Method for the treatment of amyloidoses

Abstract

The present invention relates to a method for the treatment of an amyloidosis such as Alzheimer's disease in a subject in need thereof, characterized in that it comprises administering an agonist of the P/Q type voltage-gated presynaptic calcium channel to said subject.

Description

CROSS-REFERENCE

[0001] This application is the National Stage of International Application No. PCT/EP2008/001549, filed on Feb. 27, 2008, which claims the benefit of European Application Serial No. 07020257.7, filed Oct. 16, 2007, European Application Serial No. 08000325.4, filed Jan. 9, 2008, and U.S. Provisional Application Ser. No. 60/903,700, filed Feb. 27, 2007, all of which are incorporated herein by reference in its entirety.

[0002] The present invention relates to a method for the treatment of an amyloidosis such as Alzheimer's disease.

[0003] Alzheimer's disease (AD), the most frequent cause for dementia among the aged with an incidence of about 10% of the population above 65 years, is a dementing disorder characterized by a progressive loss of cognitive abilities and by characteristic neuropathological features comprising extracellular amyloid deposits, intracellular neurofibrillary tangles and neuronal loss in several brain regions (Mattson, M. P. Pathways towards and away from Alzheimer's disease. Nature 430, 631-639 (2004); Hardy, J. & Selkoe, D. J. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353-356 (2002)). The principal constituents of the amyloid deposits are amyloid .beta. peptides (A.beta.) which arise from the (.beta.-amyloid precursor protein (APP) by proteolytic cleavage.

[0004] Both cerebral amyloid deposits and cognitive impairments very similar to those observed in Alzheimer's disease are also hallmarks of Down's syndrome (trisomy 21), which occurs at a frequency of about 1 in 800 births. Hence, Alzheimer's disease and Down's syndrome are jointly termed "amyloidoses".

[0005] Recently, however, it was shown that in amyloidoses soluble, globular A.beta. oligomers (hereinafter referred to as A.beta. globulomers), rather than the eponymous insoluble amyloid deposits, are the causative agents for the impairment of higher-level functions, such as memory function, as indicated by its suppressing effect on long-term potentiation (WO2004/067561; Barghorn S. et al., J. Neurochem. 95: 834-847 (2005); WO2006/094724).

[0006] The term "A.beta. globulomer" here refers to a particular soluble, globular, non-covalent association of A.beta. peptides, possessing homogeneity and distinct physical characteristics. A.beta. globulomers are stable, non-fibrillar, oligomeric assemblies of A.beta. peptides which are obtainable by incubation with anionic detergents, in particular as described in WO2004/067561. In contrast to A.beta. monomer and fibrils, these globulomers are characterized by defined assembly numbers of subunits (WO2004/067561). The globulomers have a characteristic three-dimensional globular type structure ("molten globule", see Barghorn et al., J. Neurochem. 95: 834-847 (2005)). They have been shown to closely mimic the properties, behaviour and effects of naturally occurring soluble A.beta. oligomers.

[0007] Soluble A.beta. oligomer was found to impair the functioning of the central nervous system even before the onset of cytotoxicity. However, the exact mechanisms whereby soluble A.beta. oligomer causes memory failure in amyloidoses has not been elucidated so far, and a lack of understanding of such mechanisms has so far hampered the development of rational therapeutic approaches for inhibiting the further progression of the disease or compensating the damage already done.

[0008] It was thus an object of the present invention to provide a new approach to the treatment of amyloidoses such as Alzheimer's disease, in particular to rehabilitating treatment such as the restoration of cognitive abilities in amyloidoses such as Alzheimer's disease.

[0009] Surprisingly, it was now found that A.beta. globulomer exerts its detrimental effects essentially by hampering normal ion fluxes through the P/Q type presynaptic calcium channel, reducing presynaptic neurotransmitter release and inhibiting spontaneous synaptic activity and thereby interfering with the proper functioning of the central nervous system even before the onset of manifest neural cytotoxicity, and that activation of the P/Q type presynaptic calcium channel is therefore effective in compensating these effects (increasing the extracellular Ca.sup.2+ concentration (P/Q type presynaptic calcium channel agonism) was effective in reversing the inhibitory effect of A.beta. globulomer on the P/Q type voltage-gated presynaptic calcium chnnel).

[0010] The present invention thus relates to a method for the treatment of an amyloidosis, preferably Alzheimer's disease, in a subject in need thereof, comprising administering an agonist of the P/Q type voltage-gated presynaptic calcium channel to said subject.

[0011] The P/Q type voltage-gated presynaptic calcium channel (the channel is also referred to as Ca.sub.v2.1 channel and the associated currents as P/Q type currents) belongs to the group of voltage-gated calcium channels which mediate the influx of calcium ions into excitable cells. The opening state of a voltage-gated channel is controlled by the electrical state of the surrounding membrane; however, the responsiveness of the P/Q type voltage-gated presynaptic calcium channel to membrane depolarization is extensively modulated, both qualitatively and quantitatively, by and/or through its interaction partners.

[0012] As used herein, a "P/Q type voltage-gated presynaptic calcium channel" is a voltage-gated calcium channel that is functionally characterized by its sensitivity towards .omega.-agatoxin IVA (a well-known funnel web spider venom).

[0013] According to a particular embodiment, .omega.-agatoxin IVA acts as a gating modifier of the P/Q type voltage-gated presynaptic calcium channel (e.g., P type Kd=1-3 nM; Q type Kd=100-200 nM). Further, P/Q type voltage-gated presynaptic calcium channels according to the present invention may be characterized by one or more than one of the following features: [0014] (i) requires strong depolarization for activation (high-voltage activation); and [0015] (ii) no or slow inactivation.

[0016] The P/Q type voltage-gated presynaptic calcium channel according to the present invention comprises an .alpha.1 subunit. According to a particular embodiment of the invention, the .alpha.1 subunit has an amino acid sequence with at least 70%, advantageously at least 80%, preferably at least 90%, more preferably at least 95% and in particular at least 98%, e. g. at least 99%, amino acid sequence identity with the sequence SEQ ID NO:1. The .alpha.1 subunit incorporates the conduction pore, the voltage sensor and gating apparatus, and sites of channel regulation by second messengers, drugs, and toxins.

[0017] Usually, the P/Q type voltage-gated presynaptic calcium channel also comprises an .alpha.2-.delta. subunit and a .beta. subunit. It may also comprise an .gamma. subunit. In a particular embodiment of the invention, the .alpha.2-.delta. subunit, when present, has at least 70%, advantageously at least 80%, preferably at least 90%, more preferably at least 95% and in particular at least 98%, e. g. at least 99%, amino acid sequence identity with the sequence SEQ ID NO:2. In a further particular embodiment of the invention, the .beta. subunit, when present, has at least 70%, advantageously at least 80%, preferably at least 90%, more preferably at least 95% and in particular at least 98%, e. g. at least 99%, amino acid sequence identity with the sequence SEQ ID NO:3.

[0018] Further characteristic features of P/Q type voltage-gated presynaptic calcium channels are described in Catterall W A, Perez-Reyes E, Snutch T P, Striessnig J. International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels. Pharmacol Rev. 57: 411-25 (2005), which is herein incorporated by reference in its entirety.

[0019] As used herein, an "agonist of the P/Q type voltage-gated presynaptic calcium channel" is any substance that increases the flow of calcium ions through the P/Q type voltage-gated presynaptic calcium channel.

[0020] According to a particular embodiment, the agonist of the invention increases the open probability of the channel.

[0021] According to a further particular embodiment, the agonist of the invention directly interacts with the closed channel to open it.

[0022] According to a further particular embodiment, the agonist of the invention increases the duration of the open state once the channel has been opened.

[0023] According to a further particular embodiment, the agonist of the invention interacts with the time constants of voltage-gated activation, voltage-gated inactivation and voltage-gated deinactivation in a way which results in an increased net calcium flux under physiological conditions and which may be voltage-dependent in itself.

[0024] According to a further particular embodiment, the agonist of the invention changes one or more transition probabilities between the different states of the channel (closed, open, inactivated).

[0025] Hence an agonist of the P/Q type voltage-gated presynaptic calcium channel will increase ion flux through the P/Q type voltage-gated presynaptic calcium channel.

[0026] The agonist of the P/Q type voltage-gated presynaptic calcium channel is preferably a partial or complete agonist of the P/Q type voltage-gated presynaptic calcium channel, and more preferably a complete agonist of the P/Q type voltage-gated presynaptic calcium channel.

[0027] As used herein, a "partial agonist of the P/Q type voltage-gated presynaptic calcium channel" is an agonist which is not capable even at high concentration of causing the maximal activating effect of the P/Q type voltage-gated presynaptic calcium channel, as defined above. In a particular embodiment a partial agonist does not lead to maximal calcium flow through the P/Q type voltage-gated presynaptic calcium channel at saturating concentration.

[0028] As used herein, a "complete agonist of the P/Q type voltage-gated presynaptic calcium channel" is an agonist which is capable of inducing, at suitable concentrations, maximal activation of the P/Q type voltage-gated presynaptic calcium channel, as defined above.

[0029] The person skilled in the art will understand that an agonist of the P/Q type voltage-gated presynaptic calcium channel may either bind directly to the P/Q type voltage-gated presynaptic calcium channel, i. e. by binding the calcium channel molecule, e. g. by forming a covalent or non-covalent attachment to said calcium channel molecule, or exert its effect on the ion channel predominantly without direct physical contact between the agonist and said calcium channel, the effect being mediated, in this case, by any of a wide range of go-betweens. These include but are not limited to molecules with a capacity of complexing said calcium channel that is modified in the presence of the agonist; catalytically active molecules which regulate said calcium channel in a way that is influenced by the presence of the agonist; and physico-chemical effects, in particular membrane effects, subject to influences of the agonist, such as shifts in the molecular arrangement, fluidity, conductivity, etc. of the membrane. In particular, an agonist of the P/Q type voltage-gated presynaptic calcium channel may bind to both the P/Q type voltage-gated presynaptic calcium channel and components of its environment, thereby influencing the mutual interaction. In all of these cases, under otherwise identical conditions the behaviour of said calcium channel in the presence of an effective amount of the agonist will be detectably different from that in the absence of said agonist.

[0030] In the context of the present invention, the term "bind" is used generically to denote any immediate association between two molecules, which may be covalent or non-covalent, thus including covalent bonds, hydrogen bridges, ionic interactions, hydrophobic associations, van der Waals forces, etc. It will thus be understood that the term also extends to the temporary association of a first molecule with a catalytically active second molecule, wherein said second molecule performs modifications on said first molecule which, and consequently whose effects, outlast the actual contact between said first and said second molecule, e. g. generation or removal of covalent bonds.

[0031] In a particular embodiment of the invention, the agonist increases net calcium flux through the P/Q type voltage-gated presynaptic calcium channel with an EC50 of less than 120 .mu.M, preferably of less than 10 .mu.M, and in particular of less than 1 .mu.M. Here the EC50 is the agonist concentration required for obtaining 50% of a maximum effect of this agonist determined using the patch-clamp method for whole-cell recording of channel activity.

[0032] The standard method employed here for all determinations of Ca.sup.++ currents is a patch-clamp method using 120 mM NMG.Cl, 10 mM TEA.Cl, 14 mM creatine phosphate, 6 mM MgCl.sub.2, 1.mM CaCl.sub.2 10 mM NMG.HEPES, 5 mM Tris.sub.2.ATP and 11 NMG.sub.2.EGTA as internal, and 30 mM BaCl.sub.2, 100 mM NMG.Cl, 10 mM NMG.HEPES and 15 mM glucose as external solution, both adjusted to a pH of about 7.2-7.3, for measuring stably transfected BHK (Baby Hamster Kidney) cells expressing the al component together with the .alpha.2.delta. and .beta.IB components of the P/Q type voltage-gated presynaptic calcium channel.

[0033] Further details of said standard method have been described by Zafir Buraei et al., Roscovitine differentially affects CaV2 and Kv channels by binding to the open state, Neuropharmacology (2006), doi:10.1016/j.neuropharm.2006.10.006 (corresponds to issue 52, 2007, pages 883-894), which is herein incorporated by reference in its entirety.

[0034] Unexpectedly, it was further found that other presynaptic calcium channels, such as the N and R type voltage-gated presynaptic calcium channels, are not susceptible to A.beta., in particular to A.beta. globulomer, and it is known to the skilled person that these types of channels may all be present in parallel at any given synapse, or one of them may be dominant in terms of abundance and thus account for the major part of the Ca.sup.++ influx. It is thus, in the interest of reducing side effects, advantageous to employ in the method of the present invention an agonist which specifically affects the P/Q type voltage-gated presynaptic calcium channel only. Thus, it is advantageous if the agonist affects the P/Q type voltage-gated presynaptic calcium channel with lower EC50 than either the N type or the R type, or both the N and the R type voltage-gated presynaptic calcium channels. In a particular embodiment of the invention, the agonist of the P/Q type voltage-gated presynaptic calcium channel thus increases net calcium flux through the L type voltage-gated presynaptic calcium channel with an EC50 of more than 54 .mu.M, preferably of more than 100 .mu.M, and in particular of more than 1000 .mu.M, using the standard method as defined above. In a further particular embodiment of the invention, it increases net calcium flux through the N type voltage-gated presynaptic calcium channel with an EC50 of more than 54 .mu.M, preferably of more than 100 .mu.M, and in particular of more than 1000 .mu.M, using the standard method as defined above. In a further particular embodiment of the invention, it increases net calcium flux through the R type voltage-gated presynaptic calcium channel with an EC50 of more than 54 .mu.M, preferably of more than 100 .mu.M, and in particular of more than 1000 .mu.M, using the standard method as defined above.

[0035] It has been reported that inhibition of CDKs (cyclin-dependant kinases), which kinases are known to be crucial elements of the cell cycle, inhibits dedifferentiation and proliferation and thereby stimulates neurons to increase their expression of ion channels such as voltage-gated presynaptic calcium channels. However, due to the naturally highly pleiotropic effects of CDK inhibitors and in particularly their propensity to induce apoptosis, which is expected to outweigh the benefits due to the neurotoxicity caused thereby, it is preferred that the agonists for use in the methods of the present invention do not exert any significant inhibitory effect on CDKs such as CDK2 (cyclin A) and CDK5 (p35). In a particular embodiment of the invention, the agonist thus has an IC50 for CDK5, in particular for human CDK5, of more than 0.2 .mu.M, preferably of more than 10 .mu.M, and in particular of more than 100 .mu.M. In a further particular embodiment of the invention, the agonist thus has an IC50 for CDK2, in particular for human CDK2, of more than 0.7 .mu.M, preferably of more than 10 .mu.M, and in particular of more than 100 .mu.M. Preferably, the agonist has an IC50 for any CDK, in particular for any human CDK, of more than 0.5 .mu.M, preferably of more than 10 .mu.M, and in particular of more than 100 .mu.M. These IC50 values all refer to the activity of the respective CDK/cyclin complex in a radioactive kinase assay using 15 .mu.M of [.gamma.-.sup.32P]ATP as phosphate donor and an appropriate phosphorylation target protein (1 mg/ml histone H1 or retinoblastoma protein complexed with glutathione-S-transferase, respectively) as acceptor in a reaction buffer comprising 60 mM glycerol-2-phosphate, 15 mM p-nitrophenyl phosphate, 25 mM MOPS pH 7.2, 5 mM EGTA, 15 mM MgCl.sub.2 and 1 mM dithiotreitol, as described by Meijer et al., Eur. J. Biochem. 234: 527-536 (1997).

[0036] The metabolism of APP and its products such as A.beta. is complex and not yet fully understood. Therefore, in a particular embodiment of the invention, the agonist as such does not after APP expression, A.beta. formation or processing, e. g. soluble A.beta. oligomer or A.beta. fibril formation, in the central nervous system. In another particular embodiment, however, its use may be combined with a suitable treatment, e. g. a treatment to suppress the formation of soluble A.beta. oligomers and/or to promote their degradation and/or elimination from the central nervous system; essentially any such treatment may be combined with the methods of the present invention.

[0037] Roscovitine has been described as an agonist of the P/Q type voltage-gated presynaptic calcium channel (Yan Z, Chi P, Bibb J A, Ryan T A and Greengard P., J. Physiol. 540 : 761-770 (2002)). As used herein, the term "roscovitine" denotes the compound (1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine and any of its isomers, in particular 2-(R)-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine.

[0038] However, roscovitine is known to reduce APP formation by binding to and inhibiting the action of human CDKs, in particular CDK5, thereby being prone to the adverse effects outlined above. Therefore, agonists other than roscovitine or any pharmacologically useful salt or derivative of roscovitine, in particular such as may be readily converted to roscovitine in vivo (prodrug), are preferred.

[0039] According to a particular embodiment, suitable agonists of the P/Q type voltage-gated presynaptic calcium channel of the invention are selected among the roscovitine analogues of formula (Ia):

##STR00001##

wherein [0040] R.sup.1 is hydrogen or C.sub.1-C.sub.6-alkyl; [0041] R.sup.2a, R.sup.2b are independently hydrogen, C.sub.1-C.sub.6-alkyl, C.sub.2-C.sub.6-alkenyl, C.sub.3-C.sub.8-cycloalkyl, optionally substituted C.sub.6-C.sub.12-aryl or optionally substituted C.sub.6-C.sub.12-aryl-C.sub.1-C.sub.4-alkyl, or [0042] R.sup.2a, R.sup.2b together are C.sub.2-C.sub.5-alkylene; [0043] Q is NR.sup.3; [0044] R.sup.3 is hydrogen, C.sub.1-C.sub.6-alkyl or optionally substituted C.sub.6-C.sub.12-aryl; [0045] X is N or CR.sup.4; [0046] R.sup.4 is hydrogen or C.sub.1-C.sub.6-alkyl, [0047] Y is N or CR.sup.5; and [0048] R.sup.5 is hydrogen or C.sub.1-C.sub.6-alkyl, and the pharmacologically useful salts thereof.

[0049] According to a further particular embodiment, suitable agonists of the P/Q type voltage-gated presynaptic calcium channel of the invention are selected among the roscovitine analogues of formula (Ib):

##STR00002##

wherein [0050] R.sup.1 is hydrogen or C.sub.1-C.sub.6-alkyl; [0051] R.sup.2a, R.sup.2b are independently hydrogen, C.sub.1-C.sub.6-alkyl, C.sub.2-C.sub.6-alkenyl, C.sub.3-C.sub.8-cycloalkyl, optionally substituted C.sub.6-C.sub.12-aryl or optionally substituted C.sub.6-C.sub.12-aryl-C.sub.1-C.sub.4-alkyl, or [0052] R.sup.2a, R.sup.2b together are C.sub.2-C.sub.5-alkylene; [0053] Q is NR.sup.3; [0054] R.sup.3 is hydrogen, C.sub.1-C.sub.6-alkyl or optionally substituted C.sub.6-C.sub.12-aryl; [0055] X is N or CR.sup.4; [0056] R.sup.4 is hydrogen or C.sub.1-C.sub.6-alkyl, [0057] Y is N or CR.sup.5; and [0058] R.sup.5 is hydrogen or C.sub.1-C.sub.6-alkyl, and the pharmacologically useful salts thereof.

[0059] According to a further particular embodiment, suitable agonists of the P/Q type voltage-gated presynaptic calcium channel of the invention are selected among isoproterenol and isoproterenol analogues of formula (II):

##STR00003##

wherein [0060] R.sup.1 is C.sub.1-C.sub.6-alkyl or C.sub.3-C.sub.8-cycloalkyl; [0061] R.sup.2a, R.sup.2b, R.sup.2c, R.sup.2d, R.sup.2e are independently hydrogen, halogen, C.sub.1-C.sub.4-alkyl, optionally substituted phenyl, OH, SH, CN, CF.sub.3, O--CF.sub.3, C.sub.1-C.sub.4-alkoxy, NH.sub.2, NH--C.sub.1-C.sub.4-alkyl, N--(C.sub.1-C.sub.4-alkyl).sub.2, or [0062] R.sup.2b and R.sup.2c or R.sup.2b and R.sup.2d together with the carbon atoms to which they are attached form an optionally substituted anellated C.sub.5-C.sub.7 carbocyclic ring; [0063] and the pharmacologically useful salts thereof.

[0064] Provided that the compounds of the formulae (Ia), (Ib) and (II) exist in different spatial arrangements, for example if they possess one or more centers of asymmetry, polysubstituted rings or double bonds, or as different tautomers, it is also possible to use enantiomeric mixtures, in particular racemates, diastereomeric mixtures and tautomeric mixtures, preferably, however, the respective essentially pure enantiomers, diastereomers and tautomers of the compounds of formulae (Ia), (Ib) and (II) and/or of their salts.

[0065] The pharmacologically useful salts of the compounds of the formulae (Ia), (Ib) and (II) are especially acid addition salts with physiologically tolerated acids. Examples of suitable physiologically tolerated organic and inorganic acids are hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, C.sub.1-C.sub.4-alkylsulfonic acids, such as methanesulfonic acid, cycloaliphatic sulfonic acids, such as S-(+)-10-campher sulfonic acid, aromatic sulfonic acids, such as benzenesulfonic acid and toluenesulfonic acid, di- and tricarboxylic acids and hydroxycarboxylic acids having 2 to 10 carbon atoms, such as oxalic acid, malonic acid, maleic acid, fumaric acid, lactic acid, tartaric acid, citric acid, glycolic acid, adipic acid and benzoic acid. Other utilizable acids are described, e.g., in Fortschritte der Arzneimittelforschung [Advances in drug research], Volume 10, pages 224 ff., Birkhauser Verlag, Basel and Stuttgart, 1966.

[0066] The organic moieties mentioned in the above definitions of the variables are--like the term halogen--collective terms for individual listings of the individual group members. The prefix C.sub.n-C.sub.m indicates in each case the possible number of carbon atoms in the group.

[0067] Unless specified, the term "substituted" means that a radical may be substituted with 1, 2 or 3, especially 1 or 2, substituent selected from the group consisting of halogen, C.sub.1-C.sub.4-alkyl, OH, SH, CN, CF.sub.3, O--CF.sub.3, C.sub.1-C.sub.4-alkoxy, NH--C.sub.1-C.sub.4-alkyl, N--(C.sub.1-C.sub.4-alkyl).sub.2, in particular with 1, 2 oder 3 substituents selected from the group consisting of halogen, methyl, OH, CN, CF.sub.3, O--CF.sub.3, methoxy, NH.sub.2, NH--CH.sub.3, and N--(CH.sub.3).sub.2.

[0068] The term halogen denotes in each case fluorine, bromine, chlorine or iodine, in particular fluorine or chlorine.

[0069] C.sub.1-C.sub.4-Alkyl is a straight-chain or branched alkyl group having from 1 to 4 carbon atoms. Examples of an alkyl group are methyl, C.sub.2-C.sub.4-alkyl such as ethyl, n-propyl, iso-propyl, n-butyl, 2-butyl, iso-butyl or tert-butyl. C.sub.1-C.sub.2-Alkyl is methyl or ethyl, C.sub.1-C.sub.3-alkyl is additionally n-propyl or isopropyl.

[0070] C.sub.1-C.sub.6-Alkyl is a straight-chain or branched alkyl group having from 1 to 6 carbon atoms. Examples include methyl, C.sub.2-C.sub.4-alkyl as mentioned herein and also pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl and 1-ethyl-2-methylpropyl.

[0071] C.sub.3-C.sub.8-Cycloalkyl is a cycloaliphatic radical having from 3 to 12 carbon atoms. In particular, 3 to 6 carbon atoms form the cyclic structure, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The cyclic structure may be unsubstituted or may carry 1, 2, 3 or 4 C.sub.1-C.sub.4 alkyl radicals, preferably one or more methyl radicals.

[0072] C.sub.6-C.sub.12-aryl-C.sub.1-C.sub.4-alkyl is a straight-chain or branched alkyl group having 1 to 4 carbon atoms, preferably 1 to 3 carbon atoms, more preferably 1 or 2 carbon atoms, in particular 1 or two carbon atoms, wherein one hydrogen atom is replaced by C.sub.6-C.sub.12-aryl, such as in benzyl.

[0073] C.sub.2-C.sub.6-Alkenyl is a singly unsaturated hydrocarbon radical having 2, 3, 4, 5 or 6 carbon atoms, e.g. vinyl, allyl (2-propen-1-yl), 1-propen-1-yl, 2-propen-2-yl, methallyl(2-methylprop-2-en-1-yl) and the like. C.sub.3-C.sub.4-Alkenyl is, in particular, allyl, 1-methylprop-2-en-1-yl, 2-buten-1-yl, 3-buten-1-yl, methallyl, 2-penten-1-yl, 3-penten-1-yl, 4-penten-1-yl, 1-methylbut-2-en-1-yl or 2-ethylprop-2-en-1-yl.

[0074] C.sub.6-C.sub.12-Aryl is a 6- to 12-membered, in particular 6- to 10-membered, aromatic cyclic radical. Examples include phenyl and naphthyl.

[0075] C.sub.2-C.sub.7-Alkylene is straight-chain or branched alkylene group having from 2 to 7 carbon atoms. Examples include ethylene, 1,3-propylene, 1,4-butylene and 1,5-pentylene.

[0076] Particular roscovitine analogues are defined as follows.

[0077] According to a particular embodiment, R.sup.1 in formula (Ia) and formula (Ib) is hydrogen or C.sub.1-C.sub.3-alkyl. C.sub.1-C.sub.3-alkyl is in particular ethyl or isopropyl.

[0078] According to a further particular embodiment, R.sup.2a and R.sup.2b in formula (Ia) and formula (Ib) are independently hydrogen, C.sub.1-C.sub.3-alkyl, in particular ethyl, C.sub.2-C.sub.3-alkenyl, in particular allyl, C.sub.3-C.sub.8-cycloalkyl, in particular cyclohexyl, optionally substituted phenyl or optionally substituted benzyl. Substituted phenyl is in particular phenyl substituted with 1, 2 or 3 substituent which are independently selected from the group consisting of halogen, methyl, methoxy and NH.sub.2. According to a further particular embodiment, R.sup.3 is hydrogen and R.sup.2b is as defined. Alternatively, R.sup.2a and R.sup.2b in formula (Ia) and formula (Ib) together are C.sub.2-C.sub.7-alkylene, in particular 1,5-pentylene, and thus form a 3- to 8-membered, in particular 6-membered, ring including the nitrogen atom to which they are attached.

[0079] According to a further particular embodiment, R.sup.3 in formula (Ia) and formula (Ib) is C.sub.1-C.sub.6-alkyl, in particular C.sub.1-C.sub.3-alkyl, or optionally substituted C.sub.6-C.sub.12-aryl, in particular optionally substituted phenyl.

[0080] According to a further particular embodiment of formula (Ia), X is N, Y is CR.sup.5 (in particular CH) and Q is NR.sup.3 (in particular N(C.sub.1-C.sub.3-alkyl), e.g. NCH.sub.3, NCH.sub.2CH.sub.3 or NCH(CH.sub.3).sub.2, or N(phenyl)); or X is CR.sup.4 (in particular CH), Y is N and Q is NR.sup.3 (in particular N(C.sub.1-C.sub.3-alkyl) or N(phenyl)).

[0081] According to a further particular embodiment of formula (Ib), X is N, Y is CR.sup.5 (in particular CH) and Q is NR.sup.3 (in particular NCH.sub.3).

[0082] Roscovitine analogues according to the present invention, in particular, include the following compounds

##STR00004## ##STR00005## ##STR00006##

and their pharmacologically useful salts.

[0083] According to a particular embodiment, the roscovitine analogue is (1-ethyl-2-hydroxyethylamino)-6-(2-hydroxybenzyl)amino-9-isopropylpurine (hereinafter referred to as roscovitine analogue A). This roscovitine analogue is capable of reversing the inhibitory effect of A.beta. globulomer on synaptic transmission.

[0084] According to a particular embodiment, the agonists of the P/Q type voltage-gated presynaptic calcium channel of formula (II) is isoproterenol. Isoproterenol has been described as an agonist of the P/Q type voltage-gated presynaptic calcium channel (Huang C.-C., et al., The Journal of Neuroscience, 1996, 16(3): 1026-1033, Huang C.-C., et al., The Journal of Neuroscience, 1998, 18(6): 2276-2282). As used herein, the term "isoproterenol" (also known as isoprenaline) denotes 4-[1-hydroxy-2-(1-methylethylamino)ethyl]benzene-1,2-diol. Isoproterenol is capable of reversing the inhibitory effect of A.beta. globulomer on spontaneous synaptic activity, which is mediated by suppression of the P/Q type voltage-gated presynaptic calcium channel.

[0085] Particular isopreterenol analogues are defined as follows.

[0086] According to a further particular embodiment, R.sup.1 in formula (II) is C.sub.1-C.sub.4-alkyl or C.sub.3-C.sub.6-cycloalkyl. C.sub.1-C.sub.4-alkyl is in particular methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl. C.sub.3-C.sub.6-cycloalkyl is in particular cyclopropyl or cyclohexyl.

[0087] According to a further particular embodiment, at least one, two or three of R.sup.2a, R.sup.2b, R.sup.2c, R.sup.2d, R.sup.2e in formula (II) is/are different from hydrogen. In particular at least one of R.sup.2a, R.sup.2b, R.sup.2c, R.sup.2d is different from hydrogen.

[0088] According to a further particular embodiment, R.sup.2a, R.sup.2b, R.sup.2c, R.sup.2d, are independently hydrogen, halogen, methyl, optionally substituted phenyl, OH, methoxy, CN, or NH.sub.2, or R.sup.2b and R.sup.2c or R.sup.2c and R.sup.2d together with the carbon atoms to which they are attached form an optionally substituted anellated carbocyclic ring. The carbocyclic ring may be partially unsaturated (due to the benezene moiety to which R.sup.2b, R.sup.2c and R.sup.2d are attached) or aromatic. According to a particular embodiment, R.sup.2b and R.sup.2c or R.sup.2c and R.sup.2d together with the benzene moiety to which they are attached form an optionally substituted 1,2,3,4-tetrahydronaphthalene or naphthalene moiety.

[0089] Isopreterenol analogues according to the present invention, in particular, include the following compounds

##STR00007## ##STR00008##

and their pharmacologically useful salts.

[0090] The compounds of the formula (Ia), (Ib) or (II) can be prepared by analogy to methods which are well known in the art. Many of said compounds are commercially available.

[0091] Further agonists of the P/Q type voltage-gated presynaptic calcium channel may be identified among compounds known per se by screening for their capacity to act as agonists of the P/Q type voltage-gated presynaptic calcium channel, preferably by screening using a method comprising determining the effect of a candidate compound on the opening state of the P/Q type voltage-gated presynaptic calcium channel, most conveniently by determining the effect of said compound on the Ca.sup.++ flux through the P/Q type voltage-gated presynaptic calcium channel. Suitable means for determining ion fluxes such as Ca.sup.++ fluxes through the P/Q type voltage-gated presynaptic calcium channel have been described in the art (Yan Z, et al., 2002, supra; Buraei et al 2006, supra).

[0092] A method for determining whether any candidate compound is an agonist at the P/Q type voltage-gated presynaptic calcium channel comprises the steps of [0093] (I) providing the P/Q type voltage-gated presynaptic calcium channel; and [0094] (II) determining Ca.sup.++ fluxes through said P/Q type voltage-gated presynaptic calcium channel in the presence and in the absence of the candidate compound; wherein an increase of the Ca.sup.++ flux through the P/Q type voltage-gated presynaptic calcium channel in the presence relative to the Ca.sup.++ flux through the P/Q type voltage-gated presynaptic calcium channel in the absence of the candidate compound is indicative of an agonistic effect of the candidate compound at the P/Q type voltage-gated presynaptic calcium channel. Alternatively, the agonistic effect can be assessed by measuring Ba.sup.++ currents through P/Q type voltage-gated presynaptic calcium channels using methods well-known to the skilled artisan.

[0095] The P/Q type voltage-gated presynaptic calcium channel is known per se (see, e. g., WO98/13490; Qian J and Noebels J L. J Neurosci 21: 3721-3728, 2001; Yan Z, et al., 2002, supra). WO98/13490 in particular discloses the cDNA sequence for the human P/Q type voltage-gated presynaptic calcium channel, encoding a protein of 2261 amino acids. Methods for expressing a protein from a cDNA in vertebrate cells are well-documented in the art; e. g. WO96/39512 discloses a process for generating cell lines expressing voltage-gated calcium channels. It is thus within the ken of the skilled person to provide the P/Q type voltage-gated presynaptic calcium channel.

[0096] Expediently, the P/Q type voltage-gated presynaptic calcium channel is provided on a living cell, which cell may be either in its natural environment (in situ) or separated therefrom (ex vivo). In a particular embodiment, the cell to be used in the screening method is of a type that naturally expresses the P/Q type voltage-gated presynaptic calcium channel, e. g. a neuronal cell such as a hippocampal neuronal cell. In another embodiment, the cell to be used in the screening method expresses the P/Q type voltage-gated presynaptic calcium channel as a foreign gene. In this embodiment, it is preferred that the cell naturally does not express any other voltage-gated presynaptic calcium channels, e. g. a non-neural cell, e. g. a Xenopus oocyte. Conveniently, expression of the P/Q type voltage-gated presynaptic calcium channel in the cells is verified using standard methology, e. g. by Northern blotting, RT-PCR, Western blotting, cytometry, binding of P/Q-specific ligands such as .omega.-agatoxin, or pharmacological characterization, i. e. reduction of calcium current after agatoxin application.

[0097] In a further particular embodiment, said living cell further comprises an agent for the in situ detection of calcium ion levels (i. e. a calcium sensor agent), e. g. a protein with a calcium-dependent luminescence or fluorescence, such as aequorin or cameleon (Putney P W. Calcium Signaling. CRC Press Inc, 2005). Such calcium sensor agents are well-known to the skilled person, and essentially any of them may be used in the present invention. Without wishing to be bound by theory, it is believed that in suitable agents the conformation of the molecule changes in a manner that depends on the local concentration of Ca.sup.++, thereby hampering or facilitating physical processes, such as inter- or intramolecular energy transfers, that may be detected and correlated with calcium channel function by the experimentator. Thus, the fluorescence or luminescence of said calcium sensor agents is indicative of the local (e. g. intracellular) calcium levels.

[0098] Hence, when the only functional calcium channel of the cell is the P/Q type voltage-gated presynaptic calcium channel, increases in intracellular calcium concentrations

( [ Ca ++ ] t > 0 ) ##EQU00001##

indicate calcium fluxes through the P/Q type voltage-gated presynaptic calcium channel. Therefore, a rise in said increase

( [ Ca ++ ] C t > [ Ca ++ ] 0 t , ##EQU00002##

where [Ca.sup.++].sub.C is the intracellular calcium concentration in the cell in the presence and [Ca.sup.++].sub.0 in the absence of the candidate compound) indicates the P/Q agonist activity of a candidate substance and thus its potential for the treatment of amyloidoses, as described above.

[0099] Suitable methods for the direct determination of ion fluxes, such as the voltage-clamp method, are likewise known in the art (Sakmann B and Neher E. Single-Channel Recording. Springer US, 97 A.D.). Essentially, conductive microconnections with the inside and the outside of the cell membrane are established, and the electrical reactivity of the system under different conditions is observed.

[0100] Preferably, prior to the measurement irrelevant ion channels are blocked using inhibitors specific for said irrelevant channels ("pharmacological isolation" of the relevant channel or channels), eliminating the dependencies of the electrical status of the membrane on all channels except the one or ones of interest (i. e. the P/Q channel). An activator of the P/Q type voltage-gated presynaptic calcium channel and hence an agent suitable for the treatment of amyloidoses according to the present invention, as mentioned above, will thus be identified as an enhancer of Ca.sup.++ flux when only the P/Q type voltage-gated presynaptic calcium channel is expressed, or when all other calcium channels are blocked.

[0101] As all these methods for the determination of Ca.sup.++ fluxes are essentially quantitative, they are also suitable for the identification of an agonist with any particularly desired strength of agonistic effect on the P/Q type voltage-gated presynaptic calcium channel, wherein the strength of the agonistic effect is the increase in calcium influx induced by the agonist under the conditions selected.

[0102] Thus, an agent for the treatment of amyloidoses such as Alzheimer's disease can be identified by determining the effect of said agent on a cell comprising at least the P/Q type voltage-gated presynaptic calcium channel, in particular the effect on the Ca.sup.++ flux through the P/Q type voltage-gated presynaptic calcium channel of said living cell, wherein an agonist at the P/Q type voltage-gated presynaptic calcium channel is potentially a suitable agent for the treatment of amyloidoses according to the present invention.

[0103] Among the agonists identified thereby, such as those having an affinity to the N type voltage-gated presynaptic calcium channel of less than any particularly desired value may be readily selected using methods essentially known in the art, e. g. by employing the methods for detection of Ca.sup.++ fluxes disclosed above in combination with known blockers for non-N type voltage-gated presynaptic calcium channels.

[0104] Suitable methods for determining affinity between molecules are generally well-known to the person skilled the art and comprise, without being limited to, determining radiation-free energy transfer, radiolabelling of ligands and co-immunoprecipitation. Likewise, the skilled person is familiar with suitable methods for determining the inhibitory effect of a compound on any given enzyme, and will thus be able to readily select among the agonist identified as described above such as have an IC50 for any CDK, such as CDK5, of more than any particularly desired value.

[0105] As used herein, the term "administering" is used to denote delivering an agent to a subject, especially a human subject. Basically, any route of administration known in the art, e. g. buccal, sublingual, oral, rectal, transdermal, subcutaneous, intramuscular, intravenous, intraarterial, intraperitoneal, intrathecal, intralumbaginal or intradural, and any dosage regimen, e. g. as bolus or as continuous supply, may be employed to administer the agent.

[0106] The agent may be delivered simply as such or, preferably, in combination with any of a wide range of carriers and excipients, as known in the art, thereby forming a pharmaceutical composition. If desired, a convenient drug targeting and/or delivery system may be used. Expediently, the agent and at least one carrier are combined into a dosage form as known per se to those skilled in the art, e. g. into a controlled or sustained release system. Basically, any carrier and/or excipient compatible with the agent and any kind of dosage form may be used in the methods of the present invention. Suitable compounds and methods are known in the art.

[0107] Thus, the present invention will be understood to also relate to the methods and uses relating to the manufacture of pharmaceutical compositions useful in the treatment of amyloidoses. In particular, amyloidoses according to the present invention comprise Alzheimer's disease and Down's syndrome.

[0108] In a particular embodiment of the invention, the treatment is a rehabilitating and/or symptomatic treatment.

[0109] A "rehabilitating" treatment, as used herein, is, in particular, for providing a benefit with regard to the patient's overall quality of life.

[0110] As used herein, a "benefit" is any amelioration in relevant clinical parameters or decrease in subjective suffering of the subject amenable to scoring that can be causally connected to a particular therapeutic measure. Expediently, the benefit is measured by comparing the relevant clinical parameters or the subjective suffering of the subject at a time point before treatment and at least one time point during or after treatment, and expressed in terms of a gain in quality-adjusted life years or disability-adjusted life years (QALYs and DALYs).

[0111] The concept of "quality-adjusted life years" and "disability-adjusted life years" is used extensively in the art to evaluate agents and methods, in particular in the context of those diseases where morbidity and disability are medically and socially more of a concern than mortality is, such as dementing diseases. Essentially, each year the life time following treatment is multiplied with an index factor which ranges from 1.0 to indicate perfect quality of life, or zero disability, to 0.0 to indicate death, or complete disability, and the sum of these products is compared to the value obtainable without treatment. Suitable definitions and methods for determining gains and losses in QALYs and DALYs, in particular with regard to dementing diseases such as amyloidoses, have been described in the art.

[0112] Thus, a benefit is preferably an increase in the aforementioned index factor. In a particular embodiment of the invention, the treatment is hence for providing a benefit to a subject suffering from an amyloidosis.

[0113] A "symptomatic" treatment, as used herein, is, in particular, a treatment directed to the abatement or relief of the symptoms of the disease.

[0114] In a particular embodiment the present invention relates to a method for the restoration of A.beta.-impaired synaptic function and/or plasticity, in particular long-term potentiation, in the subject.

[0115] In a further particular embodiment the present invention relates to a method for the restoration of cognitive abilities, memory function and/or performance of activities of daily life (ADL) capacity in the subject.

[0116] As used herein, the terms "cognitive abilities", "synaptic function", "long-term potentiation" and "memory function" have the meanings as are widely known and used in the art, and their quantificable values are considered as "normal" or "restored" when within the range which is commonly to be expected, e. g. based on long-standing medical practice, appropriate clinical trials and/or biochemical analysis, for the individual subject under consideration when compared to a representative population of other subjects whose essential parameters otherwise agree with those of said subject under consideration (peers of said subject). In particular, memory function is considered normal in a subject when said subject upon investigation by suitable means, e. g. short- and/or long-time learning tests, shows no significant deficiencies with regard to memory in function in comparison to a control group matched in species, age, gender and optionally other factors acknowledged as relevant to mental health, which are well-known to those skilled in the art, e. g. blood cholesterol levels, and/or psycho-social factors, e. g. educational and/or occupational background.

[0117] As used herein, the term "activities of daily living", abbreviated "ADL", is used to denote the essential manual and mental tasks and chores of everyday life, in particular those involving domains of language (impairment thereof being known as "aphasia"), skilled movements (impairment being known as "apraxia" and potentially leading to total loss of control over the body in the final stages of the disease), and the use of cognitive abilities such as recognition (impairment being known as "agnosia", often accompanied by disorientation and disinhibition, and sometimes also with behavioural changes) and higher-level intellectual functions (such as decision-making and planning). These capacities can be assessed e. g. using questionnaire-based tests well-known in the art, such as the Hodgkinson test (aka. "minimental state examination" or MMSE, comprising the recital of basic facts of everyday life) and the Folstein test (aka. "abbreviated mental test score" or AMTS, comprising, remembering the time and place of the test, repeating lists of words, arithmetic, language use and comprehension, and copying a simple drawing) for basic mental functions and the John Hopkins Functioning Inventory (aka. JHFI) for basically motoric or movement-related abilities such as sitting, standing, walking, eating, washing, dressing etc.

[0118] The skilled person will be aware that in amyloidoses such as Alzheimer's disease the impairment of ADL capacity is dominated, in particular in its early and middle stages, by impairment of the intellectual rather than of motoric or sensory functions, and that even the latter, when found, is due to central rather than peripheral disturbances (e. g. "forgetting how to walk" rather than genuine organic paralysis).

[0119] According to another aspect, the present invention relates to a method for identifying an agent for the treatment of amyloidoses such as Alzheimer's disease, said method comprising determining whether a candidate compound exerts an agonistic effect on the P/Q type voltage-gated presynaptic calcium channel, as disclosed above.

[0120] The invention will now be further illustrated by way of reference to the following non-limiting examples and figures. Unless stated otherwise, the terms "A-Beta", "A.beta..sub.1-42", "A.beta.", "a.beta.", "glob" all denote the A.beta.(1-42) globulomer described in reference example 2. "Kontrolle" means "control".

DESCRIPTION OF THE FIGURES

[0121] FIG. 1: Effect of A.beta.(1-42) globulomer on spontaneous synaptic activity as recorded from rat primary cultured hippocampal neurons by voltage clamp: (A) and (C) are recordings of spontaneously occurring synaptic currents in a cultured hippocampal neuron (downward deflections indicate the postsynaptic currents which are elicited by neurotransmitter release from one or more presynaptic neurons; application of the globulomer and washout (top trace) are indicated); (B) and (D) are the cumulative probability functions.

[0122] FIG. 2: Effect of A.beta.(1-42) globulomer on the frequency of synaptic currents.

[0123] FIG. 3: Effect of A.beta.(1-42) globulomer on the frequency of mIPSCs in of cells cultivated with 0.5 .mu.M .omega.-conotoxin MVIIA to achieve synaptic P/Q predominance (n=6): Number of synaptic events during 5 min relative to non-A.beta. globulomer treated cells. Left to right: (1) non-A.beta. globulomer treated P/Q-dominated cells=reference, (2) P/Q-dominated cells treated with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer).

[0124] FIG. 4: A.beta.(1-42) globulomer has no effect on the amplitude of mIPSCs of cells cultivated with .omega.-conotoxin MVIIA to achieve synaptic P/Q predominance: Average amplitude of synaptic events relative to non-A.beta. globulomer treated cells. Left to right: (1) non-A.beta. globulomer treated P/Q-dominated cells=reference, (2) P/Q-dominated cells treated with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer).

[0125] FIG. 5: Effect of .omega.-agatoxin on the frequency of mIPSCs in of cells cultivated with 0.5 .mu.M .omega.-conotoxin MVIIA to achieve synaptic P/Q predominance (n=3): Number of synaptic events during 5 min relative to non-.omega.-agatoxin treated cells. Left to right: (1) non-.omega.-agatoxin treated P/Q-dominated cells=reference, (2) P/Q-dominated cells treated with 0.5 .mu.M .omega.-agatoxin.

[0126] FIG. 6: No additive effect on the frequency of mIPSCs in of cells cultivated with 0.5 .mu.M .omega.-conotoxin MVIIA to achieve synaptic P/Q predominance after blockade of P/Q-channels by .omega.-agatoxin (n=6): Number of synaptic events during 5 min relative to non-A.beta. globulomer treated cells. Left to right: (1) non-A.beta. globulomer treated P/Q-dominated cells (.omega.-agatoxin only)=reference, (2) P/Q-dominated cells treated with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer) after pre-treatment with 0.5 .mu.M .omega.-agatoxin.

[0127] FIG. 7: No effect of globulomer on the amplitude of mIPSCs when P/Q channels of P/Q-dominated cells are already blocked by 0.5 .mu.M .omega.-agatoxin IVA (n=6): Number of synaptic events during 5 min relative to non-A.beta. globulomer treated cells. Left to right: (1) non-AP globulomer treated P/Q-dominated cells (.omega.-agatoxin only)=reference, (2) P/Q-dominated cells treated with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer) after pre-treatment with 0.5 .mu.M .omega.-agatoxin.

[0128] FIG. 8: Agatoxin does not impair spontaneous synaptic activity in cultures that lack functional P/Q-type Ca.sup.++ channels: Number of synaptic events during 5 min was set to 100% for each cell analysed. The right bar indicates the relative number of synaptic events in each cell after application of 0.5 .mu.M .omega.-agatoxin.

[0129] FIG. 9: Globulomer does not impair spontaneous synaptic activity in cultures that lack functional P/Q-type Ca.sup.++ channels: Number of synaptic events during 5 min relative to non-A.beta. globulomer treated cells was set to 100% for each cell analysed. The right bar indicates the relative number of synaptic events in each cell after application of A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer).

[0130] FIG. 10: Suppression of spontaneous synaptic currents by A.beta.(1-42) globulomer and its reversal by the P/Q channel agonist roscovitine: Number of synaptic events during 5 min relative to non-A.beta. globulomer treated P/Q-dominated cells. Left to right: (1) non-A.beta. globulomer treated P/Q-dominated same cells=reference, (2) P/Q-dominated same cells treated with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer), (3) P/Q-dominated same cells treated simultaneously with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer) and 20 .mu.M roscovitine.

[0131] FIG. 11: No effect on the amplitude of spontaneous synaptic currents of the P/Q channel agonist roscovitine: Average amplitude of synaptic events relative to non-A.beta. globulomer treated P/Q-dominated cells. Left to right: (1) non-A.beta. globulomer treated P/Q-dominated same cells=reference, (2) P/Q-dominated same cells treated with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer), (3) P/Q-dominated same cells treated simultaneously with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer) and 20 .mu.M roscovitine.

[0132] FIG. 12: The effect of A.beta.(1-42) globulomer on spontaneous synaptic activity of P/Q-dominated cells can be reversed by the P/Q channel agonist roscovitine: Synaptic potentials over time. Arrows indicate the time points when A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer) and 20 .mu.M roscovitine, respectively, were added.

[0133] FIG. 13: Reducing effect of A.beta. globulomer on the amplitude of pharmacologically isolated P/Q-type calcium channels: Traces represent membrane currents after activation of P/Q-type channels by a depolarizing voltage step. Left to right: (1) P/Q-current under control conditions, (2) P/Q-current of the same cell after application of A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer), (3) P/Q-current of the same cell after washout of A.beta. globulomer.

[0134] FIG. 14: Effect of A.beta.(1-42) globulomer on the pharmacologically isolated P/Q current at different time points: Average amplitude of P/Q-mediated current amplitude relative to non-A.beta. globulomer treated P/Q-dominated cells. Left to right: (1) non-A.beta. globulomer treated same cells=reference, (2) same cells 10 min after treatment with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer), (3) same cells 15 min after treatment with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer).

[0135] FIG. 15: Effect of 0.5 .mu.M .omega.-agatoxin IVA on the pharmacologically isolated P/Q current at different time points: Average amplitude of P/Q currents relative to non-.omega.-agatoxin treated P/Q-dominated same cells. Left to right: (1) non-.omega.-agatoxin treated P/Q-dominated same cells =reference, (2) P/Q-dominated cells 10 min after treatment with 0.5 .mu.M .omega.-agatoxin, (3) P/Q-dominated cells 15 min after treatment with 0.5 .mu.M .omega.-agatoxin.

[0136] FIG. 16: Effect of A.beta. on the pharmacologically isolated P/Q current at different time points, revealing the effect of washing out the A.beta. globulomer: Average amplitude of P/Q-mediated current relative to non-A.beta. globulomer treated P/Q-dominated cells. Left to right: (1) non-A.beta. globulomer treated P/Q-dominated cells=reference, (2) P/Q-dominated cells 10 min after treatment with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer), (3) P/Q-dominated cells 15 min after treatment with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer), (4) P/Q-dominated cells treated with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer) after washing out the A.beta. globulomer.

[0137] FIG. 17: Effect of A.beta. on spontaneous synaptic activity in the hippocampal slice: Number of synaptic events during 5 min relative to non-A.beta. globulomer treated tissue. Left to right: (1) non-A.beta. globulomer treated same slice=reference, (2) same slice treated with A.beta. globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer).

[0138] FIG. 18: Spontaneous synaptic activity is reversibly suppressed by A.beta.(1-42) globulomer. Original recording of spontaneously occurring synaptic currents in a cultured hippocampal neuron before (top), during (middle) and after (bottom) application of A.beta.(1-42) globulomer.

[0139] FIG. 19: Effects of A.beta.(1-42) globulomer on different types of synaptic currents in cultured hippocampal neurons. White bars: effect of A.beta.(1-42) globulomer; black bars: washout for at least 10 min. A: Reduction of event frequency as percentage of previously recorded control currents (1.0). B: Effects of A.beta.(1-42) globulomer on median amplitude of the respective currents. sPSCs: spontaneously occurring pharmacologically naive postsynaptic currents; mPSCs: pharmacologically naive miniature postsynaptic currents recorded in the presence of TTX; mIPSCs: miniature inhibitory postsynaptic currents; mEPSCs: spontaneously occurring excitatory postsynaptic currents; mEPSCs: miniature excitatory postsynaptic currents.

[0140] FIG. 20: Stability of GABA.sub.A receptor-mediated currents towards A.beta.(1-42) globulomer. A: Repetitive application of 100 .mu.M GABA to a cultured hippocampal neuron yields stable inward current before, during, and after application of the oligomer. B: Enlarged view of current traces marked with * in A. Note the stability of response in the absence (left) and presence (right) of A.beta.(1-42) globulomer. C: Time course of GABA-induced currents from 5 cells recorded in control solution (dashed line) and from 3 neurons where A.beta.(1-42) globulomer was applied (continuous line, time of application indicated by bar). Amplitudes normalized to the last GABA-induced current before application of A.beta.(1-42) globulomer.

[0141] FIG. 21 Suppression of P/Q-type calcium currents by A.beta.(1-42) globulomer. A: Time course of current amplitudes upon application of globulomer. Currents were elicited by voltage steps to -10 mV. B: Example traces of P/Q-type currents before, during and after globulomer.

[0142] FIG. 22 Steady-state activation and inactivation parameters of P/Q currents. A: Current/voltage relationship before globulomer (squares) and during A.beta..sub.1-42 (triangles). A reduction of the current amplitudes over the entire voltage-range, were the current could be activated, was observed following application of the globulomer. B & C: No difference in steady-state activation (B) and inactivation curves (C) for P/Q channel-mediated barium currents in the absence and presence of A.beta.(1-42) globulomer. D: A significant decrease in maximal conductance (g.sub.max) of the P/Q channels was induced by A.beta.(1-42) globulomer.

[0143] FIG. 23 Pharmacological modulation of the effect of A.beta.(1-42) globulomer by agents interacting with P/Q-type calcium channels. A: Effects of A.beta.(1-42) globulomer on frequency of mixed synaptic currents. B: Effects on median amplitude. Values are given relative to data in control solution. Note suppression of the effect by .omega.-agatoxin and partial recovery of event frequency by roscovitine.

[0144] FIG. 24 Pharmacological modulation of the effect of A.beta.(1-42) globulomer by agents interacting with P/Q-type calcium channels. Left to right: (1) frequency of mixed synaptic currents of non-A.beta.(1-42) globulomer treated cultivated hippocampal cells, (2) frequency of mixed synaptic currents after application of A.beta.(1-42) globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer), (3) frequency of mixed synaptic currents after application of A.beta.(1-42) globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer) and 15 .mu.M isoproterenol.

[0145] FIG. 25 Enhancing P/Q calcium currents by roscovitine prevents/reverses chronic A.beta. globulomer-induced deficits on evoked synaptic transmission in hippocampal tissue (slice cultures). Recordings were performed after incubation with A.beta.(1-42) globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer), A.beta.(1-42) globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer)+20 .mu.M roscovitine, or control (SDS).

[0146] FIG. 26 Enhancing P/Q calcium currents by a roscovitine analogue prevents/reverses chronic A.beta. globulomer-induced deficits on evoked synaptic transmission in hippocampal tissue (slice cultures). Recordings were performed after incubation with A.beta.(1-42) globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer), A.beta.(1-42) globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer)+20 .mu.M roscovitine analogue A, or control (SDS).

[0147] FIG. 27 Effect of extracellular Ca.sup.2+ on sPSC frequency after treatment with A.beta.(1-42) globulomer: Original recording of sPSCs before (control in 1 mM Ca.sup.2+), after addition of A.beta.(1-42) globulomer (glob in 1 mM Ca.sup.2+) and after subsequent elevation of Ca.sup.2+-concentration (glob in 4 mM Ca.sup.2+). B: Reduction of event frequency after application of A.beta.(1-42) globulomer (p<0.05; n=6) and partial recovery after elevation of Ca.sup.2+ from 1 mM to 4 mM. Values are given as percentage of control currents. C: Event frequency of single cells (n=6) after application of A.beta.(1-42) globulomer and after subsequent elevation of Ca.sup.2+ from 1 mM to 4 mM. Values are given as percentage of control currents. D: No difference in median amplitude after application of A.beta.(1-42) globulomer (n=6) and after subsequent elevation of Ca.sup.2+. Values are given as percentage of control currents. E: Original recordings of massive discharges directly after Ca.sup.2+ elevation for the cell shown in A. These currents were rejected from analysis.

FIRST SERIES OF EXPERIMENTS

REFERENCE EXAMPLE 1

Determination of Synaptic Potentials

[0148] Neuronal cells from the rat hippocampus were obtained and cultured in accordance with methods known per se in the art (Banker G A, Cowan W M, Brain Res. 1977 May 13; 126(3):397-42). Cultured neurons show spontaneous postsynaptic currents (PSCs), i. e. spontaneous PSCs and, in the presence of the sodium channel blocker tetrodotoxin, miniature PSCs. As mentioned above, the influx of Ca.sup.++ through presynaptic ion channels such as the N, P/Q and R type voltage-gated presynaptic calcium channels is what causes the release of neurotransmitter from preformed vesicles in presynaptic terminals. The measured signal reflects the current response of the postsynaptic cell to the release of such transmitters, e.g. gamma-aminobutyric acid or glutamate.

[0149] For measurements, primary cell cultures were transferred to a recording chamber mounted on a microscope (Olympus CKX1) and were immersed at room temperature into a buffered solution consisting of 156 mM NaCl, 2 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 16.5 mM glucose and 10 mM HEPES at a pH of 7.3. The osmolarity of the solution was 330 mosmol.

[0150] Electrodes were produced by pulling from borosilicate capillaries (available from Science Products) with a horizontal pipette pulling device (P-97 from Sutter Instruments). After filling with the intracellular solution, the final resistance of the electrodes was from 2 to 5 M.OMEGA.. The intracellular solution consisted of either (for recordings of miniature PSCs) 100 mM KCl, 10 mM NaCl, 0.25 mM CaCl.sub.2, 5 mM EGTA, 40 mM glucose, 4 mM MgATP and 0.1 mM NaGTP at a pH of 7.3, or (for recording of calcium currents) 110 mM CsCl, 10 mM EGTA, 25 mM HEPES, 10 mM tris-phosphocreatine, 20 U/ml creatine phosphokinase, 4 mM MgATP and 0.3 mM NaGTP.

[0151] All test compounds were applied either by bath perfusion or by addition to the bath by means of a micropump connected to a manually guided pipette.

[0152] All recordings of miniature PSCs were made in the presence of 0.5 .mu.M tetrodotoxin (TTX; available from Tocris Bioscience) to block the Na.sup.+ and K.sup.+ channels in the neuronal cell membrane which would otherwise also influence the electrical status of the membrane. For calcium current recordings the extracellular solution contained 140 mM TEA-CI (to block K.sup.+-channels) 10 mM BaCl.sub.2, 0.5 .mu.M TTX, 10 mM HEPES and 20 mM glucose at a pH 7.3. When required, .omega.-conotoxin MVIIA (available from Alomone Labs, Jerusalem, Israel) was added to a final concentration of 0.5 .mu.M to block N type voltage-gated presynaptic Ca.sup.++ channels, thereby "pharmacologically isolating" the ion fluxes through the P/Q type voltage-gated presynaptic calcium channel. If necessary, L-type voltage-gated calcium channels were blocked by addition of 10 .mu.M nifedipine.

[0153] To mimic the effect of A.beta. globulomer as P/Q type blocker, .omega.-agatoxin IVA (available from Alomone Labs, Jerusalem, Israel) was added to a final concentration of 0.5 .mu.M to specifically block the P/Q type voltage-gated presynaptic Ca.sup.++ channels of the sample cell.

[0154] All substances were stored as lyophilized powders at -20 .degree. C. Stock solutions were prepared with vehicles appropriate for the solubility (i. e. immersion solution). Vehicle was destilled water or standard extracellular solution for all drugs except nifedipine, which was dissolved in ethanol, and roscovitine, which was dissolved in dimethyl sulfoxide (DMSO). The final concentration of the solvents in the A.beta.-globulomer solvent buffer which was applied to neurons was <1%0 and the final concentration of DMSO was <1.5%o.

[0155] Whole-cell patch-clamp recordings (sPSCs and mPSCs) were conducted in a manner essentially known per se (see, e.g., Sakmann B and Neher E. Single-Channel Recording. Springer US, 97 A.D.) at a holding potential of -70 mV using an EPC7 amplifier (available from HEKA Electronics). Signals were filtered at 3 kHz and sampled at 20 kHz.

[0156] After formation of a seal, rupture of the membrane by the electrode and establishment of the whole-cell configuration, the perfusion of the bath was stopped, and the substances to be tested were injected into the bath using a custom-made syringe pump.

[0157] The sPSCs or mPSCs were then recorded for 10 minutes giving the control values before any toxins were added.

[0158] For the selective determination of P/Q type voltage-gated presynaptic calcium channel currents, the cells were activated in a manner known per se (see Yan et al., 2002, supra) by a voltage protocol, where the cells were excited by depolarization to -10 mV for 50 ms every 20 sec. After the formation of the whole-cell configuration, currents increased steadily until they had reached a stable amplitude level. After this stable amplitude level had been established, the effects of different test compounds on the rate of ion flux were observed and expressed in terms of the normalized mean P/Q amplitude and standard error of the mean SEM. Frequency and amplitude of synaptic currents were calculated offline using a template-based algorithm (custom made routine within the Signal and Spike software, purchased from CED Inc., Cambridge, UK).

[0159] When desired, the measurement was evaluated at several timepoints and optionally after a washout. Student's t-test was applied to determine significance, p<0.05 being considered as indicative of significant differences.

REFERENCE EXAMPLE 2

Generation of A.beta. Globulomer

[0160] An A.beta.(1-42) globulomer preparation with an apparent molecular weight of 38/48 kDa as determined by SDS-PAGE was obtained as described in Example 6b of WO2004/067561. Essentially, A.beta. monomer was pretreated with HFIP for dissolving hydrogen bonds, then diluted and further incubated in the presence of 0.2% SDS, followed by isolation of the thus formed globulomer.

EXAMPLE 3

Inhibitory Effect of A.beta. Globulomer on Spontaneous Synaptic Activity

[0161] Using acute application of the P/Q channel blocker .omega.-agatoxin as a negative control and cells untreated with regard to the P/Q type voltage-gated presynaptic calcium channel as a positive control, the effects of A.beta.(1-42) globulomer on the frequency of spontaneous synaptic events in cultured hippocampal neurons treated with .omega.-conotoxin to achieve synaptic dominance of the P/Q type channel, as described in Reference Example 1, were observed.

[0162] A.beta. globulomer, obtained as described in Reference Example 2, was tested according to the procedure described in Reference Example 1 for channel function inhibitors such as .omega.-agatoxin. In the presence of .omega.-agatoxin, A.beta. globulomer had no further effect on synaptic activity, indicating that the effects of both agents involved a common mechanism. A total of 200 .mu.l A.beta.-globulomer solvent buffer comprising a A.beta.(1-42) globulomer concentration corresponding to approximately 2 .mu.M of A.beta. monomer was added to the bath (previous volume 200 .mu.l), resulting in a final A.beta.(1-42) globulomer concentration corresponding to approximately 1 .mu.M of A.beta. monomer. Based on the assumption that the A.beta.(1-42) globulomer consists of 12 A.beta.(1-42) monomers a final A.beta.(1-42) globulomer concentration of approximately 83 nM can be calculated. Measurements of synaptic activity were then taken.

[0163] Results are shown in FIGS. 1-7, demonstrating that the A.beta. globulomer inhibits the frequency of spontaneous synaptic events with an efficiency approaching that of the strong P/Q inhibitor .omega.-agatoxin but has no or little effect on the amplitude of the synaptic events. Thus, A.beta.(1-42) globulomer reduces synaptic activity, most likely by a presynaptic mechanism, which shares crucial elements with the effect of .omega.-agatoxin.

[0164] These results were verified by subjecting the A.beta.(1-42) globulomer containing A.beta.-globulomer solvent buffer to ultrafiltration with a filter having a molecular cutoff size of 5 kDa for globular proteins. The resulting solvent buffer contained no detectable amounts of A.beta. globulomer protein prior to bringing it into contact with the cells. The ultrafiltrate had no effect on the synaptic events (see FIG. 2), indicating that the agent responsible for reducing the frequency of spontaneous synaptic events was unable to pass ultrafilters.

[0165] Furthermore, the effect of A.beta.(1-42) globulomer is absent in cells predominantly expressing presynaptic N-type calcium channels. Results are shown in FIGS. 8 and 9, demonstrating that in the N-dominated cells no reduction of the frequency nor any reduction in amplitude is achieved by either .omega.-agatoxin or A.beta. globulomer, i. e. that both .omega.-agatoxin and A.beta. globulomer target the P/Q type voltage-gated presynaptic calcium channel.

EXAMPLE 4

Rescue of Spontaneous Synaptic Activity by Roscovitine

[0166] Using the A.beta.(1-42) globulomer of Reference Example 2 as a negative control and cells untreated with regard to the P/Q type voltage-gated presynaptic calcium channel as a positive control, the effects of the P/Q type voltage-gated presynaptic calcium channel activator roscovitine on the A.beta. globulomer-induced reduction of the frequency of spontaneous synaptic events in cultured hippocampal neurons treated with .omega.-conotoxin, as described in Reference Example 1, were observed.

[0167] Roscovitine was used at a final concentration of 20 .mu.M, by adding it simultaneously with A.beta.(1-42) globulomer (final concentration of A.beta. globulomer corresponding to approximately 1 .mu.M of A.beta. monomer). Roscovitine is known (Zhen Yan et al., J. Physiol. 540: 761-770 (2002)) to slow down the inactivation of the P/Q type voltage-gated presynaptic calcium channel, i. e. to extend the time for which a channel, once opened, remains in the open state, thereby increasing the calcium ion flow through the P/Q type voltage-gated presynaptic calcium channel.

[0168] Results are shown in FIGS. 10 and 11, demonstrating that a P/Q type voltage-gated presynaptic calcium channel activator is capable of restoring the frequency of spontaneous synaptic events under the influence of A.beta. globulomer to almost that of untreated cells, i. e., that a P/Q activator may be used to reverse the detrimental effects of A.beta. globulomer.

REFERENCE EXAMPLE 5

Direct Determination of the Activity of the P/Q Type Voltage-Gated Presynaptic Calcium Channel, and of Inhibitory and Activating Influences, by the Voltage-Clamp Method

[0169] Cells were prepared and subjected to measurement of membrane currents by the voltage-clamp method basically as described in Reference Example 1, the difference being essentially that all irrelevant (non-P/Q type) ion channels of the cells were blocked chemically, thereby allowing for direct determination of the ion fluxes rather than of the resulting IPSCs. Blocking of the irrelevant channels was achieved using the following additions to the bath or electrode solution:

TABLE-US-00001 Compound Concentration Channel blocked TEA-Cl 140 mM I[K.sup.+] BaCl.sub.2 10 mM CsCl (in the pipette) 110 mM Nifedipine 10 mM L-type Ca.sup.++ channel .omega.-conotoxin MVIIA 0.5 mM N-type Ca.sup.++ channel Tetrodotoxin 0.5 Na.sup.+ channels

[0170] The Ba.sup.++ also served as the charge carrier (i. e. substrate replacement) for the P/Q type voltage-gated presynaptic Ca.sup.++ channel, with the additional advantage that conductance through this channel and hence the sensitivity of the assay were thereby increased to approximately tenfold. This made it possible to directly detect ion fluxes through P/Q-channels in somatic recordings.

[0171] In order to prevent the "run down" of Ca.sup.++ currents in the samples, the electrode solution also comprised, in addition to the substances listed above, 10 mM tris-phosphocreatinine and 20 U/ml creatine phosphokinase, which together served as an ATP regenerating system preventing "run-down", i.e. decline due to a gradual loss of channel conductance, of the observed currents. ATP is needed to maintain the conductance of the calcium channels over time intervals longer than several minutes, allowing to conduct the described pharmacological experiments with sufficiently stable calcium currents.

EXAMPLE 6

Direct Effect of A.beta. Globulomer on the P/Q Type Voltage-Gated Presynaptic Calcium Channel in Cultured Cells

[0172] Using .omega.-agatoxin as a negative control and cells untreated with regard to the P/Q type voltage-gated presynaptic calcium channel as a positive control, the effects of A.beta.(1-42) globulomer of Reference Example 2 (at a concentration corresponding to approximately 1 .mu.M of A.beta.(1-42) monomers) on calcium flux in hippocampal neurons treated with .omega.-conotoxin were directly observed as described in Reference Example 5.

[0173] Recordings were taken at 10 min and 15 min and optionally after a washout. Typical results are shown in FIGS. 13-16. These findings demonstrate that A.beta. globulomer directly inhibits the activity of the P/Q type voltage-gated presynaptic calcium channel and cannot be readily washed out after binding to the P/Q type voltage-gated presynaptic calcium channel. They further demonstrate that A.beta. globulomer impedes, by decreasing the amplitude of the calcium flux, the initiation of synaptic currents.

EXAMPLE 7

Direct Effect of A.beta. Globulomer on the P/Q Type Voltage-Gated Presynaptic Calcium Channel In Situ

[0174] To verify whether the effect of the globulomer on neurons in cell cultures also takes place in the more organotypic slice-preparation of the hippocampus, synaptic currents were determined in this tissue.

[0175] 400 .mu.m thick slices were prepared from freshly dissected hippocampi of the mouse using a method known per se (Dingledine R. Brain Slices. New York: Plenum Press, 1983). CA1 pyramidal cells were patched and spontaneous synaptical currents were recorded prior and after application of A.beta.(1-42) globulomer via an Eppendorff pipette.

[0176] Typical results are shown in FIG. 17. These findings demonstrate that the mechanism for A.beta. globulomer mediated inhibition disclosed herein is also valid in situ.

SECOND SERIES OF EXPERIMENTS

REFERENCE EXAMPLE 8

Cell Culture

[0177] Primary hippocampal cell cultures were prepared from Wistar rat embryos at the embryonic age E19 in accordance with the protocol described earlier by Banker and Cowan (1977). Briefly, pregnant rats were deeply anesthetized by ether narcosis and decapitated. Embryos were rapidly removed and brains were dissected under constant cooling with chilled (.about.4.degree. C.) phosphate buffered saline (PBS). Then both hippocampi were taken out and washed twice with ice-cold PBS followed by a wash with PBS at room temperature. Hippocampi were triturated using three siliconized pipettes with decreasing tip diameters. Cells were then plated on coverslips (density 60000 cells/coverslip) coated with 0.01% poly-L-lysine solution and stored at 37.degree. C. in an incubator gassed with 5% CO.sub.2 in normal air. The culture medium contained 0.25% penicilline/streptomycine, 2% B27, 0.25% L-glutamine (Gibco, Karlsruhe, Germany). Throughout culturing, we added 0.5 .mu.M/L .omega.-conotoxin MVIIA to the culture medium to block N-type calcium channels and to stabilize the expression of P/Q-type currents. Cells were cultured for 14 to 28 days until used for experiments.

REFERENCE EXAMPLE 9

Current Recording

[0178] Currents were measured under whole-cell voltage-clamp conditions at room temperature using borosilicate pipettes of 2-4 M.OMEGA. resistance. Electrode solution contained (in mM/l): NaCl 10, KCl 100, CaCl.sub.2 0.25, EGTA 5, HEPES 10, glucose 40 (pH set at 7.3) when used for recordings of synaptic events. A low-chloride solution was used for experiments in which GABA induced currents were elicited, which consists of (mM): Cs-gluconate 130, CsCl 10, CaCl.sub.2 0.5, MgCl.sub.2 2, EGTA 10, HEPES 10, Mg-ATP 2 (pH: 7.3). Using this solution the calculated equilibrium potential for chloride-ions was -54 mV. During calcium current measurements the solution contained in (mM): CsCl 110, EGTA 10, HEPES 25, tris-phosphocreatine 10, Mg-ATP 4, Na-GTP 0.3 and 20 units/ml creatine-phosphokinase at pH 7.3. Osmolarity was adjusted to 295 mosmol/l, when necessary, by adding glucose. Bath solutions contained (in mM): NaCl 156, KCl 2, CaCl.sub.2 2, MgCl.sub.2, Glucose 16.5, HEPES 10 (pH set to 7.3) for recordings of synaptic events and TEA-Cl 140, BaCl.sub.2, 10, HEPES 10, and Glucose 20 at a pH: 7.3 for calcium currents, respectively. The bath perfusion was stopped for 10 min prior to the application of the A.beta.(1-42) globulomer and baseline activity was recorded. Subsequently, A.beta.(1-42) globulomer (164 nM in respect to the 12mer complex) was added to the bath by means of a micro pump, yielding a final concentration of 82 nM. TTX, .omega.-agatoxin IVA, .omega.-conotoxin MVIIA, roscovitine (Alomone Labs, Jerusalem, Israel), and nifedipine (Sigma, Deisenhofen, Germany) were added directly to the bath solution at the concentrations indicated.

[0179] Currents were measured with an Axopatch 200B (Axon Instruments, Union City, US) or an EPC-7 amplifier (HEKA, Lambrecht, Germany), digitized by a CED 1401 micro analog/digital converter (CED, Cambridge, UK) and stored on a PC (sample frequency 20 kHz). All recorded currents were low-pass filtered with a cut-off frequency of 3 kHz. Capacitive transients and series resistances were compensated on-line (.about.50-60% compensation) during the calcium current measurements. No compensation was performed during recordings of synaptic events. Data were evaluated off-line using Spike5 and Signal3 software (CED, Cambridge, UK). All calcium current traces were corrected for aspecific linear leak (reversal potential 0 mV) determined at holding potential using .+-.5 mV potential steps.

REFERENCE EXAMPLE 10

Current Analysis

[0180] All cells were voltage clamped at a holding potential of -80 mV, and calcium ions were substituted by Barium ions to increase the amplitude of the current flow through the calcium channels. Peak amplitudes of the currents (I) evoked with the activation protocol were plotted as a function of membrane potential (V). The resulting IV-relations were fitted with a combination of a first order Boltzmann activation function and the Goldman-Hodgkin-Katz (GHK) current-voltage relation (Kortekaas and Wadman, 1997):

I ( V ) = V g max 1 + exp ( V h - V V c ) [ Ba + ] in / [ Ba + ] out - exp ( - .alpha. V ) 1 - exp ( - .alpha. V ) with a = F / RT and g max = .alpha. FP 0 [ Ba + ] out , [ 1 ] ##EQU00003##

where g.sub.max is the maximal membrane conductance (which is proportional to the maximal permeability and the extracellular concentration of barium), V.sub.h is the potential of half maximal activation and V.sub.c is proportional to the slope of the curve at V.sub.h. F represents the Faraday constant, R the gas constant, P.sub.0 is the maximal permeability, and T the absolute temperature. The intracellular concentration of Ba.sup.2+ was assumed to be 0.01 .mu.M. Assuming higher values of up to 0.1 mM did not significantly change the resulting values of the parameters.

[0181] The voltage dependence of steady state inactivation of the barium current was estimated from the relation of peak current amplitude versus the pre-potential. This relation was well described by a Boltzmann function, which normalized the current:

N ( V ) = I ( V ) I max where I ( V ) = I max 1 + exp ( V h - V V c ) [ 2 ] ##EQU00004##

where N(V) is the level of steady state inactivation determined from the current amplitude I(V) normalized to I.sub.max, V is the pre-pulse potential, V.sub.h is the potential of half maximal inactivation and V.sub.c is a factor proportional to the slope of the curve at V.sub.h.

REFERENCE EXAMPLE 11

Synaptic Events

[0182] For these recordings, all cells were voltage clamped at a holding potential of -70 mV. Synaptic events triggered by the release of GABA were inwardly directed (E.sub.Cl.about.-10 mV) due to the use of high chloride concentrations in the pipette and the bath. Routinely, 10 min of baseline activity was acquired, serving as control data, before any drug application was started. Synaptic events were then analyzed off-line for frequency and amplitude, using a custom-made, template based algorithm.

REFERENCE EXAMPLE 12

Statistics

[0183] Values are presented as the mean.+-.standard error of the mean (SEM). Statistical comparisons were made with Student's t-test. A p-value<0.05 was used to indicate significant differences.

EXAMPLE 13

A.beta.(1-42) Globulomer Reduces Spontaneous Synaptic Activity in Hippocampal Cell Cultures

[0184] Spontaneous synaptic was measured activity in cultured hippocampal neurons using whole-cell voltage clamp techniques (V.sub.hold=-70 mV). Under our ionic conditions, all synaptic events appeared as inward currents (spontaneous postsynaptic currents; sPSCs) with a mean frequency of 189.+-.63/min (n=13). Bath-application of 82 nM A.beta.(1-42) globulomer (globulomer molarities calculated with respect to the 12mer complex) rapidly reduced the frequency of sPSCs to 38.+-.5% of control (p<0.05; n=13; FIG. 18). This effect was partially reversible upon washout in 2 of 3 cells tested (61.+-.16%). The median amplitude of events was 310.+-.168 pA and was reduced to 84.+-.10% under A.beta.(1-42) globulomer (p<0.05; n=14; FIG. 19). Similar--but slightly weaker--effects were seen after application of 8.2 nM A.beta.(1-42) globulomer (frequency reduced to 63.+-.9%; p<0.05; median amplitude 94.+-.5% of control, n=8, n.s.). Thus, the suppression of spontaneous synaptic activity by A.beta.(1-42) globulomer is dose-dependent and starts at low nanomolar concentrations. Input resistance was not affected by A.beta.(1-42) globulomer (control: 120.9.+-.13.6 MO; A.beta.(1-42): 131.6.+-.13.7 M.OMEGA.).

[0185] Suppression of synaptic currents by an agent may be caused by changes in neuronal activity or, alternatively, by specific synaptic interactions. It was therefore tested for effects of A.beta.(1-42) globulomer on active discharge properties by recording action potentials in current clamp mode. Action potentials elicited by current injection showed no difference in amplitude, shape or kinetics when compared before and after A.beta.(1-42) globulomer application. In detail, the threshold for firing was -22.5.+-.8.2 mV vs. -24.2.+-.9.8 mV, and the amplitude of the AP (baseline to peak) amounted to 119.9.+-.11.2 vs. 110.9.+-.16.7 mV. Likewise, kinetic parameters did not differ: values for the half-width time were 3.5.+-.1.6 ms vs. 4.0.+-.2.9 ms, maximal rate of rise 100.5.+-.46.4 V/s vs. 84.2.+-.50.0 V/s and maximal rate of repolarization 46.0.+-.18.6 V/s vs. 47.4.+-.19.3 V/s (n=16 action potentials from 4 cells before and after A.beta.(1-42) globulomer respectively. It thus appears that the alteration of synaptic activity by A.beta.(1-42) globulomer may be caused by a direct interaction with pre- or postsynaptic proteins, rather than by an unspecific alteration of cellular excitability.

[0186] This hypothesis was corroborated by recordings of spontaneously occurring miniature post-synaptic currents (mPSCs) in the presence of TTX. Similar to spontaneous "macroscopic" PSCs, these currents were reduced in frequency by 82 nM A.beta.(1-42) globulomer (yielding 56.+-.9% of control; p<0.05; FIG. 19). However, the amplitude of mPSCs was unaltered (median amplitude 31.1.+-.4.0 pA under control conditions vs. 30.2.+-.5.2 pA in the presence of A.beta.(1-42) globulomer, n=6). Upon washout for 10 minutes, the effect on event frequency recovered partially to 77.+-.7.6% of control, n=6, wash: 4/6). Together, these data suggest that A.beta.(1-42) globulomer interferes with the presynaptic machinery of transmitter release.

EXAMPLE 14

Effects on Spontaneous and Miniature Inhibitory Postsynaptic Currents

[0187] Pharmacologically naive synaptic currents reflect a mixture of glutamatergic (excitatory) and GABAergic (inhibitory) events. In order to differentiate between these components, inhibitory postsynaptic currents were isolated by adding CNQX (20 .mu.M) and DL-APV (30 .mu.M) to the bath solution. The frequency of spontaneously occurring IPSCs was suppressed by A.beta.(1-42) globulomer (yielding 64.+-.5% of control; p<0.05; n=12) and the median amplitude was reduced to 82.+-.2% of control (p<0.05). These reductions could be reversed to some degree following withdrawal of the globulomer (frequency: 90.+-.12%; amplitude: 94.+-.2%). Miniature inhibitory postsynaptic currents (mIPSCs, recorded in 0.5 .mu.M TTX) did also show a similar reduction of frequency after application of A.beta.(1-42) globulomer (52.+-.10% of control; p<0.05; n=6). This effect was partially reversible upon washout, yielding 68.+-.12% of control (FIG. 19). In addition, a reduction of mIPSC amplitude was observed (81.+-.6% of control; p<0.05; no washout in 3/3 cells (85.+-.6%)).

EXAMPLE 15

Effects on Postsynaptic GABA.sub.A Receptors

[0188] In order to test for potential effects of A.beta.(1-42) globulomer on postsynaptic GABA.sub.A receptors, a high (100 .mu.M) concentration of GABA was applied by brief pressure-pulses directly onto the cell. Repetitive application of GABA to cultured cells elicited large (>1 nA) inward currents which showed only minor rundown with time. This behaviour was unaltered after application of A.beta.(1-42) globulomer for 5 min, indicating that GABA.sub.A receptors are not affected by the agent (FIG. 20).

EXAMPLE 16

Effects on Spontaneous and Miniature Excitatory Postsynaptic Currents

[0189] Finally, excitatory synaptic currents (EPSCs) were isolated in the presence of 5 .mu.M gabazine (a GABA.sub.A receptor antagonist). Basal frequency of these events was 386.+-.124/min. Their frequency was reduced by A.beta.(1-42) globulomer to 14.+-.4% of control (p<0.05; n=6; FIG. 19). Likewise, the amplitude was reduced to 79.+-.4% of control (n=6; p<0.05; FIG. 19). The effect was partially reversible during washout (frequency increasing to 52.+-.19% of control, amplitude to 96.+-.6%; n=6). The frequency of miniature EPSCs was likewise suppressed to 57.+-.9% of control (n=6; p<0.05), while the amplitude of mEPSCs remained stable (96.+-.3% of control). The frequency suppression did not recover upon wash-out (54.+-.8%; n=6). Together, these data indicate that A.beta.(1-42) globulomer depresses vesicular release at GABAergic and glutamatergic synapses, most likely by decreasing the probability of vesicle exocytosis from presynaptic terminals.

EXAMPLE 17

Effects on Voltage-Activated Calcium Currents

[0190] Presynaptic vesicle release is triggered by an influx of calcium into the presynaptic terminal. Therefore, A.beta.(1-42) globulomer might act on presynaptic calcium signalling. A common pathway for release of both, glutamatergic and GABAergic vesicles is presynaptic calcium influx via N-type or P/Q-type calcium channels. Therefore, the effects of A.beta.(1-42) on whole-cell calcium currents in cultured hippocampal neurons were analyzed. Typical P/Q channel-mediated currents could be reliably elicited in somatic whole-cell recordings under our culture conditions. In these experiments, 10 mM Ba.sup.2+ was used as charge carrier in the extracellular solution (see methods). Measurements were performed in the presence of 10 .mu.M nifedipine (a L-type calcium channel blocker), .omega.-conotoxin MVIIA (a N-type calcium channel blocker) and blockers of other voltage-gated ion channels (TTX 0.5 .mu.M, TEA 140 mM, Cs.sup.+-based intracellular solution). Rundown of these currents was avoided by adding 20 U/ml phosphocreatine kinase and 10 mM tris-phosphocreatine to the pipette solution. Under these conditions, P/Q-type currents were evoked by a depolarizing voltage step to -10 mV (mean amplitude 1015.+-.145 pA; FIG. 21). A.beta.(1-42) globulomer reduced the amplitude of these currents to 62.+-.7% of control (n=10). This effect was partially reversible in 3/3 cells.

[0191] If the effect of A.beta.(1-42) globulomer on synaptic currents is mediated by block of P/Q-type calcium channels, it should be mimicked and occluded by the selective P/Q-type calcium channel blocker .omega.-Agatoxin IVA. Indeed, this toxin (0.5 .mu.M) reduced the frequency of miniature PSCs to 27.+-.7% (n=3; amplitude 90.+-.7%), similar to the effect of A.beta.(1-42) globulomer. Following pre-incubation with .omega.-Agatoxin IVA, A.beta.(1-42) globulomer had no additional effect on the synaptic currents (n=6, frequency 108.+-.15%; amplitude 102.+-.7% of currents after .omega.-Agatoxin IVA control; FIG. 23). These data suggest that .omega.-Agatoxin IVA and A.beta.(1-42) globulomer share the same molecular mechanism.

[0192] The effect of A.beta.(1-42) globulomer on P/Q-type calcium currents was further characterized by steady-state activation and -inactivation protocols (see methods). Maximal conductance (g.sub.max) was 61.7.+-.2.4 nS (control) versus 27.2.+-.3.2 nS (A.beta.(1-42) globulomer; p<0.05; n=6; FIG. 22). Thus, A.beta.(1-42) globulomer reduces the current amplitude without affecting its voltage-dependent activation. In contrast to this marked reduction in conductance (and current amplitude), other kinetic parameters were not affected by A.beta.(1-42) globulomer. Steady-state activation was characterized by V.sub.h=-15.4.+-.1.1 mV which was not changed after application of A.beta.(1-42) globulomer (V.sub.h=-17.3.+-.1.3 mV; n=6). The slope of the fitted first-order Boltzmann-equation V.sub.c was -7.8.+-.0.3 mV in control solution and -10.8.+-.0.5 mV in A.beta.(1-42) globulomer (not different, n=6). Likewise, steady-state inactivation was not affected by A.beta.(1-42) globulomer, as indicated by stable values for the voltage at half-maximal inactivation (29.2.+-.0.6 mV in control; 32.4.+-.1.2 mV in A.beta.(1-42) globulomer; n=4) and for the slope V.sub.c (-11.0.+-.0.9 mV versus -12.6.+-.1.1 mV; FIG. 22).). Thus, A.beta.(1-42) globulomer reduces the current amplitude without affecting its voltage-dependent activation or inactivation.

[0193] In addition the effects of A.beta.(1-42) globulomer on N- and L-type calcium currents were analyzed. Besides blockers for Na.sup.+- and K.sup.+-channels (see above) 0.5 .mu.M .omega.-agatoxin IVA were added to block P/Q-channels. L-type calcium currents were isolated by addition of 0.5 .mu.M .omega.-conotoxin MVIIA. Voltage pulses from -80 mV to -10 mV elicited inward currents of 597.7.+-.230.9 pA amplitude which remained stable after addition of A.beta.(1-42) globulomer (573.0.+-.225.6 pA; n=3). When N-type currents were isolated by adding nifedipine (10 .mu.M) instead of .omega.-conotoxin, the same voltage clamp protocol elicited inward currents which were, again, insensitive to A.beta.(1-42) globulomer (amplitude in control solution 1368.9.+-.332.8; amplitude in A.beta.(1-42) globulomer 1399.8.+-.376.4 pA; n=3). When all blockers were added together, the remaining calcium current (possibly R-type) was too small for a detailed analysis (<100 pA), indicating that this component was only marginally expressed in the cultured hippocampal neurons.

EXAMPLE 18

Rescue by Roscovitine

[0194] Application of roscovitine in the presence of A.beta.(1-42) globulomer did indeed partially recover the frequency of synaptic currents. While in these experiments A.beta.(1-42) globulomer reduced the frequency of spontaneous PSCs to 38.+-.10% of control, application of roscovitine (20 .mu.M) brought this parameter back to 75.+-.13% (n=5; FIG. 23).

[0195] Together, these data indicate that A.beta.(1-42) globulomer reduces the frequency of spontaneous and miniature synaptic currents by suppression of presynaptic calcium influx via P/Q-type calcium channels.

THIRD SERIES OF EXPERIMENTS

EXAMPLE 19

Rescue of Spontaneous Synaptic Activity by Isoproterenol

[0196] Isoproterenol was used at a final concentration of 15 .mu.M, by adding it simultaneously with A.beta.(1-42) globulomer (final concentration of A.beta. globulomer corresponding to approximately 1 .mu.M of A.beta. monomer). Isoproterenol is known (Huang C.-C., et al., The Journal of Neuroscience, 1996, 16(3): 1026-1033, Huang C.-C., et al., The Journal of Neuroscience, 1998, 18(6): 2276-2282) to increase the Ca.sup.2+ influx through the P/Q type voltage-gated presynaptic calcium channel.

[0197] Application of isoproterenol in the presence of A.beta.(1-42) globulomer did indeed recover the frequency of synaptic currents. While in these experiments A.beta.(1-42) globulomer reduced the frequency of spontaneous IPSCs to 57.+-.7% of control, application of isoproterenol (15 .mu.M) brought this parameter back to 122.+-.58% (n=6; FIG. 24).

[0198] This demonstrates that the P/Q type voltage-gated presynaptic calcium channel activator isoproterenol is capable of restoring the frequency of spontaneous synaptic events under the influence of A.beta. globulomer to that of untreated cells, i. e., that the P/Q activator may be used to reverse the detrimental effects of A.beta. globulomer.

EXAMPLE 20

Enhancing P/Q Calcium Currents by Roscovitine Prevents/Reverses Chronic A.beta. Globulomer-Induced Deficits on Evoked Synaptic Transmission in Hippocampal Tissue

[0199] Rat hippocampal slice cultures (9 days old Wistar rats; 15-17 DIV) were incubated over night with either A.beta.(1-42) globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer), A.beta.(1-42) globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer)+20 .mu.M roscovitine, or control (SDS). Recordings were performed (in artificial cerebrospinal fluid) from CA1 stratum radiatum after stimulation of the Schaffer collateral at different intensities.

[0200] Results are shown in FIG. 25, demonstrating that the application of globulomer strongly suppresses synaptic transmission. Co-application of 20 .mu.M roscivitine completely prevents/reverses the globulomer-induced deficit.

EXAMPLE 21

Enhancing P/Q Calcium Currents by Roscovitine Analogue A Prevents/Reverses Chronic A.beta. Globulomer-Induced Deficits on Evoked Synaptic Transmission in Hippocampal Tissue

[0201] Rat hippocampal slice cultures (11 days old Wistar rats; 23-24 DIV) were incubated over night with either A.beta.(1-42) globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer), A.beta.(1-42) globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer)+20 .mu.M roscovitine analogue A, or control (SDS). Recordings were performed (in artificial cerebrospinal fluid) from CA1 stratum radiatum after stimulation of the Schaffer collateral at different intensities.

[0202] Results are shown in FIG. 26, demonstrating that the application of globulomer strongly suppresses synaptic transmission. Co-application of 20 .mu.M roscivitine analogue A completely prevents/reverses the globulomer-induced deficit.

EXAMPLE 22

Effect of Extracellular Ca.sup.2+ on sPSC Frequency After Treatment with A.beta.(1-42) Globulomer

[0203] Spontaneous synaptic activity was measured in cultured hippocampal neurons using whole-cell voltage clamp techniques (V.sub.hold=-70 mV). Under the ionic conditions used (E.sub.Cl.about.-10 mV) all synaptic events appeared as inward currents.

[0204] The effects of A.beta.(1-42) globulomer (at a concentration corresponding to approximately 1 .mu.M of A.beta. monomer) were assessed by comparing spontaneously occurring postsynaptic currents (sPSCs) in single cells in 5 min intervals in the presence or absence of globulomer in bath solution containing 1 mM Ca.sup.2+. Currents recorded prior to the addition of the globulomer served as control describing basal synaptic transmission. Currents recorded in the interval immediately after application were analysed with respect to the control data. Afterwards, extracellular Ca.sup.2+ was elevated from 1 mM to 4 mM (leaving the concentration of globulomer unchanged). Currents in the following 5 min recording interval were again analysed with respect to control data.

[0205] Basal frequency of sPSCs in 1 mM Ca.sup.2+ was 4.2.+-.1.2 Hz. Bath-application of A.beta.(1-42) globulomer rapidly reduced the sPSC frequency to 63.+-.7% of control (p<0.05; n=6; FIG. 27 A+B). After elevation of extracellular Ca.sup.2+ to 4 mM, sPSC frequency partially recovered to 77.+-.13% of control (FIG. 27 B). In 4 of 6 cells tested, sPSC frequency increased, whereas it remained unaltered in the other 2 cells (FIG. 27 C).

[0206] Median amplitude of sPSCs under control conditions was 27.7.+-.2.2 pA and remained unaltered after addition of A.beta.(1-42) globulomer (97.+-.5%; FIG. 27 D) or subsequent elevation of extracellular Ca.sup.2+ (98.+-.6%).

[0207] In most cases, prominent currents with amplitudes up to 2000 pA occurred directly after elevation of extracellular Ca.sup.2+-concentration. These currents with multiple peaks (see FIG. 27 E) were rejected from analysis.

[0208] This clearly demonstrates that the principle of activating the P/Q type presynaptic calcium channel is effective in compensating the detrimental effects exerted by A.beta. globulomer.

Sequence CWU 1

1

312505PRTHomo sapiens 1Met Ala Arg Phe Gly Asp Glu Met Pro Ala Arg Tyr Gly Gly Gly Gly1 5 10 15Ser Gly Ala Ala Ala Gly Val Val Val Gly Ser Gly Gly Gly Arg Gly 20 25 30Ala Gly Gly Ser Arg Gln Gly Gly Gln Pro Gly Ala Gln Arg Met Tyr 35 40 45Lys Gln Ser Met Ala Gln Arg Ala Arg Thr Met Ala Leu Tyr Asn Pro 50 55 60Ile Pro Val Arg Gln Asn Cys Leu Thr Val Asn Arg Ser Leu Phe Leu65 70 75 80Phe Ser Glu Asp Asn Val Val Arg Lys Tyr Ala Lys Lys Ile Thr Glu 85 90 95Trp Pro Pro Phe Glu Tyr Met Ile Leu Ala Thr Ile Ile Ala Asn Cys 100 105 110Ile Val Leu Ala Leu Glu Gln His Leu Pro Asp Asp Asp Lys Thr Pro 115 120 125Met Ser Glu Arg Leu Asp Asp Thr Glu Pro Tyr Phe Ile Gly Ile Phe 130 135 140Cys Phe Glu Ala Gly Ile Lys Ile Ile Ala Leu Gly Phe Ala Phe His145 150 155 160Lys Gly Ser Tyr Leu Arg Asn Gly Trp Asn Val Met Asp Phe Val Val 165 170 175Val Leu Thr Gly Ile Leu Ala Thr Val Gly Thr Glu Phe Asp Leu Arg 180 185 190Thr Leu Arg Ala Val Arg Val Leu Arg Pro Leu Lys Leu Val Ser Gly 195 200 205Ile Pro Ser Leu Gln Val Val Leu Lys Ser Ile Met Lys Ala Met Ile 210 215 220Pro Leu Leu Gln Ile Gly Leu Leu Leu Phe Phe Ala Ile Leu Ile Phe225 230 235 240Ala Ile Ile Gly Leu Glu Phe Tyr Met Gly Lys Phe His Thr Thr Cys 245 250 255Phe Glu Glu Gly Thr Asp Asp Ile Gln Gly Glu Ser Pro Ala Pro Cys 260 265 270Gly Thr Glu Glu Pro Ala Arg Thr Cys Pro Asn Gly Thr Lys Cys Gln 275 280 285Pro Tyr Trp Glu Gly Pro Asn Asn Gly Ile Thr Gln Phe Asp Asn Ile 290 295 300Leu Phe Ala Val Leu Thr Val Phe Gln Cys Ile Thr Met Glu Gly Trp305 310 315 320Thr Asp Leu Leu Tyr Asn Ser Asn Asp Ala Ser Gly Asn Thr Trp Asn 325 330 335Trp Leu Tyr Phe Ile Pro Leu Ile Ile Ile Gly Ser Phe Phe Met Leu 340 345 350Asn Leu Val Leu Gly Val Leu Ser Gly Glu Phe Ala Lys Glu Arg Glu 355 360 365Arg Val Glu Asn Arg Arg Ala Phe Leu Lys Leu Arg Arg Gln Gln Gln 370 375 380Ile Glu Arg Glu Leu Asn Gly Tyr Met Glu Trp Ile Ser Lys Ala Glu385 390 395 400Glu Val Ile Leu Ala Glu Asp Glu Thr Asp Gly Glu Gln Arg His Pro 405 410 415Phe Asp Gly Ala Leu Arg Arg Thr Thr Ile Lys Lys Ser Lys Thr Asp 420 425 430Leu Leu Asn Pro Glu Glu Ala Glu Asp Gln Leu Ala Asp Ile Ala Ser 435 440 445Val Gly Ser Pro Phe Ala Arg Ala Ser Ile Lys Ser Ala Lys Leu Glu 450 455 460Asn Ser Thr Phe Phe His Lys Lys Glu Arg Arg Met Arg Phe Tyr Ile465 470 475 480Arg Arg Met Val Lys Thr Gln Ala Phe Tyr Trp Thr Val Leu Ser Leu 485 490 495Val Ala Leu Asn Thr Leu Cys Val Ala Ile Val His Tyr Asn Gln Pro 500 505 510Glu Trp Leu Ser Asp Phe Leu Tyr Tyr Ala Glu Phe Ile Phe Leu Gly 515 520 525Leu Phe Met Ser Glu Met Phe Ile Lys Met Tyr Gly Leu Gly Thr Arg 530 535 540Pro Tyr Phe His Ser Ser Phe Asn Cys Phe Asp Cys Gly Val Ile Ile545 550 555 560Gly Ser Ile Phe Glu Val Ile Trp Ala Val Ile Lys Pro Gly Thr Ser 565 570 575Phe Gly Ile Ser Val Leu Arg Ala Leu Arg Leu Leu Arg Ile Phe Lys 580 585 590Val Thr Lys Tyr Trp Ala Ser Leu Arg Asn Leu Val Val Ser Leu Leu 595 600 605Asn Ser Met Lys Ser Ile Ile Ser Leu Leu Phe Leu Leu Phe Leu Phe 610 615 620Ile Val Val Phe Ala Leu Leu Gly Met Gln Leu Phe Gly Gly Gln Phe625 630 635 640Asn Phe Asp Glu Gly Thr Pro Pro Thr Asn Phe Asp Thr Phe Pro Ala 645 650 655Ala Ile Met Thr Val Phe Gln Ile Leu Thr Gly Glu Asp Trp Asn Glu 660 665 670Val Met Tyr Asp Gly Ile Lys Ser Gln Gly Gly Val Gln Gly Gly Met 675 680 685Val Phe Ser Ile Tyr Phe Ile Val Leu Thr Leu Phe Gly Asn Tyr Thr 690 695 700Leu Leu Asn Val Phe Leu Ala Ile Ala Val Asp Asn Leu Ala Asn Ala705 710 715 720Gln Glu Leu Thr Lys Asp Glu Gln Glu Glu Glu Glu Ala Ala Asn Gln 725 730 735Lys Leu Ala Leu Gln Lys Ala Lys Glu Val Ala Glu Val Ser Pro Leu 740 745 750Ser Ala Ala Asn Met Ser Ile Ala Val Lys Glu Gln Gln Lys Asn Gln 755 760 765Lys Pro Ala Lys Ser Val Trp Glu Gln Arg Thr Ser Glu Met Arg Lys 770 775 780Gln Asn Leu Leu Ala Ser Arg Glu Ala Leu Tyr Asn Glu Met Asp Pro785 790 795 800Asp Glu Arg Trp Lys Ala Ala Tyr Thr Arg His Leu Arg Pro Asp Met 805 810 815Lys Thr His Leu Asp Arg Pro Leu Val Val Asp Pro Gln Glu Asn Arg 820 825 830Asn Asn Asn Thr Asn Lys Ser Arg Ala Ala Glu Pro Thr Val Asp Gln 835 840 845Arg Leu Gly Gln Gln Arg Ala Glu Asp Phe Leu Arg Lys Gln Ala Arg 850 855 860Tyr His Asp Arg Ala Arg Asp Pro Ser Gly Ser Ala Gly Leu Asp Ala865 870 875 880Arg Arg Pro Trp Ala Gly Ser Gln Glu Ala Glu Leu Ser Arg Glu Gly 885 890 895Pro Tyr Gly Arg Glu Ser Asp His His Ala Arg Glu Gly Ser Leu Glu 900 905 910Gln Pro Gly Phe Trp Glu Gly Glu Ala Glu Arg Gly Lys Ala Gly Asp 915 920 925Pro His Arg Arg His Val His Arg Gln Gly Gly Ser Arg Glu Ser Arg 930 935 940Ser Gly Ser Pro Arg Thr Gly Ala Asp Gly Glu His Arg Arg His Arg945 950 955 960Ala His Arg Arg Pro Gly Glu Glu Gly Pro Glu Asp Lys Ala Glu Arg 965 970 975Arg Ala Arg His Arg Glu Gly Ser Arg Pro Ala Arg Gly Gly Glu Gly 980 985 990Glu Gly Glu Gly Pro Asp Gly Gly Glu Arg Arg Arg Arg His Arg His 995 1000 1005Gly Ala Pro Ala Thr Tyr Glu Gly Asp Ala Arg Arg Glu Asp Lys 1010 1015 1020Glu Arg Arg His Arg Arg Arg Lys Glu Asn Gln Gly Ser Gly Val 1025 1030 1035Pro Val Ser Gly Pro Asn Leu Ser Thr Thr Arg Pro Ile Gln Gln 1040 1045 1050Asp Leu Gly Arg Gln Asp Pro Pro Leu Ala Glu Asp Ile Asp Asn 1055 1060 1065Met Lys Asn Asn Lys Leu Ala Thr Ala Glu Ser Ala Ala Pro His 1070 1075 1080Gly Ser Leu Gly His Ala Gly Leu Pro Gln Ser Pro Ala Lys Met 1085 1090 1095Gly Asn Ser Thr Asp Pro Gly Pro Met Leu Ala Ile Pro Ala Met 1100 1105 1110Ala Thr Asn Pro Gln Asn Ala Ala Ser Arg Arg Thr Pro Asn Asn 1115 1120 1125Pro Gly Asn Pro Ser Asn Pro Gly Pro Pro Lys Thr Pro Glu Asn 1130 1135 1140Ser Leu Ile Val Thr Asn Pro Ser Gly Thr Gln Thr Asn Ser Ala 1145 1150 1155Lys Thr Ala Arg Lys Pro Asp His Thr Thr Val Asp Ile Pro Pro 1160 1165 1170Ala Cys Pro Pro Pro Leu Asn His Thr Val Val Gln Val Asn Lys 1175 1180 1185Asn Ala Asn Pro Asp Pro Leu Pro Lys Lys Glu Glu Glu Lys Lys 1190 1195 1200Glu Glu Glu Glu Asp Asp Arg Gly Glu Asp Gly Pro Lys Pro Met 1205 1210 1215Pro Pro Tyr Ser Ser Met Phe Ile Leu Ser Thr Thr Asn Pro Leu 1220 1225 1230Arg Arg Leu Cys His Tyr Ile Leu Asn Leu Arg Tyr Phe Glu Met 1235 1240 1245Cys Ile Leu Met Val Ile Ala Met Ser Ser Ile Ala Leu Ala Ala 1250 1255 1260Glu Asp Pro Val Gln Pro Asn Ala Pro Arg Asn Asn Val Leu Arg 1265 1270 1275Tyr Phe Asp Tyr Val Phe Thr Gly Val Phe Thr Phe Glu Met Val 1280 1285 1290Ile Lys Met Ile Asp Leu Gly Leu Val Leu His Gln Gly Ala Tyr 1295 1300 1305Phe Arg Asp Leu Trp Asn Ile Leu Asp Phe Ile Val Val Ser Gly 1310 1315 1320Ala Leu Val Ala Phe Ala Phe Thr Gly Asn Ser Lys Gly Lys Asp 1325 1330 1335Ile Asn Thr Ile Lys Ser Leu Arg Val Leu Arg Val Leu Arg Pro 1340 1345 1350Leu Lys Thr Ile Lys Arg Leu Pro Lys Leu Lys Ala Val Phe Asp 1355 1360 1365Cys Val Val Asn Ser Leu Lys Asn Val Phe Asn Ile Leu Ile Val 1370 1375 1380Tyr Met Leu Phe Met Phe Ile Phe Ala Val Val Ala Val Gln Leu 1385 1390 1395Phe Lys Gly Lys Phe Phe His Cys Thr Asp Glu Ser Lys Glu Phe 1400 1405 1410Glu Lys Asp Cys Arg Gly Lys Tyr Leu Leu Tyr Glu Lys Asn Glu 1415 1420 1425Val Lys Ala Arg Asp Arg Glu Trp Lys Lys Tyr Glu Phe His Tyr 1430 1435 1440Asp Asn Val Leu Trp Ala Leu Leu Thr Leu Phe Thr Val Ser Thr 1445 1450 1455Gly Glu Gly Trp Pro Gln Val Leu Lys His Ser Val Asp Ala Thr 1460 1465 1470Phe Glu Asn Gln Gly Pro Ser Pro Gly Tyr Arg Met Glu Met Ser 1475 1480 1485Ile Phe Tyr Val Val Tyr Phe Val Val Phe Pro Phe Phe Phe Val 1490 1495 1500Asn Ile Phe Val Ala Leu Ile Ile Ile Thr Phe Gln Glu Gln Gly 1505 1510 1515Asp Lys Met Met Glu Glu Tyr Ser Leu Glu Lys Asn Glu Arg Ala 1520 1525 1530Cys Ile Asp Phe Ala Ile Ser Ala Lys Pro Leu Thr Arg His Met 1535 1540 1545Pro Gln Asn Lys Gln Ser Phe Gln Tyr Arg Met Trp Gln Phe Val 1550 1555 1560Val Ser Pro Pro Phe Glu Tyr Thr Ile Met Ala Met Ile Ala Leu 1565 1570 1575Asn Thr Ile Val Leu Met Met Lys Phe Tyr Gly Ala Ser Val Ala 1580 1585 1590Tyr Glu Asn Ala Leu Arg Val Phe Asn Ile Val Phe Thr Ser Leu 1595 1600 1605Phe Ser Leu Glu Cys Val Leu Lys Val Met Ala Phe Gly Ile Leu 1610 1615 1620Asn Tyr Phe Arg Asp Ala Trp Asn Ile Phe Asp Phe Val Thr Val 1625 1630 1635Leu Gly Ser Ile Thr Asp Ile Leu Val Thr Glu Phe Gly Asn Asn 1640 1645 1650Phe Ile Asn Leu Ser Phe Leu Arg Leu Phe Arg Ala Ala Arg Leu 1655 1660 1665Ile Lys Leu Leu Arg Gln Gly Tyr Thr Ile Arg Ile Leu Leu Trp 1670 1675 1680Thr Phe Val Gln Ser Phe Lys Ala Leu Pro Tyr Val Cys Leu Leu 1685 1690 1695Ile Ala Met Leu Phe Phe Ile Tyr Ala Ile Ile Gly Met Gln Val 1700 1705 1710Phe Gly Asn Ile Gly Ile Asp Val Glu Asp Glu Asp Ser Asp Glu 1715 1720 1725Asp Glu Phe Gln Ile Thr Glu His Asn Asn Phe Arg Thr Phe Phe 1730 1735 1740Gln Ala Leu Met Leu Leu Phe Arg Ser Ala Thr Gly Glu Ala Trp 1745 1750 1755His Asn Ile Met Leu Ser Cys Leu Ser Gly Lys Pro Cys Asp Lys 1760 1765 1770Asn Ser Gly Ile Leu Thr Arg Glu Cys Gly Asn Glu Phe Ala Tyr 1775 1780 1785Phe Tyr Phe Val Ser Phe Ile Phe Leu Cys Ser Phe Leu Met Leu 1790 1795 1800Asn Leu Phe Val Ala Val Ile Met Asp Asn Phe Glu Tyr Leu Thr 1805 1810 1815Arg Asp Ser Ser Ile Leu Gly Pro His His Leu Asp Glu Tyr Val 1820 1825 1830Arg Val Trp Ala Glu Tyr Asp Pro Ala Ala Trp Gly Arg Met Pro 1835 1840 1845Tyr Leu Asp Met Tyr Gln Met Leu Arg His Met Ser Pro Pro Leu 1850 1855 1860Gly Leu Gly Lys Lys Cys Pro Ala Arg Val Ala Tyr Lys Arg Leu 1865 1870 1875Leu Arg Met Asp Leu Pro Val Ala Asp Asp Asn Thr Val His Phe 1880 1885 1890Asn Ser Thr Leu Met Ala Leu Ile Arg Thr Ala Leu Asp Ile Lys 1895 1900 1905Ile Ala Lys Gly Gly Ala Asp Lys Gln Gln Met Asp Ala Glu Leu 1910 1915 1920Arg Lys Glu Met Met Ala Ile Trp Pro Asn Leu Ser Gln Lys Thr 1925 1930 1935Leu Asp Leu Leu Val Thr Pro His Lys Ser Thr Asp Leu Thr Val 1940 1945 1950Gly Lys Ile Tyr Ala Ala Met Met Ile Met Glu Tyr Tyr Arg Gln 1955 1960 1965Ser Lys Ala Lys Lys Leu Gln Ala Met Arg Glu Glu Gln Asp Arg 1970 1975 1980Thr Pro Leu Met Phe Gln Arg Met Glu Pro Pro Ser Pro Thr Gln 1985 1990 1995Glu Gly Gly Pro Gly Gln Asn Ala Leu Pro Ser Thr Gln Leu Asp 2000 2005 2010Pro Gly Gly Ala Leu Met Ala His Glu Ser Gly Leu Lys Glu Ser 2015 2020 2025Pro Ser Trp Val Thr Gln Arg Ala Gln Glu Met Phe Gln Lys Thr 2030 2035 2040Gly Thr Trp Ser Pro Glu Gln Gly Pro Pro Thr Asp Met Pro Asn 2045 2050 2055Ser Gln Pro Asn Ser Gln Ser Val Glu Met Arg Glu Met Gly Arg 2060 2065 2070Asp Gly Tyr Ser Asp Ser Glu His Tyr Leu Pro Met Glu Gly Gln 2075 2080 2085Gly Arg Ala Ala Ser Met Pro Arg Leu Pro Ala Glu Asn Gln Arg 2090 2095 2100Arg Arg Gly Arg Pro Arg Gly Asn Asn Leu Ser Thr Ile Ser Asp 2105 2110 2115Thr Ser Pro Met Lys Arg Ser Ala Ser Val Leu Gly Pro Lys Ala 2120 2125 2130Arg Arg Leu Asp Asp Tyr Ser Leu Glu Arg Val Pro Pro Glu Glu 2135 2140 2145Asn Gln Arg His His Gln Arg Arg Arg Asp Arg Ser His Arg Ala 2150 2155 2160Ser Glu Arg Ser Leu Gly Arg Tyr Thr Asp Val Asp Thr Gly Leu 2165 2170 2175Gly Thr Asp Leu Ser Met Thr Thr Gln Ser Gly Asp Leu Pro Ser 2180 2185 2190Lys Glu Arg Asp Gln Glu Arg Gly Arg Pro Lys Asp Arg Lys His 2195 2200 2205Arg Gln His His His His His His His His His His Pro Pro Pro 2210 2215 2220Pro Asp Lys Asp Arg Tyr Ala Gln Glu Arg Pro Asp His Gly Arg 2225 2230 2235Ala Arg Ala Arg Asp Gln Arg Trp Ser Arg Ser Pro Ser Glu Gly 2240 2245 2250Arg Glu His Met Ala His Arg Gln Gly Ser Ser Ser Val Ser Gly 2255 2260 2265Ser Pro Ala Pro Ser Thr Ser Gly Thr Ser Thr Pro Arg Arg Gly 2270 2275 2280Arg Arg Gln Leu Pro Gln Thr Pro Ser Thr Pro Arg Pro His Val 2285 2290 2295Ser Tyr Ser Pro Val Ile Arg Lys Ala Gly Gly Ser Gly Pro Pro 2300 2305 2310Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Ala Val Ala Arg 2315 2320 2325Pro Gly Arg Ala Ala Thr Ser Gly Pro Arg Arg Tyr Pro Gly Pro 2330 2335 2340Thr Ala Glu Pro Leu Ala Gly Asp Arg Pro Pro Thr Gly Gly His 2345 2350 2355Ser Ser Gly Arg Ser Pro Arg Met Glu Arg Arg Val Pro Gly Pro 2360 2365 2370Ala Arg Ser Glu Ser Pro Arg Ala Cys Arg His Gly Gly Ala Arg 2375 2380 2385Trp Pro Ala Ser Gly Pro His Val Ser Glu Gly Pro Pro Gly Pro 2390 2395 2400Arg His His Gly Tyr Tyr Arg Gly Ser Asp Tyr Asp Glu Ala Asp 2405 2410 2415Gly Pro Gly Ser Gly Gly Gly Glu Glu Ala Met Ala Gly Ala Tyr 2420 2425 2430Asp Ala Pro Pro Pro Val Arg His Ala Ser Ser Gly Ala Thr Gly 2435 2440 2445Arg Ser

Pro Arg Thr Pro Arg Ala Ser Gly Pro Ala Cys Ala Ser 2450 2455 2460Pro Ser Arg His Gly Arg Arg Leu Pro Asn Gly Tyr Tyr Pro Ala 2465 2470 2475His Gly Leu Ala Arg Pro Arg Gly Pro Gly Ser Arg Lys Gly Leu 2480 2485 2490His Glu Pro Tyr Ser Glu Ser Asp Asp Asp Trp Cys 2495 2500 250521145PRTHomo sapiensMISC_FEATURE(1)..(1145)PQ calcium channel, alpha 2/delta subunit 2 isoform a 2Met Ala Val Pro Ala Arg Thr Cys Gly Ala Ser Arg Pro Gly Pro Ala1 5 10 15Arg Thr Ala Arg Pro Trp Pro Gly Cys Gly Pro His Pro Gly Pro Gly 20 25 30Thr Arg Arg Pro Thr Ser Gly Pro Pro Arg Pro Leu Trp Leu Leu Leu 35 40 45Pro Leu Leu Pro Leu Leu Ala Ala Pro Gly Ala Ser Ala Tyr Ser Phe 50 55 60Pro Gln Gln His Thr Met Gln His Trp Ala Arg Arg Leu Glu Gln Glu65 70 75 80Val Asp Gly Val Met Arg Ile Phe Gly Gly Val Gln Gln Leu Arg Glu 85 90 95Ile Tyr Lys Asp Asn Arg Asn Leu Phe Glu Val Gln Glu Asn Glu Pro 100 105 110Gln Lys Leu Val Glu Lys Val Ala Gly Asp Ile Glu Ser Leu Leu Asp 115 120 125Arg Lys Val Gln Ala Leu Lys Arg Leu Ala Asp Ala Ala Glu Asn Phe 130 135 140Gln Lys Ala His Arg Trp Gln Asp Asn Ile Lys Glu Glu Asp Ile Val145 150 155 160Tyr Tyr Asp Ala Lys Ala Asp Ala Glu Leu Asp Asp Pro Glu Ser Glu 165 170 175Asp Val Glu Arg Gly Ser Lys Ala Ser Thr Leu Arg Leu Asp Phe Ile 180 185 190Glu Asp Pro Asn Phe Lys Asn Lys Val Asn Tyr Ser Tyr Ala Ala Val 195 200 205Gln Ile Pro Thr Asp Ile Tyr Lys Gly Ser Thr Val Ile Leu Asn Glu 210 215 220Leu Asn Trp Thr Glu Ala Leu Glu Asn Val Phe Met Glu Asn Arg Arg225 230 235 240Gln Asp Pro Thr Leu Leu Trp Gln Val Phe Gly Ser Ala Thr Gly Val 245 250 255Thr Arg Tyr Tyr Pro Ala Thr Pro Trp Arg Ala Pro Lys Lys Ile Asp 260 265 270Leu Tyr Asp Val Arg Arg Arg Pro Trp Tyr Ile Gln Gly Ala Ser Ser 275 280 285Pro Lys Asp Met Val Ile Ile Val Asp Val Ser Gly Ser Val Ser Gly 290 295 300Leu Thr Leu Lys Leu Met Lys Thr Ser Val Cys Glu Met Leu Asp Thr305 310 315 320Leu Ser Asp Asp Asp Tyr Val Asn Val Ala Ser Phe Asn Glu Lys Ala 325 330 335Gln Pro Val Ser Cys Phe Thr His Leu Val Gln Ala Asn Val Arg Asn 340 345 350Lys Lys Val Phe Lys Glu Ala Val Gln Gly Met Val Ala Lys Gly Thr 355 360 365Thr Gly Tyr Lys Ala Gly Phe Glu Tyr Ala Phe Asp Gln Leu Gln Asn 370 375 380Ser Asn Ile Thr Arg Ala Asn Cys Asn Lys Met Ile Met Met Phe Thr385 390 395 400Asp Gly Gly Glu Asp Arg Val Gln Asp Val Phe Glu Lys Tyr Asn Trp 405 410 415Pro Asn Arg Thr Val Arg Val Phe Thr Phe Ser Val Gly Gln His Asn 420 425 430Tyr Asp Val Thr Pro Leu Gln Trp Met Ala Cys Ala Asn Lys Gly Tyr 435 440 445Tyr Phe Glu Ile Pro Ser Ile Gly Ala Ile Arg Ile Asn Thr Gln Glu 450 455 460Tyr Leu Asp Val Leu Gly Arg Pro Met Val Leu Ala Gly Lys Glu Ala465 470 475 480Lys Gln Val Gln Trp Thr Asn Val Tyr Glu Asp Ala Leu Gly Leu Gly 485 490 495Leu Val Val Thr Gly Thr Leu Pro Val Phe Asn Leu Thr Gln Asp Gly 500 505 510Pro Gly Glu Lys Lys Asn Gln Leu Ile Leu Gly Val Met Gly Ile Asp 515 520 525Val Ala Leu Asn Asp Ile Lys Arg Leu Thr Pro Asn Tyr Thr Leu Gly 530 535 540Ala Asn Gly Tyr Val Phe Ala Ile Asp Leu Asn Gly Tyr Val Leu Leu545 550 555 560His Pro Asn Leu Lys Pro Gln Thr Thr Asn Phe Arg Glu Pro Val Thr 565 570 575Leu Asp Phe Leu Asp Ala Glu Leu Glu Asp Glu Asn Lys Glu Glu Ile 580 585 590Arg Arg Ser Met Ile Asp Gly Asn Lys Gly His Lys Gln Ile Arg Thr 595 600 605Leu Val Lys Ser Leu Asp Glu Arg Tyr Ile Asp Glu Val Thr Arg Asn 610 615 620Tyr Thr Trp Val Pro Ile Arg Ser Thr Asn Tyr Ser Leu Gly Leu Val625 630 635 640Leu Pro Pro Tyr Ser Thr Phe Tyr Leu Gln Ala Asn Leu Ser Asp Gln 645 650 655Ile Leu Gln Val Lys Tyr Phe Glu Phe Leu Leu Pro Ser Ser Phe Glu 660 665 670Ser Glu Gly His Val Phe Ile Ala Pro Arg Glu Tyr Cys Lys Asp Leu 675 680 685Asn Ala Ser Asp Asn Asn Thr Glu Phe Leu Lys Asn Phe Ile Glu Leu 690 695 700Met Glu Lys Val Thr Pro Asp Ser Lys Gln Cys Asn Asn Phe Leu Leu705 710 715 720His Asn Leu Ile Leu Asp Thr Gly Ile Thr Gln Gln Leu Val Glu Arg 725 730 735Val Trp Arg Asp Gln Asp Leu Asn Thr Tyr Ser Leu Leu Ala Val Phe 740 745 750Ala Ala Thr Asp Gly Gly Ile Thr Arg Val Phe Pro Asn Lys Ala Ala 755 760 765Glu Asp Trp Thr Glu Asn Pro Glu Pro Phe Asn Ala Ser Phe Tyr Arg 770 775 780Arg Ser Leu Asp Asn His Gly Tyr Val Phe Lys Pro Pro His Gln Asp785 790 795 800Ala Leu Leu Arg Pro Leu Glu Leu Glu Asn Asp Thr Val Gly Ile Leu 805 810 815Val Ser Thr Ala Val Glu Leu Ser Leu Gly Arg Arg Thr Leu Arg Pro 820 825 830Ala Val Val Gly Val Lys Leu Asp Leu Glu Ala Trp Ala Glu Lys Phe 835 840 845Lys Val Leu Ala Ser Asn Arg Thr His Gln Asp Gln Pro Gln Lys Cys 850 855 860Gly Pro Asn Ser His Cys Glu Met Asp Cys Glu Val Asn Asn Glu Asp865 870 875 880Leu Leu Cys Val Leu Ile Asp Asp Gly Gly Phe Leu Val Leu Ser Asn 885 890 895Gln Asn His Gln Trp Asp Gln Val Gly Arg Phe Phe Ser Glu Val Asp 900 905 910Ala Asn Leu Met Leu Ala Leu Tyr Asn Asn Ser Phe Tyr Thr Arg Lys 915 920 925Glu Ser Tyr Asp Tyr Gln Ala Ala Cys Ala Pro Gln Pro Pro Gly Asn 930 935 940Leu Gly Ala Ala Pro Arg Gly Val Phe Val Pro Thr Val Ala Asp Phe945 950 955 960Leu Asn Leu Ala Trp Trp Thr Ser Ala Ala Ala Trp Ser Leu Phe Gln 965 970 975Gln Leu Leu Tyr Gly Leu Ile Tyr His Ser Trp Phe Gln Ala Asp Pro 980 985 990Ala Glu Ala Glu Gly Ser Pro Glu Thr Arg Glu Ser Ser Cys Val Met 995 1000 1005Lys Gln Thr Gln Tyr Tyr Phe Gly Ser Val Asn Ala Ser Tyr Asn 1010 1015 1020Ala Ile Ile Asp Cys Gly Asn Cys Ser Arg Leu Phe His Ala Gln 1025 1030 1035Arg Leu Thr Asn Thr Asn Leu Leu Phe Val Val Ala Glu Lys Pro 1040 1045 1050Leu Cys Ser Gln Cys Glu Ala Gly Arg Leu Leu Gln Lys Glu Thr 1055 1060 1065His Cys Pro Ala Asp Gly Pro Glu Gln Cys Glu Leu Val Gln Arg 1070 1075 1080Pro Arg Tyr Arg Arg Gly Pro His Ile Cys Phe Asp Tyr Asn Ala 1085 1090 1095Thr Glu Asp Thr Ser Asp Cys Gly Arg Gly Ala Ser Phe Pro Pro 1100 1105 1110Ser Leu Gly Val Leu Val Ser Leu Gln Leu Leu Leu Leu Leu Gly 1115 1120 1125Leu Pro Pro Arg Pro Gln Pro Gln Val Leu Val His Ala Ser Arg 1130 1135 1140Arg Leu 11453598PRTHomo sapiensMISC_FEATURE(1)..(598)channel beta subunit PQ 3Met Val Gln Lys Thr Ser Met Ser Arg Gly Pro Tyr Pro Pro Ser Gln1 5 10 15Glu Ile Pro Met Glu Val Phe Asp Pro Ser Pro Gln Gly Lys Tyr Ser 20 25 30Lys Arg Lys Gly Arg Phe Lys Arg Ser Asp Gly Ser Thr Ser Ser Asp 35 40 45Thr Thr Ser Asn Ser Phe Val Arg Gln Gly Ser Ala Glu Ser Tyr Thr 50 55 60Ser Arg Pro Ser Asp Ser Asp Val Ser Leu Glu Glu Asp Arg Glu Ala65 70 75 80Leu Arg Lys Glu Ala Glu Arg Gln Ala Leu Ala Gln Leu Glu Lys Ala 85 90 95Lys Thr Lys Pro Val Ala Phe Ala Val Arg Thr Asn Val Gly Tyr Asn 100 105 110Pro Ser Pro Gly Asp Glu Val Pro Val Gln Gly Val Ala Ile Thr Phe 115 120 125Glu Pro Lys Asp Phe Leu His Ile Lys Glu Lys Tyr Asn Asn Asp Trp 130 135 140Trp Ile Gly Arg Leu Val Lys Glu Gly Cys Glu Val Gly Phe Ile Pro145 150 155 160Ser Pro Val Lys Leu Asp Ser Leu Arg Leu Leu Gln Glu Gln Lys Leu 165 170 175Arg Gln Asn Arg Leu Gly Ser Ser Lys Ser Gly Asp Asn Ser Ser Ser 180 185 190Ser Leu Gly Asp Val Val Thr Gly Thr Arg Arg Pro Thr Pro Pro Ala 195 200 205Ser Ala Lys Gln Lys Gln Lys Ser Thr Glu His Val Pro Pro Tyr Asp 210 215 220Val Val Pro Ser Met Arg Pro Ile Ile Leu Val Gly Pro Ser Leu Lys225 230 235 240Gly Tyr Glu Val Thr Asp Met Met Gln Lys Ala Leu Phe Asp Phe Leu 245 250 255Lys His Arg Phe Asp Gly Arg Ile Ser Ile Thr Arg Val Thr Ala Asp 260 265 270Ile Ser Leu Ala Lys Arg Ser Val Leu Asn Asn Pro Ser Lys His Ile 275 280 285Ile Ile Glu Arg Ser Asn Thr Arg Ser Ser Leu Ala Glu Val Gln Ser 290 295 300Glu Ile Glu Arg Ile Phe Glu Leu Ala Arg Thr Leu Gln Leu Val Ala305 310 315 320Leu Asp Ala Asp Thr Ile Asn His Pro Ala Gln Leu Ser Lys Thr Ser 325 330 335Leu Ala Pro Ile Ile Val Tyr Ile Lys Ile Thr Ser Pro Lys Val Leu 340 345 350Gln Arg Leu Ile Lys Ser Arg Gly Lys Ser Gln Ser Lys His Leu Asn 355 360 365Val Gln Ile Ala Ala Ser Glu Lys Leu Ala Gln Cys Pro Pro Glu Met 370 375 380Phe Asp Ile Ile Leu Asp Glu Asn Gln Leu Glu Asp Ala Cys Glu His385 390 395 400Leu Ala Glu Tyr Leu Glu Ala Tyr Trp Lys Ala Thr His Pro Pro Ser 405 410 415Ser Thr Pro Pro Asn Pro Leu Leu Asn Arg Thr Met Ala Thr Ala Ala 420 425 430Leu Ala Ala Ser Pro Ala Pro Val Ser Asn Leu Gln Gly Pro Tyr Leu 435 440 445Ala Ser Gly Asp Gln Pro Leu Glu Arg Ala Thr Gly Glu His Ala Ser 450 455 460Met His Glu Tyr Pro Gly Glu Leu Gly Gln Pro Pro Gly Leu Tyr Pro465 470 475 480Ser Ser His Pro Pro Gly Arg Ala Gly Thr Leu Arg Ala Leu Ser Arg 485 490 495Gln Asp Thr Phe Asp Ala Asp Thr Pro Gly Ser Arg Asn Ser Ala Tyr 500 505 510Thr Glu Leu Gly Asp Ser Cys Val Asp Met Glu Thr Asp Pro Ser Glu 515 520 525Gly Pro Gly Leu Gly Asp Pro Ala Gly Gly Gly Thr Pro Pro Ala Arg 530 535 540Gln Gly Ser Trp Glu Asp Glu Glu Glu Asp Tyr Glu Glu Glu Leu Thr545 550 555 560Asp Asn Arg Asn Arg Gly Arg Asn Lys Ala Arg Tyr Cys Ala Glu Gly 565 570 575Gly Gly Pro Val Leu Gly Arg Asn Lys Asn Glu Leu Glu Gly Trp Gly 580 585 590Arg Gly Val Tyr Ile Arg 595

Claims

1. A method for treating amyloidosis active, the method comprising administering an agonist of the P/Q type voltage-gated presynaptic calcium channel, to a subject in need there of.

2. The method of claim 1, wherein the amyloidosis is Alzheimer's disease or Down's Syndrome.

3. The method of claim 1, wherein the agonist binds to the P/Q type voltage-gated presynaptic calcium channel.

4. The method of claim 1, wherein the agonist affects the P/Q type voltage-gated presynaptic calcium channel with an EC50 of less than 120 .mu.M.

5. The method of claim 1, wherein the agonist affects the P/Q type voltage-gated presynaptic calcium channel with an EC50 that is lower than the EC50 with which it affects the N and/or R type presynaptic calcium channel.

6. The method of claim 1, wherein the agonist affects the N and/or R type presynaptic calcium channel with an EC50 of more than 54 .mu.M.

7. (canceled)

8. The method of claim 1, wherein the treatment is for the restoration of synaptic function and/or plasticity.

9. The method of claim 1, wherein the treatment is for the restoration of long-term potentiation.

10. The method of claim 1, wherein the treatment is for the restoration of memory function.

11. The method of claim 1, wherein the treatment is for the restoration of performance of activities of daily living (ADL) capacity in the subject.

12. The method of claim 1, wherein the agonist is a compound having formula selected from the group consisting of: ##STR00009## and a pharmaceutically acceptable salt thereof, wherein if the compound has formula (Ia), then R.sup.1 is hydrogen or C.sub.1-C.sub.6 alkyl; R.sup.2a, R.sup.2b are independently hydrogen, C.sub.1-C.sub.6-alkyl, C.sub.2-C.sub.6-alkenyl, C.sub.3-C.sub.8-cycloalkyl, optionally substituted C.sub.6-C.sub.12-aryl or optionally substituted C.sub.6-C.sub.12-aryl-C.sub.1-C.sub.4-alkyl, or R.sup.2a, R.sup.2b together are C.sub.2-C.sub.5-alkylene; Q is NR.sup.3; R.sup.3 is hydrogen, C.sub.1-C.sub.6-alkyl or optionally substituted C.sub.6-C.sub.12-aryl; X is N or CR.sup.4; R.sup.4 is hydrogen or C.sub.1-C.sub.6-alkyl, Y is N or CR.sup.5; and R.sup.5 is hydrogen or C.sub.1-C.sub.6-alkyl; wherein if the compound has formula (Ib), then R.sup.1 hydrogen or C.sub.1-C.sub.6 alkyl; R.sup.2a, R.sup.2b are independently hydrogen, C.sub.1-C.sub.6-alkyl, C.sub.2-C.sub.6-alkenyl, C.sub.3-C.sub.8-cycloalkyl, optionally substituted C.sub.6-C.sub.12-aryl or optionally substituted C.sub.6-C.sub.12-aryl-C.sub.1-C.sub.4-alkyl, or R.sup.2a, R.sup.2b together are C.sub.2-C.sub.5-alkylene; Q is NR.sup.3; R.sup.3 is hydrogen, C.sub.1-C.sub.6-alkyl or optionally substituted C.sub.6-C.sub.12-aryl; X is N or CR.sup.4; R.sup.4 is hydrogen or C.sub.1-C.sub.6alkyl, Y is N or CR.sup.5; and R.sup.5 is hydrogen or C.sub.1-C.sub.6-alkyl; and wherein if the compound has formula (II), then R.sup.1 is C.sub.1-C.sub.6-alkyl or C.sub.3-C.sub.8cycloalkyl; R.sup.2a, R.sup.2b, R.sup.2c, R.sup.2d, R.sup.2e are independently hydrogen, halogen, C.sub.1-C.sub.4-alkyl, optionally substituted phenyl, OH, SH, CN, CF.sub.3, O--CF.sub.3, C.sub.1-C.sub.4-alkoxy; NH.sub.2, NH--C.sub.1-C.sub.4-alkyl, alkyl).sub.2, or R.sup.2b and R.sup.2c or R.sup.2c and R.sup.2d together with the carbon atoms to which they are attached form an optionally substituted anellated C.sub.5-C.sub.7 carbocyclic ring; and the pharmacologically useful salts thereof.