In vitro and in vivo models of tacrine 1,2,3 triazole hybrids on how to improve memory loss in alzheimer’s disease

HO CHI MINH UNIVERSITY OF SCIENCE FA C U LT Y O F C H E M I S T R Y C H E M I C A L E N G I N E E R I N G A N D T E C H N O LO GY Group IN VITRO AND IN VIVO MODELS OF TACRINE-1,2,3-TRIAZOLE HYBRIDS ON HOW TO IMPROVE MEMORY LOSS IN ALZHEIMER’S DISEASE I Information on Alzheimer’s disease (AD) II In Vitro Model III In Vivo Model I Information on Alzheimer ’s disease Pathways towards Alzheimer's disease - A neurodegenerative disorder - Cognitive impairment, memory decline - In individuals above the age of 60 Fig PET scans (Glucose utilization) The target of disease - Due to age (people over 65 have a high risk of Alzheimer's disease) - Genetics (people have a family history of Alzheimer's disease; or carry a gene called APOE4) - People with a history of head injury or mild cognitive impairment - Unhealthy lifestyle (using stimulants, a diet lacking in vegetables and fruits, sedentary routine) Figure PATHOGENESIS AND ROLE OF ACETYLCHOLINESTERASE IN THE TREATMENT Amyloid protein Tau-protein Fig The pathological evolution of Alzheimer’s disease PATHOGENESIS AND ROLE OF ACETYLCHOLINESTERASE IN THE TREATMENT Treatment methods o Using acetylcholinesterase inhibitor drugs (eg: tacrine, rivastigmine, galantamine and donepezil) o Healthy nutrition o Do exercise II In Vitro Model 10 N NH2 Tacrine Fig Schematic representation of the binding sites of AChE ES: esteratic site AS: anionic substrate binding site ACS: aromatic cation binding site PAS: peripheral anionic binding site 11 Acetylcholinesterase Acetylcholinesterase Acetylcholinesterase 𝜆= 405 𝑛𝑚 Source: https://www.sigmaaldrich.com/VN/en/product/sigma/c3389 12 - Source: www.sigmaaldrich.com/VN/en/product/sigma/ cs0003?context=product - - Acetylcholinesterase (AChE, E.C 3.1.1.7, Type V-S, lyopholized powder, from electric eel, 1000 unit) Butylcholinesterase (BChE, E.C 3.1.1.8, from equine serum, 1000 unit) Acetylthiocholine iodide (ATCI), Butyrylthiocholine iodide (BTCI), and 5,5-dithiobis-(2-nitrobenzoicacid) (DTNB) Methanol Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, DMSO - 96-well plate and 96-well plate reader (BioTek ELx808) Source: https://www.biospx.com/elx808.html 13 50 potassium phosphate buffer (KH2PO4/ K2HPO4, 0.1 M, pH 8) 25 sample dissolved in 50% methanol and 50% DMSO 25 enzyme (final concentration 0.22 U/ml in buffer) Control - Sample Preincubate for 15 at room temperature Add 125 DTNB (3 mM in buffer) and 50 substrate (ATCI mM in water) and wait 20 minutes Blank - enzyme Absorbance was measured at 405 nm IC50 values were determined graphically from inhibition curves Four different concentrations were tested for each compound in triplicate to obtain the range of 20 % - 80 % inhibition for AChE 14 Table 1: In vitro results 15 III In Vivo Model 16 HO O O N O H Br Dissolved in 20% PEG 400 (Polyethylene glycol) as drug vehicle scopolamine hydrobromide O O O H Cl N Donepezil hydrochloride Scopolamine hydrobromide and Donepezil hydrochloride were obtained from Sigma Which X = H Y = Cl R = 4-OMe Intraperitoneal injection 17 Table 2: In vivo group arrangement I Control Normal saline (5mg/ kg) II Scopolamine III Vehicle IV Test sample 5l (5mg/kg) and Scopolamine hydrobromide (4mg/kg) V Test sample 5l (10mg/kg) and Scopolamine hydrobromide (4mg/kg) VI Test sample 5l (20mg/kg) and Scopolamine hydrobromide (4mg/kg) VII Positive control Donepezil (2.5mg/kg) and Scopolamine hydrobromide (4mg/kg) Scopolamine hydrobromide (4mg/kg) 20% PEG-400 (5ml/kg) Male albino Wistar rats (200-220g) were obtained from the Faculty of Pharmacy, Tehran University of Medical Sciences A 12-hrs light/12-hrs dark cycle was kept and provided food and water ad libitum The room was maintained at the temperature 25 ± oC 18 Morris water maze test Four successive days in a Morris water maze Walls were covered by different shaped visual cues A black circular pool with a diameter of about 136 cm and a depth of 35 cm The pool was filled with water and the temperature was kept at 25 ± C A circular-shaped invisible platform made of Plexiglass whose diameter is 10 cm On fifth day (probe trial test) It should be noted that the platform in the target quadrant was removed and animals did not receive treatment on test day 19 Fig In vivo results of (A) Escape latency, (B) Travel distance, (C) Swimming speed The significant increase of escape latency (Fig A) and traveled distance (Fig B) was observed compared with the control group produced by scopolamine injection Escape latency and traveled distance have improved in groups that received compound 5l plus scopolamine in comparison to scopolamine administered group All compound 5l treated groups displayed decreased escape latency in comparison to the scopolamine group The traveled distance was significantly decreased in all groups that received compound 5l in comparison to the 20 scopolamine group ...I Information on Alzheimer’s disease (AD) II In Vitro Model III In Vivo Model I Information on Alzheimer ’s disease Pathways towards Alzheimer''s disease - A neurodegenerative... PATHOGENESIS AND ROLE OF ACETYLCHOLINESTERASE IN THE TREATMENT Treatment methods o Using acetylcholinesterase inhibitor drugs (eg: tacrine, rivastigmine, galantamine and donepezil) o Healthy nutrition... exercise II In Vitro Model 10 N NH2 Tacrine Fig Schematic representation of the binding sites of AChE ES: esteratic site AS: anionic substrate binding site ACS: aromatic cation binding site PAS: