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.

Absorption, Distribution and Excretion
Donepezil is slowly absorbed from the gastrointestinal tract after oral administration. The time to peak concentration (Tmax) is 3 to 4 hours, with 100% bioavailability. Steady-state plasma concentrations are reached within 15 to 21 days after administration. A pharmacokinetic study determined a Tmax of 4.1 ± 1.5 hours. According to a Canadian monograph, the Cmax of a 5 mg donepezil tablet is estimated to be 8.34 ng/mL. The AUC of a 5 mg donepezil tablet is 221.90–225.36 ng·hr/mL. In a study of radiolabeled donepezil administration in healthy adults, 57% of the radioactive material was detected in urine and 5% in feces. The volume of distribution for a 5 mg donepezil dose is 11.8 ± 1.7 L/kg, and for a 10 mg dose, it is 11.6 ± 1.91 L/kg. It is primarily distributed in the extravascular space. Donepezil can cross the blood-brain barrier, and at the above-mentioned doses, the concentration in cerebrospinal fluid is 15.7%. According to the FDA drug information leaflet, the steady-state volume of distribution of donepezil is 12-16 L/kg. The mean apparent plasma clearance of this drug is 0.13-0.19 L/hr/kg, according to the FDA drug information leaflet. In healthy subjects, after taking 5 mg of donepezil, the plasma clearance was 0.110 ± 0.02 L/h/kg. The plasma clearance in 10 patients with alcoholic cirrhosis was 20% lower than that in 10 healthy subjects. No significant change in plasma clearance was observed in 4 patients with severe renal impairment compared to 4 healthy subjects. Donepezil is well absorbed, with a relative oral bioavailability of 100%, reaching peak plasma concentration in 3-4 hours. Pharmacokinetics are linear in the once-daily dose range of 1-10 mg. Food and the time of administration (morning or evening) do not affect the rate or extent of absorption of donepezil hydrochloride tablets.
...The mean apparent plasma clearance (Cl/F) was 0.13 L/hr/kg. Following multiple doses, donepezil accumulated in plasma at a rate of 4 to 7 times, reaching steady state within 15 days. The steady-state volume of distribution was 12 L/kg.
In a study of 10 patients with stable alcoholic cirrhosis, donepezil hydrochloride clearance was 20% lower than in 10 age- and sex-matched healthy subjects.
In a study of 11 patients with moderate to severe renal impairment (creatinine clearance < 18 mL/min/1.73 m²), donepezil hydrochloride clearance was not different from that in 11 age- and sex-matched healthy subjects.
For more complete data on absorption, distribution, and excretion of donepezil (out of 12), please visit the HSDB records page.

---

Metabolism/Metabolites
Donepezil is primarily metabolized via first-pass metabolism in the liver, mainly by CYP3A4, with CYP2D6 also involved. Subsequently, O-dealkylation, hydroxylation, N-oxidation, hydrolysis, and O-glucuronidation occur, generating various metabolites with half-lives similar to the parent drug. A pharmacokinetic study of radiolabeled donepezil showed that approximately 53% of plasma radioactivity was present in the parent drug form, and 11% was identified as the metabolite 6-O-demethyldonepezil, which has similar inhibitory efficacy against acetylcholinesterase as the parent drug. The drug is primarily metabolized into four major metabolites, two of which are considered pharmacologically active, as well as several inactive and unidentified metabolites. Donepezil is excreted parentally in the urine and is extensively metabolized into the four major metabolites (two of which are known to be active) and several minor metabolites (not all of which have been identified). Donepezil is metabolized by CYP450 isoenzymes 2D6 and 3A4 and undergoes glucuronidation. Following administration of 14C-labeled donepezil, plasma radioactivity (expressed as a percentage of the administered dose) was primarily in the form of intact donepezil (53%) and 6-O-desmethyl donepezil (11%). 6-O-desmethyl donepezil has been reported to inhibit acetylcholinesterase (AChE) to the same extent as donepezil in vitro, and its plasma concentration is approximately 20% that of donepezil. This study aimed to investigate the metabolism and elimination of donepezil hydrochloride following a single oral dose of 5 mg (liquid) containing a mixture of unlabeled and 14C-labeled donepezil. …In each matrix, the largest proportion of donepezil unchanged from the administered dose was recovered. The study identified three metabolic pathways: (i) O-dealkylation and hydroxylation to generate metabolites M1 and M2, followed by glucuronidation to generate metabolites M11 and M12; (ii) hydrolysis to generate metabolite M4; and (iii) N-oxidation to generate metabolite M6. In plasma, the parent compound accounted for approximately 25% of the recovered dose per sampling period and 25% of the cumulative recovered dose. The recovered residues contained higher concentrations of hydroxylated metabolites M1 and M2 than their glucuronide conjugates M11 and M12, respectively. In urine, the parent compound accounted for an average of 17% of the recovered dose per mixed sample and 17% of the total recovered dose. The dominant metabolite was hydrolysis product M4, followed by glucuronide conjugates M11 and M12. In feces, the parent compound also predominated, but accounted for only 1% of the recovered dose. The majority of the radioactive material in feces consisted of unidentified, highly polar metabolites, which were retained in situ by thin-layer chromatography. Among the extracted metabolites, hydroxylation products M1 and M2 were the most abundant, followed by hydrolysis product M4 and N-oxidation product M6. Donepezil is primarily metabolized in the liver, and the main route of excretion for the parent drug and its metabolites is the kidneys, as 79% of the recovered dose is found in urine, and the remaining 21% in feces. Furthermore, the parent compound donepezil is the main excreted product in urine. The major metabolites of donepezil include M1 and M2 (generated via O-dealkylation and hydroxylation, respectively), M11 and M12 (generated via glucuronidation of M1 and M2, respectively), M4 (generated via hydrolysis), and M6 (generated via N-oxidation). Known human metabolites of donepezil include 6-O-demethyldone-dopezil, 5,6-dimethoxy-2-(piperidin-4-ylmethyl)-2,3-dihydroindene-1-one, and 5-O-demethyldone-dopezil. Donepezil is metabolized in the liver by CYP450 isoenzymes 2D6 and 3A4, and undergoes glucuronidation. The major metabolite, 6-O-demethyldonepezil, has been reported to have the same inhibitory effect on acetylcholinesterase (AChE) as donepezil in vitro. Elimination pathway: Donepezil is excreted unchanged in the urine and extensively metabolized into four major metabolites (two of which are known to be active) and several minor metabolites (not all of which have been identified). Half-life: 70 hours. Based on multiple studies and the FDA's donepezil label, the mean elimination half-life of donepezil is approximately 70 hours. One pharmacokinetic study determined its mean terminal half-life to be 81.5 ± 22.0 hours. The elimination half-life of donepezil is approximately 70 hours. A 79-year-old woman with Alzheimer's disease was admitted to the hospital with acute cholinergic symptoms after an overdose of donepezil (45 mg). Her plasma DPZ concentration upon admission was 54.6 ng/mL, which gradually decreased to normal levels over approximately 90 hours. The calculated half-life of DPZ is approximately 55 hours.

Toxicity Summary
Donepezil is a cholinesterase, or acetylcholinesterase (AChE) inhibitor. Cholinesterase inhibitors (or "anticholinesterases") inhibit the activity of acetylcholinesterase. Because acetylcholinesterase plays a vital physiological role, chemicals that interfere with its activity are potent neurotoxins; even low doses can cause excessive salivation and lacrimation, followed by muscle spasms and ultimately death. Substances used in nerve gases and many pesticides have been shown to exert their effects by binding to serine residues at the active site of acetylcholinesterase, thus completely inhibiting the enzyme's activity. Acetylcholinesterase breaks down the neurotransmitter acetylcholine, which is released at the neuromuscular junction, causing muscle or organ relaxation. Inhibition of acetylcholinesterase results in the accumulation and sustained action of acetylcholine, leading to the continuous transmission of nerve impulses and an inability to stop muscle contractions. The most common acetylcholinesterase inhibitors are phosphorus-containing compounds designed to bind to the enzyme's active site. Its structural requirements include a phosphorus atom with two lipophilic groups, a leaving group (e.g., a halide or thiocyanate), and a terminal oxygen atom.
Hepatotoxicity
In several large clinical trials, donepezil treatment did not increase the incidence of elevated serum enzymes compared to placebo. Furthermore, the incidence of ALT elevation did not increase after increasing the dose from 10 mg to 23 mg daily compared to patients maintaining a lower dose. However, since donepezil was introduced clinically, several cases of clinically significant hepatotoxicity have been reported. Onset is short (1 to 6 weeks), with serum enzyme elevations in a cholestatic or mixed pattern. The condition can be severe, with persistent jaundice and pruritus (Case 1), but no deaths have been reported. Immune hypersensitivity and autoimmune features are uncommon.
Probability score: D (likely a rare cause of clinically significant liver injury). Protein Binding Donepezil has a protein binding rate of 96%, with approximately 75% bound to albumin and approximately 21% bound to α1-glycoprotein. Interactions
Ketoconazole and quinidine are inhibitors of CYP450 3A4 and 2D6, respectively, and they inhibit the metabolism of donepezil in vitro. The clinical significance of quinidine is unclear. In a 7-day crossover study, the mean concentrations of donepezil (AUC0-24 and Cmax) increased by 36% in 18 healthy volunteers after ketoconazole administration. The clinical significance of this increase is unclear. Cholinesterase inhibitors may have a synergistic effect when used in combination with succinylcholine, similar neuromuscular blocking agents, or cholinergic agonists (such as betanidol).
CYP2D6 and CYP3A4 inducers (e.g., phenytoin sodium, carbamazepine, dexamethasone, rifampin, and phenobarbital) may increase the clearance of donepezil hydrochloride.
A 75-year-old male patient with Alzheimer's disease, who had been treated with the cholinesterase inhibitor donepezil for 14 months, was scheduled for a left hemicolectomy under general anesthesia. During the surgery, the duration of succinylcholine-induced muscle relaxation was prolonged, and even with atracurium at a dose higher than the recommended dose for the patient's body weight, the effect was insufficient. Blood tests for cholinesterase at 10 months, 1 month, and 10 days prior to surgery showed a gradually shortening duration of enzyme activity. This effect has been reported with cholinesterase inhibitors such as neostigmine and donepezil, which could explain the prolonged duration of succinylcholine action. After ruling out other causes of atracurium resistance, we concluded that donepezil or its metabolites act on muscle plaques, blocking the hydrolysis of acetylcholine, thereby antagonizing the effects of atracurium. For more complete data on donepezil drug interactions (7 items in total), please visit the HSDB record page.

[1]. 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]. 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]. Donepezil protects endothelial cells against hydrogen peroxide-induced cell injury. CNS Neurosci Ther, 2012. 18(2): p. 185-7.

Therapeutic Uses
Cholinesterase inhibitor; nootropic agent. Donepezil hydrochloride tablets are indicated for the treatment of Alzheimer's disease-related dementia. It has been shown to be effective in patients with mild to moderate Alzheimer's disease. /US product label contains/ /EXPTL Ther:/ ... Donepezil is officially approved for the treatment of mild to moderate and severe Alzheimer's disease (AD) and has been shown to be effective in early Alzheimer's disease, vascular dementia, Parkinson's dementia/Lewy body dementia, and cognitive symptoms associated with multiple sclerosis. Furthermore, one study showed that donepezil may delay the onset of Alzheimer's disease in patients with mild cognitive impairment (a prodromal symptom of Alzheimer's disease). Drug Warnings Donepezil hydrochloride tablets are contraindicated in patients with known hypersensitivity to donepezil hydrochloride or piperidine derivatives. Due to its pharmacological action, cholinesterase inhibitors may have a vagal excitatory effect on the sinoatrial node and atrioventricular node. This effect may manifest as bradycardia or cardiac conduction block, regardless of whether the patient has a known underlying cardiac conduction abnormality. Syncope has been reported with donepezil hydrochloride. The primary action of cholinesterase inhibitors is to increase cholinergic activity, which may increase gastric acid secretion. Therefore, patients should be closely monitored for signs of active or occult gastrointestinal bleeding, especially those with a history of peptic ulcer disease or those at increased risk of ulceration, such as those taking nonsteroidal anti-inflammatory drugs (NSAIDs). The pharmacological properties of donepezil hydrochloride can cause diarrhea, nausea, and vomiting, which are predictable side effects. These side effects are more common in the 10 mg daily dose group than in the 5 mg daily dose group. In most cases, these side effects are mild and transient, sometimes lasting one to three weeks, and subside with continued use of donepezil hydrochloride. For more complete data on donepezil (12 total), please visit the HSDB records page.

---

Pharmacodynamics
Donepezil improves cognitive and behavioral symptoms in Alzheimer's disease patients, including apathy, aggression, confusion, and psychosis, by inhibiting acetylcholinesterase.

C24H29NO3

379.492

379.214

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

3152

White to off-white solid powder

1.1±0.1 g/cm3

527.9±50.0 °C at 760 mmHg

207ºC

273.1±30.1 °C

0.0±1.4 mmHg at 25°C

1.578

4.71

0

4

6

28

510

0

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.

---

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.

---

View More

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.

---

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