WO2007062862A2

WO2007062862A2 - Use of calmodulin inhibitors for the treatment of neurodegenerative disorders

Info

Publication number: WO2007062862A2
Application number: PCT/EP2006/011553
Authority: WO
Inventors: Stefan Lichtenthaler; Stefan Mitterreiter
Original assignee: Ludwig Maximilians Universität München
Priority date: 2005-12-02
Filing date: 2006-12-01
Publication date: 2007-06-07
Also published as: WO2007062862A3

Abstract

The present invention relates to the use of a calmodulin inhibitor/antagonist, like bepridil, phenoxybenzamine, cetiedil, chlorpromazine or W7, for the preparation of a pharmaceutical composition for the treatment, amelioration and/or prevention of neurodegenerative disorders. Also means and methods for the prevention, amelioration and/or treatment of neurodegenerative disorders are described, wherein said calmodulin inhibitor/antagonist is to be administered to a subject in need of such a an prevention, amelioration and/or treatment. Preferably said subject is human and said calmodulin inhibitor/antagonist to be employed in the uses and methods of the invention is bepridil (or a pharmaceutically acceptable salt thereof).

Description

Use of calmodulin inhibitors for the treatment of neurodegenerative disorders

The present invention relates to neurodegenerative disorder, like Alzheimer's disease. Particularly the present invention relates to the use of a calmodulin inhibitor, like bepridil, phenoxybenzamine, cetiedil, chlorpromazine or W7, for the preparation of a pharmaceutical composition for the treatment, amelioration and/or prevention of neurodegenerative disorders, like Alzheimer' disease. As documented herein and in particular in the appended examples, the calmodulin inhibitor/antagonist to be employed in the uses and methods of the invention is in one embodiment bepridil (or a pharmaceutically acceptable salt thereof).

Alzheimer' disease (AD) is the most frequent cause of death in the industrialized countries besides cardiovascular diseases and cancer. AD (used herein as abbreviation of the term "Alzheimer' disease") affects roughly one million people in Germany and about 4 million in the US. About 70% of all cases of dementia are due to Alzheimer's disease which is associated with selective damage of brain regions and neural circuits critical for cognition. Alzheimer's disease is characterized by neurofibrillary tangles in particular in pyramidal neurons of the hippocampus and numerous amyloid plaques containing mostly a dense core of amyloid deposits and defused halos.

The extracellular neuritic plaques contain large amounts of a pre-dominantly fibrillar peptide termed "amyloid β", "A-beta", "Aβ4", "β-A4" or "Aβ"; see Selkoe (1994), Ann. Rev. Cell Biol. 10, 373-403, Koo (1999), PNAS Vol. 96, pp. 9989-9990, US 4,666,829 or Glenner (1984), BBRC 12, 1131. This amyloid β is derived from "Alzheimer precursor protein/β-amyloid precursor protein" (APP). APPs are integral membrane glycoproteins (see Sisodia (1992), PNAS Vol. 89, pp. 6075) and are endoproteolytically cleaved within the Aβ sequence by a plasma membrane protease, α-secretase (see Sisodia (1992), loc. cit.). Furthermore, further secretase activity, in particular β-secretase and γ-secretase activity leads to the extracellular release of amyloid-β (Aβ) comprising either 39 amino acids (Aβ39), 40 amino acids (Aβ40), 42 amino acids (Aβ42) or 43 amino acids (Aβ43); see Sinha (1999), PNAS 96, 11094- 1053; Price (1998), Science 282, 1078 to 1083; WO 00/72880 or Hardy (1997), TINS 20, 154.

It is of note that Aβ has several naturally occurring forms, whereby the human forms are referred to as the above mentioned Aβ39, Aβ40, Aβ41 , Aβ42 and Aβ43. The most prominent form, Aβ42, has the amino acid sequence (starting from the N- terminus): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGWIA (SEQ ID NO: 1). Since Aβ42 is the most prominent form, the term "Aβ" as employed in context of this invention refers in particular to this form. Yet, also the other naturally occurring isoforms are comprised in the term "Aβ", for example the Aβ41-, Aβ40- or Aβ39-form, wherein the C-terminal amino acids A, IA and VIA are missing, respectively. Also comprised as "Aβ" is for example, the Aβ43-form which has an additional threonine residue at the C-terminus of the above depicted sequence.

As pointed out above, the pathogenesis of AD starts with the generation of the amyloid β peptide (Aβ), in particular in the most prominent form, Aβ42. Two proteases cleave Aβ out of the much larger amyloid precursor protein (APP) (Fig. 1). Subsequently, Aβ forms aggregates, which are neurotoxic and lead to nerve cell loss in the brain and finally to death (Selkoe and Schenk, 2003). Currently, there are often only drugs available that ameliorate the symptoms of Aβ related disorders, like Alzheimer's disease (AD). Hardly any compounds interfering with the disease- causing process are known. One attempt to medically interfere with Aβ generation/plaque formation and/or related Aβ pathologies were immunization approaches. For example, Vaccination of transgenic mice overexpressing mutant human APP WI7F (PDAPP mice) with Aβ1-42 resulted in an almost complete prevention of amyloid deposition in the brain when treatment was initiated in young animals, i. e. before the onset of neuropathologies, whereas in older animals a reduction of already formed plaques was observed suggesting antibody-mediated clearance of plaques (Schenk et al., (1999), Nature 400,173-177). Also passive immunization has been described, have confirmed that antibodies can enter the central nervous system, decorate plaques and induce clearance of preexisting amyloid plaques in APP transgenic mice ( e.g. PDAPP mice) (Bard et al., (2000) Nat. Med. 6, 916-919; WO 00/72880). However, these vaccination approaches have been of limited success since microbleeding and corresponding negative side-effects have been noticed; see Pfeifer (2002). Science, 298, p.1379.

Thus, further efforts are required in the development of means and methods for stopping the first steps in the APP pathology process, namely the generation and aggregation of Aβ.

The APP is a membrane protein and consists of a large extracellular domain, a transmembrane and a cytoplasmic domain (Fig. 1). The two proteases, which cleave APP and generate Aβ, are referred to as β- and γ-secretase (Fig. 1). β-secretase cleaves first, γ-secretase cleaves second. Alternatively, α-secretase cleaves APP within the Aβ-domain and thus precludes Aβ-generation. Pathological APP processing and/or the generation of extracellular plaques containing proteinaceous depositions are not only known from Alzheimer's pathology but also from subjects suffering from other neurological and/or neurodegenerative disorders. These disorders comprise, inter alia, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson's disease, ALS (amyotrophic lateral sclerosis), Creutzfeld Jacob disease, HIV-related dementia and motor neuropathy Therapeutically it would be highly desirable to inhibit β- or γ-secretase, either by directly targeting these enzymes or by indirectly targeting them (e.g. targeting modulators of their activity). Pharmaceutical companies have put much effort into developing specific inhibitors of β- and γ-secretase - without success for many years. Difficulties in developing β- and γ-inhibitors are multiple, β-secretase seems to be an ideal drug target (the knock-out mouse suggests that there are no major unwanted side-effects), but has a large active site. Many compounds that inhibited this enzyme were simply too large (molecular weight > 500 Dalton) to be used for drug development. For γ-secretase, the situation was the opposite. Although many small molecule inhibitors of γ-secretase were rapidly identified, these compounds seem to have severe side effects as γ-secretase is required for the cleavage of many other membrane proteins. One of them is Notch that is required during embryonic development as well as during T-cell maturation and in the gastro-intestinal tract of adults. Thus, it is crucial to provide means and methods for effective enzyme inhibition.

Thus, the technical problem underlying the present invention is the provision of reliable means and methods to treat, ameliorate or prevent AD.

The solution to the above technical problem is achieved by providing the embodiments characterized in the claims.

The present invention solves the above identified technical problem since, as documented herein below and in the appended examples, calmodulin inhibitors, e.g. bepridil, trifluoperazine, chlorpromazine, cetiedil or W7, were surprisingly identified as inhibitors of the AD β-secretase cleavage. In particular, it was found that calmodulin inhibitors, e.g. bepridil, inhibit the AD β-secretase cleavage in a dose-dependent manner. An inhibition of this enzyme activity is a key goal in therapeutic approaches against AD. Thus, it is proposed to use calmodulin inhibitors, e.g. bepridil, trifluoperazine, chlorpromazine, cetiedil, W7, zaldaride maleate (also known in the art as CGS 9343B), promazine, desipramine, flunarizine, or promethazine, to treat neurodegenerative disorders, in particular AD.

Particularly, it is intended in context of the present invention to use calmodulin inhibitors/antagonists for the preparation of a pharmaceutical composition for the treatment, amelioration and/or prevention of neurodegenerative disorders, in particular disorders that show a linkage to modified/pathological APP processing, like Down's syndrome and in particular AD. Therefore, the present invention provides for the use of a calmodulin inhibitor/antagonist for the preparation of a pharmaceutical composition for the treatment, amelioration and/or prevention of a neurodegenerative disease.

In the prior art, folic acid was proposed to be used for the treatment of stroke and AD (US 6,369,058). However, it was also demonstrated in the prior art the folate/folic acid does not seem to protect against AD (Morris (2006)) and may even be contra- indicated (Schneider (2006)). Moreover, in US 6,369,058 the use of calcium channel antagonists like, inter alia, bepridil was supposed for the amelioration of symptoms after stroke or ischemia. In US 6,369,058, bepridil was not mentioned in context of AD.

By contrast, in context of the present invention, bepridil is described being a calmodulin inhibitor, particularly useful in the therapeutic intervention of AD.

A person skilled in the art is able to test whether a certain compound acts as a calmodulin inhibitor. Test systems for calmodulin activity of certain compounds are known in the art. For instance, such test systems are described in Agre (1984; Binding of ¹²⁵l-Calmodulin to erythrocyte membranes,), ltoh (1986; Competition experiment, which measures, whether novel compounds competes with ³H bepridil for calmodulin binding, Myosin light chain kinase activity), Roberson (2005; Inhibition of Gonadotropoin-releasing hormone induction of the kinase ERK) and Kahn (1998; Calmodulin inhibitors should induce the proteolytic cleavage of L-selectin (as measured by Western Blot or by FACS)). In terms of the present invention inhibition of myosin light chain kinse (ltoh et al., loc.cit) is preferred. More details on useful test systems are given herein below.

Accordingly, the person skilled in the art is readily in a position to elucidate by means and methods known in the art whether a given compound is a calmodulin inhibitor/antagonist. In context of this is invention, it is of note that the term "calmodulin inhibitor" is employed as a synonym for "calmodulin antagonist".

The calmodulin inhibitors/antagonists to be employed in the means and methods of the present invention are capable of stabilizing membrane bound APP and/or inhibiting the release of soluble APP in vivo as well in vivo.

A person skilled in the art is readily in a position to test whether a certain compound, for example a calmodulin inhibitor, acts as an inhibitor of APP β-secretase cleavage and Aβ generation.

For example, the compound to be tested can be administered to a suitable animal model for AD, such as a model from mice, rats or guinea pigs, by oral or nasal route or by injection (for example i.p. or i.v.). Other routes of administration are also possible and are well known in the art. At different time points after administration of the compound, such as 4h and/or 8h, blood or CSF can be taken or the whole brain can be isolated. Detection and quantification of Aβ or APPsβ can be carried out by ELISA or by immunoblot (as, for example, described in the appended examples) or by immunoprecipitation followed by immunoblot (as also described in the appended examples). A reduction of APPsβ and Aβ indicates that the tested compound is an inhibitor of β-secretase cleavage and, accordingly, might be useful for lowering Aβ generation in vivo.

An alternative test, whether a certain compound can act as an inhibitor of APP β- secretase cleavage and Aβ generation, comprises repeated administration of the compound to be tested chronically over a certain period of time. For example such repeated administration may be once or several times per month over a period of one or several month. After that repeated administration, the reduction of plaque numbers or plaque size or a reduction of Aβ burden in the brain can be tested. A reduction in any of these parameters indicates that the compound might be useful for lowering Aβ generation in vivo. For example, such an alternative test system as described above has recently been published in McLaurin (2006).

Methods to test whether a certain compound, for example a calmodulin inhibitor, inhibits the cleavage of γ-secretase are also known in the art. For example, such methods take advantage of mouse models, like those described in Anderson (2005) and Dovey (2001).

Several animal models for AD, suitable to be employed in test systems for APP β- secretase cleavage inhibition and Aβ generation, are also known in the art. For example, guinea pigs, suitable as animal models to test compounds for a reduction of Aβ-production and for changes in APP secretion by α- or β-secretase, are described in Fassbender (2001). Moreover, as such above-mentioned suitable animal models for AD, the in vivo models useful in testing the influence of a given calmodulin antagonist on APP processing described herein below may also be employed.

As shown in the appended examples, it was surprisingly found that calmodulin inhibitors also interfere with pathological APP processing. As a test system, whether a given or a deduced calmodulin antagonist can be employed in the medical intervention of neurodegenerative disorders, in vitro test systems, as provided by Lichtenthaler (2003) may, inter alia, be employed. The Lichtenthaler system is described in detail in the appended examples and is based on the use of stably transfected COS7 or transfected human embryonic kidney 293 cells, i.e. suitable cells transfected with APP695. Also common neuro-cell lines may be employed, like neuroglioma cell lines, e. g. H4 (see also appended examples) However, also other in vitro systems may be employed, which may comprise, but are not limited to 293 cells, COS7 cells, CHO cells, SH-SY5Y cells, N2a cells expressing endogenous APP or transfected with different APP isoforms, such as APP695 APP751 or APP770, or primary neurons.

However, also in vivo models are useful in testing the influence of a given calmodulin antagonist on APP processing. Said in vivo models comprise, but are not limited to animal models, like mouse models or guinea pig models, which comprise mutations in genes relevant in the pathogenesis of amyloid disorders. Such models are e.g. presenilin mutations, like the PS1 model , as described in Borchelt (1998), Neurobiol Aging 19, S15-S18. Also useful are in vivo models which express heterologously human APP, either normal APP or modified/mutated APP, like APP of the "Swedish" or the "London"-type. Corresponding animal models are well known in the art; see e.g the gene-targeted mice bearing the Swedish familial Alzheimer's disease mutations and a "humanized" Aβ sequence as described by Reaume (1996) J Biol Chem 271 ,233380, the APP-model ("Swedish") as described in WO95/11968, the APP23 mice, Swedish double mutation, as described by Gartner (2003) Acta Neuropathol 106, 535-544. Further useful models comprise: the PDAPP mouse model expressing human APP with the mutation V717F (Games et al. 1995 Nature 373: 523-527), the Tg2576 mouse model expressing human APP with the Swedish mutation (Hsiao et al. 1996 Science 274: 99-102) or the TgCRNDδ mouse model expressing human APP with the Swedish mutation and the V717I mutation (Chishti et al. 2001 J Biol Chem 276: 21562-21570) or the J20 mouse model expressing human APP with the Swedish mutation and the V717F mutation (Hsia et al. PNAS 1999, 96: 3228-3233). A suitable guinea pig model is, for example, described in Fassbender (2001). Also mice harboring two transgenes, like e.g. familial AD-linked genes (human APP Swedish and presenilin1-DeltaE9 as descried in Sheng 2002, J Neurosci 22(22):9794-9799; double Swedish/London mutation).

As pointed out above, also the PDAPP transgenic mouse model of Alzheimer's disease is very useful in screening/determining calmodulin inhibitors to be employed in context of the present invention. The PDAPP model was, inter alia, described in Johnson-Wood (1997), Proc Natl Acad Sci U S A, 94(4): 1550-1555, Su (1998)J Neurosci Res., 53(2): 177-186. or in Bard (2000), Nat Med. 6(8):916-919. Further mouse models to be employed in the screening fro useful calmodulin inhibitors have been reviewed in Bornemann (2000) Ann N Y Acad Sci.908:260-266.

In a preferred embodiment of this invention, calmodulin inhibitors/antagonists are to be employed, wherein said calmodulin inhibitors/antagonists have an IC50 value for the inhibition of calmodulin of less than 80 μM or less than 50 μM, for example less than 20 μM. The general meaning of the term "IC50" is known in the art. The term refers to the "inhibitory concentration 50". This means the concentration of a compound (here calmodulin inhibitor/antagonist), where 50 % of the activity targeted by the compound (here calmodulin activity) is inhibited. The person skilled in the art is readily in the position to determine the IC50 value for the inhibition of calmodulin of the calmodulin inhibitors/antagonists to be used herein by his general knowledge and the methods described herein and as illustrated in the appended Figures and Examples.

Inhibition of calmodulin may, inter alia, be determined in the following in vitro assay, which measured the calmodulin-dependent activation of myosin light chain kinase (MLCK). Activated MLCK phosphorylates chicken gizzard myosin light chain. If calmodulin is inhibited the rate of myosin light chain phosphorylation is reduced. To test this, the following experiment is carried out (according to ltoh et al. Biochem. Pharm. 1986, 35:217-220). The reaction mixture (0.2 ml) contains 20 mM Tris-HCI (pH 7.5), 0.05 mM [γ-³²P] ATP (1μCi/assay tube), 5 mM MgCI₂, 10 μM myosin light chain, 24 nM calmodulin and 0.1 mM CaCI₂. MLCK (specific activity: 4.5 moles/min/mg) concentration from chicken gizzard is 0.1 μg/ml. The incubation is carried out at 3O⁰C for 4 min. The reaction is terminated by addition of 1 ml of 20% trichloroacetic acid. Then 0.1 ml of bovine serum albumin (1 mg/ml) is added to the reaction mixture. The sample is then centrifuged at 200Og for 10 min, the pellet is resuspended in 5% trichloroacetic acid. The final pellet is dissolved in 2 ml of 1 N NaOH and the radioactivity measured in a liquid scintillation counter. Trypsin-treated MLCK can be prerared as described in ltoh et al. J Pharmacol. Exp. Ther. 1984, 230, p737. The reaction is initiated by the addition of the ATP and is carried out in the presence of the potential inhibitors or - as a control - in the presence of their solvent. Different concentrations of the compounds will be tested in the above assay. The concentration of the compound which results in 50% decrease of kinase activity will be the IC50 concentration.

An alternative method is the method as modified from Kahn et al. Cell 1998, 92:809- 818: As a read-out the inhibition of Gonadotropin-releasing hormone (GnRH) induced ERK Phosphorylation in αT3-1 cells as measured. αT3-1 cells are serum-starved for 2h, pretreated with control solvent or increasing concentrations of the compounds to be tested for 30 min. Then GnRH is administered for 60 min. Cell lysates are prepared and resolved by SDS-PAGE. Western blot analysis is used to determine the phosphorylation status of ERKs using a phospho-specific antibody (cell signaling technologies). As a control, total ERK2 will also be determined using an ERK specific antibody (Santa Cruz Biotech). Western-Blot fluorescence of phospho-ERK and total ERK2 will be quantified. The ratio of phospho-ERK/total ERK2 will be plotted against the concentration of the compound to be tested. The estimated concentration, at which a 50% reduction of ERK phosphorylation (rel. to total ERK2) occurs, will be used as the IC50 value for this compound.

As documented in the appended examples, the calmodulin inhibitors/antagonists to be employed in context of the present invention inhibit the generation of soluble amyloid precursor protein APP/APPsβ (Aβ as defined herein above which leads, in a pathological condition, to the accumulation of amyloid plaques) and/or stabilizes membrane bound APP. Accordingly, it is desired that the calmodulin inhibitors/antagonists is capable of inhibition the generation of soluble APP wherein, preferably, said inhibition of the generation of soluble APP/ APPsβ comprises an IC50 value of less than 20μM, preferably less than 10μM. The person skilled in the art is readily in a position to determine said IC50 value by the methods described herein and as illustrated in the appended Figures and Examples. For example, for the determination of the IC50 value of a given compound, different concentrations of the compound can be assayed for their effect on APPsβ or Aβ secretion. The concentration of the compound which results in 50% decrease of APPsβ or Aβ secretion (compared to the APPsβ or Aβ secretion of a corresponding control, which was not treated with the compound) will then be the IC50 concentration.

In particular, the use of the calmodulin inhibitors bepridil (also known as bepridil- hydrochloride, Vascor^®, Unicordium^®, Cordium^®, Bepricor^® and CERM-1978 (mainly used in publications from the late 1970s)), phenoxybenzamine (i.a. marketed as Benzpyran®) cetiedil (also known as Stratene^® and Vasocet^®) and/or W7 (also known as N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (Hidaka et al, J Pharmacol Exp Ther 1978 207(1):8-15 and Hidaka (1981) PNAS, 78, 4354-4357 is envisaged. Moreover, the use of the calmodulin inhibitors zaldaride maleate (also known in the art as CGS 9343B) and chlorpromazine (also known as Propaphenin^®, Largactil^®, Epokuhl^® and Thorazine^®) is envisaged in context of the present invention. These calmodulin inhibitors are, for example, described in Norman, 1987 and Khan, 2000, respectively.

Further compounds to be used as calmodulin inhibitors in context of the present invention may be compounds like promazine, desipramine, flunarizine, or promethazine. For example, these compounds are described in US 2006/0009506 and have structural similarity to compounds known to act as calmodulin inhibitors.

Also derivatives of said compounds are useful in context of the present invention, for example W7-derivatives, like N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride or N-(6-aminohexyl)-5-chloro-2-naphthalenesulfonamide.

In in vitro experiments and in animals bepridil has been shown to influence a large number of processes, including many ion channel currents (calcium, potassium and sodium channels) such as delayed rectifier K⁺ current (Yumoto et al., 2004), HERG (Chouabe et al., 1998), Na⁺/Ca²⁺ exchanger (Calabresi et al., 1999). Bepridil can even bind actin (Cramb and Dow, 1983). The IC50 for these processes is typically in the low micromolar range and thus, similar to what was observed in context of this invention for β-cleavage inhibition. The molecular mechanism (direct or indirect inhibition) is mostly unknown. Bepridil is currently used for the treatment of angina and other forms of heart disease. Chlorpromazine or trifluoperazine are old drugs against psychotic disorders. Bepridil has anti-anginal properties and (less well characterized) anti-arrhythmic and anti-hypertensive properties. Bepridil has also been reported to ameliorate experimental autoimmune encephalomyelitis in mice (Brand-Schieber and Werner, 2004). Chemically, it is not related to other calcium channel blockers, such as nifedipine, verapamil or diltiazem. Furthermore, bepridil is known as a calcium antagonist (Hollingshead et al., 1992), but the molecular mechanism of bepridil's cellular actions is not well understood.

In terms of the present inventions, the use of the calmodulin inhibitors bepridil, phenoxybenzamine, cetiedil and/or W7 are preferred in the uses and methods provided herein. As documented in the appended examples, however, particular impressive results can be obtained with bepridil (or pharmaceutically acceptable salts of these compounds).

The calmodulin inhibitors to be used in terms of the present invention may have an overall high degree of hydrophobicity and/or may comprise an amino group linked through a spacer to an aromatic system. Thereby, the amino group may be a heterocyclic amine. The spacer may be an aliphatic hydrocarbon chain, but may also include ester linkages or side chains. The aromatic system maximally comprise 1 , 2 or 3 aromatic rings, even heterocycles may also be employed. The aromatic rings may be directly fused to each other or may also or be separate (e. g. such as in bepridil). The aromatic rings may also carry substitutents, such as chlorine.

Preferably, said calmodulin inhibitor/antagonist to be employed in context of this invention is a compound of the general formula (I):

wherein:

A at each occurrence is independently selected from 6 to 10-membered aromatic or 5 to 6-membered heteroaromatic rings, which may be optionally substituted by 1- 5 substituents selected from halogen, hydroxy, C-] -Ce alkyl, optionally substituted amino, or C-|-C6 alkoxy if p=0, then the two A together with N may form a tricyclic system via a -CH2- or heteroatom brigde, such that a 6-membered ring is formed in the center of the tricyclic system;

Y is H or a 3- to 7-membered carbo-or 5-6 membered heterocyclus containing 1-2 heteroatoms selected from N, S or O;

R-I , R2 and R3 may be the same or different and are independently selected from hydrogen, C1-C6 alkyl, halogen, hydroxy, optionally substituted amino, or

C 1-C6 alkoxy; m and s are selected from an integer of 0 to 4; and n are independently selected from an integer of 1 to 4; r is and integer of 0 to 2; p is an integer of 0 or 1 ; with the proviso that if r=0, then W is NH₂, CH₃ or OH; if r=1 , then W is NHB, CH₂B or OB; if r=2, then W is NB₂ or CHB₂; and B at each occurrence is independently a group a the formula:

R¹

(CH₂),

R^ and pharmaceutically acceptable salts of said formula (i).

In a preferred embodiment of the calmodulin inhibitor as defined above by formula (I), said calmodulin inhibitor/antagonist is a compound of the general formula (I), wherein A at each occurrence is phenyl; one p is 0, the other p is 1 Y is a 5-membered heteroaromatic ring containing 1 heteroatom selected from N, S or O; m is 1 , n is 1 , s is 1 ;R3 is H and wherein R^ and R2 are C<|-Cβ alkyl. Particular preferred in this context are compounds wherein R-] and R2 are both methyl.

In this context it is furthermore preferred that W is OB, wherein both R-| and R2 are both methyl and s is 1.

In an even more preferred embodiment of the invention, a compound as defined above under formula (I) is to be employed, wherein Y is 1 -pyrrol id inyl

A distinct and preferred example of a compound of the general formula (I) is bepridil or a pharmaceutically acceptable salt thereof.

Also envisaged in the here described uses and methods is the use a calmodulin inhibitor/antagonist is a compound of the general formula (M):

wherein:

-A and B are the same or different and are independently selected from 6- to 10- membered aromatic or 5 to 6-membered heteroaromatic rings;

-R-I and R2 are independently selected from hydrogen, C-i-Ce alkyl, C-j-Ce alkoxy, hydroxy, optionally substituted amino, or halogen,

-m is an integer selected from 0 to 4 and n is an integer from 1 to 4;and pharmaceutically acceptable salts thereof. In a preferred embodiment, a calmodulin inhibitor/antagonist of the general formula (II) is a compound wherein A and B are phenyl, R-| is C-i-Ce alkyl, preferably methyl,, F*2 is halogen, preferably chloride, wherein and n and m are 1. Such a preferred calmodulin inhibitor/antagonist is phenoxybenzamine or a pharmaceutically acceptable salt thereof.

In another embodiment, uses and methods are described, wherein said calmodulin inhibitor/antagonist to be employed is a compound of the general formula(lll):

wherein:

-X is selected from S, O, NH and CH₂; -m is an integer from 0-4;

-R is a C-| to CQ alkyl or a 6-or 10-membered aromatic or 5 to 6-membered heteroaromatic ring, wherein a aromatic or heteroaromatic ring, in particular a heteroaromatic ring is preferred and a pharmaceutically acceptable salt thereof.

A preferred calmodulin inhibitor/antagonist to be employed is a compound of the general formula(lll)wherein X is O or NH, m is 0 or 1 and R is CH3 or thiophenyl.

Such a compound is cetiedil or a pharmaceutically acceptable salt thereof.

The term ,,C-I-Ce ^a|M" ^{as usecl} herein alone or in combination with other terms such as in ,,alkoxy" refers to a C^-CQ, preferably C1-C4 straight or branched alkyl group such as methyl, ethyl, iso-propyl, n-propyl, iso-butyl, n-butyl sec-butyl, tert.-butyl, pentyl and hexyl.

The term "C<\-CQ alkoxy" as used herein refers to a C-|-C6, preferably C-1-C4 straight or branched alkoxy group such as methoxy, ethoxy, iso-propoxy, n-propoxy, iso- butoxy, n-butoxy, sec.-butoxy, ter.-butoxy, pentoxy and hexoxy. The terms "halogen" or "halo" as used herein alone or in combination with other terms refers to a halogen atom selected from fluorine, chlorine, bromine and iodine.

The term "6 to 10 membered aromatic ring" as used herein refers to a mono- or bicyclic aromatic group having 6 to 10 backbone carbon atoms, wherein optionally one of the rings of the bicyclic structure is aromatic and the other is a carbocyclic group, such as phenyl, 1-naphthyl, 2-naphthyl, indenyl, indanyl, azulenyl, fluorenyl, and 1 ,2,3,4-tetrahydronaphthyl.

The term "5 to 6 membered heteroaromatic ring" as used herein refers to a moncyclic aromatic group with 1 to 4 hetero atoms selected from N, S and O, with the remainder of the ring atoms being carbon atoms, such as furyl, thienyl, pyrrolyl, pyridinyl, pyridazinyl, pyrazinyl, pyrimidinyl, oxazolyl, thiazolyl, isooxazolyl, isothiazolyl, pyrazolyl, imidazolyl

The term "3 to 7 membered carbocyclus" as used herein refers to a carbocyclic alkyl substituent or group having 3 to 7 ring atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cyclopentyl.

The term "5 to 6 membered heterocyclus" as used herein refers to monocyclic saturated heterocyclyl groups with 1 to 4 hetero atoms selected from N, S and O, with the remainder of the ring atoms being carbon atoms such as morpholino, piperazinyl, piperidinyl, pyridyl, tetrahydrofuryl, tetrahydropyrrolyl, pyrrolidinyl, or imidazolidinyl.

The term "optionally substituted amino" as used herein refers to a amino group, wherein optionally one or both hydrogen atoms are substituted by a Ci-Ce alkyl, C-| to CQ alkoxy group or halogen atom as defined above.

The term "heteroatom" as used herein refers to a heteroatom selected from N, S, O or P, preferably to N, S or O. Pharmaceutically acceptable salts of the compounds of the invention can be formed with numerous organic and inorganic acids and bases. Exemplary acid addition salts including acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphersulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethane sulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethane sulfonate, lactate, maleate, methane sulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate, 3-phenyl sulfonate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluene sulfonate such as tosylate, undecanoate, or the like.

Basic nitrogen-containing moieties can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromide and iodide; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long-chain alkyl halides such as decyl, lauryl, myristyl and stearyl chloride, bromide and iodide, or aralkyl halides like benzyl and phenethyl bromides, or others. Water soluble or dispersible products are thereby obtained.

Pharmaceutically acceptable basic addition salts include but are not limited to cations based on the alkaline and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, aluminum salts and the like, as well as non toxic ammonium quarternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like. Other representative amines useful for the formation of base addition salts include benzazethine, dicyclohexyl amine, hydrabine, N-methyl-D-glucamine, N-methyl-D-glucamide, t-butyl amine, diethylamine, ethylendiamine, ethanolamine, diethanolamine, piperazine and the like and salts with amino acids such as arginine, lysine, or the like.

Compounds of the present invention can be present as tautomers. The present invention comprises all tautomeric forms. Furthermore, the present invention also comprises all stereoisomers of the compounds according to the invention, including its enantiomers and diastereomers. Individual stereoisomers of the compounds according to the invention can be substantially present pure of other isomers, in admixture thereof or as racemates or as selected stereoisomers.

Further useful calmodulin inhibitors/antagonists to be employed in context of this invention are selected from the group consisting of a phenothiazine compound, a butyrophenone compound, a diphenylbutylamine compound, or a pharmaceutically acceptable salt or ester of these compounds.

Accordingly, in context of the present invention, it is also envisaged to employ substances like R24571 (calmodazolium chloride or 1-[bis-(p-Cholorophenyl)methyl]- 3-[2,4-dichloro-β-(2,40dichlorobenzyloxy)phenethyl]-imidazolium chloride; as well as compound R24571 chloride; as described in Adunyah (1982), FEBS Lett, 143: 65-68.

The phenothiazine compound to be employed may be, thioridazine, or fluphenazine, said butyrophenone compound may be selected from the group consisting penfluridol, benperidol and spiroperidol, or said diphenylbutylamine may be pimozide.

Yet, the calmodulin inhibitor/antagonist may also be selected from the group consisting of (+) and (-) butaclamol, clozapine, cis-and trans-chlorprothixene, cis- and trans-flupenthixol, and W7.

In a more preferred embodiment, the calmodulin inhibitor/antagonist to be employed in the context of the present invention is a compound of the general formula (I):

γ1a

A^1a— (CH₂)_ma-N-(CH₂)_na-CH-(CH₂)oa-O-B^{1 a} A^2a

(I)

or a pharmaceutically acceptable salt thereof. A^1a is a 6- to 10-membered aromatic ring or a 5- or 6-membered heteroaromatic ring, each of which may be optionally substituted by one or more, preferably one, substituents selected from halogen, hydroxyl, C-i-₆ alkyl, optionally substituted amino and C-₁-₆ alkoxy. Preferably, A^1a is optionally substituted phenyl, more preferably unsubstituted phenyl.

A^2a is a 6- to 10-membered aromatic ring, a 5- or 6-membered heteroaromatic ring or Ci_-4 alkyl, each of which may be optionally substituted by one or more, preferably one, substituents selected from halogen, hydroxyl, Ci_-6 alkyl, optionally substituted amino and Ci_-6 alkoxy. Preferably, A^2a is optionally substituted phenyl, more preferably unsubstituted phenyl. In another preferred embodiment, A^2a is C1-4 alkylene-halogen, preferably Ci_-4 alkylene-CI.

Y^1a is Ci_-4 alkyl, a 3- to 7-membered carbocylic ring or a 5- or 6- membered heterocyclic ring containing one or two heteroatoms selected from N, S and O. Preferably, Y^1a is a 5- or 6- membered heterocyclic ring containing one or two heteroatoms selected from N, S and O. In a more preferred embodiment, Y^1a is a 5- or 6- membered heterocyclic ring containing one nitrogen atom, wherein the heterocyclic ring is attached to the neighboring carbon atom via the nitrogen atom. In another preferred embodiment, Y^1a is Ci_-4 alkyl.

B^1a is C_3-6 alkyl or a 6- to 10-membered aromatic ring, which may be optionally substituted by one or more, preferably one, substituents selected from halogen, hydroxyl, Ci_-6 alkyl, optionally substituted amino and Ci_-6 alkoxy. Preferably, B^1a is branched C_4-6 alkyl, in particular CH₂CH(CH₃)₂. In another preferred embodiment, B^1a is optionally substituted phenyl, more preferably unsubstituted phenyl.

ma is an integer of 0 to 4. Preferably, ma is 1 or 2, more preferably 1.

na is an integer of 0 to 4. In one preferred embodiment, na is 1. In another preferred embodiment, na is 0.

oa is an integer of 0 to 4. Preferably, oa is 1. In a particular preferred embodiment, the compound of formula (I) is bepridil or phenoxybenzamine, or a pharmaceutically acceptable salt thereof, such as bepridil hydrochloride. In a most preferred embodiment, the compound of formula (I) is bepridil or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the calmodulin inhibitor/antagonist to be employed in the context of the present invention is a phenothiazine compound. Preferably, the calmodulin inhibitor/antagonist to be employed in the context of the present invention is a compound of the general formula (II):

(H)

or a pharmaceutically acceptable salt thereof.

R^1a is H, halogen, 0(Ci_-4 alkyl), S(Ci_-4 alkyl), Ci_-4 alkyl or Ci_-4 fluoroalkyl. Preferably, R^1a is H, halogen, S(Ci_-4 alkyl), or Ci_-4 fluoroalkyl; more preferably, H, Cl, SCH₃ or CF₃. Even more preferably, R^1a is chloro or CF₃.

The moiety X _/2^za^a- γY_/2^za^a is N-Ci_-5 alkylene or C=Ci_-5 alkylene. Preferably, the moiety X 2a-

V _/2a^a is N-C₃ alkylene or C=C₃ alkylene.

R^2a is optionally substituted amino or a 5- or 6-membered heterocyclic ring containing one or two nitrogen atoms, wherein the nitrogen atoms may be substituted by Ci-6 alkyl, Ci_-6 alkylene-OH or Ci_-6 alkylene-halogen. Preferably, R^2a is substituted amino or substituted piperidinyl. More preferably, R^2a is C₁-₄ dialkyl amino or substituted piperidinyl, wherein the piperidinyl moiety is attached to the neighboring carbon atom via a nitrogen atom. Even more preferably, R^2a is N(CH₃)₂ or N-(N'-hydroxyethyl)- piperidinyl.

In a particularly preferred embodiment the compound of formula (II) is trifluoperazine, chlorpromazine, thioridazine, fluphenazine, promazine, promethazine, cis- or trans- chlorprothixene or cis- or trans-flupenthixol, or a pharmaceutically acceptable salt thereof, such as trifluoperazine dihydrochloride. Even more preferably, the compound of formula (II) is trifluoperazine or chlorpromazine, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the calmodulin inhibitor/antagonist to be employed in the context of the present invention is a butyrophenone compound. Preferably, the butyrophenone compound is benperidol or spiroperidol, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the calmodulin inhibitor/antagonist to be employed in the context of the present invention is a diphenylbutylamine compound or flunarizine or a pharmaceutically acceptable salt thereof. Preferably, the diphenylbutylamine compound is pimozide or penfluridol, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the calmodulin inhibitor/antagonist to be employed in the context of the present invention is a compound of the general formula (III):

or a pharmaceutically acceptable salt thereof.

X^1a is S, O, NH or CH₂. Preferably, X^1a is O or NH, more preferably O. pa is an integer of 0 to 4, preferably 0 or 1 , more preferably 0.

R^3a is C-ι-₆ alkyl or a 6- to 10-membered aromatic or 5- or 6-heteroaromatic ring. Preferably, R^3a is a Ci_-4 alkyl or a 5- or 6-heteroaromatic ring with one heteroatom selected from N, S and O. More preferably, R^3a is CH₃ or thiophenyl, in particular 3- thiophenyl.

In a particularly preferred embodiment, the compound of formula (III) is cetiedil or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the calmodulin inhibitor/antagonist to be employed in the context of the present invention is a compound of formula (IV)

(IV)

or a pharmaceutically acceptable salt thereof.

R^4a is H, halogen, 0(Ci_-4 alkyl), S(Ci_-4 alkyl), Ci_-4 alkyl or Ci_-4 fluoroalkyl. Preferably, R^4a is halogen, more preferably chloro.

qa is an integer of 2 to 10, preferably from 4 to 8, more preferably 6.

R^5a is optionally substituted amino, more preferably unsubstituted amino.

In a particularly preferred embodiment, the compound of formula (IV) is W7 (N-(6- aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride) or the free base thereof or another pharmaceutically acceptable salt thereof. In another preferred embodiment, the calmodulin inhibitor/antagonist to be employed in the context of the present invention is (+)- or (-)-butaclamol, clozapine or desipramine, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the calmodulin inhibitor/antagonist to be employed in the context of the present invention is zaldaride or a pharmaceutically acceptable salt thereof, in particular, zaldaride maleate.

It is preferred that the pharmaceutical composition to be prepared in accordance with this invention, optionally comprises a pharmaceutically acceptable carrier and/or diluent. The herein disclosed pharmaceutical composition may be particularly useful for the treatment of neurological and/or neurodegenerative disorders. Said disorders comprise, but are not limited to AD, amyothrophic lateral sclerosis (ALS), hereditary cerebral hemorrhage with amyloidosis Dutch type, Down's syndrome, HIV-dementia, Parkinson's disease and neuronal disorders related to aging The pharmaceutical composition of the invention is, inter alia, envisaged as potent inhibitors of amyloid plaque formation. Therefore, the present invention provides for means and methods for the provision of pharmaceutical compositions comprising the compounds of the invention, namely the herein defined calmodulin inhibitors, like bepridil, trifluoperazine, chlorpromazine, cetiedil or W7 (or pharmaceutically acceptable salts thereof) to be used for the treatment of diseases/disorders associated with pathological APP proteolysis and/or amyloid plaque formation.

Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. It is particularly preferred that said administration is carried out by injection and/or delivery, e.g., to a site in a brain artery or directly into brain tissue. The compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like the brain. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A person skilled in the art is aware of and is able to test the relevant doses, the compounds to be used in terms of the present invention are to be administered in.

For instance, Hollingshead (1992), describes useful dosages of bepridil for the medical intervention in human patients. The pharmaceutical effective concentrations of the compounds as employed herein at the site of action and/or within the blood (plasma) are also known in the art or can be achieved by a person skilled in the art. For instance, this doses and concentrations are described in Hollingshead (1992), Orringer (1986), Roufogalis (1983) and Benjamin (1986). E.g, in case of Cetiedil, the plasma concentration may be 70 -200 ng/ml after infusion (Orringer, 1986) and the dosage may be 0.4 mg/kg body weight.

In context of the invention, it is of note that the subject to be treated is a mammalian subject, most preferably a human being in need of medical intervention, either in form of prophylaxis or in from of a curative treatment/amelioration. Accordingly, the present invention also relates to A method for the prevention, amelioration or treatment of a neurodegenerative disease in a subject in need of such a prevention, amelioration or treatment, said method comprising the step of administering to said subject a pharmaceutically active amount of a calmodulin inhibitor/antagonist as defined herein above. Most preferably, said calmodulin inhibitor/antagonist is selected from the group consisting of bepridil or a pharmaceutically acceptable salt thereof; trifluoperazine or a pharmaceutically acceptable salt thereof, chlorpromazine or a pharmaceutically acceptable salt thereof, cetiedil or a pharmaceutically acceptable salt thereof, and W7 or a pharmaceutically acceptable salt thereof. As documented in the appended examples, particularly preferred is bepridil or a pharmaceutically acceptable salt thereof. Suitable modes of administration of the calmodulin inhibitors or pharmaceutical compositions comprising them to be employed in context of the present invention are well known in the art. For example, such modes of administration may be selected from the group consisting of administrations by blood infusion(s) (like intravenous infusion(s)), intraperitoneal administrations, intravesical administrations, subcutaneous administrations, intramuscular administrations, intrathecal administrations, transmucosal administrations, transpulmonal administrations, subdural administrations, sublingual administrations, administrations by inhalation, transdermal administrations, oral administrations, and rectal administrations (e.g. in form of enemas or suppositories), and the like. Preferred modes of administration are nasal administrations, for example via aerosols/sprays.

The present invention is further described by reference to the following non-limiting figures and examples.

The Figures show:

Figure 1 : Proteolytic cleavage of APP. by α-, β- and γ-secretase. The Alzheimer protein APP is a membrane protein and consists of a large extracellular domain, a transmembrane and a cytoplasmic domain. The two proteases, which cleave APP and generate Aβ, are referred to as β- and γ-secretase. β-secretase cleaves first, γ- secretase cleaves second. Alternatively, α-secretase cleaves APP within the Aβ- domain and thus precludes Aβ-generation.

Figure 2: Bepridil hydrochloride is a modulator of APP shedding. Clonal 293- EBNA cells stably expressing a fusion protein of alkaline phosphatase (AP) and APP (AP-APP cells) were treated for 4h with the indicated concentrations of bepridil hydrochloride or PMA (positive control). The amount of secreted AP-APP in the conditioned medium was analysed by measuring the AP activity in a colorimetric reaction. Shown are the mean and standard deviation out of four (bepridil 10μM) or five (bepridil 50μM and PMA 10OnM) independent experiments. Figure 3: Bepridil hydrochloride is not toxic to 293E-APP695 cells at concentrations up to 20μM. 293E-APP695 cells were treated for 24h with the indicated concentrations of bepridil hydrochloride or solvent (DMSO) alone. The levels of lactate dehydrogenase (LDH) released by dying cells was determined using the CytoTox^® assay from Promega. As a positive control for cell death Tamoxifen (50μM) and its solvent (Ethanol) were included. Given are the mean and standard deviation out of two independent experiments.

Figure 4: Bepridil hydrochloride inhibits the formation of APPsβ and Aβ in different cell types in a concentration-dependent manner. 293E-APP695 (A), COS7-APP695 (B), wt293E (C; expressing endogenous APP) and H4-APP751 (D and E) cells were pretreated with the indicated concentrations of bepridil hydrochloride for 45min. The medium was changed for fresh medium containing the substance and cells were incubated for another 4h (A₁B, D&E) or 24h (C). The levels of the indicated, different APP species in the conditioned medium (APPsα, APPsβ and Aβ) and cell lysates (total APP) were analysed by western blotting using the antibodies named in brackets. For Aβ detection an immunoprecipitation was performed prior to western blotting. Shown are representative blots of 2-4 independent experiments. In 293-APP695 cells, bepridil stimulates APP α-cleavage at 50 μM (A, detected with antibody W02), but not at lower concentrations.

Figure 5: Quantification of the effects of bepridil hydrochloride on APPsβ and Aβ formation. The intensities of the bands in Figure 4 were quantified. Given are the mean and standard deviation out of four (293E-APP695/APPsβ), three (H4-APP751/ APPsβ) or two (293E-APP695/Aβ, COS7-APP695/APPsβ and H4-APP751/Aβ) independent experiments. The IC50 value of bepridil for the inhibition of APPsβ or Aβ formation is the estimated concentration at which bepridil reduces APPsβ or Aβ formation to 50% of the control cells, which were not treated with bepridil. A similar test may be employed for any compound to be determined/identified or screened. In said test, bepridil will be replaced by the compound to be tested for its IC50 value in the inhibition of APPsβ or Aβ. Figure 6: Bepridil hydrochloride does not directly inhibit BACE1 activity. The effect of different concentrations of bepridil hydrochloride on the activity of soluble BACE 1 was determined by a fluorometric BACE 1 activity assay (see Materials and methods). As a control the well-characterized BACE1 inhibitor GL-189 (Capell et al., 2002) was used, which completely inhibited BACE1 activity.

Figure 7: Increasing or decreasing the cellular calcium concentration does not phenocopy the inhibitory effect of bepridil hydrochloride on APPsβ formation.

293E-APP695 cells were pretreated for 45min with the indicated concentrations of the cell-permeable calcium chelator BAPTA-AM (A) or the Ca⁺⁺-ATPase inhibitor thapsigargin (TG) (B). The medium was changed for fresh medium containing the substances and incubated for additional 4h. The levels of the different APP species in the conditioned medium and cell lysates were determined by western blotting using the same antibodies as in Figure 4. Shown are representative blots of two independent experiments. The vertical lines (*) indicate, that the samples were run on the same gel but not in neighbouring lanes. Note that BAPTA-AM treatment leads to a slightly different running behaviour of APPsα/β and full-length APP. This might be due to changes in maturation of APP caused by the depletion of calcium from the cells.

Figure 8: Inhibition of calmodulin leads to a reduction of APPsβ and Aβ formation in 293E-APP695 cells. 293E-APP695 cells were pretreated for 45min with the indicated concentrations of the calmodulin antagonists trifluoperazine dihydrochloride (TFP) (A), chlorpromazine hydrochloride (B) and N-(6-Aminohexyl)-5- chloro-1-naphthalenesulfonamide hydrochloride (W-7) (C). The medium was replaced with fresh medium containing the substances and incubated for another 4h. The levels of the different APP species in the conditioned medium and cell lysates were determined by western blotting using the antibodies already described in Figure 4. For Aβ detection an immunoprecipitation was performed prior to western blotting. Shown are representative blots of two independent experiments. For chlorpromazine hydrochloride and W-7 only the blots for APPsβ are displayed. The vertical lines (^*) indicate, that the samples were run on the same gel but not in neighbouring lanes. Figure 9: Structure of bepridil hydrochloride (A), trifluoperazine dihydrochloride (B), chlorpromazine (C) and W7 (D).

The Examples illustrate the invention.

Example 1 : Materials and methods Materials

Bepridil hydrochloride, trifluoperazine dihydrochloride (TFP), chlorpromazine hydrochloride, N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), thapsigargin (TG) and Tamoxifen (Tarn) were purchased from SIGMA-Aldrich Germany. Phorbol-12-myristate-13-acetate (PMA) was purchased from Calbiochem. BAPTA-AM was purchased from SIGMA-Aldrich Germany. Bepridil, chlorpromazine hydrochloride, N-(6-Aminohexyl)-5-chloro-1- naphthalenesulfonamide hydrochloride (W-7), Thapsigargin, PMA and BAPTA-AM were dissolved in DMSO, Tamoxifen was dissolved in ethanol and TFP was dissolved in water. CytoTox 96^® Non-Radioactive Cytotoxicity Assay was purchased from Promega. Tropix Western-Star Protein Detection Kit was purchased from Applied Biosystems.

Antibodies

For detection of the different APP species the following antibodies were used: Mouse monoclonal W02 (detecting APPsα; Ida et al., 1996); rabbit polyclonal 192wt (detecting APPsβ; Seubert et al., 1993); mouse monoclonal 22C11 (Chemicon, cat. no. MAB348); rabbit polyclonal 6687 (against the C-terminus of APP, detecting total APP; Steiner et al., 2000); mouse monoclonal 6E10 (detecting Aβ; Senetek, cat. no. 9320-02). For immunoprecipitation of Aβ polyclonal rabbit antibody 3552 (raised against synthetic peptide Aβ-_Mo; Eurogentec) was used.

Cell culture

293-EBNA cells were cultured in Dulbecco's modified eagles medium (D-MEM) supplemented with 10% fetal calf serum, 1 % penicillin/streptomycin and are referred to as wt293E cells. Clonal 293-EBNA cells expressing AP-APP and Bcl-X_L/CrmA (clone SABC70) were generated and cultured as described (Lichtenthaler et al., 2003) and are referred to as AP-APP cells. 293-EBNA cells stably transfected with the construct pCEP4/APP695 (Lichtenthaler et al., 1999) were cultured in D-MEM supplemented with 10% fetal calf serum, 1 % penicillin/streptomycin and 100μg/ml Hygromycin and are referred to as 293E-APP695 cells. COS7 cells stably transfected with the construct peak12/APP695 (Lichtenthaler et al., 2003) were cultured in D- MEM supplemented with 10% fetal calf serum, 1 % penicillin/streptomycin and 3.0μg/ml Puromycin and are referred to as COS7-APP695 cells. Clonal H4 cells stably transfected with the construct pRC/CMV hAPPwt were generously provided by Cornelia Dorner-Ciossek (Boehringer Ingelheim) and cultured in D-MEM supplemented with 10% fetal calf serum, 1 % penicillin/streptomycin and 0,2mg/ml G418 and are referred to as H4-APP751 cells.

Screen for chemical modifiers of amyloid precursor protein (APP) shedding

AP-APP cells were plated into poly-D-lysine coated 384-well plates at a density of 2.75 x 10⁴ cells/well. On the following day the medium was replaced with fresh medium containing heat inactivated serum. Compound stocks (dissolved in DMSO) from a 480-member library of known bioactive compounds (BIOMOL) were transferred by using a robot controlled stainless-steel pin array. After 4 and 8h samples of the supernatants were taken and subjected to an AP reaction to determine the amount of secreted AP-APP. The library was screened in duplicate.

Treatment of cells with chemical compounds

AP-APP cells were plated into a poly-D-lysine coated 24-well plate at a density of 3.25-4 x 10⁵ cells/well. On the following day the medium was changed for fresh medium containing the chemical substances or solvent alone. After incubation for 4h conditioned medium and cell lysates (in 5OmM Tris pH 7.5, 15OmM NaCI, 1 % NP40) were collected. Aliquots of the conditioned medium were treated for 30 min at 65 ⁰C to heat-inactivate the endogenous alkaline phosphatase activity. AP activity in the conditioned medium was measured as described previously (Lichtenthaler et al., 2003) and normalised to the protein concentration in the cell lysate. Alternatively wt293E, 293E-APP695, COS7-APP695 and H4-APP751 cells were plated into poly-L-lysine coated 6cm dishes at a density of 0.75-3.5 x 10⁶ cells/dish (depending on cell type). As a second alternative cells were plated into a poly-D- lysine coated 24-well plate at a density of 2.25-3.5 x 10⁵ cells/well. One day after plating, the medium was replaced with fresh medium. Two days after plating, the medium was changed for fresh medium containing different concentrations of the chemical compounds or solvent alone. Dishes were preincubated for 45min. After preincubation the medium was replaced with fresh medium containing the compounds or solvent and incubated for additional 4h. The conditioned medium and cell lysates were collected. For H4-APP751 cells treatment was done one day after plating.

lmmunoprecipitation and western blot analysis

For detection of the different APP species in the conditioned medium and cell lysates, aliquots of the samples were boiled for 5min with SDS-sample buffer and subjected to SDS-PAGE on an 8% SDS-gel (loading volumes normalised for protein concentration). The proteins were transferred to a PVDF-membrane and detected with the respective antibodies.

For detection of Aβ, the conditioned medium was subjected to immunoprecipitation with anti-Aβ antibody 3552 (1 :400) and Protein-A-Sepharose (PAS) beads overnight. After washing, the beads were mixed with SDS-sample buffer and boiled for 5min. Samples were then subjected to SDS-PAGE on a 10-16.5% Tris/Tricine-gel. The proteins were transferred to a nitrocellulose membrane and Aβ was detected using antibody 6E10. The Tropix Western-Star Protein Detection Kit was used for developing the signals. Quantification was performed using the digital camera system Fluorchem™8900 (Alpha Innotech) and AlphaEase FC software. For the determination of the IC50 value of a given compound, different concentrations of the compound will be assayed for their effect on APPsβ or Aβ secretion. The concentration of the compound which results in 50% decrease of APPsβ or Aβ secretion will be the IC50 concentration. Determination of cytotoxicity of bepridil hydrochloride by the lactate dehydrogenase (LDH)-assay

293E-APP695 cells were plated into a poly-D-lysine coated 96-well plate at a density of 2 x 10⁴ cells/well in medium containing heat-inactivated serum. After overnight incubation the medium was replaced with fresh medium containing different concentrations of bepridil hydrochloride or DMSO (solvent control; 8 wells/condition). Tamoxifen was used as a positive control with ethanol as solvent control. The plate was incubated for another 24h. 45min before the end of the incubation time, 0.8% Triton X-100 was added to three wells of each condition to achieve maximal LDH release (LDH_max). After 24h of incubation the plate was centrifuged for 5min at 25Og. Samples of the supernatants were taken and diluted 1 :5 with fresh medium. LDH measurement was performed as described in the CytoTox® manual (Promega). The degree of cytotoxicity for each condition was calculated as follows:

% cytotoxicity = average LDH_sampies / average LDH_maχ The results are presented as cytotoxicity values relative to the solvent control.

Fluorometric BACE1 Activity Assay (modified from Capell et al., 2002)

A quenched fluorescence substrate Cy3-SEVNLDAEFK(Cy5Q)-NH2 (SEQ ID NO. 2) containing the Swedish APP mutation (in boldface) was synthesized by Amersham Biosciences, Inc., UK. Citron et al. Nature 1992, 360: 672-674.The reaction was carried out in 100μl preparations containing 1 μl purified soluble BACE1 ectodomain, 1μM peptide substrate, 4OmM sodium acetate (pH 4.4) and various concentrations of bepridil hydrochloride. The well-characterised BACE1 inhibitor GL-189 (Capell et al., 2002) was used as a control. Fluorescence was measured continuously over a period of 2h (cycle time 30sec) at room temperature (Fluoroskan Ascent, Labsystems, Finland, excitation 530 nm, emission 590 nm).

Example 2: Identification of bepridil in a chemical screen

When APP is cleaved by α- and/or β-secretase, the large, soluble extracellular domain of APP is secreted and can be detected in the conditioned medium (Fig. 1). To identify chemical compounds stimulating or inhibiting APP α- and/or β-cleavage, a reporter cell line was used, by which APP shedding can be monitored. This reporter cell line is known in the art (Lichtenthaler et al., 2003). It was found that bepridil (chemical structure shown in Fig. 9) is an activator of APP secretion, which slightly increased APP secretion 1.5 fold at a concentration of 50 μM (Fig. 2). This increase may be due to cellular toxicity observed at this concentration (as measured in the LDH assay Fig. 3).

Example 3: Use of bepridil as an inhibitor of β-secretase cleavage

At lower, non-toxic concentrations (10 μM; Fig. 3) bepridil no longer stimulated APP secretion (Fig. 2). However, it was surprisingly found that at the lower concentrations (and also at the higher concentrations) bepridil inhibited β-secretase cleavage in a dose-dependent manner. This was found in two different sets of experiments. First, using a cleavage site-specific antibody, the soluble APP (cleaved by β-secretase, APPsβ) in the supernatant of our cells (human embryonic kidney 293 cells expressing endogenous or transfected APP as well as COS7 and H4 cells stably transfected with APP; Fig. 4) was detected. Second, it was tested for Aβ peptide generation (Fig. 4). The approximate IC50 value for inhibition of APPsβ and Aβ is 6 μM (Fig. 5), and thus is in the range of the physiological plasma concentration (2.5 - 3 μM) found of patients treated with bepridil for angina (Hollingshead et al., 1992; Massingham and Van Zwieten, 1989).

Bepridil does not directly inhibit the β-secretase enzyme BACE1 (β-site APP cleaving enzyme), as determined in an in vitro BACE1 assay (Fig. 6).

Example 4: Mechanism of bepridil's action and use of calmodulin antagonists as inhibitors of β-secretase cleavage

As mentioned before, bepridil is known as a calcium antagonist (Hollingshead et al., 1992), but the molecular mechanism of bepridil's cellular actions is not well understood. Herein, it is assumed that it does not directly inhibit L-type calcium channels (as speculated previously), because they are not even present in the human embryonic kidney cells used in this study. Moreover, two unrelated compounds known to increase (thapsigargin) or decrease (BAPTA-AM) intracellular calcium concentrations, slightly increased but did not decrease β-secretase cleavage (as bepridil did) (Fig. 7). Bepridil has been reported to bind to a number of different cellular proteins (see above), including calmodulin (Agre et al., 1984; ltoh et al., 1986). To test the possibility that bepridil inhibits calmodulin and thereby β-secretase cleavage, the known calmodulin inhibitors trifluoperazine (TFP), chlorpromazine and W-7 were tested for an inhibition of β-secretase cleavage. Like bepridil, all three compounds inhibited β-secretase cleavage and Aβ generation in a concentration dependent manner (Fig. 8; chemical structure of TFP shown in Fig. 9). From these experiments it was concluded that bepridil and calmodulin inhibitors can be used therapeutically to inhibit Aβ generation and to prevent and treat AD.

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Brand-Schieber (2004) Exp Neurol 189, 5-9.

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Chouabe (1998) MoI Pharmacol 54, 695-703.

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Yumoto (2004) J Cardiovasc Pharmacol 43, 178-182. The present invention refers to the following sequences:

SEQ ID No. 1 :

Human Aβ:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGWIA

SEQ ID No. 2 fluorescence substrate Cy3-SEVNLDAEFK(Cy5Q)-NH2, containing the Swedish APP mutation:

SEVNLDAEFK

Claims

1. Use of a calmodulin inhibitor/antagonist for the preparation of a pharmaceutical composition for the treatment, amelioration and/or prevention of a neurodegenerative disease.

2. The use of claim 1 , wherein said calmodulin inhibitor/antagonist inhibits the generation of soluble amyloid precursor protein APP/APPsβ.

3. The use of claim 1 or 2, wherein the calmodulin inhibitor/antagonist is a compound of the general formula (I):

,1a

A^1a— (CH₂)_ma-N-(CH₂)_na-CH-(CH₂)_oa-O-B^1a

A^2a

(I) wherein

A^1a is a 6- to 10-membered aromatic ring or a 5- or 6-membered heteroaromatic ring, each of which may be optionally substituted by one or more substituents selected from halogen, hydroxy!, C-ι-₆ alkyl, optionally substituted amino and C1-₆ alkoxy;

A^2a is a 6- to 10-membered aromatic ring, a 5- or 6-membered heteroaromatic ring or Ci_-4 alkyl, each of which may be optionally substituted by one or more substituents selected from halogen, hydroxyl, C-ι-₆ alkyl, optionally substituted amino and C-ι-₆ alkoxy;

Y^1a is C-ι-₄ alkyl, a 3- to 7-membered carbocylic ring or a 5- or 6- membered heterocyclic ring containing one or two heteroatoms selected from N, S and O;

B^1a is C₃-₆ alkyl or a 6- to 10-membered aromatic ring, which may be optionally substituted by one or more substituents selected from halogen, hydroxyl, Ci_-6 alkyl, optionally substituted amino and Ci_-6 alkoxy; ma is an integer of 0 to 4; na is an integer of 0 to 4; and oa is an integer of 0 to 4; or a pharmaceutically acceptable salt thereof.

4. The use of claim 3, wherein the compound of formula (I) is bepridil or phenoxybenzamine, or a pharmaceutically acceptable salt thereof.

5. The use of claim 1 or 2, wherein the calmodulin inhibitor/antagonist is a phenothiazine compound.

6. The use of any of claims 1 , 2 and 5, wherein the calmodulin inhibitor/antagonist is a compound of the general formula (II):

(II) wherein

R^1a is H, halogen, 0(Ci_-4 alkyl), S(Ci_-4 alkyl), Ci_-4 alkyl or Ci_-4 fluoroalkyl; the moiety X^2a-Y^2a is N-Ci_-5 alkylene or C=Ci_-5 alkylene.; and

R^2a is optionally substituted amino or a 5- or 6-membered heterocyclic ring containing one or two nitrogen atoms, wherein the nitrogen atoms may be substituted by Ci_-6 alkyl, Ci_-6 alkylene-OH or Ci_-6 alkylene-halogen; or a pharmaceutically acceptable salt thereof.

The use of claim 6, wherein the phenothiazine compound is trifluoperazine, chlorpromazine, thioridazine, fluphenazine, promazine, promethazine, cis- or trans-chlorprothixene or cis- or trans-flupenthixol, or a pharmaceutically acceptable salt thereof.

8. The use of claim 1 or 2, wherein the calmodulin inhibitor/antagonist is a butyrophenone compound.

9. The use of claim 8, wherein the butyrophenone compound is benperidol or spiroperidol, or a pharmaceutically acceptable salt thereof.

10. The use of claim 1 or 2, wherein the calmodulin inhibitor/antagonist is a diphenylbutylamine compound or flunarizine or a pharmaceutically acceptable salt thereof.

11. The use of claim 10, wherein the diphenylbutylamine compound is pimozide or penfluridol, or a pharmaceutically acceptable salt thereof.

12. The use of claim 1 or 2, wherein the calmodulin inhibitor/antagonist is a compound of the general formula (III):

(III) wherein

X /^■1ⁱa^a is S, O, NH or CH₂; pa is an integer of 0 to 4; and

R^3a is Ci_-6 alkyl or a 6- to 10-membered aromatic or 5- to 6-membered heteroaromatic ring; or a pharmaceutically acceptable salt thereof.

13. The use of claim 12, wherein the compound of formula (III) is cetiedil or a pharmaceutically acceptable salt thereof.

14. The use of claim 1 or 2, wherein the calmodulin inhibitor/antagonist is a compound of the general formula (IV):

NH(CH₂)_qaR^5a (IV) wherein

R^4a is H, halogen, 0(Ci_-4 alkyl), S(Ci_-4 alkyl), Ci_-4 alkyl or Ci_-4 fluoroalkyl; qa is an integer of 2 to 10; and

R^5a is optionally substituted amino; or a pharmaceutically acceptable salt thereof.

15. The use of claim 14, wherein the compound of formula (IV) is W7 (N-(6- aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride) or the free base thereof or another pharmaceutically acceptable salt thereof.

16. The use of claim 1 or 2, wherein the calmodulin inhibitor/antagonist is (+)- or (-)-butaclamol, clozapine or desipramine, or a pharmaceutically acceptable salt thereof.

17. The use of claim 1 or 2, wherein the calmodulin inhibitor/antagonist is zaldaride or a pharmaceutically acceptable salt thereof.

18. The use of any one of claims 2 to 17, wherein said inhibition of the generation of soluble APP/ APPsβ comprises an IC50 value of less than 20μM.

19. The use of any one of claims 2 to 18, wherein said inhibition of the generation of soluble APP/ APPsβ comprises an IC50 value of less than 10μM.

20. A method for the prevention, amelioration or treatment of a neurodegenerative disease in a subject in need of such a prevention, amelioration or treatment, said method comprising the step of administering to said subject a pharmaceutically active amount of a calmodulin inhibitor/antagonist as defined in any one of claims 1 to 19.

21. The method of claim 20, wherein said calmodulin inhibitor/antagonist is selected from the group consisting of bepridil or a pharmaceutically acceptable salt thereof; trifluoperazine or a pharmaceutically acceptable salt thereof, chlorpromazine or a pharmaceutically acceptable salt thereof, W7 or a pharmaceutically acceptable salt thereof and cetiedil or a pharmaceutically acceptable salt thereof.

22. The method of claim 20, wherein said calmodulin inhibitor/antagonist is selected from the group consisting of a phenothiazine compound, a butyrophenone compound, a diphenylbutylamine compound, (+) and (-) butaclamol, clozapine, cis-and trans-chlorprothixene and cis- and trans- flupenthixol or a pharmaceutically acceptable salt or a pharmaceutically acceptable ester thereof.

23. The use of any one of claims 1 to 19 or the method of any one of claims 20 to 22, wherein said neurodegenerative disease is a disorder associated or caused by pathological processing of amyloid precursor protein (APP).

24. The use or the method of claim 23, wherein said neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), Down's syndrome, hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson's disease, ALS (amyotrophic lateral sclerosis), Creutzfeld Jacob disease, HIV-related dementia and motor neuropathy.

25. The use or the method of claim 24, wherein said neurodegenerative disease is AD.