A Robust and Scalable High-Throughput Compatible Assay for Screening Amyloid-ß-Binding Compounds.

A robust fluorescent readout assay using topologically-sensitive dyes improves the screening of novel amyloid-binding molecules. One of the key components that make this assay more realistic is the use of endogenous amyloid obtained from 5XFAD mouse brains. The assay conditions were optimized for high throughput screening operation with Z-prime values >0.6. Using a combination of library of 3,500 compounds including known drugs, natural-derived molecules and random organic molecules, 8 unique molecules were identified as potential amyloid-binding agents.

Lipopolysaccharide Induced Opening of the Blood Brain Barrier on Aging 5XFAD Mouse Model.

The development of neurotherapeutics for many neurodegenerative diseases has largely been hindered by limited pharmacologic penetration across the blood-brain barrier (BBB). Previous attempts to target and clear amyloid-ß (Aß) plaques, a key mediator of neurodegenerative changes in Alzheimer's disease (AD), have had limited clinical success due to low bioavailability in the brain because of the BBB. Here we test the effects of inducing an inflammatory response to disrupt the BBB in the 5XFAD transgenic mouse model of AD. Lipopolysaccharide (LPS), a bacterial endotoxin recognized by the innate immune system, was injected at varying doses. 24 hours later, mice were injected with either thioflavin S, a fluorescent Aß-binding small molecule or 30 nm superparamagnetic iron oxide (SPIO) nanoparticles, both of which are unable to penetrate the BBB under normal physiologic conditions. Our results showed that when pretreated with 3.0 mg/kg LPS, thioflavin S can be found in the brain bound to Aß plaques in aged 5XFAD transgenic mice. Following the same LPS pretreatment, SPIO nanoparticles could also be found in the brain. However, when done on wild type or young 5XFAD mice, limited SPIO was detected. Our results suggest that the BBB in aged 5XFAD mouse model is susceptible to increased permeability mediated by LPS, allowing for improved delivery of the small molecule thioflavin S to target Aß plaques and SPIO nanoparticles, which are significantly larger than antibodies used in clinical trials for immunotherapy of AD. Although this approach demonstrated efficacy for improved delivery to the brain, LPS treatment resulted in significant weight loss even at low doses, resulting from the induced inflammatory response. These findings suggest inducing inflammation can improve delivery of small and large materials to the brain for improved therapeutic or diagnostic efficacy. However, this approach must be balanced with the risks of systemic inflammation.

In vivo evaluation of IGF1R/IR PET ligand [18F]BMS-754807 in rodents.

In vivo evaluation of [18F]BMS-754807 binding in mice and rats using microPET and biodistribution methods is described herein. The radioligand shows consistent binding characteristics, in vivo, in both species. Early time frames of the microPET images and time activity curves of brain indicate poor penetration of the tracer across the blood brain barrier (BBB) in both species. However, microPET experiments in mice and rats show high binding of the radioligand outside the brain to heart, pancreas and muscle, the organs known for higher expression of IGF1R/1R. Biodistribution analysis 2h after injection of [18F]BMS-754807 in rats show negligible [18F]defluorination as reflected by the low bone uptake and clearance from blood. Overall, the data indicate that [18F]BMS-754807 can potentially be a radiotracer for the quantification of IGF1R/IR outside the brain using PET.

Aerosol Delivery of Curcumin Reduced Amyloid-ß Deposition and Improved Cognitive Performance in a Transgenic Model of Alzheimer's Disease.

We report a novel approach for the delivery of curcumin to the brain via inhalation of the aerosol for the potential treatment of Alzheimer's disease. The percentage of plaque fraction in the subiculum and hippocampus reduced significantly when young 5XFAD mice were treated with inhalable curcumin over an extended period of time compared to age-matched nontreated counterparts. Further, treated animals demonstrated remarkably improved overall cognitive function, no registered systemic or pulmonary toxicity associated with inhalable curcumin observed during the course of this work.

Toxicity studies of coumarin 6-encapsulated polystyrene nanospheres conjugated with peanut agglutinin and poly(N-vinylacetamide) as a colonoscopic imaging agent in rats.

We are investigating an imaging agent that detects early-stage primary colorectal cancer on the mucosal surface in real time under colonoscopic observation. The imaging agent, which is named the nanobeacon, is fluorescent nanospheres conjugated with peanut agglutinin and poly(N-vinylacetamide). Its potential use as an imaging tool for colorectal cancer has been thoroughly validated in numerous studies. Here, toxicities of the nanobeacon were assessed in rats. The nanobeacon was prepared according to the synthetic manner which is being established as the Good Manufacturing Practice-guided production. The rat study was performed in accordance with Good Laboratory Practice regulations. No nanobeacon treatment-related toxicity was observed. The no observable adverse effect levels (NOAEL) of the nanobeacon in 7-day consecutive oral administration and single intrarectal administration were estimated to be more than 1000mg/kg/day and 50mg/kg/day, respectively. We concluded that the nanobeacon could be developed as a safe diagnostic agent for colonoscopy applications. FROM THE CLINICAL EDITOR: Colon cancer remains a major cause of death. Early detection can result in early treatment and thus survival. In this article, the authors tested potential systemic toxicity of coumarin 6-encapsulated polystyrene nanospheres conjugated with peanut agglutinin (PNA) and poly(N-vinylacetamide) (PNVA), which had been shown to bind specifically to colonic cancer cells and thus very promising in colonoscopic detection of cancer cells.

Fluorescence-based endoscopic imaging of Thomsen-Friedenreich antigen to improve early detection of colorectal cancer.

Thomsen-Friedenreich (TF) antigen belongs to the mucin-type tumor-associated carbohydrate antigen. Notably, TF antigen is overexpressed in colorectal cancer (CRC) but is rarely expressed in normal colonic tissue. Increased TF antigen expression is associated with tumor invasion and metastasis. In this study, we sought to validate a novel nanobeacon for imaging TF-associated CRC in a preclinical animal model. We developed and characterized the nanobeacon for use with fluorescence colonoscopy. In vivo imaging was performed on an orthotopic rat model of CRC. Both white light and fluorescence colonoscopy methods were utilized to establish the ratio-imaging index for the probe. The nanobeacon exhibited specificity for TF-associated cancer. Fluorescence colonoscopy using the probe can detect lesions at the stage which is not readily confirmed by conventional visualization methods. Further, the probe can report the dynamic change of TF expression as tumor regresses during chemotherapy. Data from this study suggests that fluorescence colonoscopy can improve early CRC detection. Supplemented by the established ratio-imaging index, the probe can be used not only for early detection, but also for reporting tumor response during chemotherapy. Furthermore, since the data obtained through in vivo imaging confirmed that the probe was not absorbed by the colonic mucosa, no registered toxicity is associated with this nanobeacon. Taken together, these data demonstrate the potential of this novel probe for imaging TF antigen as a biomarker for the early detection and prediction of the progression of CRC at the molecular level.

Inhalable curcumin: offering the potential for translation to imaging and treatment of Alzheimer's disease.

Curcumin is a promising compound that can be used as a theranostic agent to aid research in Alzheimer's disease. Beyond its ability to bind to amyloid plaques, the compound can also cross the blood-brain barrier. Presently, curcumin can be applied only to animal models, as the formulation needed for iv injection renders it unfit for human use. Here, we describe a novel technique to aerosolize a curcumin derivative, FMeC1, and facilitate its safe delivery to the brain. Aside from the translational applicability of this approach, a study in the 5XFAD mouse model suggested that inhalation exposure to an aerosolized FMeC1 modestly improved the distribution of the compound in the brain. Additionally, immunohistochemistry data confirms that following aerosol delivery, FMeC1 binds amyloid plaques expressed in the hippocampal areas and cortex.

Identification of promethazine as an amyloid-binding molecule using a fluorescence high-throughput assay and MALDI imaging mass spectrometry.

The identification of amyloid-binding compounds is a crucial step in the development of imaging probes and therapeutics for the detection and cure of Alzheimer's disease. Unfortunately, the process typically lags during the translation from in vitro to in vivo studies due to the impenetrable nature of the blood brain barrier (BBB). Here, we integrate fluorescence assay with MALDI imaging mass spectrometry to screen known compounds and repurpose their properties to enable the second function of binding to amyloid plaques. Through this approach, we identified an antihistamine compound, promethazine, that can bind to amyloid plaques. Finally, we demonstrate that promethazine is retained in the amyloid-burdened brain compared to a normal brain and that its distribution within the brain corroborates with that of amyloid plaques.

Randomized controlled trial of the Breast Cancer Recovery Program for women with breast cancer-related lymphedema.

Evidence-based exercise and relaxation recommendations for people with breast cancer-related lymphedema (BCRL) are needed. We report a randomized controlled study of one program, designed to achieve synergistic improvements in physical and emotional BCRL symptoms. People in the treatment group received an exercise and relaxation program, The Breast Cancer Recovery Program (N=16). The control participants (N=16) continued with health professionals' recommendations. Participants were tested at entry, 2.5 weeks, 5 weeks, and 3 months. Treatment group participants, compared with control participants, demonstrated significant treatment effects for improved bioimpedance z, arm flexibility, quality of life, mood at 3 months, and weight loss. Adherence was high for this safe and effective program, which improved lymphedema physical and emotional symptoms.

Molecular neurodevelopment: an in vivo 31P-1H MRSI study.

Synaptic development and elimination are normal neurodevelopmental processes, which if altered could contribute to various neuropsychiatric disorders. 31P-1H magnetic resonance spectroscopic imaging (MRSI) and structural magnetic resonance imaging (MRI) exams were conducted on 105 healthy children ages 6-18 years old to identify neuromolecular indices of synaptic development and elimination. Over the age range studied, age-related changes in high-energy phosphate (phosphocreatine), membrane phospholipid metabolism (precursors and breakdown products), and percent gray matter volume were found. These neuromolecular and structural indices of synaptic development and elimination are associated with development of several cognitive domains. Monitoring of these molecular markers is essential for devising treatment strategies for neurodevelopmental disorders.

Effect of intraperitoneal acetyl-L-carnitine (ALCAR) on anxiety-like behaviours in rats.

Acetyl-L-carnitine (ALCAR) is an acetyl derivative of carnitine, an endogenous molecule synthesized in vivo and supplemented by diet (mainly via meat and dairy products). Several parallel, double-blind, placebo-controlled studies have demonstrated that ALCAR treatment produces beneficial effects in geriatric depression. Since most antidepressants also have anti-anxiety effects we examined whether ALCAR shows anti-anxiety effects in a rat model of anxiety. Compared to a saline-injected control group, chronic administration of ALCAR at doses of 10 and 100 mg/kg (tested 24 h after the last dose administration) showed no effects, whereas doses of 50 and 75 mg/kg significantly reduced anxiety-like behaviours in the elevated plus-maze. Acute ALCAR (100 mg/kg), on the other hand (tested 6 h after administration), demonstrated anxiogenic effects. Our data suggest that chronic ALCAR administration may produce an inverted U-shaped curve of dose-dependent changes in anxiety-like behaviour. The precise mechanism by which ALCAR decreases anxiety-like behaviour after peripheral administration remains to be determined.

Interactions of Abeta(1-40) with glycerophosphocholine and intact erythrocyte membranes: fluorescence and circular dichroism studies.

Deposition of amyloid beta peptide in human brain in the form of senile plaques is a neuropathological hallmark of Alzheimer's disease (AD). Levels of a phospholipid breakdown product, glycerophosphocholine (GPC), also increase in AD brain. The effect of GPC on amyloid beta(1-40) peptide (Abeta) aggregation in PBS buffer was investigated by circular dichroism and fluoresence spectroscopy; interactions of Abeta and GPC with the intact erythrocyte membrane was examined by fluoresence spectroscopy. Fluorescamine labeled Abeta studies indicate GPC enhances Abeta aggregation. CD spectroscopy reveals that Abeta in the presence of GPC adopts 14% more beta-sheet structure than does Abeta alone. Fluorescamine anisotropy measurements show that GPC and Abeta interact in the phospholipid head-group region of the erythrocyte membrane. In summary, both soluble Abeta and GPC insert into the phospholipid head-group region of the membrane where they interact leading to beta-sheet formation in soluble Abeta which enhances Abeta aggregation.

31P-MRS study of acetyl-L-carnitine treatment in geriatric depression: preliminary results.

OBJECTIVE: This 12-week study of two elderly, depressed subjects investigated the effect of acetyl-L-carnitine (ALCAR) treatment on the Hamilton Depression Rating Scale (HDRS) and on measures of high-energy phosphate and membrane phospholipid metabolism. METHODS: Two mildly depressed (HDRS 15-20), non-demented male subjects 70 and 80 years old were compared with six non-demented controls (all males, mean age of 73.6 +/- 3.6 years). High-energy and membrane phospholipid metabolites were measured by phosphorus magnetic resonance spectroscopic imaging (31P MRSI) analysis. HDRS and 31P MRSI measurements were taken at entry, 6 and 12 weeks for the depressed subjects. RESULTS: 31P MRSI analysis revealed elevated levels of phosphomonesters [PME(s - tau(c))] in the prefrontal region of these mildly depressed subjects, which decreased with ALCAR treatment and showed a trend for correlation of the PME(s - tau(c)) levels with HDRS. ALCAR treatment also resulted in increasing levels of the prefrontal phosphocreatine (PCr), which correlated with HDRS. CONCLUSIONS: In the prefrontal region, the mildly depressed subjects compared with controls had elevated PME(s - tau(c)) levels which normalized after 12 weeks of ALCAR and increased PCr levels after ALCAR treatment. These preliminary findings suggest further studies are warranted.

Psychosis in Alzheimer disease: postmortem magnetic resonance spectroscopy evidence of excess neuronal and membrane phospholipid pathology.

BACKGROUND: The presence of psychotic symptoms in Alzheimer Disease subjects (AD+psychosis, AD+P) is a marker for a phenotype characterized by more severe cognitive impairment and a more rapidly deteriorating course. Although AD+P has been inconsistently associated with more severe neuropathology, no prior studies have examined measures of neuronal and synaptic integrity. OBJECTIVE: To determine whether AD+P is associated with evidence of disrupted neuronal and synaptic integrity, as indicated by magnetic resonance spectroscopy (MRS) measurement of N-acetyl-L-aspartate and the membrane breakdown products, glycerophosphocholine and glycerophosphoethanolamine. METHODS: 31P and 1H MRS studies of perchloric acid extract from postmortem brain of AD subjects with and without a history of psychotic symptoms. All subjects were characterized for the presence of comorbid cortical Lewy body pathology and for history of neuroleptic use. Brain tissue from dorsolateral prefrontal, superior temporal, inferior parietal, and occipital cortex, amygdala, and cerebellum were examined in all subjects. Statistical analysis accounted for correlated observations across brain regions within-subjects. RESULTS: AD+P subjects demonstrated significant elevations of glycerophosphoethanolamine and significant reductions of N-acetyl-L-aspartate. Between group differences were greatest in neocortical brain regions. CONCLUSION: Excess impairment of neocortical neuronal and synaptic integrity may provide the structural substrate underlying AD+P. Confirmation of these findings using in vivo MRS measures is indicated.

Acetyl-l-carnitine as a possible therapy for Alzheimer's disease.

Alterations have been demonstrated in membrane phospholipid metabolism in Alzheimer's disease. Alterations in membrane phospholipid metabolite levels have been observed by 31P magnetic resonance spectroscopy 4 years before onset of cognitive changes. Implications of phospholipid membrane changes in Alzheimer's disease are presented. Augmentation of brain levels of acetyl-l-carnitine, an endogenous molecule, may help to reverse membrane phospholipid changes in Alzheimer's disease. Neurobiological effects of acetyl-l-carnitine and potential molecular mechanisms of acetyl-l-carnitine activity are reviewed. Clinical trials of acetyl-l-carnitine indicate that it is well-tolerated and may be effective in younger Alzheimer's disease patients and may be an effective adjuvant treatment. Acetyl-l-carnitine may especially benefit those Alzheimer's disease patients with depressive disorders.