Multitargeted Imidazoles: Potential Therapeutic Leads for Alzheimer's and Other Neurodegenerative Diseases

Alzheimer’s disease (AD) is a complex, multifactorial disease in which different neuropathological mechanisms are likely involved, including those associated with pathological tau and Aβ species as well as neuroinflammation. In this context, the development of single multitargeted therapeutics directed against two or more disease mechanisms could be advantageous. Starting from a series of 1,5-diarylimidazoles with microtubule (MT)-stabilizing activity and structural similarities with known NSAIDs, we conducted structure−activity relationship studies that led to the identification of multitargeted prototypes with activities as MT-stabilizing agents and/or inhibitors of the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways. Several examples are brain-penetrant and exhibit balanced multitargeted in vitro activity in the low μM range. As brain-penetrant MT-stabilizing agents have proven effective against tau-mediated neurodegeneration in animal models, and because COX- and 5-LOX-derived eicosanoids are thought to contribute to Aβ plaque deposition, these 1,5-diarylimidazoles provide tools to explore novel multitargeted strategies for AD and other neurodegenerative diseases

Evaluation of the brain-penetrant microtubule-stabilizing agent, dictyostatin, in the PS19 tau transgenic mouse model of tauopathy.

Neurodegenerative disorders referred to as tauopathies, which includes Alzheimer's disease (AD), are characterized by insoluble deposits of the tau protein within neuron cell bodies and dendritic processes in the brain. Tau is normally associated with microtubules (MTs) in axons, where it provides MT stabilization and may modulate axonal transport. However, tau becomes hyperphosphorylated and dissociates from MTs in tauopathies, with evidence of reduced MT stability and defective axonal transport. This has led to the hypothesis that MT-stabilizing drugs may have potential for the treatment of tauopathies. Prior studies demonstrated that the brain-penetrant MT-stabilizing drug, epothilone D, had salutary effects in transgenic (Tg) mouse models of tauopathy, improving MT density and axonal transport, while reducing axonal dystrophy. Moreover, epothilone D enhanced cognitive performance and decreased hippocampal neuron loss, with evidence of reduced tau pathology. To date, epothilone D has been the only non-peptide small molecule MT-stabilizing agent to be evaluated in Tg tau mice. Herein, we demonstrate the efficacy of another small molecule brain-penetrant MT-stabilizing agent, dictyostatin, in the PS19 tau Tg mouse model. Although dictyostatin was poorly tolerated at once-weekly doses of 1 mg/kg or 0.3 mg/kg, likely due to gastrointestinal (GI) complications, a dictyostatin dose of 0.1 mg/kg was better tolerated, such that the majority of 6-month old PS19 mice, which harbor a moderate level of brain tau pathology, completed a 3-month dosing study without evidence of significant body weight loss. Importantly, as previously observed with epothilone D, the dictyostatin-treated PS19 mice displayed improved MT density and reduced axonal dystrophy, with a reduction of tau pathology and a trend toward increased hippocampal neuron survival relative to vehicle-treated PS19 mice. Thus, despite evidence of dose-limiting peripheral side effects, the observed positive brain outcomes in dictyostatin-treated aged PS19 mice reinforces the concept that MT-stabilizing compounds have significant potential for the treatment of tauopathies

Estimating drug potency in the competitive target mediated drug disposition (TMDD) system when the endogenous ligand is included.

Predictions for target engagement are often used to guide drug development. In particular, when selecting the recommended phase 2 dose of a drug that is very safe, and where good biomarkers for response may not exist (e.g. in immuno-oncology), a receptor occupancy prediction could even be the main determinant in justifying the approved dose, as was the case for atezolizumab. The underlying assumption in these models is that when the drug binds its target, it disrupts the interaction between the target and its endogenous ligand, thereby disrupting downstream signaling. However, the interaction between the target and its endogenous binding partner is almost never included in the model. In this work, we take a deeper look at the in vivo system where a drug binds to its target and disrupts the target’s interaction with an endogenous ligand. We derive two simple steady state inhibition metrics (SSIMs) for the system, which provides intuition for when the competition between drug and endogenous ligand should be taken into account for guiding drug development

Use of fluorescent ANTS to examine the BBB-permeability of polysaccharide

Recently, some polysaccharides showed therapeutic potentials for the treatment of neurodegenerative diseases while the most important property, their permeability to the blood brain barrier (BBB) that sheathes the brain and spinal cord, is not yet determined. The determination has been delayed by the difficulty in tracking a target polysaccharide among endogenous polysaccharides in animal. We developed an easy way to examine the BBB-permeability and, possibly, tissue distribution of a target polysaccharide in animal. We tagged a polysaccharide with fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS) for tracking. We also developed a simple method to separate ANTS-tagged polysaccharide from unconjugated free ANTS using 75% ethanol. After ANTS-polysaccharide was intra-nasally administered into animals, we could quantify the amounts of ANTS-polysaccharide in the brain and the serum by fluorocytometry. We could also separate free ANTS-polysaccharide from serum proteins using trichloroacetic acid (TCA) and 75% ethanol. Our method will help to track a polysaccharide in animal easily. • ANTS-labeling is less tedious than but as powerful as radiolabeling for tracking a target polysaccharide in animal. • Our simple method can separate structurally intact ANTS-polysaccharide from animal serum and tissues. • This method is good for the fluorometry-based measurement of ANTS-conjugated macromolecules in tissues

BBB-Permeable, Neuroprotective, and Neurotrophic Polysaccharide, Midi-GAGR

<div><p>An enormous amount of efforts have been poured to find an effective therapeutic agent for the treatment of neurodegenerative diseases including Alzheimer’s disease (AD). Among those, neurotrophic peptides that regenerate neuronal structures and increase neuron survival show a promise in slowing neurodegeneration. However, the short plasma half-life and poor blood-brain-barrier (BBB)-permeability of neurotrophic peptides limit their <i>in vivo</i> efficacy. Thus, an alternative neurotrophic agent that has longer plasma half-life and better BBB-permeability has been sought for. Based on the recent findings of neuroprotective polysaccharides, we searched for a BBB-permeable neuroprotective polysaccharide among natural polysaccharides that are approved for human use. Then, we discovered midi-GAGR, a BBB-permeable, long plasma half-life, strong neuroprotective and neurotrophic polysaccharide. Midi-GAGR is a 4.7kD cleavage product of low acyl gellan gum that is approved by FDA for human use. Midi-GAGR protected rodent cortical neurons not only from the pathological concentrations of co-/post-treated free reactive radicals and Aβ₄₂ peptide but also from activated microglial cells. Moreover, midi-GAGR showed a good neurotrophic effect; it enhanced neurite outgrowth and increased phosphorylated cAMP-responsive element binding protein (pCREB) in the nuclei of primary cortical neurons. Furthermore, intra-nasally administered midi-GAGR penetrated the BBB and exerted its neurotrophic effect inside the brain for 24 h after one-time administration. Midi-GAGR appears to activate fibroblast growth factor receptor 1 (FGFR1) and its downstream neurotrophic signaling pathway for neuroprotection and CREB activation. Additionally, 14-day intranasal administration of midi-GAGR not only increased neuronal activity markers but also decreased hyperphosphorylated tau, a precursor of neurofibrillary tangle, in the brains of the AD mouse model, 3xTg-AD. Taken together, midi-GAGR with good BBB-permeability, long plasma half-life, and strong neuroprotective and neurotrophic effects has a great therapeutic potential for the treatment of neurodegenerative diseases, especially AD.</p></div

Midi-GAGR reverses neurite atrophy caused by 4HNE and H₂O₂.

<p>(A-E) Differentiated N2A cells were treated with different concentrations of 4HNE (0, 1, 5, 10 and 25 μM) for 48 h or H₂O₂ (0, 1, 10, 50, 100 and 200 μM) for 24 h and immunostained with α-tubulin antibody. The representative images of N2A cells treated with H₂O (vehicle) (A), 25 μM 4HNE (B), or 200 μM H₂O₂ (C). Scale bar = 75 μm. Bar graphs represent the average total neurite lengths of N2A cells in response to different concentrations of either 4HNE (D) or H₂O₂ (E). *, p < 0.05 and **, p < 0.001 compared to control. (F-I) Differentiated N2A cells were pre-treated with different concentrations (0, 0.001, 0.01, 0.1, 1, and 10 μM) of midi-GAGR for 24 h, followed by incubation with either 25 μM 4HNE for 48 h or 200 μM H₂O₂ for 24 h and then immunostained with anti-α-tubulin antibody. The representative images of N2A cells pretreated with 1 μM midi-GAGR and then incubated with either 25 μM 4HNE (F) or 200 μM H₂O₂ (G). Scale bar = 75 μm. Bar graphs show the average total neurite lengths of N2A cells pre-treated with different concentrations of midi-GAGR followed by treatment with 25 μM 4HNE (H) or 200 μM H₂O₂ (I). Dotted lines correspond to the average total neurite lengths of N2A cells without any treatment. Data represent mean ± SEM of, at least, 40 cells/group from two independent experiments. *, p<0.05 and **, p<0.001 compared to 4HNE alone.</p

Midi-GAGR enhances neuritogenesis in mouse cortical neurons.

<p>(A-F) Mouse cortical neurons (E17, DIV4) were treated with H₂O (vehicle) (A), 1 μM midi-GAGR (B), 0.1 μM LA-GAGR (C), 0.01 μM HA-GAGR (D), 1 μM alginate (C), or 1 μM dextran (F) for 48 h and immunostained with anti-α-tubulin antibody. Scale bar = 100 μm. (G) Bar graphs show average fold changes in the total neurite length of mouse cortical neurons in response to different polysaccharides (mean ± SEM of, at least, three independent experiments). **, p<0.01 compared to control.</p

Midi-GAGR protects rodent cortical neurons from co-treated 4HNE, Aβ₄₂ peptide and activated microglial cells.

<p>(A) Rat cortical neurons (E17) at DIV5 were co-treated with either 10 μM 4HNE (for 24 h) or 2 μM Aβ₄₂ (for 48 h) and either water or 1 μM midi-GAGR. After the treatment, neurons were processed for live/dead assay using calcein AM and ethidium homodimer-I. Live and dead cells were imaged using a fluorescence microscope. The numbers of live and dead cells were counted using Metamorph software. Bar graphs show the percents of dead neurons after co-treatment with either 4HNE or Aβ₄₂ plus/minus midi-GAGR. Data represent mean ± SEM of three independent experiments. *, p<0.05. (B) The co-cultures of rat cortical neurons and microglia cells were treated with 2 μM Aβ₄₂ plus/minus 1 μM midi-GAGR. After 48 h, transwell filters containing microglial cells were removed and neurons in bottom wells were processed for live/dead assay. Live and dead cells were imaged using a fluorescence microscope. The numbers of live and dead neurons were counted using Metamorph software. Bar graphs show the percent of dead neurons. Data represent mean ± SEM of three independent experiments. *, p<0.05.</p

Intranasally administered midi-GAGR increases the expression of NF200 and GAP-43 in the brains of live rats.

<p>SD rats were intranasally administered with either vehicle or midi-GAGR and processed to obtain brains at 6h, 24h or 48h after the administration. Brains were dissected to the frontal cortex, hippocampus, and rest of the brain. (A) Brain tissue lysates were processed for immunoblotting using the antibody to NF200 (upper panel), GAP-43 (middle panel), or GAPDH (lower panel). ‘C’ is control and ‘M’ is midi-GAGR. The band densities of NF200 and GAP-43 were measured using image J software and normalized to those of GAPDH. Bar graphs show fold changes in the level of NF200 (B) and GAP-43 (C) in the different parts of brains at given time points. Data represents mean ± SEM (n = 4 animals/group). *, p<0.05.</p