Download VRX-03011, a novel 5-HT4 agonist, enhances memory and

Neuropharmacology 53 (2007) 563e573
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VRX-03011, a novel 5-HT4 agonist, enhances memory
and hippocampal acetylcholine efflux
Eric G. Mohler a,b,1, Sharon Shacham c, Silvia Noiman c,
Frank Lezoualc’h d,e, Sylvain Robert d,e, Monique Gastineau d,e, Joseph Rutkowski c,
Yael Marantz c, Aline Dumuis f, Joel Bockaert f, Paul E. Gold b,g, Michael E. Ragozzino a,h,*
a
Department of Psychology, University of Illinois at Chicago, Chicago, IL 60607, USA
Department of Psychology, University of Illinois UrbanaeChampaign, Champaign, IL 68210, USA
c
Epix Pharmaceuticals, Lexington, MA 02421, USA
d
INSERM U769, IFR-141, Faculté de Pharmacie, F-92296 Châtenay-Malabry, France
e
Université Paris-Sud, IFR-141, Faculté de Pharmacie, F-92296 Châtenay-Malabry, France
f
Institut de Génomique Fonctionnelle, Université de Montpellier, CNRS UMR5203; INSERM U661, F-34094 Montpellier, France
g
Neuroscience Program, University of Illinois UrbanaeChampaign, Champaign, IL 68210, USA
h
Laboratory of Integrative Neuroscience, University of Illinois at Chicago, Chicago, IL 60607, USA
b
Received 27 March 2007; received in revised form 11 June 2007; accepted 20 June 2007
Abstract
Recent evidence suggests that 5-hydroxytryptamine (5-HT)4 receptor activity enhances cognition and provides neuroprotection. Here we
report the effects of VRX-03011, a novel partial 5-HT4 agonist, that is both potent (Ki w 30 nM) and highly selective (Ki > 5 mM for all other
5-HT receptors tested). In separate experiments, rats received VRX-03011 (0.1e10 mg/kg i.p.) 30 min prior to spontaneous alternation testing in
a no-delay or a 30-s delay condition. VRX-03011 (1, 5 and 10 mg/kg, but not 0.1 mg/kg) significantly enhanced delayed spontaneous alternation
performance while none of the doses enhanced performance in the no-delay test. VRX-03011 (1 and 5 mg/kg) concomitantly enhanced hippocampal acetylcholine output and delayed spontaneous alternation scores compared to that of vehicle controls, but had no effect on hippocampal
acetylcholine release under a resting condition. Moreover, suboptimal doses of VRX-03011 and the acetylcholinesterase inhibitor galanthamine
combined to enhance memory. VRX-03011 also regulated amyloid precursor protein (APP) metabolism by inducing a concentration-dependent
increase in the non-amyloidogenic soluble form of APP (sAPPa) with an EC50 w 1–10 nM. VRX-03011 had no effect on contractile properties
in guinea pig ileum or colon preparations with an EC50 > 10 mM and did not alter rat intestinal transit at doses up to 10 mg/kg. These findings
suggest that VRX-03011 may represent a novel treatment for Alzheimer’s disease that reduces cognitive impairments and provides neuroprotection without gastrointestinal side effects.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Serotonin; Acetylcholine; Hippocampus; Memory; 5-HT4; Alzheimer’s disease; Microdialysis; Behavior
1. Introduction
* Corresponding author. Department of Psychology, University of Illinois
Chicago, 1007 West Harrison Street MC 285, Chicago, IL 60607, USA.
Tel.: þ1 312 413 2630; fax: þ1 312 413 4122.
E-mail address: [email protected] (M.E. Ragozzino).
1
Present address: Abbott Laboratories, Abbott Park, IL 60064, USA.
0028-3908/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2007.06.016
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that results in severe cognitive impairment (Ho
et al., 2002; Hodges et al., 1992). Dysfunction and death of cholinergic neurons comprise the central neuropathology observed
in AD. Reductions in cholinergic markers are commonly observed in hippocampal and cortical areas, as well as being
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E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573
correlated with the severity of dementia (Hyman et al., 1984;
Wilcock et al., 1982). Accordingly, the predominant treatment
for AD focuses on increasing cholinergic tone through the use
of acetylcholinesterase inhibitors (AChE-I) (Aruglia et al.,
2004; Venneri et al., 2005). Treatment with AChE-I in AD
has led to modest effects in cognition and sometimes unwanted
side effects, e.g. depression (Frankfort et al., 2006).
Another potential treatment approach is to target other
neurotransmitter system ligands that modulate acetylcholine
(ACh) release and may be used alone or in combination with
AChE-I. One potential neurotransmitter to target is 5-HT. There
is some evidence that 5-HT neurotransmission is altered in AD
(Garcia-Alloza et al., 2005; Meltzer et al., 1998). More specifically, post-mortem binding studies in AD reported decreased
density of 5-HT4 receptors in the hippocampus and neocortex
(Reynolds et al., 1995; Wong et al., 1996, but see Lai et al.,
2003). Furthermore, an increasing body of evidence indicates
that the 5-HT4 receptor modulates ACh release, and is a promising target for the development of novel therapies for AD
(Bockaert et al., 2004).
The 5-HT4 receptor was first cloned from rat (Gerald et al.,
1995). In human, eight splice variants have been identified (a,
b, c, d, e, f, g, and n). With few exceptions, the pharmacology
of all of the splice variants is nearly identical (reviewed in
Bockaert et al., 2004), though the level of constitutive activity
of the receptor has been found to differ between isoforms, with
longer C-terminals generally associated with diminished constitutive activity (Claeysen et al., 1999). 5-HT4 receptors are
localized in the heart (Kaumann, 1990), intestine (Craig and
Clarke, 1990), adrenal cortex (Idres et al., 1991), bladder (Tonini and Candura, 1996), and the central nervous system (Patel
et al., 1995). Within the brain, highest expression is found in
the hippocampus, basal ganglia, and amygdala (Medhurst
et al., 2001), regions associated with learning and memory.
Developing pharmacological agents that target the 5-HT4 receptor may also be valuable because 5-HT4 receptor activation
produces secretion of the soluble amyloid precursor protein
(sAPPa) (Maillet et al., 2004; Robert et al., 2001). sAPPa
may have neuroprotective effects by potentially blocking the
formation and the toxic effect of b-amyloid peptide (Ab), a major component of the senile plaques associated with AD (Lezoualc’h and Robert, 2003). Cho and Hu (2006) demonstrated
that a partial 5-HT4 agonist can inhibit the formation of Ab in
primary cortical cultures from Tg2576 mice. Moreover, stimulation of central 5-HT4 receptors increases sAPPa in the mouse
hippocampus and cortex (Chacard-Castel et al., 2007).
The importance of 5-HT4 receptors in mnemonic processes
is further supported by several studies in rodents which found
that partial 5-HT4 agonists enhance learning and memory,
either alone (Lamirault and Simon, 2001; Lelong et al.,
2003; Matsumoto et al., 2001; Moser et al., 2002) or in combination with an AChE-I (Lamirault et al., 2003; Moser et al.,
2002). Although there is some evidence suggesting that 5-HT4
receptor activity modulates ACh release (see Bockaert et al.,
2004 for a review), some partial 5-HT4 receptor agonists
when administered centrally have been found to increase brain
ACh levels under non-mnemonic conditions (Consolo et al.,
1994; Matsumoto et al., 2001). Thus, these drugs may indiscriminately enhance cholinergic tone. Related to this issue,
there is no evidence that a partial 5-HT4 receptor agonist
enhances ACh output under conditions that require learning
and memory. This limitation is compounded by some previously developed 5-HT4 receptor agonists producing adverse
drug reactions in gastrointestinal functions (Briejer et al.,
2001; Wooltorton, 2004). Thus, a partial 5-HT4 agonist that
more selectively modulates ACh output during mnemonic
conditions without peripheral side effects could prove to be
of significant therapeutic value in dementia.
The findings described above suggest that partial 5-HT4
agonists may provide effective and novel treatments to ameliorate cognitive and neuropathological deficits in conditions
associated with memory impairments and neurodegeneration.
The present experiments examined the profile of VRX-03011,
a novel partial 5-HT4 receptor agonist (see Fig. 1). Because
previous experiments did not investigate whether a partial 5HT4 agonist enhances hippocampal ACh output during a memory test, VRX-03011 was tested in a spontaneous alternation
task in which rodents choose the least recently visited arm
in a maze and thus must remember recent arm selections.
Previous experiments demonstrated that hippocampal ACh
efflux is important for spontaneous alternation performance
and memory-enhancing agents concomitantly augment spontaneous alternation performance and hippocampal ACh output
(Ragozzino et al., 1996, 1998). Because earlier investigations
showed synergistic effects with partial 5-HT4 agonists and
AChE-Is (Lamireault et al, 2003; Moser et al., 2002), subefficacious doses of VRX-03011 were examined in combination with the AChE-I, galanthamine. To investigate whether
VRX-03011 modified sAPPa levels, the effects of VRX03011 on the induction of sAPPa secretion were tested in vitro.
Finally, to determine whether VRX-03011 affected gastric
motility, VRX-03011 was studied in gastrointestinal models.
2. Methods
All studies were carried out in accordance with the Declaration of Helsinki
and the Guide for the Care and Use of Laboratory Animals adopted by the U.S.
National Institutes of Health.
2.1. Radioligand binding studies
All radioligand binding studies were carried out by CEREP (Paris, France)
under contract with Predix Pharmaceuticals Inc. (now EPIX Pharmaceuticals
K+ O-
O
N
H
S
N
N
O
Fig. 1. VRX-03011, Potassium salt of 6,7-dihydro-4-hydroxy-7-isopropyl-6oxo-N-(3-(piperidin-1-yl)propyl)thieno[2,3-b]pyridine-5-carboxamide.
E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573
Inc.). Binding was initially tested using 1 mM VRX-03011. A specific Ki was
determined in all cases where greater than 50% displacement was observed at
1 mM. The binding affinities for other 5-HT receptors as well as 55 other proteins, including other G-protein coupled receptors, ion channels and transporters, were evaluated. When available, the experiments were carried out
using recombinant human receptors cloned into various cell types as shown
in Table 1.
2.2. Cell culture and transfection for splice variants of the
5-HT4 receptor
The cDNAs coding for the 5-HT4(a), 5-HT4(b) and/or 5-HT4(e) receptors
were subcloned into the pRK5 vector and, were introduced into COS-7 cells
by transfection as previously described (Claeysen et al., 1999). IMR32 cells
and Chinese hamster ovary (CHO) cells stably expressing the h5-HT4(d) or
the h5-HT4(e) receptor isoform were cultured as previously described (Mialet
et al., 2000; Robert et al., 2001). For transient transfection experiments, CHO
cells stably expressing the h5-HT4(d) receptor isoform were transfected with
the cDNA encoding the human APP695 using jetPEIÔ (Polyplus-transfection,
Illkirch, France) according to the manufacturer instructions.
2.3. Membrane preparation and radioligand binding assay for
splice variants of the 5-HT4 receptor
In competitive binding analyses against 5-HT, the binding affinities of VRX03011 to 5-HT4 receptor isoforms were tested using membranes prepared from
COS-7 cells transiently transfected with the recombinant 5-HT4(a) receptor
splice variants or 5-HT4(e) receptor isoforms. Membranes were prepared from
transiently transfected cells plated on 15 cm dishes and grown in DMEM
with 10% FBSd for 6 h and 20 h in DMEM without FBSd. Radioligand binding
studies with [3H]GR 113808 were performed on membrane preparation of
guinea pig striatum that express the endogenous 5-HT4 receptors as previously
described (Ansanay et al., 1996). Protein concentration in the samples was
determined with the Bio-Rad protein assay. Competition and saturation experiments were analyzed by non-linear regression using the computer program
LIGAND (Murson and Rodbard, 1980).
2.4. cAMP production in transfected cells
To test 5-HT4 receptor variants, COS-7 cells were transiently transfected
with 5-HT4(a), 5-HT4(b) or 5-HT4(e) receptor isoforms. COS-7 cells were
transfected with the appropriate cDNA and plated into 24-well clusters
(70,000 cells/well). Twenty-four hours post-transfection, cAMP production
levels were determined as follows. Cells were stimulated for 5 min with the
565
appropriate concentrations of 5-HT, 0.1 mM L-ascorbic acid and 0.1 mM
RO-20-1724, a phosphodiesterase inhibitor, in 250 ml of HEPES-buffered
saline (20 mM HEPES, 150 mM NaCl, 4.2 mM KCl, 0.9 mM CaCl2,
0.5 mM MgCl2, 0.1% glucose, 0.1% BSA) at 37 C. The same volume of
Triton X-100 (0.1%) was added to stop the reaction, and then the cells were
incubated 30 min at 37 C. Quantification of cAMP production was performed
by Homogenous Time Resolved FluorescenceÔ (HTRF) using the cAMP
Dynamic kit (CIS Bio International, Bagnols sur Cèze, France) according to
the manufacturer’s instructions.
2.5. Behavioral pharmacology experiments
2.5.1. Animals
Male LongeEvans rats (Charles River Laboratories, Indianapolis, IN)
weighing approximately 350 g were used for all experiments. Subjects were
individually housed in plastic cages in a temperature controlled room with
a 12 h light/dark cycle (lights on at 07:00 h). Rats had free access to food
and water except for the 24 h preceding testing, at which time they were
restricted to approximately 12 g of food.
2.5.2. Maze apparatus
A four-arm cross-maze made of 0.6 cm thick black plastic was used for all
behavioral testing. The maze was placed on a table that was 72 cm above the
floor. Each arm was 55 cm long 10 cm wide. The height of the arm walls
was 15.0 cm. Each arm contained a food well (3.2 cm diameter 1.6 cm
high) that was 3 cm away from the end wall. Each food-well hole was
2.3 cm in diameter and 1.6 cm deep.
2.5.3. Spontaneous alternation (no-delay) and drug injection procedures
In the spontaneous alternation test, rats were placed in the testing room
5 min before testing. Rats were allowed to freely explore the maze for
12 min. The number and sequence of arm entries was recorded. An arm entry
was recorded when all four paws entered an arm. An alternation was defined as
entry into four different arms on overlapping quadruple sets of arm entries;
e.g., a quadruple set consisting of arm choices A,D,C,B was an alternation
but a quadruple set consisting of arm choices A,D,A,C was not. The percent
alternation score is equal to the ratio of (actual alternations/possible
alternations) multiplied by 100; chance performance on this task is 22.2%.
Rats that made less than 15 arm entries in 12 min were excluded from the
analysis. This criterion is similar to that used in a previous experiment (Ragozzino et al., 1996).
Thirty minutes before testing, each rat received either VRX-03011 (0.1, 1,
5, or 10 mg/kg, i.p.) (Epix Pharmaceuticals Inc., Lexington, MA) or vehicle
(5% DMSO/sterile water). All doses were based on the weight of the salt.
Each rat received a total injection volume of 5 ml/kg. The number of rats in
Table 1
Serotonin receptor binding profile for VRX-03011
Receptor
Ligand
Ligand
concentration (nM)
Cells/Tissue
Affinity for
VRX-03011 (nM)
5-HT4a
5-HT4e
5-HT4
5-HT1A
5-HT1B
5-HT1D
5-HT2A
5-HT2B
5-HT2C
5-HT3
5-HT5A
5-HT6
5-HT7
[3H]GR 113808
[3H]GR 113808
[3H]GR 113808
[3H]8-OH-DPAT
[125I]Cyanopindolol (CYP)
[3H]Serotonin
[3H]Ketanserin
[3H]LSD
[3H]Mesulergine
[3H]BRL 43694
[3H]LSD
[3H]LSD
[3H]LSD
0.42
0.42
0.1
0.5
0.1
2
0.5
1.2
1.0
0.5
1.0
2.0
4.0
COS-7 cells expressing human cDNA
COS-7 cells expressing human cDNA
Guinea pig striatum
HEK293 cells expressing human cDNA
Rat cerebral cortex
Bovine caudate
HEK293 cells expressing human cDNA
CHO cells expressing human cDNA
CHO cells expressing human cDNA
HEK293 cells expressing human cDNA
HEK293 cells expressing human cDNA
HEK293 cells expressing human cDNA
CHO cells expressing human cDNA
31
17
36
>5 mMa
>5 mMa
>5 mMa
>5 mMa
>5 mMa
>5 mMa
>5 mMa
>5 mMa
>5 mMa
>5 mMa
All binding studies were carried out by CEREP except for 5-HT4a and 5-HT4e receptor. LSD, lysergic acid diethylamide.
a
Binding affinity estimated based on % displacement at 1 mM VRX-03011.
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E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573
each group was 6, except the vehicle group (n ¼ 10) and the VRX-03011
(5 mg/kg) group (n ¼ 9).
2.5.4. Spontaneous alternation (30-s delay) and drug injection procedures
A different set of rats was used in this experiment. In the delayed version,
each rat was allowed to freely choose an arm, but after making a choice, a rat
was blocked into that arm for 30 s. The block was a 21.5 cm 12 cm piece of
plastic. After the 30-s delay, the block was removed and a rat was allowed to
enter another arm. The test session lasted 15 min. Rats that made fewer than
11 arm entries in 15 min were excluded from the analysis.
Each rat received an intraperitoneal injection 30e32 min before testing.
Three separate experiments were carried out using spontaneous alternation
with a 30-s delay. A separate set of rats was used for each experiment. In
the first experiment, a rat received one of the following treatments: vehicle;
VRX-03011 (0.1, 1, 5, or 10 mg/kg); GR 125487 1 mg/kg (Tocris, Ellisville,
MO) or a combination of GR 125487 (1 mg/kg) and VRX-03011 (1 mg/kg).
In the second experiment, delayed spontaneous alternation testing was
combined with in vivo microdialysis using the following treatments: vehicle
or VRX-03011 (1 or 5 mg/kg). In the third experiment the interaction between
VRX-03011 and the AChE-I, galanthamine was investigated. This experiment
had the following treatment groups: vehicle; VRX-03011 (0.03 mg/kg);
galanthamine (0.3 or 2.5 mg/kg) (Tocris); a combination of VRX-03011 and
galanthamine (0.3 mg/kg). All doses were based on the weight of the salt.
Rats received a total injection volume of 5 ml/kg. In the experiment testing
VRX-03011 in 30-s delayed spontaneous alternation, the number of rats in
each group was 6. In the combined in vivo microdialysis and delayed spontaneous alternation experiment the number of rats in each group ranged from
7 to 9. In the experiment examining VRX-03011 and galanthamine, the number of rats in each group ranged from 6 to 8.
2.5.5. Surgery
Each rat for the in vivo microdialysis experiment received stereotaxic
surgery to implant cannulae into the hippocampus. Rats were injected with
atropine sulfate (0.2 ml; of a 250 mg/ml solution, i.p.) and the anesthetic
sodium pentobarbital (50 mg/kg, i.p.). The rat was then placed in the stereotaxic frame and 10-mm guide cannulae (CMA microdialysis) were implanted
bilaterally at stereotaxic coordinates 5.2 posterior to bregma, 5.2 lateral to
the midline, and 3.7 ventral to the surface of the skull. The cannulae were
secured in place with skull screws and dental acrylic. Rats were allowed to
recover for 1 week after surgery.
2.5.6. Microdialysis procedures
The day before testing, each rat was placed in a large plastic bowl and a microdialysis probe was inserted into the cannula for 3 min. The purpose of this
was to preclude the possibility that levels of ACh at the time of testing were
abnormal as a result of damage from initial probe insertion (Westerink, 1995).
On the day of testing, a 3-mm dialysis probe (CMA, North Chelmsford, MA)
was inserted through the guide cannula into the hippocampus. The dialysis
probe was connected to polyethylene tubing (CMA), which was connected
to a quartz swivel (Instech, Plymouth Meeting, PA), an additional length of
tubing, and then to an infusion pump (Harvard Apparatus, Holliston, MA).
The probe was perfused continuously at a rate of 1.5 ml/min with artificial
cerebrospinal fluid, which consisted of 128 mM NaCl, 2.5 mM KCl, 1.3 mM
CaCl2, 2.1 mM MgCl2, 0.9 mM NaH2PO4, 2.0 mM Na2HPO4, 1.0 mM
dextrose and adjusted to pH 7.4 by NaOH. To reliably detect ACh levels in
the dialysate, the reversible AChE-I, neostigmine bromide (0.1 mM) (Sigmae
Aldrich, St. Louis, MO) was added to the artificial cerebrospinal fluid. A
previous study demonstrated that 0.1 mM concentration of neostigmine in
the artificial cerebrospinal fluid is a minimal amount needed to reliably detect
behaviorally-induced changes in brain ACh efflux (Chang et al., 2006).
2.5.7. Drug injection and microdialysis sample collection
On the day of testing, a microdialysis probe was inserted into the hippocampus and the rat was placed in a large plastic bowl. The first 60 min
of perfusate was not analyzed to allow for equilibration between the brain
tissue and perfusion solution before testing. Subsequently, 12-ml samples
were collected at 8-min intervals. Three baseline samples were collected,
and then rats were injected i.p. with VRX-03011 (1 or 5 mg/kg) or vehicle
(5% DMSO/sterile water) with an injected volume of 5 ml/kg. Four additional baseline samples were collected followed by the placing of the rat
in the maze to begin delayed spontaneous alternation testing as described
above. Delayed spontaneous alternation testing lasted 16 min. Thus, the
testing session begin 32 min after receiving the drug or vehicle. After
testing was over, each rat was returned to the plastic bowl and two posttest samples were collected. The sample size in each group ranged from
7 to 9.
2.5.8. Drug injection and resting condition procedure
Resting condition measurements were made in rats that had completed
delay spontaneous alternation task with in vivo microdialysis collection. There
was a washout period of at least 1 week in between sessions. Microdialysis
samples were collected from the hemisphere opposite that used during maze
testing to ensure that tissue damage from the previous dialysis session did
not affect ACh output. Previous observations revealed diminishing ACh levels
from the same site with repeated dialysis sessions in the hippocampus (Gold
and Ragozzino, unpublished observations). The microdialysis procedure was
identical to the behavioral testing procedure, except that a rat was not placed
in the maze. Each rat received a different treatment in the resting condition
than in the behavioral testing condition. For this experiment the sample size
in each group was 6.
2.5.9. ACh assay
Samples (10 ml) were assayed for ACh using high-pressure liquid chromatography with electrochemical detection (Bioanalytical Systems, West
Lafayette, IN). Samples were loaded on a microbore analytical column for
separation of ACh and choline. Following separation, an enzymatic post-column
reactor containing acetylcholinesterase and choline oxidase converted ACh to
choline and acetate and choline to betaine and hydrogen peroxide. ACh and
choline were further broken down into stoichiometric quantities of hydrogen
peroxide. Hydrogen peroxide was broken down and detected by a glassy carbon
wired electrode coated with horseradish peroxidase at þ100 mV versus and
Ag/AgCl reference electrode. The mobile phase consisting of 50 mM Na2HPO4,
0.3 mM EDTA and 0.005% ProClin was delivered at a rate of 100 mL/min by
a solvent delivery system.
2.5.10. Histology
After completion of behavioral testing, each rat received a lethal dose of
sodium pentobarbital. A probe dipped in 2.5% Chicago blue stain was inserted
in each guide cannula to highlight the location of the probe. All rats were perfused intracardially with 0.9% phosphate-buffered saline, followed by a 4%
formaldehyde solution. Brains were removed and stored in a 4% formaldehyde
solution. The brains were frozen and cut in coronal sections (40 mm) on a cryostat. The brain sections were mounted on slides, dried, and stained with cresyl
violet to assess the location of the cannula tips or probes. Rats with placements
extending beyond the hippocampus were excluded from all analyses.
2.5.11. Statistical analysis
For all behavioral experiments, the threshold for statistical significance
was set at P < 0.05. In each version of the spontaneous alternation task,
a one-way analysis of variance (ANOVA) was used to identify differences
in percent alternation scores and number of arm entries across groups. A
Fisher Least Significant Difference post-hoc test was used to compare treatment and control group measures. For ACh measurements, data was converted
to a percentage of the baseline measurement. Specifically, the first three baseline data points were averaged, and all subsequent data points were expressed
as a percentage of that average. Changes in ACh output were expressed as
a percentage of baseline output. For the measurement of hippocampal ACh
efflux during spontaneous alternation testing and the resting condition, an
ANOVA with repeated measures was used to identify differences in ACh efflux
across the different test samples (seven baseline periods, two behavioral testing periods and two post-test periods). A Dunnett post-hoc test was used to
determine whether ACh output differed in the testing periods compared to
that of baseline for the different groups.
E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573
2.6. Measurement of sAPPa
CHO cells stably expressing the h5-HT4(e) receptor and APP695 and
IMR32 cells were cultured overnight in 5% dialyzed FCS-containing medium,
followed by a serum free medium for a period of 4 h. Then, 5-HT4 ligands
were added for 30 min in CHO cells or 4 h in IMR32 cells. After this period
of incubation, conditioned supernatants were collected and sAPPa was determined as previously described (Robert et al., 2001).
2.7. Guinea pig colon functional assay
The activity of VRX-03011 at the 5-HT4 receptor in the guinea pig colon
bioassay was determined as previously described (Gale et al., 1994). Briefly,
segments of guinea pig distal colon were suspended between two stainless-steel hooks in 20-ml organ baths. The baths containing an oxygenated
(95% O2 and 5% CO2) and pre-warmed (37 C) physiological salt solution
of 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 1.2 mM
KH2PO4, 25 mM NaHCO3 and 11 mM glucose, pH 7.4. Pyrilamine
(1 mM), methysergide (1 mM) and ondansetron (10 mM) were also present
throughout the experiments to block the histamine H1, 5-HT2 and 5-HT3
receptors, respectively. The tissues were stretched to a resting tension of
1 g then allowed to equilibrate for 60 min during which time they were
washed repeatedly and the tension readjusted. To test for agonist activity
the tissues were exposed to increasing concentrations of VRX-03011. To
test for antagonist activity, tissues were exposed to increasing concentrations of VRX-03011 or the reference antagonist GR 113808, 30 min
before exposure to a single effective concentration of the reference agonist
5-HT (0.3 mM). GR113808 was generously donated by Glaxo (Ware, Herts,
UK).
2.8. Intestinal transit in rats
The effects of VRX-03011 on gastrointestinal motility were determined in
male OFA-SD IGS rats weighing 144 to 208 g (n ¼ 8 per treatment group).
Rats were fasted for 24 h prior to i.v. administration of either vehicle
(12.5% DMSO in saline) or VRX-03011 (3 or 10 mg/kg i.v., expressed as
base) on the test day. Forty minutes later, a pulp consisting of 10% vegetable
charcoal in 2% Arabic gum was given by gavage (10 ml/kg body weight).
Twenty minutes after charcoal meal, animals were euthanized by CO2 inhalation. The whole gut (from stomach to cecum) was removed as rapidly as
possible and laid out on a centimeter measuring stick. The whole gut was
examined in coded order and the length of the intestine from the pyloric
sphincter to the ileocecal junction as well as the distance traveled by the charcoal front were both measured and recorded. For each animal, the intestinal
transit was determined as a percentage of the distance traveled by the charcoal
front, relative to the total length of the small intestine. Treatment effects
relative to controls were analyzed by one-way ANOVA followed by a pairwise
multiple comparisons (Dunnett’s method) where necessary. Differences
between groups were considered significant at P < 0.05.
3. Results
3.1. Binding affinity and specificity for the 5-HT4
receptor
VRX-03011 demonstrated high affinity for the two
recombinant 5-HT4 receptor isoforms, 5-HT4 (a) and (e)
(Kis ¼ 31 and 17 nM, respectively), and for the striatal 5HT4 receptor (Ki ¼ 36 nM). Experiments testing competitive
displacement by VRX-03011 using several proteins revealed
considerable 5-HT4 receptor selectivity, with a Ki > 5 mM
for all other 5-HT receptors (Table 1). Furthermore, VRX03011 exhibited no binding activity for 55 other proteins
tested (Kis > 1 mM), including other G-protein coupled
567
receptors, ion channels and transporters (See supplementary
information at end of manuscript). VRX-03011 was only
active at s1 and s2 receptors (Kis ¼ 79 and 40 nM, respectively). In COS-7 cells transfected with 5-HT4 (a), (b) and
(e) variants, VRX-03011 increased cAMP formation in a concentration-dependent manner with high potency (EC50 of
15e93 nM) and intrinsic agonist activity between 41% and
61% relative to 5-HT (Table 2). Taken together these results
demonstrate that VRX-03011 is a selective partial agonist
for the 5-HT4 receptor.
3.2. Spontaneous alternation testing
In the no-delay spontaneous alternation task, an ANOVA
revealed there was not a significant treatment effect,
F(4,32) ¼ 2.36, P > 0.05. Although the effect was not significant, only the 5 mg/kg of VRX-03011 showed a trend for improved alternation scores of w12% (see Fig. 2a). The mean
number of arm entries among the groups was comparable, suggesting that general activity levels are not affected by the drug,
F(4,32) ¼ 0.48, P > 0.05 (see Fig. 2b).
In this experiment, 13 rats were excluded from the analyses
because an insufficient number of arm entries were completed
during the behavioral test. The distribution of these rats among
the treatment groups was as follows: vehicle (n ¼ 2); VRX03011 0.1 mg/kg (n ¼ 1); VRX-03011 1 mg/kg (n ¼ 2);
VRX-03011 5 mg/kg (n ¼ 3); VRX-03011 10 mg/kg (n ¼ 5).
The results from the 30-s delay spontaneous alternation test
are illustrated in Fig. 3. In the delayed spontaneous alternation
task, VRX-03011 treatment significantly enhanced alternations
in a dose-dependent manner, F(6,35) ¼ 4.01, P < 0.005. A
Fisher LSD post-hoc test indicated that VRX-03011 significantly improved delayed alternation scores at the 1, 5 and
10 mg/kg doses; P’s < 0.05 vs. vehicle controls. When rats
received the 5-HT4 receptor antagonist GR 125487 (1 mg/kg)
co-administered with VRX-03011 (1 mg/kg), the antagonist
reduced the alternation scores back down to control levels
(P > 0.05 vs. vehicle controls). GR 125487 (1 mg/kg) alone
was without effect. The mean number of arm entries among
all groups was comparable, F(6,35) ¼ 0.70, P > 0.05, suggesting that VRX-03011 alone or in combination with GR 125487
did not affect general activity.
In this experiment, six rats were excluded from the analyses
because an insufficient number of arm entries were completed
during the behavioral test. The distribution of these rats among
the treatment groups was as follows: vehicle (n ¼ 4) and
VRX-03011 0.1 mg/kg (n ¼ 2).
Table 2
Stimulation of cAMP production by VRX-03011 in COS-7 cells expressing
recombinant 5-HT4 receptors
Receptor
VRX-03011
EC50 (nM)
VRX-03011
Emax (%)
5-HT4(a)
5-HT4(e)
5-HT4(b)
15e93
17e52
18e42
41e53
30e61
39e59
E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573
70
60
(a)
70
*
60
Percent Alternation
(mean ± SEM)
(a)
Percent Alternation
(mean ± SEM)
568
50
40
30
20
10
0
Veh
0.1
1.0
5.0
50
40
†
30
20
0
Vehicle
40
0.1
mg/kg
1
mg/kg
5
mg/kg
VRX-03011
35
30
25
(b)
20
15
10 GR 125487 VRX-03011
mg/kg 1 mg/kg
1 mg/kg
+
GR 125487
1 mg/kg
30
25
10
5
0
Veh
0.1
1.0
5.0
10
VRX-03011
Fig. 2. Effect of VRX-03011 treatment on standard spontaneous alternation
performance in a four arm cross-maze. (a) Effect of VRX-03011 (0.1e
10 mg/kg) treatment on spontaneous alternation performance in a four arm
cross-maze. VRX-03011 did not significantly improve performance compared
to that of vehicle controls. The dashed line denotes chance performance
(approximately 22%) (b) Mean number of arm entries in the delayed spontaneous alternation test. All groups made comparable number of arm entries in
the test session.
3.3. Hippocampal ACh efflux and spontaneous
alternation testing
To determine whether enhancement of memory by VRX03011 is accompanied by modulation of brain ACh output,
the effects of VRX-03011 (1 mg/kg and 5 mg/kg) on delayed
spontaneous alternation performance and hippocampal ACh
efflux were assessed simultaneously (Fig. 4a). Probe placements for rats included in the analyses were concentrated in
the CA3 and CA2 regions of the hippocampal formation at
the level of the splenium of the corpus callosum along the
anterioreposterior plane. Three rats were excluded from the
analyses because cannulae were outside of the hippocampal
formation. These probe placements were found in the fimbria
and/or external capsule.
Comparable to the results described above, VRX-03011 (1
and 5 mg/kg) significantly enhanced delayed spontaneous
alternation scores to 48.6 6.3% and 44.9 4.2% (S.E.M.),
respectively, compared to that of vehicle control-treated rats
which had a mean score of 16.6 3.9%, F(2,20) ¼ 12.30,
P < 0.05. Again, the memory enhancement with VRX03011 treatment occurred without altering the number of
arm entries, F(2,20) ¼ 0.14, P > 0.05. The mean number of
arm entries for the 1 mg/kg, 5 mg/kg and vehicle control
groups was 14.4 1.4, 15 1.2 and 14 1.5, respectively.
Number of Entries
(mean ± SEM)
Numberof Entries
(mean ± SEM)
*
10
10
VRX-03011
(b)
*
20
15
10
5
0
Vehicle
0.1
mg/kg
1
mg/kg
5
mg/kg
VRX-03011
10 GR 125487 VRX-03011
mg/kg 1 mg/kg
1 mg/kg
+
GR 125487
1 mg/kg
Fig. 3. Effect of VRX-03011 treatment on delayed spontaneous alternation
performance in a four-arm cross-maze. (a) Effect of VRX-03011 (0.1e
10 mg/kg) treatment on delayed spontaneous alternation performance in
a four arm cross-maze. VRX-03011 (1 mg/kg) significantly enhanced performance compared to that of vehicle controls. *P < 0.05 vs. vehicle controls.
y
P < 0.05 vs. VRX-03011 (1, 5 and 10 mg/kg). n ¼ 6 for each group. The
dashed line denotes chance performance (approximately 22%) (b) Mean number of arm entries in the delayed spontaneous alternation test. All groups made
comparable number of arm entries in the test session.
For those rats who had an accurate cannula placement,
seven were excluded from the analyses because of an insufficient number of arm entries during the behavioral test. The
distribution of these rats among the treatment groups was as
follows: vehicle (n ¼ 1); VRX-03011 1 mg/kg (n ¼ 3) and
VRX-03011 5 mg/kg (n ¼ 3).
The average absolute value of hippocampal ACh in baseline
samples was 889.4 126.8 fmol per 10 ml sample for the
vehicle group, 745.6 20.9 fmol per 10 ml sample for the
VRX-03011 1 mg/kg group, and 601.6 72.5 fmol per 10 ml
sample for the VRX-03011 5 mg/kg group; this difference
was not significant: F(2,21) ¼ 1.61, P > 0.05. An ANOVA
with repeated measures was employed to determine whether
hippocampal ACh efflux was different among the groups across
the different test samples. The analysis revealed there was a significant group effect, F(2,21) ¼ 4.95, P < 0.05, a significant
effect of test, F(10,210) ¼ 64.47, P < 0.05, as well as a significant group test interaction, F(20,210) ¼ 2.03, P < 0.05. A
E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573
225
200
Vehicle
VRX-03011 1 mg/kg
VRX-03011 5 mg/kg
175
150
125
100
75
8 16 24
Bselinea
32
40 48 56
Pre-Testing
Drug
Injection
Percent of Baseline
Hippocampal
ACh Efflux (Mean ± s.e.m.)
(b)
3.4. Combined treatment with VRX-03011 and the
AChE-I galanthamine
*
250
64 72 80 88
Testing Post-Testing
Time (Min)
250
225
200
Vehicle
VRX-03011 1 mg/kg
VRX-03011 5 mg/kg
175
150
The interaction between suboptimal doses of VRX-03011
and the AChE-I galanthamine was tested in the delayed spontaneous alternation task and compared with a known effective
dose of galanthamine (2.5 mg/kg). An ANOVA revealed a significant treatment effect on alternation scores, F(4,27) ¼ 7.82,
P < 0.05 (see Fig. 5a). A post-hoc analyses revealed that the
combination of VRX-03011 (0.03 mg/kg) and galanthamine
(0.3 mg/kg), that were individually ineffective for enhancing
memory, resulted in a significant increase in delayed alternation scores compared to that of vehicle controls (P < 0.05).
Galanthamine (2.5 mg/kg) also significantly improved alternation scores compared to that of vehicle controls (P < 0.05).
The enhancement observed with the combination of VRX03011 and galanthamine was similar in magnitude to that of
(a)
125
70
60
100
75
8
16 24
Baseline
32
40
48 56 64 72
Resting Condition
80
88
Drug
Injection Time (Min)
Fig. 4. Effect of VRX-03011 treatment on hippocampal ACh efflux during
delayed spontaneous alternation testing and resting conditions (a) Effect of
VRX-03011 (1 and 5 mg/kg) treatment on ACh release during delayed spontaneous alternation testing. VRX-03011 at 1 and 5 significantly enhanced
efflux compared to that of vehicle controls during maze testing, *P < 0.05
vs. vehicle controls. No significant differences were found during baseline,
pre-test or post-test periods. (b) ACh efflux during resting condition. VRX03011 treatment did not affect ACh efflux at any point.
Percent Alternation
(mean ± SEM)
Percent of Baseline
Hippocampal
ACh Efflux (Mean ± s.e.m.)
(a)
569
*
40
30
20
10
0
Vehicle
(b)
VRX-03011 Galanthamine VRX-03011 Galanthamine
0.03 mg/kg
0.3 mg/kg 0.03 mg/kg 2.5 mg/kg
+
Galanthamine
0.3 mg/kg
30
25
Number of Entries
(mean ± SEM)
Dunnett test revealed that vehicle controls, as well as both drug
groups exhibited a significant increase in hippocampal ACh efflux during the behavioral test periods compared to their respective baseline levels (P’s < 0.05). To better understand the
interaction, the groups differences in ACh output were analyzed in the different conditions. A Bonferroni t-test revealed
that ACh output among the groups was comparable during
the baseline periods (P’s > 0.05). In contrast, VRX-03011
at 1 and 5 mg/kg significantly increased ACh output during
both test samples compared to that of vehicle controls
(P’s < 0.05). In the first post-test period, ACh output remained significantly elevated in the VRX-03011 5 mg/kg
compared to that of vehicle controls (P < 0.05). In the final
post-test sample, ACh output among the groups was comparable (P’s > 0.05).
To determine the specificity of VRX-03011 treatment
potentiating hippocampal ACh output during memory testing,
the effects of VRX-03011 (1 and 5 mg/kg) on hippocampal
ACh output was further examined while a rat remained in
a holding chamber (Fig. 4b). In this non-mnemonic condition,
ACh efflux in the hippocampus was no different in rats receiving vehicle or VRX-03011 (1 and 5 mg/kg) at any time point,
F(2,15) ¼ 0.32, P > 0.05.
*
50
*
*
20
*
15
10
5
0
Vehicle
VRX-03011 Galanthamine VRX-03011 Galanthamine
0.03 mg/kg 0.3 mg/kg 0.03 mg/kg 2.5 mg/kg
+
Galanthamine
0.3 mg/kg
Fig. 5. Effects of treatment with VRX-03011 (0.03 mg/kg), galanthamine (0.3
and 2.5 mg/kg), and combination of the galanthamine (0.3 mg/kg) and VRX03011 (0.03 mg/kg) on delayed spontaneous alternation performance in a four
arm cross-maze. (a) VRX-03011 at 0.03 mg/kg or galanthamine 0.3 mg/kg
alone did not significantly enhance performance compared to that of vehicle
controls. In contrast, VRX-03011 0.03 mg/kg and galanthamine 0.3 mg/kg
significantly enhanced performance in a manner similar to that of galanthamine 2.5 mg/kg, *P < 0.05 vs. vehicle. (b) Mean number of arm entries in
the delayed spontaneous alternation test. Galanthamine 2.5 mg/kg, VRX03011 0.03 mg/kg, and the combination of galanthamine 0.3 mg/kg and
VRX-03011 0.03 significantly increased arm entries, *P < 0.05 vs. vehicle.
E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573
570
galanthamine (2.5 mg/kg) alone, suggesting a synergistic
effect of the two drugs.
The number of arm entries for this experiment is illustrated
in Fig. 5b. There was also a significant overall effect on arm
entries, F(4,27) ¼ 9.94, P < 0.05. A post-hoc test found that
galanthamine (2.5 mg/kg), VRX-03011 (0.03 mg/kg), and
the combination of galanthamine (0.3 mg/kg) and VRX-03011
(0.03 mg/kg) resulted in significantly more entries than vehicle
alone (P’s < 0.05).
VRX-03011 resulted in a concentration-dependent weak inhibition of the 5-HT-induced contraction, consistent with an antagonist activity (IC50 ¼ 2.2 mM in ileum and IC50 ¼ 280 nM
in colon). When VRX-03011 was administered to rats, doses
up to 10 mg/kg had no effect on the intestinal transit compared
with that of controls (62.2 1.0% of total intestinal length for
10 mg/kg VRX-03011-treated vs. 60.2 2.2% for control).
3.5. sAPPa secretion
The present findings indicate that VRX-03011 is a highly
selective and potent partial 5-HT4 receptor agonist with
adequate brain penetration (brain [ng/g] to serum [g/ml] ratio
of 0.93) in rats following intravenous administration of 1 mg/
kg. VRX-03011 did not affect performance in the no-delay
condition, but produced a robust enhancement in the delayed
spontaneous alternation task. Furthermore, the VRX-03011induced enhancement occurred in a dose-dependent manner.
Although VRX-03011 also binds to s1 and s2 receptors, as
do the 5-HT4 agonists BIMU1 and RS67333 (Lelong et al.,
2003), the 5-HT4 receptor antagonist GR125487 reversed the
VRX-03011-induced memory enhancement, consistent with
the idea that these effects are a result of 5-HT4 receptor
activation. VRX-03011 enhancement of memory performance
is comparable to past findings showing that other partial 5-HT4
receptor agonists enhance short-term memory (Lamirault and
Simon, 2001; Marchetti et al., 2000, 2004; Marchetti-Gauthier
et al., 1997; Moser et al., 2002) and reverse memory deficits in
aged rats (Moser et al., 2002). Taken together, the results
suggest that activation of central 5-HT4 receptors enhances
memory.
One issue about the interpretation of the present results
related to memory is whether VRX-03011-induced improvement in delayed spontaneous alternation is principally due to
affecting mnemonic processes. The spontaneous alternation
test takes advantage of rodents’ natural tendency to alternate
among spatial locations (Dember and Fowler, 1958). In order
for a rat to alternate in this test, a rat must always enter a different arm in consecutive sets of four arm entries. This alternation of arm entries requires a memory for recent arm
choices. Consistent with the idea that this task has a mnemonic
component, inserting a delay between arm choices decreases
alternation scores as observed in a previous study (Ragozzino
et al., 1996). Furthermore, increasing the number of arms used
and thus to remember decreases alternation scores (Ragozzino
et al., 1996; Walker et al., 1991). Moreover, alternations did
not occur because rats exhibit a fixed response pattern,
whether it be a simple response sequence, e.g. always turning
right or a more complex response sequence, e.g. go straight,
turn right, turn left, go straight. If this was the case, then alternation scores would be perfect or near perfect. However, in the
delay task controls performed near chance performance
(22.2%). In addition, even under conditions in which VRX030111 enhanced performance, alternation scores did not
approach 100% nor lead rats to produce a fixed response pattern. Finally, removal of extramaze cues decreases alternation
performance indicating that rats are using extramaze spatial
The effect of VRX-03011 on sAPPa secretion was tested in
CHO cells co-expressing the human APP695 and the human 5HT4(e) receptor (h5-HT4(e)), a neuronal h5-HT4 isoform which
is one of the most highly expressed isoform in the human brain
(Medhurst et al., 2001). VRX-03011 induced a concentrationdependent increase in sAPPa levels (Fig. 6a), with an EC50
w 1e10 nM. Moreover, VRX-03011 had a comparable effect
on sAPPa to the full 5-HT4 receptor agonist, prucalopride,
which was used at 10 mM as a positive control (Fig. 6a). A
dose-dependent study has previously shown that prucalopride
used at this concentration of 10 mM induced a maximal effect
on sAPPa secretion in CHO cells expressing the 5-HT4(e)
receptor (Lezoualc’h and Robert, 2003). In addition, VRX03011 strongly increased the level of extracellular sAPPa at
native h5-HT4 receptors in IMR32 human neuroblastoma cells
(Fig. 6b).
3.6. Gastrointestinal functioning
To explore possible side effects related to the gastrointestinal systems, the activity of VRX-03011 in guinea pig colon
and ileum as well as rat intestine was investigated. VRX03011 had no contractile effect on guinea pig ileum or colon
preparations (EC50 > 10 mM). However, preincubation with
(a)
Prucalo
pride
HCl salt of VRX-03011 (M)
CT
10-10
10-9
10-8
10-7
10-6
10-5
10-5
4.4
4.0
9.0
9.4
7.1
4.1
Secreted
sAPPα
Relative
Secretion
1.0
1.4
(b)
Prucalo
pride
HCl salt of VRX-03011 (M)
CT
10-10
10-9
10-8
10-7
10-6
10-5
2.7
3.1
3.9
1.2
10-5
Secreted
sAPPα
Relative
Secretion
1.0
1.0
1.4
2.5
Fig. 6. Effects of treatment with VRX-03011 on secretion levels of non-amyloidogenic sAPPa and Ab. Representative immunoblot showing the effects
of increasing concentrations of VRX-03011 on the cellular release of sAPPa
in (a) CHO cells stably expressing the h5-HT4(e) receptor isoform and (b)
IMR32 neuroblastoma cells.
4. Discussion
E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573
cues to guide their arm entries as opposed to using some consistent response sequence (Ragozzino and Gold, unpublished
observations). These findings suggest that the spontaneous
alternation task requires the use of spatial working memory.
The present findings demonstrating that VRX-03011 enhances delayed spontaneous alternation may have implications
for the development of novel treatments in AD. Recent reports
suggest that working memory deficits are among the first cognitive impairments manifested in AD or its precursor, Mild
Cognitive Impairment (MCI) (Bäckman et al., 2005). The
memory test used in the behavioral experiments is analogous
to some of the memory tests employed for testing AD and
MCI patients, e.g. Corsi block-tapping test to assess visuospatial working memory (Gron et al., 2006; Pasquier et al., 2001).
One possibility is that activation of central 5-HT4 receptors
may facilitate working memory in MCI and AD. Clearly,
more studies are needed to examine the effects of VRX03011 in a broader array of animal models, as well as learning
and memory tests to more completely characterize the potential benefits of this novel drug in alleviating cognitive deficits.
VRX-03011 treatment not only enhanced delayed spontaneous alternation performance, but also concomitantly potentiated an increase in hippocampal ACh output. Previous
studies found that partial 5-HT4 agonists enhanced brain
ACh output under baseline conditions (Consolo et al., 1994;
Matsumoto et al., 2003). However, previous experiments did
not demonstrate whether partial 5-HT4 agonists modulate
brain ACh output under mnemonic demands. An important aspect concerning the pattern of the present findings is that activation of 5-HT4 receptors improved spontaneous alternation
performance and increased hippocampal ACh output only under the delayed spontaneous condition but did not significantly
modify ACh output in the resting condition. The lack of effect
observed in the resting condition is comparable to findings
showing that intracerebroventricular injections of BIMU 1 or
BIMU 8 do not affect hippocampal ACh output (Consolo
et al., 1994). This selective modulation of hippocampal ACh
output by VRX-03011 may allow an increase in cholinergic
tone that corresponds to specific cognitive demands (improving signal-to-noise), rather than a global increase in ACh induced with AChE-Is (generally increasing ‘‘gain’’ in the
system) (Sarter et al., 2005).
One issue related to VRX-03011 potentiating hippocampal
ACh output during spontaneous alternation testing is whether
the drug modulates ACh output only under mnemonic conditions that engage the hippocampal cholinergic system or also
affect hippocampal ACh levels when increased pharmacologically, e.g. AChE-I treatment. The latter situation is unlikely
because the perfusion solution used in the in vivo microdialysis experiment contained the AChE-I, neostigmine. Despite
this, in the resting condition VRX-03011 did not increase
hippocampal ACh output compared to pre-drug baseline levels.
Because a single concentration of neostigmine was used in the
perfusion solution, it is possible that a different concentration
of neostigmine would have led to VRX-03011 potentiating
hippocampal ACh efflux in a resting condition. Because neostigmine was also in the perfusion solution for the combined
571
spontaneous alternation and in vivo microdialysis test, another
possibility is that the combination of an AChE-I with a memory test that activates the hippocampal cholinergic system led
to a partial 5-HT4 agonist augmenting ACh efflux. However,
the present results are consistent with previous results demonstrating that agents which concomitantly enhance hippocampal ACh output and spontaneous alternation performance do
not affect hippocampal ACh output in a resting condition
even when using a range of different concentrations of neostigmine in the perfusion solution (Ragozzino et al., 1996,
1998). Thus, a parsimonious interpretation of the results is
that VRX-03011 administration potentiated hippocampal
ACh output during a memory test that engages the hippocampal cholinergic system. In the future, this question can be further addressed by examining systemic injections of an AChE-I
and VRX-03011 on hippocampal ACh output in a resting condition without using a AChE-I in the perfusion solution.
At a behavioral level, the present experiment did observe an
interaction between an AChE-I and a partial 5-HT4 agonist.
More specifically, suboptimal doses of VRX-03011 and
galanthamine co-administered enhanced delayed spontaneous
alternation performance. At present, AChE-Is are the most
common treatment for dementia. This treatment approach
has limited effects in reducing the cognitive deficits associated
with AD (Winblad and Jelic, 2004). One possible reason for
this may be that AChE-Is cause a global increase in ACh
release (generally increasing ‘‘gain’’ in the system) rather
than specifically improving the signal-to-noise ratio (Sarter
et al., 2005). There is clearly a need for novel approaches in
alleviating cognitive impairments in dementia. Because activation of 5-HT4 receptors modulates ACh output and may have
neuroprotective effects, treatments that target central 5-HT4
receptors may prove beneficial in alleviating the severe
mnemonic deficits found in AD and other types of dementia.
The selective enhancement of ACh output during a mnemonic
condition suggests that VRX-03011 activation of 5-HT4 receptors may be an efficacious strategy for improving cholinergic
function. In addition, combining low doses of 5-HT4 agonists
with low doses of AChE-Is may allow greater flexibility in the
treatment of AD. Titrating doses of VRX-03011 and galanthamine may allow adjustments of both signal-to-noise ratios
through 5-HT4 receptor activation and increasing gain through
AChE-Is, maximizing therapeutic effectiveness in AD patients. Furthermore, AChE-Is induce secondary peripheral
effects, such as nausea, vomiting, and muscle cramping (Cummings, 2003). It is possible that a low dose of VRX-03011
combined with a low dose of an AChE-I may result in less serious side effects than a therapeutic dose of either drug alone,
although this was not examined in the present study.
In the experiment investigating the combination of VRX03011 and galanthamine, some groups exhibited an increase
in the number of arm entries during spontaneous alternation
testing. Unclear is why a low dose of VRX-03011 enhanced
arm entries in this study. Importantly, there is not a relationship
in arm entries and spontaneous alternation performance as
some treatments did not enhance memory but increased arm
entries while other treatments enhanced memory and also
572
E.G. Mohler et al. / Neuropharmacology 53 (2007) 563e573
increased arm entries. This can even be observed with the
same drug treatment. For example, in the case of VRX-03011
0.03 mg treatment, this dose enhanced arm entries when
administered alone or in combination with galanthamine
0.3 mg. When VRX-03011 0.3 mg was administered alone
it had no effect on alternation scores and when administered
with galanthamine 0.3 mg it enhanced alternation scores.
Moreover, this lack of relationship between alternation performance and number of arm entries is consistent with a large
series of experiments that examined a wide variety of drugs
on spontaneous alternation (Ragozzino and Gold, 1991;
Walker et al., 1991).
Another important finding supporting the potential
therapeutic utility of VRX-03011 is that the drug exhibited
possible neuroprotective activity by increasing secretion of
the non-amyloidogenic sAPPa at native and recombinant
5-HT4 receptors. VRX-03011 had no effect on sAPPa in
CHO cells expressing only the human APP695 receptor
(data not shown). However, in CHO cells co-expressing the
human APP695 and the human 5-HT4(e) receptor, VRX03011 induced a dose-dependent increase in sAPPa. This suggests that reduction of sAPPa by VRX-03011 is mediated by
the 5-HT4(e) receptor. Non-amyloidogenic secreted sAPPa has
potent neuroprotective functions against neurotoxic insults
such as glutamate and Ab. Furthermore, sAPPa promotes
neurite outgrowth, regulates neuronal excitability and has
memory-enhancing effects in animal models of dementia
(Bour et al., 2004; Lezoualc’h and Robert, 2003). These possible neuroprotective actions suggest that VRX-03011 may be
a useful new treatment for AD. As a next step, it will be interesting to test whether VRX-03011 may slow down disease
progression in transgenic animal models of AD. Interestingly,
it has been recently shown that 5-HT4 may increase sAPPa in
a transgenic mouse line over-expressing the ‘‘London’’ mutant
of human APP (Cachard-Chastel et al., 2007).
One last important consideration for potential AD treatments is the absence of adverse peripheral actions. The human
5-HT4 receptor is expressed in the brain as well as the gastrointestinal system. Therefore, potential therapeutic limitations
of 5-HT4 receptor agonists come from possible adverse drug
reactions involving gastrointestinal organs (Briejer et al.,
2001). VRX-03011 had no agonist effects on the activity of
the guinea pig colon and ileum or rat intestine. These data suggest VRX-03011 is a highly selective partial 5-HT4 receptor
agonist that does not affect gastrointestinal functions. Overall,
the findings suggest that VRX-03011 has a unique set of properties that could provide both symptomatic relief of cognitive
impairments as well as possible neuroprotective therapeutic
effects in patients with AD in the absence of adverse gastrointestinal actions.
Acknowledgments
This work was supported by Epix Pharmaceuticals, Inc.
S.S., S.N., J.R., and Y.M. are employees of Epix Pharmaceuticals, which conducted tests of gastrointestinal functioning.
CEREP (Paris, France) was contracted by Predix
Pharmaceuticals (Currently Epix Pharmaceuticals) for radioligand binding assays. Work examining splice variants of the 5HT4 Receptor was completed by A.D. and J.B. Spontaneous
alternation testing and in vivo microdialysis studies were completed by E.G.M., P.E.G., and M.E.R. Measurement of sAPPa
was completed by F.L., S.R., and M.G. E.G.M. is currently
employed by Abbott Laboratories. Abbott Laboratories is
not affiliated with this work in any way.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.neuropharm.2007.06.016.
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