Alzheimer's Drug PBT2 Interacts with the Amyloid ß 1-42 Peptide Differently than Other 8-Hydroxyquinoline Chelating Drugs.

Although Alzheimer's disease (AD) was first described over a century ago, it remains the leading cause of age-related dementia. Innumerable changes have been linked to the pathology of AD; however, there remains much discord regarding which might be the initial cause of the disease. The "amyloid cascade hypothesis" proposes that the amyloid ß (Aß) peptide is central to disease pathology, which is supported by elevated Aß levels in the brain before the development of symptoms and correlations of amyloid burden with cognitive impairment. The "metals hypothesis" proposes a role for metal ions such as iron, copper, and zinc in the pathology of AD, which is supported by the accumulation of these metals within amyloid plaques in the brain. Metals have been shown to induce aggregation of Aß, and metal ion chelators have been shown to reverse this reaction in vitro. 8-Hydroxyquinoline-based chelators showed early promise as anti-Alzheimer's drugs. Both 5-chloro-7-iodo-8-hydroxyquinoline (CQ) and 5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline (PBT2) underwent unsuccessful clinical trials for the treatment of AD. To gain insight into the mechanism of action of 8HQs, we have investigated the potential interaction of CQ, PBT2, and 5,7-dibromo-8-hydroxyquinoline (B2Q) with Cu(II)-bound Aß(1-42) using X-ray absorption spectroscopy (XAS), high energy resolution fluorescence detected (HERFD) XAS, and electron paramagnetic resonance (EPR). By XAS, we found CQ and B2Q sequestered ∼83% of the Cu(II) from Aß(1-42), whereas PBT2 sequestered only ∼59% of the Cu(II) from Aß(1-42), suggesting that CQ and B2Q have a higher relative Cu(II) affinity than PBT2. From our EPR, it became clear that PBT2 sequestered Cu(II) from a heterogeneous mixture of Cu(II)Aß(1-42) species in solution, leaving a single Cu(II)Aß(1-42) species. It follows that the Cu(II) site in this Cu(II)Aß(1-42) species is inaccessible to PBT2 and may be less solvent-exposed than in other Cu(II)Aß(1-42) species. We found no evidence to suggest that these 8HQs form ternary complexes with Cu(II)Aß(1-42).

Synthesis of new clioquinol derivatives as potent α-glucosidase inhibitors; molecular docking, kinetic and structure-activity relationship studies.

Diabetes mellitus is a chronic metabolic disorder with increasing prevalence and long-term complications. The aim of this study was to identify α-glucosidase inhibitory compounds with potential anti-hyperglycemic activity. For this purpose, a series of new clioquinol derivatives 2a-11a was synthesized, and characterized by various spectroscopic techniques. The enzyme inhibitory activities of the resulting derivatives were assessed using an in-vitro mechanism-based assay. All the tested compounds 2a-11a of the series showed a significant α-glucosidase inhibition with IC50 values 43.86-325.81 µM, as compared to the standard drug acarbose 1C50: 875.75 ± 2.08 µM. Among them, compounds 4a, 5a, 10a, and 11a showed IC50 values of 105.51 ± 2.41, 119.24 ± 2.37, 99.15 ± 2.06, and 43.86 ± 2.71 µM, respectively. Kinetic study of the active analogues showed competitive, non-competitive, and mixed-type inhibitions. Furthermore, the molecular docking study was performed to elucidate the binding interactions of most active analogues with the various sites of α-glucosidase enzyme. The results indicate that these compounds have the potential to be further studied as new anti-diabetic agents.

Electrocatalytic evaluation of graphene oxide warped tetragonal t-lanthanum vanadate (GO@LaVO4) nanocomposites for the voltammetric detection of antifungal and antiprotozoal drug (clioquinol).

Metastable and rarely reported GO warped tetragonal phase t-lanthanum vanadate nanocomposites (GO@LaVO4-NCs) are reported for the sensitive electrochemical determination of antifungal drug Clioquinol (CQ). The hydrothermal method was adopted for synthesis of GO@LaVO4-NCs. The electrochemical performance of CQ was examined using cyclic voltammetry (CV) and differential plus voltammetry (DPV) at GO@LaVO4-NCs modified glassy carbon electrode (GCE). The electrocatalytic oxidation of CQ at the GO@LaVO4-NCs/GCE shows the highest anodic peak current at a potential of +0.51 V vs. Ag/AgCl. The proposed sensor provides excellent sensitivity of 4.1894 µA µM-1 cm-2, a very low detection limit (LOD) of 2.44 nM, and a wide range of 25 nM to 438.52 µM towards CQ detection. Finally, the detection of CQ in biological media was successfully done using the GO@LaVO4-NCs/GCE and possesses recoveries of 94.67-98.0%.

Copper(II) Binding to PBT2 Differs from That of Other 8-Hydroxyquinoline Chelators: Implications for the Treatment of Neurodegenerative Protein Misfolding Diseases.

PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) is a small Cu(II)-binding drug that has been investigated in the treatment of neurodegenerative diseases, namely, Alzheimer's disease (AD). PBT2 is thought to be highly effective at crossing the blood-brain barrier and has been proposed to exert anti-Alzheimer's effects through the modulation of metal ion concentrations in the brain, specifically the sequestration of Cu(II) from amyloid plaques. However, despite promising initial results in animal models and in clinical trials where PBT2 was shown to improve cognitive function, larger-scale clinical trials did not find PBT2 to have a significant effect on the amyloid plaque burden compared with controls. We propose that the results of these clinical trials likely point to a more complex mechanism of action for PBT2 other than simple Cu(II) sequestration. To this end, herein we have investigated the solution chemistry of Cu(II) coordination by PBT2 primarily using X-ray absorption spectroscopy (XAS), high-energy-resolution fluorescence-detected XAS, and electron paramagnetic resonance. We propose that a novel bis-PBT2 Cu(II) complex with asymmetric coordination may coexist in solution with a symmetric four-coordinate Cu(II)-bis-PBT2 complex distorted from coplanarity. Additionally, PBT2 is a more flexible ligand than other 8HQs because it can act as both a bidentate and a tridentate ligand as well as coordinate Cu(II) in both 1:1 and 2:1 PBT2/Cu(II) complexes.

Design, synthesis and evaluation of flurbiprofen-clioquinol hybrids as multitarget-directed ligands against Alzheimer's disease.

A series of novel flurbiprofen-clioquinol hybrids were designed and synthesized as multifunctional agents for Alzheimer's disease therapy, and their potential was evaluated through various biological experiments. In vitro studies showed that most target compounds exhibited significant ability to inhibit self- and Cu2+-induced ß-amyloid aggregation. Furthermore, some target compounds, especially 7i and 7r, also showed biometal chelating abilities, antioxidant activity, anti-neuroinflammatory activity and appropriate BBB permeability. These biological activities indicated that the representative compound 7i and 7r might be promising multifunctional agents for AD treatment.

Using zinc ion-enhanced fluorescence of sulfur quantum dots to improve the detection of the zinc(II)-binding antifungal drug clioquinol.

A turn on-off fluorometric assay for clioquinol (CQ) is described here. It is based on modulation of the fluorescence of sulfur quantum dots (SQDs; best measured at excitation/emission wavelengths of 360/426 nm) by using the Zn2+-CQ affinity pair. Although the fluorescence enhancement effect of Zn2+ on SQDs was not obvious, a good quenching modulation effect was observed in the presence of CQ. This resulted in a linear analytical range that is increased by two orders of magnitude (from 0.024 µM to 0.24 µM, and 0.62 µM to 30 µM), with a detection limit (3 s) of 0.015 µM. The selectivity of the method is also improved. Graphical abstractSchematic illustration of the turn on-off fluorometric assay for for clioquinol (CQ) based on Zn2+-modulated sulfur quantum dots (SQDs).

Effect of metal chelators on the aggregation of beta-amyloid peptides in the presence of copper and iron.

Amyloid ß (Aß) fibrils and amorphous aggregates are found in the brain of patients with Alzheimer's disease (AD), and are implicated in the etiology of AD. The metal imbalance is also among leading causes of AD, owing to the fact that Aß aggregation takes place in the synaptic cleft where Aß, Cu(II) and Fe(III) are found in abnormally high concentrations. Aß40 and Aß42 are the main components of plaques found in afflicted brains. Coordination of Cu(II) and Fe(III) ions to Aß peptides have been linked to Aß aggregation and production of reactive oxygen species, two key events in the development of AD pathology. Metal chelation was proposed as a therapy for AD on the basis that it might prevent Aß aggregation. In this work, we first examined the formation of Aß40 and Aß42 aggregates in the presence of metal ions, i.e. Fe(III) and Cu(II), which were detected by fluorescence spectroscopy and atomic force microscopy. Second, we studied the ability of the two chelators, ethylenediaminetetraacetic acid and 5-chloro-7-iodo-8-hydroxyquinoline (clioquinol), to investigate their effect on the availability of these metal ions to interact with Aß and thereby their effect on Aß accumulation. Our findings show that Fe(III), but not Cu(II), promote aggregation of both Aß40 and Aß42. We also found that only clioquinol decreased significantly iron ion-induced aggregation of Aß42. The presence of ions and/or chelators also affected the morphology of Aß aggregates.

Possible role of metal ionophore against zinc induced cognitive dysfunction in D-galactose senescent mice.

Metal ionophores are considered as potential anti-dementia agents, and some are currently undergoing clinical trials. Many metals are known to accumulate and distribute abnormally in the aging brain. Alterations in zinc metal homeostasis in the glutaminergic synapse could contribute to ageing and the pathophysiology of Alzheimer's disease (AD). The present study was designed to investigate the effect of metal ionophores on long term administration of zinc in D-galactose induced senescent mice. The ageing model was established by combined administration of zinc and D-galactose to mice for 6 weeks. A novel metal ionophore, PBT-2 was given daily to zinc-induced d-galactose senescent mice. The cognitive behaviour of mice was monitored using the Morris Water Maze. The anti-oxidant status and amyloidogenic activity in the ageing mouse was measured by determining mito-oxidative parameters and deposition of amyloid ß (Aß) in the brain. Systemic administration of both zinc and D-galactose significantly produced memory deficits, mito-oxidative damage, heightened acetylcholinesterase enzymatic activity and deposition of amyloid-ß. Treatment with PBT-2 significantly improved behavioural deficits, biochemical profiles, cellular damage, and curbed the deposition of APP in zinc-induced senescent mice. These findings suggest that PBT-2, acting as a metal protein attenuating compound, may be helpful in the prevention of AD or alleviation of ageing.

New Hydroxyquinoline-Based Derivatives as Potent Modulators of Amyloid-ß Aggregations.

Copper and zinc have been found to contribute to the burden of amyloid-ß (Aß) aggregations in neurodegenerative Alzheimer's disease (AD). Dysregulation of these metals leads to the generation of reactive oxygen species (ROS) and eventually results in oxidative damage and accumulation of the Aß peptide, which are the key elements of the disease. Aiming to pursue the discovery of new modulators for the disease, we here rationally focused on conjugating the core hydroxyquinoline of the metal-protein attenuating compound PBT2 and the N-methylanilide analogous moiety of the Aß imaging agent to build a new type of multi-target modulators of Aß aggregations. We found that the N,N-dimethylanilinyl imines 7a, 8a, and the corresponding amines 7b, 8b exerted efficient inhibition of Cu(2+) - or Zn(2+) -induced Aß aggregations and significant disassembly of metal-mediated Aß aggregated fibrils. Further, 7a and 7b also exhibited significant ROC scavenging effects compared to PBT2. The results suggested that 7a and 7b are promising lead compounds for the development of a new therapy for AD.

Coordination properties of a metal chelator clioquinol to Zn(2+) studied by static DFT and ab initio molecular dynamics.

Several lines of evidence supporting the role of metal ions in amyloid aggregation, one of the hallmarks of Alzheimer's disease (AD), have turned metal ion chelation into a promising therapeutic treatment. The design of efficient chelating ligands requires proper knowledge of the electronic and molecular structure of the complexes formed, including their hydration properties. Among various potential chelators, clioquinol (5-chloro-7-iodo-8-hydroxyquinoline, CQH) has been evaluated with relative success in in vitro experiments and even in phase 2 clinical trials. Clioquinol interacts with Zn(ii) to lead to a binary metal/ligand 1 : 2 stoichiometric complex in which the phenolic group of CQH is deprotonated, resulting in Zn(CQ)2 neutral complexes, to which additional water molecules may coordinate. In the present work, the coordinative properties of clioquinol in aqueous solution have been analyzed by means of static, minimal cluster based DFT calculations and explicit solvent ab initio molecular dynamics simulations. Results from static calculations accounting for solvent effects by means of polarized continuum models suggest that the preferred metal coordination environment is tetrahedral Zn(CQ)2, whereas ab initio molecular dynamics simulations point to quasi degenerate penta Zn(CQ)2(H2O) and hexa Zn(CQ)2(H2O)2 coordinated complexes. The possible reasons for these discrepant results are discussed.

Introduction of a fluorescent probe to amyloid-ß to reveal kinetic insights into its interactions with copper(II).

The kinetics of the interactions between amyloid-ß (Aß) and metal ions are crucial to understanding the physiological and pathological roles of Aß in the normal brain and in Alzheimer's disease. Using the quenching of a fluorescent probe by Cu(2+), the mechanism of Aß/Cu(2+) interactions in physiologically relevant conditions has been elucidated. Cu(2+) binds to Aß at a near diffusion-limited rate, initially forming component I. The switching between component I and II occurs on the second timescale, with a significant energy barrier. Component I is much more reactive towards Cu(2+) ligands and likely responsible for initial Aß dimer formation. Clioquinol (CQ) is shown to sequester Cu(2+) more effectively than other tested ligands. These findings have implications for the potential roles of Aß in regulating neurotransmission, and for the screening of small molecules targeting Aß-metal interactions.

Small bifunctional chelators that do not disaggregate amyloid ß fibrils exhibit reduced cellular toxicity.

Multifunctional metal chelators that can modulate the amyloid ß (Aß) peptide aggregation and its interaction with metal ions such as copper and zinc hold considerable promise as therapeutic agents for Alzheimer's disease (AD). However, specific rather than systemic metal chelation by these compounds is needed in order to limit any side effects. Reported herein are two novel small bifunctional chelators, 2-[2-hydroxy-4-(diethylamino)phenyl]benzothiazole (L1) and 2-(2-hydroxy-3-methoxyphenyl)benzothiazole (L2), in which the metal-binding donor atoms are integrated within a molecular framework derived from the amyloid-binding fluorescent dye thioflavin T (ThT). The metal-binding properties of L1 and L2 were probed by pH spectrophotometric titrations to determine their pKa values and the corresponding metal complex stability constants, and the isolated metal complexes were structurally characterized. The amyloid-fibril-binding properties of L1 and L2 were investigated by fluorescence titrations and ThT competition assays. Interestingly, L1 and L2 do not lead to the formation of neurotoxic Aß42 oligomers in the presence or absence of metal ions, as observed by native gel electrophoresis, Western blotting, and transmission electron microscopy. In addition, L1 and L2 were able to reduce the cell toxicity of preformed Aß42 oligomers and of the copper-stabilized Aß42 oligomers. Given their ability to reduce the toxicity of soluble Aß42 and Cu-Aß42 species, L1 and L2 are promising lead compounds for the development of chemical agents that can control the neurotoxicity of soluble Aß42 species in AD.

New multi-target-directed small molecules against Alzheimer's disease: a combination of resveratrol and clioquinol.

Alzheimer's disease (AD) is currently one of the most difficult and challenging diseases to treat. Based on the 'multi-target-directed ligands' (MTDLs) strategy, we designed and synthesised a series of new compounds against AD by combining the pharmacophores of resveratrol and clioquinol. The results of biological activity tests showed that the hybrids exhibited excellent MTDL properties: a significant ability to inhibit self-induced ß-amyloid (Aß) aggregation and copper(II)-induced Aß aggregation, potential antioxidant behaviour (ORAC-FL value of 0.9­3.2 Trolox equivalents) and biometal chelation. Among these compounds, (E)-5-(4-hydroxystyryl)quinoline-8-ol (10c) showed the most potent ability to inhibit self-induced Aß aggregation (IC50 = 8.50 µM) and copper(II)-induced Aß aggregation and to disassemble the well-structured Aß fibrils generated by self- and copper(II)-induced Aß aggregation. Note that 10c could also control Cu(I/II)-triggered hydroxyl radical (OH˙) production by halting copper redox cycling via metal complexation, as confirmed by a Cu­ascorbate redox system assay. Importantly, 10c did not show acute toxicity in mice at doses of up to 2000 mg kg−1 and was able to cross the blood­brain barrier (BBB), according to a parallel artificial membrane permeation assay. These results indicate that compound 10c is a promising multifunctional compound for the development of novel drugs for AD.

The use of a triple quadrupole linear ion trap mass spectrometer with electrospray ionisation for fragmentation studies of selected antifungal drugs.

Fragmentation studies of three antifungal drugs, clotrimazole, fluconazole and clioquinol ,were performed. A triple quadrupole linear ion trap mass spectrometer was used for this purpose. This type of equipment enables MS(3) spectra to be obtained which lead to better understanding of fragmentation pathways. Nevertheless, it is rarely used for fragmentation studies. The results obtained here for the antifungal drugs gave further insight into fragmentation pathways of clotrimazole and fluconazole. Moreover, fragmentation of clioquinol was investigated which had not been presented before.

Small-molecule aggregates inhibit amyloid polymerization.

Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited beta-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus.

A clioquinol-containing Pluronic® F127 polymeric micelle system is effective in the treatment of visceral leishmaniasis in a murine model.

A clioquinol (ICHQ)-containing Pluronic® F127 polymeric micelle system (ICHQ/Mic) was recently shown to be effective against Leishmania amazonensis infection in a murine model. In the present study, ICHQ/Mic was tested against L. infantum infection. BALB/c mice (n = 12 per group) were infected with L. infantum stationary promastigotes through subcutaneous injection and, 45 days after challenge, received saline or were treated via the subcutaneous route with empty micelles, ICHQ or ICHQ/Mic. In addition, animals were treated with miltefosine by the oral route, as a drug control. Half of the animals were euthanized 1 and 15 days after treatment, aiming to evaluate two endpoints after therapy, when parasitological and immunological parameters were investigated. Results showed that the treatment using miltefosine, ICHQ or ICHQ/Mic induced significantly higher anti-parasite IFN-γ, IL-12, GM-CSF, nitrite and IgG2a isotype antibody levels, which were associated with low IL-4 and IL-10 production. In addition, a higher frequency of IFN-γ and TNF-α-producing CD4+ and CD8+ T-cells was found in these animals. The parasite load was evaluated in distinct organs, and results showed that the treatment using miltefosine, ICHQ or ICHQ/Mic induced significant reductions in organic parasitism in the treated and infected mice. A comparison between the treatments suggested that ICHQ/Mic was the most effective in inducing a highly polarized Th1-type response, as well as reducing the parasite load in significant levels in the treated and infected animals. Data obtained 15 days after treatment suggested maintenance of the immunological and parasitological responses. In conclusion, ICHQ/Mic could be considered in future studies for the treatment of visceral leishmaniasis.

TITLE: Un système à micelles polymériques Pluronic® F127 contenant du clioquinol est efficace pour le traitement de la leishmaniose viscérale dans un modèle murin. ABSTRACT: Un système à micelles polymériques Pluronic® F127 (ICHQ/Mic) contenant du clioquinol (ICHQ) s'est récemment révélé efficace contre l'infection à Leishmania amazonensis dans un modèle murin. Dans la présente étude, l'ICHQ/Mic a été testé contre l'infection à L. infantum. Les souris BALB/c (n = 12 par groupe) ont été infectées par des promastigotes stationnaires de L. infantum par injection sous-cutanée et ont reçu 45 jours après l'épreuve une solution saline ou ont été traitées par voie sous-cutanée avec des micelles vides, ICHQ ou ICHQ/Mic. De plus, les animaux ont été traités avec de la miltefosine par voie orale, comme contrôle médicamenteux. La moitié des animaux ont été euthanasiés 1 et 15 jours après le traitement, dans le but de mesurer deux critères d'évaluation après la thérapie, lorsque les paramètres parasitologiques et immunologiques ont été étudiés. Les résultats ont montré que le traitement par miltefosine, ICHQ ou ICHQ/Mic induisait des niveaux d'anticorps anti-parasite IFN-γ, IL-12, GM-CSF, nitrite et IgG2a significativement plus élevés, associés à de faibles productions d'IL-4 et IL-10. De plus, une fréquence plus élevée de cellules T CD4+ et CD8+ produisant de l'IFN-γ and TNF-α a été trouvée chez ces animaux. La charge parasitaire a été évaluée dans des organes distincts et les résultats ont montré que le traitement utilisant la miltefosine, ICHQ ou ICHQ/Mic induisait des réductions significatives du parasitisme des organes chez les souris traitées et infectées. Une comparaison entre les traitements a suggéré qu'ICHQ/Mic était le plus efficace pour induire une réponse de type Th1 polarisée, ainsi que pour réduire la charge parasitaire à des niveaux significatifs chez les animaux traités et infectés. Les données obtenues 15 jours après le traitement suggèrent le maintien des réponses immunologiques et parasitologiques. En conclusion, ICHQ/Mic pourrait être envisagé dans de futures études pour le traitement contre la leishmaniose viscérale.

Unravelling the interaction mechanism between clioquinol and bovine serum albumin by multi-spectroscopic and molecular docking approaches.

Clioquinol has recently been proposed for the treatment of Alzheimer's disease. It is able to diminish ß-amyloid protein aggregation and to restore cognition of Alzheimer's mice. However, its therapeutic benefits for Alzheimer's disease in human remain controversy and need further confirmation. Herein, we have explored the interaction mechanism of clioquinol toward bovine serum albumin (BSA) by means of multi-spectroscopic and docking simulation approaches. Clioquinol interacts with BSA by a combined mechanism of static and dynamic processes. Application of the Hill's equation to fluorescence quenching experiment revealed that the binding constant of the BSA-clioquinol complex is extremely high at 108 M-1 level. Competitive displacement and docking analysis consistently suggested that there are the multiple binding modes of clioquinol toward BSA. Competitive binding study showed that clioquinol shares the binding sites with ibuprofen and digitoxin on albumin, referring to be site II and site III binding compounds. Besides, partial binding in site I was also observed. Docking simulation confirmed that clioquinol favors to bind in site I, site II, site III, fatty acid binding site 5, and the protein cleft between subdomain IB and IIIB of the BSA. Due to its small size and electric dipole property, clioquinol may easily fit in multiple pockets of the BSA. Our finding suggests the potential role of BSA as a clioquinol carrier in the vascular system. Nonetheless, clioquinol-induced BSA aggregation has been observed by the three-dimensional fluorescence technique. This phenomenon may not only impair the BSA, but may also affect other endogenous proteins, which eventually causes adverse effects to human. Therefore, the redesigned or modified molecular structure of clioquinol may reduce its toxicity and improve its bioavailability.

WBQ5187, a Multitarget Directed Agent, Ameliorates Cognitive Impairment in a Transgenic Mouse Model of Alzheimer's Disease and Modulates Cerebral ß-Amyloid, Gliosis, cAMP Levels, and Neurodegeneration.

Previously, we designed, synthesized, and evaluated a series of quinolone-benzofuran derivatives as multitargeted anti-Alzheimer's disease (anti-AD) compounds, and we discovered that WBQ5187 possesses superior anti-AD bioactivity. In this work, we investigated the pharmacokinetics of this new molecule, as well as its therapeutic efficacy in restoring cognition and neuropathology, in the APP/PS1 mouse model of AD. Pharmacokinetic analyses demonstrated that WBQ5187 possessed rational oral bioavailability, metabolic stability, and excellent blood-brain barrier (BBB) permeability. Pharmacodynamics studies indicated that a 12-week treatment with the lead compound at doses of 40 mg/kg or higher significantly enhanced the learning and memory performance of the APP/PS1 transgenic mice, and the effect was more potent than that of clioquinol (CQ). Furthermore, WBQ5187 notably reduced cerebral ß-amyloid pathology, gliosis, and neuronal cell loss and increased the levels of cAMP in the hippocampus of these mice. The surrogate measures of emesis indicated that WBQ5187 had no effect at its cognitive effective doses. Overall, our results demonstrated that this compound markedly improves cognitive and spatial memory functions in AD mice and represents a promising pharmaceutical agent with potential for the treatment of AD.

Cholesterol and Clioquinol modulation of A beta(1-42) interaction with phospholipid bilayers and metals.

The beta-sheet plaques that are the most obvious pathological feature of Alzheimer's disease are composed of amyloid-beta peptides and are highly enriched in the metal ions Zn, Fe and Cu. The interaction of the full-length amyloid peptide, A beta(1-42), with phospholipid lipid bilayers was studied in the presence of the metal-chelating drug, Clioquinol (CQ). The effect of cholesterol and metal ions was also determined using solid-state 31P and 2H NMR. CQ modulated the effect of metal ions on the integrity of the bilayer and although CQ perturbed the phospholipid membrane, the bilayer integrity was maintained. Model membranes enriched in cholesterol were studied under conditions of peptide association and incorporation. Solid-state NMR showed that the bilayer integrity was preserved in cholesterol-enriched membranes in comparison to phosphatidylcholine-phosphatidylserine bilayers. Changes in peptide structure, consistent with an increase in beta-sheet, were observed using specifically 13C-labelled A beta(1-42) by magic angle spinning NMR. Results using aligned phosphatidylcholine bilayers and completely 15N-labelled peptide indicated that the peptide aggregated. The results are consistent with oligomeric beta-sheet structured peptides only partially penetrating the bilayer and cholesterol reducing the membrane disruption.

Safety, efficacy, and biomarker findings of PBT2 in targeting Abeta as a modifying therapy for Alzheimer's disease: a phase IIa, double-blind, randomised, placebo-controlled trial.

BACKGROUND: PBT2 is a metal-protein attenuating compound (MPAC) that affects the Cu2(+)-mediated and Zn2(+)-mediated toxic oligomerisation of Abeta seen in Alzheimer's disease (AD). Strong preclinical efficacy data and the completion of early, clinical safety studies have preceded this phase IIa study, the aim of which was to assess the effects of PBT2 on safety, efficacy, and biomarkers of AD. METHODS: Between December 6, 2006, and September 21, 2007, community-dwelling patients over age 55 years were recruited to this 12-week, double-blind, randomised trial of PBT2. Patients were randomly allocated to receive 50 mg PBT2, 250 mg PBT2, or placebo. Inclusion criteria were early AD (mini-mental state examination [MMSE] score between 20 and 26 points or Alzheimer's disease assessment scale-cognitive subscale (ADAS-cog) score between 10 and 25 points), taking a stable dose of acetylcholinesterase inhibitor (donepezil, galantamine, or rivastigmine) for at least 4 months, a modified Hachinski score of 4 points or less, and CT or MRI results that were consistent with AD. The principal outcomes were safety and tolerability. Secondary outcomes were plasma and CSF biomarkers and cognition. Analysis was intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00471211. FINDINGS: 78 patients were randomly assigned (29 to placebo, 20 to PBT2 50 mg, and 29 to PBT2 250 mg) and 74 (95%) completed the study. 42 (54%) patients had at least one treatment emergent adverse event (10 [50%] on PBT2 50 mg, 18 [62%] on PBT2 250 mg, and 14 [48%] on placebo). No serious adverse events were reported by patients on PBT2. Patients treated with PBT2 250 mg had a dose-dependent (p=0.023) and significant reduction in CSF Abeta(42) concentration compared with those treated with placebo (difference in least squares mean change from baseline was -56.0 pg/mL, 95% CI -101.5 to -11.0; p=0.006). PBT2 had no effect on plasma biomarkers of AD or serum Zn(2+) and Cu(2+) concentrations. Cognition testing included ADAS-cog, MMSE, and a neuropsychological test battery (NTB). Of these tests, two executive function component tests of the NTB showed significant improvement over placebo in the PBT2 250 mg group: category fluency test (2.8 words, 0.1 to 5.4; p=0.041) and trail making part B (-48.0 s, -83.0 to -13.0; p=0.009). INTERPRETATION: The safety profile is favourable for the ongoing development of PBT2. The effect on putative biomarkers for AD in CSF but not in plasma is suggestive of a central effect of the drug on Abeta metabolism. Cognitive efficacy was restricted to two measures of executive function. Future trials that are larger and longer will establish if the effects of PBT2 on biomarkers and cognition that are reported here translate into clinical effectiveness.