Liposome nanoparticle conjugation and cell penetrating peptide sequences (CPPs) enhance the cellular delivery of the tau aggregation inhibitor RI-AG03.

Given the pathological role of Tau aggregation in Alzheimer's disease (AD), our laboratory previously developed the novel Tau aggregation inhibitor peptide, RI-AG03. As Tau aggregates accumulate intracellularly, it is essential that the peptide can traverse the cell membrane. Here we examine the cellular uptake and intracellular trafficking of RI-AG03, in both a free and liposome-conjugated form. We also characterize the impact of adding the cell-penetrating peptide (CPP) sequences, polyarginine (polyR) or transactivator of transcription (TAT), to RI-AG03. Our data show that liposome conjugation of CPP containing RI-AG03 peptides, with either the polyR or TAT sequence, increased cellular liposome association three-fold. Inhibition of macropinocytosis modestly reduced the uptake of unconjugated and RI-AG03-polyR-linked liposomes, while having no effect on RI-AG03-TAT-conjugated liposome uptake. Further supporting macropinocytosis-mediated internalization, a 'fair' co-localisation of the free and liposome-conjugated RI-AG03-polyR peptide with macropinosomes and lysosomes was observed. Interestingly, we also demonstrate that RI-AG03-polyR detaches from liposomes following cellular uptake, thereby largely evading organellar entrapment. Collectively, our data indicate that direct membrane penetration and macropinocytosis are key routes for the internalization of liposomes conjugated with CPP containing RI-AG03. Our study also demonstrates that peptide-liposomes are suitable nanocarriers for the cellular delivery of RI-AG03, furthering their potential use in targeting Tau pathology in AD.

Structural study of the recognition mechanism of tau antibody Tau2r3 with the key sequence (VQIINK) in tau aggregation.

One of the histopathological features of Alzheimer's disease (AD) is higher order neurofibrillary tangles formed by abnormally aggregated tau protein. The sequence 275VQIINK280 in the microtubule-binding domain of tau plays a key role in tau aggregation. Therefore, an aggregation inhibitor targeting the VQIINK region in tau may be an effective therapeutic agent for AD. We have previously shown that the Fab domain (Fab2r3) of a tau antibody that recognizes the VQIINK sequence can inhibit tau aggregation, and we have determined the tertiary structure of the Fab2r3-VQIINK complex. In this report, we determined the tertiary structure of apo Fab2r3 and analyzed differences in the structures of apo Fab2r3 and Fab2r3-VQIINK to examine the ligand recognition mechanism of Fab2r3. In comparison with the Fab2r3-VQIINK structure, there were large differences in the arrangement of the constant and variable domains in apo Fab2r3. Remarkable structural changes were especially observed in the H3 and L3 loop regions of the complementarity determining regions (CDRs) in apo Fab2r3 and the Fab2r3-VQIINK complex. These structural differences in CDRs suggest that formation of hydrophobic pockets suitable for the antigen is important for antigen recognition by tau antibodies.

The antidepressant drug; trazodone inhibits Tau amyloidogenesis: Prospects for prophylaxis and treatment of AD.

Tau protein, characterized as "natively unfolded", is involved in microtubule assembly/stabilization in physiological conditions. Under pathological conditions, Tau dysfunction leads to its accumulation of insoluble toxic amyloid aggregates and thought to be involved in the degeneration and neuronal death associated with neurodegenerative diseases. Trazodone (TRZ), a triazolopyridine derivative, is a selective serotonin reuptake inhibitor (SSRI) which increases serotonin levels in synaptic cleft and potentiating serotonin activity, with antidepressant and sedative properties. This drug is more effective and tolerable than other therapeutic agents. In this study, the 1N4R isoform of Tau protein was purified and the effect of TRZ on the protein fibrillation was investigated using multi-spectroscopic techniques as well as computational methods. The results showed that TRZ is not only able to affect formation of Tau amyloid fibrils in vitro but also attenuates Tau oligomerization within SH-SY5Y cell line resulting in more cells surviving. Moreover, membrane disrupting activity of Tau aggregates decreased upon TRZ treatment. The binding forces involved in TRZ-Tau interaction were also explored using both experimental as well as theoretical docking/molecular dynamics approaches. The results of the current work may open new insights for applying therapeutic potential of TRZ against Alzheimer's disease.

Reducing tau aggregates with anle138b delays disease progression in a mouse model of tauopathies.

Pathological tau aggregation leads to filamentous tau inclusions and characterizes neurodegenerative tauopathies such as Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Tau aggregation coincides with clinical symptoms and is thought to mediate neurodegeneration. Transgenic mice overexpressing mutant human P301S tau exhibit many neuropathological features of human tauopathies including behavioral deficits and increased mortality. Here, we show that the di-phenyl-pyrazole anle138b binds to aggregated tau and inhibits tau aggregation in vitro and in vivo. Furthermore, anle138b treatment effectively ameliorates disease symptoms, increases survival time and improves cognition of tau transgenic PS19 mice. In addition, we found decreased synapse and neuron loss accompanied by a decreased gliosis in the hippocampus. Our results suggest that reducing tau aggregates with anle138b may represent an effective and promising approach for the treatment of human tauopathies.

Assays for the Screening and Characterization of Tau Aggregation Inhibitors.

Aggregation of tau protein is a pathological hallmark of Alzheimer's disease and other neurodegenerative tauopathies. Inhibition of tau aggregation may provide a method for treatment of these disorders. Methods to identify tau aggregation inhibitors (TAIs) in vitro are useful and here we describe assays for TAIs using purified recombinant tau protein fragments in a cell-free immunoassay format and in a stably transfected cell model to create a more physiological environment.

Tau-aggregation inhibitors derived from Streptomyces tendae MCCC 1A01534 protect HT22 cells against okadaic acid-induced damage.

Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by tau aggregating into neurofibrillary tangles. Targeting tau aggregation is one of the most critical strategies for AD treatment and prevention. Herein, a high-throughput screening of tau-aggregation inhibitors was performed by thioflavin T (ThT) fluorescence assay and tauR3 peptides. According to bioactivity-guided isolation, homoprejadomycin (1) was obtained from the marine bacterium Streptomyces tendae MCCC 1A01534. Two new stable derivatives, 2 and 3, were yielded in a one-step reaction. By ThT assay, transmission electron microscopy, and circular dichroism, we demonstrated that the angucyclinones 2 and 3 inhibited tau aggregation and disaggregated tau fibrils. In the presence of 2, native tauR3 peptides maintained the disorder conformation, whereas the tauR3 aggregates reduced ß-sheet structures. And compound 2 was confirmed to inhibit the aggregation of full-length 2N4R tau protein. Furthermore, 2 with low cytotoxicity protected HT22 cells from okadaic acid-induced damage by suppressing tau aggregates. These results indicated that 2 was a promising lead structure with tau therapeutic potency for AD treatment.

HMTM-Mediated Enhancement of Brain Bioenergetics in a Mouse Tauopathy Model Is Blocked by Chronic Administration of Rivastigmine.

The tau protein aggregation inhibitor hydromethylthionine mesylate (HMTM) was shown recently to have concentration-dependent pharmacological activity in delaying cognitive decline and brain atrophy in phase 3 Alzheimer's disease (AD) clinical trials; the activity was reduced in patients receiving symptomatic therapies. The methylthionine (MT) moiety has been reported to increase the clearance of pathological tau and to enhance mitochondrial activity, which is impaired in AD patients. In line 1 (L1) mice (a model of AD), HMTM (5/15 mg/kg) was administered either as a monotherapy or as an add-on to a chronic administration with the cholinesterase inhibitor rivastigmine (0.1/0.5 mg/kg) to explore mitochondrial function and energy substrate utilization as potential targets of drug interference. Compared with wild-type NMRI mice, the L1 mice accumulated greater levels of l-lactate and of the LDH-A subunit responsible for the conversion of pyruvate into l-lactate. In contrast, the levels of LDH-B and mitochondrial ETC subunits and the activity of complexes I and IV was not altered in the L1 mice. The activity of complex I and complex IV tended to increase with the HMTM dosing, in turn decreasing l-lactate accumulation in the brains of the L1 mice, despite increasing the levels of LDH-A. The chronic pre-dosing of the L1 mice with rivastigmine partially prevented the enhancement of the activity of complexes I and IV by HMTM and the increase in the levels of LDH-A while further reducing the levels of l-lactate. Thus, HMTM in combination with rivastigmine leads to a depletion in the energy substrate l-lactate, despite bioenergetic production not being favoured. In this study, the changes in l-lactate appear to be regulated by LDH-A, since neither of the experimental conditions affected the levels of LDH-B. The data show that HMTM monotherapy facilitates the use of substrates for energy production, particularly l-lactate, which is provided by astrocytes, additionally demonstrating that a chronic pre-treatment with rivastigmine prevented most of the HMTM-associated effects.

Oral Tau Aggregation Inhibitor for Alzheimer's Disease: Design, Progress and Basis for Selection of the 16 mg/day Dose in a Phase 3, Randomized, Placebo-Controlled Trial of Hydromethylthionine Mesylate.

BACKGROUND: Hydromethylthionine mesylate is a tau aggregation inhibitor shown to have exposure-dependent pharmacological activity on cognitive decline and brain atrophy in two completed Phase 3 trials in mild/moderate Alzheimer's disease (AD). OBJECTIVES: The present report summarises the basis for selection of 16 mg/day as monotherapy as the optimal treatment regime and the design rationale of a confirmatory Phase 3 trial (LUCIDITY). DESIGN: The trial comprises a 12-month double-blind, placebo-controlled phase followed by a 12-month modified delayed-start open-label treatment phase. SETTING: 76 clinical research sites in North America and Europe. PARTICIPANTS: 545 patients with probable AD or MCI-AD in the final version of the protocol. INTERVENTION: Participants were assigned randomly to receive hydromethylthione mesylate at doses of 16 mg/day, 8 mg/day or placebo at a 4:1:4 ratio during the double-blind phase. All participants in the open-label phase receive the 16 mg/day dose. MEASUREMENTS: Co-primary clinical outcomes are the 11-item Alzheimer's Disease Assessment Scale (ADAS-cog11) and the 23-item Alzheimer's Disease Cooperative Study - Activities of Daily Living (ADCS-ADL23). Secondary biomarker measures include whole-brain atrophy and temporal lobe 18F-fluorodeoxyglucose positron emission tomography. RESULTS: 446 participants are expected to complete the 12-month placebo-controlled phase in March 2022. CONCLUSIONS: If the primary end points are met, the data will provide confirmatory evidence of the clinical and biomarker benefits of hydromethylthionine mesylate in minimal to moderate AD. As low-dose oral hydromethylthionine mesylate is simple to use clinically, does not cause amyloid-related imaging abnormalities and has a benign safety profile, it would likely improve AD management.

Small molecule therapeutics for tauopathy in Alzheimer's disease: Walking on the path of most resistance.

Alzheimer's disease (AD) is the most common form of dementia characterized by presence of extracellular amyloid plaques and intracellular neurofibrillary tangles composed of tau protein. Currently there are close to 50 million people living with dementia and this figure is expected to increase to 75 million by 2030 putting a huge burden on the economy due to the health care cost. Considering the effects on quality of life of patients and the increasing burden on the economy, there is an enormous need of new disease modifying therapies to tackle this disease. The current therapies are dominated by only symptomatic treatments including cholinesterase inhibitors and N-methyl-D-aspartate receptor blockers but no disease modifying treatments exist so far. After several failed attempts to develop drugs against amyloidopathy, tau targeting approaches have been in the main focus of drug development against AD. After an overview of the tauopathy in AD, this review summarizes recent findings on the development of small molecules as therapeutics targeting tau modification, aggregation, and degradation, and tau-oriented multi-target directed ligands. Overall, this work aims to provide a comprehensive and critical overview of small molecules which are being explored as a lead candidate for discovering drugs against tauopathy in AD.

An evaluation of hydromethylthionine as a treatment option for Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is a major cause of morbidity worldwide and its prevalence is expected to rise. Previous studies involving compounds that target the accumulation of amyloid ß protein have been unsuccessful, renewing interest in therapies directed against intracellular deposits of tau proteins. Derived from methylene blue, hydromethylthionine is a tau aggregation inhibitor that recently emerged as a promising disease-modifying treatment for AD. AREAS COVERED: Herein, the authors cover the chemistry, pharmacodynamics and pharmacokinetics of hydromethylthionine and its oxidized form methylthionine chloride (MTC) that was first studied, as well as clinical efficacy and safety of hydromethylthionine in the treatment of mild to moderate AD. EXPERT OPINION: Randomized clinical trials with hydromethylthionine failed to show any impact of the doses used on the disease course. Data analysis from a non-randomized cohort showed that a smaller dose of the drug previously thought to be ineffective and used as placebo, prescribed as monotherapy rather than as add-on to AD approved symptomatic therapies may slow cognitive decline. This finding was further confirmed by a pharmacokinetic analysis study showing a dose/response relationship with doses around 16 mg daily. Future trials need to study the pharmacological properties of hydromethylthionine and ascertain the optimal safe and effective dose to be used.

Mechanisms of Anticholinesterase Interference with Tau Aggregation Inhibitor Activity in a Tau-Transgenic Mouse Model.

BACKGROUND: Symptomatic treatments of Alzheimer's Disease (AD) with cholinesterase inhibitors and/or memantine are relatively ineffective and there is a need for new treatments targeting the underlying pathology of AD. In most of the failed disease-modifying trials, patients have been allowed to continue taking symptomatic treatments at stable doses, under the assumption that they do not impair efficacy. In recently completed Phase 3 trials testing the tau aggregation inhibitor leuco-methylthioninium bis (hydromethanesulfonate) (LMTM), we found significant differences in treatment response according to whether patients were taking LMTM either as monotherapy or as an add-on to symptomatic treatments. METHODS: We have examined the effect of either LMTM alone or chronic rivastigmine prior to LMTM treatment of tau transgenic mice expressing the short tau fragment that constitutes the tangle filaments of AD. We have measured acetylcholine levels, synaptosomal glutamate release, synaptic proteins, mitochondrial complex IV activity, tau pathology and Choline Acetyltransferase (ChAT) immunoreactivity. RESULTS: LMTM given alone increased hippocampal Acetylcholine (ACh) levels, glutamate release from synaptosomal preparations, synaptophysin levels in multiple brain regions and mitochondrial complex IV activity, reduced tau pathology, partially restored ChAT immunoreactivity in the basal forebrain and reversed deficits in spatial learning. Chronic pretreatment with rivastigmine was found to reduce or eliminate almost all these effects, apart from a reduction in tau aggregation pathology. LMTM effects on hippocampal ACh and synaptophysin levels were also reduced in wild-type mice. CONCLUSION: The interference with the pharmacological activity of LMTM by a cholinesterase inhibitor can be reproduced in a tau transgenic mouse model and, to a lesser extent, in wild-type mice. Long-term pretreatment with a symptomatic drug alters a broad range of brain responses to LMTM across different transmitter systems and cellular compartments at multiple levels of brain function. There is, therefore, no single locus for the negative interaction. Rather, the chronic neuronal activation induced by reducing cholinesterase function produces compensatory homeostatic downregulation in multiple neuronal systems. This reduces a broad range of treatment responses to LMTM associated with a reduction in tau aggregation pathology. Since the interference is dictated by homeostatic responses to prior symptomatic treatment, it is likely that there would be similar interference with other drugs tested as add-on to the existing symptomatic treatment, regardless of the intended therapeutic target or mode of action. The present findings outline key results that now provide a working model to explain interference by symptomatic treatment.

Assays for the Screening and Characterization of Tau Aggregation Inhibitors.

Aggregation of tau protein is a pathological hallmark of Alzheimer's disease and other neurodegenerative tauopathies. Inhibition of tau aggregation could provide a method for the treatment of these disorders. Methods to identify tau aggregation inhibitors (TAIs) in vitro are useful and here we describe assays for TAIs using purified recombinant tau protein fragments in a cell-free immunoassay format and a stably transfected cell model to create a more physiological environment.