2024 Volume 47 Issue 1 Pages 328-333
Alzheimer’s disease (AD) is accompanied by behavioral and psychological symptoms of dementia (BPSD), which is often alleviated by treatment with psychotropic drugs, such as antidepressants, hypnotics, and anxiolytics. If these drugs also inhibit acetylcholinesterase (AChE) activity, they may contribute to the suppression of AD progression by increasing brain acetylcholine concentrations. We tested the potential inhibitory effects of 31 antidepressants, 21 hypnotics, and 12 anxiolytics on recombinant human AChE (rhAChE) activity. At a concentration of 10−4 M, 22 antidepressants, 19 hypnotics, and 11 anxiolytics inhibited rhAChE activity by <20%, whereas nine antidepressants (clomipramine, amoxapine, setiptiline, nefazodone, paroxetine, sertraline, citalopram, escitalopram, and mirtazapine), two hypnotics (triazolam and brotizolam), and one anxiolytic (buspirone) inhibited rhAChE activity by ≥20%. Brotizolam (≥10−6 M) exhibited stronger inhibition of rhAChE activity than the other drugs, with its pIC50 value being 4.57 ± 0.02. The pIC50 values of the other drugs were <4, and they showed inhibitory activities toward rhAChE at the following concentrations: ≥3 × 10−6 M (sertraline and buspirone), ≥10−5 M (amoxapine, nefazodone, paroxetine, citalopram, escitalopram, mirtazapine, and triazolam), and ≥3 × 10−5 M (clomipramine and setiptiline). Among these drugs, only nefazodone inhibited rhAChE activity within the blood concentration range achievable at clinical doses. Therefore, nefazodone may not only improve the depressive symptoms of BPSD through its antidepressant actions but also slow the progression of cognitive symptoms of AD through its AChE inhibitory actions.
Alzheimer’s disease (AD) is a neurodegenerative disease that causes dementia, and its prevalence is increasing significantly worldwide.1) The main treatment for AD is drug therapy using cholinesterase inhibitors that suppresses the progression of AD symptoms by increasing the amount of acetylcholine (ACh) in the brain.1) The main symptom of AD is dementia, but behavioral and psychological symptoms of dementia (BPSD) such as hallucinations, agitation, depression, anxiety, and sleep disturbances also develop.2) Patients with AD and BPSD are often treated with psychotropic drugs such as antipsychotics, antidepressants, hypnotics, and anxiolytics to alleviate BPSD.2) If these drugs also have acetylcholinesterase (AChE) inhibitory activity, they may contribute to the suppression of AD progression by increasing brain ACh concentrations. Recently, we evaluated the inhibitory actions of 26 clinically available antipsychotics on recombinant human AChE (rhAChE) and found that aripiprazole may inhibit AChE within its clinically achievable blood concentration range (CABCR).3) Other than antipsychotics, some psychotropic drugs used for BPSD have also been reported to inhibit AChE,4–9) but the AChE inhibitory actions of all clinically available psychotropic drugs have not been fully tested. In this study, we tested the potential inhibitory effects of 31 antidepressants, 21 hypnotics, and 12 anxiolytics on rhAChE activity. For each drug, the concentration at which its AChE inhibitory activity begins to be observed (CAIA) was also compared with its CABCR.
In this study, 31 antidepressants, 21 hypnotics, and 12 anxiolytics were used (Supplementary Tables 1–3). All other chemicals were obtained from generic suppliers and were reagent grade.
All tested drugs were dissolved in pure dimethyl sulfoxide (DMSO) to prepare 2 × 10−2 M stock solutions and diluted further with DMSO to the desired concentrations. Acetylthiocholine (ATCh)/5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was dissolved in 0.1 M phosphate buffer (PB, pH 7.4) to obtain a 20 mM/5 mM stock solution.
Inhibitory Actions of Antidepressants, Hypnotics, and Anxiolytics on rhAChE ActivityrhAChE activity was measured using a plate reader (Infinite® F200 PRO; Tecan, Männedorf, Switzerland) as previously described.3) Briefly, rhAChE (C1682; Sigma-Aldrich Co., LLC, St. Louis, MO, U.S.A.) was dissolved in 0.1 M PB (0.1 mg/mL) and then diluted 200-fold with modified Hanks’ Balanced Salt Solution (HBSS) of the following composition (in mM): NaCl, 136.9; KCl, 5.37; CaCl2, 1.26; MgCl2, 0.81; Na2HPO4, 0.77; KH2PO4, 0.44; NaHCO3, 4.17; and glucose, 5.55. The diluted solution (50 µL) and 5 mM DTNB (149 µL) were added to each well of a 96-well plate and incubated for 10 min at 30 °C. Subsequently, the tested drugs (2 × 10−6–2 × 10−2 M, 1 µL) were applied to their corresponding wells (final concentrations:10−8–10−4 M) and incubated for 30 min at 30 °C.
After the incubation, 20 mM ATCh (10 µL) was added to each well, and the absorbance at 415 nm was measured every 1 min for 10 min. All measurements were performed in duplicate. The absorbance changes were converted to absorbance changes per minute (ΔA), and the extent of AChE inhibition (%) was calculated according to equation (1):
 | (1) |
where ΔAx is ΔA in the presence of the tested drugs, ΔAcontrol is ΔA in the presence of the vehicle (DMSO, 1 µL), and ΔAblank is ΔA in the absence of rhAChE (HBSS, 50 µL) and presence of DMSO (1 µL).
Concentration–inhibition curves were generated according to equation (2):
 | (2) |
where Y is the extent of AChE inhibition (%), X is the drug concentration (M), nH is the Hill coefficient, and IC50 is the drug concentration that inhibits AChE activity by 50%.
Curve fitting was performed using GraphPad Prism (version 6) (GraphPad Software, San Diego, CA, U.S.A.). Data are expressed as means ± standard error of the mean (S.E.M.), and n refers to the number of replicates.
Figure 1 depicts the inhibitory actions of 31 antidepressants at 10−4 M on rhAChE activity. Twenty-two of the tested antidepressants inhibited rhAChE activity by <20%, whereas nine (clomipramine, amoxapine, setiptiline, nefazodone, paroxetine, sertraline, citalopram, escitalopram, and mirtazapine) inhibited rhAChE activity by ≥20%.
The tested antidepressants are tricyclic antidepressants (imipramine, desipramine, amitriptyline, trimipramine, clomipramine, nortriptyline, amoxapine, dibenzepin, doxepin, dosulepin, tianeptine, and opipramol), tetracyclic antidepressants (maprotiline, mianserin, and setiptiline), serotonin 5-HT2A blockers (trazodone and nefazodone), selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, citalopram, and escitalopram), serotonin and noradrenaline reuptake inhibitors (SNRIs) (milnacipran, levomilnacipran, duloxetine, venlafaxine, and desvenlafaxine), a noradrenaline and dopamine reuptake inhibitor (NDRI) (bupropion), a noradrenergic and specific serotonergic antidepressant (NaSSA) (mirtazapine), and a serotonin reuptake inhibitor and serotonin modulator (S-RIM) (vortioxetine). The extent of AChE inhibition is expressed as a percentage of the control value in the absence of each drug (0%). Data are shown as means ± standard error of the mean (n = 6 each).
Figure 2 depicts the inhibitory effects of 21 hypnotics and 12 anxiolytics at 10−4 M on rhAChE activity. Nineteen of the tested hypnotics and eleven of the tested anxiolytics inhibited rhAChE activity by <20%, whereas two hypnotics (triazolam and brotizolam) and one anxiolytic (buspirone) inhibited rhAChE activity by ≥20%.
The tested hypnotics are barbiturate hypnotics (thiamylal, thiopental, pentobarbital, secobarbital, amobarbital, and phenobarbital), benzodiazepine hypnotics (triazolam, etizolam, brotizolam, lormetazepam, estazolam, flunitrazepam, nitrazepam, and flurazepam), non-benzodiazepine hypnotics (zolpidem and zopiclone), and others (ramelteon, suvorexant, bromovalerylurea, chloral hydrate, and hydroxyzine). The tested anxiolytics are benzodiazepine anxiolytics (tofisopam, clotiazepam, alprazolam, lorazepam, bromazepam, oxazolam, chlordiazepoxide, diazepam, clonazepam, and ethyl loflazepate) and serotonin 5-HT1A partial agonists (tandospirone and buspirone). The extent of AChE activity inhibition is expressed as a percentage of the control value in the absence of each drug (0%). Data are shown as means ± standard error of the mean (n = 6 each).
Figure 3 depicts concentration-inhibition curves for the nine antidepressants (Figs. 3A–I), two hypnotics (Figs. 3J, K), and one anxiolytic (Fig. 3L) with strong inhibition (≥20%) of rhAChE activity at 10−4 M (Figs. 1, 2). Brotizolam (Fig. 3K) showed the strongest inhibition of rhAChE activity compared to that of the other drugs, and this effect was observed at ≥10−6 M. The pIC50 of brotizolam was 4.57 ± 0.02 (n = 6). For the other drugs, the pIC50 values were <4, and they exhibited inhibitory actions on rhAChE at the following concentrations: ≥3 × 10−6 M (sertraline, Fig. 3F; buspirone, Fig. 3L), ≥10−5 M (amoxapine, Fig. 3B; nefazodone, Fig. 3D; paroxetine, Fig. 3E; citalopram, Fig. 3G; escitalopram, Fig. 3H; mirtazapine, Fig. 3I; triazolam, Fig. 3J), and ≥3 × 10−5 M (clomipramine, Fig. 3A; setiptiline, Fig. 3C).
The extent of AChE activity inhibition is expressed as a percentage of the control value in the absence of each drug (0%). Data are shown as means ± standard error of the mean (n = 6 each).
Among the tested drugs (31 antidepressants, 21 hypnotics, and 12 anxiolytics), only nefazodone inhibited rhAChE activity within its CABCR. Therefore, nefazodone may suppress the progression of cognitive symptoms in patients with AD by inhibiting AChE activity. However, almost none of the drugs tested inhibited AChE within their CABCR if used within recommended dose ranges.
First, we investigated the rhAChE inhibitory activities of 31 antidepressants at 10−4 M, a higher concentration than their CABCR10–14) (Supplementary Table 4). We found that nine (clomipramine, amoxapine, setiptiline, nefazodone, paroxetine, sertraline, citalopram, escitalopram, and mirtazapine) inhibited rhAChE activity by ≥20%. Although clomipramine, paroxetine, sertraline, and citalopram (escitalopram) were reported to inhibit AChE,5–7) amoxapine, setiptiline, nefazodone, and mirtazapine were not. To our knowledge, this is the first report demonstrating the AChE inhibitory potency of the latter four antidepressants. We also compared their CAIA (expressed as −logM) with their standard CABCR (Supplementary Table 4), and the results were as follows: clomipramine, CAIA: 4.5, CABCR: 5.85–6.32; amoxapine, CAIA: 4.5, CABCR: 5.80–6.20; setiptiline, CAIA: 5, CABCR: 7.61–9.51; nefazodone, CAIA: 5, CABCR: 5.27–7.19; paroxetine, CAIA: 5, CABCR: 6.52–7.04; sertraline, CAIA: 5.5, CABCR: 6.31–7.49; citalopram, CAIA: 5, CABCR: 6.31–6.60; escitalopram, CAIA: 4.5, CABCR: 6.61–6.91; and mirtazapine, CAIA: 5, CABCR: 6.17–6.83. Therefore, because the tested antidepressants other than nefazodone exhibited AChE inhibitory actions at concentrations much higher than their CABCR, they are unlikely to exhibit AChE inhibitory actions within their CABCR. Regarding nefazodone, its CAIA (−logM: 5) and standard CABCR (−logM: 5.27–7.19) are similar, and we searched its actual CABCR, which was reported to reach 7269 ng/mL (−logM: 4.81).15) Thus, nefazodone could exhibit AChE inhibitory actions within its CABCR. In addition, nefazodone was reported to possess low affinity for muscarinic receptors and showed few adverse events because of anticholinergic actions.16) This report indicates that the AChE inhibitory actions of nefazodone could not be counteracted by its potential anticholinergic actions.
Patients with AD have disorders involving brain cholinergic neurons, which are involved in cognitive and learning functions. For example, cholinergic neuron numbers and ACh synthase activity are decreased in patients’ brains.17) AChE inhibitors used for AD treatment (donepezil, rivastigmine, and galantamine) are considered to suppress the decline in cognitive and learning functions by recovering cholinergic transmission through their AChE inhibitory actions. Nefazodone may act similarly. To our knowledge, there is no information regarding the extent to which nefazodone increases the amount of ACh in the brain under clinical conditions. Nevertheless, nefazodone has been reported to (1) dose-dependently improve psychomotor performance and complex memory performance in healthy subjects,18) and (2) moderately improve highway driving ability in healthy adults and older individuals.19) Based on these reports and our present results, nefazodone is expected to improve cognitive function by increasing ACh levels in the brain through AChE inhibition. However, the AChE inhibitory actions of nefazodone were weaker than those of drugs used for AD treatment (donepezil, rivastigmine, and galantamine), except for memantine (Supplementary Fig. 1), suggesting that nefazodone’s effects on the cognitive symptoms of AD are limited. Therefore, although nefazodone itself may improve AD cognitive symptoms, its therapeutic use against AD should be a combination treatment with other anti-AD drugs possessing AChE inhibitory actions, such as donepezil.
The reason why nefazodone showed AChE inhibitory effects within its CABCR may be due to its chemical structure in addition to its high CABCR. Nefazodone has a chemical structure similar to that of trazodone (Supplementary Fig. 2). However, the inhibitory effect of nefazodone on AChE was more potent than that of trazodone (Fig. 1). Both nefazodone and trazodone have 1,2,4-triazol-3-one rings with different chemical moieties. In trazodone, the benzene ring is directly bonded to the 1,2,4-triazol-3-one ring, which decreases electron density of the 1,2,4-triazol-3-one ring by resonance between these structures. In contrast, in nefazodone, the 1,2,4-triazol-3-one ring is not directly bound to the benzene ring; therefore, the electron density of the 1,2,4-triazol-3-one ring does not decrease. It is likely that the high electron density of the 1,2,4-triazol-3-one ring of nefazodone will bind to the electrophilic region in the active site of AChE,20) and thus, nefazodone shows more potent inhibitory effects against AChE than trazodone. However, nefazodone has been discontinued in many countries, except the United States, because of its rare side effects such as liver injury. Therefore, nefazodone may remain unused clinically. However, the chemical and structural characteristics of nefazodone are expected to be useful for the design of new AChE inhibitors. In fact, the chemical and structural characteristics of AChE inhibitors that are similar to those of nefazodone have been studied in recent years.21,22)
Next, we investigated the rhAChE inhibitory activities of 21 hypnotics and 12 anxiolytics at 10−4 M, which is higher than their CABCR (Supplementary Tables 5, 6).11–13,23–28) We found that two hypnotics (triazolam and brotizolam) and one anxiolytic (buspirone) inhibited rhAChE activity by ≥20%. To our knowledge, triazolam, brotizolam, and buspirone have not been reported to inhibit AChE activity, and this is the first report indicating their AChE inhibitory potency. However, all of them inhibited AChE at much higher concentrations than their standard CABCR (Supplementary Tables 5, 6), as follows: triazolam, CAIA: 5.5, CABCR: 7.23–8.23; brotizolam, CAIA: 6, CABCR: 7.60–7.99; and buspirone, CAIA: 5.5, CABCR: 6.42–6.64. Thus, the tested hypnotics and anxiolytics are unlikely to exhibit AChE inhibitory activity within their CABCR.
Brotizolam showed much stronger inhibitory effects on AChE than the other drugs tested. Brotizolam has a chemical structure in which the ethyl group of etizolam is replaced with a Br (Supplementary Fig. 3). At 10−4 M, the AChE inhibitory rate of brotizolam was 74.7 ± 3.1%, whereas that of etizolam was 14.8 ± 2.4%. Therefore, the Br moiety in brotizolam may play an important role in AChE inhibition. The bonding of a halogen to an aromatic ring reduces the electron density of the aromatic ring owing to the negative inductive effects of the halogen but creates a region with high electron density owing to resonance effects.29) The high electron density region of brotizolam created by Br-induced resonance is more likely to bind to the electrophilic region present in the active site of AChE.20) Therefore, Br bonding to the thienodiazepine ring may lead to relatively strong AChE inhibition.
Finally, our study had a few limitations. (1) For many psychotropic drugs, their blood concentrations correlate with their brain concentrations.30) However, for others, their brain concentrations may be higher than their blood concentrations.31) In other words, even drugs that do not inhibit AChE within their CABCR may exhibit AChE inhibitory activity in the brain. The metabolites of these drugs may also possess AChE inhibitory activities. (2) We previously reported that the AChE inhibitory action of distigmine, a typical AChE inhibitor, reached a steady state within 30 min.32) Thus, the drug treatment time in this study was set at 30 min. The pIC50 values of the tested drugs in this study were consistent with those reported previously.4–8,33–36) This consistency strongly supports the hypothesis that a pretreatment time of 30 min is appropriate for evaluating the AChE inhibitory effects of drugs. Additionally, if these drugs are administered orally or intravenously, their maximum blood concentrations do not persist beyond 30 min. Therefore, a drug treatment duration of 30 min was judged to be clinically reasonable. However, regarding rivastigmine, which is used as a sustained-release preparation for AD treatment in Japan, our results showed that it required ≥3 h to reach a steady state of AChE inhibition (Supplementary Fig. 4). Thus, for drugs with sustained therapeutic blood concentrations, the drug treatment time should be designed considering their pharmacokinetics/pharmacodynamics. Specifically, for sustained-release antidepressants or long-acting anxiolytics/hypnotics, their AChE inhibitory actions may be more potent under clinical conditions.
The authors declare no conflict of interest.
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