present invention or the channel coding sequences can be designed with signal sequences which direct secretion of the agent of the present invention or the channel coding sequences through a particular prokaryotic or eukaryotic cell membrane.
  • the vectors of the present invention may be transformed or transfected into a suitable host cell and/or a target cell as described below to provide for expression of an agent of the present invention or a channel of the present invention.
  • This process may comprise culturing a host cell and/or target cell transformed with an expression vector under conditions to provide for expression by the vector of a coding sequence encoding an agent of the present invention or the channel and optionally recovering the expressed agent of the present invention or channel.
  • the vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector.
  • the expression of an agent of the present invention or an channel of the invention may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression.
  • inducible expression production of an agent of the present invention or a channel can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG.
  • the channel or an agent of the present invention may be expressed as a fusion protein to aid in extraction and purification and/or delivery of the agent of the present invention or the channel to an individual and/or to facilitate the development of a screen for agents capable of modulating channel activity.
  • fusion protein partners include glutathione-S- fransferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the target.
  • the fusion protein may comprise an antigen or an antigenic determinant fused to the substance of the present invention.
  • the fusion protein may be a non- naturally occurring fusion protein comprising a substance which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system.
  • the antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the substance.
  • the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence for example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a heterologous epitope that is recognized by a commercially available antibody.
  • host cells can be employed for expression of the nucleotide sequences encoding the agent - such as an agent of the present invention or an channel of the present invention. These cells may be both prokaryotic and eukaryotic host cells. Suitable host cells include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof.
  • suitable expression hosts within the scope of the present invention are fungi such as Aspergillus species (such as those described in EP-A-0184438 and EP-A-0284603) and Trichoderma species; bacteria such as Bacillus species (such as those described in EP-A- 0134048 and EP-A-0253455), Streptomyces species and Pseudomonas species; and yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A- 0301670) and Saccharomyces species.
  • typical expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var.
  • Preferred host cells are able to process the expression products to produce an appropriate mature polypeptide.
  • processing includes but is not limited to glycosylation, ubiquitination, disulfide bond formation and general post-translational modification. SCREENS
  • the present invention encompasses a range of screening methods.
  • Screening can be performed in vitro. This may be done for example by using neurons or parts thereof.
  • calcium or potassium ion channels or membranes comprising such channels may be used.
  • the calcium and potassium, channels are preferably channels of a dopaminergic neuron, more preferably of a Substantia nigra dopaminergic neuron. Desirably they are T-type calcium channels and SK (preferably SK3) potassium channels.
  • Non-human animals e.g. primates
  • transgenic nonhuman animals e.g. rodents
  • Techniques for producing transgenic animals are well known and are described e.g. in US-A-4870009 and US-A4873191.
  • a nucleic acid encoding a desired polypeptide may be microinjected into a pronucleus of a fertilised oocyte.
  • the oocyte may then be allowed to develop in a pseudopregnant female foster animal.
  • the animal resulting from development of the oocyte can be tested (e.g.
  • a transgenic animal can be used as a founder animal, which may be bred from in order to produce further transgenic animals. Two transgenic animals may be crossed. For example, in some cases transgenic animals may be haploid for a given gene and it may be desired to try to provide a diploid offspring via crossing.
  • a transgenic animal may be cloned, e.g. by using the procedures set out in WO-A- 97/07668 and WO-A-97/07699 (see also Nature 385:810-813 (1997)).
  • a quiescent cell can be provided and combined with an oocyte from which the nucleus has been removed combined. This can be achieved using electrical discharges. The resultant cell can be allowed to develop in culture and can then be transferred to a pseudopregnant female.
  • Computer models can be used. They include models of the channels as described above, particularly models predicting the effect of agents on the opening or closing of said channels andor of changes in firing mode. Computer models may also be used to identify binding sites for binding agents. Computer-generated models include computer generated images. The model may be two or three dimensional, although this is not essential. It may comprise a plurality of co-ordinates (e.g. it may be a crystallographic image, such as an X-ray cystallographic image). It may be arranged to be rotatable or otherwise movable to enable different views to be taken.
  • Test agents capable of modulating the channel activity of targets may be screened in assays which are well known in the art. Screening may be carried out, for example in vitro, in cell culture, and/or in vivo. Biological screening assays may be based on but not limited -to - channel activity-based response models, binding assays (which measure how well an agent modulates channel activity), and bacterial, yeast and animal cell lines. The assays can be automated for high capacity-high throughput screening (HTS) in which large numbers of compounds can be tested to identify compounds with the desired channel modulating activity (see, for example WO 84/03564).
  • HTS high capacity-high throughput screening
  • one method of the present invention comprises providing an agent and determining whether or not it is capable of causing a change in the firing mode of a dopaminergic neuron, or of preventing a change that would otherwise occur.
  • the agent may cause the dopaminergic neuron to enter bursting mode or may prevent it from leaving bursting mode.
  • a further method comprises providing an agent and determining whether or not it is capable of reducing calcium efflux through a calcium channel or of reducing potassium efflux through a potassium channel. This can be used to identify channel blockers.
  • the channel is preferably a channel of a dopaminergic neuron, more preferably of a Substantia nigra dopaminergic neuron. Desirably the channel is a T-type calcium channel or an SK (preferably SK3) potassium channel.
  • screening can be performed by using individual neurons or parts thereof.
  • suction may be used to attach a micropipette to a part of a membrane.
  • Current through ion channels in the membrane can then be recorded.
  • a patch of membrane may be removed with the micropipette and used for screening (e.g. for measuring calcium or potassium flux).
  • the advantage of this technique is that the ionic composition either side of the membrane can be readily adjusted. Membrane patches comprising only a single ion channel or only a few ion channels can therefore be obtained and used in this manner.
  • screens may be based upon binding studies.
  • binding agents to calcium or potassium channels or to moieties operatively associated with them e.g. moieties capable of causing opening or closing of channels
  • binding agents to calcium or potassium channels or to moieties operatively associated with them may be useful as channel activators or blockers.
  • Binding studies are therefore useful in identifying agents that may then be subjected to further screening as discussed above. Such studies include providing a binding agent and determining whether or not it binds to an ion channel or to a moiety operatively associated with the channel. If desired, the binding agent may be labelled to aid in identification.
  • any one or more of appropriate targets - such as an amino acid sequence and/or nucleotide sequence - may be used for identifying an agent capable of modulating T-type channel activity and/or SK (preferably SK3) channel activity and/or the coupling thereof in any of a variety of drug screening techniques.
  • the target employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of target activity or the formation of binding complexes between the target and the agent being ested may be measured.
  • Techniques for drug screening may be based on the method described in Geysen, European Patent Application 84/03564, published on September 13, 1984.
  • large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with a suitable target or fragment thereof and washed. Bound entities are then detected - such as by appropriately adapting methods well known in the art.
  • a purified target can also be coated directly onto plates for use in a drug screening techniques.
  • non-neutralising antibodies can be used to capture the peptide and immobilise it on a solid support.
  • This invention also contemplates the use of competitive drug screening assays in which neutralising antibodies capable of binding a target specifically compete with a test compound for binding to a target.
  • the assay methods of the present invention will be suitable for both small and large-scale screening of test compounds as well as in quantitative assays.
  • the present invention provides a method of identifying agents that selectively modulating channel activity in the Substantia nigra of an individual, the method comprising contacting a suitable target from (or obtainable from) the Substantia nigra of an individual and then measuring the channel activity and/or extent of dopamine release.
  • This immunohistochemistry kit permits localization of a channel in tissue sections and cultured cells using both light and electron microscopy which may be used for both research and clinical purposes. Such information may be useful for diagnostic and possibly therapeutic purposes in the detection and/or prevention and/or treatment of a neurodegenerative disorders, such as Parkinoson's disease.
  • a neurodegenerative disorders such as Parkinoson's disease.
  • Intraassay and interassay variation is established at 20%, 50% and 80% points on the standard curves of displacement or activity. DIAGNOSTIC TESTING
  • normal or standard values from a target should be established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with, for example, an antibody to a target under conditions suitable for complex formation which are well known in the art.
  • the amount of standard complex formation may be quantified by comparing it to a dilution series of positive controls where a known amount of antibody is combined with known concentrations of a purified target.
  • standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by a neurodegenerative disorder. Deviation between, standard and subject values establishes the presence of the disease state.
  • a target itself, or any part thereof, may provide the basis for a diagnostic and/or a prophylactic and/or therapeutic compound.
  • target polynucleotide sequences may be used to detect and quantify gene expression in conditions, disorders or diseases in which neurodegenerative disorder may be implicated.
  • the target encoding polynucleotide sequence may be used for the diagnosis of a neurodegenerative disorder resulting from expression of the target.
  • polynucleotide sequences encoding a target may be used in hybridisation or PCR assays of tissues from biopsies or autopsies or biological fluids, to detect abnormalities in target expression.
  • the form of such qualitative or quantitative methods may include Southern or northern analysis, dot blot or other membrane-based technologies; PCR technologies; dip stick, pin or chip technologies; and ELISA or other multiple sample formal technologies. All of these techniques are well known in the art and are in fact the basis of many commercially available diagnostic kits.
  • Such assays may be tailored to evaluate the efficacy of a particular therapeutic treatment regime and may be used in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for target expression should be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with the target or a portion thereof, under conditions suitable for hybridisation or amplification. Standard hybridisation may be quantified by comparing the values obtained for normal subjects with a dilution series of positive controls run in the same experiment where a known amount of purified target is used. Standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by a disorder or disease related to expression of the target coding sequence.
  • Deviation between standard and subject values establishes the presence of the disease state. If disease is established, an existing therapeutic agent is administered, and treatment profile or values may be generated. Finally, the assay may be repeated on a regular basis to evaluate whether the values progress toward or return to the normal or standard pattern.- Successive treatment profiles may be used to show the efficacy of treatment over a period of several days or several months.
  • the present invention relates to the use of a target polypeptide, or variant, homologue, fragment or derivative thereof, to produce anti-target antibodies which can, for example, be used diagnostically to detect and quantify target levels in neurodegenerative disorder states.
  • the present invention further provides diagnostic assays and kits for the detection of a target in cells and tissues comprising a purified target which may be used as a positive control, and anti-target antibodies.
  • a purified target which may be used as a positive control, and anti-target antibodies.
  • Such antibodies may be used in solution-based, membrane-based, or tissue-based technologies to detect any disease state or condition related to the expression of target protein or expression of deletions or a variant, homologue, fragment or derivative thereof.
  • compositions and/or kits comprising these entites may be used for a rapid, reliable, sensitive, and specific measurement and localization of appropriate channel activity in appropriate tissue extracts.
  • the present invention can be used in diagnosis.
  • a binding agent as discussed above may be used to determine whether or not a patient has an epitope or other structure that is not present in a healthy individual but is present in an individual with a disorder as described herein.
  • the binding agent may be used to identify an epitope or other structure that is not present in an individual with the disorder, but is present in a healthy individual.
  • the epitope or other structure may, for example, be part of an SK (preferably SK3) or T-type channel, or may be part of a moiety that is operatively associated with the channel (e.g. it may be part of a moiety that causes channel opening or closing).
  • a nucleic acid molecule may be used in attempted hybridisation studies to determine whether or not a patient carries a genetic defect (e.g. a defective gene) that may give rise to one or more of the disorders discussed above.
  • a genetic defect affects the structure and/or function of an SK (preferably SK3) channel, a T-type channel, or a moiety operatively associated therewith (e.g. a moiety that causes channel opening or closing).
  • the nucleic acid may be provided as a probe or primer (e.g. in a diagnostic kit). It may be labelled.
  • the probe or primer may be used for PCR-based amplification. It may also be used for reverse PCR.
  • Hybridisation studies may be performed under stringent conditions.
  • Hybridisation conditions are discussed in detail at pp 1.101 -1.110 and 11.45 — 11.61 of Sambrook et al [Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory Press (1989)].
  • One example of stringent hybridisation conditions involves using a pre- washing solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and attempting hybridisation overnight at 55°C using 5 X SSC.
  • 5 X SSC 0.5% SDS
  • 1.0 mM EDTA pH 8.0
  • Hybridisation can be followed by washes of increasing stringency. Thus initial washes may be under conditions of low stringency, but these can be followed with higher stringency washes, up to the stringency of the conditions under which hybridisation was performed.
  • reporter gene may encode an enzyme which catalyses a reaction which alters light absorption properties.
  • the production of the reporter molecule is measured by the enzymatic activity of the reporter gene product, such as ⁇ -galactosidase.
  • a variety of protocols for detecting and measuring the expression of the target such as by using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site, monoclonal-based immunoassay utilising monoclonal antibodies reactive to two non-interfering epitopes on the target is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R et al (1990, Serological Methods, A Laboratory Manual, APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 15 8:121 1).
  • reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.
  • Patents teaching the use of such labels include US-A-3817837; US-A-3850752; US-A-3939350; US-A-3996345; US-A-4277437; US-A- 4275149 and US-A-4366241.
  • recombinant immunoglobulins may be produced as shown in US-A-4816567.
  • thejpresence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed.
  • the nucleotide sequence is inserted within a marker gene sequence, recombinant cells containing the same may be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a target coding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the target as well.
  • host cells which contain the coding sequence for the target and express the target coding regions may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridisation and protein bioassay or immunoassay techniques which include membrane- based, solution-based, or chip-based technologies for the detection and/or quantification of the nucleic acid or protein.
  • In vivo models may be used to investigate and/or design therapies or therapeutic agents to freat neurodegenerative disorders, such as Parkinson's disease.
  • the models could be used to investigate the effect of various tools/lead compounds on a variety of parameters which indicate dopamine release.
  • the invention further provides transgenic nonhuman animals capable of expressing the nucleotide sequence encoding the channel of the present invention or a variant, homologue, derivative or fragment thereof and/or a transgenic nonhuman animal having one or more nucleotide sequence encoding the channel of the present invention or a variant, homologue, derivative or fragment thereof inactivated.
  • Biologically active substances of the present invention may be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated. Thus for example they may be substantially free of one or more potentially contaminating polypeptides- and/or- nucleic- acid molecules. They may be provided in a form that is substantially free of other cell components (e.g. of cell membranes, of cytoplasm, etc.). When a composition is substantially free of a given contaminant, the contaminant will be at a low level (e.g. at a level of less than 10%, less than 5%, or less than 1% on the dry weight/dry weight basis set out above) ALLEVIATED
  • the increase in dopamine release could be utilised in a number of clinical/medical fields - such as in the therapy of schizophrenia and Parkinson's disease.
  • aspects of the present invention relate to:
  • an agent in the manufacture of a medicament, wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode.
  • an agent in the manufacture of a medicament wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron.
  • an agent in the manufacture of a medicament wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode, wherein said agent modulates: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • said agent modulates: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • an agent in the manufacture of a medicament wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron, wherein said agent modulates: a T-type channel and/or an S (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • an agent in the manufacture of a medicament wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in dopamine release from said dopaminergic neuron, wherein said agent modulates: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • an agent in the manufacture of a medicament wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode, wherein said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • an agent in the manufacture of a medicament wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron, wherein said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • an agent in the manufacture of a medicament wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in dopamine release from said dopaminergic neuron, wherein said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • a method of treatment comprising administering to a subject in need of same an agent, wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode.
  • a method of treatment comprising administering to a subject in need of same an agent, wherein said agent is capable of causing a change in the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron.
  • a method of treatment comprising administering to a subject in need of same an agent, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in dopamine release from said dopaminergic neuron.
  • a method of treatment comprising administering to a subject in need of same an agent, wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode, wherein said agent modulates: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • said agent modulates: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • a method of treatment comprising administering to a subject in need of same an agent, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron, wherein said agent modulates: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • a method of freatment comprising administering to a subject in need of same an agent, wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode, wherein said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • a method of treatment comprising administering to a subject in need of same an agent, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron, wherein said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • a method of treatment comprising administering to a subject in need of- same an agent, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in dopamine release from said dopaminergic neuron, wherein said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T-type channel with an SK (preferably SK3) channel.
  • An assay method for identifying and/or improving the effect of an agent wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron.
  • An assay method for identifying and/or improving the effect of an agent wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in dopamine release from said dopaminergic neuron.
  • said agent blocks: a T-type channel and/or an SK (preferably SK3) channel and/or the coupling of a T- type channel with an SK (preferably SK3) channel.
  • the agent can be a mixture of suitable agents.
  • Highly preferred aspects of the present invention relate to: Use of an agent in the manufacture of a medicament, wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode, wherein said agent modulates: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • an agent in the manufacture of a medicament wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron, wherein said agent modulates: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • an agent in the manufacture of a medicament wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in dopamine release from said dopaminergic neuron, wherein said agent modulates: a T-type channel and/or an SK3 channel and/or the coupling of a T- type channel with an SK3 channel.
  • an agent in the manufacture of a medicament wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode, wherein said agent blocks: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • an agent in the manufacture of a medicament wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron, wherein said agent blocks: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • an agent in the manufacture of a medicament wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in dopamine release from said dopaminergic neuron, wherein said agent blocks: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • a method of freatment comprising administering to a subject in need of same an agent, wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode.
  • a method of freatment comprising administering to a subject in need of same an agent, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron.
  • a method of freatment comprising administering to a subject in need of same an agent, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a ⁇ ficial increase in dopamine release from said dopaminergic neuron.
  • a method of freatment comprising administering to a subject in need of same an agent, wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode, wherein said agent modulates: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • a method of freatment comprising administering to a subject in need of same an agent, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron, wherein said agent modulates: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • a method of treatment comprising administering to a subject in need of same an agent, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in dopamine release from said dopaminergic neuron, wherein said agent modulates: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • a method of treatment comprising administering to a subject in need of same an agent, wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode, wherein said agent blocks: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • a method of treatment comprising administering to a subject in need of same an agent, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron, wherein said agent blocks: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • An assay method for identifying and/or improving the effect of an agent wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron.
  • T-type channel with an SK3 channel T-type channel with an SK3 channel.
  • An assay method for identifying and/or improving the effect of an agent wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in dopamine release from said dopaminergic neuron, wherein said agent blocks: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • An assay method for identifying and/or improving the effect (such as by using the assay in a drug development program to improve efficacy) of an agent wherein said assay utilises: a T- type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel, wherein said agent is capable of causing a dopaminergic neuron to enter bursting mode and/or of preventing it from leaving bursting mode.
  • An assay method for identifying and/or improving the effect of an agent wherein said assay utilises: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial change in dopamine release from said dopaminergic neuron.
  • An assay method for identifying and/or improving the effect of an agent wherein said assay utilises: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel, wherein said agent is capable of affecting (in particular causing a change in) the firing mode of a dopaminergic neuron that causes a beneficial increase in dopamine release from said dopaminergic neuron, wherein said agent modulates: a T-type channel and/or an SK3 channel and/or the coupling of a T-type channel with an SK3 channel.
  • the agent can be a mixture of suitable agents.
  • the agent is for the freatment of Parkinson's disease.
  • Nifedipine (lO ⁇ M, L-type) did not affect I-AHPs.
  • FTX-3.3 (l ⁇ M, P/Q-type) had no effect on I-AHPs (see also agatoxin, e).
  • Fig. 3 Use-dependent inactivation of SK and T-type currents display similar kinetics, (a) AHP currents (I-AHP) evoked with hybrid-clamp depolarizations (100ms, +60mV) at a frequency of 1Hz using the standard whole-cell configuration (recording potential -80mV). Successive AHP currents decreased reaching a steady state level at 38% of the initial amplitude. The time constant of cumulative inactivation was 1.26s. Scale bars 0.5s and 50pA. (b) Recording of low voltage-activated currents (I-LVA) evoked by the same voltage pulse protocol as in (a) using the standard whole-cell configuration and calcium channel recording solutions (see methods).
  • I-AHP low voltage-activated currents
  • Nickel-sensitive T-type and apamin-sensitive SK channels maintain the high precision of pacemaker spiking in dopaminergic neurons
  • (a-c) Perforated current-clamp recordings during control (a), lOO ⁇ M nickel (b) and 300nM apamin (c) application.
  • Left panels show a 4s recording trace representative of 5min recording for each condition.
  • Interspike interval (ISI) frequency distributions are displayed in the right panels for each recording.
  • ISI Interspike interval
  • CV coefficient of variation
  • Fig. 5 Inhibition of T-type channels evokes bursting in a subpopulation of dopaminergic midbrain neurons,
  • (a-c) Perforated current-clamp recordings during control (a), nickel (b) and washout (c) conditions.
  • a 20s recording trace representative of 5min recording is shown for each condition.
  • CV coefficient of variation
  • the coefficient of variation (CV) was calculated from gaussian fits_of ISI histograms (n ⁇ Lshown, see fig. 4). Dotted line -50mV. Scale bars Is and lOmV.
  • (a) This neuron showed pacemaker spiking at the lower end of firing precision (CV 20%, compare fig.4).
  • Low voltage-activated calcium channels were recorded in the standard whole-cell configuration and patch pipettes filled with a solution containing (in mM) 140 TEACl, 10 HEPES, 10 EGTA, 2 MgCl 2 , pH 7.3. Recorded neurons were locally perfused with an application solution containing (in mM) 145 TEACl, 2.5 CsCl, 10 HEPES, 2 CaCl 2 , 2 MgCl 2 , 25 glucose, pH 7.4.
  • control solution contained 50 ⁇ M picrotoxin, 5 ⁇ M kynurenic acid, 0.5 ⁇ M tetrodptoxin, 4mM 4-AP, lO ⁇ M nifedipine j lOnM ⁇ -conotoxim Drugs were diluted in the respective application solutions and applied locally ( ⁇ 50 ⁇ m) at 50- lOO ⁇ l/min under visual control.
  • IgorPro For analysis and plotting the software IgorPro was used. Shown voltage-clamp traces were filtered at 100Hz and averaged from 3-10 traces at steady state levels. For spiking pattern analysis a burst detector was programmed in IgorPro. The burst detector compared all interspike intervals (ISIs) of a trace with its mean spiking rate and detected the coincidence of a short ISI ( ⁇ 0.5*mean rate) with a long ISI (>1.25*mean rate) within 2-7 consecutive spikes and marked it as a "burst". Intervals within the burst were additionally tested by a poisson surprise mechanism that compared ISIs with the poisson distribution of all ISIs of a recording (12). The spikes within bursts were summed up and normalized to the total number of spikes in the trace. All values are mean ⁇ S.E.M. Significance values are p ⁇ 0.05 (*). EXPERIMENTAL DISCUSSION
  • Dopaminergic midbrain (DA) neurons are important for movement, cognition, reward and are implicated in disorders such as schizophrenia and Parkinson's disease.
  • SK3 a member of the small-conductance, calcium-activated potassium channel family, controls the frequency and precision of pacemaker spiking in DA neurons of the substantia nigra (SN), but not the ventral tegmental area (1).
  • the aim of the present study was to determine the calcium source of SK channel activation in SN DA neurons of mice.
  • SK channels are assumed to be activated via voltage-sensitive calcium channels (VSCCs) and in hippocampal neurons it may be specifically the L-type VSCC that activates SK channels (2).
  • VSCCs voltage-sensitive calcium channels
  • T-type channel blockers during perforated patch-clamp recording of hybrid pulse evoked AHP currents reduced 85-94% of the calcium-sensitive I-AHP, whereas other VSCC blockers either had no effect (L-type, P/Q-type) or blocked only minor components (26%, N-type).
  • SK and T-type currents display a similar use-dependent inactivation that defines temporal AHP behaviour.
  • SK and T-type channels are equally important to maintain high precision of pacemaker spiking in DA neurons, as shown with perforated current clamp recordings.
  • T-type calcium and small-conductance, calcium-activated potassium (SK) channels constitute a functional signalling complex in mouse DA neurons which stabilizes pacemaker spiking and prevents burst firing.
  • In vivo DA neurones exert their function by integrating synaptic inputs with their prominent intrinsic pacemaker activity to generate patterns of electrical activity that control dendritic and synaptic dopamine release.
  • the intrinsic pacemaker of DA neurones which is preserved in in vitro brain slice preparations, is likely to be orchestrated by a specific set of coexpressed ion channels. Previous studies have highlighted a number of ionic conductances that influence pacemaker activity, but the molecular identity of the respective ion channels was unknown.
  • Ion channels that are active in the subthreshold range are good candidates for tuning the pacemaker frequency and precision in DA midbrain neurones (Grace, 1991; Amini et al., 1999; Kitai et al., 1999).
  • the action potential in DA neurones initiates - at least in DA SN neurones - a prominent, calcium-dependent afterhyperpolarisation that is followed by a spontaneous slow pacemaker-depolarisation that leads the membrane potential back to threshold.
  • As the first of these two phases, which constitute the interspike interval, is sensitive to calcium and apamin, we studied function as well as mRNA and protein expression of the small-conductance calcium-activated potassium (SKI -4) channel family (Bond et al., 1999).
  • Quantitative real-time single-cell RT-PCR linear correlation between pacemaker frequency and ion channel transcript number
  • the single-cell RT-PCR expression profiles for A-type K channel candidate genes were less complex.
  • DA SN neurones that displayed fast-inactivating A-type K currents with varying amplitudes but homogeneous biophysical gating properties which was verified in nucleated outside-out patches, only the long splice, variant of Kv4.3 of all candidate genes tested was expressed.
  • the pharmacological and biophysical properties of native A-currents in DA SN neurones were consistent with the expression of Kv4 channels.
  • the presence of Kv4.3 protein in TH-positive DA SN neurones was confirmed by immunohistochemistry.
  • the classical calbindin- negative SN neurones are Ih channels directly involved in pacemaker confrol.
  • diversity within the DA system is not restricted to diverging axonal projections and differences in synaptic connectivity, but essentially involves differences between DA neurones in their somato- dendritic domains.
  • K-ATP ATP-sensitive potassium
  • DA neurones in the substantia nigra respond differently to low nanomolar concentrations of the complex I inhibitor rotenone.
  • One highly sensitive population of DA SN neurones hyperpolarized and completely lost its spontaneous activity in the presence of rotenone, while another population was not affected.
  • This differential response was mediated by the selective activation of K-ATP channels.
  • Single-cell RT-multiplex PCR we showed that it was correlated to the differential mRNA expression of K-ATP subunits with SUR1/Kir6.2 expression being found in the rotenone- sensitive DA subpopulation (Liss et al., 1999b).
  • hypothalamic K-ATP channels are essential for glucose homeostasis
  • GABAA receptors The pathophysiology of guanidinoacetate methyltransferase (GAMT) deficiency: the accumulating metabolite guanidinoacetate activates GABAA receptors
  • GAMT deficiency is a recently identified human disease of creatine biosynthesis that manifests postnatally with developmental delay and a chorea-like movement disorder.
  • MRI imaging studies have highlighted selective lesions in the globus pallidus.
  • GAA neurotoxic action of the accumulating metabolite guanidinoacetate
  • GAA evoked GABAA receptor mediated chloride currents with an EC 50 of 164 ⁇ M.
  • millimolar concentrations of creatine had no effect on neuroneal activity.
  • the GABA-mimetic action of GAA in brain and especially in the globus pallidus may be a candidate mechanism explaining the extrapyramidal dysfunction in patients with GAMT deficiency (Neu et al., 2000).
  • the aim of this aspect was to understand the genotype-phenotype correlation for single dopaminergic (DA) neurones combining functional electrophysiological with single-cell molecular-biological and immunohistochemical techniques.
  • the two central topics are: a) the characterisation of differentially expressed ion channel genes', which determine the functional differences of DA neurone subpopulations; and b) the identification of differentially expressed ion channel genes involved in the selective vulnerability of degenerating DA neurones in Parkinson's disease.
  • Parkinson's disease (Liss et al., 1999a).
  • DA neurones exert their function by integrating synaptic inputs in the context of their intrinsic pacemaker to generate pattern of elecfrical activity that control dopamine release and their functional effects on their respective target cells.
  • potential diversity within the DA system might originate both from differences in axonal projections and _synaptic_connectivity as well _as_ from, diverging properties of the somato-dendritic integrator and pacemaker.
  • This aspect of the present invention interconnects these distinct anatomical, physiological and pathophysiological maps of DA midbrain neurones on the level of individual neurones. It aims to define the molecular basis and to understand the functional implications of the observed diversity of DA neurones with respect to their electrophysiological function, anatomical connections, and vulnerability to neurodegeneration. Our combination of techniques on the single-cell level is of great advantage as it creates a compendium of single cell genotype-phenotype correlations. Post-genomic biology aimes to define molecular modules that correspond to a particular function as a cluster of co-expressed and co-regulated genes, whose products are likely to physically interact and localise to similar cellular compartments. This aspect addresses the molecular and functional definition of three important modules in DA neurones: 1. the pacemaker generation, intrinsic frequency control, and synaptic integration
  • Neuronal function is intimately coupled to cell-specific patterns of somatodendritic integration and generation of electrical activity. This cellular excitability is orchestrated in turn by a specific set of coexpressed ion channels.
  • DA midbrain neurones mainly as a single population (Sanghera et al., 1984; Grace and Onn, 1989; Lacey et al., 1989; Richards et al., 1997).
  • HCN2, HCN3, HCN4 transcripts and immunocytochemical analysis are carried out with suitable antibodies. These experiments define, which of the coexpressed HCN2-4 subunits are relevant for somato-dendritic Ih channels in DA neurones and whether on a transcriptional level one or more HCN subunits correlate with the large differences in Ih current densities in DA subpopulations that we have identified.
  • Single-cell electroporation (Haas et al., 2001) and ribozyme-based gene suppression techniques (Goodchild, 2000; Liu et al., 2000) for functional validation of the defined HCN genes are used. Ih currents are compared before and after HCN subtype- specific gene suppression in the same neurone. For comparison, heterologous expression of recombinant HCN channels are usefu.
  • the analysis of emerging HCN transgenic or knockout mice may be integrated.
  • Kv4 and KChiP genes Single-cell electroporation (Haas et al., 2001) and ribozyme-based gene suppression techniques (Goodchild, 2000; Liu et al., 2000) for functional validation of the defined Kv4 and KChiP genes are used.
  • A-type potassium currents are compared before and after Kv4/KChip subtype-specific gene suppression in the same neurone.
  • heterologous expression of heteromeric Kv4 + KChiP channels are used.
  • DA neurones are essential in feeding (Zhou and Palmiter, 1995; Szczypka et al., 2000; Wang et al., 2001) and might directly control cortical microcirculation (Krimer et al., 1998).
  • a combination of perforated- patch clamp recordings to define glucose sensitivity with subsequent cell labelling and immunocytochemistry (Neuhoff et al., 2000; Wolfart et al., 2001) to define the topography of glucose-sensing DA neurones are used.
  • mRNA expression profiling is used to define the set of differentially expressed genes including K-ATP channel subunits that render DA subpopulation glucose-sensitive. The functional role of a presumably mitochondrial K-ATP channel is studied in Kir6.1 knockout mice.
  • VGCC voltage-gated calcium channels
  • VGCCs In addition to their direct generator role in rhythmogenesis, VGCCs also provide the calcium influx that controls the activity of calciumrsensitive potassium channels like SK and BK (Marrion and Tavalin, 1998). For SK3 channels, we have recently shown that this mechanism is important for frequency and precision control of the DA pacemaker (Wolfart et al., 2001).
  • BK calcium- and voltage-activated potassium
  • iberiotoxin a highly selective BK peptide blocker is applied in perforated-patch current-clamp recordings of DA neurones. If interesting functional differences between DA populations are discovered, further mRNA expression studies are initiated.
  • DA midbrain neurones The function of DA midbrain neurones is controlled by the interaction of the intrinsic pacemaker mechanism with extrinsic fast (ionotropic) and slow (metabotropic) synaptic input (Kitai et al., 1999).
  • the importance of synaptic integration for DA function maybe best exemplified by the synaptic generation of phasic burst discharge in DA neurones, an activity that dramatically diverges from that of the single spike pacemaker (Overton and Clark, 1997; Lokwan et al., 1999). Burst activity is coupled to dopamine release in a highly non-linear fashion and is believed to be the cellular equivalent for phasic DA signalling in the brain that is critical in reward, cognition and motor learning (Schultz, 1998).
  • Distinct DA subpopulations in vivo show large differences in their frequency of burst firing (Greenhoff et al., 1988), which might depend both, on differential synaptic input and a different set of somato-dendritic conductances (Neuhoff et ak, 2,001).
  • Subthreshold ion channels like Ih (Magee, 1998, 1999), A-type (Hoffman et al., 1997; Schoppa and Westbrook, 1999) and other potassium channels, sodium and calcium channels have been shown to shape the temporal and spatial integration of Synaptic inputs ⁇ and amplify or filter the inputs in- nroften non-linear fashion-in other neurones (Bennett and Wilson,- 1998; Hausser et al., 2001; Stuart and Hausser, 2001; Fricker and Miles, 2002). We have defined significant differences in the repertoire of these channels in distinct DA populations (Neuhoff et al., 2001) they are likely to possess different modes of synaptic integration.
  • DA SN neurones possess very efficient dendritic backpropagation of action potentials (Hausser et al., 1995) implicating the somato-dendritic spatial distribution of ion channel species as a significant component.
  • the integration of stimulated and simulated injection of synaptic input are investigated focussing on the conditions of burst initiation combining standard and/or dendritic whole-cell recordings with stimulation of pharmacologically isolated excitatory or inhibitory input (Kitai et al., 1999).
  • stereotactically guided retrograde tracer (initially fluorescent beads) are injected in the different target areas of dopaminergic axons starting with the prefrontal cortex, nucleus accumbens (core/shell) and dorsolateral striatum in mice (Mattiace et al., 1989). After 1-2 postoperative days the animals are sacrificed and coronal midbrain slices prepared. Fluorescently labelled individual neurones are identified by fluorescence microscopy. The electrophysiological properties of these DA neurones are studied in patch- clamp recordings and the cells labelled for immunohistochemical processing. This may generate a functional map of DA neurones with identified axonal projections and target the important question to what extent the distinct functional properties of DA neurones (see I &II) are co-segregated with their axonal projections.
  • the present invention seeks- to precise understanding of the physiology - of DA midbrain neurones in the context of their different functional roles in the brain. This knowledge may also provide a detailed framework to identify relevant pathophysiological changes occurring in particular DA subpopulations during brain disease states. These might include altered pacemaker activity, altered synaptic input and integration as well as altered responses to metabolic or modulatory pathways. For a genetic model of dopaminergic neurodegeneration, the weaver mouse, we have demonstrated how dramatic these changes can be (Liss et al., 1999a). Furthermore, the definition of single-cell genotype-phenotype correlations may facilitate the identification of those genes that confribute to the pathophysiology.
  • dopamine neurochemistry TH-immunohistochemistry, stereological non-biased cell counts, rotarod tests
  • the functional properties of DA neurones in in vitro brain slices during different stages of the PD model in order to identify the chronological order of pathophysiological changes that occur in different types of DA neurones is investigated. Based on previous work (Neuhoff et al., 2000, 2001), the phenotypic plasticity in the four DA population in SN and VTA is compared. The highly vulnerable (ventral tier calbindin-negative DA SN neurones) are compared with the more resistant DA neurones (dorsal tier calbindin-positive DA SN and DA VTA neurones).
  • the limiting factor is not primarily the DNA array technology that is now widely available and well established (Epstein and Butow, 2000; Hughes et al., 2000) but a reliable method of linear amplification of the single cell mRNA or cDNA pool (Kacharmina et al., 1999). With the current array technology, this is necessary to generate sufficient starting material for the hybridisation analysis on DNA arrays (oligonucleotides or cDNA clones) for up to several thousand genes.
  • kits comprising, in separate containers, an agent capable of causing increased dopamine release and an agent capable of causing reduced dopamine release. This can be useful in regulating dopamine levels.
  • Kitai ST Shepard PD, Callaway JC, Scroggs R (1999) Afferent modulation of dopamine neuron firing patterns. Curr Opin Neurobiol 9:690-697.
  • Tzschentke TM (2001) Pharmacology and behavioral pharmacology of the mesocortical dopamine system. Prog Neurobiol 63:241-320. Verhoeff NP (1999) Radiofracer imaging of dopaminergic fransmission in neuropsychiatric disorders. Psychopharmacology (Berl) 147:217-249.
  • Lacey MG Mercuri NB, North RA (1989) Two cell types in rat substantia nigra zona compacta distinguished by membrane properties and the actions of dopamine and opioids. J Neurosci 9:1233-1241.
  • Verhoeff NP (1999) Radiofracer imaging of dopaminergic transmission in neuropsychiafric disorders. Psychopharmacology (Berl) 147:217-249.
  • Damier P Hirsch EC, Agid Y, Graybiel AM (1999a) The substantia nigra of the human brain.
  • I Nigrosomes and the nigral matrix, a compartmental organization based on calbindin D(28K) immunohistochemistry [see comments].
  • Trpcl and trpc5 form a novel cation channel in mammalian brain. Neuron 29:645-655.
  • Tzschentke TM (2001) Pharmacology and behavioral pharmacology ° of the mesocortical dopamine system. Prog Neurobiol 63:241-320. Van Goor F, Krsmanovic LZ, Catt KJ, Stojilkovic SS (1999) Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion. Proc Natl Acad Sci U S A 96:4101-4106.
  • Verhoeff NP (1999) Radiofracer imaging of dopaminergic transmission in neuropsychiafric disorders. Psychopharmacology (Berl) 147:217-249.

Landscapes

  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Endocrinology (AREA)
  • Diabetes (AREA)
  • Pathology (AREA)
  • Rheumatology (AREA)
  • Toxicology (AREA)
  • Urology & Nephrology (AREA)
  • Psychology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
EP02770126A 2001-11-07 2002-10-30 Modulierung von dopaminergischen neuronen Withdrawn EP1465609A2 (de)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GBGB0126781.4A GB0126781D0 (en) 2001-11-07 2001-11-07 Modulation
GB0126781 2001-11-07
US10/216,128 US20030087799A1 (en) 2001-11-07 2002-08-09 Modulation
US216128 2002-08-09
PCT/GB2002/004880 WO2003039449A2 (en) 2001-11-07 2002-10-30 Modulation of dopaminergic neurons

Publications (1)

Publication Number Publication Date
EP1465609A2 true EP1465609A2 (de) 2004-10-13

Family

ID=26246750

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02770126A Withdrawn EP1465609A2 (de) 2001-11-07 2002-10-30 Modulierung von dopaminergischen neuronen

Country Status (3)

Country Link
EP (1) EP1465609A2 (de)
JP (1) JP2005518346A (de)
WO (1) WO2003039449A2 (de)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003271548A1 (en) * 2002-10-13 2004-05-04 Neurosearch A/S Use of skca channel blocking drugs for combating parkinson's disease
US20170056352A1 (en) 2015-08-25 2017-03-02 Rgenix, Inc. PHARMACEUTICALLY ACCEPTABLE SALTS OF beta-GUANIDINOPROPIONIC ACID WITH IMPROVED PROPERTIES AND USES THEREOF
WO2018160178A1 (en) 2017-03-01 2018-09-07 Rgenix, Inc. Pharmaceutically acceptable salts of b-guanidinopropionic acid with improved properties and uses thereof
IL293721A (en) 2019-12-11 2022-08-01 Inspirna Inc Methods of treating cancer

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4981867A (en) * 1989-12-08 1991-01-01 The Board Of Trustees Of The Leland Stanford Junior University Treatment of tremor using compounds that depress calcium currents in thalamic and other central nervous system neurons
HU226635B1 (en) * 1995-01-23 2009-05-28 Asubio Pharma Co Piperidine and piperazine derivatives, medicines containing them as active ingredient and their use
US6083932A (en) * 1997-04-18 2000-07-04 Cv Technologies Inc. Pharmaceutical compositions derived from ginseng and methods of treatment using same
AU754656B2 (en) * 1997-10-31 2002-11-21 Asubio Pharma Co., Ltd. Arylpiperidinopropanol and arylpiperazinopropanol derivatives and pharmaceuticals containing the same
AU760309B2 (en) * 1997-12-03 2003-05-15 Merck & Co., Inc. Low-voltage activated calcium channel compositions and methods
DE69924493T2 (de) * 1998-07-02 2005-09-15 Neurosearch A/S Kaliumkanal-blockierende mittel
DE60005517T2 (de) * 1999-06-29 2004-07-01 Neurosearch A/S Kaliumkanal-blockierende mittel
WO2001002561A2 (en) * 1999-07-02 2001-01-11 Neuromed Technologies, Inc. Mammalian calcium channels and related probes, cell lines and methods
US6358706B1 (en) * 1999-10-26 2002-03-19 Ortho-Mcneil Pharmaceutical, Inc. DNA encoding human alpha1G-C T-Type calcium channel

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03039449A2 *

Also Published As

Publication number Publication date
JP2005518346A (ja) 2005-06-23
WO2003039449A2 (en) 2003-05-15
WO2003039449A3 (en) 2004-04-15

Similar Documents

Publication Publication Date Title
Borjigin et al. A novel pineal night-specific ATPase encoded by the Wilson disease gene
US8410054B2 (en) Methods for treating pain by inhibition of the SCN9A channel
US5658786A (en) DNA encoding rat taurine transporter and uses thereof
US8703125B2 (en) Modulation of the Vps10p-domain receptor family for the treatment of mental and behavioural disorders
US6720175B1 (en) Nucleic acid molecule encoding homer 1B protein
US20030087799A1 (en) Modulation
Ibi Role of interaction of mGlu2 and 5-HT2A receptors in antipsychotic effects
US8871443B2 (en) Schizophrenia-related isoform of KCNH2 and development of antipsychotic drugs
US20080311578A1 (en) System for screening eukaryotic membrane proteins
US9278088B2 (en) Methods for identifying analgesic agents
JP2011229529A (ja) 糖尿病および肥満調節におけるｇｐｒ１００受容体の使用
EP1465609A2 (de) Modulierung von dopaminergischen neuronen
CA2467206A1 (en) Genes encoding g-protein coupled receptors and methods of use therefor
US8038995B2 (en) Human N-type calcium channel isoform and uses thereof
WO2003033672A2 (en) Nr3b nmda receptor subunit compositions and related methods
AU2002335985A1 (en) Modulation of dopaminergic neurons
JP2002523056A (ja) Ｈｏｍｅｒ相互作用タンパク質
JP2007530069A (ja) イオンチャネル
JP2007530069A6 (ja) イオンチャネル
EP1662862A1 (de) Rezeptor
KR20060129411A (ko) 이온 채널
KR20070018987A (ko) 수용체 ｇｐｒ86의 용도
Silva et al. A meta-analysis of the biomarkers in late-onset Spinal Muscular Atrophy patients treated with Nusinersen
US20030008823A1 (en) DNA encoding a human 5-HT1F receptor and uses thereof
Friedman Alterations of Expression of the Serotonin 5-HT4 Receptor in Brain Disorders

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20040602

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

RIN1 Information on inventor provided before grant (corrected)

Inventor name: ROEPER, JOCHEN

Inventor name: WOLFART, JAKOB

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20041221