Neuroprotective effects of photobiomodulation by hormesis on scopolamine induced neurodegenerative diseases of memory disorders in rats a paradigm shift.

The loss and progressive dysfunction of neurons are hallmarks of neurodegenerative diseases. The aim of the current study is to explore the effects of photobiomodulation at 460-660 nm (100-1000 lux units) on the progression of scopolamine-induced cognitive dysfunctions in Wistar male rats. Photobiomodulation (PBM) is defined as "the use of monochromatic or quasi-monochromatic light from a low-power laser or light-emitting diode (LED) source to modify or modulate biological functions." Neuroprotective activity was assessed by in vivo models such as the Morris water maze, the elevated plus maze (EPM), and the T-maze. After using scopolamine (1 mg/kg/day) as a dementia induction model for 21 days, the induction was primarily due to impairments in cholinergic transmission, oxidative stress, and inflammation. The in vitro determinations, including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), reduced glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor-alpha (TNF-α), Interleukin 1 beta (IL-1ß), and alkaline phosphatase (ALP), were assessed biochemicals and biomarkers. The structural and morphological integrity of the cortex and hippocampus was investigated through histopathology. In vivo studies of exteroceptive behavior models such as the Morris water maze, the EPM, and the T-maze revealed that administration of scopolamine resulted in enhanced escape latency time (ELT), transfer latency (TL), and decreased percentage alternation, respectively. The levels of AChE, BChE, reduced, GSH, SOD, TNF-α, IL-1ß and ALP were increased, while MDA level was decreased. In contrast to normal and control groups with treatment groups, histopathology of the cortex and hippocampus examination revealed the maintenance of structural integrity and densities of CA1 and CA3 neuronal cells. However, network pharmacology predicted Ca+2 modulation of various pathways, among the treatments with red LED light showed highly significant amelioration compared with normal and control groups. Photobiomodulation by hormesis, chromophores in cells, and tissues excitation can influence neuroprotective effect mainly by scavenging of ROS, variation in the level of GSH MDA and SOD mitochondrial electron transfer, the improved abscopal effects on improved in gut microbiome by resembles the of fecal ALP level correlation of intestinal microbiome, cholinergic neurotransmissions, anti-inflammatory, and antioxidant activities.

Effects of cadmium chloride and biofertilizer (Bacilar) on biochemical parameters of freshwater fish, Alburnus mossulensis.

Fish in wild are often faced with various types of xenobiotics, that may display synergistic or antagonistic effects. In this study, we aim to examine how exposure to agrochemical compound (Bacilar) and cadmium (CdCl2) alone and in combination affect biochemical parameters (lactate dehydrogenase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, alanine aminotransferase; creatine phosphokinase (CKP), cholinesterase) and oxidative stress (total antioxidant capacity, catalase, malondialdehyde and protein carbonyl concentrations) of freshwater fish Alburnus mossulensis. Fish were exposed to two concentrations of Bacilar (0.3, and 0.6 mL L-1) and to 1 mg L-1 cadmium chloride alone and in combination for 21 days. Results showed that fish accumulate Cd in their body, with the highest rate in individuals exposed to Cd in combination with Bacilar. Both xenobiotics in fish liver induced the activation of liver enzymes suggesting hepatotoxic effects, with the greatest impact in co-exposed groups. A significant decrease in the hepatocyte's total antioxidant capacity indicates the collapse of the antioxidant defense in fish exposed to Cd and Bacilar. A decrease in the antioxidant biomarkers was followed by increased oxidative damage of lipids and proteins. We also reported altered function in the muscle of individuals exposed to Bacilar and Cd seen as decreased activities in CKP and butyrylcholinesterase. Overall, our results point to the toxicity of both Bacilar and Cd on fish but also to their synergistic effects on Cd bioaccumulation, oxidative stress, and liver and muscle damage. This study highlights the need for evaluating the use of agrochemicals and their possible additive effects on non-target organisms.

Green Synthesis of Silver Nanoparticles from Allium cepa L. Peel Extract, Their Antioxidant, Antipathogenic, and Anticholinesterase Activity.

The present work deals with the green synthesis and characterization of silver nanoparticles (AgNPs) using Allium cepa (yellowish peel) and the evaluation of its antimicrobial, antioxidant, and anticholinesterase activities. For the synthesis of AgNPs, peel aqueous extract (200 mL) was treated with a 40 mM AgNO3 solution (200 mL) at room temperature, and a color change was observed. In UV-Visible spectroscopy, an absorption peak formation at ~439 nm was the sign that AgNPs were present in the reaction solution. UV-vis, FE-SEM, TEM, EDX, AFM, XRD, TG/DT analyses, and Zetasizer techniques were used to characterize the biosynthesized nanoparticles. The crystal average size and zeta potential of AC-AgNPs with predominantly spherical shapes were measured as 19.47 ± 1.12 nm and -13.1 mV, respectively. Pathogenic microorganisms Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans were used for the Minimum Inhibition Concentration (MIC) test. When compared to tested standard antibiotics, AC-AgNPs demonstrated good growth inhibitory activities on P. aeuruginosa, B. subtilis, and S. aureus strains. In vitro, the antioxidant properties of AC-AgNPs were measured using different spectrophotometric techniques. In the ß-Carotene linoleic acid lipid peroxidation assay, AC-AgNPs showed the strongest antioxidant activity with an IC50 value of 116.9 µg/mL, followed by metal-chelating capacity and ABTS cation radical scavenging activity with IC50 values of 120.4 µg/mL and 128.5 µg/mL, respectively. The inhibitory effects of produced AgNPs on the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes were determined using spectrophotometric techniques. This study provides an eco-friendly, inexpensive, and easy method for the synthesis of AgNPs that can be used for biomedical activities and also has other possible industrial applications.

Bioactivity Profiles on 15 Different Effect Mechanisms for 15 Golden Root Products via High-Performance Thin-Layer Chromatography, Planar Assays, and High-Resolution Mass Spectrometry.

Planar chromatography has recently been combined with six different effect-directed assays for three golden root (Rhodiola rosea L.) samples. However, the profiles obtained showed an intense tailing, making zone differentiation impossible. The profiling was therefore improved to allow for the detection of individual bioactive compounds, and the range of samples was extended to 15 commercial golden root products. Further effect-directed assays were studied providing information on 15 different effect mechanisms, i.e., (1) tyrosinase, (2) acetylcholinesterase, (3) butyrylcholinesterase, (4) ß-glucuronidase, and (5) α-amylase inhibition, as well as endocrine activity via the triplex planar yeast antagonist-verified (6-8) estrogen or (9-11) androgen screen, (12) genotoxicity via the planar SOS-Umu-C bioassay, antimicrobial activity against (13) Gram-negative Aliivibrio fischeri and (14) Gram-positive Bacillus subtilis bacteria, and (15) antioxidative activity (DPPH• radical scavengers). Most of the golden root profiles obtained were characteristic, but some samples differed substantially. The United States Pharmacopeia reference product showed medium activity in most of the assays. The six most active compound zones were further characterized using high-resolution mass spectrometry, and the mass signals obtained were tentatively assigned to molecular formulae. In addition to confirming the known activities, this study is the first to report that golden root constituents inhibit butyrylcholinesterase (rosin was tentatively assigned), ß-glucuronidase (rosavin, rosarin, rosiridin, viridoside, and salidroside were tentatively assigned), and α-amylase (stearic acid and palmitic acid were tentatively assigned) and that they are genotoxic (hydroquinone was tentatively assigned) and are both agonistic and antagonistic endocrine active.

Assessment of hypolipidemic and anti-inflammatory properties of walnut (Juglans regia) seed coat extract and modulates some metabolic enzymes activity in triton WR-1339-induced hyperlipidemia in rat kidney, liver, and heart.

Atherosclerosis and cognitive impairment are both influenced by hyperlipidemia. Due to their high margin of safety and low cost, natural chemicals have recently attracted particular attention in the context of the treatment of disease. Hence, the purpose of this study was to investigate the possible amendatory impact of ethanol extract walnut (Juglans regia) seed coat (E-WSC) on some metabolic enzymes (glutathione reductase (GR), paraoxonase-1 (PON1), aldose reductase (AR), sorbitol dehydrogenase (SDH), acetylcholinesterase (AChE), glutathione S-transferase (GST), and butyrylcholinesterase (BChE)) activity in the liver, kidney, and heart of rats with Triton WR-1339-induced hyperlipidemia. Rats were divided into five groups: control group, HL-Control group (Triton WR-1339 400 mg/kg, i.p administered group), E- WSC + 150 (150 mg/kg,o.d given group), E- WSC + 300 (E- WSC 300 mg/kg, o.d given group) and HL+ E-WSC + 300 (Group receiving E- WSC 300 mg/kg, o.d 30 min prior to administration of Triton WR-1339 400 mg/kg, i.p). In HL-Control, AR, SDH, and BChE enzyme activity was significantly increased in all tissues compared to the control, while the activity of other studied enzymes was significantly decreased. The effects of hyperlipidemia on balance were improved and alterations in the activity of the investigated metabolic enzymes were prevented by E-WSC. As a result, promising natural compounds that can be used as adjuvant therapy in the treatment of cognitive disorders and hyperlipidemia may be found in E-WSC powder.

Composición química y actividad AChE-BuChE del aceite esencial de palo santo Bursera graveolens (Kunth) Triana & Planch de Jipijapa, Ecuador / Chemical composition and AChE-BuChE activities of the essential oil of palo santo Bursera graveolens (Kunth) Tr iana & Planch from Jipijapa, Ecuador

Abstract: This article describes the chemical composition, physical properties and acetylcholinesterase (A ChE) and butyrylcholinesterase (BuChE) activity of stem - distilled essential oil (E O ) from Bursera graveolens wood chips, Burseraceae. The plant material was acquired in Quimis (Bosque de Sancán), city of Jipijapa in the province of Manabí, coastal region o f Ecuador. Thirty - six components were identified by CG - MS, which represented 98.54% of the volatile oil. The main components were limonene (68.52%) and mentofuran (20.37%). The hydrocarbon monoterpenes constituted the most abundant fractions. The average y ield of the E O was 1.26%. Regarding the physical properties of E O , the following values were obtained: relative density (1,029 g/mL), refractive index (1,477) and specific rotation (+4,567). The E O presented IC 50 inhibition values of 47.2 and 51.9 µg/mL fo r the enzymes AChE and BuChE, respectively.

Resumen: Este artículo describe la composición química, propiedades físicas y actividad acetilcolinesterasa (AChE) y butirilcolinesterasa (BuChE) del aceite esencial (AE) destilado a vapor de astillas de madera de Bursera graveolens , Burseraceae. La materia vegetal fue adquirida en Quimis (Bosque de Sancán), ciudad de Jipijapa en la provincia de Manabí, región costera d e Ecuador. Treinta y seis componentes fueron identificados por CG - MS, que representaron al 98.54 % del aceite volátil. Los componentes principales fueron limoneno (68.52%) y mentofurano (20.37%). Los monoterpenos hidrocarburos constituyeron las fracciones m ás abundantes. El rendimiento medio del AE fue de 1.26%. Con respecto a las propiedades físicas del AE se obtuvo los siguientes valores, densidad relativa (1.029 g/mL), índice de refracción (1.477) y rotación específica (+4.567). El AE presentó valores de inhibición IC 50 de 47.2 y 51.9 µg/mL para las enzimas AChE y BuChE, respectivamente.

Experimental validation of Vitex negundo leaves hydroalcoholic extract for neuroprotection in haloperidol induced parkinson's disease in rat.

Parkinsonism is a neurodegenerative disease, mainly imbalance in dopamine and acetylcholine neurotransimitter in mid brain, which manifestation of dysfunctions of extrapyramidal like akinesia, tremor, rigidity and catalepsy etc., even cognitive and memory loss. The current study is framed to evaluate the effect of Vitex negundo (VNL) leaf extract in Haloperidol induced PD in rats. In vitro studies of antioxidant capacity were checked via DPPH and NO assays and identified its Acetylcholinesterase (AChE) inhibitory activity. Secondly the In vivo study of anti-PD activity in Haloperidol induced in rats were evaluated by Rotarod, morris water maze (MWM), cooks pole climb (CPC), actophotometer, novel object recognition (NOR), and T-maze were utilized to assess extrapyramidal, cognitive and memory function. Thirdly, changes in biomarker level viz. (AChE), butyrylcholinesterase. (BChE) in hippocampus and cortex, reduced glutathione (GSH), malondialdehyde (MDA), total protein (TP), superoxide dismutase (SOD), catalase (CAT), and dopamine level in the whole brain were measured. Finally, histopathology of hippocampus and cortex was examined at 40x magnification to access restoring integrity and maintaining the architecture of neuronal cell in the treatment group compared to control group and L-DOPA as a standard treatment group. V. negundo showed potent antioxidant potency on scavenging of DPPH (IC50 84.81 µg/ml) and NO (IC50 133.20 µg/ml) and possess AChE inhibitory potency (IC50 114.35 µg/ml) by in vitro studies. The Rotarod, MWM, CPC, Actophotometer, NOR, T-maze demonstrated that Haloperidol group administration declines performance time, ELT, TL and decreases locomotion, cognitive and memory respectively. The treatment of VNL 100, 200, and 400 mg/kg p.o. significantly (p < 0.05 to p < 0.0001) reversed. Whole brain AChE, BChE, and MDA level were significantly raised and GSH, TP, SOD, CAT and Dopamine were significantly declined in Haloperidol treated group rats, especially V. negundo 400 mg/kg p.o. highly significantly ameliorate the Haloperidol group altered pathological changes through the restoration of the cholinergic function, enhancing the antioxidant defense and by increasing the dopaminergic function. The current study provides validation of V. negundo for its anti-PD activity and could be a valuable source for the treatment of PD in future.

The biological activities of butyrylcholinesterase inhibitors.

Acetylcholinesterase (AChE) inhibitor is the first choice for the treatment of Alzheimer's disease (AD), but it has some defects, such as dose limitation and unsatisfactory long-term treatment effect. Recent studies have shown that butyrylcholinesterase (BuChE) inhibitors or double acetyl and butyryl cholinesterase inhibitors have better curative effects on AD, and the side effects are lower than those of specific AChE inhibitors. Dual target cholinesterase inhibitors have become a new hotspot in the research of anti-AD drugs. Herein, the synthesis and bioactivities of BuChE inhibitors were reviewed.

Phytol loaded PLGA nanoparticles ameliorate scopolamine-induced cognitive dysfunction by attenuating cholinesterase activity, oxidative stress and apoptosis in Wistar rat.

OBJECTIVE: Alzheimer's disease (AD) is an acquired neurological disorder of cognitive and behavioral impairments, with a long and progressive route. Currently, efforts are being made to develop potent drugs that target multiple pathological mechanisms that drive the successful treatment of AD in human beings. The development of nano-drug delivery systems has recently emerged as an effective strategy to treat AD. METHODS: In the present study, the protective effect of Phytol and Phytol loaded Poly Lactic-co-Glycolic Acid nanoparticles (Phytol-PLGANPs) were evaluated in Wistar rat scopolamine model of AD. RESULTS AND DISCUSSION: The consumption of Phytol and Phytol-PLGANPs significantly ameliorated the cognitive deficits caused by scopolamine on spatial and short term memory. Phytol and Phytol-PLGANPs significantly enhanced the cholinergic effect by inhibiting both acetylcholinesterase and butyrylcholinesterase (AChE & BuChE), ß-secretase 1 (BACE1) activity, attenuating macromolecular damage, reducing reactive oxygen species (ROS) and reactive nitrogen species (RNS) level by activating antioxidative defense system (Superoxide dismutase and catalase) and restoring glutathione metabolizing enzyme systems (Glutathione S-transferase) and also regulating the apoptotic mediated cell death. Moreover, in vivo toxicity study suggests that Phytol and Phytol-PLGANPs did not cause any adverse pathological alteration in rats treated with a higher concentration of Phytol-PLGANPs (200 mg/kg). Pharmacokinetic study revealed that Phytol-PLGANPs enhanced the biodistribution and sustained the release profile of phytol in the brain and plasma. CONCLUSION: Overall, the outcome of the study suggests that Phytol and Phytol-PLGANPs act as a potent candidate with better anti-amnesic effects and multi-faceted neuroprotective potential against scopolamine-induced memory dysfunction in Wistar rats.

Anticholinesterase activity of ß-carboline-1, 3, 5-triazine hybrids

Abstract The ß-carboline-1,3,5-triazine hydrochlorides 8-13 were evaluated in vitro against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The analysed compounds were selective to BuChE, with IC50 values in the range from 1.0-18.8 µM being obtained. The N-{2-[(4,6-dihydrazinyl-1,3,5-triazin-2-yl)amino]ethyl}-1-phenyl-ß-carboline-3-carboxamide (12) was the most potent compound and kinetic studies indicate that it acts as a competitive inhibitor of BuChE. Molecular docking studies show that 12 strongly interacts with the residues of His438 (residue of the catalytic triad) and Trp82 (residue of catalytic anionic site), confirming that this compound competes with the same binding site of the butyrylthiocholine

Chemical Composition and Biological Activities of Essential Oils from the Leaves, Stems, and Roots of Kadsura coccinea.

The chemical composition and biological activities of the essential oils from the leaves, stems, and roots of Kadsura coccinea (K. coccinea) were investigated. The essential oils were extracted by hydro distillation and analyzed by gas chromatography mass spectrometry (GC-MS) and gas chromatography with flame ionization detector (GC-FID). Antioxidant activities of the essential oils were examined with DPPH radical scavenging assay, ABTS cation radical scavenging assay, and ferric reducing antioxidant power assay. Antimicrobial activities were evaluated by determining minimum inhibitory concentrations (MIC) and minimum microbiocidal concentrations (MMC). Acetylcholinesterase and butyrylcholinesterase inhibitory activity of the essential oils were also tested. A total of 46, 44, and 47 components were identified in the leaf, stem, and root oils, representing 95.66%, 97.35%, and 92.72% of total composition, respectively. The major compounds of three essential oils were α-pinene (16.60-42.02%), ß-pinene (10.03-18.82%), camphene (1.56-10.95%), borneol (0.50-7.71%), δ-cadinene (1.52-7.06%), and ß-elemene (1.86-4.45%). The essential oils were found to have weak antioxidant activities and cholinesterase inhibition activities. The essential oils showed more inhibitory effects against Staphylococcus aureus (S. aureus) than those of other strains. The highest antimicrobial activity was observed in the root oil against S. aureus, with MIC of 0.78 mg/mL. Therefore, K. coccinea essential oils might be considered as a natural antibacterial agent against S. aureus with potential application in food and pharmaceutical industries.

Amygdalus spinosissima root extract enhanced scopolamine-induced learning and memory impairment in mice.

The Amygdalus spinosissima (Rosaceae) plant has been used in the Iranian folk medicine as a remedy for the burn wound. Hence, in this study, we aimed to determine the possible medicinal potential of the plant focusing on the root part. The bioactive phenolic and flavonoid compounds present in the root extract of the Amygdalus spinosissima plant as well as its antioxidant and anti-inflammatory properties were determined. Moreover, the effects of root extract on learning and memory in mice were evaluated. The results revealed that the root methanolic extract contained phenolic and flavonoid compounds including apigenin, quercetin, rutin, kaempferol, gallic acid, syringic acid, ferulic acid, and ellagic acid. The extract possessed antioxidant, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) inhibitory activities in vitro. These biological activities were attributed to the presence of phenolics and flavonoids. The A. spinosissima root extract improved learning and memory function in scopolamine-induced memory dysfunction in mice as determined using the Morris water maze task. The extract modulated the AChE, BChE, and inflammatory genes and enhanced the expression of the antioxidant enzymes in the brain. Consequently, A. spinosissima root extract could be considered as a promising source of potent bioactive compounds in the retarding the development of neurodegenerative diseases such as Alzheimer's disease.

Combination of donepezil and gallic acid improves antioxidant status and cholinesterases activity in aluminum chloride-induced neurotoxicity in Wistar rats.

The present study compared the effect of donepezil only and combination of donepezil and gallic acid on oxidative status and cholinesterase activity in the brain of Wistar rats administered AlCl3 for 60 days. Twenty-eight rats (180 - 200 g) were arbitrarily distributed into four groups of seven animals apiece. Group 1 served as normal control and received distilled water throughout the study. Group 2 animals received only AlCl3 throughout the study while animals in groups 3 and 4 were administered donepezil only (10 mg/kg) and combination of donepezil (10 mg/kg) and gallic acid (50 mg/kg), respectively, in addition to AlCl3. Treatments were administered orally by gavage. At the end of the study, animals were sacrificed and activities of acetylcholinesterase, butyrylcholinesterase, superoxide dismutase (SOD) and catalase as well as levels of malondialdehyde (MDA), total thiol and nitric oxide (NO) were evaluated in the brain. Histopathological study was conducted on the hippocampus of experimental animals. Results showed that AlCl3 significantly (p < 0.05) increased brain activities of cholinesterases and levels of MDA and NO with a concomitant decrease in total thiol level as well as activities of SOD and catalase. Donepezil only and combination of donepezil and gallic acid reversed these alterations. Also, combination of donepezil and gallic acid significantly (p < 0.05) improved antioxidant status better than donepezil only. It could be concluded that a synergy might exist between gallic acid and donepezil especially in ameliorating oxidative stress associated with AlCl3-induced neurotoxicity.

Three-dimensional brain-on-chip model using human iPSC-derived GABAergic neurons and astrocytes: Butyrylcholinesterase post-treatment for acute malathion exposure.

Organophosphates (OPs) induce acute and chronic neurotoxicity, primarily by inhibiting acetylcholinesterase (AChE) activity as well as by necrosis, and apoptosis. Butyrylcholinesterase (BuChE), an exogenous bioscavenger of OPs, can be used as a treatment for OP exposure. It is prerequisite to develop in vitro brain models that can study BuChE post-treatment for acute OP exposure. In this study, we developed a three-dimensional (3D) brain-on-chip platform with human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes to simulate human brain behavior. The platform consists of two compartments: 1) a hydrogel embedded with human iPSC-derived GABAergic neurons and astrocytes and 2) a perfusion channel with dynamic medium flow. The brain tissue constructs were exposed to Malathion (MT) at various concentrations and then treated with BuChE after 20 minutes of MT exposure. Results show that the iPSC-derived neurons and astrocytes directly interacted and formed synapses in the 3D matrix, and that treatment with BuChE improved viability after MT exposure up to a concentration of 10-3 M. We conclude that the 3D brain-on-chip platform with human iPSC-derived brain cells is a suitable model to study the neurotoxicity of OP exposure and evaluate therapeutic compounds for treatment.

TV-1380 attenuates cocaine-induced changes in cardiodynamic parameters in monkeys and reduces the formation of cocaethylene.

BACKGROUND: TV-1380 is a rationally mutated, human BChE fused to human serum albumin that has high hydrolytic enzymatic activity against cocaine and as well as an extended elimination half-life. OBJECTIVE: The present studies examined the safety of TV-1380 and its protective effect when given to monkeys alone or concomitantly with cocaine and ethanol. METHODS: A set of studies was conducted in monkeys with TV-1380. The parameters tested included telemetric assessment of cardiovascular parameters, clinical pathology, plasma analysis of cardiac troponin I, ex-vivo analyses of cocaethylene and PK analysis of serum concentrations of TV-1380, cocaine and its metabolites, and histopathological examinations. RESULTS: TV-1380 treatment in monkeys was well tolerated. TV-1380 pretreatment prior to cocaine significantly attenuated the cardiac effects of cocaine and reduced cocaine-induced elevations in serum cardiac troponin I. TV-1380 changed the metabolic fate of cocaine resulting in decreased exposure to benzoylecgonine, while increasing the exposure to ecgonine methyl ester in plasma.TV-1380 reduced the plasma levels of the toxic metabolite cocaethylene formed after co-administration of ethanol and cocaine. CONCLUSION: The results of this study demonstrate that TV-1380 not only accelerates the elimination of cocaine, but also protects the treated animal from the cardiac effects of cocaine, and inhibits the formation of the toxic cocaethylene metabolite when cocaine is given together with ethanol, supporting further clinical development of modified BChE products as possible treatments for cocaine abuse.

Novel Multitarget-Directed Ligands Aiming at Symptoms and Causes of Alzheimer's Disease.

Alzheimer's disease (AD) is a major public health problem, which is due to its increasing prevalence and lack of effective therapy or diagnostics. The complexity of the AD pathomechanism requires complex treatment, e.g. multifunctional ligands targeting both the causes and symptoms of the disease. Here, we present new multitarget-directed ligands combining pharmacophore fragments that provide a blockade of serotonin 5-HT6 receptors, acetyl/butyrylcholinesterase inhibition, and amyloid ß antiaggregation activity. Compound 12 has displayed balanced activity as an antagonist of 5-HT6 receptors ( Ki = 18 nM) and noncompetitive inhibitor of cholinesterases (IC50 hAChE = 14 nM, IC50 eqBuChE = 22 nM). In further in vitro studies, compound 12 has shown amyloid ß antiaggregation activity (IC50 = 1.27 µM) and ability to permeate through the blood-brain barrier. The presented findings may provide an excellent starting point for further studies and facilitate efforts to develop new effective anti-AD therapy.

Design, Synthesis, and Biological Evaluation of 1-Benzylamino-2-hydroxyalkyl Derivatives as New Potential Disease-Modifying Multifunctional Anti-Alzheimer's Agents.

The multitarget approach is a promising paradigm in drug discovery, potentially leading to new treatment options for complex disorders, such as Alzheimer's disease. Herein, we present the discovery of a unique series of 1-benzylamino-2-hydroxyalkyl derivatives combining inhibitory activity against butyrylcholinesterase, ß-secretase, ß-amyloid, and tau protein aggregation, all related to mechanisms which underpin Alzheimer's disease. Notably, diphenylpropylamine derivative 10 showed balanced activity against both disease-modifying targets, inhibition of ß-secretase (IC50  hBACE-1 = 41.60 µM), inhibition of amyloid ß aggregation (IC50 Aß = 3.09 µM), inhibition of tau aggregation (55% at 10 µM); as well as against symptomatic targets, butyrylcholinesterase inhibition (IC50  hBuChE = 7.22 µM). It might represent an encouraging starting point for development of multifunctional disease-modifying anti-Alzheimer's agents.

Butyrylcholinesterase regulates central ghrelin signaling and has an impact on food intake and glucose homeostasis.

BACKGROUND: Ghrelin is the only orexigenic hormone known to stimulate food intake and promote obesity and insulin resistance. We recently showed that plasma ghrelin is controlled by butyrylcholinesterase (BChE), which has a strong impact on feeding and weight gain. BChE knockout (KO) mice are prone to obesity on high-fat diet, but hepatic BChE gene transfer rescues normal food intake and obesity resistance. However, these mice lack brain BChE and still develop hyperinsulinemia and insulin resistance, suggesting essential interactions between BChE and ghrelin within the brain. METHODS: To test the hypothesis we used four experimental groups: (1) untreated wild-type mice, (2) BChE KO mice with LUC delivered by adeno-associated virus (AAV) in combined intravenous (i.v.) and intracerebral (i.c.) injections, (3) KO mice given AAV for mouse BChE (i.v. only) and (4) KO mice given the same vector both i.v. and i.c. All mice ate a 45% calorie high-fat diet from the age of 1 month. Body weight, body composition, daily caloric intake and serum parameters were monitored throughout, and glucose tolerance and insulin tolerance tests were performed at intervals. RESULTS: Circulating ghrelin levels dropped substantially in the KO mice after i.v. AAV-BChE delivery, which led to normal food intake and healthy body weight. BChE KO mice that received AAV-BChE through i.v. and i.c. combined treatments not only resisted weight gain on high-fat diet but also retained normal glucose and insulin tolerance. CONCLUSIONS: These data indicate a central role for BChE in regulating both insulin and glucose homeostasis. BChE gene transfer could be a useful therapy for complications linked to diet-induced obesity and insulin resistance.

In vitro characterization of cationic copolymer-complexed recombinant human butyrylcholinesterase.

Effective use of exogenous human BChE as a bioscavenger for organophosphorus toxicants (OPs) is hindered by its limited availability and rapid clearance. Complexes made from recombinant human BChE (rhBChE) and copolymers may be useful in addressing these problems. We used in vitro approaches to compare enzyme activity, sensitivity to inhibition, stability and bioscavenging capacity of free enzyme and copolymer-rhBChE complexes (C-BCs) based on one of nine different copolymers, from combinations of three molecular weights (MW) of poly-L-lysine (PLL; high MW, 30-70 kDa; medium MW, 15-30 kDa; low MW, 4-15 kDa) and three grafting ratios of poly(ethylene glycol) (PEG; 2:1, 10:1, 20:1). Retarded protein migration into acrylamide gels stained for BChE activity was noted with all copolymers as the copolymer-to-protein ratio was increased. BChE activity of C-BCs was lower relative to free enzyme, with the 2:1 grafting ratio showing generally greater reduction. Free enzyme and C-BCs showed relatively similar in vitro sensitivity to inhibition by paraoxon, but use of the 20:1 grafting ratio led to lower potencies. Through these screening assays we selected three C-BCs (high, medium and low MW; 10:1 grafting) for further characterizations. BChE activity was higher in C-BCs made with the medium and low compared to high MW-based copolymer. C-BCs generally showed higher stability than free enzyme when maintained for long periods at 37 °C or following incubation with chymotrypsin. Free enzyme and C-BCs were similarly effective at inactivating paraoxon in vitro. While these results are promising for further development, additional studies are needed to evaluate in vivo performance.

Pharmacokinetics and immunogenicity of a recombinant human butyrylcholinesterase bioscavenger in macaques following intravenous and pulmonary delivery.

Recombinant (r) and native butyrylcholinesterse (BChE) are potent bioscavengers of organophosphates (OPs) such as nerve agents and pesticides and are undergoing development as antidotal treatments for OP-induced toxicity. Because of the lethal properties of such agents, regulatory approval will require extensive testing under the Animal Rule. However, human (Hu) glycoprotein biologicals, such as BChE, present a challenge for assessing immunogenicity and efficacy in heterologous animal models since any immune responses to the small species differences in amino acids or glycans between the host and biologic may alter pharmacodynamics and preclude accurate efficacy testing; possibly underestimating their potential protective value in humans. To establish accurate pharmacokinetic and efficacy data, an homologous animal model has been developed in which native and PEGylated forms of CHO-derived rMaBChE were multiply injected into homologous macaques with no induction of antibody. These now serve as controls for assessing the pharmacokinetics and immunogenicity in macaques of multiple administrations of PEGylated and unmodified human rBChE (rHuBChE) by both intravenous (IV) and pulmonary routes. The results indicate that, except for maximal concentration (Cmax), the pharmacokinetic parameters following IV injection with heterologous PEG-rHuBChE were greatly reduced even after the first injection compared with homologous PEG-rMaBChE. Anti-HuBChE antibody responses were induced in all monkeys after the second and third administrations regardless of the route of delivery; impacting rates of clearance and usually resulting in reduced endogenous MaBChE activity. These data highlight the difficulties inherent in assessing pharmacokinetics and immunogenicity in animal models, but bode well for the efficacy and safety of rHuBChE pretreatments in homologous humans.