Neuropharmacological effects of brexpiprazole on animal ... · recognition, in untreated mice. Moreover, we investigated the role of serotonin 5-HT 1A receptor on the effect of brexpiprazole

Neuropharmacological effects of brexpiprazole

on animal models of cognitive impairment

（認知機能障害における新規抗精神病薬ブ

レクスピプラゾールの神経薬理学的研究）

千葉大学大学院医学薬学府

先端医学薬学専攻

（主任：橋本謙二教授）

吉見典子

CONTENTS

I. GENERAL INTRODUCTION……………………………………………………1

II. CHAPTER 1

Improvement of dizocilpine-induced social recognition deficits in mice by

brexpiprazole, a novel serotonin-dopamine activity modulator……………… 8

1. ABSTRACT…………………………………………………………………….8

2. INTRODUCTION……………………………………………………………….9

3. EXPERIMENTAL PROCEDURES…………………………………………...10

3.1. Animals……………………………………………………………10

3.2. Drugs……………………………………………………………….11

3.3. Apparatus………………………………………………………….11

3.4. Behavioral procedures………………………………………………12

3.4.1. Habituation…………………………………………………………12

3.4.2. Sociability test……………………………………………………12

3.4.3. Social recognition test……………………………………………12

3.5. Statistical analyses…………………………………………………13

4. RESULTS………………………………………………………………………13

4.1. Effects of brexpiprazole on dizocilpine-induced social recognition

deficits………………………………………………………………13

4.2. Brexpiprazole alone did not affect social recognition deficits………14

4.3. Risperidone and olanzapine had no effect on dizocilpine-induced

social recognition deficits…………………………………………16

4.4. Role of the 5-HT1A receptor in brexpiprazole's effects on

dizocilpine-induced social recognition deficits……………………19

5. DISCUSSION…………………………………………………………………21

6. REFERENCES…………………………………………………………………25

III. CHAPTER 2

Effects of brexpiprazole, a novel serotonin-dopamine activity modulator, on

phencyclidine-induced cognitive deficits in mice: A role for serotonin 5-HT1A

receptors………………………………………………………………………….. 34

1. ABSTRACT…………………………………………………………………35

2. INTRODUCTION……………………………………………………………37

3. EXPERIMENTAL PROCEDURES………………………………………….37

3.1. Animals……………………………………………………………………37

3.2. Drugs…………………………………………………………………….37

3.3. Drug administration……………………………………………………….38

3.4. Novel object recognition test (NORT)……….……….……….…………39

3.5. Statistical analysis………………………………………………………..40

4. RESULTS…………………………………………………………………….41

4.1. Effects of brexpiprazole on PCP-induced cognitive deficits in mice……41

5. DISCUSSION…………………………………………………………….……43

6. REFERENCES……………………………………………………………….45

IV. CONCLUSIONS………………………………………………………………..54

V. AKNOWLEGEMENTS…………………………………………………………..55

1

I. GENERAL INTRODUCTION

Cognitive deficits in patients of psychiatric disorders including schizophrenia and

major depressive disorder are recognized as a serious social problem in recent years.

Especially in schizophrenia, it is thought that cognitive deficits are not epiphenomena

arising from positive and negative symptoms but one of the main symptoms of

schizophrenia. The Measurement and Treatment Research to Improve Cognition in

Schizophrenia (MATRICS) developed by the US National Institute of Mental Health

(NIMH) defined that there are several main cognitive domains easily impaired by

schizophrenia such as attention/vigilance, working memory, verbal learning and

memory, visual learning and memory, reasoning and problem solving, speed of

processing and social cognition (Table 1).

Cognitive domains Example

attention/vigilance ability to response appropriately to series of stimuli appeared

quickly

ex) reading book, watching movie

working memory ability to memorize information for short-time (about 5-20

seconds) and process it

ex) memorizing phone number newly taught

verbal learning

verbal memory

ability to memorize verbal information for long-time (a few

minutes- a few years)

ex) memorizing someone’s request to buy in the store

visual learning

visual memory

ability to memorize visual information for long-time (a few

minutes- a few years)

2

ex) memorizing what and where to store

reasoning

problem solving

ability to carry out a project effectively

ex) getting workplace in time despite of changing time of the

usual bus

speed of processing ability to process the information exactly and response it

quickly

ex) serving a customer with touch panel operation

social cognition ability to process and memorize social information such as

expressions, emotions and meaning of social interaction

ex) memorizing someone’s face or ability of recognizing

someone’s feeling from his/her expression

Table 1. Cognitive domains impaired in patients with schizophrenia

These cognitive domains are necessary for working. Therefore cognitive deficits

constitute a major cause of low employment rate in patients of psychiatric disorders

(Mueser et al, 2002). Social isolation by disemployment might decrease patient’s

motivation, and has possibility to result in worsening of symptoms. Additionally,

cognitive deficits cause a decrease in the drug adherence rate of patients and might

prevent improvement of symptoms (Patterson et al, 2002). Treatment for cognitive

deficits is needed for not only improvement of cognitive deficits itself, but also

improvement of other symptoms of psychiatric disorders. There is no antipsychotic drug

having enough therapeutic effect for cognitive deficits despite of challenging for

development of novel drugs. Development of effective drugs for cognitive deficits is

critically-needed by psychiatric patients.

3

Brexpiprazole , 7-{4-[4-(1-benzothiophen-4-yl)piperazin-1-yl]butoxy}

quinolin-2(1H)-one (Figure 1) , is a novel serotonin-dopamine activity modulator

developed within recent years. Brexpiprazole has higher serotonin 5-HT1A receptor and

dopamine D2 receptor binding affinity than an atypical antipsychotic drug, aripiprazole

(Figure 1, Table 2). Brexpiprazole has a slightly higher binding affinity for h5-HT1A

receptors than hD2 receptors, whereas aripiprazole has reverse effect (Table 2).

Additionally, the inhibitory activity for hD2L receptor on brexpiprazole was a little

weaker than that on aripiprazole which indicates that the intrinsic activity for D2 on

brexpiprazole is slightly lower than that on aripiprazole (Figure 2). As brexpiprazole

has lower intrinsic activity at D2 receptor compared with aripiprazole, it can expect to

have fewer adverse effects by D2 receptor agonism and antagonism than aripiprazole. It

is also an antagonist at 5-HT2A receptors and adrenergic α1B/2C receptors (Maeda et al.,

2014a). The 5-HT1A receptor agonism is focused on the ability for improvement of

several clinical and non-clinical reports.

4

Figure 1. Chemical structure of brexpiprazole and aripiprazole

aData from Maeda et al., 2014a.

bData from Shapiro et al., 2003.

5

Table 2. Binding affinities for cloned human receptors in vitro (Maeda et al. 2014a)

Data are calculated by nonlinear regression analysis using data from three assays performed in

duplicate or triplicate and expressed as mean values.

Figure 2. Partial agonist activity of brexpiprazole and reference drugs on human

cloned D2L receptors in vitro (Maeda et al. 2014a)

Concentration-response curves are shown for brexpiprazole and aripiprazole on forskolin induced

cAMP accumulation in hD2L receptor–expressing CHO cells. Data are mean 6 S.D. of three assays

performed in duplicate. Cyclic AMP accumulation was normalized to the percentage of

forskolin-induced cAMP accumulation (set at 100%).

The research in animal model reflecting clinical cognitive deficits is critically important

to evaluate the effects of drugs. However, there is no unified classification whether

which animal model for testing cognitive deficits match which domains in listed in

Table 1. Multiple testing for one drug can resolve this problem.

6

In the present study, we investigated the therapeutic effects of a novel

antipsychotic drug brexpiprazole on social recognition impaired by the

N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine, and cognitive deficits

in mice after administration of the NMDA receptor antagonist phencyclidine (PCP). We

divided our study into the following two chapters.

CHAPTER 1

We investigated the effects of brexpiprazole, olanzapine and risperidone, on

dizocilpine-induced social recognition impairment, in mice. Furthermore, we also

investigated the effect of brexpiprazole, olanzapine, and risperidone on social

recognition, in untreated mice. Moreover, we investigated the role of serotonin 5-HT1A

receptor on the effect of brexpiprazole on dizocilpine-induced social recognition

impairment.

CHAPTER 2

Using the novel object recognition test (NORT), we investigated the effects of

subsequent subchronic brexpiprazole on PCP -induced cognitive deficits in mice.

Furthermore, we investigated the role of 5-HT1A receptor on the effect of brexpiprazole

in this model.

REFERENCES

Maeda, K., Sugino, H., Akazawa, H., Amada, N., Shimada, J., Futamura, T., Yamashita,

H., Ito, N., McQuade, R.D., Mork, A., Pehrson, A.L., Hentzer, M., Nielsen, V.,

Bundgaard, C., Arnt, J., Stensbol, T.B., Kikuchi, T., 2014a. Brexpiprazole I: in

7

vitro and in vivo characterization of a novel serotonin-dopamine activity

modulator. The Journal of pharmacology and experimental therapeutics 350,

589-604.

Mueser, K.T., Salyers, M.P., Mueser, P.R., 2001. A prospective analysis of work in

schizophrenia. Schizophrenia bulletin 27, 281-296.

Patterson, T.L., Lacro, J., McKibbin, C.L., Moscona, S., Hughs, T., Jeste, D.V., 2002.

Medication management ability assessment: results from a performance-based

measure in older outpatients with schizophrenia. Journal of clinical

psychopharmacology 22, 11-19.

Shapiro, D.A., Renock, S., Arrington, E., Chiodo, L.A., Liu, L.X., Sibley, D.R., Roth,

B.L., Mailman, R., 2003. Aripiprazole, a novel atypical antipsychotic drug with a

unique and robust pharmacology. Neuropsychopharmacology : official

publication of the American College of Neuropsychopharmacology 28,

1400-1411.

8

II. CHAPTER 1

Improvement of dizocilpine-induced social recognition deficits in mice by

brexpiprazole, a novel serotonin-dopamine active modulator

1. ABSTRACT

Cognitive impairment, including impaired social cognition, is largely responsible for the

deterioration in social life suffered by patients with psychiatric disorders, such as

schizophrenia and major depressive disorder (MDD). Brexpiprazole

(7-{4-[4-(1-benzothiophen-4-yl)piperazin-1-yl]butoxy}quinolin-2(1H)-one), a novel

serotonin-dopamine activity modulator, was developed to offer efficacious and tolerable

therapy for different psychiatric disorders, including schizophrenia and adjunctive

treatment of MDD. In this study, we investigated whether brexpiprazole could improve

social recognition deficits (one of social cognition deficits) in mice, after administration

of the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (dizocilpine).

Dosing with dizocilpine (0.1 mg/kg) induced significant impairment of social

recognition in mice. Brexpiprazole (0.01, 0.03, 0.1 mg/kg, p.o.) significantly

ameliorated dizocilpine-induced social recognition deficits, without sedation or a

reduction of exploratory behavior. In addition, brexpiprazole alone had no effect on

social recognition in untreated control mice. By contrast, neither risperidone (0.03

mg/kg, p.o.) nor olanzapine (0.03 mg/kg, p.o.) altered dizocilpine-induced social

recognition deficits. Finally, the effect of brexpiprazole on dizocilpine-induced social

recognition deficits was antagonized by WAY-100,635, a selective serotonin 5-HT1A

antagonist. These results suggest that brexpiprazole could improve dizocilpine-induced

social recognition deficits via 5-HT1A receptor activation in mice. Therefore,

9

brexpiprazole may confer a beneficial effect on social cognition deficits in patients with

psychiatric disorders.

2. INTRODUCTION

Cognitive impairment describes a diverse range of deficits, seen in psychiatric

disorders. Of these, impaired social cognition greatly hampers everyday life, resulting

in poor work productivity or underemployment of patients with schizophrenia and

major depressive disorder (MDD) (Tandberg et al, 2011; Tse et al, 2013; Horan et al,

2012; Lo and Siu, 2014). It is generally accepted that reducing social cognitive

dysfunction is an important factor in assisting psychiatric patients to make healthy

adjustments in their social lives (Tandberg et al, 2011; Tse et al, 2013; Horan et al,

2012; Trapp et al, 2013). Current reports suggest that training of social cognition may

help to improve functional outcome in patients with schizophrenia (Henderson, 2013).

To complement this, the development of novel drugs to improve social cognition

deficits in patients with schizophrenia is also imperative.

Social recognition testing is designed to measure the propensity of a mouse to

make contact with a novel rather than familiar mouse. This testing therefore represents a

cognitive model that reflects innate ability to communicate with others (Moy et al,

2004; van der Kooij and Sandi, 2012). Social recognition test in rodents is also one of

the assays for evaluating social cognition in humans (Millan and Bales, 2013). Based on

the N-methyl-D-aspartate (NMDA) hypofunction hypothesis of schizophrenia

(Hashimoto, 2006; 2014; Hashimoto et al, 2013), the NMDA receptor antagonist,

(+)-MK-801 (dizocilpine), is widely used to induce schizophrenia-like behavioral

abnormalities, including positive and negative symptoms and cognitive deficits in

10

rodents (Hashimoto et al, 2009; Karasawa et al, 2008; Rajagopal et al, 2013; Meltzer et

al, 2011; Okamura et al, 2004; Rogoz, 2013; Zhang et al, 2007). Furthermore,

dizocilpine also impairs social interaction and social recognition (Maehara et al, 2011;

Oh et al, 2013; Hikichi et al, 2013).

Brexpiprazole, 7-{4-[4-(1-benzothiophen-4-yl)piperazin-1-yl]butoxy}

quinolin-2(1H)-one, is a novel serotonin-dopamine activity modulator with high affinity

for serotonin, dopamine and noradrenaline receptors (Maeda et al, 2014a). It is a partial

agonist at serotonin 5-HT1A and dopamine D2 receptors, with a relatively equal potency,

and an antagonist at 5-HT2A receptors and adrenergic α1B/2C receptors. Brexpiprazole is

currently under clinical evaluation and expected to show efficacy and tolerability when

used as therapy for different psychiatric disorders, including schizophrenia and

adjunctive treatment for MDD. Very recently, brexpiprazole was shown to improve the

NMDA receptor antagonist phencyclidine-induced cognitive deficits in the novel object

recognition test in rodents (Maeda et al, 2014b; Yoshimi et al, 2014). To date, the effect

of brexpiprazole on social recognition has not been investigated. In this study, we

evaluated the effects of brexpiprazole on dizocilpine-induced social recognition deficits

in mice.

3. EXPERIMENTAL PROCEDURES

3.1. Animals

Male C57BL/6NCrSlc mice (Japan SLC Inc., Shizuoka, Japan) aged between 4

and 5 weeks old were selected as stranger mice, while animals between 8 and 10 weeks

old were used for this study. All mice were housed in groups of five per cage, in a room

maintained at 23 ± 2 °C and 60 ± 10 % humidity, with a 12/12 hour light/dark cycle

11

(lights on at 7:00 a.m.). The mice were given free access to food and water. Animal care

and use were conducted in accordance with the Institutional Guidelines for Animal Care

and Use (Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan).

3.2. Drugs

(+)-MK-801 hydrogen maleate (dizocilpine) was purchased from Sigma-Aldrich

Co., Ltd (Tokyo, Japan). Brexpiprazole, risperidone, olanzapine and WAY-100,635

were synthesized at Otsuka Pharmaceutical Co., Ltd (Tokyo, Japan). Dizocilpine was

dissolved in saline and administered intraperitoneally (i.p.) at 10 ml/kg, twice daily, on

the day before and 30 minutes prior to sociability testing. Brexpiprazole, risperidone,

olanzapine and WAY-100,635 were dissolved in 5% (w/v) gum Arabic and

administered orally (p.o.), at 10 ml/kg, 1 hour prior to sociability testing. The doses of

dizocilpine (0.1 mg/kg) and WAY-100,635 (1.0 mg/kg) were selected from previously

published reports in mice (Hagiwara et al, 2008; Hashimoto et al, 2009; Takatsu et al,

2011; Yoshimi et al, 2014; Zhang et al, 2007). The doses of antipsychotic drugs were

selected based on doses that did not impact locomotion (data not shown).

3.3. Apparatus

The test apparatus consisted of a rectangular, three-chambered box, and a lid with

an attached infrared video camera (O’Hara & Co., Tokyo, Japan). The apparatus was

610W x 400D x 220H mm, and the dividing walls were made from clear Plexiglass,

with small square openings (3 x 5 cm) allowing access into each chamber. The stranger

mouse was enclosed in a small, round wire cage (diameter, 10 cm; height, 12 cm),

allowing olfactory, visual, auditory, and tactile contact, but no deep contact. Using a

CCD camera, measures were taken of the amount of time spent around the wire cage.

The total distance traveled was calculated based on traced mice movement and

12

presented as the locomotor activity in this study. All data were computerized. Activity

was monitored and analyzed using applications based on the public domain NIH Image

or Image J program (developed by Wayne Rasband at the U.S. National Institute of

Mental Health and available on the Internet at http://rsb.info.nih.gov/nih-image/)

(O’Hara & Co., Tokyo, Japan).

3.4. Behavioral procedures

The measurement of sociability and social recognition was performed using the

same procedure described in previous reports (Moy et al, 2007; Nakatani et al, 2009;

Matsuo et al, 2009; Riedel et al, 2009). In this study, we focused on a narrowly defined

set of parameters.

3.4.1. Habituation

Mice were randomly assigned to groups. Mice were first placed in the middle

compartment of the apparatus and allowed to explore freely for 6 minutes. All other

compartments were empty during this habituation period.

3.4.2. Sociability test

An unfamiliar male (stranger) that had no prior contact with the mouse was placed

in one of the side chambers. After the habituation period, the unfamiliar male juvenile

mouse (stranger 1) was placed inside the round wire cage, in one of the side

compartments (randomly selected and counterbalanced for each group). The opposite

compartment was empty. The mice were able to freely explore all three compartments

of the apparatus for 6 minutes. The time spent around cages (stranger 1 or empty) was

calculated as direct contact.

3.4.3. Social recognition test

After sociability testing, a novel unfamiliar juvenile mouse (stranger 2) was

13

placed inside a new round wire cage and introduced to the ‘empty compartment’ of the

apparatus. Mice were confined to the central compartment and were able to explore

freely in all compartments for 10 min.

The recognition index (RI) was calculated using parameters measured during

social recognition sessions and was defined as the quotient of the time the mouse spent

around the novel juvenile (stranger 2) divided by the sum of the time spent around the

familiar (stranger 1) and novel (stranger 2) juvenile mice.

3.5. Statistical analyses

All statistical analyses were performed using Prism 5 (GraphPad Software Inc., La

Jolla, USA). Data are presented as mean ± standard error of the mean (S.E.M.). To

determine the effects of drug treatment, one-way ANOVA, followed by the post hoc

Bonferroni test (for RI and total distance) or a two-tailed paired t-test (for RI and time

spent around cage), was used. P values of less than 0.05 were considered statistically

significant.

4. RESULTS

4.1 Effects of brexpiprazole on dizocilpine-induced social recognition deficits

Mice in all groups spent significantly more time around stranger 1, than the empty

area in sociability sessions (data not shown). A one-way ANOVA on RI in social

recognition session showed a significant effect of treatment (F (4, 56) = 5.83, p <

0.001)(Figure 1A). Saline-treated control mice had a significant preference for

spending more time with the novel unfamiliar stranger 2, in comparison with familiar

stranger 1, in the social recognition session (Figure 1B). Treatment with dizocilpine

(0.1 mg/kg, i.p.) significantly decreased the total time spent around stranger 2 in social

14

recognition sessions. A paired t-test showed a significant (p < 0.01) impairment of RI in

the dizocilpine-treated group. Mice spent the same amount of time interacting with

stranger 1 and stranger 2 mice (Figure 1A, and 1B). The total distance moved

(exploratory behavior), indicative of locomotor activity, was significantly increased by

dizocilpine administration (Figure 1C).

Next, we examined whether brexpiprazole attenuated dizocilpine-induced social

recognition deficits in mice. Neither dizocilpine (0.1 mg/kg, i.p.) nor brexpiprazole

(0.01, 0.03 and 0.1 mg/kg, p.o) altered the amount of time spent around stranger 1 mice

in sociability sessions (data not shown). However, at all three doses of brexpiprazole,

mice treated with brexpiprazole showed a significant preference for spending more time

with novel unfamiliar stranger 2 mice, compared with familiar stranger 1 mice in social

recognition sessions (Figure 1B), and additionally, impaired RI was significantly

restored by treatment with brexpiprazole at the same doses used in sociability testing

(Figure 1A). Furthermore, brexpiprazole (0.01, 0.03 and 0.1 mg/kg) did not temper the

increased locomotor activity induced by dizocilpine in social recognition sessions

(Figure 1C).

4.2 Brexpiprazole alone did not affect social recognition or exploratory behavior

We examined the effect of brexpiprazole on social recognition in untreated control

mice. Treatment with brexpiprazole (0.01, 0.03 and 0.1 mg/kg) had no effect on the

time spent around stranger 1 mice in either sociability (data not shown) or social

recognition sessions (Figure 1D, and 1E). Brexpiprazole did not alter RI in social

recognition sessions (Figure 1D), nor did it have an effect on exploratory behavior

during either session (Figure 1F).

15

Figure 1. Effects of brexpiprazole on dizocilpine-induced social recognition

deficits

Dizocilpine (0.1 mg/kg, i.p.) or saline (10 ml/kg, i.p.) was administered twice daily on the day

before and 30 minutes prior to sociability testing. Brexpiprazole (0.01, 0.03, 0.1 mg/kg, p.o.) or

vehicle (5% (w/v) gum Arabic, p.o.) was administrated 1 hour prior to sociability testing. (A)

Social recognition was measured by the recognition index (RI), that is, the quotient of time the

subject mouse spent around the novel juvenile (stranger 2) mouse divided by the sum of the time

spent around the familiar (stranger 1) and novel juvenile (stranger 2) mice during social

recognition sessions. (B) Duration of time spent around each stranger during the social

recognition sessions and, (C) exploratory behavior during social recognition sessions. Values are

the mean S.E.M (n = 11-15). *p < 0.05, **p < 0.01, ***p < 0.001 compared with dizocilpine +

vehicle group (A and C, one-way ANOVA, followed by post hoc Bonferroni test). **p < 0.01,

***p < 0.001 compared with stranger 1 mice (B, two-tailed paired t-test). Brexpiprazole (0.01,

0.03, 0.1 mg/kg, p.o.) or vehicle (5% (w/v) gum Arabic, p.o.) was administrated 1 hour prior to

sociability testing. The effects of brexpiprazole on the RI (D), duration of time spent around each

stranger mouse (E), and exploratory behavior (F), during social recognition sessions. Values are

the mean S.E.M (n = 7-14). *p < 0.05, **p < 0.01, ***p < 0.001 compared with stranger 1 (E,

two-tailed paired t-test). N.S.: not significant.

16

4.3 Risperidone and olanzapine had no effect on dizocilpine-induced social

recognition deficits

We examined whether the atypical antipsychotic drugs, risperidone and

olanzapine, affected dizocilpine-induced social recognition deficits in mice. A one-way

ANOVA on RI in social recognition session showed a significant effect of treatment (F

(2, 31) = 4.60, p < 0.05)(Figure 2A). Pot-hoc analysis showed that risperidone (0.03

mg/kg, p.o.) did not ameliorate dizocilpine-induced social recognition deficits (Figure

2A, 2B), and dizocilpine-induced hyperlocomotion in the recognition sessions (Figure

2C). When administered alone, risperidone (0.03 mg/kg, p.o.) significantly decreased

both RI and locomotor activity in untreated control mice (Figure 2D, 2E, and 2F).

17

A one-way ANOVA on RI in social recognition session showed a significant

effect of treatment (F (2, 27) = 7.72, p < 0.01)(Figure 3A). Post-hoc analysis showed that

olanzapine (0.03 mg/kg, p.o.) did not affect time spent around stranger 2 mice, nor did it

reverse dizocilpine-induced impairment of the RI (Figure 3A, and 3B). Furthermore,

olanzapine had no effect on dizicilpine-induced hyperlocomotion in the recognition

sessions (Figure 3C). Olanzapine (0.03 mg/kg, p.o.) alone was incapable of altering RI,

the time spent around stranger 2 mice, or exploratory behavior in untreated control mice

(Figure 3D, 3E, and 3F).

Figure 2. Effect of risperidone on dizocilpine-induced social recognition deficits


before and 30 minutes prior to sociability testing. Risperidone (0.03 mg/kg, p.o.) or vehicle (5%

(w/v) gum Arabic, p.o.) was administrated 1 hour prior to sociability testing. The effects of

risperidone on RI (A), duration of time spent around each stranger mouse (B), and exploratory

behavior (C) during social recognition sessions. Values are the mean S.E.M (n = 11 or 12). *p

< 0.05 compared with dizocilpine + vehicle group (A and C, one-way ANOVA, followed by post

hoc Bonferroni test). N.S.: not significant. **p < 0.01 compared with stranger 1 mice (B,

two-tailed paired t-test). Risperidone (0.03 mg/kg, p.o.) or vehicle (5% (w/v) gum Arabic, p.o.)

was administrated 1 hour prior to sociability testing. The effects of risperidone on RI (D),

duration of time spent around each stranger mouse (E) and exploratory behavior (F) during

social recognition sessions. Values are the mean S.E.M (n=7). *p < 0.05 compared with the

vehicle-treated control group (D and F, two-tailed paired t-test). *p < 0.05, ***p < 0.001

compared with stranger 1 mice (E, two-tailed paired t-test).

18

Figure 3. Effect of olanzapine on dizocilpine-induced social recognition deficits

Dizocilpine (0.1 mg/kg, i.p.) or saline (10 ml/kg, i.p.) administered twice daily on the day before

and 30 minutes prior to sociability testing. Olanzapine (0.03 mg/kg, p.o.) or vehicle (5% (w/v)

gum Arabic, p.o.) was administrated 1 hour prior to sociability testing. The effects of olanzapine

on RI (A), duration of time spent around each stranger mouse (B) and exploratory behavior (C)

during social recognition sessions. Values are the mean S.E.M (n=10). **p < 0.01, ***p <

0.001 compared with vehicle-treated control group for (A) and (C) (one-way ANOVA, followed

by post hoc Bonferroni test). ***p < 0.001 compared with stranger 1 mice for (B) (two-tailed

paired t-test). Olanzapine (0.03 mg/kg, p.o.) or vehicle (5% (w/v) gum Arabic, p.o.) was

administrated 1 hour prior to sociability testing. The effects of olanzapine on RI (D), duration of

time spent around each stranger mouse (E) and exploratory behavior (F) during social

recognition sessions. Values are the mean S.E.M (n=7). **p < 0.01, ***p < 0.001 compared

with stranger 1 mice for (E) (two-tailed paired t-test). N.S.: not significant.

19

4.4 Role of the 5-HT1A receptor in brexpiprazole's effects on dizocilpine-induced

social recognition deficits

To investigate the function of serotonin 5-HT1A receptors in the pharmacological

effect of brexpiprazole on dizocilpine-induced social recognition deficits, we examined

the effect of WAY-100,635, a selective 5-HT1A receptor antagonist. A one-way

ANOVA on RI in social recognition session showed a significant effect of treatment (F

(4, 57) = 7.82, p < 0.0001)(Figure 4A). Post-hoc analysis showed that WAY-100,635 (1.0

mg/kg, p.o.) significantly antagonized the effect of brexpiprazole on

dizocilpine-induced social recognition deficits (Figure 4A, and 4B). Treatment with

WAY-100,635 alone did not alter RI and locomotion in dizocilpine-treated mice

(Figure 4A, and 4C). Furthermore, treatment with WAY-100,635 alone did not affect

RI, the time spent around stranger 2 mice, or exploratory behavior in untreated control

mice (Figure 4D, 4E and 4F).

20

Figure 4. Effect of a 5-HT1A antagonist on brexpiprazole's action in the

dizocilpine-induced social recognition deficits model


before and 30 minutes prior to sociability testing. Brexpiprazole (Brex: 0.03 mg/kg, p.o.),

WAY-100,635 (WAY: 1 mg/kg, p.o.), or vehicle (5% (w/v) gum Arabic, p.o.) was administrated 1

hour prior to sociability testing. The effects of brexpiprazole on RI (A), duration of time spent

around each stranger mouse (B) and exploratory behavior (C) during social recognition sessions.

Values are the mean S.E.M (n = 11-13). *p < 0.05, ***p < 0.001 compared with dizocilpine

plus vehicle group, ###p < 0.001 compared with the dizocilpine plus brexpiprazole-treated group

(A, one-way ANOVA, followed by post hoc Bonferroni test). *p < 0.05, ***p < 0.001 compared

with stranger 1 mice (B, two-tailed paired t-test). N.S.: not significant. WAY-100,635 (WAY: 1.0

mg/kg, p.o.), or vehicle (5% (w/v) gum Arabic, p.o.) was administrated 1 hour prior to sociability

testing. The effects of WAY-100,635 on RI (D), duration of time spent around each stranger

mouse (E) and exploratory behavior (F) during social recognition sessions were shown. Values

are the mean S.E.M (n = 7). ***p < 0.001 compared with stranger 1 mice (E, two-tailed paired

t-test). N.S.: not significant.

21

5. DISCUSSION

The two major findings of this study are that the novel serotonin-dopamine

activity modulator brexpiprazole, but not risperidone or olanzapine, improved social

recognition deficits in mice after the administration of dizocilpine, and that 5-HT1A

receptors are critical to this beneficial effect. Antipsychotic drugs, such as risperidone

and olanzapine, are known to show limited efficacy in cognitive impairment, including

social recognition in schizophrenia and depression (Sergi et al, 2007; Krakowski and

Czobor, 2011; Suzuki et al, 2011; Remberk et al, 2012), despite improving various

cognitive functions in rodent disease models (Wang et al, 2007; Gumuslu et al, 2013;

Mutlu et al, 2011; 2012; Wolff and Leander, 2003). It is of considerable clinical

importance to find new ways of improving social recognition deficits in patients with

psychiatric diseases, such as schizophrenia and MDD, because of their huge negative

impact on the social functioning of patients (Tandberg et al, 2011; Tse et al, 2013;

Horan et al, 2012). There are noted discrepancies in antipsychotic drug effects between

rodents and humans, increasing the need for further detailed clinical studies on the

effects of brexpiprazole on social recognition in patients with psychiatric diseases.

Social recognition testing is a paradigm capable of evaluating social recognition,

based on the propensity of an individual mouse to spend more time with an unfamiliar

mouse than with a familiar mouse (Riedel et al, 2009; Nakatani et al, 2009). In other

cognitive tests performed in rodents, mice discriminate objects by vision in the novel

objective recognition test, by hearing in the fear conditioning test and by odor in the

social transmission food preference test (Hashimoto et al, 2005; 2007; Boix-Trelis et al,

2007; Amann et al, 2010). However, since social recognition testing incorporates

various factors such as visual, auditory and olfactory function and some tactile stimuli

22

needed for mouse sociability, this test provides a comprehensive evaluation of

cognition in rodents, compared with other cognitive tests (Nadler et al, 2004; Moy et al,

2004; 2007). Therefore, the social recognition test in mice may represent a translational

rodent model for human social cognition, since the model utilizes multiple aspects of

mice socialization.

Here, we evaluated social recognition using an automated three-chambered

apparatus system. This apparatus is capable of measuring the time and distance a subject

mouse covers in approaching a novel mouse, thereby evaluating the subject’s

motivation to interact with the novel mouse. Furthermore, this system can also measure

exploratory behavior by producing a representative trace of locomotor activity. This

means that effects on locomotion, such as, sedation by a drug, can be easily detected.

Since stranger mice were isolated individually in a wire cage, the subject mouse could

interact with a stranger mouse without attack or aversive stimuli. This makes the social

recognition test in mice a potential tool for assessing the efficacy of new drugs on social

recognition.

We found that the 5-HT1A antagonist, WAY-100,635, reversed the effects of

brexpiprazole on dizocilpine-induced social recognition deficits, indicating that 5-HT1A

receptors are critical to brexpiprazole’s mechanism of action. A report suggests that

5-HT1A receptor agonism is related to social recognition in rats (Millan et al, 2004). In

addition, it is reported that 5-HT1A receptors are important in the action of other

antipsychotic drugs (perospirone, aripiprazole, blonanserin), when tested in a

phencyclidine-induced cognitive deficits model (Hagiwara et al, 2008; Nagai et al,

2009; Horiguchi and Meltzer, 2012; 2013). In contrast, WAY-100,635 could alleviate

cognitive deficits in monkeys after administration of dizocilpine (Harder and Ridley,

23

2000), suggesting pro-cognitive effect of 5-HT1A receptor antagonist. However, in this

study, WAY-100,635 alone was no effect in social recognition in both dizocilpine-treated

and vehicle-treated control mice. Further studies of the effects of 5-HT1A receptor

agonists and antagonists on social recognition are needed. Previously, Sumiyoshi et al.

(2001a; 2001b; 2007) reported that treatment with tandospirone (or buspirone), 5-HT1A

receptor agonists, improved cognition including executive function and verbal learning

in patients with schizophrenia, indicating a key role of 5-HT1A receptor in cognition in

patients (Meltzer and Sumiyoshi, 2008; Sumiyoshi et al, 2013). Taken together, it seems

that brexpiprazole can ameliorate cognitive deficits associated with NMDA receptor

dysfunction, via its 5-HT1A agonistic activity. Further studies are needed to clarify the

effects of other antipsychotic drugs with 5-HT1A agonistic activity, on social cognition

in patients.

Previous reports demonstrated that 5-HT1A receptor antagonists impair sociability

in rodents, and that 5-HT1A receptor agonists improved sociability in the social

interaction in rodents (File and Seth, 2003; Bruins Slot et al, 2005; Snigdha and Neil,

2008; Gould et al, 2011). These findings suggest an important role of 5-HT1A receptors

in social behaviors. A recent study showed that the rewarding properties of social

interaction in mice require the coordinated activity of oxytocin and 5-HT1B receptor in

the nucleus accumbens, which are implicated in the social cognitive dysfunction in

patients with a number of psychiatric diseases (Dölen et al, 2013). Since brexpiprazole

has a moderate affinity at 5-HT1B receptor (Ki = 32 nM) (Maeda et al, 2014a), it is

possible that 5-HT1B receptor may, in part, be involved in the mechanisms of action of

brexpiprazole on social recognition deficits. Nonetheless, further studies on the role of

5-HT1B receptors in the mechanisms of action of brexpiprazole would be needed to

24

confirm this hypothesis.

In conclusion, brexpiprazole ameliorated social recognition deficits in mice after

administration of dizocilpine, and the 5-HT1A receptor antagonist WAY-100,635

reversed the effects of brexpiprazole in this model. Our results imply that brexpiprazole

could potentially serve as a therapeutic drug to treat social cognitive deficits in patients

with schizophrenia and MDD.

25

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34

III. CHAPTER 2


phencyclidine-induced cognitive deficits in mice: a role for serotonin 5-HT1A

receptors.

1. ABSTRACT

Brexpiprazole, a serotonin-dopamine activity modulator, is currently being

tested in clinical trials as a new therapy for a number of neuropsychiatric diseases,

including schizophrenia and major depressive disorder. Accumulating evidence suggests

that 5-hydroxytryptamine (5-HT)1A receptors play a role in cognition. This study was

undertaken to examine whether brexpiprazole, a novel drug with 5-HT1A receptor partial

agonism, could improve cognitive deficits in mice, induced by repeated administration

of the N-methyl-D-aspartate (NMDA) receptor antagonist, phencyclidine (PCP).

Subsequent subchronic (14 days) oral administration of brexpiprazole (0.3, 1, or 3.0

mg/kg/day) significantly attenuated PCP (10 mg/kg/day for 10 days)-induced cognitive

deficits in mice, in a dose-dependent manner. The effects of brexpiprazole (3.0 mg/kg)

were significantly antagonized by co-administration of the selective 5-HT1A receptor

antagonist, WAY-100,635 (1.0 mg/kg), although WAY-100,635 alone was not effective

in this model. These findings suggest that brexpiprazole can ameliorate PCP-induced

cognitive deficits in mice via 5-HT1A receptors. Therefore, brexpiprazole could

ameliorate cognitive deficits as seen in schizophrenia and other neuropsychiatric

diseases.

35

2. INTRODUCTION

Cognitive deficits are a core feature in people with schizophrenia, leading to both

vocational and social disabilities (Coyle and Tsai, 2004; Freedman, 2003; Green et al,

2004; Niitsu et al, 2012; Sumiyoshi et al, 2013; Yoshida et al, 2012b). Current evidence

suggests that dysfunctional glutamatergic neurotransmission, via N-methyl-D-aspartate

(NMDA) receptors is instrumental in the pathophysiology of schizophrenia (Coyle and

Tsai, 2004; Hashimoto et al, 2004; 2005b; 2013; Heresco-levy and Javitt, 1998; Krystal

et al, 1999; Javitt and Zukin, 1991; Javitt et al, 2012). Antagonists at the NMDA

receptor, such as phencyclidine (PCP), are known to induce schizophrenia-like

symptoms, including cognitive deficits in healthy subjects (Javitt and Zukin, 1991). This

has led to the use of sub-chronic administration of PCP, as an animal model of cognitive

deficits in schizophrenia (Fujita et al, 2008; Hagiwara et al, 2008; Hashimoto et al,

2005a; 2006; 2007a; 2007b; Kunitachi et al, 2009; Javitt et al, 201; Jentsch and Roth,

1999; Tanibuchi et al, 2009). In the novel object recognition test (NORT), PCP (10

mg/kg/day for 10 days)-induced cognitive deficits were significantly ameliorated by

subsequent, subchronic (14 days) doses of clozapine, but not haloperidol (Hashimoto et

al, 2005a). These findings suggest that this reversal of PCP-induced cognitive deficits as

measured by the NORT, may represent a potential animal model of atypical

antipsychotic activity, in the amelioration of schizophrenia related cognitive deficits

(Hagiwara et al., 2008; Hashimoto et al, 2005a; 2006; 2007a; 2007b; 2007c; Tanibuchi

et al, 2009).

Several lines of evidence show that 5-hydroxytryptamine (5-HT)1A receptors play

a role in the pathophysiology of neuropsychiatric diseases including schizophrenia, and

that 5-HT1A receptors are important targets for emotion and cognition (Bantick et al,

36

2001; Meltzer, 1999; Ogren et al, 2008; Sumiyoshi et al, 2013). Early evidence comes

from findings that the density of 5-HT1A receptor binding is altered in the hippocampus

and cerebral cortex of postmortem brains from schizophrenia patients (Burnet et al,

1996; Gurevich and Joyce, 1997; Joyce et al, 1993; Lopez-Figueroa et al, 2004). A

positron emission tomography study demonstrated increased cortical 5-HT1A receptor

binding in drug-naive patients with schizophrenia (Borg, 2008; Tauscher et al, 2002). In

contrast, other studies found no changes in 5-HT1A receptor density between

schizophrenia and control samples (Bantick et al, 2004; Borg, 2008; Frankle et al, 2006).

Second, atypical antipsychotic drugs, such as clozapine, ziprasidone, aripiprazole and

quetiapine are all partial 5-HT1A receptor agonists, a property which may be relevant for

their therapeutic actions in schizophrenia (Jordan et al, 2002; Newman-Tancredi et al,

2001; Rollema et al, 2000; Sprouse et al, 1999). Third, Sumiyoshi and colleagues

(2001a; 2001b) reported that adjunctive treatment with tandospirone, a selective 5-HT1A

receptor agonist, induced improvements in some types of memory function, as well as

cognitive performance in schizophrenia. Taken together, findings to date suggest

5-HT1A receptor agonists as potential therapeutic drugs for treating cognitive deficits in

schizophrenia (Meltzer and Sumiyoshi, 2008; Newman-Tancredi, 2010; Yoshida et al.,

2012b; Sumiyoshi et al, 2013).

Brexpiprazole,

7-{4-[4-(1-benzothiophen-4-yl)piperazin-1-yl]butoxy}quinolin-2(1H)-one (Figure 1), is

a potent partial agonist at human 5-HT1A (Ki = 0.12 nM), and dopamine D2L (Ki = 0.3

nM), and an antagonist at 5-HT2A receptors (Ki = 0.47 nM)(Maeda et al, 2014).

Considering the high affinity of brexpiprazole for 5-HT1A receptors, it would be of

interest to study the effects of brexpiprazole on PCP-induced cognitive deficits in mice.

37

In the present study, using the NORT, we examined the effects of subchronic treatment

(14 days) with brexpiprazole on cognitive deficits in mice, induced after repeated

administration of PCP. Furthermore, we examined the role of 5-HT1A receptors in the

action of brexpiprazole on the PCP-induced cognitive deficit model.

3. EXPERIMENTAL PROCEDURES

3.1. Animals

Male ICR mice (6 weeks old) weighing 25–30 g were purchased from SLC Japan

(Hamamatsu, Shizuoka, Japan). The mice were housed in clear polycarbonate cages

(22.5×33.8×14.0 cm) in groups of 5 or 6 individuals under a controlled 12/12-h

light–dark cycle (light from 7:00 AM to 7:00 PM), with the room temperature kept at 23

± 1°C and humidity at 55 ± 5%. The mice were given free access to water and food

pellets specifically designed for mice. The experimental procedure was approved by the

Animal Care and Use Committee of Chiba University Graduate School of Medicine.

3.2. Drugs

PCP hydrochloride was synthesized in our laboratory at Chiba University.

Brexpiprazole was provided from Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan).

WAY-100,635 maleate, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-

(2-pyridinyl)cyclohexanecarboxamide maleate salt), was purchased from Sigma-Aldrich

Corporation (St. Louis, MO, USA). Other drugs were purchased from commercial

sources.

38

3.3. Drug administration

Saline (10 ml/kg/day) or PCP (10 mg/kg/day expressed as a hydrochloride salt)

was administered subcutaneously (s.c.) for 10 days (once daily on days 1-5, 8-12) with

no treatment on days 6, 7, 13, and 14 (Figure 5). The treatment schedule was designed

in the manner of a previous PCP-induced cognitive deficit model (Hashimoto et al,

2005a; 2006; 2007a; 2007b; 2007c). After the final administration of saline or PCP,

vehicle (10 ml/kg/day; 0.5% methylcellulose), brexpiprazole (0.3, 1, or 3.0 mg/kg/day),

brexpiprazole (3.0 mg/kg/day) plus WAY-100,635 (1 mg/kg/day), or WAY-100,635 (1

mg/kg/day) alone was administered orally for 14 consecutive days (once daily on days

15-28) (Figure 5). The dose (1 mg/kg) chosen for WAY-100,635 was selected because

this dose was found to attenuate PCP-induced cognitive deficits via 5-HT1A receptor

mechanisms in vivo (Hagiwara et al, 2008).

39

3.4. Novel object recognition test (NORT)

The NORT was administered as previously reported (Hashimoto et al, 2005a;

2006; 2007a; 2007b; 2007c). As shown in Figure 5, the training session of NORT was

carried out 1 day (day 29) after the final administration of vehicle, brexpiprazole, or

WAY-100,635. The apparatus for this task consisted of a black open-field box

(50.8×50.8×25.4 cm). Before the test, mice were habituated to the box for 3 days.

During the training session, two objects (various objects were used that differed with

respect to shape and color, but that were similar in size) were placed in the box at a

35.5-cm distance from each other, and in a symmetrical fashion, and each animal was

Figure 5. Treatment schedule

Saline (10 ml/kg/day) or PCP (10 mg/kg/day) was administered subcutaneously (s.c.) to mice for

10 days (once daily, on days 1-5, 8-12). Three days (day 15) after the final dose of saline or PCP,

vehicle (10 ml/kg/day), brexpiprazole (0.3, 1.0, or 3.0 mg/kg/day), brexpiprazole (3.0 mg/kg/day)

plus WAY-100,635 (1.0 mg/kg/day), or WAY-100,635 (1.0 mg/kg/day) was administered orally,

for 14 consecutive days (once daily on days 15-28). On days 29 and 30, the novel object

recognition test (NORT) was carried out.

40

allowed to explore the interior of the box for 10 min (5 min x 2). The animals were

considered to be investigating the object when the head of the animal was either facing

the object and was located within an inch of the object, or if any part of the body, except

for the tail, was touching the object. The time that the mice spent exploring each object

was recorded. After the training session, the mice were immediately returned to their

home cages, and the box and objects were cleaned with 75% ethanol to avoid any

possible pheromonal cues. The retention test session was carried out 1 day after the

respective training sessions. During each retention test session, each mouse was placed

back into the same box it had previously encountered, but in which one of the two

objects used during training had been replaced by a novel object. The mice were then

allowed to freely explore the interior for 5 min, and the time spent exploring each object

was recorded. Throughout the experiments, the objects were used in a counter-balanced

manner in terms of their physical complexity. In order to measure memory performance,

a preference index was used, i.e., the ratio of the amount of time the mouse spent

exploring any one of the two objects (training session) or the novel object (retention

session) to the total time spent exploring both objects.

3.5. Statistical analysis

The data are expressed as mean S.E.M. Statistical analysis was performed using

one-way analysis of variance (ANOVA) and the post hoc Bonferroni test. The P values

of less than 0.05 were considered statistically significant.

41

4. RESULTS

4.1. Effects of brexpiprazole on PCP-induced cognitive deficits in mice

In the NORT, repeated administration of PCP (10 mg/kg/day for 10 days) led to

cognitive deficits in mice, consistent with previous reports (Hashimoto et al, 2005a;

2006; 2007a; 2007b). During the training session, there were no differences in

exploratory preferences among the eight groups of mice (F (7, 96) = 0.444, p=0.872)

(Figure 6A). Subsequent subchronic (14 days) administration of brexpiprazole

significantly attenuated PCP-induced cognitive deficits in mice, in a dose-dependent

manner. However, during the test session, ANOVA analysis revealed significant

differences in the exploratory preferences among the eight groups (F (7, 96) = 15.52, p

< 0.001) (Figure 6B). Post hoc Bonferroni test results indicated that subchronic dosing

with brexpiprazole increased exploratory preferences in the PCP-treated group, in a

dose dependent manner (Figure 6B). In addition, the effect of brexpiprazole (3.0

mg/kg) on PCP-induced cognitive deficits was significantly antagonized by

co-administration of the selective 5-HT1A receptor antagonist, WAY-100,635 (1.0

mg/kg/day) (Figure 6B). Moreover, a 14 day dosing with WAY-100,635 (1.0

mg/kg/day) alone did not reverse PCP-induced cognitive deficits in mice (Figure 6B).

Neither did a 14 day dosing with brexpiprazole (3.0 mg/kg/day) alone alter exploratory

preferences in control (saline-treated) mice (Figure 6B).

42

Figure 6. Effects of subchronic administration of brexpiprazole on PCP-induced

cognitive deficits in mice

Treatments were performed as shown in Figure 2. On days 29 and 30, the NORT was performed.

Values are the mean S.E.M (n = 10-16). N.S.: not significant, Brex: brexpiprazole, WAY:

WAY-100,635. **P < 0.01, ***P < 0.001 compared with the PCP plus vehicle-treated group. ###

P

< 0.001 compared with the PCP plus brexpiprazole (3.0 mg/kg)-treated group.

43

5. DISCUSSION

This study found that brexpiprazole was able to ameliorate PCP-induced

cognitive deficits in mice, via 5-HT1A receptors. Previously, we reported that

PCP-induced cognitive deficits as demonstrated on the NORT could be improved by

subsequent subchronic (14 days) administration of clozapine, but not haloperidol. This

suggested that the reversal of these deficits may represent a potential animal model of

atypical antipsychotic activity, for use in the ongoing investigations aimed at restoring

cognitive deficits in schizophrenia (Hashimoto et al, 2005a). Given the role of 5-HT1A

receptors in cognition (Bantick et al, 2001; Meltzer, 1999; Ogren et al, 2008; Yoshida et

al., 2012b; Sumiyoshi et al, 2013), it is likely that brexpiprazole confers a beneficial

effect on cognitive deficits in patients with neuropsychiatric diseases, such as

schizophrenia.

In an earlier report, we found that repeated dosing with PCP (10 mg/kg/day for 10

days) significantly reduced the density of 5-HT1A receptors in mouse hippocampus,

although the precise mechanisms underlying this action are currently unknown

(Hagiwara et al., 2008). Furthermore, we found that repeated PCP administration

significantly altered the response to 8-OH-DPAT (8-hydroxy-2-(dipropylamino)tetralin

hydrobromide: a 5-HT1A agonist)-induced hypothermia in mice (Hagiwara et al., 2008).

We also reported that perospirone, an antipsychotic drug with 5-HT1A receptor agonism,

could attenuate PCP-induced cognitive deficits via 5-HT1A receptors (Hagiwara et al.,

2008). Together, these findings imply that altered 5-HT1A receptor function resulting

from repeated PCP treatment may add to the dysregulation of glutamatergic

neurotransmission in the brain.

Sumiyoshi et al. (2001a; 2001b) reported that adjunctive treatment with the

44

5-HT1A receptor agonist, tandospirone, improves certain types of memory function, as

well as cognitive performance in patients with schizophrenia. In a later study,

Sumiyoshi et al. (2007) also reported that adjunctive treatment with buspirone, a widely

available 5-HT1A receptor agonist, significantly enhanced attention compared with the

placebo control group, highlighting a possible benefit for buspirone augmentation of

atypical antipsychotic drugs, to enhance attention. These findings suggest that 5-HT1A

receptor agonists, including brexpiprazole, may be useful for treating cognitive deficits

in schizophrenia.

Cognitive deficits are well documented in patients with other neuropsychiatric

diseases, including major depressive disorder (Porter et al., 2003; Hindmarch and

Hashimoto, 2010; Hasselbalch et al., 2011; Yoshida et al., 2012a), bipolar disorder

(Harvey et al., 2010; Bourne et al., 2013), and substance abuse (Dean et al., 2013). In

some ways, rodent NORT performance is analogous to human declarative (episodic)

memory, one of the seven cognitive domains impaired in patients with schizophrenia

(Rajagopal et al., 2014). It is therefore possible that brexpiprazole could be an equally

useful therapeutic drug in these patients.

In conclusion, our findings showed that PCP-induced cognitive deficits in mice

could be improved by subchronic treatment with brexpiprazole. Therefore,

brexpiprazole which acts as a 5-HT1A receptor partial agonist, could potentially serve as

a therapeutic drug for the treatment of cognitive deficits in patients with schizophrenia

and other neuropsychiatric diseases.

45

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54

IV. CONCLUSION

Brexpiprazole improved social recognition deficits impaired by dizocilpine in

social recognition test. This effect was antagonized by 5-HT1A receptor antagonist

WAY-100,635, suggesting that brexpiprazole can improve social recognition deficits

impaired by N-methyl-D-aspartate antagonist via 5-HT1A agonistic activity. Additionally,

brexpiprazole improved cognitive deficits impaired by PCP in NORT. This effect was

also antagonized by WAY-100,635, suggesting that brexpiprazole can improve cognitive

deficits impaired by N-methyl-D-aspartate antagonist throughout 5-HT1A agonistic

activity. Taken together, these studies suggest that brexpiprazole has ability to improve

cognitive deficits in animal models of psychiatric disorders. Therefore, brexpiprazole

would have beneficial effects for cognitive deficits in the patients with a number of

psychiatric disorders.

55

V. ACKNOWLEGEMENT

I owe my gratitude to all the people who helped me, though it will not be enough

to express my gratitude in words. First of all, I would like to gratefully and sincerely

thank my supervisor Professor Kenji Hashimoto for his continuous guidance and

understanding. I have learned a lot from him and felt motivated by his thoughtful

message. He provided me a new challenge and gave me a chance to grow as a

researcher. Additionally, I also gratefully acknowledge the work of past and present

members in his laboratory. Especially, Dr. Yuko Fujita and Dr. Tamaki Ishima

supported my work and gave me warm words of encouragement. I also appreciate the

clerical support of Ms. Rie Kamijo.

I am also deeply grateful to Dr. Tetsuro Kikuchi for actively supporting attempts

to my taking a doctoral degree. Special thanks are also given to Dr. Takashi Futamura

for helping me to find my supervisor. He always gave me valuable advice and a

precious time on discussions. I also express my gratitude to Dr. Kenji Maeda for telling

me about pharmacological profile of brexpiprazole and helpful discussions. I really

hope brexpiprazole will help many patients to improve their symptoms. I would also

like to thank Dr. Yuta Ohgi for helpful advice and telling me details for completing a

doctoral degree. I also convey my gratitude to the members in Otsuka pharmaceutical

company who gave me warm and useful messages. I really feel that I want to contribute

to development of a novel drug with them.

On more personal note, I would like to thank my husband Mr. Nozomu Yoshimi,

my father Mr. Akinori Yoshimi, my mother Mrs. Takako Yoshimi, my two sisters Mrs.

Naoko Maeda and Mrs. Masako Abe. Without their love, support and constant

encouragement, this work would not have been possible.

56

European Neuropsychopharmacology vol. 25 No. 3

平成27年 3月1日公表済

Pharmacology Biochemistry and Behavior vol. 124

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