Patent Application
20100311767
The present invention relates to a method for the treatment of an amyloidosis such as Alzheimer's disease.
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 β peptides (Aβ) which arise from the (β-amyloid precursor protein (APP) by proteolytic cleavage.
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”.
Recently, however, it was shown that in amyloidoses soluble, globular Aβ oligomers (hereinafter referred to as Aβ 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).
The term “Aβ globulomer” here refers to a particular soluble, globular, non-covalent association of Aβ peptides, possessing homogeneity and distinct physical characteristics. Aβ globulomers are stable, non-fibrillar, oligomeric assemblies of Aβ peptides which are obtainable by incubation with anionic detergents, in particular as described in WO2004/067561. In contrast to Aβ 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 Aβ oligomers.
Soluble Aβ oligomer was found to impair the functioning of the central nervous system even before the onset of cytotoxicity. However, the exact mechanisms whereby soluble Aβ 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.
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.
Surprisingly, it was now found that Aβ 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 Ca2+ concentration (P/Q type presynaptic calcium channel agonism) was effective in reversing the inhibitory effect of Aβ globulomer on the P/Q type voltage-gated presynaptic calcium chnnel).
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.
The P/Q type voltage-gated presynaptic calcium channel (the channel is also referred to as Cav2.1 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.
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 ω-agatoxin IVA (a well-known funnel web spider venom).
According to a particular embodiment, ω-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:
The P/Q type voltage-gated presynaptic calcium channel according to the present invention comprises an α1 subunit. According to a particular embodiment of the invention, the α1 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 α1 subunit incorporates the conduction pore, the voltage sensor and gating apparatus, and sites of channel regulation by second messengers, drugs, and toxins.
Usually, the P/Q type voltage-gated presynaptic calcium channel also comprises an α2-δ subunit and a β subunit. It may also comprise an γ subunit. In a particular embodiment of the invention, the α2-δ 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 β 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.
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.
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.
According to a particular embodiment, the agonist of the invention increases the open probability of the channel.
According to a further particular embodiment, the agonist of the invention directly interacts with the closed channel to open it.
According to a further particular embodiment, the agonist of the invention increases the duration of the open state once the channel has been opened.
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.
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).
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.
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.
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.
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.
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.
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.
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 μM, preferably of less than 10 μM, and in particular of less than 1 μM. 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.
The standard method employed here for all determinations of Ca++ currents is a patch-clamp method using 120 mM NMG.Cl, 10 mM TEA.Cl, 14 mM creatine phosphate, 6 mM MgCl2, 1.mM CaCl2 10 mM NMG.HEPES, 5 mM Tris2.ATP and 11 NMG2.EGTA as internal, and 30 mM BaCl2, 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 α2δ and βIB components of the P/Q type voltage-gated presynaptic calcium channel.
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.
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 Aβ, in particular to Aβ 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++ 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 μM, preferably of more than 100 μM, and in particular of more than 1000 μM, 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 μM, preferably of more than 100 μM, and in particular of more than 1000 μM, 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 μM, preferably of more than 100 μM, and in particular of more than 1000 μM, using the standard method as defined above.
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 μM, preferably of more than 10 μM, and in particular of more than 100 μM. 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 μM, preferably of more than 10 μM, and in particular of more than 100 μM. Preferably, the agonist has an IC50 for any CDK, in particular for any human CDK, of more than 0.5 μM, preferably of more than 10 μM, and in particular of more than 100 μM. These IC50 values all refer to the activity of the respective CDK/cyclin complex in a radioactive kinase assay using 15 μM of [γ-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 MgCl2 and 1 mM dithiotreitol, as described by Meijer et al., Eur. J. Biochem. 234: 527-536 (1997).
The metabolism of APP and its products such as Aβ is complex and not yet fully understood. Therefore, in a particular embodiment of the invention, the agonist as such does not after APP expression, Aβ formation or processing, e. g. soluble Aβ oligomer or Aβ 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 Aβ 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.
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.
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.
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):
wherein
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):
wherein
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):
wherein
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.
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, C1-C4-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.
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 Cn-Cm indicates in each case the possible number of carbon atoms in the group.
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, C1-C4-alkyl, OH, SH, CN, CF3, O—CF3, C1-C4-alkoxy, NH—C1-C4-alkyl, N—(C1-C4-alkyl)2, in particular with 1, 2 oder 3 substituents selected from the group consisting of halogen, methyl, OH, CN, CF3, O—CF3, methoxy, NH2, NH—CH3, and N—(CH3)2.
The term halogen denotes in each case fluorine, bromine, chlorine or iodine, in particular fluorine or chlorine.
C1-C4-Alkyl is a straight-chain or branched alkyl group having from 1 to 4 carbon atoms. Examples of an alkyl group are methyl, C2-C4-alkyl such as ethyl, n-propyl, iso-propyl, n-butyl, 2-butyl, iso-butyl or tert-butyl. C1-C2-Alkyl is methyl or ethyl, C1-C3-alkyl is additionally n-propyl or isopropyl.
C1-C6-Alkyl is a straight-chain or branched alkyl group having from 1 to 6 carbon atoms. Examples include methyl, C2-C4-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.
C3-C8-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 C1-C4 alkyl radicals, preferably one or more methyl radicals.
C6-C12-aryl-C1-C4-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 C6-C12-aryl, such as in benzyl.
C2-C6-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. C3-C4-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.
C6-C12-Aryl is a 6- to 12-membered, in particular 6- to 10-membered, aromatic cyclic radical. Examples include phenyl and naphthyl.
C2-C7-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.
Particular roscovitine analogues are defined as follows.
According to a particular embodiment, R1 in formula (Ia) and formula (Ib) is hydrogen or C1-C3-alkyl. C1-C3-alkyl is in particular ethyl or isopropyl.
According to a further particular embodiment, R2a and R2b in formula (Ia) and formula (Ib) are independently hydrogen, C1-C3-alkyl, in particular ethyl, C2-C3-alkenyl, in particular allyl, C3-C8-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 NH2. According to a further particular embodiment, R3 is hydrogen and R2b is as defined. Alternatively, R2a and R2b in formula (Ia) and formula (Ib) together are C2-C7-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.
According to a further particular embodiment, R3 in formula (Ia) and formula (Ib) is C1-C6-alkyl, in particular C1-C3-alkyl, or optionally substituted C6-C12-aryl, in particular optionally substituted phenyl.
According to a further particular embodiment of formula (Ia), X is N, Y is CR5 (in particular CH) and Q is NR3 (in particular N(C1-C3-alkyl), e.g. NCH3, NCH2CH3 or NCH(CH3)2, or N(phenyl)); or X is CR4 (in particular CH), Y is N and Q is NR3 (in particular N(C1-C3-alkyl) or N(phenyl)).
According to a further particular embodiment of formula (Ib), X is N, Y is CR5 (in particular CH) and Q is NR3 (in particular NCH3).
Roscovitine analogues according to the present invention, in particular, include the following compounds
and their pharmacologically useful salts.
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 Aβ globulomer on synaptic transmission.
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 Aβ globulomer on spontaneous synaptic activity, which is mediated by suppression of the P/Q type voltage-gated presynaptic calcium channel.
Particular isopreterenol analogues are defined as follows.
According to a further particular embodiment, R1 in formula (II) is C1-C4-alkyl or C3-C6-cycloalkyl. C1-C4-alkyl is in particular methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl. C3-C6-cycloalkyl is in particular cyclopropyl or cyclohexyl.
According to a further particular embodiment, at least one, two or three of R2a, R2b, R2c, R2d, R2e in formula (II) is/are different from hydrogen. In particular at least one of R2a, R2b, R2c, R2d is different from hydrogen.
According to a further particular embodiment, R2a, R2b, R2c, R2d, are independently hydrogen, halogen, methyl, optionally substituted phenyl, OH, methoxy, CN, or NH2, or R2b and R2c or R2c and R2d 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 R2b, R2c and R2d are attached) or aromatic. According to a particular embodiment, R2b and R2c or R2c and R2d together with the benzene moiety to which they are attached form an optionally substituted 1,2,3,4-tetrahydronaphthalene or naphthalene moiety.
Isopreterenol analogues according to the present invention, in particular, include the following compounds
and their pharmacologically useful salts.
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.
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++ flux through the P/Q type voltage-gated presynaptic calcium channel. Suitable means for determining ion fluxes such as Ca++ 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).
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
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.
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 ω-agatoxin, or pharmacological characterization, i. e. reduction of calcium current after agatoxin application.
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++, 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.
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
indicate calcium fluxes through the P/Q type voltage-gated presynaptic calcium channel. Therefore, a rise in said increase
where [Ca++]C is the intracellular calcium concentration in the cell in the presence and [Ca++]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.
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.
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++ flux when only the P/Q type voltage-gated presynaptic calcium channel is expressed, or when all other calcium channels are blocked.
As all these methods for the determination of Ca++ 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.
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++ 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.
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++ fluxes disclosed above in combination with known blockers for non-N type voltage-gated presynaptic calcium channels.
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.
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.
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.
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.
In a particular embodiment of the invention, the treatment is a rehabilitating and/or symptomatic treatment.
A “rehabilitating” treatment, as used herein, is, in particular, for providing a benefit with regard to the patient's overall quality of life.
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).
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.
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.
A “symptomatic” treatment, as used herein, is, in particular, a treatment directed to the abatement or relief of the symptoms of the disease.
In a particular embodiment the present invention relates to a method for the restoration of Aβ-impaired synaptic function and/or plasticity, in particular long-term potentiation, in the subject.
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.
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.
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.
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).
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.
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”, “Aβ1-42”, “Aβ”, “aβ”, “glob” all denote the Aβ(1-42) globulomer described in reference example 2. “Kontrolle” means “control”.
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++ 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.
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 CaCl2, 1 mM MgCl2, 16.5 mM glucose and 10 mM HEPES at a pH of 7.3. The osmolarity of the solution was 330 mosmol.
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 MΩ. The intracellular solution consisted of either (for recordings of miniature PSCs) 100 mM KCl, 10 mM NaCl, 0.25 mM CaCl2, 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.
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.
All recordings of miniature PSCs were made in the presence of 0.5 μM tetrodotoxin (TTX; available from Tocris Bioscience) to block the Na+ and K+ 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+-channels) 10 mM BaCl2, 0.5 μM TTX, 10 mM HEPES and 20 mM glucose at a pH 7.3. When required, ω-conotoxin MVIIA (available from Alomone Labs, Jerusalem, Israel) was added to a final concentration of 0.5 μM to block N type voltage-gated presynaptic Ca++ 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 μM nifedipine.
To mimic the effect of Aβ globulomer as P/Q type blocker, ω-agatoxin IVA (available from Alomone Labs, Jerusalem, Israel) was added to a final concentration of 0.5 μM to specifically block the P/Q type voltage-gated presynaptic Ca++ channels of the sample cell.
All substances were stored as lyophilized powders at −20 ° 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 Aβ-globulomer solvent buffer which was applied to neurons was <1%0 and the final concentration of DMSO was <1.5%o.
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.
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.
The sPSCs or mPSCs were then recorded for 10 minutes giving the control values before any toxins were added.
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).
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.
An Aβ(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, Aβ 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.
Using acute application of the P/Q channel blocker ω-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 Aβ(1-42) globulomer on the frequency of spontaneous synaptic events in cultured hippocampal neurons treated with ω-conotoxin to achieve synaptic dominance of the P/Q type channel, as described in Reference Example 1, were observed.
Aβ 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 ω-agatoxin. In the presence of ω-agatoxin, Aβ globulomer had no further effect on synaptic activity, indicating that the effects of both agents involved a common mechanism. A total of 200 μl Aβ-globulomer solvent buffer comprising a Aβ(1-42) globulomer concentration corresponding to approximately 2 μM of Aβ monomer was added to the bath (previous volume 200 μl), resulting in a final Aβ(1-42) globulomer concentration corresponding to approximately 1 μM of Aβ monomer. Based on the assumption that the Aβ(1-42) globulomer consists of 12 Aβ(1-42) monomers a final Aβ(1-42) globulomer concentration of approximately 83 nM can be calculated. Measurements of synaptic activity were then taken.
Results are shown in
These results were verified by subjecting the Aβ(1-42) globulomer containing Aβ-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 Aβ globulomer protein prior to bringing it into contact with the cells. The ultrafiltrate had no effect on the synaptic events (see
Furthermore, the effect of Aβ(1-42) globulomer is absent in cells predominantly expressing presynaptic N-type calcium channels. Results are shown in
Using the Aβ(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 Aβ globulomer-induced reduction of the frequency of spontaneous synaptic events in cultured hippocampal neurons treated with ω-conotoxin, as described in Reference Example 1, were observed.
Roscovitine was used at a final concentration of 20 μM, by adding it simultaneously with Aβ(1-42) globulomer (final concentration of Aβ globulomer corresponding to approximately 1 μM of Aβ 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.
Results are shown in
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:
The Ba++ also served as the charge carrier (i. e. substrate replacement) for the P/Q type voltage-gated presynaptic Ca++ 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.
In order to prevent the “run down” of Ca++ 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.
Using ω-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 Aβ(1-42) globulomer of Reference Example 2 (at a concentration corresponding to approximately 1 μM of Aβ(1-42) monomers) on calcium flux in hippocampal neurons treated with ω-conotoxin were directly observed as described in Reference Example 5.
Recordings were taken at 10 min and 15 min and optionally after a washout. Typical results are shown in
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.
400 μm 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 Aβ(1-42) globulomer via an Eppendorff pipette.
Typical results are shown in
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 (˜4° 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° C. in an incubator gassed with 5% CO2 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 μM/L ω-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.
Currents were measured under whole-cell voltage-clamp conditions at room temperature using borosilicate pipettes of 2-4 MΩ resistance. Electrode solution contained (in mM/l): NaCl 10, KCl 100, CaCl2 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, CaCl2 0.5, MgCl2 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, CaCl2 2, MgCl2, Glucose 16.5, HEPES 10 (pH set to 7.3) for recordings of synaptic events and TEA-Cl 140, BaCl2, 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 Aβ(1-42) globulomer and baseline activity was recorded. Subsequently, Aβ(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, ω-agatoxin IVA, ω-conotoxin MVIIA, roscovitine (Alomone Labs, Jerusalem, Israel), and nifedipine (Sigma, Deisenhofen, Germany) were added directly to the bath solution at the concentrations indicated.
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 (˜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.
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):
where gmax is the maximal membrane conductance (which is proportional to the maximal permeability and the extracellular concentration of barium), Vh is the potential of half maximal activation and Vc is proportional to the slope of the curve at Vh. F represents the Faraday constant, R the gas constant, P0 is the maximal permeability, and T the absolute temperature. The intracellular concentration of Ba2+ was assumed to be 0.01 μM. Assuming higher values of up to 0.1 mM did not significantly change the resulting values of the parameters.
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:
where N(V) is the level of steady state inactivation determined from the current amplitude I(V) normalized to Imax, V is the pre-pulse potential, Vh is the potential of half maximal inactivation and Vc is a factor proportional to the slope of the curve at Vh.
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 (ECl˜−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.
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.
Spontaneous synaptic was measured activity in cultured hippocampal neurons using whole-cell voltage clamp techniques (Vhold=−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 Aβ(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;
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 Aβ(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 Aβ(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 Aβ(1-42) globulomer respectively. It thus appears that the alteration of synaptic activity by Aβ(1-42) globulomer may be caused by a direct interaction with pre- or postsynaptic proteins, rather than by an unspecific alteration of cellular excitability.
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 Aβ(1-42) globulomer (yielding 56±9% of control; p<0.05;
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 μM) and DL-APV (30 μM) to the bath solution. The frequency of spontaneously occurring IPSCs was suppressed by Aβ(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 μM TTX) did also show a similar reduction of frequency after application of Aβ(1-42) globulomer (52±10% of control; p<0.05; n=6). This effect was partially reversible upon washout, yielding 68±12% of control (
In order to test for potential effects of Aβ(1-42) globulomer on postsynaptic GABAA receptors, a high (100 μM) 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 Aβ(1-42) globulomer for 5 min, indicating that GABAA receptors are not affected by the agent (
Finally, excitatory synaptic currents (EPSCs) were isolated in the presence of 5 μM gabazine (a GABAA receptor antagonist). Basal frequency of these events was 386±124/min. Their frequency was reduced by Aβ(1-42) globulomer to 14±4% of control (p<0.05; n=6;
Presynaptic vesicle release is triggered by an influx of calcium into the presynaptic terminal. Therefore, Aβ(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 Aβ(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 Ba2+ was used as charge carrier in the extracellular solution (see methods). Measurements were performed in the presence of 10 μM nifedipine (a L-type calcium channel blocker), ω-conotoxin MVIIA (a N-type calcium channel blocker) and blockers of other voltage-gated ion channels (TTX 0.5 μM, TEA 140 mM, Cs+-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;
If the effect of Aβ(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 ω-Agatoxin IVA. Indeed, this toxin (0.5 μM) reduced the frequency of miniature PSCs to 27±7% (n=3; amplitude 90±7%), similar to the effect of Aβ(1-42) globulomer. Following pre-incubation with ω-Agatoxin IVA, Aβ(1-42) globulomer had no additional effect on the synaptic currents (n=6, frequency 108±15%; amplitude 102±7% of currents after ω-Agatoxin IVA control;
The effect of Aβ(1-42) globulomer on P/Q-type calcium currents was further characterized by steady-state activation and -inactivation protocols (see methods). Maximal conductance (gmax) was 61.7±2.4 nS (control) versus 27.2±3.2 nS (Aβ(1-42) globulomer; p<0.05; n=6;
In addition the effects of Aβ(1-42) globulomer on N- and L-type calcium currents were analyzed. Besides blockers for Na+- and K+-channels (see above) 0.5 μM ω-agatoxin IVA were added to block P/Q-channels. L-type calcium currents were isolated by addition of 0.5 μM ω-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 Aβ(1-42) globulomer (573.0±225.6 pA; n=3). When N-type currents were isolated by adding nifedipine (10 μM) instead of ω-conotoxin, the same voltage clamp protocol elicited inward currents which were, again, insensitive to Aβ(1-42) globulomer (amplitude in control solution 1368.9±332.8; amplitude in Aβ(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.
Application of roscovitine in the presence of Aβ(1-42) globulomer did indeed partially recover the frequency of synaptic currents. While in these experiments Aβ(1-42) globulomer reduced the frequency of spontaneous PSCs to 38±10% of control, application of roscovitine (20 μM) brought this parameter back to 75±13% (n=5;
Together, these data indicate that Aβ(1-42) globulomer reduces the frequency of spontaneous and miniature synaptic currents by suppression of presynaptic calcium influx via P/Q-type calcium channels.
Isoproterenol was used at a final concentration of 15 μM, by adding it simultaneously with Aβ(1-42) globulomer (final concentration of Aβ globulomer corresponding to approximately 1 μM of Aβ 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 Ca2+ influx through the P/Q type voltage-gated presynaptic calcium channel.
Application of isoproterenol in the presence of Aβ(1-42) globulomer did indeed recover the frequency of synaptic currents. While in these experiments Aβ(1-42) globulomer reduced the frequency of spontaneous IPSCs to 57±7% of control, application of isoproterenol (15 μM) brought this parameter back to 122±58% (n=6;
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 Aβ globulomer to that of untreated cells, i. e., that the P/Q activator may be used to reverse the detrimental effects of Aβ globulomer.
Rat hippocampal slice cultures (9 days old Wistar rats; 15-17 DIV) were incubated over night with either Aβ(1-42) globulomer (at a concentration corresponding to approximately 1 μM of Aβ monomer), Aβ(1-42) globulomer (at a concentration corresponding to approximately 1 μM of Aβ monomer)+20 μM roscovitine, or control (SDS). Recordings were performed (in artificial cerebrospinal fluid) from CA1 stratum radiatum after stimulation of the Schaffer collateral at different intensities.
Results are shown in
Rat hippocampal slice cultures (11 days old Wistar rats; 23-24 DIV) were incubated over night with either Aβ(1-42) globulomer (at a concentration corresponding to approximately 1 μM of Aβ monomer), Aβ(1-42) globulomer (at a concentration corresponding to approximately 1 μM of Aβ monomer)+20 μM 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.
Results are shown in
Spontaneous synaptic activity was measured in cultured hippocampal neurons using whole-cell voltage clamp techniques (Vhold=−70 mV). Under the ionic conditions used (ECl˜−10 mV) all synaptic events appeared as inward currents.
The effects of Aβ(1-42) globulomer (at a concentration corresponding to approximately 1 μM of Aβ 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 Ca2+. 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 Ca2+ 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.
Basal frequency of sPSCs in 1 mM Ca2+ was 4.2±1.2 Hz. Bath-application of Aβ(1-42) globulomer rapidly reduced the sPSC frequency to 63±7% of control (p<0.05; n=6;
Median amplitude of sPSCs under control conditions was 27.7±2.2 pA and remained unaltered after addition of Aβ(1-42) globulomer (97±5%;
In most cases, prominent currents with amplitudes up to 2000 pA occurred directly after elevation of extracellular Ca2+-concentration. These currents with multiple peaks (see
This clearly demonstrates that the principle of activating the P/Q type presynaptic calcium channel is effective in compensating the detrimental effects exerted by Aβ globulomer.
| Number | Date | Country | Kind |
|---|---|---|---|
| 070202577 | Oct 2007 | EP | regional |
| 080003254 | Jan 2008 | EP | regional |
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.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2008/001549 | 2/27/2008 | WO | 00 | 7/28/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60903700 | Feb 2007 | US |
