Characterization of the 5-HT6 receptor antagonist, SB-742457, on cognitive test performance in the rat.
Guy A. Higgins1,2, Leo Silenieks1, Winnie Lau1, Sandy Thevarkunnel1, Elijus Undzys1, Alan Henderson3, Nicholas Moore3 (1 CanCog Technologies, Toronto ON, 2 Dept. of Pharmacology,
University of Toronto, 3 Albany Molecular Research Inc., Albany, NY)
Introduction
B. Comparison of cholinergic related effects of donezepil (Aricept®) and SB-742457 in the SD rat
1
b.
*
**
3
Treatm ent (mg/kg)
6
4
*
3
Vehicle
1
3
d.
b.
SB-742457 - Distance travelled
c.
SB-742457 - Rearing count
1000
MES threshold (mA)
750
Rearing count
6000
3000
500
250
0
4
3
2
1
0
Veh
300
4000
200
c.
30
60
Treatm ent (mg/kg)
Veh
d.
SB-742457 - Distance travelled
30
60
Treatm ent (mg/kg)
SB-742457 - Rearing count
100
2100
*
100
*
0
1
3
6
Treatment (mg/kg)
10
Vehicle
1
3
6
Treatment (mg/kg)
Rearing count
*
Distance travelled (cm)
*
2000
Rearing count
75
1400
700
25
0
0
Vehicle
10
50
30
Vehicle
60
30
Treatment (mg/kg)
Figure 3. (a) Peak temperature changes recorded over a 6h period after the administration of
donepezil (Aricept®). (b) Average count of cholinergic signs recorded over the same period.
(c) Effect of donepezil on distance travelled and (d) rearing count. Donepezil (3-10mg/kg i.p.)
produced hypolocomotion, hypothermia and increased incidence of cholinomimetic signs.
*P<0.05 vs. vehicle control.
60
Treatment (mg/kg)
Figure 4. (a) Peak temperature changes over a 6h period after the administration of SB-742457.
(b) Average count of cholinergic effects over the same period. (c) Effect of SB-742457 on distance
travelled and (d) rearing count. SB-742457 failed to induce any obvious cholinomimetic signs.
Interestingly, while activity was decreased at 30mg/kg, consistent with trend evident at 10mg/kg
dose in the previous study, at the 60mg/kg dose no hypolocomotor effect was evident.
C. Effect of donepezil (Aricept®) and SB-742457 on novel object recognition in the SD rat
Donepezil (Aricept®)
D1
D1(sec)
(sec)
D2
D2
D1(sec)
(sec)
D1
0.4
5
Concentration of SB-742457
e.
e.
0.6
250
10
0.4
5
0.2
SB-742457 - NOR Index of discrimination
D2 (D1/a+b)
d.
*
15
0.6
10
SB-742457 - NOR Index of discrimination
D1 (b-a)
c.
Concentration (ng/mL)
15
SB-742457
Aricept® - NOR Index of discrimination
D2 (D1/a+b)
Concentration (ng/mL)
b.
b.
D2
D2
Aricept® - NOR Index of discrimination
D1 (b-a)
a.
0.2
200
150
100
50
0
0
0.0
Vehicle
Aricept
(0.3mg/kg)
Aricept
(1mg/kg)
Aricept
(3mg/kg)
0
Vehicle
Aricept
(0.3mg/kg)
Aricept
(1mg/kg)
Aricept
(3mg/kg)
f.
0.0
Vehicle
g.
Aricept
(1mg/kg)
SB-742457
(3mg/kg)
SB-742457
(10mg/kg)
SB (3mg/kg)
Vehicle
Aricept
(1mg/kg)
SB-742457 SB-742457
(3mg/kg)
(10mg/kg)
SB (10mg/kg)
Tre atm ent
h.
Experiementer comparison-Choice Trial
y = 0 .930 9x + 0 .088 4
2
R = 0.91 95
3 0.0
2 5.0
Expe rimenter 2 (s )
2 0.0
1 5.0
0.0
0.0
Vehicle
3
Treatment (mg/kg)
SB-742457 - Distance travelled
b.
3mg/kg
10mg/kg
15
Figure 1. Locomotor activity & MES threshold: Mouse
10
5
10
VPA 100
VPA 300
VPA 600
Treatment
SB-742457 - Rearing count
c.
250
SB742457
(a,b) Dose response for SB-742457 (3-10mg/kg oral) on total distance travelled
and rearing count in CD-1 mice recorded over a 60min activity test session. (c)
Effect of SB-742457 (10mg/kg oral) on MES threshold in CD-1 mice. Sodium
valproate (100-600mg/kg oral) was included in these experiments as a +ve
control. (d) Representative path plots for individual mice treated with either
vehicle, or SB-742457 at 3 or 10mg/kg from the activity study (a). In each study
SB-742457 (3-10mg/kg) had no significant effect compared to vehicle controls.
150
100
50
0
3
Treatment (mg/kg)
10
Vehicle
3mg/kg
10mg/kg
50
40
Figure 2. Locomotor activity & MES threshold: Rat
30
20
10
0
d.
*
60
ME S threshold (m A)
2000
MES threshold: Rat
70
200
*
*
4000
0
Vehicle
3
Treatment (mg/kg)
10
1 5.0
20 .0
2 5.0
30 .0
Expe rimenter 1 ( s)
Sprague-Dawley rat: SB-742457
6000
10 .0
20
Veh
10
Vehicle
5 .0
Figure 5. (a) Effect of donepezil (Aricept®) on the index of discrimination D1 (b-a). (b) Effect of donepezil on index of discrimination D2 ((b-a)/(a+b)). (c, d) Effect of SB-742457 on D1 and D2 respectively. (e) Plasma
concentration of SB-742457 (3-10mg/kg) measured from blood plasma taken immediately post choice test phase. (f, g) Photographs to illustrate the objects used in this test and the arena used to conduct these studies. Time
spent exploring each object is independently assessed by 2 trained observers, the graph (h) shows good inter-experimenter reliability.
*
0
0
3
Treatment (mg/kg)
d.
MES threshold: Mouse
25
Rearing count
Distance travelled (cm)
1
5
5.0
9000
Distance travelled (cm)
6
1 0.0
CD-1 mouse: SB-742457
Vehicle
2
10
Aricept - Rearing count
A. Comparison between SB-742457 on locomotor activity & MES threshold in mouse vs. rat
a.
6
SB-742457 - Total count of
cholinomimetic signs
7
3
Treatm ent (mg/kg)
Results
Vehicle
*
b.
SB-742457 - Peak temperature
change (°C)
0
6000
Vehicle
a.
4
2
10
Aricept - Distance travelled
c.
Distance travelled (cm)
1
0
Locomotor activity: Male, CD-1 mice and SD rats were used as test subjects. Following a defined pretreatment time the
animals are singly placed within test chambers where locomotor activity (defined as distance travelled) is measured by photocell
interruptions. The spatial distribution of activity can also be measured.
MES threshold test: Male, CD-1 mice and SD rats were used as test subjects. Following a defined pretreatment time the
animals were tested for MES seizure threshold using the ‘up and down’ method (Upton et al, 1997). In the mouse studies,
sodium valproate was included as a positive control.
Cholinergic side effects: Male SD rats served as test subjects. Animals were checked for cholinomimetic signs (lacrimation,
salivation, diarrhoea, tremor and hypothermia) at specific time points for up to 24 hours after drug treatment. General activity
was measured at 1 hour after treatment (represented by distance travelled and rearing count).
Novel Object recognition: Male SD rats served as test subjects. Animals were habituated twice in the test chambers prior to test
sessions. Each experiment was comprised of two trials, the sample trial (T1) and the choice trial (T2). Every exposure to the
test chamber was preceded by a defined pretreatment. During T1 animals were singly placed within test chambers where two
identical objects were presented. In the choice trial the animals were exposed to a duplicate of the object from T1 as well as a
novel object. During both trials, animals were tracked digitally using video cameras and an automated tracking software (ANYmaze®). Comparing the amount of time spent interacting with either object during T2, it is possible to asses how well rats are
able to discern between the familiar (a) and the novel objects (b). Due to the rat’s spontaneous tendency to explore and interact
with unfamiliar objects, this model is widely used as a measure of recognition memory, an important facet of human declarative
memory (Winters et al, 2008), which is compromised in AD patients.
5
a.
*
0
Vehicle
Methods
6
1
0
In the present studies we have evaluated SB-742457 in a rodent model of cognition, the novel object recognition (NOR) task, and compared
its profile to donepezil (Aricept®). Furthermore, we have conducted preliminary studies to followup on the finding that 5-HT6 receptor
antagonists may increase maximal electroshock seizure threshold in rats (Routledge et al, 2000; Stein et al, 2002). These studies were
extended to the mouse as despite significant sequence homology between the rat, mouse and human 5-HT6 receptor, the pharmacological
profile of the mouse receptor appears distinct to that of the rat and human (Hirst et al, 2003). Also species differences may exist with
respect to the regional expression of the 5-HT6 receptor in mouse compared to rat and human (Hirst et al, 2003). Consequently,
comparison of drug effect between these species offers a potential means to examine target based mechanism of action.
Aricept - Total count of
cholinomimetic signs
7
Cholinomimetic signs
Temperature change(°C)
2
Temperature change (°C)
3
**
Cholinomimetic signs
Aricept - Peak temperature
change (°C)
4
Temperature change
(°C)
a. a.
Although considerable effort is currently directed toward identifying treatments that modify the neurodegeneration that contributes to
Alzheimer’s Disease (AD) aetiology, there is also a need to identify treatments that can relieve the symptoms of this disease. One approach
that has gained significant recent interest is selective 5-HT6 receptor antagonists. Indeed one 5-HT6 antagonist, SB-742457, has
demonstrated clinical efficacy in mild-moderate AD patients as measured using ADAS-cog and CIBIC+ scales, with an effect size
equivalent to donepezil (see Upton et al, 2008). However results published from a recent Phase II study of 6 month duration conducted in a
total of 371 patients randomized to treatment showed improvements in only a subset of efficacy endpoints compared to placebo (MaherEdwards et al, 2010). In both studies, SB-742457 was reported to be well tolerated, even at exposures predicted to attain ~97% occupancy
of central 5-HT6 receptors.
Veh
SB-742457
Treatment
(a,b) Dose response for SB-742457 (3-10mg/kg oral) on total distance travelled
and rearing count in Sprague-Dawley rats recorded over a 60min test session.
(c) Effect of SB-742457 (10mg/kg oral) on MES threshold in SD rats. (d)
Representative path plots for individual rats treated with either vehicle, or SB742457 at 3 or 10mg/kg from the activity study (a). *P<0.05 vs. vehicle
pretreatment. SB-742457 (10mg/kg) significantly reduced activity and increased
MES threshold compared to vehicle controls.
References:
Hirst et al (2003) Mol. Pharmacology 64: 1295-1308.
Maher-Edwards et al (2010) Curr. Alzheimer Res. 7: 374-385.
Routledge et al (2000) Br. J. Pharmacology 130: 1606-1612.
Stean et al (2002) Pharmacol. Biochem. Behav. 71: 645-654.
Upton et al (1997) Br. J. Pharmacology 121: 1679-1686.
Upton et al (2008) Neurotherapeutics 5: 458-469.
Winters et al (2008) Neurosci. Biobehav. Reviews 32: 1055-1070
Summary and conclusions
1. SB-742457 (3-10mg/kg oral) reduced motor activity and increased MES threshold in Sprague Dawley
rats. The effect on MES threshold confirms previous studies by Routledge et al (2000); Stein et al (2002)
using other structurally distinct 5-HT6 antagonists.
2. Interestingly, SB-742457 (3-10 mg/kg oral) did not reliably reduce activity or increase MES threshold in
CD mice. This may reflect species differences in affinity of SB-742457 for the rat vs. murine 5-HT6
receptor, and/or regional species differences in expression pattern (Hirst et al, 2003).
3. In a 24h delay novel object recognition test, young vehicle pretreated rats failed to show a robust novel
object preference.
4. SB-742457 (10mg/kg oral) pretreatment during the sample and choice test phases, resulted in a
significant novel object preference. Mean plasma exposure immediately post test was measured at
143±15 ng/ml. In comparison, donepezil (0.3-1mg/kg i.p.) showed a modest, but inconsistent trend to
produce an equivalent effect.
5. At doses from 3-10mg/kg, donepezil produced clear cholinomimetic signs which likely contributed to a
decline in novel object test performance. In contrast, SB-742457 (30-60mg/kg oral) produced no
cholinomimetic or other overt behavioral signs. These observations are consistent with a good
tolerability reported in the clinic (Maher-Edwards et al, 2010).
6. The present studies support the hypothesis that 5-HT6 receptor antagonists have pro-cognitive effects
although current clinical reports seem mixed (Upton et al, 2008; Maher-Edwards et al, 2010).