The Effects of Donepezil, an Acetylcholinesterase Inhibitor, on Impaired Learning and Memory in Rodents

Original Article Biomol Ther 26(3), 274-281 (2018) The Effects of Donepezil, an Acetylcholinesterase Inhibitor, on Impaired Learning and Memory in Rodents Chang Yell Shin1,2, HaeSun Kim1, KwangHo Cha1, Dong Han Won1, JiYun Lee2, Sun Woo Jang1,* and Uy Dong Sohn2,* 1Research Institute of Dong-A ST Co., Ltd., Yongin 17073, 2College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea Abstract A previous study in humans demonstrated the sustained inhibitory effects of donepezil on acetylcholinesterase (AChE) activity; however, the effective concentration of donepezil in humans and animals is unclear. This study aimed to characterize the effective concentration of donepezil on AChE inhibition and impaired learning and memory in rodents. A pharmacokinetic study of donepezil showed a mean peak plasma concentration of donepezil after oral treatment (3 and 10 mg/kg) of approximately 1.2 ± 0.4 h and 1.4 ± 0.5 h, respectively; absolute bioavailability was calculated as 3.6%. Further, AChE activity was inhibited by increasing plasma concentrations of donepezil, and a maximum inhibition of 31.5 ± 5.7% was observed after donepezil treatment in hairless rats. Plasma AChE activity was negatively correlated with plasma donepezil concentration. The pharmacological effects of donepezil are dependent upon its concentration and AChE activity; therefore, we assessed the effects of donepezil on learning and memory using a Y-maze in mice. Donepezil treatment (3 mg/kg) significantly prevented the progression of scopolamine-induced memory impairment in mice. As the concentration of donepezil in the brain increased, the recovery of spontaneous alternations also improved; maximal improvement was observed at 46.5 ± 3.5 ng/g in the brain. In conclusion, our findings suggest that the AChE inhibitory activity and pharmacological effects of donepezil can be predicted by the concentration of donepezil. Further, 46.5 ± 3.5 ng/g donepezil is an efficacious target concentration in the brain for treating learning and memory impairment in rodents. Key Words: Acetylcholinesterase activity, Donepezil, Pharmacokinetic, Pharmacodynamic, Y-maze INTRODUCTION As acetylcholinesterase (AChE) is found predominantly in the brain, striated muscle, and blood, AD treatment strategies Alzheimer’s disease (AD) is a progressive neurological dis- are mainly directed toward AChE inhibition (Sozio et al., 2012). order and is the most common cause of senile dementia, as Pharmacological effects result from the integration of phar- characterized by loss of memory and other intellectual abilities macokinetics (PK) and pharmacodynamics (PD) (Ito et al., that interfere with daily life (Small et al., 1997). It has been 2010). The relationship between PK and PD is usually ad- estimated that 35.6 million people lived with dementia world- dressed by measuring plasma drug concentrations and PD wide in 2010; these numbers are expected to nearly double markers. AChE inhibition in blood has been used as a periph- every 20 years to 65.7 million in 2030 and 115.4 million in eral marker for the activity of centrally acting AChE inhibitors 2050 (Prince et al., 2013). Although little is known regard- in the treatment of AD (Sramek and Cutler, 2000). Further, ing the cause of AD, it is generally accepted that many of its AChE inhibition in blood reflects the central PD activity of the symptoms are related to a cholinergic deficit, and the extent of compound and demonstrates a relationship with cognitive or neuropathological features have been found to correlate with global improvement in AD (Rogers et al., 1998). cholinergic loss in the central nervous system (Davis et al., Donepezil (C24H29NO3, MW: 379.492) is a reversible, selec- 1995; Rogers et al., 1998). tive AChE inhibitor that is currently approved for the symp- Open Access https://doi.org/10.4062/biomolther.2017.189 Received Sep 25, 2017 Revised Oct 25, 2017 Accepted Nov 7, 2017 Published Online Feb 21, 2018 This is an Open Access article distributed under the terms of the Creative Com- mons Attribution Non-Commercial License (http://creativecommons.org/licens- *Corresponding Authors es/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, E-mail: [email protected] (Jang SW), [email protected] (Sohn UD) and reproduction in any medium, provided the original work is properly cited. Tel: +82-31-280-1324 (Jang SW), +82-2-820-5614 (Sohn UD) Fax: +82-31-282-8564 (Jang SW), +82-2-826-8752 (Sohn UD) Copyright © 2018 The Korean Society of Applied Pharmacology www.biomolther.org 274 Shin et al. Correlation of Donepezil and Learning and Memory A O old) were obtained from Central Lab Animal Inc. (Seoul, Ko- rea) and Dae Han Biolink (Eumseong, Korea), respectively. Animals were acclimated for one week, maintained at 23 ± O 2°C on a 12:12-h light-dark cycle (lights on 07:00-19:00), and N allowed free access to food and water. O Donepezil Pharmacokinetic study of donepezil in hairless rats B Hairless rats, with an average weight of 300 g, were ran- Donepezil oral 60 domly assigned to a treatment group (4-7 animals per group). PO3mg/kg Donepezil HCl (10 mg/kg free drug dissolved in saline) was PO 10 mg/kg 50 administered orally, and blood (250 µl) was collected through the tail vein. Sampling continued at the indicated time points (ng/ml) 40 until 24 h; samples were maintained at -80°C until liquid chro- matography-tandem mass spectrometry (LC-MS/MS) analy- conc. 30 sis. To assess absolute bioavailability, donepezil HCl (3 mg/kg free drug dissolved in saline) was administered intravenously 20 via the tail vein. Donepezil 10 AChE activity assay in rat plasma 0 Blood samples for the AChE assay were collected from in- 0 61218 24 tact hairless rats and placed into heparin-filled tubes. Blood Time (h) was centrifuged (1,000 × g, 4°C, 10 min) to obtain plasma, and the samples were then prepared by adding 900 µl assay buf- Fig. 1. Structure of donepezil (A) and plasma donepezil concen- fer to 100 µl plasma. Samples were aliquoted into tubes (80 trations (mean ± SD) following an oral (3 or 10 mg/kg) treatment in µl), and different concentrations of donepezil (from 0 to 1500 hairless rats. ng/ml, with a final volume of 20 µl) were incubated with the plasma samples. AChE activity was measured using a colo- rimetric enzyme-linked immunoassay (ELISA) kit from Abcam tomatic treatment of AD (Fig. 1A); it is believed to inhibit the (Cambridge, MA, USA). breakdown of the neurotransmitter acetylcholine (ACh) and compensate for the deficiency of ACh in the brain (Colovic et PK and PD study in hairless rats al., 2013). Donepezil was proven to improve cognitive function To assess the correlation between PK and PD, hairless rats in mild to severely moderate AD patients in clinical trials and with an average weight of 300-320 g were randomly assigned showed excellent tolerability without hepatotoxic effects (Rog- to a treatment group (6 rats per group). Donepezil (5 mg free ers et al., 1998). drug per rat [approximately equal to 17.9 mg/kg]) was admin- Although previous studies in humans (Rogers et al., 1998) istered orally. Blood was collected from the tail vein at each and rats (Goh et al., 2011) demonstrated sustained inhibitory indicated time point (0, 0.5, 1, 2, 4, 6, 8, and 24 h) in a heparin- effects of donepezil on AChE, the effective concentration of filled tube; the plasma was then separated by centrifugation. donepezil resulting in improved efficacy in the brain, as well Plasma was maintained at -80°C until donepezil concentra- as its relation to changes in the concentration of plasma done- tions or AChE activity was analyzed. pezil are still unclear. In this study, we analyzed the PK and PD of donepezil to PK and PD study in mice predict the effective concentration of donepezil for AChE in- To assess the correlation between PK and PD, ICR mice hibition. Furthermore, we evaluated donepezil-associated (average weight of 30 g) were randomly assigned to a treat- improvements to spatial memory deficits caused by scopol- ment group (6-8 animals per group). Donepezil (approximate- amine in mice to determine the brain concentration at which ly 0.1-10 mg/kg free drug) was administered orally. The mice significant improvements in behavioral pharmacology can be were anesthetized after 1 h, and blood was collected from the observed. orbital venous sinus in a heparin-filled tube; whole brain samples were then immediately extracted and briefly rinsed with water, dissected into halves, and stored in tubes (Geerts et MATERIALS AND METHODS al., 2005; Goh et al., 2011). Mouse brains were transferred to 2-ml capped, round-bottomed Eppendorf (E)-tubes that were Materials pre-labeled. Purified water (4 fold the brain weight) was added Donepezil HCl was obtained from Aurobindo Pharma to each tube, which was subsequently vortexed for approxi- (Telangana, India). All other chemicals were purchased from mately 15 s and then sonicated for 3 min. Methanol (1 fold the Sigma-Aldrich unless otherwise noted (St. Louis, MO, USA). brain weight) was then added; the tube was again vortexed for approximately 5 s and then rotary mixed for 30 min. All Ethics statement tubes were kept at -80°C until donepezil concentrations were All animal studies were approved by the Institutional Animal analyzed. Care and Use Committee of Dong-A ST Research Institute (IACCU approval No. I-1506197, I-1701030). Male hairless Analysis of plasma and brain donepezil levels rats and male imprinting control region (ICR) mice (6 weeks Analysis of plasma and brain donepezil concentrations was 275 www.biomolther.org Biomol Ther 26(3), 274-281 (2018) Donepezil intravenous Table 1. Donepezil pharmacokinetic parameters after oral treatment to 10,000 IV (3 mg/kg) hairless rats (mean ± SD) Parameter PO (3 mg/kg) PO (10 mg/kg) 1,000 Tmax (h) 1.2 ± 0.4 1.4 ± 0.5 (ng/ml) Cmax (ng/ml) 17.9 ± 2.4 44.1 ± 7.9 conc.

Load more