QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.097394v1 317/2/606    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Maurice, T. Articles by Monaghan, D. T. Search for Related Content PubMed PubMed Citation Articles by Maurice, T. Articles by Monaghan, D. T.

NEUROPHARMACOLOGY

Interaction with ₁ Protein, but Not N-Methyl-D-aspartate Receptor, Is Involved in the Pharmacological Activity of Donepezil

Unité 710 de l'Institut National de la Santé et de la Recherche Médicale, Ecole Pratique des Hautes Etudes, Université de Montpellier II, Montpellier, France (T.M., J.M.); Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska (B.F., D.T.M.); and Eisai Inc., Teaneck, New Jersey (J.I.)

Received October 19, 2005; accepted January 4, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we examined the interaction of (±)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]-methyl]-1H-inden-1-one hydrochloride (donepezil), a potent cholinesterase inhibitor, with two additional therapeutically relevant targets, N-methyl-D-aspartate (NMDA) and ₁ receptors. Donepezil blocked the responses of recombinant NMDA receptors expressed in Xenopus oocytes. The blockade was voltage-dependent, suggesting a channel blocker mechanism of action, and was not competitive at either the L-glutamate or glycine binding sites. The low potency of donepezil (IC₅₀ = 0.7–3 mM) suggests that NMDA receptor blockade does not contribute to the therapeutic actions of donepezil. Of potential therapeutic relevance, donepezil binds to the ₁ receptor with high affinity (K_i = 14.6 nM) in an in vitro preparation (Neurosci Lett 260:5–8, 1999). Thus, we sought to determine whether an interaction with the ₁ receptor may occur in vivo under physiologically relevant conditions by evaluating the ₁ receptor dependence effects of donepezil in behavioral tasks. Donepezil showed antidepressant-like activity in the mouse-forced swimming test as did the ₁ receptor agonist igmesine. This effect was not displayed by the other cholinesterase inhibitors, rivastigmine and tacrine. The donepezil and igmesine effects were blocked by preadministration of the ₁ receptor antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine (BD1047) and an in vivo antisense probe treatment. The memory-enhancing effect of donepezil was also investigated. All cholinesterase inhibitors attenuated dizocilpine-induced learning impairments. However, only the donepezil and igmesine effects were blocked by BD1047 or the antisense treatment. Therefore, donepezil behaved as an effective ₁ receptor agonist on these behavioral responses, and an interaction of the drug with the ₁ receptor must be considered in its pharmacological actions.

The compound, however, may have other pharmacological properties other than cholinesterase inhibition, and NMDA receptors and ₁ receptors are two potential sites of interaction for donepezil that may contribute to its therapeutic effects. NMDA receptors are a class of receptors for the neurotransmitter glutamate, and their blockade has been shown to be neuroprotective in a variety of neuropathological conditions such as in animal models of stroke (Choi, 1990). In addition, as evidenced by memantine, NMDA receptor blockade can have therapeutic benefits in Alzheimer's disease (Fleischhacker et al., 1986; Rossom et al., 2004). Interestingly, the structure of donepezil bears some general similarities to the NMDA receptor antagonist ifenprodil and to some of the NMDA receptor channel blocker agents. Donepezil inhibits the binding of [³H]MK-801 to NMDA receptor channels with low affinity (IC₅₀ = 135 ± 15 µM; Wang et al., 1999). However, interactions at the channels of individual NMDA receptor subtypes or at other sites on the NMDA receptor complex have not yet been evaluated. Furthermore, the functional significance of donepezil interactions at specific NMDA receptor subtypes has also not yet been defined. Thus, NMDA receptor blockade represents a potential mechanism for the neuroprotective action of donepezil, evidenced in in vitro cell toxicity models (Takada et al., 2003).

The ₁ receptor is a 25-kDa intracellular protein, bearing a transmembrane domain and an endoplasmic reticulum retention signal. Its activation induces both a rapid modulation of ion channels and neurotransmitter responses, including inositol trisphosphate-gated calcium stores, NMDA receptors, and K⁺ channels (Maurice et al., 1999, 2001b; Hayashi and Su, 2005). After activation, the protein also translocates from the vicinity of the endoplasmic reticulum toward plasma and organelles membranes, where it participates in the reconstitution of lipid microdomains (lipid rafts) and remodeling of plasma membrane composition (Hayashi and Su, 2005). Therefore, modulation of the ₁ receptor leads to a variety of neuromodulatory effects that may differ after acute or chronic activation. Although donepezil has previously been shown to have a high affinity for ₁ receptors in radioligand binding experiments, with a K_i = 14.6 nM (Kato et al., 1999), it is not clear whether donepezil can access this intracellular binding site in vivo under physiological conditions.

In the present study, we examined the putative activity of donepezil at both NMDA and ₁ receptors. First, we used two-electrode voltage clamp to examine the effect of donepezil on recombinant NMDA receptors expressed in Xenopus laevis oocytes. Such an approach will allow for testing activity at any of a growing number of sites on the NMDA receptor complex, in a physiologically relevant manner. Second, because it is already known that donepezil binds to ₁ receptor, we examined whether such an interaction occurs in vivo. The effect of donepezil was evaluated in ₁ receptor-mediated behavioral responses (Maurice et al., 1999, 2001b). The antidepressant-like effect of donepezil was investigated using the forced swim test and compared with the effects of the reference ₁ receptor agonist igmesine and other cholinesterase inhibitors, namely, tacrine and rivastigmine. The involvement of the ₁ receptor in the donepezil response was checked in animals pretreated with the ₁ receptor antagonist BD1047 or with an antisense oligodeoxynucleotide targeting the protein (Maurice et al., 2001a). In addition, the antiamnesic effect of donepezil was examined in mice treated with the noncompetitive NMDA receptor antagonist dizocilpine, a learning impairment model known to be alleviated by selective ₁ receptor agonists. Two memory tests were used, the spontaneous alternation in the Y-maze, assessing spatial working memory, and the step-through passive-avoidance response, assessing contextual long-term memory (Maurice et al., 1994).

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
In Vitro Transcription and Translation of NMDA Receptors in Xenopus Oocytes. Plasmids were linearized with NotI (NR1a) or SalI (NR2B) and transcribed with T7 (NR1a) or SP6 (NR2B) in vitro using the mMessage mMachine kit (Ambion, Austin, TX). X. laevis female frogs were obtained from Xenopus I (Ann Arbor, MI), and oocytes were isolated and prepared as described previously (Monaghan and Larsen, 1997). The handling of frogs was performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Oocytes were injected with 2 to 30 ng of NR1/NR2 RNA (1:3) in 9 to 50 nl and then incubated in ND-96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.6) at 17°C for 1 to 4 days.

Oocyte Electrophysiology. Electrophysiological responses of recombinant receptors expressed in Xenopus oocytes were measured by two-electrode voltage clamp (OC-725B oocyte clamp; Warner Instruments, Hamden, CT) at a holding potential of –60 mV. Unless indicated otherwise, NMDA receptor responses were evoked by bath application of 10 µM (S)-glutamate/10 µM glycine. Recordings were made in barium Ringer's solution (116 mM NaCl, 2 mM KCl, 2 mM BaCl₂, and 5 mM HEPES, pH 7.4) to eliminate calcium-activated chloride currents. Only cells that generated stable plateau responses were used (usually in the 50- to 250-nA range). Antagonist responses were determined after three or four agonist-alone pulses established a stable baseline, and agonist alone was applied after antagonist to confirm that there was not a significant response run-down or run-up. Current responses to drug application were recorded on both a strip-chart and by digital capture using an ITC-16 computer interface (InstruTECH Corporation, Great Neck, NY) and a MacIntosh computer with AxoData software (Axon Instruments Inc., Foster City, CA). Dose-response curves for antagonist blockade of responses were fit (GraphPad Prism; ISI Software, San Diego, CA) to the equation I = I_max/[1 + (IC₅₀/A)], where I is the current response, I_max is the current response in the absence of antagonist, and A is the concentration of antagonist.

Animals. Male Swiss mice (breeding center of the Faculty of Pharmacy, Montpellier, France), aged 5 weeks and weighing 35 ± 2 g, were used in this study. Animals were housed in plastic cages in groups. They had free access to food and water, except during behavioral experiments, and they were kept in a regulated environment (23 ± 1°C, 40–60% humidity) under a 12-h light/dark cycle (light on at 8:00 AM). Experiments were carried out between 9:00 AM and 5:00 PM in a soundproof and air-regulated experimental room, and mice were habituated 30 min before each experiment. All animal procedures were conducted in strict adherence of European Community Council Directives of 24 November 1986 (86-609).

Drugs. (±)-2,3-Dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]methyl]-1H-inden-1-one hydrochloride (E2020, donepezil) was provided by Eisai Co. Ltd. (Tokyo, Japan). MK-801 (dizocilpine) and 1,2,3,4-tetrahydro-9-aminoacridine chlorohydrate (tacrine) were purchased from Sigma-Aldrich (St-Quentin Fallavier, France). (S)-N-Ethyl-N-methyl-3-[1-(dimethylamino)ethyl]-phenyl carbamate hydrogen-(2R,3R)-tartrate (rivastigmine, Exelon) was purchased from Novartis (Basel, Switzerland). JO-1784 (igmesine) was a gift from Dr. François J. Roman (Pfizer GRD, Fresnes, France), and BD1047 was a gift from Dr. Wayne D. Bowen (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD). All drugs were solubilized in physiological saline solution or distilled water and administered i.p. in a volume of 100 µl/20 g body weight.

Design and Administration of Oligodeoxynucleotides. Based on the mouse cDNA sequences for the ₁ receptor, 16-mer phosphorothioate-modified oligodeoxynucleotide (ODN) sequences were designed, as described previously (Maurice et al., 2001a). They were targeted to the area from +15 to +1 around the initiation codon, 5'-CGCGGCCCACGGCATT-3' (= antisense oligodeoxynucleotide). This sequence was selected because no homology was found with any of the other known cDNA sequences in the GenBank database (Bainbridge Island, WA). As a control, a mismatched analog, including randomly designed defects, 5'-CACGTCCCTCTCCATT-3', was designed (= mismatch oligodeoxynucleotide). The ODNs were synthesized and purified by high-pressure liquid chromatography by Eurobio Laboratoires (Les Ulis, France). They were dissolved in sterile saline solution and stored at –20°C until use.

Under sodium pentobarbital anesthesia, mice were implanted with a polyethylene cannula (0.75 mm inner diameter; 6 mm in length), fixed using acrylic cement. The tip of the cannula was placed onto the right ventricle, with stereotaxic coordinates from the bregma being A, –0.5 mm; L, –1 mm; and V, 2.5 mm. Injections began 24 h after surgery. Under light ether anesthesia, the needle of a Hamilton microsyringe was inserted through the cannula, and ODNs (1 µl) were slowly injected over 1 min, followed by an additional 1-min wait before removing the needle. Animals received two i.c.v. injections per day at 12-h intervals during 3 days. These animals were used for experiments in vivo binding assays or behavioral observations 10 h after the last injection, i.e., 4 days after cannulation.

Forced Swim Test. Each mouse was placed in a glass cylinder (12 cm in diameter; 24 cm in height) filled with water at a height of 12 cm. Water temperature was maintained at 22 to 23°C. The animal was forced to swim for 15 min on the first day. The animal behavior was not recorded during this first session. On the second day, each mouse was placed again into the water and forced to swim for 6 min. This session was videotaped, and the duration of immobility during the last 5 min was measured. The mouse was considered as immobile when it stopped struggling and moved only to remain floating in the water. Drugs were administered 30 min before the session on the second day.

Spontaneous Alternation Performances. The Y-maze (three arms; 50 cm in length; 60° separation) was placed on the floor of the experimental room and indirectly lit with a 60-W lamp placed 150 cm above the maze. The trial consisted of a single 8-min session. Each mouse was placed at the end of one arm and allowed to move freely through the maze. The series of arm entries, including possible returns into the same arm, was recorded using an Apple IIe computer. An alternation was defined as entries into all three arms on consecutive trials. Therefore, the number of maximal alternations was the total number of arm entries minus 2, and the percentage of alternation was calculated as (actual alternations/maximal alternations) x 100. The compounds were administered 30 min before the session or 10 min before dizocilpine, given 20 min before the session.

Step-Through Passive-Avoidance Test. The apparatus consisted of an illuminated compartment with white polyvinyl chloride walls (15 x 20 x 15 grid floor and a black compartment with a grid floor) separated by a guillotine door. A 60-W lamp was positioned 40 cm above the floor of the white compartment during the experimental period. Scrambled footshocks (0.3 mA) could be delivered to the grid floor using a shock generator scrambler (Lafayette Instruments, Lafayette, MA). The guillotine door was initially closed. Each mouse was placed into the white compartment, and after 5 s, the door was raised. When the mouse entered the darkened compartment, the door was gently closed, and the scrambled footshock was delivered for 3 s. The step-through latency, i.e., the time spent to enter the dark compartment, and the number of vocalizations were recorded. The retention test was carried out 24 h after training. Each mouse was placed again into the white compartment, and after 5 s, the door was raised. The step-through latency was recorded up to 300 s. The compounds were administered 30 min before the training session or 10 min before dizocilpine, given 20 min before the training session. Injections were not repeated before the retention session (preacquisition protocol).

View larger version (9K): [in this window] [in a new window]   Fig. 1. A representative recording of donepezil blockade of NMDA receptor-mediated responses in Xenopus oocytes. NR1/NR2A RNA-injected oocytes were voltage-clamped at –60 mV, and inward currents were evoked by bath application of 10 µM L-glutamate plus 10 µM glycine (indicated by the bars on top). Increasing concentrations of donepezil caused correspondingly greater levels of current inhibition.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Donepezil Low-Affinity Blockade of NMDA Receptor Activity. NMDA receptor NR1a subunits were coexpressed in Xenopus oocytes with each of the NR2 subunits. Using two-electrode voltage clamp, cells were held at –60 mV, and NMDA receptors were activated by bath application of 10 µM L-glutamate and 10 µM glycine. Agonist-evoked currents were then tested for inhibition by donepezil. As shown in Fig. 1, high-micromolar concentrations of donepezil inhibit NMDA receptor responses. Dose-response analysis for each of the NR1a-NR2 subunit combinations showed that donepezil is weakly selective among the different NR2-containing NMDA receptors (Fig. 2A). Donepezil IC₅₀ values (95% confidence interval) were as follows: 0.68 (0.54–0.85) mM for the NR1a/NR2A combination, 1.2 (0.95–1.59) mM for NR1a/NR2B, 3.16 (2.9–3.4) mM for NR1a/NR2C, and 1.6 (1.3–2.0) mM for NR1a/NR2D (Fig. 2A).

View larger version (22K): [in this window] [in a new window]   Fig. 2. A, donepezil inhibits each of the NR2-containing NMDA receptors. NR1/NR2A (A), NR1/NR2B (B), NR1/NR2C (C), and NR1/NR2D (D) receptors were activated by 10 µM L-glutamate plus 10 µM glycine and tested for inhibition by increasing concentrations of donepezil. A 100% control response represents the current response to agonist alone. B, comparison of ifenprodil and donepezil potency at NR1/NR2B NMDA receptors. C, donepezil was tested as an antagonist of 10 µM L-glutamate/10 µM glycine (10 µM Glu/Gly) and of 300 µM L-glutamate/300 µM glycine (300 µM Glu/Gly). D, NMDA receptor responses blockade by 1 mM donepezil at various membrane holding potentials. Donepezil blockade was greater at larger negative holding potentials. For all experiments, only cells with stable plateau responses were used.

We next sought to determine whether donepezil is a competitive antagonist with either L-glutamate or glycine at the respective L-glutamate or glycine binding sites on the NMDA receptor complex. Donepezil potency was determined for inhibition against moderate agonist concentrations (10 µM L-glutamate plus 10 µM glycine) and against high-agonist concentrations (300 µM L-glutamate plus 300 µM glycine). If donepezil blocks NMDA receptor responses by binding at either the L-glutamate or glycine binding sites, then we would expect a significant reduction in donepezil potency when tested against high-agonist concentrations. Donepezil potency was not reduced by high-L-glutamate and glycine concentrations (Fig. 2C).

View larger version (58K): [in this window] [in a new window]   Fig. 3. Antidepressant-like profile of donepezil in comparison with other cholinesterase inhibitors and 1 receptor ligands. Mice were submitted to a 15-min duration forced swimming on day 1 and to a 6-min duration swimming on day 2. Donepezil (1–30 mg/kg), rivastigmine (1–30 mg/kg), tacrine (1–30 mg/kg), igmesine (2–40 mg/kg), and/or BD1047 (1–10 mg/kg) was administered i.p. 30 min before the second session. Immobility duration was determined during the last 5 min of the second session. A, dose-response effect of donepezil and blockade by BD1047 (KW = 47.70; p < 0.0001). B, lack of effect of rivastigmine or tacrine (KW = 3.04; p > 0.05). C, dose-response effect of igmesine and blockade by BD1047 (KW = 27.62; p < 0.0001). D, effect of donepezil (30 mg/kg) in mice centrally administered with the 1 receptor anti-sense ODN probe (KW = 11.03; p < 0.05). The number of animals is indicated within the columns. *, p < 0.05; **, p < 0.01 versus the vehicle (V)-treated group; ##, p < 0.01 versus the donepezil (30 mg/kg)- or igmesine (40 mg/kg)-treated group; Dunn's test.

Involvement of the ₁ Receptor in the Antidepressant-Like Effect of Donepezil. Swiss mice submitted to forced swimming rapidly developed a marked immobility response in the 220- to 240-s range during the last 5 min of the 6-min duration session (Fig. 3). Donepezil (3–30 mg/kg) significantly decreased the immobility duration (Fig. 3A). This effect was fully blocked in a dose-dependent manner by simultaneous administration of BD1047, which by itself had no effect at the highest dose tested (10 mg/kg) (Fig. 3A). Rivastigmine and tacrine, tested in the 1- to 30-mg/kg range, failed to affect the immobility duration in the same experimental conditions (Fig. 3B). Igmesine, the reference ₁ receptor agonist, dose-dependently decreased the immobility duration, and its effect at the highest dose tested was antagonized by BD1047 (Fig. 3C). Moreover, animals treated repeatedly with the ₁ antisense ODN donepezil, at 30 mg/kg, failed to decrease the immobility duration, contrarily to mice treated with the mismatch ODN (Fig. 3D).

View larger version (58K): [in this window] [in a new window]   Fig. 4. Antiamnesic effect of donepezil against dizocilpine-induced learning impairments and blockade by BD1047. Donepezil (0.12–1 mg/kg), BD1047 (0.5–1 mg/kg), or both were administered i.p. 30 min before the Y-maze session or passive-avoidance training. Mice were submitted to the spontaneous alternation test in the Y-maze (A and C) or to the step-through passive-avoidance training, the retention session being performed after 24 h (B and D). Dose-response effect of donepezil in control and dizocilpine (0.15 mg/kg)-treated animals in the Y-maze alternation test (KW = 50.67; p < 0.0001) (A) and in the passive-avoidance test (KW = 49.92; p < 0.0001) (B). Blockade by BD1047 of the donepezil effect in the Y-maze alternation test (KW = 59.58; p < 0.0001) (C) and in the passive-avoidance test (KW = 54.67, p < 0.0001) (D). The number of animals is indicated within the columns in A and C. **, p < 0.01 versus the vehicle (V)-treated group; ##, p < 0.01 versus the dizocilpine (0.15 mg/kg)-treated group; , p < 0.05 and , p < 0.01 versus the donepezil (0.5 mg/kg) + dizocilpine (0.15 mg/kg)-treated group; Dunn's test.

View larger version (41K): [in this window] [in a new window]   Fig. 5. Antiamnesic effect of the selective 1 receptor agonists igmesine against the dizocilpine-induced learning impairments and blockade by BD1047. Igmesine (0.1–3 mg/kg), BD1047 (0.5–1 mg/kg), or both were administered i.p. 30 min before the Y-maze session or passive-avoidance training. Mice were submitted to the spontaneous alternation test in the Y-maze (A and C) or to the step-through passive-avoidance training, the retention session being performed after 24 h (B and D). Dose-response effect of igmesine in control and dizocilpine (0.15 mg/kg)-treated animals in the Y-maze alternation test (KW = 49.65; p < 0.0001) (A) and in the passive-avoidance test (KW = 35.28; p < 0.0001) (B). Blockade by BD1047 of the igmesine effect in the Y-maze alternation test (KW = 51.87; p < 0.0001) (C) and in the passive-avoidance test (KW = 41.09; p < 0.0001) (D). The number of animals is indicated within the columns in A and C. **, p < 0.01 versus the vehicle (V)-treated group; ##, p < 0.01 versus the dizocilpine (0.15 mg/kg)-treated group; , p < 0.05 and , p < 0.01 versus the igmesine (1 mg/kg) + dizocilpine (0.15 mg/kg)-treated group; Dunn's test.

The effects of the ODN treatments on the antiamnesic effect of donepezil at 0.5 mg/kg were then examined. Dizocilpine treatment led to significant diminution in the spontaneous alternation behavior in animals treated centrally with mismatch ODN (Fig. 6A). Donepezil did not affect the alternation behavior by itself, but it allowed a significant attenuation of the dizocilpine-induced deficits in mismatch ODN-treated animals (Fig. 6A). In antisense ODN-treated animals, dizocilpine induced deficits of similar extent, but donepezil failed to attenuate the dizocilpine-induced deficit (Fig. 6A). Long-term memory capacity was assessed using passive-avoidance response. In mismatch ODN-treated animals, donepezil did not affect the latencies by itself, but it attenuated the dizocilpine-induced deficits (Fig. 6B). In antisense ODN-treated animals, the dizocilpine-induced decrease in latency was unchanged but remained unaffected by donepezil. It must be noted that neither the cannulation nor the ODN administration affected the ability of animals to perform the test. This was checked during the training session by measuring the step-through latency or shock sensitivity (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 6. Effect of the 1 antisense ODN treatment on the antiamnesic effect of donepezil against the dizocilpine-induced learning impairments: spontaneous alternation in the Y-maze (A) and passive-avoidance retention (B). Donepezil (0.5 mg/kg) was administered 10 min before dizocilpine (0.15 mg/kg), which was given 20 min before the Y-maze session or passive-avoidance training. KW = 36.03, p < 0.0001 in A and KW = 32.15, p < 0.0001 in B. The number of animals is indicated within the columns in A. **, p < 0.01 versus the vehicle (V)-treated group; ##, p < 0.01 versus the dizocilpine (0.15 mg/kg)-treated group; Dunn's test.

View larger version (56K): [in this window] [in a new window]   Fig. 7. Antiamnesic effect of rivastigmine (A and B) or tacrine (C and D) against the dizocilpine-induced learning impairments. Rivastigmine (0.3–1 mg/kg), tacrine (0.3–1 mg/kg), BD1047 (0.5–1 mg/kg), or a combination were administered i.p. 30 min before the Y-maze session or passive-avoidance training. Mice were submitted to the spontaneous alternation test in the Y-maze (A and C) or to the step-through passive-avoidance training, the retention session being performed after 24 h (B and D). Dose-response effect of rivastigmine in the Y-maze alternation test (KW = 35.92; p < 0.0001) (A) and in the passive-avoidance test (KW = 31.53; p < 0.0001) (B). Dose-response effect of tacrine in control and dizocilpine (0.15 mg/kg)-treated animals in the Y-maze alternation test (KW = 43.32; p < 0.0001) (C) and in the passive-avoidance test (KW = 32.68; p < 0.0001) (D). The number of animals is indicated within the columns in A and C. **, p < 0.01 versus the vehicle (V)-treated group; ##, p < 0.01 versus the dizocilpine (0.15 mg/kg)-treated group; Dunn's test.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Donepezil displays some general structural similarities to NMDA receptor channel blocker drugs, such as phencycline or CNS-1102. It has a positive charge center surrounded by hydrophobic ring groups, a piperidine and an aromatic ring. Consistent with the actions of a channel blocker, donepezil blockade was voltage-dependent. Thus, the higher negative membrane potentials favor the entering and binding of donepezil in the channel.

Recently, memantine, an NMDA receptor channel blocker drug was approved for use in severe Alzheimer's disease patients. Thus, NMDA receptor channel blockers can have therapeutic benefits in Alzheimer's disease and also present the possibility of being neuroprotective. It is unlikely, however, that a significant component the clinical actions of donepezil are due to NMDA receptor blockade. Therapeutic levels of donepezil are in the low-micromolar range, whereas NMDA receptor blocker activity requires millimolar concentrations. Donepezil, however, may have therapeutically relevant actions on NMDA receptors via indirect mechanisms. Very recently, it has been reported that low concentrations of donepezil (10–1000 nM) have a stimulatory action on NMDA receptor function in neurons (Moriguchi et al., 2005) by an indirect pertussis toxin-sensitive effect. The absence of direct effects of donepezil on NMDA receptor function in the present study is consistent with such an indirect action of donepezil.

The second key point of this study is the observation that the antidepressant-like and antiamnesic effects of donepezil involve an interaction with the ₁ receptor. The antidepressant-like activity was measured using the forced swim test. Donepezil is able to shorten the immobility duration of animals dose-dependently but at relatively high doses. This effect was shared by the reference ₁ agonist igmesine, as described previously (Matsuno et al., 1996; Urani et al., 2001), but not by other cholinesterase inhibitors, such as tacrine or rivastigmine. The donepezil effect could be blocked by a selective ₁ receptor antagonist, BD1047, and by a subchronic pretreatment with an antisense ODN probe, which has been shown to down-regulate ₁ receptor expression in vivo (Maurice et al., 2001a). These observations clearly established that donepezil interacts with the ₁ receptor to exert its anti-immobility response during forced swimming. Noteworthy, the dose range for antidepressant-like activity is markedly higher than the doses effective in learning and memory tests. This was previously observed for numerous selective ₁ receptor agonists (Matsuno et al., 1996; Urani et al., 2001). The requirement of high concentrations of ₁ receptor agonists to elicit antidepressant actions seems to be due to the competing effects of stress-induced release of progesterone, a ₁ receptor antagonist (Urani et al., 2001). Thus, this behavioral effect of donepezil may not be therapeutically relevant since the dose range is close to donepezil LD₅₀ in mice, approximately 45 mg/kg.

Donepezil was also tested at a second behavioral response known to be mediated by selective ₁ receptor agonists, the blockade of learning impairment caused by acute administration of dizocilpine, a highly potent NMDA receptor antagonist (Maurice et al., 1994, 2001a; Ohno and Watanabe, 1995; Zou et al., 1998). Two behavioral tests were used in parallel: a short-term memory procedure involving a spatial working memory component, the spontaneous alternation in the Y-maze, and a long-term memory test involving negatively reinforced contextual memory processes, the step-through-type passive-avoidance procedure. Results obtained with both tests were similar. Donepezil, administered in the 0.12- to 1-mg/kg dose range, failed to affect the learning and memory processes when administered alone but significantly blocked the dizocilpine-induced deficits at the highest doses tested, 0.5 and 1 mg/kg. The donepezil effects were blocked by pretreatment with BD1047 or repeated injections of the antisense ODN probe. These observations demonstrated that the antiamnesic activity of donepezil involves an interaction with the ₁ receptor. Igmesine showed a very similar BD1047-sensitive profile of efficacy. Moreover, the reversal of the dizocilpine-induced deficits by rivastigmine and tacrine were not blocked by BD1047. The highest donepezil dose that was tested (1 mg/kg) is a dose that should lead to less than 1 µM in the brain (Geerts et al., 2005), a dose that is well below that necessary to have direct NMDA receptor actions.

The antiamnesic effects of cholinesterase inhibitors against dizocilpine-induced learning deficits in mice have been described previously (Walker and Gold, 1992; Csernansky et al., 2005). In the latter study, physostigmine and donepezil, but not galanthamine, ameliorated the dizocilpine-induced deficits in spatial reversal learning and in contextual and cued memory. Consistent with the wide-spread distribution of cholinergic neurons and the variety of cholinergic receptor subtypes in multiple brain regions related to learning (Levin et al., 2003; Tisch et al., 2004), cholinergic drugs exert a complex effect on learning and memory processes. Donepezil, rivastigmine, tacrine, physostigmine, and galanthamine act both as cholinesterase inhibitors, thus provoking nonselective activations at all types of nicotinic and muscarinic receptors and directly as allosteric modulators of nicotinic receptors with varied efficacy. Galanthamine, particularly, is a weak cholinesterase inhibitor (Thomsen et al., 1991). Therefore, these drugs show marked pharmacological differences in terms of cholinergic activity. Considering that donepezil and physostigmine, but not galanthamine, attenuated the dizocilpine-induced deficits in their study, Csernansky et al. (2005) proposed that such antiamnesic effect may be the result of acetylcholinesterase inhibition and increased synaptic levels of acetylcholine, rather than allosteric nicotinic receptor modulation. We observed that tacrine and rivastigmine were also effective against the dizocilpine-induced learning impairments, in agreement with Csernansky's hypothesis. Moreover, the observation that direct application of nicotine alleviated the dizocilpine-induced learning deficits (Ciamei et al., 2001) is in line with such hypothesis.

The ₁ receptor is also a target for the pharmacological action of donepezil at therapeutically relevant doses. Interestingly, this ₁ receptor-mediated effect of donepezil may in turn affect the NMDA receptor, since the modulation exerted by ₁ receptors on NMDA receptor activation is now well documented. The ₁ receptor agonists are able to modulate different NMDA-mediated responses, such as enhancing the NMDA-induced excitatory response of CA3 pyramidal neurons in the rat hippocampal formation in vivo (Monnet et al., 1990; Debonnel and de Montigny, 1996). Thus, donepezil through its interaction with the ₁ receptor may exert a facilitation of NMDA responses, contrary to our initial hypothesis. Furthermore, the interaction with the ₁ receptor may also act to indirectly affect cholinergic systems. Indeed, activation of the ₁ receptor has been shown to exert a direct modulation of cholinergic systems. Selective ₁ receptor agonists modulate acetylcholine release in striatal and hippocampal slice preparations in vitro (Leventer and Johnson, 1984; Junien et al., 1991) and increase extracellular acetylcholine levels in the rat frontal cortex and hippocampus in vivo (Kobayashi et al., 1996a,b). Interestingly, striatal acetylcholine content is not affected, suggesting that selective ₁ agonists may present lower cholinomimetic side effects than that observed for cholinesterase inhibitors (Matsuno et al., 1997).

Finally, the observation that the antiamnesic effect of donepezil is fully blocked by antagonism of the ₁ receptor activation is consistent with the intracellular neuromodulatory role of the protein. The ₁ receptor is found both pre- and postsynaptically in the hippocampus. Its activation modulates Ca²⁺ mobilizations and therefore facilitates acetylcholine release and cholinergic signaling pathways (Junien et al., 1991; Kobayashi et al., 1996a,b). However, the ₁ receptor is a pure neuromodulatory receptor, and its activation is not a prerequisite for effective learning ability, as shown in antisense studies (Maurice et al., 2001a). Therefore, selective ₁ receptor agonists and cholinomimetic drugs share different modes of action. Drugs acting nonselectively through neurotransmitter receptors and ₁ receptor activation present a complex pharmacological mechanism of action, with their ₁ component sustaining and constraining the primary effect on neurotransmitter systems. It was previously observed that the antiamnesic effect induced by the neurosteroid dehydroepiandrosterone sulfate (a negative modulator of GABA_A receptors, a positive modulator of NMDA receptors, and a ₁ receptor agonist) is blocked in mice treated with a ₁ receptor antisense probe (Maurice et al., 2001a). In a coherent manner, donepezil effect involves both indirect cholinergic activity and ₁ receptor activation. Its effect is blocked by ₁ receptor antagonism, in contrast to the more selective cholinesterase inhibitors that are devoid of ₁ activity.

In conclusion, donepezil is a weak NMDA receptor blocker, with this direct activity unlikely to contribute to its therapeutic actions. However, an in vivo interaction of the drug with the ₁ receptor has now been demonstrated and must be considered in its pharmacological actions. The symptomatic and potential neuroprotective effects of donepezil in Alzheimer's disease (Francis et al., 2005) may therefore result from a both direct and indirect cholinergic mechanisms and an interaction with the ₁ receptor, known to provide neuroprotection against glutamate and amyloid toxicities.

	Footnotes

 
This work was supported by Eisai Inc. (Teaneck, NJ).

doi:10.1124/jpet.105.097394.

ABBREVIATIONS: NMDA, N-methyl-D-aspartate; MK-801, 5H-dibenzo[a,d]cyclohepten-5,10-imine; BD1047, N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine; JO-1784, (+)-N-cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride; ODN, oligodeoxynucleotide; KW, Kruskal-Wallis; CNS-1102, 1-(m-ethylphenyl)-1-methyl-3-(1-naphthyl)guanidine hydrochloride.

Address correspondence to: Dr. Tangui Maurice, INSERM U.710, EPHE, University of Montpellier II, c.c. 105, place Eugène Bataillon, 34095 Montpellier cedex 5, France. E-mail: maurice{at}univ-montp2.fr

	References

Top Abstract Materials and Methods Results Discussion References

 

Choi D (1990) The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu Rev Neurosci 13: 171–182.[CrossRef][Medline]

Ciamei A, Aversano M, Cestari V, and Castellano C (2001) Effects of MK-801 and nicotine combinations on memory consolidation in CD1 mice. Psychopharmacology 154: 126–130.[CrossRef][Medline]

Csernansky JG, Martin M, Shah R, Bertchume A, Colvin J, and Dong H (2005) Cholinesterase inhibitors ameliorate behavioral deficits induced by MK-801 in mice. Neuropsychopharmacology 30: 2135–2143.[CrossRef][Medline]

Debonnel G and de Montigny C (1996) Modulation of NMDA and dopaminergic neurotransmissions by sigma ligands: possible implications for the treatment of psychiatric disorders. Life Sci 58: 721–734.[CrossRef][Medline]

Fleischhacker WW, Buchgeher A, and Schubert H (1986) Memantine in the treatment of senile dementia of the Alzheimer type. Prog Neuropsychopharmacol Biol Psychiatry 10: 87–93.[CrossRef][Medline]

Francis PT, Nordberg A, and Arnold SE (2005) A preclinical view of cholinesterase inhibitors in neuroprotection: do they provide more than symptomatic benefits in Alzheimer's disease? Trends Pharmacol Sci 26: 104–111.[CrossRef][Medline]

Geerts H, Guillaumat P-O, Grantham C, Bode W, Anciaux K, and Sachak S (2005) Brain levels and acetylcholinesterase inhibition with galantamine and donepezil in rats, mice and rabbits. Brain Res 1033: 186–193.[CrossRef][Medline]

Hayashi T and Su TP (2005) The potential role of sigma-1 receptors in lipid transport and lipid raft reconstitution in the brain: implication for drug abuse. Life Sci 77: 1612–1624.[CrossRef][Medline]

Jane D, Tse HW, Skifter DA, Christie JM, and Monaghan DT (2000) Glutamate receptor ion channels: activators and inhibitors, in Handbook of Experimental Pharmacology (Endo M, Kurachi Y, and Kurachi M eds) pp 413–476, Springer-Verlag, Heidelberg.

Junien JL, Roman FJ, Brunelle G, and Pascaud X (1991) JO1784, a novel ligand, potentiates [³H]acetylcholine release from rat hippocampal slices. Eur J Pharmacol 200: 343–345.[CrossRef][Medline]

Kato K, Hayako H, Ishihara Y, Marui S, Iwane M, and Miyamoto M (1999) TAK-147, an acetylcholinesterase inhibitor, increases choline acetyltransferase activity in cultured rat septal cholinergic neurons. Neurosci Lett 260: 5–8.[CrossRef][Medline]

Kobayashi T, Matsuno K, and Mita S (1996a) Regional differences of the effect of sigma receptor ligands on the acetylcholine release in the rat brain. J Neural Transm 103: 661–669.[CrossRef][Medline]

Kobayashi T, Matsuno K, Nakata K, and Mita S (1996b) Enhancement of acetylcholine release by SA4503, a novel ₁ receptor agonist, in the rat brain. J Pharmacol Exp Ther 279: 106–113.[Abstract/Free Full Text]

Kosasa T, Kuriya Y, Matsui K, and Yamanishi Y (1999) Effect of donepezil hydrochloride (E2020) on basal concentration of extracellular acetylcholine in the hippocampus of rats. Eur J Pharmacol 380: 101–107.[CrossRef][Medline]

Leventer SM and Johnson KM (1984) Phencyclidine-induced inhibition of striatal acetylcholine release: comparisons with µ, and opiate agonists. Life Sci 34: 793–801.[CrossRef][Medline]

Levin ED, Sledge D, Baruah A, and Addy NA (2003) Ventral hippocampal NMDA blockade and nicotinic effects on memory function. Brain Res Bull 61: 489–495.[CrossRef][Medline]

Matsuno K, Kobayashi T, Tanaka MK, and Mita S (1996) ₁ Receptor subtype is involved in the relief of behavioural despair in the mouse forced swimming test. Eur J Pharmacol 312: 267–271.[CrossRef][Medline]

Matsuno K, Senda T, Kobayashi T, Okamoto K, Nakata K, and Mita S (1997) SA4503, a novel cognitive enhancer, with ₁ receptor agonistic properties. Behav Brain Res 83: 221–224.[CrossRef][Medline]

Maurice T, Hiramatsu M, Itoh J, Kameyama T, Hasegawa T, and Nabeshima T (1994) Behavioral evidence for a modulating role of ligands in memory processes. I. Attenuation of dizocilpine (MK-801)-induced amnesia. Brain Res 647: 44–56.[CrossRef][Medline]

Maurice T, Phan VL, Urani A, and Guillemain I (2001a) Differential involvement of the sigma₁ (₁) receptor in the anti-amnesic effect of neuroactive steroids, as demonstrated using an in vivo antisense oligodeoxynucleotide strategy in the mouse. Br J Pharmacol 134: 1731–1741.[CrossRef][Medline]

Maurice T, Phan VL, Urani A, Kamei H, Noda Y, and Nabeshima T (1999) Neuroactive neurosteroids as endogenous effector for the sigma₁ (₁) receptor: pharmacological evidences and therapeutic opportunities. Jpn J Pharmacol 81: 125–155.[CrossRef][Medline]

Maurice T, Urani A, Phan VL, and Romieu P (2001b) The interaction between neuroactive steroids and the sigma₁ (₁) receptor function: behavioral consequences and therapeutic opportunities. Brain Res Rev 37: 116–132.[CrossRef][Medline]

Mohs RC, Doody RS, Morris JC, Ieni JR, Rogers SL, Perdomo CA, Pratt RD, and "312" Study Group (2001) A 1-year, placebo-controlled preservation of function survival study of donepezil in AD patients. Neurology 57: 481–488.[Abstract/Free Full Text]

Monaghan DT and Larsen H (1997) NR1 and NR2 subunit contributions to NMDA receptor channel blocker pharmacology. J Pharmacol Exp Ther 280: 614–620.[Abstract/Free Full Text]

Monnet FP, Debonnel G, Junien JL, and De Montigny C (1990) N-Methyl-D-aspartate-induced neuronal activation is selectively modulated by receptors. Eur J Pharmacol 179: 441–445.[CrossRef][Medline]

Moriguchi S, Zhao X, Marszalec W, Yeh JZ, and Narahashi T (2005) Modulation of N-methyl-D-aspartate receptors by donepezil in rat cortical neurons. J Pharmacol Exp Ther 315: 125–135.[Abstract/Free Full Text]

Ohno M and Watanabe S (1995) Intrahippocampal administration of (+)-SKF-10,047, a sigma ligand, reverses MK-801-induced impairment of working memory in rats. Brain Res 684: 237–242.[CrossRef][Medline]

Rogers SL, Doody RS, Mohs RC, and Friedhoff LT (1998) Donepezil improves cognition and global function in Alzheimer disease: a 15-week, double-blind, placebo-controlled study. Donepezil Study Group. Arch Intern Med 158: 1021–1031.[Abstract/Free Full Text]

Rogers SL, Doody RS, Pratt RD, and Ieni JR (2000) Long-term efficacy and safety of donepezil in the treatment of Alzheimer's disease: final analysis of a US multicentre open-label study. Eur Neuropsychopharmacol 10: 195–203.[Medline]

Rogers SL and Friedhoff LT (1996) The efficacy and safety of donepezil in patients with Alzheimer's disease: results of a US Multicentre, Randomized, Double-Blind, Placebo-Controlled Trial. The Donepezil Study Group. Dementia 7: 293–303.[Medline]

Rossom R, Adityanjee and Dysken M (2004) Efficacy and tolerability of memantine in the treatment of dementia. Am J Geriatr Pharmacother 2: 303–312.[CrossRef][Medline]

Takada Y, Yonezawa A, Kume T, Katsuki H, Kaneko S, Sugimoto H, and Akaike A (2003) Nicotinic acetylcholine receptor-mediated neuroprotection by donepezil against glutamate neurotoxicity in rat cortical neurons. J Pharmacol Exp Ther 306: 772–777.[Abstract/Free Full Text]

Thomsen T, Kaden B, Fischer JP, Bickel U, Barz H, Gusztony G, Cervos-Navarro J, and Kewitz H (1991) Inhibition of acetylcholinesterase activity in human brain tissue and erythrocytes by galanthamine, physostigmine and tacrine. Eur J Clin Chem Clin Biochem 29: 487–492.[Medline]

Tisch S, Silberstein P, Limousin-Dowsey P, and Jahanshahi M (2004) The basal ganglia: anatomy, physiology and pharmacology. Psychiatr Clin North Am 27: 757–799.[CrossRef][Medline]

Tune L, Tiseo PJ, Ieni J, Perdomo C, Pratt RD, Votaw JR, Jewart RD, and Hoffman JM (2003) Donepezil HCl (E2020) maintains functional brain activity in patients with Alzheimer's disease. Am J Geriatr Psychiatry 11: 169–177.[Abstract/Free Full Text]

Urani A, Roman FJ, Phan VL, Su TP, and Maurice T (2001) The antidepressant-like effect induced by sigma₁ (₁) receptor agonists and neuroactive steroids in mice submitted to the forced swimming test. J Pharmacol Exp Ther 298: 1269–1279.[Abstract/Free Full Text]

Walker DL and Gold PE (1992) Impairment of spontaneous alternation performance by an NMDA antagonist: attenuation with non-NMDA treatments. Behav Neural Biol 58: 69–71.[CrossRef][Medline]

Wang XD, Chen XO, Yang HH, and Hu GY (1999) Comparison of the effects of cholinesterase inhibitors on [³H]MK-801 binding in rat cerebral cortex. Neurosci Lett 272: 21–24.[CrossRef][Medline]

Winblad B, Engedal K, Soininen H, Verhey F, Waldemar G, Wimo A, Wetterholm AL, Zhang R, Haglund A, Subbiah P, et al. (2001) A 1-year, randomized, placebo-controlled study of donepezil in patients with mild to moderate AD. Neurology 57: 489–495.[Abstract/Free Full Text]

Zou LB, Yamada K, and Nabeshima T (1998) Sigma receptor ligands (+)-SKF10,047 and SA4503 improve dizocilpine-induced spatial memory deficits in rats. Eur J Pharmacol 355: 1–10.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.105.097394v1 317/2/606    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Maurice, T. Articles by Monaghan, D. T. Search for Related Content PubMed PubMed Citation Articles by Maurice, T. Articles by Monaghan, D. T.