EP2797591A1 - Use of calcilytic drugs as a pharmacological approach to the treatment and prevention of alzheimer's disease, alzheimer's disease-related disorders, and down's syndrome neuropathies - Google Patents

Use of calcilytic drugs as a pharmacological approach to the treatment and prevention of alzheimer's disease, alzheimer's disease-related disorders, and down's syndrome neuropathies

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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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Prior art keywords
alzheimer
disease
casr
drug
tau
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German (de)
French (fr)
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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

A pharmacological treatment of both familial early onset and sporadic late onset Alzheimer's disease (AD), AD-related disorders and Down's syndrome-coupled neuropathies involves the use of a class of drugs, the calcilytics, which by inhibiting the calcium-sensing receptor (CaSR)signaling in all types of brain cells prevent: (i) the overproduction of cell-harming nitric oxide (NO) and peroxynitrite (ONOO-), and most importantly (ii) the intracellular overproduction, accumulation, and secretion of Amyloid β (Aβ) peptides in response to the extracellular presence of exogenous Aβpeptides and/or proinflammatory cytokines, and (iii) the Aβ peptide-related hyperphosphorylation of the Tau (τ) protein on the part of an Aβ/Ca SR-signaling activated glycogen synthase kinase-(GSK)-3β w i t h the resulting formation of neurofibrillary tangles (NTFs), the latter known to cause such severe dysfunctioning of the microtubular cytoskeleton as to eventually favor (iv) the death of human cerebral cortex neurons.

Description

USE OF CALCILYTIC DRUGS AS A PHARMACOLOGICAL APPROACH TO THE TREATMENT AND PREVENTION OF ALZHEIMER'S DISEASE, ALZHEIMER'S DISEASE-RELATED DISORDERS, AND DOWN'S SYNDROME NEUROPATHIES
TECHNICAL FIELD
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.
More in particular, 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:
(i) the overproduction of cell-harming nitric oxide (NO) and peroxynitrite (ONOO ), and most importantly
(ii) the intracellular overproduction, accumulation, and secretion of Amyloid β (Αβ) peptides in response to the extracellular presence of exogenous Αβ peptides and/or proinflammatory cytokines, and
(iii) the Αβ peptide-related hyperphosphorylation of the Tau (τ) protein on the part of an Αβ/CaSR-signaling activated glycogen synthase kinase-(GSK)^ with the resulting formation of neurofibrillary tangles (NTFs), the latter known to cause such severe dysfunctioning of the microtubular cytoskeleton as to eventually favor cell death.
Hence, calcilytics block at least three pathogenetic mechanisms favoring the development and progression of AD, i.e.
(i) cell damaging NO/ONOO overproduction;
(ii) extracellular and intracellular Αβ peptide accumulation and its cytotoxic effects, and
(iii) intracellular Tau protein hyperphosphorylation by GSK-Ββ and its cytoskeletal dysfunctional consequences. These three operative mechanisms cooperate in inducing neuronal damage and death and in evoking chronic inflammatory responses in the central nervous system (CNS).
It must be stressed here that, previously, the therapeutic uses suggested for calcilytics were human osteoporosis, autoimmune antibody-induced hypocalcemia, and autosomal dominant hypocalcemia (ADH). Therefore, this invention proposes a totally new and different set of therapeutic indications for calcilytics, namely AD, AD-related disorders, and Down's syndrome neuropathies.
Explicitly, 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
(i) blocking the Αβ and or proinflammatory cytokines-induced overproduction of NO/ONOO-and its cell- and tissue-damaging consequences;
(ii) hindering the de novo intracellular synthesis, accumulation, and the secretion into the extracellular space of Αβ peptides induced by preexistent exogenous Αβ peptides and/or proinflammatory cytokines released by any brain cell type, thereby reducing their toxic effects on nerve cell metabolism, synapse function, adult hippocampal neurogenesis, adult brain neoangiogenesis, and nerve cell stress responses and, most important of all, preventing the induction by the extracellularly accumulating Αβ peptides of the further synthesis and secretion of Αβ on the part (mainly) of both neurons and astrocytes, thereby breaking the noxious vicious cycle that underlies AD onset and progression; and
(iii) preventing the hyperphosphorylation of the Tau protein by an accumulating Αβ/CaSR-signaling increased activity of the 05Κ-3β, thereby averting NFTs formation and its consequent pathology (tauopathies) linked to critical microtubular cytoskeleton dysfunction in all types of brain cells, mainly the neurons and the astrocytes. 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.
The calcilytics (CaSR inhibitors) 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.
Definitions
The term "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.
As used herein, "treatment" of an ailment comprises the prevention/prophylaxis, therapy, delay or cutback of symptoms provoked by the ailment. The term 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.
The term "inhibition" 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 name of specific compounds within the context of this invention is meant to include not only the specifically named molecules or class of molecules, but also any pharmaceutically acceptable salt, hydrate, ester, ether, isomers, racemate, conjugates or pro-drugs thereof of any degree of purity.
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. In a combined therapy according to the present invention, the at least two drugs may be administered together or separately, at the same time or in succession. Moreover, 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.
References and Abbreviations
The following discussion of the background art underlying the present invention is based on several publications as specifically listed here below. The reader may refer to these publications in order to obtain a more accurate disclosure of some issues mentioned in the introductory part of the present application.
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Furthermore, some abbreviations and acronymes have been used throughout the present application. The list of these abbreviations and acronymes is reported here below.
Αβ, amyloid β
AD, Alzheimer's disease
ADAM, a-disintegrin and metalloproteinase
ApoE, apolipoprotein E
APP, amyloid precursor protein
ADT, amino-terminal domain
ADH, autosomal dominant hypocalcemia
BACEl/ S, β-site APP cleaving enzyme 1
BBB, blood brain barrier
BH4, tetrahydrobiopterin
BMS, Bristol-Meyers-Squibb
CaMK, calmodulin kinase
CaSR, calcium-sensing receptor
Cdk-5, cyclin-dependent kinase-5
CK, casein kinase
CM, cytokine mixture
CNS, central nervous system
CRR, cysteine-rich region
EOAD, early (familial) onset Alzheimer's disease
fA , fibrillary Αβ FHH/HHCl, familial hypocalciuric hypercalcemia
FIH, familial isolated hypoparathyroidism
GCH1, GTP cyclohydrolase-1
γ-S, γ-secretase
GABABR, γ-aminobutyric acidB receptor
GPR, G-protein coupled receptor
GSK-3 , glycogen synthase kinase-3
GTP, guanosin triphosphate
HIF, hypoxia-inducible transcription factor
HRE, hypoxia response element
LOAD, late onset (sporadic) Alzheimer's disease
LTP, long term potentiation
MBP, myelin basic protein
MCI, mild cognitive impairment
mGluR, metabotropic glutamate receptor
NAHA, normal adult human cortical astrocyte
NAHN, normal adult human cortical neuron
NMDA, N-methyl-D-aspartate
NOS-2, (inducible) nitric oxide synthase-2
NSHPT, neonatal severe (primary) hyperparathyroidism
NTF, neurofibrillary tangle
PHPT, primary hyperparathyroidism
PMF, peptide mass fingerprinting
PSD-95, postsynaptic density protein-95
PS1 or PSEN1, presenilin-1
PS2 or PSEN2, presenilin-2
sAfi, soluble Αβ (oligomers)
sAPP, soluble APP
Ser, serine
SHPT, secondary hyperparathyroidism
SUT2, mammalian solute transport protein-2 tg, transgenic
TM, transmembrane a-helix
Tyr, tyrosine
VEGF, vascular endothelial growth factor
BACKGROUND ART
Alzheimer's disease (AD), particularly its sporadic late onset (LOAD) form, is the most common type of progressive dementia affecting humans. It is estimated that LOAD patients are more than 5 million of the aging population in the United States of America (USA). In its course, AD causes a progressive, dramatic loss of cognitive functions, which implies health care costs for the AD patients nearing US$ 150 billion per year, to which the further impact of AD on single patients, their families and the society in general should be added. Due to the increasing duration of lifespan, a projection of about 13 million AD cases by 2050 in USA only is more than plausible. And, since the clinical symptoms of AD become manifest only in the later stages of the illness, many more pre-AD cases affected by mild cognitive impairment (MCI) or by pre-MCI conditions should also be taken into due account. Similar figures concerning present (frank) and predicted (MCI and pre-MCI) AD cases also concern European Union (EU) countries. It may be assumed that more than 20 million frank AD cases are actually current worldwide. Thus, AD is already and is bound to become even more one of the most pressing world health worries as hitherto no AD-aimed therapy has been shown to be beneficial for AD patients. Of note, the symptomatic stage of AD lasts between 5 and 15 years (1-11).
The pathological hallmarks of AD include synaptic dysfunction and loss, death of cholinergic neurons in the hippocampus and neocortex, accumulation of Αβ and of neurofibrillary tangles (NFTs) containing hyperphosphorylated, poorly soluble Tau protein inside neurons and astrocytes, and the extracellular buildup of plaques containing fibrillary (ί)Αβ (5-14). AD neuropathologic lesions typically progress according to a neurotransmitter-specific way, which is via cholinergic (the most vulnerable ones)→ glutamatergic→ GABAergic synaptic terminals (15).
Two major hypotheses on the pathogenetic factors causing AD have been put forward:
(i) 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 Αβ42 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. NFTs, in their turn, induce microtubular dysfunctioning, thereby altering several critical nerve cell functions, like axonal vesicular transport, thereby expediting Αβ cytotoxicity and, eventually, neuronal and astrocytic death (19-23).
Hitherto, AD researchers have principally paid attention to the mechanisms of Αβ cytotoxicity, whereas Tau protein role(s) has(ve) received less attention. Yet, and notably, Αβ peptides can also induce Tau protein hyperphosphorylation via the activation of GSK-Ββ (22,23). Importantly, the known several substrates of GSK-Ββ 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).
In conclusion, the best working hypothesis seems to be that both Αβ peptides and Tau proteins interact in the production of AD neuropathology and, hence, the mechanisms producing both kinds of agents may stand as legitimate targets for AD-specific drugs to be developed.
Albeit most cases are of the LOAD type, sporadic early onset AD (EOAD) cases with familiar prevalence are due to mutations within three genes, i.e. APP (26), presenilin-1 (PS1 or PSEN1), and presenilin-2 (PSE2 or PSEN2) (27-30). 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. Mutations in APP, PSl, and PS2 increase the production and aggregation of Αβ peptides. This, together with the neurotoxicity of insoluble ίΑβ aggregates in vitro and in vivo has lent support to the "amyloid cascade hypothesis" (14,16). According to a recent view, soluble Αβ (≤Αβ) oligomers (but not monomers), rather than ίΑβ 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). Conversely, in LOAD cases, only the presence of the ΑΡ0ε4 allele of apolipoprotein E (ApoE) embodies the most important genetic risk factor, together with increasing age and mid-life hypercholesterolemia (see for references: 37). In conclusion, 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 ίΑβ 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). On the other hand, sAfi oligomer levels correlate better with dementia severity and are more toxic than ίΑβ aggregates in vitro (38-41).
Several lines of evidence indicate that an increased production and/or accumulation and/or a decreased elimination of Αβ peptides from the brain tissue are crucial triggers in the pathogenesis of AD. Αβ peptide generation from APP occurs via the sequential proteolytic cleavage by β- and γ-secretases: this is the amyloidogenic pathway; byproducts of this pathway are the secreted ectodomain sAPPa and the intracellular APP domain (38). A third enzyme, a-secretase, when acting on APP in concert with γ-secretase, prevents Αβ synthesis: and this is the non-amyloidogenic pathway. The physiologic roles of APP are still mostly undetermined; in APP knockout mice, anatomic, physiologic, and behavioral shortcomings were reported that were rescued via the knock-in expression of sAPPa. Thus, the APP ectodomain, sAPPa derives from, may mediate the physiologic activities of APP (42,43). The a- secretase activity co-localizes with various membrane-bound ADAM (a-disintegrin and metalloprotease) metalloproteases, particularly ADAM9, ADAM 10, and ADAM17 (44). An aspartic protease intensely expressed in brain neurons and resident in the Golgi apparatus and endosomes, the β-site APP cleaving enzyme 1 (or BACE1), is endowed with a β-secretase activity; when BACE1 is downregulated, Αβ production declines (45,46). Recently, however, it has been proposed that catepsin B be the true β-secretase candidate, because of its greater efficiency and specificity in cleaving the wild-type β-secretase site (47). Then again, the catalytic subunit of γ-secretase is constructed by a tetrameric complex, in which the proteins PS1, PS2, Aph-1, Pen-2, and Nicastrin associate (48,49).
Interestingly, PS1 is also involved in lysosomal-endosomal trafficking, a function distinct from the γ-secretase activity. PS1 deficiency impairs lysosomal- endosomal trafficking increasing epidermal growth factor (EGF) levels in fibroblasts (50) and telencephalin levels in hippocampal neurons (51). In addition, 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. 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 Αβ synthesis via a decreased APP proteolysis by β- secretase and an enhanced proteolysis of APP C-terminal fragment (53). Even a redistribution of PS1 coupled with an aberrant cholesterol transport can enhance Αβ production (54). Notably, sA 4kDa monomers assemble first in sAfi oligomers of increasing size (from 8 kDa to > 100 kDa) and next in insoluble ίΑβ aggregates forming the extracellular amyloid plaques in the brains of AD patients (55). Even truncated Αβ peptides are found in postmortem AD brains (55,56) as only a fraction of Αβ is full-length Αβι-4ο or Αβι-42/43; in fact, N-terminally truncated variants of Αβ (Αβ3-42 and Αβιι-42) prevail in senile plaques of AD and Down's syndrome brains (57,58). In comparison with Αβι-42, truncated Αβ3-42 exhibits a greater aggregation proclivity and is more cytotoxic in vitro (59-61). Truncated Αβ3-42 is neurotoxic even in vivo (62). Initially, the belief prevailed that only extracellular Αβ could elicit cytotoxic effects, but current evidence shows that Αβ 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 Αβ42 before any extracellular Αβ deposition and intracellular NFT formation occur (66-68). The most cytotoxic sAfi oligomers are synthesized intracellularly and found within cytoplasmic processes and synapses of neurons (69,70) and astrocytes (65) . In all transgenic (tg) mouse models, any significant neuronal losses were headed by sizeable accretions of intraneuronal Αβ peptides, predominantly Αβ42 (71). Neuronal death could also be caused by a progressive intraneuronal accrual of N-truncated Αβ3-42 (72). Moreover, prior to neuronal death, intraneuronally accumulated Αβ42 causes the loss of synaptic terminals, and this damage correlates better with cognitive decline than plaque formation or NFT load or neuronal death. Hence, the notion has become increasingly accepted that the key event leading to initial cognitive dysfunction in AD is the Αβ^-induced loss of synaptic terminals (73-77),
Remarkably, elevated levels of extracellular sAfi oligomers can cause at least part of the loss of synaptic terminals observed in brain regions free of ίΑβ deposits in various tg AD mouse models (78), Interestingly, extracellular sAfi oligomers can bind the cellular prion protein (PrPc) with high affinity: hence PrPc 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). As a matter of fact, both sAfi oligomers and ίΑβ aggregates do bind several distinct cell membrane receptors, i.e. the p75 neurotrophin receptor (p75NTR) (82- 84) - incidentally, even PrPc can bind p75NTR 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). Notably, 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). In APP tg mice, Αβ immunotherapy has impeded the development of neuropathy and cognitive deficits (101) and LTP inhibition engendered by exposure to sAfi oligomers (102).
Regarding the above mentioned link between Αβ and hyperphosphorylated Tau/NFTs formation, it is worth mentioning here that in tg TAPP mice (bearing P301L mutant Tau and K670N/ M671L mutant APP), at variance with tg JNPL3 mice (having only a P301L mutant Tau), NFTs accumulate particularly in limbic regions, mostly the amygdala, suggesting that NFTs growth is affected by surging levels of APP or Αβ peptides (103). Additionally, anti-Αβ antibodies intracerebrally injected into the hippocampi of 3xtg-AD mice not only reduced Αβ buildup but resulted even in the clearance of early-stage, but not late-stage, hyperphosphorylated Tau NFTs; hence, Tau pathology is a consequence of increased Αβ generation and accumulation (104). Under normal conditions, Tau is a soluble microtubule-associated phosphoprotein (MAP) strongly expressed in neurons (105). 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). Under neurodegenerative conditions, including AD and various tauopathies, 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. In human adult CNS, six 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). Conversely, 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-γ, thereby affecting neurodegenerative processes (114). In tg mouse AD models, the interaction of Tau with Fyn (a non-receptor tyrosine kinase) increases Fyn localization in dendrites eventually enhancing neurotoxicity via Αβ-activated Fyn phosphorylation of subunit 2B (NR2B) of NMDA receptors, which thereby form stable complexes with the PSD-95 protein (115). Moreover, 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). Through changes in phosphorylation causing both loss and gain of function 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). Hence, GSK-3 plays a major role in pathological (and physiological) phosphorylation of Tau protein. Reportedly, 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). While 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 Αβ peptides (Figs. 3 and 4): hence, GSK-3P is the principal Tau protein kinase in adult human brain neurons and astrocytes.
Reportedly, GSK-3 β 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 Tyr279; conversely, the phosphorylation at Ser9 downregulates its enzymatic activity (see for references: 126) and reduces Tau phosphorylation levels (127). After an exposure to exogenous Αβ25-35 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). Moreover, 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).
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). Most interesting, blocking GSK-3 activity with a specific inhibitor, SB-415286, decreases Tau phosphorylation levels and protects cultured primary neurons from dying (133). Hence, 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). 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). Interestingly, a small size synthetic GSK-3 inhibitor, named Tideglusib (NP-12), has reached phase II development for two indications, i.e. AD and progressive sopranuclear palsy (PSP), a tauopathy (141,142).
From the clinical standpoint, AD becomes symptomatic once it has reached the point of discrete brain tissue damage. This rises two problems about the pathogenesis of AD: first, not only frank AD cases but even all persons affected by MCI or pre-MCI conditions should be clinically followed for years, a difficult to implement and rather expensive task; second, testing novel therapies in braindamaged patients affected by late-stage LOAD has never resulted in a favorable upshot (i.e. brain function and cognitive rescue) due to the very limited regenerative capabilities afforded by human adult neurogenesis. These facts imply the need to start the therapy for AD in its pre-symptomatic stages (i.e. pre-MCI and MCI) to prevent a further progression of the disease and improve cognitive functions.
From these two problems endeavors have stemmed to produce tg animal models of AD. The identification of the genes causing familial EOAD has led to tg mouse models overexpressing APP and/or PS1 or PS2 with mutations like those detected in EOAD cases. However, hitherto none of these models has seamlessly reproduced all the pathological aspects of human AD, but the several tg AD mouse models set up have helped single out the effects of the different species of Αβ peptides, the pathogenetic roles of sAfi oligomers, and the link between ίΑβ accumulation and tauopathies (see for references: 143).
Conversely, various APP tg mice models accumulating large amounts of ίΑβ 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 ίΑβ plaque accumulation might be the upshot of APP-mutated mouse neurons being bereft of pathways crucial for Αβ toxicity; alternatively, the processes of Αβ production and aggregation proper of sporadic LOAD cases and their pathological consequences cannot be replicated in such tg mouse models (148).
In contrast, other tg AD mouse models have revealed links between ίΑβ plaque deposition and cerebral amyloid angiopathy (CAA) (149), thereby supporting the "amyloid cascade hypothesis" of AD (14,16,150) by showing that many pathological and functional changes induced by sAfi oligomers in mouse brains precede ίΑβ plaques deposition (151,152) and intraneuronali NFT pathology (88,153).
In conclusion, a variety of tg AD mouse models has been generated overexpressing APP and/or the mutated presenilins found in familial EOAD. None of these models has perfectly reproduced the human AD, yet they have helped clarify the pathogenetic roles of sAfi oligomers in AD, the relationship between Αβ and Tau pathologies, and the fact that the manifestation and profusion of the ίΑβ deposits are directly proportional to the levels of ≤Αβ42 oligomers. Moreover, accumulating intraneuronal Αβ42 triggers the loss of synaptic terminals prior to the occurrence of toxic neuronal death (153), indicating that synaptic terminal loss is one of the earliest pathogenetic events in AD. In addition, Αβ may, directly or indirectly, interact with Tau to accelerate NFTs formation and microtubular dysfunction. It should be recalled here that the mice of the tg AD models have their own APP and APP-processing enzymes, which may tamper with the production of the different Αβ-related peptides encoded by the human transgenes. Furthermore, the genetic backgrounds of the different tg AD mice models may muddle crucial aspects of human AD. Hence, although the tg AD mice have been useful, 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). In particular, dogs are affected by an age-related syndrome of cognitive impairment mimicking key aspects of human AD, including cerebral cortex Αβ-elicited damage, neuronal degeneration, and cognitive disabilities. Thus, natural models of AD may also function as assay systems for novel AD-targeted drugs or therapeutic strategies.
In addition, 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. 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).
Although technically challenging, the just mentioned in vitro experimental systems hold great promise as they can reveal the true metabolic pathways operating in the adult human brain (pathways that may differ from those of other mammals or of tg AD models). NAHAs and NAHNs responses in the presence of Αβ 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). As an example, once challenged with proinflammatory cytokines, particularly with mixtures of IL-Ιβ, TNF-a, and IFNy, the cytokine mixture (CM)- trio— or with exogenous Αβ peptides, NAHAs response was quite complex as it involved:
(i) the parallel induction of the nitric oxide synthase-2 (NOS-2) and GTP cyclohydrolase 1 (GCH1)— in turn, the production of tetrahydrobiopterin (BH4) by activated GCH1 induces the dimerization and activation of NOS-2 molecules and, hence, the synthesis and release of significant amounts of NO (156,157). Using proteomic approaches (MALDI-TOF/MS, peptide mass fingerprinting [PMF], immunoblotting, and Kinexus protein microarrays) 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 Ca2+- calmodulin kinases (CaMKs), casein kinase Ila (CK-IIa), cAMP-dependent kinases A (PKAs), and mitogen-activated protein kinases (MAPKs) (158). In addition, exposure to the CM-trio significantly changed, within the 48-72 h required for the induction and activation of GCH1, the levels and identities of some of the 0 h-associated proteins: after 72 h CK-IIa tended to dissociate from, whereas MAPK12 and c-Jun N-terminal kinase (JNK)-3 were strongly associated with fully active GCH1 (158). Even sA peptides and myelin basic protein (MBP), a compound release by damaged myelin sheaths, strongly enhanced, in synergism with the CM-trio, the induction of NOS-2, and the production and release of NO by NAHAs (157);
(ii) the nuclear translocation of the stabilized heterodimeric hypoxia-inducible HIFla»HIFi /ARNTl transcription factor and its binding to vascular endothelial growth factor [VEGFJ-A gene hypoxia-response DNA elements (HREs), followed by the enhancement of the mRNA synthesis for three VEGF-A splice variants (VEGF-Am, VEGF-Aies, and VEGF-Aisg), and the increased synthesis and secretion of VEGF-A165 protein; reverse Αβ35-25 was ineffective under these respects (159); and most important for the present invention,
the synthesis, intracellular accumulation, and secretion of Αβ42, particularly after the exposure to exogenous Αβ25-35, whose levels peaked at 48 h (2.8- fold versus 0 h, p < 0.001) (65); such a surge of endogenous Αβ42 production and secretion is preceded by an increased binding of HIF- la'HIF-Ιβ complexes with nuclear DNA HREs and surges first of BACE-1/β- S) mRNA expression and of BACE-l/^-S activity levels at 24-h (1.4-fold versus 0 h, p = 0.001), and then of γ-secretase (γ-S) activity cresting at 48 h (1.6-fold versus 0 h, p < 0.001), which cleave the Αβ42 peptides from APP (65).
Again most crucially, the present applicants have subsequently observed a similar induction of Αβ42 synthesis and secretion in response to the challenge with exogenous Αβ25 35 (but not with reverse sequence Αβ35 J in cultured human cerebral cortex NAHNs.
Since the BACE-1/β-Ξ and γ-S genes have the same HIF-la»HIF-^-binding HREs in their promoters as has the VEGF-A gene, the present applicants' observations suggested that the Αβ42 released from neurons in the AD brain can recruit associated astrocytes via HIF-lot'HIF-Ιβ signaling into the pool of Αβ42- producing and Αβ^-releasing cells. To this regard, it must be pointed out here that astrocytes are 10-fold more numerous that neurons in the human brain: hence, once exposed to Αβ42 the astrocytes could most effectively contribute their share to the accruing of extracellular 5Αβ42 oligomers and of ίΑβ42 plaques, thus favoring AD development and progression.
In summary, Αβ^-exposed astrocytes, besides neurons, could significantly help start and maintain a vicious circle leading to progressive Αβ42 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.
Definition, structure, and pharmacology of the Calcium-Sensing Receptor (CaSR)
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 γ-aminobutyric acidB receptors (GABABRS), various taste receptors, and the promiscuous GPRC6A receptor (162). 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). In its cell membrane-bound form, 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. Ca2+. 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). Other ligands besides Ca2+, like Mg2+, Gd3+, Ba2+, 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, Ca2+ 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). As shown by point mutation studies, the aromatic L-a-amino acids bind an allosteric site nearby the orthosteric site in the ATD (171), and in the presence of Ca2+ act as true allosteric potentiators of CaSR signaling (170).
Last, but not least, the CaSR can bind Αβ peptides being activated by them (92,93).
Pharmacologically, the CaSR can be antithetically modulated by synthetic allosteric modulators belonging to two classes:
(i) the calcimimetics (potentiators or positive allosteric modulators) mimicking Ca2+ actions, examples of which are the compounds NPS R-467, NPS R-568, Cinacalcet, Calindol, etc. (172-175); and
(ii) the calcilytics (inhibitors or negative allosteric modulators), examples of which are NPS 89626, NPS 2143, Calhex 231, BMS (Bristol-Meyers-Squibb) compound 1, and JKJ05 (173-182). Both NPS 2143 and Calhex 231 bind largely overlapping extracellular portions of the 7TM, in which Glu837 is a crucial residue (182).
Moreover, the aromatic rings of NPS 2143 form hydrophobic contacts and π-stacking with Phe668 residue in TM2, Phe684, Phe688, Arg680 in TM3, and He841 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 He841 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).
As Ca2+ acts both as a primary messenger and a secondary messenger, the extracellular Ca2+ level ([Ca2+]e) is tightly controlled at the gut (uptake), bone (storage), and kidney (excretion) levels via the signaling of the respective CaSRs (187). By sensing acute or sustained changes in circulating Ca2+or [Ca2+]e levels, the CaSR activates several intracellular signal transduction pathways, through which it modulates a wide spectrum of cellular activities (186-188). Of maximal clinical relevance are the CaSR-mediated modulation of parathyroid hormone (PTH) secretion and of renal cation handling to safeguard mineral ion homeostasis (187). Notwithstanding elevated serum Ca2+ levels, 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).
In human NAHAs and NAHNs (65,92,93,156,157), CaSR also reacts with exogenous Αβ peptides thereby inducing through its signaling the de novo synthesis and secretion of NO, of endogenous Αβ42, and Tau hyperphosphorylation by an Αβ/CaSR-activated GSK-3p.
In humans, the hitherto identified mutations of the CaSR gene have been shown to underlie the following ailments:
(i) Neonatal Severe (primary) Hyperparathyroidism (NSHPT), an autosomal recessive disorder due to loss-of-function mutations in the CASR gene on chromosome 3ql3 (191).
(ii) Familial Hypocalciuric Hypercalcemia (HHC1; FHH) due to a lessened sensitivity to Ca2+ at the CaSR (192).
(iii) Familial Isolated Hypoparathyroidism (FIH) due to a gain of function mutation of the CaSR (193).
Moreover, anti-CaSR autoantibodies can inhibit or activate CaSR signaling producing clinical syndromes like FHH or Autosomal Dominant Hypocalcemia (ADH), respectively (194,195). 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).
Notably, clinical trials have shown calcimimetics to efficiently reduce high PTH and Ca2+ levels and to prevent bone disease and other complications (198). Thus, the 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) .
By sharp contrast, only a few preclinical studies have so far been performed with calcilytics (e.g. NPS 2143, BMS compound 1, etc.). The first proposed use of calcilytics was the treatment of human osteoporosis. However, 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). On the other hand, 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
Extensive areas of the human and other mammals central nervous system (CNS), such as:
• in the telencephalon, several olfactory structures and the hippocampal formation;
• in the diencephalon, the dorsal thalamus and hypothalamus;
· in the mesencephalon, the colliculi, raphe, and central gray matter;
• in the metencephalon, the cerebellum and pontine nuclei;
• in the spinal cord, the gray matter; and
• the diffuse circumventricular structures
express various levels of CaSR mRNA (200,201). In such CNS areas, 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. Moreover, indications have been emerging that, CaSRs play roles in neuroinflammatory and/or neurodegenerative conditions in the human CNS, including AD (93).
In fact, besides Ca2+, CaSRs also bind L-amino acids, and this causes specific patterns of intracellular Ca2+ oscillations (205,206). Thus, heightened concentrations of L-phenylalanine activate the CaSR, inducing neuronal cytotoxicity in cases of phenylketonuria (207).
But it is the expression of the CaSR on the part of the glial cells i.e. astrocytes (156-160,204), oligodendrocytes (208), microglia (209), and 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).
In particular, 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). And the above mentioned results of our own experimental work carried out using cultures of phenotypically locked-in NAHAs and of NAHNs have further stressed the exogenous Αβ/CaSR signaling modulation of NO/ONOO synthesis, VEGF-Ai65 synthesis and release, and, most important, of Αβ42 synthesis and release and Tau protein hyperphosphorylation by an activated GSK-3 — all events relevant for AD onset and progression (65,156-160).
The accumulation of extracellular and intracellular sA and ίΑβ peptides and of intracellular NFTs is typical of AD, AD-related conditions, and Down syndrome neuropathies (214). Notably, Αβ 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 ίΑβ aggregates possess, like polyamines, a regular array of positive charges to which anionic dyes bind (e.g., Congo Red) (216). It is believed that such positive charges of the sA or ίΑβ peptides also allow the interaction with and the activation of the CaSRs (92) and the consequent elevation of intracellular Ca2+ levels [(Ca2+)i]. Notably, 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 ίΑβ aggregates (215).
SUMMARY OF THE INVENTION 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.
Accordingly, 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.
Furthermore, 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 Αβ 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. Yet, 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 Αβ-elicited cytotoxic effects for simultaneous, isolated or sequential administration to subjects in need thereof.
In accordance with the above defined purposes, 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 dependent claims outline advantageous forms of embodiment of the present invention.
DESCRIPTION OF THE INVENTION As discussed above, 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 Αβ 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. Through the all-inclusive integration of experimental data covering results of human nerve cell biology studies describing different aspects of AD with the results of their own experiments carried out using phenotypically locked-in normal human adult cortical astrocytes and normal human adult cortical neurons set into in vitro cultures, the inventors have found that the use of specific allosteric inhibitors of the CaSR (calcilytics) prevents the CaSR signaling activation on the part of sA oligomers and ίΑβ aggregates accumulating in the brain extracellular spaces. In its own turn, this inhibition prevents:
(i) the induction of the endogenous synthesis, accumulation, and release of an excess of Αβ42 elicited in neurons and astrocytes by exogenously accumulated sA oligomers and ίΑβ aggregates as illustrated in the examples;
(ii) the hyperphosphorylation of Tau protein on the part of an Αβ/CaSR-activated GSK-3 , thereby blocking the formation of NFTs and consequent critical dysfunctions of microtubular cytoskeleton in neurons and astrocytes.
Therefore, 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 ίΑβ 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 (calcilytic) 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.
Taking the preceding disclosure into due account, the present applicants could further observe that NOS-2 enzyme activation absolutely requires CaSR signaling in NAHAs and NAHNs, being totally blocked by the calcilytic (CaSR allosteric inhibitor) NPS 89626. These findings lend strong support to the view that a properly administered calcilytic (CaSR-inhibiting) drug should prevent the damage and death of human neurons in AD due to an excess production of NO on the part of astrocytes and microglia. Moreover, the present applicants could ascertain that CaSR signaling in neurons and astrocytes plays a crucial role in AD onset and progression. In fact, CaSR can bind exogenous Αβ peptides and be activated by this bondage.
Taking stock of this background, the present applicants could surprisingly verify that the administration of the calcilytic (CaSR allosteric inhibitor) drug NPS 2143 totally suppresses in NAHAs and NAHNs:
1. the exogenous NO hyperproduction and its damaging actions, even via ONOO formation, on nerve cells;
2. the exogenous synthesis, accumulation, and secretion of endogenous Αβ42;
3. the exogenous enhancement of the activity of GSK-3 , thereby preventing Tau-protein hyperphosphorylation and the resulting microtubular dysfunction;
4. the exogenous death of NAHNs.
According to the present invention, 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 Αβ peptide accumulation in the brain tissue.
Most importantly, according to the present invention, the calcilytic drug breaks the vicious circle through which exogenous Αβ begets endogenous Αβ, thereby further increasing exogenous Αβ levels and further stimulating endogenous Αβ synthesis and secretion, NFT accrual via Tau hyperphosphorylation by an Αβ/CaSR-activated 05Κ-3β, 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. By suppressing the harming effects elicited by exogenous Αβ 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
Αβ peptides like Αβ25 35 do induce the de novo synthesis and secretion of endogenous Αβ42 not only by NAHNs (our unpublished results), but even by NAHAs. In the human CNS, this Αβ-triggered release of Αβ42 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 Αβ42 production and release, i.e. exogenous (extracellular) Αβ and endogenous (intracellular) Αβ can reciprocally heighten their levels, the upshot of which is the Αβ self-induced, self-sustaining, progressive accumulation of sAfi oligomers first and of ίΑβ aggregates later in the human brain eventually leading to clinically symptomatic and step-by-step worsening AD.
The increasing levels of Αβ 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 05Κ-3β, 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.
Even more importantly, the present applicants found that administering the calcilytic (CaSR-inhibiting) NPS 2143 together with exogenous Αβ25 35 to both NAHAs and NAHNs (not shown) cultures prevented the surge in de novo synthesis and release of NO, the synthesis accumulation, and secretion of endogenous Αβ42 (Figs. 1 and 2) and the increase in activity of the main Tau protein kinase 05Κ-3β (Figs. 3 and 4), and NAHNs death— all events favoring the development and progression of AD and otherwise induced by exogenous Αβ25 35 alone. These results imply that calcilytics can become the mainstay of a novel therapy hindering the onset and progression of AD, AD-related disorders, and Down's syndrome neuropathies. That calcilytic can be beneficial in AD patients (provided that the therapy is started in early stages of the ailment) is further strengthened by the findings that the drugs exerting contrary effects with respect to calcilytics, that is the calcimimetic (CaSR -stimulating) agents, like NPS R 568, which when administered by themselves, do increase Αβ42 synthesis (not shown) and secretion on the part of NAHAs and NAHNs (not shown) at least as effectively as does exogenously administered Αβ25 35 alone (Table 1).
Table 1. Stimulation of Αβ 2 secretion by the calcimimetic NPS R 568 in NAHAs
*Values have been normalized with respect to untreated 0-h growth medium samples. NAHA cultures were set up, treated and sampled as indicated in the Materials and Methods. The mean values shown are from two distinct experiments carried out in triplicate. SEMs, not shown were within ± 11% of the corresponding mean values.
Therefore, known calcimimetic agents like NPS R 568, stimulate de novo Αβ42 synthesis and secretion on the part of human astrocytes and neurons.
Conversely, paradigmatic calcilytic agents, like NPS 2143, by hindering CaSR signaling, totally suppress the exogenous Αβ25 35-induced increase in endogenous Αβ42 synthesis and secretion, the concurring surge in the activity of the main Tau protein kinase, Θ5Κ-3β, and the hyperproduction and release of ONOO -generating NO. In simpler words, calcimimetics favor, whereas calcilvtics strongly contrast AD development and progression.
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 Αβ42 further begetting ever more Αβ42, 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.
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.
Thus, 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 Αβ42 favored by exogenous Αβ42, and (ii) the NFT-generating hyperphosphorylation of Tau protein on the part of a strongly activated GSK-3 , ultimately leading to neuronal death. These effects of the CaSR-inhibiting calcilytic drugs also entail the suppression of the collateral effects brought about by progressively accumulating NO/ONOO , sA oligomers and/or ίΑβ aggregates, like neuroinflammation, neurocytotoxic and effects, and nerve cell and endothelial cell damage and apoptosis.
Therefore, by breaking the viciously recursive self-amplifying stimulation of the synthesis and release of Αβ42 and of NFTs formation with aggregated poorly soluble hyperphosphorylated Tau proteins, both elicited by the exogenously accumulating Αβ42, 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.
Clearly, the entity of the clinical gains for the patients will be inversely proportional to the actual duration of the brain disease, which emphasizes the importance of an early diagnosis of pre-MCI, MCI, AD and related ailments and of an early start of the treatment with calcilytics. Thus, 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.
Furthermore, the just mentioned 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 such as olanzapine, risperidone, mirtazapine, quetiapine, etc.).
ILLUSTRATION OF DRAWINGS
In the drawings:
• 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≤Αβ42 oligomers and ίΑβ42 aggregates elicited by the exposure to exogenous Αβ25-35 on the part of early passage normal adult human astrocytes (NAHAs) set into in vitro cultures;
• Fig. 2 shows how the calcilytic NPS 2143 added together with exogenous Αβ25-35 (20 μΜ) totally suppresses the increases in extracellular secretion of Αβ42 (Αβχ-42 in the Figure) on the part of the NAHAs otherwise elicited by the exposure to Αβ25-35 alone;
• Fig. 3 shows how the activity of the main Tau protein Kinase, 05Κ-3β, is increased in NAHAs exposed to exogenous Αβ25-35 (20 μΜ) alone; and
• Fig. 4 shows the activity of 05Κ-3β, the main Tau protein Kinase, as indicated by the time- and treatment-corresponding ratios of the activating phosphorylation levels at Tyr216 with respect to the inactivating phosphorylation levels at Ser9.
DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
With reference to figures 1 to 4, the present invention has been developed by implementing the following materials and methods. a) NAHAs and NAHNs culture models
Small samples were taken from fragments of temporal lobe cortices from patients with perforating head injuries. The samples were immersed in MCDB 153 medium (Sigma-Aldrich, Italy), put into a Dewar flask at 4° C, and carried to the tissue culture laboratory. There they were cut into tiny pieces, the cells in which were released by mild treatment with 0.25%w/v trypsin (Sigma-Aldrich) in Hanks' Basal Salt Solution (BSS; Lonza, France) at 18° C, and triturated with a series of Pasteur pipettes with bore diameters decreasing from 5 to 1 mm. 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). Insulin-like growth factor-I (IGF-I; 20 ng/ml; PeproTech), and platelet-derived growth factor (PDGF; 20 ng/ml; PeproTech) were added to the medium to enhance the initial adhesion, proliferation, and selection of the astrocytes in the mixed cell population. This complete medium was replaced every 2-3 days. When the primary mixed cultures became 70% confluent (1-4 weeks), the cells were detached from the flask surfaces with 0.25%w/v trypsin and 0.02%w/v EDTA (Lonza) in Hanks' BSS, split 1:4 and planted into new flasks. After the third subculture, a homogeneous population of astrocytes appeared and the four growth factors were no longer needed. 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. 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 4th to the 8th subculture were used because the response of the cells to proinflammatory cytokines and/or Αβ 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. b) Αβ peptides, calcilytic and calcimimetic agents
Αβ peptides and reversed sequence peptides were obtained from Bachem (Torrance, CA); prior to use the lyophilized Αβ 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 μg/m\ into the growth medium. DMSO (0.5%v/v) 10%v/v serum in the growth medium helped keep Αβ peptides in solution. Alternatively, Αβ 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. Human myelin basic protein (MBP) was also purchased from Bachem and used at a final concentration of 10 g/ml in the growth medium. The proinflammatory cytokines making the cytokine mixture (CM)-trio, i.e. IL-Ιβ (20 ng/ml), TNF-a (20 ng/ml), and IFN-γ (70 ng/ml) (all from PeproTech) were added to the growth medium at 0 h experimental time by themselves or in association with either Αβ peptides or MBP. c) Experimental protocol
Since astrocytes and neurons are normally not proliferating in the adult human brain when they are assailed by Αβ, MBP and/or proinflammatory cytokines, we employed for our studies proliferatively quiescent NAHAs and NAHNs cultures. 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 μΜ of either Αβ25-35 or the reverse sequence Αβ35-25 (not biotin-labeled), added to their medium. The doses we used had been found to be optimal in previous studies. For immunofluorescence (IF) analysis NAHAs were treated with 20 μΜ of biotin- labeled Αβ25-35 (AnaSpec Inc., Fremont, CA).
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 μΜ. 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.
Cultures were sampled 24-, 48-, 72-, and 96-h after the simultaneous addition of Αβ25 35 and phosphoramidon (10 μΜ; Sigma, Milan, Italy), the latter being an inhibitor of thermolysin and other proteases. It is important to note that we added the Ap42-mimicking Αβ25 35 instead of Αβ42 itself to enable us to distinguish between internally produced and externally added Αβ peptides. d) Western immunoblotting (WB)
At selected time points, control and treated NAHAs were scraped into cold PBS, sedimented at 200 x g for 10-min, and homogenized in T-PER™ tissue protein extraction reagent (Pierce, Biotechnology, Inc., Rockford, IL, USA) containing a complete EDTA-free protease inhibitor cocktail (Roche, Milan, Italy). 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 samples were next loaded on a NuPAGE Novex 10% Bis-Tris polyacrylamide gel (Invitrogen) (for β-S detection) or on NuPAGE Novex 4-12% Bis-Tris polyacrylamide gel (Invitrogen) (for Αβ42 detection). After electrophoresis in NuPAGE MES SDS Running Buffers using the Xcell SureLock™ Mini-Cell (Invitrogen) (50-min runtime at 200 V constant), proteins were blotted onto nitrocellulose membranes (0.2 μιη; Pall Life Sciences, Milan, Italy).
To ensure efficient and reproducible binding to the membrane, transfer proceeded under low power conditions (30 V constant) for 1-h in IX NuPAGE transfer buffer containing 10% methanol. Immunoblots were performed using the SNAP i.d. protein detection system (Millipore, Milan, Italy) and the membranes were probed with the specific rabbit polyclonal antibody against human β-S IgG polyclonal antibody (Santa Cruz Biotechnology, Heidelberg, Germany) or with the specific mouse monoclonal 8G7 antibody against Αβ42 (Acris), both at a final dilution of 1.0 g ml-1. Subsequent assessments of the specific bands integrated intensities were carried out using Sigmagel™ Qandel Corp., Erkrath, Germany) as previously detailed.
To assess the specificity of the mouse monoclonal 8G7 antibody (Acris) against Αβ42, the synthetic Αβ peptides used as controls were Αβ25 35 (Bachem) and Αβ1 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, ίΑβ25 35 did not react at all with the 8G7 antibody, whereas both sA , 42 and ίΑβ1 42 did react with it. e) ELISA for HIF-Ια transcriptional activity, expression of NOS-2, GCH-1, HIF-Ια, BACEl (β-S ) mRNAs assessed via RT-PCR, assays of NOS-2, GCH-1, BACE1/ -S and γ-S activities, immunocytochemistry (IC),
These specific studies were performed as minutely detailed in previous papers (65,85,156-160,215). f) ELISA assay of Αβ 2 released into NAHA- and NAHN-conditioned growth medium samples
To assess the secretion (if any) of Αβ42 into conditioned media by NAHAs, we first used a commercial ELISA kit (SIG-38956; Covance Research Products Inc. Dedham, MA). Alternatively, growth media samples were tested with the Human/Rat Αβ ELISA Kit, High-Sensitive (from Wako, Japan) according to the instructions of the seller, with a dynamic range of 0.1-20.0 pM L 1 and a sensitivity of 0.024 pM L 1. Briefly, the NAHA-conditioned media samples were centrifuged for 10-min at 13,000 rpm to remove cellular debris after adding a protease inhibitor cocktail (Roche) and then tested in triplicate according to the manufacturer's protocol. To also assess Αβ42 secretion, we immunoprecipitated Αβ42 from NAHAs-conditioned media as follows. Briefly, 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. Αβ42 was immunoprecipitated by incubating for 2-h at 4°C these conditioned media samples using the human- specific monoclonal antibody 8G7 recognizing Αβ42 (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
The data were analyzed using SigmaStat 3.5 Advisory Statistics for Scientists (Systat Software, Richmond, CA). For RT-PCRs, data were normalized to GAPDH and next analyzed by one-way ANOVA. For immunoblotting, data were normalized to lamin Bl and next analyzed by one-way ANOVA. Post hoc Holm-Sidak's test was used for multiple pair-wise comparisons. Null hypotheses were rejected when p > .05.
Taking the preceding considerations into due account, figure 1 in the left panel shows how the Exposure to Αβ25-35 (20 μΜ) alone increases the intracellular synthesis and accumulation of Αβ42 oligomers with an Mr of up to 17 kDa; the amount of such very small oligomers gives an estimate of the actual synthetic rate of new Αβ42 moieties. In sharp contrast, the addition of NPS 2143 and exogenous Αβ25-35 to NAHA cultures totally curtails any increase in the intracellular synthesis and accumulation of Αβ42 oligomers with a Mr up to 17 kDa and, hence, any surge in the actual synthetic rate of Αβ42 moieties.
In the right panel of figure 1, exposure to exogenous Αβ25-35 (20 μΜ) by itself also increases the intracellular accumulation of Αβ42 oligomers and aggregates with an Mr of > 17 kDa, a phenomenon significantly curtailed in the simultaneous presence of both NPS 2143 and exogenous Αβ25-35.
NAHAs were cultured and treated as detailed above, see the Materials and
Methods section. Total cell lysates were immunoblotted and challenged with a specific anti Αβ42 antibody. Specific protein bands (not shown) underwent densitometric analysis. Points in the curves are means ± SEMs of 8 distinct experiments. In both panels levels of statistical significance of the means of Αβ25-35 alone vs. Αβ25-35 + NPS 2143 are p<0.001 at both 24 and 48 hours; in the left panel, the levels of statistical significance of the means of Αβ25-35 + NPS 2143 vs. 0-h untreated control values are p>0.05 (i.e. not significant) at both 24 and 48 h; in the right panel, the levels of statistical significance of the means of Αβ25-35 + NPS 2143 vs. 0-h are p<0.05 at both 24 and 48 h.
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. Αβ42 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 Αβ25-35 alone vs. Αβ25-35 + NPS 2143 are p<0.001 at all the time points examined. Levels of statistical significance of the means of Αβ25-35 + NPS 2143 vs. 0-h untreated control values are p<0.05 at 48 and 72 h.
On the other hand, it must be mentioned here that reverse sequence to Αβ35- 25 (20 μΜ) given to NAHA cultures by itself or with NPS 2143 added was totally ineffective in relation to both Αβ42 production, accumulation, and secretion by the NAHAs with respect to untreated astrocytes (data not shown).
In figure 3, the activity of the main Tau protein Kinase, 05Κ-3β, is increased in NAHAs exposed to exogenous Αβ25-35 (20 μΜ) alone, as indicated by the augmented activating phosphorylation at Tyr216 (left panel) and by the simultaneously lessened inactivating phosphorylation at Ser9 (right panel). But Θ5Κ-3β activity is significantly downregulated when the calcilytic NPS 2143 (NPS in the Figure) is added together with exogenous Αβ25-35 to NAHA cultures, as shown by the decrease in the activating phosphorylation at Tyr216 (left panel] and by the increase in inactivating phosphorylation at Ser9 (right panel). Hence, in addition to suppressing the increased synthesis and secretion of Αβ42, 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-Tyr216 and phospho-Ser9 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.
Figure 3. left panel. Levels of statistical significance of the means of Αβ25-35 alone vs. Αβ25-35 + NPS 2143 are p<0.001 at both 24 and 48 h; levels of statistical significance of the means of Αβ25-35 alone vs. 0-h untreated controls are also p<0.001 at both 24 and 48 h.
Figure 3. right panel. Levels of statistical significance of the mean ratios of phospho-GSK-3 from Αβ25-35 alone vs. Αβ25-35 + NPS 2143 are p<0.001 at both 24 and 48 h. Levels of statistical significance of the means of Αβ25-35 alone vs. 0-h untreated controls are also p<0.001 at 48 h.
Finally, the activity of GSK^, the main Tau protein Kinase, as indicated by the time- and treatment-corresponding ratios of the activating phosphorylation levels at Tyr216 with respect to the inactivating phosphorylation levels at Ser9 reported in detail in Figure 4 (q.v.).
By this way of representing the data in figure 4 it is easily visible that GSK- 3β activity levels are significantly increased in NAHAs exposed to exogenous Αβ25- 35 (20 μΜ) alone, whereas GSK^ activity levels are neatly downregulated when the calcilytic (CaSR-inhibiting) NPS 2143 is added together with exogenous Αβ25-35 to NAHA cultures. Data were normalized taking as 1.0 the values found in untreated 0-h control cultures. Points in the curves are mean values; SEMs, not shown, have sizes quite close to the symbols representing the means. The points at both 24-h and 48-h all significant (p<0.01 at least) with respect to both 0-h mean value and to time-corresponding different treatments.
The invention has been described with reference to a specific form of embodiment of the same. However, the invention encompasses several other forms of embodiment falling within the terms of the following claims. In particular, although the invention has been described with specific reference to the calcilytics as CaSR inhibiting drugs, it is obvious that other allosteric modulators targeted to the CaSR must be considered as part of the present invention. Furthermore, naturally occurring mutations of the CaSR alter CaSR signaling and simultaneously indicate that the entire CaSR protein is a target susceptible to allosteric modulation.

Claims

A calcilytic, i.e. a Calcium-Sensing Receptor (CaSR) inhibiting, drug for use in the treatment of Alzheimer's disease, Alzheimer's disease-related disorders and Down's Syndrome neuropathies.
A calcilytic - CaSR-inhibiting - drug according to claim 1, whereby said Alzheimer's disease-related disorders include 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.
A calcilytic - CaSR-inhibiting - drug according to any one of the preceding claims, which is administered via oral and/or intranasal and/or subcutaneous and/or intramuscular and/or intestinal-rectal routes, and/or via cutaneous patches and/or transepidermal and/or transdermal routes, and/or via aerosols.
A calcilytic - CaSR-inhibiting - drug according to claim 3, wherein said drug is in the form of salts or pro-drugs or derivatives or as sustained release formulations thereof, that, by effectively crossing the Brain Blood Barrier (BBB), permeate the brain tissue and selectively antagonize the CaSRs expressed by nerve cells of all types and by the central nervous system endothelial cells.
5. A calcilytic - CaSR-inhibiting - drug according to claim 1, wherein said drug is allosteric or orthosteric and binds any portion of amino acid sequence of the Calcium-Sensing Receptor (CaSR) molecule.
6. A calcilytic - CaSR-inhibiting - drug according to claim 5, wherein said drug is used in combination with additional drugs used in the treatment of Alzheimer's disease, Alzheimer's disease-related disorders and Down's Syndrome Neuropathies, in particular the stimulators of adult neurogenesis like 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, 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, and atypical antipsychotics such as olanzapine, risperidone, mirtazapine, quetiapine.
7. A method of treating Alzheimer's disease, Alzheimer's disease-related disorders and Down's Syndrome neuropathies, the method comprising simultaneously, separately or sequentially administering to a subject in need thereof a calcilytic - Calcium-Sensing Receptor (CaSR) inhibiting - drug.
8. A method according to claim 7, wherein said calcilytic - Calcium-Sensing Receptor (CaSR) inhibiting - drug is administered in combination with additional drugs used in the treatment of Alzheimer's disease, Alzheimer's disease-related disorders and Down's Syndrome neuropathies, in particular the stimulators of adult neurogenesis like 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, 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 such as olanzapine, risperidone, mirtazapine, quetiapine.
9. A method of producing drugs for treating Alzheimer's disease, Alzheimer's disease-related disorders and Down's Syndrome neuropathies, the method involving a step of testing candidate drug(s) for activity as inhibitor of Calcium-Sensing Receptor (CaSR) signaling and selecting candidate drug(s) that by blocking Calcium-Sensing Receptor (CaSR) signaling curtails the endogenous overproduction and secretion of NO by glial cells and of Αβ peptides by neurons and astrocytes, thereby preventing or attenuating inflammatory tissue responses and cytotoxic effects on nerve cells and brain endothelial cells proper of Alzheimer's disease, Alzheimer's disease- related disorders and Down's Syndrome neuropathies.
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