Introduction

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Alzheimer's disease (AD) is a complex and multifaceted neurodegenerative disease affecting aged populations. The pathogenesis and the etiology remain unknown, although a "cholinergic deficit hypothesis" has been suggested (Perry, 1986). In fact, among the multiple transmitter deficits that have been described in AD, one of the most specific and consistent features is an early and severe degeneration of forebrain cholinergic system, as revealed by the correlation observed between the cholinergic pathology and dementia (Geula and Mesulam, 1994). Therefore, the enhancement of brain cholinergic transmission in AD remains a major goal for many putative therapeutic agents that are in use or under development.

Acetylcholinesterase (AChE) has long been an attractive target for the rational design of mechanism-based inhibitors because of the pivotal role it plays in the central nervous system. Currently, only the AChE inhibition approach, which enhances the function of central cholinergic neurons by permitting acetylcholine (ACh) to remain in the synaptic cleft longer through reducing ACh hydrolysis, seems to produce encouraging symptomatic improvements in clinical trials. In fact, the resulting increase in extracellular ACh concentrations might reverse central cholinergic hypofunction and improve cognitive functions in AD (Kelly, 1999).

To date, most of the drugs used therapeutically have proved to ameliorate AD symptomatically, but it is controversial whether there is an effect on the disease progression (Giacobini, 1998).

The clinical usefulness of AChE inhibitors (AChEIs) has been limited by either an extremely short or an excessive long half-life, hepatotoxicity, and severe peripheral cholinergic side effects (Giacobini, 1998; Kelly, 1999). To obtain greater therapeutic benefit, newer AChEIs that circumvent these problems are needed.

This study describes the biochemical and neurobehavioral profile of CHF2819 (Fig. 1), a novel geneserine derivative with AChE inhibitory activity (Pietra et al., 1999). The characterization of CHF2819 started with an in vivo (microdialysis technique) investigation of the effects of this compound, administered by the oral route or by local perfusion via the dialysis probe, on the extracellular concentrations of ACh in young adult rat hippocampus, the main target region, together with the cerebral cortex, for symptomatic treatment of AD. This was followed by an investigation of behavioral correlates of central cholinergic function in young adult rats (scopolamine-induced amnesia in a passive avoidance task).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Chemical structure of CHF2819, a novel AChEI.

A functional observational battery (FOB) of tests was used to assess functional domains (sensory, motor, and autonomic) in young adult rats to investigate potential neurotoxic effects of CHF2819. Moreover, because a recovery of extracellular ACh levels in aged rats has been obtained after the administration of AChEIs as well as drugs acting on brain cholinergic neurons with different mechanisms (Quirion et al., 1995; Scali et al., 1997a; Vannucchi et al., 1997), the effects of CHF2819 on extracellular ACh levels in the hippocampus of aged rats were also measured in this study.

However, noncholinergic neurochemical abnormalities that may contribute to the behavioral and cognitive disorders associated with AD have been identified (Zubenko et al., 1990; Camacho et al., 1996). Furthermore, experimental and clinical data have shown interactions between central cholinergic and catecholaminergic, serotonergic, and -aminobutyric acid (GABA)ergic systems (Bianchi et al., 1982; Memo et al., 1988; Robinson et al., 1989; Decker and McGaugh, 1991). Therefore, it is likely that the efficacy of AChEIs in the treatment of demented patients could be due not only to cholinesterase inhibition but also to other neurochemical effects. In this study, we therefore also investigated, in addition to ACh, the effects of CHF2819 on extracellular concentrations of dopamine (DA), 5-hydroxytryptamine (5-HT), norepinephrine (NE), and GABA in the rat hippocampus.

	Materials and Methods

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Animals. Young adult (2-3 months old) and aged (20-22 months old) male Wistar rats (Harlan, S. Pietro al Natisone, Udine, Italy) were used. They were housed at constant room temperature (22 ± 1°C) and relative humidity (55 ± 5%) under a 12-h light/dark cycle (lights on from 8:00 AM to 8:00 PM). Food and water were freely available.

Chemicals. CHF2819 (Fig. 1) was provided by Chiesi Farmaceutici S.p.A. (Parma, Italy). The drug was dissolved in saline and administered p.o. in a volume of 2 ml/kg. All doses refer to the salt form (HCl). All other chemicals were obtained from commercial sources.

Dialysis Procedure. As previously described (Cagiano et al., 1998), rats were anesthetized with 3 ml/kg i.p. of a solution containing 1.2 g of pentobarbital, 5.3 g of chloral hydrate, 2.7 g of MgSO₄, 49.5 ml of propylene glycol, 12.5 ml of ethanol, and 58 ml of distilled water.

HPLC Analysis. ACh concentrations were determined by HPLC using a microbore column and enzyme reactor coupled with electrochemical detection in reduction mode and a glassy carbon electrode (6 mm) coated with peroxidase with +0.0 V applied (wired electrode system; Bioanalytical Systems, West Lafayette, IN; Sepstik 530- mm × 1-mm analytical column and ACh/Ch IMER). The mobile phase used was 80 mM sodium phosphate with 5 ml/l Kathon (Bioanalytical Systems) at pH 8.5. The flow rate used was 125 µl/min, and detection sensitivity for ACh was 0.1 nM (10 µl injected).

Passive Avoidance Behavior. A stepdown-type apparatus was used. It consisted of a compartment (25 × 24 × 24 cm) constructed of black Plexiglas and equipped with a grid floor to which an elevated compartment (13 × 24 × 16 cm) with solid Plexiglas floor was attached. The opening between the elevated compartment and the large compartment was separated by a guillotine door (9 × 10 cm). A 25-W lamp illuminated the elevated compartment, whereas the large compartment remained dark. Scrambled foot shocks were delivered from a Letica shock generator (LI 2750 U; Barcelona, Spain). The experiments were performed in a sound-attenuating chamber (Amplifon G-type cabin) that was dark except for the illumination of the elevated compartment of the apparatus. Each animal was removed from the home cage and placed in a holding cage adjacent to the apparatus. Two minutes later, the rat was placed in the illuminated compartment, and after a 10-s delay, the guillotine door was raised; thereafter, its latency (approach latency) to enter the dark compartment was recorded and a single 2-s unavoidable scrambled foot shock (0.8 mA) was immediately delivered after entering the dark compartment. The retention of the passive avoidance response was tested 24 h after the learning trial. The animal was placed on the elevated compartment, and the latency to reenter (avoidance latency) the dark compartment was recorded. Both acquisition and retention trials lasted for a maximal observation time of 180 s. CHF2819 (0.5, 1.5, and 4.5 mg/kg) was administered orally 90 min before the acquisition trial. Scopolamine hydrobromide (0.75 mg/kg) was dissolved in saline and injected s.c. 30 min before the acquisition trial.

FOB. The FOB consisted of measures of sensory, motor, and autonomic function. The evaluation of the end points considered and the scoring criteria used have been extensively described elsewhere (Moser, 1997). Briefly, the rat was placed on a flat surface (open field with a perimeter barrier, 60 × 60 cm) covered with a clean absorbent pad. The rat was observed for 3 min, and during that time, the frequency of rearing responses was recorded. At the same time, gait characteristics were noted and ranked; the ease with which the rat moved about was also ranked, and arousal, tremor, convulsions, and abnormal postures were evaluated. At the end of the 3 min, the number of the fecal boluses and urine pools on the absorbent pad were recorded. Reflex testing then consisted of recording the responses of each rat to the approach of a blunt object such as a pencil, a touch of an object to the posterior flank, and an auditory click stimulus. Responsiveness to a pinch on the tail and the ability of the pupil to constrict to light were then assessed. These measures were followed by a test for the righting reaction and then followed by measures of forelimb and hindlimb grip strength, body weight and rectal temperature, and, finally, hindlimb landing foot splay. The entire battery of tests required approximately 6 to 8 min per rat. Animals were subjected to FOB screening 90 min and 24 h after dosing.

Data Analysis. Neurochemical data were expressed as percentages of baseline, which was defined as the average of at least three consecutive samples with stable level of neurotransmitters. Actual data were analyzed by two-way ANOVA for repeated measures with treatment (tr) as the between-subject factor and time (t) as the within-subject factor. Conservative F tests using the Greenhouse-Geisser correction were performed to account for possible violations of the sphericity assumption. Post hoc comparisons were made by Dunnett's and Tukey's tests where appropriate. The adoption of nonparametric tests (Wilcoxon's paired signed rank test) was due to the nonhomogeneity of variances, as shown by Bartlett's test.

Animal Care. The experiments were conducted in accordance with guidelines released by Italian Ministry of Health (D.L. 116/92), the Declaration of Helsinki, and the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health.

	Results

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Effects of CHF2819 Administration on Extracellular ACh Concentrations in Young Adult and Aged Rats. Constant extracellular concentrations of ACh were detectable in the 20-min baseline samples collected for 6 h from the hippocampus of conscious, freely moving young adult rats (mean ± S.E. = 1.2 ± 0.2 nM; n = 36) (Fig. 2). The effects of the oral administration of CHF2819 on the basal extracellular concentrations of ACh were determined for 4 h after administration of the drug (Fig. 2). Two-way ANOVA for repeated measures showed the following differences: [F(tr)_3,240 = 13.11, P < .0001; F(t)_12,240 = 3.94, P < .01; F(tr × t)_36,240 = 3.93, P < .01]. The post hoc test showed that CHF2819 (0.5, 1.5, and 4.5 mg/kg p.o.) dose dependently increased ACh levels. The maximal stimulatory effect of CHF2819 was found at 4.5 mg/kg 120 min after its administration (732% increase above the baseline). The increase in ACh concentrations was still significant 4 h after treatment (Fig. 2).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of oral 0.5 (), 1.5 (), and 4.5 () mg/kg CHF2819 and saline () administration on extracellular levels of ACh in microdialysis samples from hippocampus of conscious, freely moving young adult rats. CHF2819 was administered at time 0 (  ). Data are mean ± S.E. (n = 6 rats). *P < .05 and #P < .01 versus saline (Tukey's test).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effect of local 1 µM () and 10 µM () CHF2819 perfusion (horizontal bar) on extracellular levels of ACh in microdialysis samples from hippocampus of conscious, freely moving young adult rats. Data are mean ± S.E. (n = 5 or 6 rats). *P < .05 versus basal values (Wilcoxon's paired signed rank test).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effect of oral 4.5 mg/kg CHF2819 () and saline () administration on extracellular levels of ACh in microdialysis samples from hippocampus of conscious, freely moving aged rats. CHF2819 was administered at time 0 (  ). Data are mean ± S.E. (n = 5 rats). *P < .001 versus basal levels and #P < .001 versus saline (Tukey's test). Inset, basal ACh concentrations in young adult (hatched column) and aged (open column) rat hippocampus. *P < .05 versus young adult ACh levels (Dunnett's test).

Effects of CHF2819 Administration on Extracellular DA, DOPAC, HVA, 5-HT, 5-HIAA, NE, and GABA Concentrations. Data are reported in Fig. 5. For DA, the two-way ANOVA for repeated measures showed that significant differences between treatments were time-independent [F(tr)_3,240 = 12.27, P < .001; F(t)_12,240 = 1.02, N.S.; F(tr × t)_36,240 = 1.26, N.S.], and therefore individual comparisons between marginal means (pooled data of 13 samples for each treatment) were made. Results showed that at the highest dose, CHF2819 significantly decreased DA levels (baseline, 0.67 ± 0.39 nM) (Fig. 5, inset). Concentrations of DA metabolites DOPAC and HVA were not affected by any of the doses tested: DOPAC: [F(tr)_3,240 = 0.71, N.S.; F(t)_12,240 = 1.29, N.S.; F(tr × t)_36,240 = 0.69, N.S.]; HVA: [F(tr)_3,240 = 0.23, N.S.; F(t)_12,240 = 1.56, N.S.; F(tr × t)_36,240 = 1.86, N.S.].

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effect of oral 0.5 (), 1.5 (), and 4.5 () mg/kg CHF2819 and saline () administration on extracellular levels of DA, HVA, DOPAC, 5-HT, 5-HIAA, NE, and GABA in microdialysis samples from hippocampus of conscious, freely moving young adult rats. CHF2819 was administered at time 0 (arrowheads). Data are mean (n = 5 or 6 rats). Inset, columns represent marginal mean (pooled data of 13 samples for each treatment) ± S.E. *P < .05 versus saline (Dunnett's test).

Effects of CHF2819 Administration on Passive Avoidance Behavior. Kruskal-Wallis ANOVA for approach latencies showed no significant differences (H = 0.87; df = 4, N.S.) among groups (data not shown). Conversely, Kruskal-Wallis ANOVA for avoidance latencies showed the following significant differences: H = 11.78; df = 4; P < .05. Individual comparisons between groups indicated that CHF2819, at a dose of 1.5 mg/kg, significantly attenuated scopolamine-induced decrease of avoidance latencies, whereas both the lowest (0.5 mg/kg) and the highest (4.5 mg/kg) doses did not affect this behavioral end point (Fig. 6).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effect of oral CHF2819 administration on scopolamine-induced impairment of passive avoidance behavior. Median stepdown latencies in the retention trial. Number of animals given in parentheses. *P < .05 versus vehicle plus vehicle and #P < .05 versus vehicle plus scopolamine (Mann-Whitney U test).

FOB Assessment. Results of neurobehavioral screening battery showed that oral administration of CHF2819 (1.5 and 4.5 mg/kg) did not significantly affect activity, excitability, autonomic, neuromuscular, and sensorimotor domains (data not shown). In particular, the following end points were not influenced by this AChEI: handling (ease of removal, handling, lacrimation, palpebral closure, piloerection, salivation), open field (rears, urination, defecation, gait, gait score, mobility score, arousal, vocalizations, stereotypy), reflexes (approach response, touch response, click response, tail pinch response, pupil response, righting reflex, landing foot splay), grip strength (forelimb, hindlimb), and physiological (body weight, body temperature). However, at 90 min after dosing, this compound induced involuntary motor movements (ranging from mild tremors to myoclonic jerks) in a dose-dependent manner (Fig. 7). These alterations were not observed 24 h after treatment.

View larger version (9K): [in this window] [in a new window]   Fig. 7.   FOB: involuntary motor movements detected 90 min after oral CHF2819 (1.5 and 4.5 mg/kg) administration. Number of animals given in parentheses. *P < .05 and #P < .01 versus vehicle (Fisher's exact test).

	Discussion

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The severity of cognitive decline in AD has been shown to be mainly correlated with alterations of the cholinergic function (Mountjoy et al., 1984), and this has led to the hypothesis that impaired learning and memory would be ameliorated by the restoration of cholinergic neurotransmission. In this regard, an overwhelming amount of evidence suggests the importance of hippocampal cholinergic transmission in cognitive processes. It has been demonstrated that a number of cholinomimetic agents enhance memory (Bartus, 1987); furthermore, it has been shown that several AChEIs stabilize most AD patients at their present cognitive and behavioral state for a period of at least 1 year (Giacobini, 1998).

At present, a novel AChEI that is orally administrable, efficacious, tolerable, and relatively safe remains a major therapeutic goal for further validation of the cholinergic deficit hypothesis of AD and successfully treatment of AD patients. This study suggests that the novel geneserine derivative CHF2819 may be an important candidate in this respect.

Our results have shown clearly that in the hippocampus of both young and aged rats, CHF2819 given either systemically or directly potently increases extracellular ACh concentrations. The highly sensitive detection methods used for determining ACh concentrations in the microdialysis samples made it possible to determine these effects without having to add other AChEIs. The use of such inhibitors has often restricted interpretation of previous studies on ACh release where neostigmine or physostigmine was required to be added to dialysates for efficient detection of basal ACh levels. The addition of these inhibitors has been reported previously to alter ACh release profiles (de Boer et al., 1990) and complicate the interpretation of systemically administered AChEI effects (Messamore et al., 1993). Incorporation of neostigmine in the dialysis medium also modifies the calcium dependence and tetrodotoxin sensitivity of extracellular ACh levels (Damsma et al., 1988). Furthermore, in several studies, it has been demonstrated that the increase in ACh levels induced by retrodialysis application of neostigmine or physostigmine induces a muscarinic autoregulatory mechanism that appears to be quiescent under basal conditions (de Boer et al., 1990; Kawashima et al., 1991).

The increase in endogenous hippocampal ACh levels after the oral administration of CHF2819 in young adult rats was dose-dependent. At the maximum dose used (4.5 mg/kg), this compound induced a 732% increase in ACh concentrations above baseline between 80 and 120 min after its administration orally. Furthermore, the ACh increase was long lasting, with levels of this neurotransmitter still being significantly elevated above baseline 240 min after treatment.

This long-lasting effect was not found after the oral administration of other AChEIs, such as metrifonate or tacrine. In fact, metrifonate (80 mg/kg) and tacrine (3 mg/kg) induced a significant elevation of hippocampal ACh, which lasted 140 and 100 min, respectively (Scali et al., 1997a,b).

Infusions of CHF2819 through the dialysis probe allowed us to confirm its direct action on in vivo hippocampal neurotransmitter concentrations. Under these circumstances, elevated ACh levels occurred immediately in the sample where the drug was first infused and were initially higher in the hippocampus than after systemic administration (1970% increase above baseline at 20 min). However, ACh levels remained significantly elevated for only 20 min after the end of the infusion, although preinfusion levels were not completely restored for 80 to 100 min. These neurochemical changes were paralleled by behavioral effects showing that CHF2819 significantly attenuated scopolamine-induced amnesia in a passive avoidance task.

The attenuation of scopolamine-induced amnesia elicited by CHF2819 occurred at a lower dose level (1.5 mg/kg) than that (4.5 mg/kg) producing the peak effect on extracellular concentrations of ACh, thus suggesting that the lower increase in ACh levels elicited by 1.5 mg/kg CHF2819 is sufficient to antagonize scopolamine-induced memory impairment. Literature data have clearly shown that the administration of AChEIs is followed by AChE inhibition and an increase in extracellular ACh levels in various brain areas. In turn, this is accompanied by an improvement in learning and memory deficits associated with cholinergic hypofunction (Pepeu, 2000). However, no straightforward correlation can be established between the intensity of AChE inhibition, the increase in ACh, and the beneficial effect on cognitive function. In some cases, improvements have been observed with doses of AChEIs with effects on AChE and ACh levels that were undetectable. For example, previous findings have shown that scopolamine-induced impairment of the passive avoidance response was prevented by metrifonate also at doses (10-15 mg/kg p.o.) that did not significantly increase cortical ACh levels in rats (Itoh et al., 1997). Therefore, it has been assumed that small undetectable increases in ACh levels at critical synapses are sufficient to ameliorate the cognitive impairment induced by scopolamine (Pepeu, 2000).

On the other hand, at the highest dose (4.5 mg/kg) used in this study, CHF2819 did not significantly affect scopolamine-induced amnesia. These results are in agreement with those reported in recent studies (Wang and Tang, 1998) showing the effectiveness of other AChEI agents [()-huperazine, donepezil, and tacrine] in antagonizing the disruptive effect of scopolamine on memory in rats (bell-shaped dose-effect curve). Interestingly, more than 20 years ago, when discussing the possible role of the cholinergic system in memory, Deutsch (1973) proposed that "as physostigmine prolongs the effects of ACh by inhibiting AChE, then this drug should strengthen weak memories but weaken strong memories because there will be excessive ACh which will result in a depolarization block". Moreover, because CHF2819 induced involuntary motor movements (ranging from mild tremors to myoclonic jerks) in a dose-dependent manner, the lack of antiamnestic effect at the highest dose (4.5 mg/kg) could be in part due to the marked neurotoxic alteration caused by this AChEI.

Cognitive impairment in aged rats seems to be in part due to cholinergic hypofunction, and activation of the brain cholinergic system under these circumstances is often accompanied by an improvement in cognitive dysfunction (Riekkinen et al., 1991; Ikari et al., 1995). Our data have demonstrated that in aged rats, there is a significant (73%) reduction in basal ACh concentrations in the hippocampus and that CHF2819 is also capable of producing marked elevations in levels in these animals that were of a similar magnitude to those found in young animals.

Findings from previous electrophysiological, biochemical, and pharmacological experiments are consistent with the hypothesis that there is a close functional interaction between central cholinergic and monoaminergic and GABAergic neurotransmitter systems (Bianchi et al., 1982; Robinson et al., 1989; Decker and McGaugh, 1991).

Our current results show that CHF2819 can increase 5-HT concentrations and decrease those of DA in the hippocampus of freely moving rats. These data are in agreement with previous in vitro studies showing that tacrine, an AChEI agent, evokes the release of radiolabeled 5-HT in rodent brain (Robinson et al., 1989).

To our knowledge, this is the first in vivo report showing a concomitant increase of ACh and 5-HT levels in rat hippocampus after AChEI administration. Indeed, previous studies have reported no consistent changes in rat brain of 5-HT or 5-HIAA efflux after the systemic or local administration of various AChEIs (Cuadra et al., 1994; Mori et al., 1995; Giacobini et al., 1996; Warpman et al., 1996). Increased 5-HT levels produced by CHF2819 could be of particular interest in AD treatment. Indeed, depressive disorders have been reported in some AD patients (Gottfries, 1996). Furthermore, significant decreases in 5-HT and 5-HIAA levels have been shown in discrete areas of the brain of the AD patient as well as a reduced number of 5-HT nerve terminals (Gottfries et al., 1983; Gottfries, 1990).

As far as the dopaminergic and noradrenergic system is concerned, our present data show that CHF2819 does not affect NE concentrations, whereas it significantly decreases DA levels, without affecting DA metabolism. Previous findings have demonstrated that AChEI-induced stimulation of cholinergic activity induces an increase in DA levels (Grenhoff and Svensson, 1992; Warpman et al., 1996), whereas other studies have shown no significant effect (Westerink et al., 1990; Dajas-Bailador et al., 1996). However, interactions between cholinergic and dopaminergic systems seem to play a role in the modulation of memory processes. In fact, a specific cholinergic control of the DA system located in brain areas involved in cognitive functions (hippocampus and cerebral cortex) has been shown (Memo et al., 1988). Cholinergic agonists increase DA turnover and release in vivo (Xu et al., 1989), and the systemic administration of muscarinic antagonists impairs performance on cognitive tests and reduces DA turnover in frontal cortex (Memo et al., 1988). Moreover, previous studies have shown a reciprocal interaction of DA and ACh, as well as an elevation of cortical DA levels, after physostigmine administration (Day and Fibiger, 1992; Cuadra et al., 1994).

Furthermore, tacrine administered to rats at doses that did not elevate ACh levels caused a modest increase in DOPAC levels in the whole brain (Nielsen et al., 1989). Conversely, a clear-cut increase in extracellular NE concentrations and a smaller increase in DA were shown after the systemic administration of heptylphysostigmine (Cuadra et al., 1994). The systemic administration of a large dose of metrifonate induced an increase in NE levels, whereas the systemic administration of small doses of metrifonate induced an elevation of DA concentrations in rat cortex (Mori et al., 1995). The systemic administration of donepezil at the dose of 2 mg/kg i.p., which in rat cortex caused a 35% AChE inhibition associated with a 2100% increase in ACh extracellular concentrations, was accompanied by increases of 100 and 80% in extracellular levels in the cortex of NE and DA, respectively (Giacobini et al., 1996). It has been shown that NE inhibits ACh release in the cerebral cortex by acting on presynaptic ₂-heteroreceptors (Beani et al., 1986). The possibility of enhancing the effects of AChE inhibition either by blocking the ₂-receptors or by inhibiting NE release has been previously investigated. It has been shown that only the combination of AChEIs with a selective ₂ antagonist, such as idazoxan, is able to potentiate the effect of AChE inhibition on extracellular concentrations of ACh (Cuadra et al., 1994; Cuadra and Giacobini, 1995).

Finally, this study shows that CHF2819 does not affect extracellular GABA concentrations in rat hippocampus. To our knowledge, there is no information available on the effects of AChEIs on GABAergic function, even though this neurotransmitter system is affected by AD (Chu et al., 1987).

In conclusion, the neurochemical and behavioral profile of CHF2819 (i.e., marked increase in ACh levels in the hippocampus of both young adult and aged rats, attenuation of scopolamine-induced amnesia, increase in hippocampal 5-HT concentrations) suggests that this orally active novel AChEI agent could be of clinical interest mainly for the symptomatic treatment of AD patients in which the cognitive impairment is accompanied by a depressive syndrome.