Biochemical and neurobehavioral profile of CHF2819, a novel, orally active acetylcholinesterase inhibitor for Alzheimer's disease

Biochemical and neurobehavioral profile of CHF2819, a novel, orally active acetylcholinesterase inhibitor for Alzheimer's disease
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  See discussions, stats, and author profiles for this publication at: Biochemical and neurobehavioral profile of CHF2819, a novel, orally activeacetylcholinesterase inhibitor for Alzheimer's...  Article   in  Journal of Pharmacology and Experimental Therapeutics · August 2000 Source: PubMed CITATIONS 32 READS 31 7 authors , including:Tommaso CassanoUniversità degli studi di Foggia 77   PUBLICATIONS   1,878   CITATIONS   SEE PROFILE Luca SteardoSapienza University of Rome 211   PUBLICATIONS   3,649   CITATIONS   SEE PROFILE Claudio PietraHelsinn Group 83   PUBLICATIONS   2,334   CITATIONS   SEE PROFILE All content following this page was uploaded by Tommaso Cassano on 02 March 2015. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  Biochemical and Neurobehavioral Profile of CHF2819, a Novel,Orally Active Acetylcholinesterase Inhibitor for Alzheimer’sDisease 1 LUIGIA TRABACE, TOMMASO CASSANO, LUCA STEARDO, CLAUDIO PIETRA, 2 GINO VILLETTI, 2 KEITH M. KENDRICK, 3 and VINCENZO CUOMO Department of Pharmacology and Human Physiology, University of Bari, Bari, Italy   Accepted for publication March 21, 2000 This paper is available online at  ABSTRACT 1,2,3,3a,8,8a-Hexahydro-1,3a,8-trimethylpyrrolo[2,3-  b ]indol-5-ol 2-ethylphenylcarbamate  N  -oxide hydrochloride (3a S - cis  )(CHF2819) is a novel acetylcholinesterase inhibitor that pro-duces central cholinergic stimulation after oral administration inrats. In vivo studies show that CHF2819 (0.5, 1.5, and 4.5mg/kg p.o.) significantly increases acetylcholine levels in youngadult rat hippocampus in a dose-dependent manner. Moreover,aged animals, which show a significant decrease in basal ace-tylcholine levels with respect to young adult rats, also exhibit amarked increase in the hippocampal concentrations of thisneurotransmitter after the administration of CHF2819. Thiscompound (1.5 mg/kg p.o.) significantly attenuates scopol-amine-induced amnesia in a passive avoidance task. Further-more, CHF2819 induces a significant decrease in dopaminelevels and a significant elevation of extracellular concentrationsof 5-hydroxytryptamine, whereas it does not modify norepi-nephrine and    -aminobutyric acid levels in the hippocampus ofyoung adult rats. Functional observational battery screeningdemonstrates that CHF2819 (1.5 and 4.5 mg/kg p.o.) does notaffect activity, excitability, autonomic, neuromuscular, and sen-sorimotor domains, as well as physiological end points (bodyweight and temperature). However, this compound inducesinvoluntary motor movements (ranging from mild tremors tomyoclonic jerks) in a dose-dependent manner. These findingssuggest that the anti-amnestic properties of CHF2819, togetherwith its stimulatory effect on cholinergic and serotonergic func-tions, might have a therapeutic potential mainly for the symp-tomatic treatment of Alzheimer’s disease patients in which thecognitive impairment is accompanied by a depressive syn-drome.  Alzheimer’s disease (AD) is a complex and multifacetedneurodegenerative disease affecting aged populations. Thepathogenesis and the etiology remain unknown, although a“cholinergic deficit hypothesis” has been suggested (Perry,1986). In fact, among the multiple transmitter deficits thathave been described in AD, one of the most specific andconsistent features is an early and severe degeneration of forebrain cholinergic system, as revealed by the correlationobserved between the cholinergic pathology and dementia(Geula and Mesulam, 1994). Therefore, the enhancement of brain cholinergic transmission in AD remains a major goalfor many putative therapeutic agents that are in use or underdevelopment. Acetylcholinesterase (AChE) has long been an attractivetarget for the rational design of mechanism-based inhibitorsbecause of the pivotal role it plays in the central nervoussystem. Currently, only the AChE inhibition approach, whichenhances the function of central cholinergic neurons by per-mitting acetylcholine (ACh) to remain in the synaptic cleftlonger through reducing ACh hydrolysis, seems to produceencouraging symptomatic improvements in clinical trials. Infact, the resulting increase in extracellular ACh concentra-tions might reverse central cholinergic hypofunction and im-prove cognitive functions in AD (Kelly, 1999).To date, most of the drugs used therapeutically haveproved to ameliorate AD symptomatically, but it is contro- versial whether there is an effect on the disease progression(Giacobini, 1998).The clinical usefulness of AChE inhibitors (AChEIs) hasbeen limited by either an extremely short or an excessivelong half-life, hepatotoxicity, and severe peripheral cholin-ergic side effects (Giacobini, 1998; Kelly, 1999). To obtain Received for publication February 3, 2000. 1 This work was supported by Chiesi Farmaceutici S.p.A. and Biotechnologyand Biological Sciences Research Council. 2 Current address: Pharmacology Department, Chiesi Farmaceutici S.p.A., Via Palermo 26/A, 43100 Parma, Italy. 3 Current address: Department of Neurobiology, The Babraham Institute,Babraham, Cambridge, CB2 4AT UK.  ABBREVIATIONS:  AD, Alzheimer’s disease; AChE, acetylcholinesterase; AChEI, AChE inhibitor; ACh, acetylcholine; NE, norepinephrine; DA,dopamine; DOPAC, 3,4-dihydroxyphenilacetic acid; HVA, homovanillic acid; 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindolacetic acid;GABA,    -aminobutyric acid; FOB, functional observational battery. 0022-3565/00/2941-0187$03.00/0T HE  J OURNAL OF  P HARMACOLOGY AND  E  XPERIMENTAL  T HERAPEUTICS  Vol. 294, No. 1Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics  Printed in U.S.A. JPET 294:187–194, 2000 /2570/830413 187   a t  B r i   s  c  o eL i   b r  ar  y - U T H  S  C  S A  onA  pr i  l  2 7  ,2  0  0  9  j   p e t  . a s  p e t   j   o ur n al   s . or  gD  ownl   o a d  e d f  r  om   greater therapeutic benefit, newer AChEIs that circumventthese problems are needed.This study describes the biochemical and neurobehavioralprofile of CHF2819 (Fig. 1), a novel geneserine derivativewith AChE inhibitory activity (Pietra et al., 1999). The char-acterization of CHF2819 started with an in vivo (microdialy-sis technique) investigation of the effects of this compound,administered by the oral route or by local perfusion via thedialysis probe, on the extracellular concentrations of ACh inyoung adult rat hippocampus, the main target region, to-gether with the cerebral cortex, for symptomatic treatment of  AD. This was followed by an investigation of behavioral cor-relates of central cholinergic function in young adult rats(scopolamine-induced amnesia in a passive avoidance task). A functional observational battery (FOB) of tests was usedto assess functional domains (sensory, motor, and autonomic)in young adult rats to investigate potential neurotoxic effectsof CHF2819. Moreover, because a recovery of extracellular ACh levels in aged rats has been obtained after the admin-istration of AChEIs as well as drugs acting on brain cholin-ergic 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 abnormalitiesthat may contribute to the behavioral and cognitive disordersassociated with AD have been identified (Zubenko et al.,1990; Camacho et al., 1996). Furthermore, experimental andclinical data have shown interactions between central cho-linergic and catecholaminergic, serotonergic, and   -aminobu-tyricacid(GABA)ergicsystems(Bianchietal.,1982;Memoetal., 1988; Robinson et al., 1989; Decker and McGaugh, 1991).Therefore, it is likely that the efficacy of AChEIs in thetreatment of demented patients could be due not only tocholinesterase inhibition but also to other neurochemical ef-fects. In this study, we therefore also investigated, in addi-tion to ACh, the effects of CHF2819 on extracellular concen-trations of dopamine (DA), 5-hydroxytryptamine (5-HT),norepinephrine (NE), and GABA in the rat hippocampus. Materials and Methods  Animals.  Young adult (2–3 months old) and aged (20–22 monthsold) 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 freelyavailable. Chemicals.  CHF2819 (Fig. 1) was provided by Chiesi Farmaceu-tici S.p.A. (Parma, Italy). The drug was dissolved in saline andadministered p.o. in a volume of 2 ml/kg. All doses refer to the saltform (HCl). All other chemicals were obtained from commercialsources. Dialysis Procedure.  As previously described (Cagiano et al.,1998), rats were anesthetized with 3 ml/kg i.p. of a solution contain-ing 1.2 g of pentobarbital, 5.3 g of chloral hydrate, 2.7 g of MgSO 4 ,49.5 ml of propylene glycol, 12.5 ml of ethanol, and 58 ml of distilledwater.Rats were placed on a stereotaxic apparatus (David Kopf Instru-ments, Tujunga, CA), and a dialysis fiber was positioned in thedorsal hippocampus. Stereotaxic coordinates were as follows: AP   5.7, H    6.6 (young adult rats) and AP    5.2, H    7.0 (aged rats)from the interaural line with the incisor bar set at  2.4 mm accord-ing to a stereotaxic atlas (Paxinos and Watson, 1982). A short pieceof dialysis fiber made of copolymer of acrylonitrile sodium methallylsulfonate (AN69 Hospal S.p.A; 20,000 Da cutoff) was covered withepoxy glue to confine dialysis to the area of interest (6-mm glue-freezone). The skull of the rat was exposed, and two holes were made onthe lateral surface at the level of the head of the dorsal hippocampus. A dialysis fiber, held straight by a tungsten wire inside, was insertedtransversely into the brain so that the glue-free zone was locatedexactly in the target area. The tungsten wire was withdrawn, andstainless steel cannulas (22-gauge diameter, 15 mm long) were gluedto the ends of the fibers. These ends were bent up and fixed verticallyto the skull using dental cement and modified Eppendorf tips. Fi-nally, the skin was sutured, and the rats were allowed to recoverfrom anesthesia for at least 15 h before the neurotransmitter releasestudy. On the day of the experiment, the fibers were perfused with aKrebs-Ringer solution containing 138 mM NaCl, 11 mM KCl, 1.5 mMCaCl 2 , 1 mM MgCl 2  and 11 mM NaHCO 3  in distilled water. Thesolution was buffered at pH 7.4 with a 2 mM sodium-phosphatebuffer, filtered (0.22   m), and degassed. No AChEIs were adminis-tered through the dialysis probe to increase ACh detection capabil-ity. The fibers were perfused at a constant flow rate of 2   l/min witha CMA/100 microinjection pump (CMA Microdialysis, Stockholm,Sweden). After a 60-min washout period, consecutive 20-min sam-ples of perfusate were collected, and neurotransmitter concentra-tions were assayed by HPLC. Once a stable basal neurotransmitteroutput was obtained (no more than 10% difference between threeconsecutive samples), rats were administered the drug. The positionof the microdialysis probe was verified by histological procedures atthe end of each experiment. Only rats in which probe tracks wereexactly located in the target area were considered in  Results . HPLC Analysis.  ACh concentrations were determined by HPLCusing a microbore column and enzyme reactor coupled with electro-chemical detection in reduction mode and a glassy carbon electrode(6 mm) coated with peroxidase with  0.0 V applied (wired electrodesystem; Bioanalytical Systems, West Lafayette, IN; Sepstik 530-mm    1-mm analytical column and ACh/Ch IMER). The mobilephase used was 80 mM sodium phosphate with 5 ml/l Kathon (Bio-analytical Systems) at pH 8.5. The flow rate used was 125   l/min,and detection sensitivity for ACh was 0.1 nM (10   l injected).DA, 3,4-dihydroxyphenilacetic acid (DOPAC), homovanillic acid(HVA), 5-HT, 5-hydroxyindolacetic acid (5-HIAA), and NE levelswere determined by microbore HPLC using a Spherisorb 15-cm  2-mm column (3-  m packing). The detection was accomplished with aUnijet cell (BAS) with a 6-mm-diameter glassy carbon electrode at  0.65 V, connected to a Waters 460 electrochemical detector, aspreviously described (Kendrick et al., 1996). Detection limits for a10-  l injection volume were 100 to 200 pM. GABA concentrationswere measured by HPLC with fluorescence detection (Gilson, 157)after derivatization with  o -phthaldialdehyde as previously described(Kendrick et al., 1996) and with a detection limit of 5 nM for a 10-  lsample volume. Passive Avoidance Behavior.  A stepdown-type apparatus wasused. It consisted of a compartment (25  24  24 cm) constructed of  Fig. 1.  Chemical structure of CHF2819, a novel AChEI. 188  Trabace et al.  Vol. 294   a t  B r i   s  c  o eL i   b r  ar  y - U T H  S  C  S A  onA  pr i  l  2 7  ,2  0  0  9  j   p e t  . a s  p e t   j   o ur n al   s . or  gD  ownl   o a d  e d f  r  om   black Plexiglas and equipped with a grid floor to which an elevatedcompartment (13    24    16 cm) with solid Plexiglas floor wasattached. The opening between the elevated compartment and thelarge compartment was separated by a guillotine door (9  10 cm). A 25-W lamp illuminated the elevated compartment, whereas the largecompartment remained dark. Scrambled foot shocks were deliveredfrom a Letica shock generator (LI 2750 U; Barcelona, Spain). Theexperiments were performed in a sound-attenuating chamber (Am-plifon G-type cabin) that was dark except for the illumination of theelevated compartment of the apparatus. Each animal was removedfrom the home cage and placed in a holding cage adjacent to theapparatus. Two minutes later, the rat was placed in the illuminatedcompartment, and after a 10-s delay, the guillotine door was raised;thereafter, its latency (approach latency) to enter the dark compart-ment was recorded and a single 2-s unavoidable scrambled foot shock(0.8 mA) was immediately delivered after entering the dark compart-ment. The retention of the passive avoidance response was tested24 h after the learning trial. The animal was placed on the elevatedcompartment, and the latency to reenter (avoidance latency) thedark compartment was recorded. Both acquisition and retentiontrials 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 ac-quisition trial. Scopolamine hydrobromide (0.75 mg/kg) was dis-solved in saline and injected s.c. 30 min before the acquisition trial. FOB.  The FOB consisted of measures of sensory, motor, andautonomic function. The evaluation of the end points considered andthe scoring criteria used have been extensively described elsewhere(Moser, 1997). Briefly, the rat was placed on a flat surface (open fieldwith a perimeter barrier, 60  60 cm) covered with a clean absorbentpad. The rat was observed for 3 min, and during that time, thefrequency of rearing responses was recorded. At the same time, gaitcharacteristics were noted and ranked; the ease with which the ratmoved about was also ranked, and arousal, tremor, convulsions, andabnormal postures were evaluated. At the end of the 3 min, thenumber of the fecal boluses and urine pools on the absorbent padwere recorded. Reflex testing then consisted of recording the re-sponses 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 clickstimulus. Responsiveness to a pinch on the tail and the ability of thepupil to constrict to light were then assessed. These measures werefollowed by a test for the righting reaction and then followed bymeasures of forelimb and hindlimb grip strength, body weight andrectal temperature, and, finally, hindlimb landing foot splay. Theentire battery of tests required approximately 6 to 8 min per rat. Animals were subjected to FOB screening 90 min and 24 h afterdosing. Data Analysis.  Neurochemical data were expressed as percent-ages of baseline, which was defined as the average of at least threeconsecutive samples with stable level of neurotransmitters. Actualdata were analyzed by two-way ANOVA for repeated measures withtreatment (tr) as the between-subject factor and time ( t ) as thewithin-subject factor. Conservative  F   tests using the Greenhouse-Geisser correction were performed to account for possible violationsof the sphericity assumption. Post hoc comparisons were made byDunnett’s and Tukey’s tests where appropriate. The adoption of nonparametric tests (Wilcoxon’s paired signed rank test) was due tothe nonhomogeneity of variances, as shown by Bartlett’s test.Statistical analysis of behavioral data (passive avoidance task)was based on Kruskal-Wallis ANOVA. The Mann-Whitney  U   testwas used for individual comparisons between groups.The data collected with FOB assessment fall into these differentclasses: categorical (i.e., presence or absence of a sign), ordinal (i.e.,ranking of the severity of a sign), or continuous (i.e., a range of motoractivity counts) values. Analyses of the individual FOB measures, aswell as the physiological measures (body weight and body tempera-ture), were conducted as previously described (Moser, 1997).  Animal Care.  The experiments were conducted in accordancewith 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 NationalInstitutes of Health. Results Effects of CHF2819 Administration on Extracellular ACh Concentrations in Young Adult and Aged Rats. Constant extracellular concentrations of ACh were detect-able in the 20-min baseline samples collected for 6 h from thehippocampus 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 extracellu-lar concentrations of ACh were determined for 4 h afteradministration of the drug (Fig. 2). Two-way ANOVA forrepeated 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 thatCHF2819 (0.5, 1.5, and 4.5 mg/kg p.o.) dose dependentlyincreased ACh levels. The maximal stimulatory effect of CHF2819 was found at 4.5 mg/kg 120 min after its adminis-tration (732% increase above the baseline). The increase in ACh concentrations was still significant 4 h after treatment(Fig. 2).CHF2819 (1 and 10   M), administered via the dialysisprobe in freely moving young adult rats, also produced asignificant increase in ACh levels at the highest dose used. As shown in Fig. 3, the percentage of increase was 1970% (20min) and 1211% (40 min).In aged animals, extracellular ACh levels measured in thehippocampus before the administration of CHF2819 weresignificantly lower than in young adult rats (  73%) (Fig. 4,inset). The post hoc test showed that CHF2819, administeredp.o. at the dose of 4.5 mg/kg, induced a significant increase inextracellular ACh concentrations that peaked 20 min aftertreatment and disappeared within 60 min [  F  (tr) 1,8    14.93,  P  .005;  F  ( t ) 9,72  3.37,  P  .005;  F  (tr  t ) 9,72  3.29,  P  .005] (Fig. 4). Effects of CHF2819 Administration on ExtracellularDA, DOPAC, HVA, 5-HT, 5-HIAA, NE, and GABA Con-centrations.  Data are reported in Fig. 5. For DA, the two-way ANOVA for repeated measures showed that significantdifferences between treatments were time-independent Fig. 2.  Effect of oral 0.5 ( f ), 1.5 (  ), and 4.5 (  ) mg/kg CHF2819 andsaline ( F ) administration on extracellular levels of ACh in microdialysissamples from hippocampus of conscious, freely moving young adult rats.CHF2819 was administered at time 0 ( 1 ). Data are mean  S.E. ( n  6 rats). *  P  .05 and  #  P  .01 versus saline (Tukey’s test).  2000  Biochemical and Neurobehavioral Profile of CHF2819  189   a t  B r i   s  c  o eL i   b r  ar  y - U T H  S  C  S A  onA  pr i  l  2 7  ,2  0  0  9  j   p e t  . a s  p e t   j   o ur n al   s . or  gD  ownl   o a d  e d f  r  om   [  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 comparisonsbetween marginal means (pooled data of 13 samples for eachtreatment) were made. Results showed that at the highestdose, CHF2819 significantly decreased DA levels (baseline,0.67  0.39 nM) (Fig. 5, inset). Concentrations of DA metab-olites DOPAC and HVA were not affected by any of the dosestested: 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.]. As far as the effects of CHF2819 on 5-HT levels, becausetwo-way ANOVA for repeated measures showed that signif-icant differences between treatments were time-indepen-dent, [  F  (tr) 3,228  4.45,  P  .02;  F  ( t ) 12,228  2.48, N.S.;  F  (tr  t ) 36,228    0.55, N.S.], individual comparisons between mar-ginal mean values (pooled data of 13 samples for each treat-ment) were made. Results showed that CHF2819, at a doselevel of 4.5 mg/kg, significantly increased extracellular con-centrations of 5-HT (baseline, 3.8    1.5 nM) (Fig. 5, inset).Concentrations of the 5-HT metabolite 5-HIAA were not af-fected by any dose of CHF2819: [  F  (tr) 3,240    0.38, N.S.;  F  ( t ) 12,240  1.82, N.S.;  F  (tr  t ) 36,240  1.90, N.S.]. Moreover,levels of GABA were not affected either: [  F  (tr) 3,240    2.82,N.S.;  F  ( t ) 12,240  0.78, N.S.;  F  (tr  t ) 36,240  1.05, N.S.] (Fig.5). As far as NE, two-way ANOVA for repeated measuresshowed the following differences: [  F  (tr) 3,240  3.50,  P  .05;  F  ( t ) 12,240  2.08, N.S.;  F  (tr  t ) 36,240  2.13,  P  .02]. How-ever, post hoc test showed no significant differences betweenCHF2819-treated and control animals (Fig. 5). Effects of CHF2819 Administration on Passive AvoidanceBehavior. Kruskal-Wallis ANOVA for approachlatencies 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. Individualcomparisons between groups indicated that CHF2819, at adose of 1.5 mg/kg, significantly attenuated scopolamine-in-duced decrease of avoidance latencies, whereas both the low-est (0.5 mg/kg) and the highest (4.5 mg/kg) doses did notaffect this behavioral end point (Fig. 6). FOB Assessment.  Results of neurobehavioral screening batteryshowedthatoraladministrationofCHF2819(1.5and4.5 mg/kg) did not significantly affect activity, excitability,autonomic, neuromuscular, and sensorimotor domains (datanot shown). In particular, the following end points were notinfluenced by this AChEI: handling (ease of removal, han-dling, lacrimation, palpebral closure, piloerection, saliva-tion), open field (rears, urination, defecation, gait, gait score,mobility score, arousal, vocalizations, stereotypy), reflexes(approach response, touch response, click response, tail pinchresponse, pupil response, righting reflex, landing foot splay),grip strength (forelimb, hindlimb), and physiological (bodyweight, body temperature). However, at 90 min after dosing,this compound induced involuntary motor movements (rang-ing from mild tremors to myoclonic jerks) in a dose-depen-dent manner (Fig. 7). These alterations were not observed24 h after treatment. Discussion The severity of cognitive decline in AD has been shown tobe mainly correlated with alterations of the cholinergic func-tion(Mountjoyetal.,1984),andthishasledtothehypothesisthat impaired learning and memory would be ameliorated bythe restoration of cholinergic neurotransmission. In this re-gard, an overwhelming amount of evidence suggests the im-portance of hippocampal cholinergic transmission in cogni-tive processes. It has been demonstrated that a number of cholinomimetic agents enhance memory (Bartus, 1987); fur-thermore, it has been shown that several AChEIs stabilizemost AD patients at their present cognitive and behavioralstate 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 majortherapeutic goal for further validation of the cholinergic def-icit hypothesis of AD and successfully treatment of AD pa-tients. This study suggests that the novel geneserine deriv-ative CHF2819 may be an important candidate in thisrespect.Our results have shown clearly that in the hippocampus of both young and aged rats, CHF2819 given either systemi- Fig. 3.  Effect of local 1   M ( F ) and 10   M ( f ) CHF2819 perfusion(horizontal bar) on extracellular levels of ACh in microdialysis samplesfrom hippocampus of conscious, freely moving young adult rats. Data aremean  S.E. ( n  5 or 6 rats). *  P  .05 versus basal values (Wilcoxon’spaired signed rank test). Fig. 4.  Effect of oral 4.5 mg/kg CHF2819 (  ) and saline ( F ) administra-tion on extracellular levels of ACh in microdialysis samples from hip-pocampus of conscious, freely moving aged rats. CHF2819 was adminis-tered at time 0 ( 1 ). Data are mean  S.E. ( n  5 rats). *  P  .001 versusbasal levels and  #  P  .001 versus saline (Tukey’s test). Inset, basal AChconcentrations in young adult (hatched column) and aged (open column)rat hippocampus. *  P    .05 versus young adult ACh levels (Dunnett’stest). 190  Trabace et al.  Vol. 294   a t  B r i   s  c  o eL i   b r  ar  y - U T H  S  C  S A  onA  pr i  l  2 7  ,2  0  0  9  j   p e t  . a s  p e t   j   o ur n al   s . or  gD  ownl   o a d  e d f  r  om 
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