EP2797591A1 - Utilisation de médicaments calcilytiques en tant qu'approche pharmacologique vis-à-vis du traitement et de la prévention de la maladie d'alzheimer, de troubles associés à la maladie d'alzheimer et de neuropathies associées au syndrome de down - Google Patents

Utilisation de médicaments calcilytiques en tant qu'approche pharmacologique vis-à-vis du traitement et de la prévention de la maladie d'alzheimer, de troubles associés à la maladie d'alzheimer et de neuropathies associées au syndrome de down

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EP2797591A1
EP2797591A1 EP11817420.0A EP11817420A EP2797591A1 EP 2797591 A1 EP2797591 A1 EP 2797591A1 EP 11817420 A EP11817420 A EP 11817420A EP 2797591 A1 EP2797591 A1 EP 2797591A1
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alzheimer
disease
casr
drug
tau
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Ubaldo Armato
Ilaria Pierpaola DAL PRA'
Anna Maria CHIARINI
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to a novel pharmacological treatment of both familial early onset and sporadic late onset Alzheimer's disease (AD), AD-related disorders and Down's syndrome-coupled neuropathies.
  • the present invention concerns a novel therapeutic use for treating such illnesses of a class of drugs, the calcilytics, which by inhibiting the calcium-sensing receptor (CaSR) signaling in all types of brain cells prevent:
  • NTFs neurofibrillary tangles
  • calcilytics block at least three pathogenetic mechanisms favoring the development and progression of AD, i.e.
  • this invention proposes a totally new and different set of therapeutic indications for calcilytics, namely AD, AD-related disorders, and Down's syndrome neuropathies.
  • this invention is about a class of chemical compounds, the calcilytics, which by themselves can effectively inhibit or negatively modulate the functioning of the CaSR in all types of human brain cells, namely neurons, astrocytes, oligodendrocytes, microglia, ependimocytes, brain vascular endothelial cells, and brain stem cells, thereby
  • the present invention also relates to allosteric and/or orthosteric CaSR inhibitors (calcilytics) so chemically modified as to be able, whatever be their way of administration, to cross the brain blood barrier (BBB) efficiently and thus to reach the CaSRs located on the outer or inner membranes of all types of human nerve cells, thereby constituting a new method for the treatment of AD or AD- related disorders or Down's syndrome neuropathies.
  • allosteric and/or orthosteric CaSR inhibitors so chemically modified as to be able, whatever be their way of administration, to cross the brain blood barrier (BBB) efficiently and thus to reach the CaSRs located on the outer or inner membranes of all types of human nerve cells, thereby constituting a new method for the treatment of AD or AD- related disorders or Down's syndrome neuropathies.
  • calcilytics may be combined, though not obligatorily so, with any other current or future therapies of AD, of AD-related disorders, and of Down's syndrome neuropathies.
  • AD related disorder includes senile dementia of AD type (SDAT), frontotemporal dementias, vascular dementia, Parkinson's disease, Lewis body dementia, mild cognitive impairment (MCI), pre-MCI conditions, age-associated memory impairment (AAMI) and problems linked to ageing, post-encephalitic Parkinsonism, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Down's syndrome neuropathies.
  • SDAT senile dementia of AD type
  • frontotemporal dementias vascular dementia
  • Parkinson's disease Lewis body dementia
  • MCI mild cognitive impairment
  • pre-MCI conditions pre-MCI conditions
  • AAMI age-associated memory impairment
  • problems linked to ageing post-encephalitic Parkinsonism
  • ALS amyotrophic lateral sclerosis
  • MS multiple sclerosis
  • Down's syndrome neuropathies include senile dementia of AD type (SDAT), frontotemporal dementias, vascular dementia, Parkinson's disease, Lewis body dementia, mild cognitive impairment (MCI), pre-MC
  • treatment incorporates in particular the control of illness progression and accompanying symptoms.
  • the term "increase” comprises any upsurge in level of the studied biological parameter as compared to the existing level in the patient. Such an improvement may comprise a return to normal levels or a lesser increase still adequate to recover the patient condition. Such an increase can be assessed or substantiated using accepted biological tests, such as illustrated in the experimental section.
  • inhibitor indicates any diminution in the contemplated biological parameter as related to the existing activity in the subject. Such reduction may involve a partial lessening, e.g., from 5-to-30%, which is adequate to better the patient's complaint, as well as more substantial reductions, e.g., from 30- 70% or more complete inhibition, e.g., above 50%. The inhibition can be appraised or substantiated using established specific biological tests, such as reported in the experimental section.
  • the term "combination or combinatorial treating/therapy” designates a treatment in which at least two or more drugs are co-administered to a patient to elicit a biological effect.
  • the at least two drugs may be administered together or separately, at the same time or in succession.
  • the at least two drugs may be given via different routes and protocols. As a result, although they may be formulated together, the drugs of a combination may also be formulated separately.
  • DeKosky ST Scheff SW. Synapse loss in frontal cortex biopsies in Alzheimer disease: correlation with cognitive severity. Ann Neurol. 1990; 27(5) :457 -464. 10. Scheff SW, Price DA. Synapse loss in the temporal lobe in Alzheimer disease.
  • AD & frontotemporal dementia mutation database Univ Antwerp. Accessed on September 20, 2011.
  • ADAM9 Asai MC, Hattori B, Szabo N, et al. Putative function of ADAM9, ADAM 10, and ADAM17 as APP alpha-secretase. Biochem Biophys Res Commun. 2003;
  • Vassar R Bennett BD, Babu-Khan S, et al. Beta-secretase cleavage of Alzheimer amyloid precursor protein by the transmembrane aspartic protease BACE. Science. 1999; 286(5440) :735 -741.
  • 46. Yan R, Bienkowski MJ, Shuck ME, et al. Membrane-anchored aspartyl protease with Alzheimer disease beta-secretase activity. Nature. 1999; 402 (6761) :533 - 537.
  • Presenilin 1 regulates epidermal growth factor receptor turnover and signaling in the endosomal-lysosomal pathway. /
  • Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway.
  • Alzheimer's and Parkinson's diseases Apoptosis. 2010; 15(ll):1354-63.
  • Drechsel DN Hyman AA, Cobb MH, et al. Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau. Mol Biol Cell. 1992; 3(10):1141-54.
  • mice develop age-related A beta deposits and neuropil abnormalities, but no neuronal loss in CA1. / Neuropathol Exp Neurol 1997; 56:965-73. 10.1097/00005072- 199709000-00002.
  • Brown EM The calcium-sensing receptor: physiology, pathophysiology and
  • AD Alzheimer's disease
  • ADAM ADAM, a-disintegrin and metalloproteinase
  • BBB blood brain barrier
  • CNS central nervous system
  • GCH1 GTP cyclohydrolase-1
  • GABABR ⁇ -aminobutyric acidB receptor
  • GPR G-protein coupled receptor
  • GSK-3 glycogen synthase kinase-3
  • MBP myelin basic protein
  • NAHA normal adult human cortical astrocyte
  • NAHN normal adult human cortical neuron
  • NSHPT neonatal severe (primary) hyperparathyroidism
  • VEGF vascular endothelial growth factor
  • AD Alzheimer's disease
  • LOAD sporadic late onset
  • AD neuropathologic lesions typically progress according to a neurotransmitter-specific way, which is via cholinergic (the most vulnerable ones) ⁇ glutamatergic ⁇ GABAergic synaptic terminals (15).
  • the "amyloid cascade hypothesis” indicates as the primary triggers of the neurodegenerative process the buildup and aggregation of the amyloid precursor protein (APP) -derived, whole or truncated ⁇ 42 peptides (14,16); this has been the most broadly accepted explanation of AD onset and progression; conversely, (ii) the "neuronal cytoskeletal degeneration hypothesis" (17, 18) conjectures that the accretion of hyperphosphorylated and/or truncated (mutated) microtubule-associated Tau proteins causes the accumulation of poorly soluble NFTs on the microtubules.
  • APP amyloid precursor protein
  • NFTs in their turn, induce microtubular dysfunctioning, thereby altering several critical nerve cell functions, like axonal vesicular transport, thereby expediting ⁇ cytotoxicity and, eventually, neuronal and astrocytic death (19-23).
  • AD researchers have principally paid attention to the mechanisms of ⁇ cytotoxicity, whereas Tau protein role(s) has(ve) received less attention. Yet, and notably, ⁇ peptides can also induce Tau protein hyperphosphorylation via the activation of GSK- ⁇ (22,23).
  • the known several substrates of GSK- ⁇ include presenilins and hypoxia inducible factor (HIF)-l (21,24). Additionally, the amount of synaptic losses and the distribution and number of NFTs correlate with the severity and duration of cognitive impairment in AD patients (18,25).
  • EOAD sporadic early onset AD
  • PS1 or PSEN1 presenilin-1
  • PSE2 or PSEN2 presenilin-2
  • EOAD cases of which about 50% are due to mutations within PSl and/or PS2 genes, are around 5% of all AD cases.
  • Identified mutations in PSl and PS2 genes amount to more than 180, and to 50 in the APP gene (31).
  • APP locus duplication (32) and polymorphisms in the APP promoter site (33) can also cause EOAD.
  • soluble ⁇ ( ⁇ ) oligomers but not monomers, rather than ⁇ aggregates, are the true neurotoxins capable of inhibiting hippocampal long-term potentiation (LTP), a synaptic correlate of memory and learning (34), possibly via a disproportionate activation of extra-synaptic N-methyl-D-aspartate (NMDA) receptors (35,36).
  • LTP hippocampal long-term potentiation
  • NMDA N-methyl-D-aspartate
  • apolipoprotein E apolipoprotein E
  • various lines of genetic and molecular evidence support the "amyloid cascade hypothesis" for EOAD, but the picture about LOAD is less clear.
  • the numbers of amyloid plaques containing ⁇ may not parallel dementia severity and even abundant plaques have been observed in the brains of subjects with no sign of cognitive impairment (38,39).
  • sAfi oligomer levels correlate better with dementia severity and are more toxic than ⁇ aggregates in vitro (38-41).
  • ⁇ peptide generation from APP occurs via the sequential proteolytic cleavage by ⁇ - and ⁇ -secretases: this is the amyloidogenic pathway; byproducts of this pathway are the secreted ectodomain sAPPa and the intracellular APP domain (38).
  • APP ectodomain, sAPPa derives from, may mediate the physiologic activities of APP (42,43).
  • ADAM a-disintegrin and metalloprotease
  • PS1 is also involved in lysosomal-endosomal trafficking, a function distinct from the ⁇ -secretase activity.
  • PS1 deficiency impairs lysosomal- endosomal trafficking increasing epidermal growth factor (EGF) levels in fibroblasts (50) and telencephalin levels in hippocampal neurons (51).
  • EGF epidermal growth factor
  • telencephalin levels in hippocampal neurons
  • autophagic-lysosomal proteolysis is entirely and specifically stuck by PS1 deletion (52).
  • the acidification of lysosomal contents also needs PS1 as, by acting like an endoplamic reticulum (ER) chaperone protein, PS1 targets a vacuolar-type proton pump (v-ATPase) to lysosomes.
  • ER endoplamic reticulum
  • Non-lysosomal localization of v-ATPase due to PS1 mutations causes an autophagic/lysosomal dysfunction leading to EOAD independently of APP proteolysis (52).
  • Aging and disruption of lysosomal- endosomal activity by a block of cholesterol transport also modifies APP processing, leading to a surge in ⁇ synthesis via a decreased APP proteolysis by ⁇ - secretase and an enhanced proteolysis of APP C-terminal fragment (53).
  • Even a redistribution of PS1 coupled with an aberrant cholesterol transport can enhance ⁇ production (54).
  • sA 4kDa monomers assemble first in sAfi oligomers of increasing size (from 8 kDa to > 100 kDa) and next in insoluble ⁇ aggregates forming the extracellular amyloid plaques in the brains of AD patients (55).
  • Even truncated ⁇ peptides are found in postmortem AD brains (55,56) as only a fraction of ⁇ is full-length ⁇ - 4 ⁇ or ⁇ - 42 /43; in fact, N-terminally truncated variants of ⁇ ( ⁇ 3- 42 and ⁇ - 42 ) prevail in senile plaques of AD and Down's syndrome brains (57,58).
  • Truncated ⁇ 3- 42 exhibits a greater aggregation proclivity and is more cytotoxic in vitro (59-61). Truncated ⁇ 3- 42 is neurotoxic even in vivo (62). Initially, the belief prevailed that only extracellular ⁇ could elicit cytotoxic effects, but current evidence shows that ⁇ accumulated inside neurons and astrocytes can also act as a neurotoxin (63-65). In regions affected by AD and elsewhere in AD brains, neurons stockpile ⁇ 42 before any extracellular ⁇ deposition and intracellular NFT formation occur (66-68).
  • cytotoxic sAfi oligomers are synthesized intracellularly and found within cytoplasmic processes and synapses of neurons (69,70) and astrocytes (65) .
  • any significant neuronal losses were headed by sizeable accretions of intraneuronal ⁇ peptides, predominantly ⁇ 42 (71).
  • Neuronal death could also be caused by a progressive intraneuronal accrual of N-truncated ⁇ 3- 42 (72).
  • intraneuronally accumulated ⁇ 42 causes the loss of synaptic terminals, and this damage correlates better with cognitive decline than plaque formation or NFT load or neuronal death.
  • the notion has become increasingly accepted that the key event leading to initial cognitive dysfunction in AD is the ⁇ -induced loss of synaptic terminals (73-77),
  • extracellular sAfi oligomers can cause at least part of the loss of synaptic terminals observed in brain regions free of ⁇ deposits in various tg AD mouse models (78), Interestingly, extracellular sAfi oligomers can bind the cellular prion protein (PrP c ) with high affinity: hence PrP c may act as a receptor for sAfi oligomers (79) and provoke an inhibition of long- term potentiation (LPT), a toxic mechanism underlying synaptic loss and memory dysfunction in AD (80,81).
  • LPT long- term potentiation
  • both sAfi oligomers and ⁇ aggregates do bind several distinct cell membrane receptors, i.e.
  • PrP c can bind p75 NTR and cause neuronal death (85) - the Frizzled receptor (86), the insulin receptor (87), the NMDA receptor (88,89), the nicotinic acetylcholine receptor (90), the receptor for advanced glycation endproducts (RAGE) (91), and the calcium-sensing receptor (CaSR) (92,93, and see also below), the aberrant signalings of which alter various neuronal and astrocytic functions and may help induce cell death.
  • sA oligomers can also interact with scaffold proteins, like Homerlb and
  • Shankl that couple postsynaptic density (PSD)-95 protein with ionotropic and metabotropic glutamate receptors in the postsynaptic density regions (94,95), inducing transmembrane channel formation (96,97), and mitochondrial dysfunction (98).
  • a dysfunctioning lysosomal-autophagic system would cause the accrual of aged and damaged mitochondria, which may release proapoptotic factors (99).
  • Both mammalian brain ageing and AD onset and progression in human patients associate with altered expression and function of genes at the mitochondrial level (100).
  • ⁇ immunotherapy has impeded the development of neuropathy and cognitive deficits (101) and LTP inhibition engendered by exposure to sAfi oligomers (102).
  • Tau pathology is a consequence of increased ⁇ generation and accumulation (104).
  • Tau is a soluble microtubule-associated phosphoprotein (MAP) strongly expressed in neurons (105).
  • MAP microtubule-associated phosphoprotein
  • Tau structure encompasses a C-terminal repeat domain binding microtubules, a central proline- rich domain, and an N-terminal domain interacting with membranes and/or other proteins. Tau is rapidly and reversibly phosphorylated by various protein kinases and phosphatases (106).
  • Tau is typically hyperphosphorylated and is deposited as poorly soluble intracellular aggregates, the NFTs, in which Tau predominates, and hyperreacts to anti-phospho-Tau specific antibodies (107, 108).
  • the main Tau kinase is GSK-3 in both normal and AD brains.
  • 6 Tau isoforms are found stemming from an alternatively spliced single gene, of which 4RTau and 3RTau are the most intensely expressed and phosphorylated ones (109,110).
  • Hyperphosphorylated Tau detaches from tubulin increasing microtubule instability and easing microtubule disassembly (111,112).
  • dephosphorylated Tau binds more forcefully to tubulin, speeds microtubule elongation up, stabilizes microtubules, and also associates with plasma membranes (113).
  • Membrane-associated Tau may interact with Src-family kinases and phospholipase C- ⁇ , thereby affecting neurodegenerative processes (114).
  • Fyn a non-receptor tyrosine kinase
  • Fyn a non-receptor tyrosine kinase
  • NR2B subunit 2B
  • Tau associates with mammalian solute transport protein-2 (SUT2) mainly located in SC35-positive nuclear speckles, where it plays a role in mRNA processing (116).
  • SUT2 mammalian solute transport protein-2
  • SUT2 may affect the pathogenesis of tauopathies (117,118).
  • Tau protein is endowed with 80 serine and threonine sites that can be phosphorylated: 37 of such sites are phosphorylated by GSK-3 (119), as many others by casein kinase 1 (CK1), and but a few by cyclin-dependent kinase-5 (cdk-5) (109).
  • GSK-3 plays a major role in pathological (and physiological) phosphorylation of Tau protein.
  • GSK-3 is found in two isoforms, GSK-3a (483 amino acids, 51 kDa in humans) and GSK-3 (433 amino acids, 47 kDa in humans), each encoded by a distinct gene (on chromosome 19 and 3, respectively) (120). Both GSK-3 isoforms phosphorylate Tau within the pyramidal neurons of the hippocampus (121). Two variants of GSK-3 exist, GSK-3 i and GSK-3 2: the latter abounds within somata, dendrites and axons of neurons (122). GSK-3a and GSK-3P play distinct functional roles even though they share some substrates and may partially compensate for each other (123).
  • GSK-3a plays a unique role in glucose metabolism (124), life-essential GSK-3P (125) is strongly expressed in neurons and astrocytes, and its activity-regulating phosphorylations are affected by an exposure to extracellular ⁇ peptides (Figs. 3 and 4): hence, GSK-3P is the principal Tau protein kinase in adult human brain neurons and astrocytes.
  • GSK-3 ⁇ kinase activity is controlled through the phosphorylation and de-phosphorylation of some of its serine and tyrosine residues.
  • the constitutive activity of GSK-3 is kept up by the phosphorylation of Tyr 279 ; conversely, the phosphorylation at Ser 9 downregulates its enzymatic activity (see for references: 126) and reduces Tau phosphorylation levels (127).
  • GSK-3 activity is enhanced in cultured hippocampal neurons (128) as well as in normal adult human astrocytes (NAHAs) (see Figs. 3 and 4) and normal adult human neurons (NAHNs) isolated from the temporal cerebral cortex (unpublished results from our laboratory).
  • exogenous GSK-3 phosphorylates nuclear SC35 protein, which favors the splicing of Tau exon 10 and reduces the expression of 4RTau— events suppressed by lithium, an inhibitor of GSK-3 (and of other enzymes) activity (129).
  • GSK-3 The promotion of normal microtubule assembly by Tau is diminished when Tau is phosphorylated by GSK-3 (130). But the aggregated Tau proper of diseased brains is poorly soluble, forms NFTs, and reacts more intensely to specific phospho-Tau antibodies (131). In AD brains, 45 phosphorylation sites have been recognized on Tau vs. 16 Tau sites in control brains; such sites partially differ (109,110). GSK-3 is the main candidate kinase for some 27 of the more than 40 phosphorylation sites recognized in poorly soluble Tau, since GSK-3P co-localizes with NFTs in AD and AD-related disorders (119,132).
  • GSK-3 activity decreases Tau phosphorylation levels and protects cultured primary neurons from dying (133).
  • increases in Tau phosphorylation due to GSK-3 activity are indicative of reduced nerve cell healthy functions (e.g., axonal transport in neurons, etc.) (134).
  • Tau phosphorylation by GSK-3 has been investigated in various tg mouse models (135). In mice, overexpressed GSK-3 heightens Tau phosphorylation, reactive gliosis, neuronal death, all features proper of human tauopathies (136).
  • GSK-3 -phosphorylated Tau The pathological role of GSK-3 -phosphorylated Tau is also supported by the results obtained in mouse tg AD or tauopathies models, in which GSK-3 inhibition lessens Tau phosphorylation and aggregation and axonal degeneration (137-140).
  • GSK-3 inhibition lessens Tau phosphorylation and aggregation and axonal degeneration (137-140).
  • Tideglusib NP-12
  • PSP progressive sopranuclear palsy
  • tauopathy 141,142.
  • AD becomes symptomatic once it has reached the point of discrete brain tissue damage.
  • pre-MCI and MCI pre-symptomatic stages
  • mice models accumulating large amounts of ⁇ plaques in their brains do not show any sign of concurrent neurodegeneration.
  • Such PDAPP (144), Tg2576 (145), TgCRND8 (146), and APP23 (147) mice do not endorse the "amyloid cascade hypothesis" (14,16).
  • These negative findings in the face of ⁇ plaque accumulation might be the upshot of APP-mutated mouse neurons being bereft of pathways crucial for ⁇ toxicity; alternatively, the processes of ⁇ production and aggregation proper of sporadic LOAD cases and their pathological consequences cannot be replicated in such tg mouse models (148).
  • AD cerebral amyloid angiopathy
  • ⁇ may, directly or indirectly, interact with Tau to accelerate NFTs formation and microtubular dysfunction.
  • mice of the tg AD models have their own APP and APP-processing enzymes, which may tamper with the production of the different ⁇ -related peptides encoded by the human transgenes.
  • the genetic backgrounds of the different tg AD mice models may muddle crucial aspects of human AD.
  • the notion has been surmised that additional relevant information on AD pathophysiology should be gained from other natural, i.e. non-tg, animal models, like chick embryos and dogs, the enzymatic machineries for APP processing of which are almost identical to human ones (154).
  • AD Alzheimer's disease
  • AD pathophysiology can be investigated via still other experimental models, the ideal ones being human nerve cells, particularly normal adult human astrocytes (NAHAs) and normal adult human neurons (NAHNs), isolated from tissue fragments of the temporal cerebral cortex and set into in vitro cultures.
  • NAHAs normal adult human astrocytes
  • NAHNs normal adult human neurons
  • Astrocytes and neurons isolated from AD brains could be of use as well, keeping in mind that their prolonged permanence and survival in a chronically cytotoxic and inflammatory environment might have caused epigenetic changes in their biological features and responding capabilities to specific stimuli.
  • Even isolated brain stem cells or even engineered iPS cells might be induced to differentiate into any kind of brain nerve cells, including neurons and astrocytes (155).
  • NAHAs and NAHNs responses in the presence of ⁇ peptides and/or proinflammatory cytokines should help elucidate critical steps in the pathophysiology of AD.
  • the present applicants have set up such cultures from brain tissue fragments of the temporal lobes of people with perforating head injuries and performed several kinds of experiments with the aims just mentioned (65,156- 160).
  • CM- trio— or with exogenous ⁇ peptides, NAHAs response was quite complex as it involved:
  • GCH1 was shown to associate with adaptor/regulator molecules involved in G-protein-coupled receptor signaling, protein serine/threonine phosphatase 2Cb (PP2Cb), and serine-threonine kinases, like Ca 2+ - calmodulin kinases (CaMKs), casein kinase Ila (CK-IIa), cAMP-dependent kinases A (PKAs), and mitogen-activated protein kinases (MAPKs) (158).
  • CaMKs Ca 2+ - calmodulin kinases
  • CK-IIa casein kinase Ila
  • PDAs cAMP-dependent kinases
  • MAPKs mitogen-activated protein kinases
  • the present applicants' observations suggested that the ⁇ 42 released from neurons in the AD brain can recruit associated astrocytes via HIF-lot'HIF- ⁇ signaling into the pool of ⁇ 42 - producing and ⁇ -releasing cells.
  • astrocytes are 10-fold more numerous that neurons in the human brain: hence, once exposed to ⁇ 42 the astrocytes could most effectively contribute their share to the accruing of extracellular 5 ⁇ 42 oligomers and of ⁇ 42 plaques, thus favoring AD development and progression.
  • ⁇ -exposed astrocytes besides neurons, could significantly help start and maintain a vicious circle leading to progressive ⁇ 42 accumulation in the AD brain and, increasingly, to its aftereffects, i.e. loss of synaptic terminals, synapse-deprived neurons held incommunicado (so called “undead” neurons), NO overproduction leading to highly toxic peroxynitrite (HNOO ) formation, activation of astrocytes and of microglia, chronic neuroinflammation, CAA, apoptosis of neurons, astrocytes, oligodendrocytes, myelin sheaths dissolution releasing toxic MBP, etc.
  • HNOO highly toxic peroxynitrite
  • CaSR Calcium-Sensing Receptor
  • the CaSR (or CAR; FHH; FIH; HHC; EIG8; HHC1; NSHPT; PCAR1; GPRC2A; MGC138441) gene is highly conserved from zebrafish to humans (161). It encodes a protein belonging to family C of G protein-coupled receptors (GPRs), which also includes 8 metabotropic glutamate receptors (mGluRs), 2 ⁇ -aminobutyric acidB receptors (GABABRS), various taste receptors, and the promiscuous GPRC6A receptor (162).
  • GPRs G protein-coupled receptors
  • Family C GPCRs have no amino acid sequence homology with the remaining GPCR families (163,164).
  • Family C GPCRs are made up by an extracellular amino (N) -terminal domain (ATD), seven transmembrane a-helices (TM1-TM7) connected by loops placed inside and outside the cell (altogether indicated as the 7TM region), and an intracellular carboxy (C)-terminus; a cysteine-rich region (CRR) including 9 conserved cysteine residues joins the ATD with 7TM domains (161).
  • the CaSR constitutively forms homodimers (CaSR/CaSR) or heterodimeric (CaSR/mGluR) complexes joined by noncovalent and covalent bonds (165).
  • the huge (-600 amino acids) extracellular ATDs of the CaSR homodimer comprise the binding (or orthosteric) site for the specific ligand, i.e. Ca 2+ .
  • This orthosteric site is placed between the two lobes of a clam shelf structure indicated as the "Venus flytrap" domain (166); the filamin-binding intracellular C-termini have 10 sites assumed to be phosphorylated by protein kinase C (PKC) (167).
  • PKC protein kinase C
  • ligands besides Ca 2+ like Mg 2+ , Gd 3+ , Ba 2+ , polyamines, and neomycin (an antibiotic), bind to the cleft between the two ATD lobes (the specific polar amino acid residues involved have been identified) of the homodimeric CaSR complexes: this bond twists the conformation of the homodimer rearranging the two 7TM regions and allowing G proteins to link to the intracellular CaSRs tails (168). Moreover, Ca 2+ also binds a second orthosteric site in the 7TM domain of the CaSR (169).
  • CaSRs undergo allosteric modulation by a lot of endogenous ligands and factors, like pH, ionic strength, Na + concentration, and aromatic L-a-amino acids (170).
  • endogenous ligands and factors like pH, ionic strength, Na + concentration, and aromatic L-a-amino acids (170).
  • aromatic L-a-amino acids bind an allosteric site nearby the orthosteric site in the ATD (171), and in the presence of Ca 2+ act as true allosteric potentiators of CaSR signaling (170).
  • the CaSR can bind ⁇ peptides being activated by them (92,93).
  • the CaSR can be antithetically modulated by synthetic allosteric modulators belonging to two classes:
  • calcilytics examples of which are NPS 89626, NPS 2143, Calhex 231, BMS (Bristol-Meyers-Squibb) compound 1, and JKJ05 (173-182).
  • NPS 2143 and Calhex 231 bind largely overlapping extracellular portions of the 7TM, in which Glu 837 is a crucial residue (182).
  • NPS 2143 forms hydrophobic contacts and ⁇ -stacking with Phe 668 residue in TM2, Phe 684 , Phe 688 , Arg 680 in TM3, and He 841 in TM7 (183,184).
  • Calhex 231 interacts with part of these residues.
  • NPS 2143 and Calhex 231 are structurally related phenylalkylamines endowed with an NL + and bind to a common allosteric site at the 7TM.
  • BMS compound 1 and its correlated JKJ05 have a different site of interaction and the He 841 residue is crucial for their inhibitory activity (179,185).
  • the four transmembrane helices TM3, TM5, TM6 and TM7 form the binding pocket for CaSR allosteric modulators (186).
  • the extracellular Ca 2+ level ([Ca 2+ ] e ) is tightly controlled at the gut (uptake), bone (storage), and kidney (excretion) levels via the signaling of the respective CaSRs (187).
  • the CaSR activates several intracellular signal transduction pathways, through which it modulates a wide spectrum of cellular activities (186-188).
  • PTH parathyroid hormone
  • renal cation handling safeguard mineral ion homeostasis
  • CaSR knock-out mice exhibit highly increased PTH levels and parathyroid cellular hyperplasia, thereby revealing a direct control of the CaSR on parathyroid cell growth and PTH release (189). Being broadly expressed, the CaSR plays other physiological roles, e.g. in gut hormone secretion control (190).
  • CaSR In human NAHAs and NAHNs (65,92,93,156,157), CaSR also reacts with exogenous ⁇ peptides thereby inducing through its signaling the de novo synthesis and secretion of NO, of endogenous ⁇ 42 , and Tau hyperphosphorylation by an ⁇ /CaSR-activated GSK-3p.
  • Neonatal Severe primary
  • Hyperparathyroidism an autosomal recessive disorder due to loss-of-function mutations in the CASR gene on chromosome 3ql3 (191).
  • HHC1 Familial Hypocalciuric Hypercalcemia (HHC1; FHH) due to a lessened sensitivity to Ca 2+ at the CaSR (192).
  • anti-CaSR autoantibodies can inhibit or activate CaSR signaling producing clinical syndromes like FHH or Autosomal Dominant Hypocalcemia (ADH), respectively (194,195).
  • CaSR signaling producing clinical syndromes like FHH or Autosomal Dominant Hypocalcemia (ADH), respectively (194,195).
  • ADH Autosomal Dominant Hypocalcemia
  • Reduced expression of the CaSR inside parathyroid glands occurs in primary (parathyroid cancer) or in secondary (uremic) hyperparathyroidism (PHPT or SHPT, respectively) with excessive PTH secretion (196,197).
  • calcimimetic Cinacalcet has been approved by the FDA for the treatment of PHPT and SHPT, but its approved use is likely to be extended to other forms of hyperparathyroidism, like FHH and NSPHP, and to hypercalcemia due to CaSR- inhibiting autoantibodies (198) .
  • calcilytics e.g. NPS 2143, BMS compound 1, etc.
  • the first proposed use of calcilytics was the treatment of human osteoporosis.
  • the calcilytic NPS 2143 was found to increase PTH levels for several hours: this action of NPS 2143 does not change actual bone density as it accelerates both bone formation and bone destruction (199).
  • the calcilytic BMS compound 1 was reported to induce shorter-lasting (1 hour) blood PTH surges, which might only stimulate bone formation and hence counter osteoporosis. But, further either preclinical or clinical investigations on the effects of BMS compound 1 were neither carried out nor published.
  • Other suggested therapeutic uses of calcilytics were hypocalcemia due to CaSR-activating autoantibodies and ADH (198). Calcium-Sensing Receptor, the brain and Alzheimer's Disease
  • CNS central nervous system
  • CaSR mRNA express various levels of CaSR mRNA (200,201).
  • all types of nerve cells i.e. neurons, astrocytes, oligodendrocytes, microglia, neural stem cells, ependymal cells and brain vascular endothelial cells— are engaged in CaSR expression.
  • the complex physiological roles played by CaSR in the human CNS like oligodendocyte development (202), dendrites and axon growth (203), and secretion of MCP-1, MCP-3, and CXCL10 by GnRH neurons (200,204), are still being unraveled.
  • CaSRs play roles in neuroinflammatory and/or neurodegenerative conditions in the human CNS, including AD (93).
  • CaSRs also bind L-amino acids, and this causes specific patterns of intracellular Ca 2+ oscillations (205,206).
  • heightened concentrations of L-phenylalanine activate the CaSR, inducing neuronal cytotoxicity in cases of phenylketonuria (207).
  • astrocytes 156-160,204
  • oligodendrocytes 208
  • microglia 209
  • brain vascular endothelial cells 210) that hints the most significant neuropathologic implications. It should be recalled here that these non-neuronal cell types not only are 10-fold more numerous than neurons, but by themselves are also directly involved in neuroinflammatory and neurodegenerative processes (211).
  • adult human astrocytes once considered to be the brain's "gluons” merely supporting neurons via the control of the BBB, not only actively partake in key physiological processes like the coordinated firing of groups of neurons and the local stimulation of the blood flow needed to sustain this coordinated firing (which is the basis of MRI functional imaging) (212), but even play significant roles in neuroinflammatory and neurodegenerative diseases (160,212,213).
  • sA and ⁇ peptides interact with the plasma membranes not only of neurons residing in the hippocampi and in other brain areas, but even of astrocytes, oligodendrocytes, microglial cells, ependimocytes, neural stem cells, and endothelial cells, which altogether are about 10-fold more numerous than neurons (215).
  • sA oligomers and ⁇ aggregates possess, like polyamines, a regular array of positive charges to which anionic dyes bind (e.g., Congo Red) (216).
  • CaSRs are expressed by all the nerve cell types: hence, they all (and not the neurons only) are the targets of the cytotoxic effects of sA oligomers and ⁇ aggregates (215).
  • the present invention is based on the observation that no consideration or suggestion so far has been given for the use of calcilytics in Alzheimer's Disease, AD-related neurodegenerative disorders, Down Syndrome neuropathies or other neurodegenerative disorders of any kind.
  • a purpose the present invention relates to providing method of treating Alzheimer's disease or a related disorder, the method comprising simultaneously, separately or sequentially administering to a subject in need thereof a drug combination that inhibits CaSR signaling and/or a drug that modulates synaptic transmission and/or a drug that modulates angiogenesis and/or a drug that reduces cholesterol levels and/or a drug that modulates cell stress response.
  • a purpose of the present invention resides in providing a method of producing drug(s) for treating Alzheimer's disease or an AD-related disorder or Down's syndrome neuropathologies, the method involving a step of testing candidate drug(s) for activity as inhibitor of CaSR signaling and selecting candidate drug(s) that by blocking CaSR signaling curtails the endogenous overproduction and secretion of NO by glial cells and of ⁇ peptides by neurons and astrocytes, thereby preventing or attenuating inflammatory tissue response and cytotoxic effects on nerve cells and brain endothelial cells proper of AD, AD- related disorders, and Down's syndrome neuropathologies.
  • another purpose of the present invention concerns a method of delivering a drug association for treating Alzheimer's disease or an AD-related disorder or Down's syndrome neuropathology, the method encompassing the combination of a drug that acts as a CaSR inhibitor on all types of human brain nerve cells and one or more drugs improving synaptic transmission and/or favoring brain neurogenesis (e.g.
  • leptin or leptin-mimicking compounds and nerve growth factor (NGF), or brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3), or Neurotrophin-4 (NT-4 or NT5, NTF4, and NT-4/5) and any compound/drug mimicking their activities) and/or brain neoangiogenesis and/or preventing or mitigating ⁇ -elicited cytotoxic effects for simultaneous, isolated or sequential administration to subjects in need thereof.
  • nerve growth factor or brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3), or Neurotrophin-4 (NT-4 or NT5, NTF4, and NT-4/5) and any compound/drug mimicking their activities
  • NTF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • Neurotrophin-4 Neurotrophin-4 or NT5, NTF4, and NT-4/5) and any compound/drug mimicking their activities
  • brain neoangiogenesis and/or preventing or
  • the present invention relates to a class of drugs, the calcilytics, for use in the treatment of Alzheimer's disease, AD-related disorders and Down's syndrome-coupled neuropathies as recited in claim 1.
  • the present invention relates to compositions and methods for treating AD or AD-related disorders or Down's syndrome neuropathies in a subject in need thereof, using particular drugs or drug combinations that by preventing the accumulation of ⁇ oligomers and fibrils in the affected regions of the brain and its collateral effects (synaptic deactivation, activation of astrocytes and microglia, migration of blood leukocytes into the brain, inflammatory responses, nerve cell stress responses, neuron apoptosis, decline of cognitive functions, tec.) ameliorate synapse functioning and/or increase neurogenesis and/or angiogenesis and/or prevent Alzheimer's disease progression.
  • the upshot of the pharmacological inhibition of the nerve cells CaSRs is the breaking up of an otherwise vicious self-amplifying cycle that would cause a progressive accumulation of sA oligomers and ⁇ aggregates in the brain extracellular spaces and intracellularly, and simultaneously the intracellular accumulation of NFTs, thereby preventing AD onset, development, and progression.
  • the specific (allosteric or orthosteric) inhibitors of the CaSR may also be combined with other kinds of present or forthcoming drugs used to treat AD, AD-related disorders, and Down's syndrome neuropathology, thereby embodying novel approaches to the treatment of the just mentioned ailments.
  • the pharmacological blockage of CaSR signaling in human brain neurons and astrocytes brought about by calcilytic drugs exerts several simultaneous beneficial and anti-AD effects on the two most representative types of human brain cells, i.e. neurons and astrocytes, by curtailing both the cytotoxic, proapoptotic, and proinflammatory actions engendered by a progressive ⁇ peptide accumulation in the brain tissue.
  • the calcilytic drug breaks the vicious circle through which exogenous ⁇ begets endogenous ⁇ , thereby further increasing exogenous ⁇ levels and further stimulating endogenous ⁇ synthesis and secretion, NFT accrual via Tau hyperphosphorylation by an ⁇ /CaSR-activated 05 ⁇ -3 ⁇ , NO overproduction and release by NOS-2 and ONOO formation, neuroinflammation, synaptic loss, neuronal death, and so on and so on, eventually leading to frank AD with increasing loss of cognitive functions that advances up to patient's death.
  • the calcilytic By suppressing the harming effects elicited by exogenous ⁇ and proinflammatory cytokines, the calcilytic also obviously attenuates the no longer needed anti- cytotoxic and pro-neo-angiogenic release of significant amounts of VEGF165 by the NAHAs. Importantly, the present applicants found that exogenously administered
  • ⁇ peptides like ⁇ 25 35 do induce the de novo synthesis and secretion of endogenous ⁇ 42 not only by NAHNs (our unpublished results), but even by NAHAs.
  • this ⁇ -triggered release of ⁇ 42 by such large pool of neurons (about 1:11 of total CNS cells) and astrocytes (about 10:11 of total CNS cells) can engender viciously recursive loops of self-amplifying ⁇ 42 production and release, i.e.
  • exogenous (extracellular) ⁇ and endogenous (intracellular) ⁇ can reciprocally heighten their levels, the upshot of which is the ⁇ self-induced, self-sustaining, progressive accumulation of sAfi oligomers first and of ⁇ aggregates later in the human brain eventually leading to clinically symptomatic and step-by-step worsening AD.
  • ⁇ peptides also enhance the production and release of NO and the formation of ONOO with their severe cell-damaging effects, and increase the activity of 05 ⁇ -3 ⁇ , the main Tau protein kinase, which results in nearly insoluble hyperphosphorylated Tau proteins forming microtubule- associated NFTs that cause critical microtubular dysfunctions, e.g. deep alterations of vesicular transport, in neurons and astrocytes.
  • the purpose of the present invention is to provide a new therapeutic approach for treating AD, AD-related disorders, and Down's syndrome neuropathies through the breaking of these viciously recursive loops of released ⁇ 42 further begetting ever more ⁇ 42 , the hyperproduction and release of cell- damaging NO/ONOO , and the progressive accumulation of GSK-3p- hyperphosphorylated Tau protein in NFTs, and the death of neurons.
  • This therapeutic target will be achieved through the administration, via whichever route (via oral, intranasal, subcutaneous and/or intramuscular and/or intestinal-rectal routes, via cutaneous patches and transepidermal and transdermal routes, via aerosols, etc.) of calcilytic (CaSR-inhibiting) drugs, be they given in the form of salts or pro-drugs or derivatives or as sustained release formulations thereof, that, by effectively crossing the BBB, can permeate the brain tissue and selectively antagonize the CaSRs expressed by all types of nerve cells and by the CNS endothelial cells.
  • calcilytic (CaSR-inhibiting) drugs be they given in the form of salts or pro-drugs or derivatives or as sustained release formulations thereof, that, by effectively crossing the BBB, can permeate the brain tissue and selectively antagonize the CaSRs expressed by all types of nerve cells and by the CNS endothelial cells.
  • Such CaSR-inhibiting calcilytics can be of entirely novel conception and synthesis or those already published (like NPS 89626, NPS 2143, Calhex 231, BMS compound 1, JKJ05) or other CaSR-inhibiting structurally similar but as yet unpublished compounds hitherto or in future synthesized with the aim to treat osteoporosis or hypocalcemia due to CaSR-activating autoimmune antibodies or ADH.
  • the inhibition the CaSR signaling by any of the calcilytic not only prevents cell damaging NO hyperproduction but even, and most importantly, concurrently blocks the two main mechanisms that are believed to favor AD onset and progression, that is (i) the synthesis, accumulation, and secretion of ⁇ 42 favored by exogenous ⁇ 42 , and (ii) the NFT-generating hyperphosphorylation of Tau protein on the part of a strongly activated GSK-3 , ultimately leading to neuronal death.
  • the CaSR-inhibiting calcilytic drugs will block the onset and/or halt the progression of the neuroinflammatory and neurocytotoxic events otherwise leading step by step to frank AD or AD-related diseases.
  • CaSR-inhibiting calcilytics of any kind be they allosteric or even orthosteric and binding any portion or amino acid sequence of the CaSR molecule, will represent by themselves the mainstay of new and effective therapeutic regimens for the treatment of AD, of AD-related disorders, and of the neurotoxic injuries accompanying Down's syndrome.
  • calcilytics of any kind may also be used in further combination with additional drugs, like stimulators of adult neurogenesis (e.g. leptin and leptin action mimicking drugs, and nerve growth factor (NGF), or brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3), or Neurotrophin-4 (NT-4 or NT5, NTF4, and NT-4/5) and any compound/drug mimicking their activities), or treatments presently used for AD (like the acetylcholinesterase inhibitors memantine, rivastigmine, galantamine, estrogen, antioxidants like selegiline, a-tocopherol [vitamin E], Ginkgo biloba extract, antidepressants like selective serotonin reuptake inhibitors [SSRIs], nonsteroidal antinflammatory drugs [NSAIDs],and HMG-CoA reductase inhibitors like statins, anticonvulsivants like phenytoin or carbamazepine, atypical antipsychotics
  • Fig. 1 shows the inhibitory effects of a paradigmatic CaSR-inhibiting (calcilytic) agent like NPS 2143 (abbreviated as NPS in the Figure) on the endogenous production and accumulation of ⁇ 42 oligomers and ⁇ 42 aggregates elicited by the exposure to exogenous ⁇ 2 5-35 on the part of early passage normal adult human astrocytes (NAHAs) set into in vitro cultures;
  • a paradigmatic CaSR-inhibiting (calcilytic) agent like NPS 2143 (abbreviated as NPS in the Figure)
  • NPS 2143 abbreviated as NPS in the Figure
  • Fig. 2 shows how the calcilytic NPS 2143 added together with exogenous ⁇ 2 5-35 (20 ⁇ ) totally suppresses the increases in extracellular secretion of ⁇ 42 ( ⁇ ⁇ - 42 in the Figure) on the part of the NAHAs otherwise elicited by the exposure to ⁇ 2 5-35 alone;
  • Fig. 3 shows how the activity of the main Tau protein Kinase, 05 ⁇ -3 ⁇ , is increased in NAHAs exposed to exogenous ⁇ 2 5-35 (20 ⁇ ) alone;
  • Fig. 4 shows the activity of 05 ⁇ -3 ⁇ , the main Tau protein Kinase, as indicated by the time- and treatment-corresponding ratios of the activating phosphorylation levels at Tyr 216 with respect to the inactivating phosphorylation levels at Ser 9 .
  • the isolated cells were planted into culture flasks (BD Biosciences, France) containing a medium consisting of 89% v / v of a 1:1 mixture of Ham's F-12 and MCDB 153 media (Sigma- Aldrich), 10% v / v heat-inactivated (at 56° C for 30 min) fetal bovine serum (FBS; BioWhittaker Europe, Belgium), and 1% V / V of a penicillin-streptomycin solution (Lonza, Italy).
  • a medium consisting of 89% v / v of a 1:1 mixture of Ham's F-12 and MCDB 153 media (Sigma- Aldrich), 10% v / v heat-inactivated (at 56° C for 30 min) fetal bovine serum (FBS; BioWhittaker Europe, Belgium), and 1% V / V of a penicillin-streptomycin solution (Lonza, Italy).
  • IGF-I Insulin-like growth factor-I
  • PDGF platelet-derived growth factor
  • the cells of these pure cultures were stably "locked” into the astrocyte phenotype; they only expressed astrocyte- specific markers such as glial fibrillary acid protein (GFAP) and glutamine synthase (GS). None of the cells expressed neuronal (enolase), oligodendrocytes' (galactocerebroside), microglia's (CD-68), or endothelial cells' (factor VIII) markers. These astrocytes proliferated slowly without added growth factors in serum-enriched Ham's F-12/MCDB 153 medium. But the serum was still needed and withdrawing it caused the astrocytes to self-destruct by apoptosis.
  • GFAP glial fibrillary acid protein
  • GS glutamine synthase
  • the proliferatively quiescent cells in confluent astrocyte cultures started cycling again when subcultured. At least 15-18 subcultures could be obtained over 2.5 years from a piece of normal cortex. Only astrocytes from the 4 th to the 8 th subculture were used because the response of the cells to proinflammatory cytokines and/or ⁇ peptides became erratic with further subculturing.
  • NAHNs either isolated from cerebral cortex fragments or obtained through ATCC, were cultured, experimentally treated, and processed just as NAHAs were.
  • ⁇ peptides and reversed sequence peptides were obtained from Bachem (Torrance, CA); prior to use the lyophilized ⁇ peptides were first dissolved at 1.0 mg/ml in DMSO (100% v / v ) and, after 1 h, were directly diluted (1:200) to a final concentration of 20.0 ⁇ g/m ⁇ into the growth medium.
  • DMSO (0.5% v / v ) 10% v / v serum in the growth medium helped keep ⁇ peptides in solution.
  • ⁇ was resuspended in PBS and next its fibrillization degree was assessed according to fluorescence intensity measurements after staining with Thioflavine T. Reversed sequence peptides did not form fibrils.
  • CM cytokine mixture
  • TNF-a 20 ng/ml
  • IFN- ⁇ 70 ng/ml
  • NAHAs and NAHNs were isolated, grown, and propagated under 'normoxic' conditions as previously described. At "0-h”, some of such cultures served as untreated controls while others had 20 ⁇ of either ⁇ 2 5-35 or the reverse sequence ⁇ 35-25 (not biotin-labeled), added to their medium. The doses we used had been found to be optimal in previous studies.
  • IF immunofluorescence
  • the calcilytic agent NPS 2143 (Tocris) was dissolved in DMSO prior to be diluted in the growth medium at a final concentration of 100 nm.
  • the calcimimetic NPS R 568 (also from Tocris) was also dissolved in DMSO according to the seller's instructions and used at a final concentration of 1.0 ⁇ . Starting at 0-h experimental time and every 24-h thereafter NAHAs and NAHNs were exposed for 30 min to either NPS 2143 or NPS R 568 dissolved in fresh medium; thereafter fresh (at 0-h) or the previously conditioned (at 24-, 48- and 72-h) medium was added again to the cultures.
  • control and treated NAHAs were scraped into cold PBS, sedimented at 200 x g for 10-min, and homogenized in T-PERTM tissue protein extraction reagent (Pierce, Biotechnology, Inc., Rockford, IL, USA) containing a complete EDTA-free protease inhibitor cocktail (Roche, Milan, Italy).
  • T-PERTM tissue protein extraction reagent Pieris, Biotechnology, Inc., Rockford, IL, USA
  • the protein contents of the samples were assayed according to Bradford using BSA as standard.
  • Equal amounts (10-30 g) of protein from the samples were heat- denatured for 10-min at 70°C in an appropriate volume of IX NuPAGE LDS Sample Buffer supplemented with IX NuPAGE Reducing Agent (Invitrogen).
  • the synthetic ⁇ peptides used as controls were ⁇ 25 35 (Bachem) and ⁇ 1 42 (Biopeptide Co. Inc., San Diego, CA). Three g of each control peptide were electrophoresed on NuPAGE Novex 4-12% Bis/Tris polyacrylamide gel (Invitrogen) and then subjected to silver staining and Western immunoblotting analysis. Though visible in the gel as protein, ⁇ 25 35 did not react at all with the 8G7 antibody, whereas both sA , 42 and ⁇ 1 42 did react with it.
  • ⁇ 42 secretion was assessed by Western blotting.
  • media samples were collected, centrifuged at 1,000 x g for 10-min to remove cells or debris, mixed with a protease inhibitor cocktail (Roche), and then concentrated with an Ultracel YM- 3 (3,000 MWCO, Millipore) Centricon filter column.
  • ⁇ 42 was immunoprecipitated by incubating for 2-h at 4°C these conditioned media samples using the human- specific monoclonal antibody 8G7 recognizing ⁇ 42 (Acris) bound to Immunopure- immobilized Protein A (Pierce). Following centrifugation at 1,000 x g for 5-min and several washes in Tris-buffered saline, the immunoprecipitated peptides were resolved by SDS-PAGE and immunoblotting.
  • g) Statistical analysis
  • figure 1 in the left panel shows how the Exposure to ⁇ 2 5-35 (20 ⁇ ) alone increases the intracellular synthesis and accumulation of ⁇ 42 oligomers with an M r of up to 17 kDa; the amount of such very small oligomers gives an estimate of the actual synthetic rate of new ⁇ 42 moieties.
  • the addition of NPS 2143 and exogenous ⁇ 2 5-35 to NAHA cultures totally curtails any increase in the intracellular synthesis and accumulation of ⁇ 42 oligomers with a M r up to 17 kDa and, hence, any surge in the actual synthetic rate of ⁇ 42 moieties.
  • NAHAs were cultured and treated as detailed above, see the Materials and
  • NAHAs shown in figure 2 were cultured and treated as detailed in the Materials and Methods. NAHA-conditioned media were sampled, stored, and processed as indicated in the Materials and Methods. ⁇ 42 levels in the samples were determined via a specific hyper-sensitive ELISA assay (Wako). Points in the curves are means ⁇ SEMs from 8 distinct experiments each carried out in triplicate Levels of statistical significance of the means of ⁇ 2 5-35 alone vs. ⁇ 2 5-35 + NPS 2143 are p ⁇ 0.001 at all the time points examined. Levels of statistical significance of the means of ⁇ 2 5-35 + NPS 2143 vs. 0-h untreated control values are p ⁇ 0.05 at 48 and 72 h.
  • the administered calcilytic NPS 2143 prevents the activation of GSK-3 and the consequent hyperphosphorylation of microtubule-associated Tau proteins, which would otherwise lead to the intracellular accumulation of NFTs and to critical microtubular cytoskeleton dysfunction, the second main pathogenetic mechanism of AD.
  • NAHAs were cultured and treated as detailed in the Materials and Methods.
  • Total cell lysates were immunoblotted and challenged with specific anti total GSK-3 and corresponding phospho-Tyr 216 and phospho-Ser 9 GSK-3 antibodies.
  • Specific protein bands (not shown) underwent densitometric analysis. Points in the curves express the mean ratios between the specific phosphorylated sites and total GSK-3 ⁇ SEMs from 8 distinct experiments.
  • FIG. 3 left panel. Levels of statistical significance of the means of ⁇ 2 5-35 alone vs. ⁇ 2 5-35 + NPS 2143 are p ⁇ 0.001 at both 24 and 48 h; levels of statistical significance of the means of ⁇ 2 5-35 alone vs. 0-h untreated controls are also p ⁇ 0.001 at both 24 and 48 h.
  • FIG. 3 right panel. Levels of statistical significance of the mean ratios of phospho-GSK-3 from ⁇ 2 5-35 alone vs. ⁇ 2 5-35 + NPS 2143 are p ⁇ 0.001 at both 24 and 48 h. Levels of statistical significance of the means of ⁇ 2 5-35 alone vs. 0-h untreated controls are also p ⁇ 0.001 at 48 h.

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Abstract

L'invention concerne un traitement pharmacologique à la fois de la maladie d'Alzheimer (AD) familiale à apparition précoce et sporadique à apparition tardive, de troubles associés à AD et de neuropathies associées au syndrome de Down, qui met en jeu l'utilisation d'une classe de médicaments, les calcilytiques, qui, par l'inhibition de la signalisation par le récepteur sensible au calcium (CaSR) dans tous les types de cellules cérébrales, empêchent : (i) la surproduction d'oxyde nitrique (NO) et de peroxynitrite (ONOO-) dangereux pour les cellules, et surtout, (ii) la surproduction, l'accumulation et la sécrétion intracellulaire de peptides Amyloïde β (Aβ) en réponse à la présence extracellulaire de peptides Aβ exogènes et/ou de cytokines proinflammatoires, et (iii) l'hyperphosphorylation associée au peptide Aβ de la protéine Tau (τ) par une glycogène synthase kinase (GSK)-3β activée par la signalisation Aβ/Ca SR avec la formation résultante d'enchevêtrements de neurofibrilles (NTF), celles-ci étant connues comme provoquant un dysfonctionnement tellement sévère du cytosquelette microtubulaire tel qu'il favorise finalement (iv) la mort des neurones du cortex cérébral humain.
EP11817420.0A 2011-12-27 2011-12-27 Utilisation de médicaments calcilytiques en tant qu'approche pharmacologique vis-à-vis du traitement et de la prévention de la maladie d'alzheimer, de troubles associés à la maladie d'alzheimer et de neuropathies associées au syndrome de down Ceased EP2797591A1 (fr)

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ILARIA DAL PRA ET AL: "Roles of Ca2+ and the Ca2+-sensing receptor (CASR) in the expression of inducible NOS (nitric oxide synthase)-2 and its BH4 (tetrahydrobiopterin)-dependent activation in cytokine-stimulated adult human astrocytes", JOURNAL OF CELLULAR BIOCHEMISTRY, vol. 96, no. 2, 1 October 2005 (2005-10-01), US, pages 428 - 438, XP055373660, ISSN: 0730-2312, DOI: 10.1002/jcb.20511 *
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