Acetylcholinesterase (AChE)

The muscarinic antagonist activity of donepezil (E2020 free base) is demonstrated by its concentration-dependent inhibition of the carbachol-stimulated increase in intracellular Ca2+ concentration in human SHSY5Y neuroblastoma cells. Once rats received donepezil intraperitoneally, there was a dose-dependent rise in tremor and salivation, indicating overt cholinergic behavior, with an ED50 of 6 μmol/kg. With an ED50 of 50 μmol/kg, donepezil was found to be marginally less effective when taken orally [2]. According to a recent study, donepezil shields human umbilical vein endothelial cells (HUVEC) from cellular damage brought on by H2O2. This could potentially be used as a treatment for oxidative stress in diseases related to the heart and brain [3].

The in vitro and in vivo effects of the novel acetylcholinesterase inhibitors donepezil and NXX-066 have been compared to tacrine. Using purified acetylcholinesterase from electric eel both tacrine and donepezil were shown to be reversible mixed type inhibitors, binding to a similar site on the enzyme. In contrast, NXX-066 was an irreversible non-competitive inhibitor. All three compounds were potent inhibitors of rat brain acetylcholinesterase (IC50 [nM]; tacrine: 125 +/- 23; NXX-066: 148 +/- 15; donepezil: 33 +/- 12). Tacrine was also a potent butyrylcholinesterase inhibitor. Donepezil and tacrine displaced [3H]pirenzepine binding in rat brain homogenates (IC50 values [microM]; tacrine: 0.7; donepezil: 0.5) but NXX-066 was around 80 times less potent at this M1-muscarinic site. Studies of carbachol stimulated increases in [Ca2+]i in neuroblastoma cells demonstrated that both donepezil and tacrine were M1 antagonists. Ligand binding suggested little activity of likely pharmacological significance with any of the drugs at other neurotransmitter sites. Intraperitoneal administration of the compounds to rats produced dose dependent increases in salivation and tremor (ED50 [micromol/kg]; tacrine: 15, NXX-066: 35, donepezil: 6) with NXX-066 having the most sustained effect on tremor. Following oral administration, NXX-066 had the slowest onset but the greatest duration of action. The relative potency also changed, tacrine having low potency (ED50 [micromol/kg]; tacrine: 200, NXX-066: 30, donepezil: 50). Salivation was severe only in tacrine treated animals. Using in vivo microdialysis in cerebral cortex, both NXX-066 and tacrine were found to produce a marked (at least 30-fold) increase in extracellular acetylcholine which remained elevated for more than 2 h after tacrine and 4 h after NXX-066[2].

This study was designed to compare the in vitro inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) of donepezil and some other cholinesterase (ChE) inhibitors which have been developed for the treatment of Alzheimer's disease. The carbamate derivatives physostigmine and rivastigmine needed preincubation to exhibit appropriate anti-ChE activity. The maximum ChE inhibition by physostigmine developed within 30-60 min, while the inhibitory effect of rivastigmine on AChE and BuChE activities reached its peak after 48 and 6 h, respectively. The order of inhibitory potency (IC50) towards AChE activity under optimal assay conditions for each ChE inhibitor was: physostigmine (0.67 nM) > rivastigmine (4.3 nM) > donepezil (6.7 nM) > TAK-147 (12 nM) > tacrine (77 nM) > ipidacrine (270 nM). The benzylpiperidine derivatives donepezil and TAK-147 showed high selectivity for AChE over BuChE. The carbamate derivatives showed moderate selectivity, while the 4-aminopyridine derivatives tacrine and ipidacrine showed no selectivity. The inhibitory potency of these ChE inhibitors towards AChE activity may illustrate their potential in vivo activity[1].

The effect of cholinesterase inhibitors on calcium flux in SHSY5Y human neuroblastoma cells[2]
SHSY5Y cells were maintained in Eagles Minimum Essential Medium and Hams F-12 medium (1:1) supplemented by 10% foetal calf serum, 2% l-glutamine, 1% non-essential amino acids and 20 mM Hepes pH 7.4. To harvest the cells the medium was removed and the monolayer rinsed with 10 ml Hank’s balanced salt solution, and scraped from the base of the flask using a cell scraper. The resulting suspension was centrifuged at 250×g for 5 min at 4°C and the resulting pellet was resuspended in a loading buffer consisting of 6 ml phosphate buffered saline containing 2 mM EDTA, 10 μM Fluo-3AM and 0.02% pluronic F-127 pH 7.4. The cells were loaded with the acetoymethyl ester derivative of Fluo3 (Fluo-3AM) for 15 min at 37°C. Following centrifugation at 250×g for 5 min at 4°C the resulting pellet was resuspended in oxygenated Hepes-Ringer buffer pH 7.4 containing CaCl2 (0.5 mM) and glucose (10 mM). The cell suspension was then incubated at room temperature for a further 20 min to allow for hydrolysis of the Fluo-3AM, centrifuged at 250×g for 5 min and resuspended in oxygenated Hepes–Ringer buffer as described earlier. Aliquots (2 ml) of cell suspension were placed in quartz cuvettes nd equilibrated for 1 min with stirring at room temperature. A basal fluorescence was recorded after which test agents were added. Twenty microlitre additions were made with a Hamilton syringe, permitting continuous measurement of the fluorescence signal. The background fluorescence was unaffected by this procedure. Fluorescence was measured at excitation 505 nm: emission 530 nm. Maximal fluorescence was measured by addition of 10 μM calcium ionophore 4-bromo calcimycin. The fluorescence signal was then quenched using MnCl2 (1 mM).

Behavioural observations[2]
Tremor and salivation were assessed using the methods described by Hunter et al. (1989). Briefly, groups of animals were injected with various doses of cholinesterase inhibitor and observed. Tremor (score 0–3) and salivation (weight in 10 mg units) were noted. 2.9. Measurement of extracellular acetylcholine in rat brain using in vivo microdialysis.
Individually prepared concentric probes, essentially as described by Hutson et al. (1985), were used except that they were implanted without the use of a guide assembly and the internal glass capillary tubes were replaced by fused silica tubes (VS-150-075-1D). The dialysis membrane was 4mm long and had an approximate diameter of 0.2 mm. The in vitro efficiency of ACh recovery when the microdialysis probes were placed in an ACh (60 μM) solution at room temperature and perfused at 1.0 μl/min, was 17.9±2.4%.
Probes were implanted transversely into the cortex of rats anaesthetised with halothane (2%) in O2/N2O mixture (1: 2) and secured in Kopf stereotaxic frame with the tooth bar at −3.3 mm below interaural zero. Probes were implanted horizontally into the right cortex: +7.7 mm anterior and +4.2 mm lateral from interaural zero and −1.3 mm from the skull surface and secured to the skull with two screws and dental cement. Following surgery, the animals were housed in perspex boxes until the beginning of microdialysis procedures the next day. Placement of the probe was verified by visual inspection of the probe track at the end of each experiment by injecting Luxol Fast blue.
Dialysis probes were perfused at a rate of 1 μl/min with artificial CSF (composition mM: NaCl 125, KCl 2.5, MgCl2, 1.18 and CaCl2 1.26), or high K+CSF (composition mM: NaCl 27.5, KCl 100, MgCl2 1.18 and CaCl2 1.26) using a model 22 Harvard Microlitre syringe pump. The artificial CSF did not contain an acetylcholinesterase inhibitor. Thirty minute fractions were collected which were then stored on ice. Dialysate from the first 60 min was discarded and the next three collections of 30 min were baseline samples prior to the i.p administration of tacrine (21 μmol/kg) or NXX-066 (106 μmol/kg).
Acetylcholine was measured by a hplc method similar to that originally described by Potter et al. (1983). Following its separation on an analytical ion exchange column ACh was converted to hydrogen peroxide inside a 4.1×30 mm analytical column, filled with polymeric matrix to which AChE and choline oxidase enzymes has been covalently linked. The hydrogen peroxide formed was detected electrochemically by oxidation on a platinum electrode at +500 mV versus a Ag/AgCl reference. The mobile phase composition was 3.4 mM H3PO4 (85%) and 5 mM Kathon CG (1%); the pH was adjusted to 8.5 by addition of NaOH. The flow rate of 0.6 ml/min ensured quantitative conversion of ACh to H2O2 within an enzyme reactor. Peaks were recorded on a Kontron integrator. ACh in brain dialysates was quantified by the standard curve method. A lower level of 0.6 pmol of ACh on the column could be reliably detected.
Donepezil were dissolved in 0.9% w/v NaCl (saline) before injection.

[1]. Ogura, H., et al., Comparison of inhibitory activities of donepezil and other cholinesterase inhibitors on acetylcholinesterase and butyrylcholinesterase in vitro. Methods Find Exp Clin Pharmacol, 2000. 22(8): p. 609-13.
[2]. Snape, M.F., et al., A comparative study in rats of the in vitro and in vivo pharmacology of the acetylcholinesterase inhibitors tacrine, donepezil and NXX-066. Neuropharmacology, 1999. 38(1): p. 181-93.
[3]. Huang, Z.H., et al., Donepezil protects endothelial cells against hydrogen peroxide-induced cell injury. CNS Neurosci Ther, 2012. 18(2): p. 185-7.

C24H29NO3

379.492

379.21475

C, 75.96; H, 7.70; N, 3.69; O, 12.65

120014-06-4

(R)-Donepezil;142698-19-9;(S)-Donepezil;142057-80-5;Donepezil-d7 hydrochloride;1261394-20-0;Donepezil-d5;1128086-25-8;Donepezil Hydrochloride;120011-70-3

Typically exists as solids (or liquids in special cases) at room temperature

4.71

38.77

O=C1C2=C([H])C(=C(C([H])=C2C([H])([H])C1([H])C([H])([H])C1([H])C([H])([H])C([H])([H])N(C([H])([H])C2C([H])=C([H])C([H])=C([H])C=2[H])C([H])([H])C1([H])[H])OC([H])([H])[H])OC([H])([H])[H]

ADEBPBSSDYVVLD-UHFFFAOYSA-N

InChI=1S/C24H29NO3/c1-27-22-14-19-13-20(24(26)21(19)15-23(22)28-2)12-17-8-10-25(11-9-17)16-18-6-4-3-5-7-18/h3-7,14-15,17,20H,8-13,16H2,1-2H3

2-((1-benzylpiperidin-4-yl)methyl)-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one

HSDB 7743; HSDB-7743; HSDB7743

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~33.33 mg/mL (~87.83 mM)
H2O : ~2 mg/mL (~5.27 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.59 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.59 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)