oRIgINAL PAPERS

original papers
Adv Clin Exp Med 2012, 21, 3, 307–312
ISSN 1899–5276
© Copyright by Wroclaw Medical University
Ramin Hajikhani1, Abbas Ahmadi2, Nima Naderi3, Kayvan Yaghoobi3,
Zahra Shirazizand3, Nazereh M. Rezaee2, Babak N. Niknafs4
Effect of Phencyclidine Derivatives on Anxiety-like
Behavior Using an Elevated-plus Maze Test in Mice*
Ocena wpływu pochodnych fencyklidyny na zachowania lękowe
za pomocą uniesionego labiryntu krzyżowego u myszy
Department of Physiology, Faculty of Science, Islamic Azad University, Karaj branch, Karaj, Iran
Department of Chemistry, Faculty of Science, Islamic Azad University, Karaj branch, Karaj, Iran
3
Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4
Islamic Azad University, Karaj branch, Karaj, Iran
1
2
Abstract
Objectives. The study attempted to investigate the anti-anxiety activities of Phencyclidine (1-(1-phenylcyclohexyl)
piperidine, PCP, I) and some of its derivatives (M, F, L, B, S, P) with the elevated-plus maze (EPM) Test.
Material and Methods. Phencyclidine and its derivatives (M, F, L, B, S, P) were administrated intraperitoneally
(i.p.) at a dose of 10 mg/kg to male mice. Anxiety-like behaviors were assessed using the elevated-plus maze test.
Results. EPM results revealed an increase in open arms time spent after applying PCP and M, L, P, and B compounds at the administered dosage. Moreover, an increase in the number of open arm entries was observed with
M, P, and B compounds. The P, B and S compounds increased the locomotion of animals, too, which might be
considered as the side effect to the compounds.
Conclusions. Considering the elevated-plus maze results, it was concluded that M and L compounds could be
considered as a potential anxiolytic with less side effects due to a probable high electron donation of the methoxy
group, as well as the hydrophilic properties of hydroxyl groups on these compounds (Adv Clin Exp Med 2012,
21, 3, 307–312).
Key words: phencyclidine derivatives, anxiety-like behavior, elevated-plus maze test, mice.
Streszczenie
Cel pracy. Zbadanie przeciwlękowego działania fencyklidyny (1-(1- fenylocykloheksylo)piperydyny – PCP, I)
i niektórych jej pochodnych (M, F, L, B, S, P) za pomocą uniesionego labiryntu krzyżowego (EPM).
Materiał i metody. Fencyklidynę i jej pochodne (M, K, L, B, S, P) podawano dootrzewnowo (ip) w dawce 10 mg/kg
samcom myszy. Zachowania lękowe oceniano za pomocą uniesionego labiryntu krzyżowego.
Wyniki. Badanie EPM wykazało dłuższy czas pobytu na ramieniu otwartym po zastosowaniu PCP i związków M,
L, P i B w podanej dawce. Ponadto zaobserwowano większą liczbę wejść na ramiona otwarte po podaniu związków M, P, B. Związki P, B i S wywołały także zwiększoną ruchliwość zwierząt, co może być uważane za działanie
uboczne podanych związków.
Wnioski. Mając na uwadze wyniki badania za pomocą uniesionego labiryntu krzyżowego, stwierdzono, że związki
M i L mogą być traktowane jako potencjalnie przeciwlękowe i wykazujące mniej działań ubocznych ze względu
na prawdopodobnie duże dodawanie elektronów grupy metoksylowej, jak również właściwości hydrofilowe grup
hydroksylowych tych związków (Adv Clin Exp Med 2012, 21, 3, 307–312).
Słowa kluczowe: pochodne fencyklidyny, zachowanie lękowe, uniesiony labirynt krzyżowy, myszy.
* This article is a report made from a research project that enjoyed the financial supports of Islamic Azad
University, Karaj Branch, Iran.
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R. Hajikhani et al.
largely affect the central nervous system [3]. PCP
binds to the N-methyl-D-asparate (NMDA) receptor complex and blocks the NMDA-mediated
gating of calcium channel conductance [4]. Moreover, PCP and its derivatives demonstrate some
pharmacological and behavioral properties, such
as analgesic, stimulating, hallucinogenic, anticonvulsant, anti-depressive and anti-anxiety effects
on specific binding sites in the brain [5–10].
Anxiety as a model of behavioral effect for
many drugs, PCP and its analogues, morphine,
amphetamine, dexfenfluramine and diazepam
were examined in laboratory animals and the findings pointed to various brain systems affected by
such behavioral effects [11–13]. In this study, the
anti-anxiety activities of some PCP derivatives (M,
F, L, B, S, P) [14–19] on male NMRI mice were
investigated and the results were compared with
compound I in the control groups.
The Diagnostic and Statistical Manual of
Mental Disorders (DSM-IV-TR) characterizes
anxiety as a feeling of persistent worry that hinders an individual’s ability to relax [1]. It ranges
from the transient anxiety levels a person may feel
prior to a surgery or a menstrual cycle to a pervasive feeling of nervousness that characterizes an
anxiety disorder (e.g. generalized anxiety disorder, obsessive-compulsive disorder, panic disorder and social phobia). The impact of the anxiety
is not only limited to consistent stress which is
associated with higher risk of cardiovascular and
cerebrovascular disease, but also it has such debilitating physical manifestations as headaches,
uncontrolled trembling, sweating, muscle tension
and aches [2]. Phencyclidine (1-(1-phenylcyclohexyl) piperidine, PCP, I, Fig. 1) and its analogues
are characterized by their highly potent and widely abused psychotomimetic properties, which
OCH3
OH
N
N
M
I
CH3
OH
CH3
N
N
OH
P
S
OCH3
CH3
N
N
L
F
CH3
O
N
B
Fig. 1. Structural formulas of
PCP (I) and its derivatives (M,
F, L, B, S, P)
Ryc. 1. Wzory strukturalne PCP
(I) i jej pochodnych (M, K, L,
B, S, P)
309
Phencyclidine Derivatives and Anxiety-like Behavior
Material and Methods
Animals
The experiments were carried out with male
NMRI mice (Pasteur Institute, Karaj, Iran) weighing 20–25 g. The animals were maintained at 22°C
in a 12 h light–dark cycle with food and tap water
available ad libitum. All procedures were in accordance with Shahid Beheshti University of Medical Sciences Guidelines for the Care and Use of
Laboratory Animals, and approved by the local
Research and Medical Ethics Committee.
Drugs
Phencyclidine and its derivatives (S, M, P, B, L, F)
were dissolved in saline to deliver by intra peritoneal
(i.p.) injection at a volume of 10 ml/kg. The control
group merely received i.p. 10 ml/kg saline. All compounds were administered in five consecutive days
and the elevated-plus maze test was performed on day
five, 30 min after receiving the last dose of the drug.
Elevated-plus Maze Test
Anxiety-related behaviors were assessed using
the elevated-plus maze (EPM) test [20]. The apparatus consists of two open and two enclosed horizontal perpendicular arms (30 × 5 cm) positioned
at 40 cm above the floor. The junction of the four
arms makes a central square platform (5 × 5 cm).
Every mouse was placed in the central platform,
faced one of the open arms and allowed to remain
for 5 min. After every trial, the maze was thoroughly cleaned with 10% ethanol solution dried by cloth.
The experiments were conducted under an artificial
laboratory illumination (fluorescent lamps, 80 lx at
maze level). The sessions were recorded by a camera hung from the ceiling of the maze. Data was
obtained using Ethovision software (version 3.1) as
a video tracking system for automation of behavioral experiments (Noldus Information Technology, the Netherlands). During the 5 min trial, the
behavior of every mouse was recorded in terms of
the number of entries into open arms (OAE), the
time spent in open arms (OAT), the number of entries into closed arm (CAE) and the average velocity. Increase of both OAT and OAE was inferred as
the index of lower anxiety behavior. Velocity and
the number of entries into closed arms (CAE) reflected animal locomotion and activities.
Statistical Analysis
In this study, the findings were analyzed
and reported as the mean ± SEM, using Prism 5 (Graphpad Software Inc.). A one-way Analysis of
Variances (ANOVA) followed by Dunnett’s Multiple Comparison Test was conducted to compare
the behaviors of treatment and control groups.
A p-value < 0.05 was allocated as the level of significance.
Results
General Consideration
The observation of animal behaviors hardly
showed any rate of mortality, morbidity, irritability and other side effects due to drug administration. To evaluate the effects of drugs on motor
coordination, a rota-rod test was carried out after
the EPM test. The animals were placed on a rotarod apparatus (Ugo Basil, Italy) with a speed of
12 rpm. Falling off the rod during the 90 seconds
of the trial was considered as drug-induced motor
impairment. However, the compared data indicated no significant differences between the treatment and control mice. In fact, all the animals
passed the 90 sec trials without falling off the rod
(data not shown).
Effects of PCP and Its
Derivatives (S, M, P, B, L, F)
on Anxiety-related Behavior
The results are shown in Figure 2. One-way
ANOVA revealed a significant change of OAT between the treatment groups [F (7,71) = 10.03, p <
0.0001; Figure 2A]. A significant increase in OAT
was observed in the mice treated with PCP (p <
0.05) and compounds M (p < 0.001), L (p < 0.05),
P (p < 0.001) and B (p < 0.001) compared to the
control group. Moreover, one-way ANOVA revealed a significant change in OAE between treatment groups [F (7,73) = 8.016, p < 0.0001; Figure
2B]. A significant increase in OAE was observed in
mice treated with compounds P (p < 0.001), M (p
< 0.01), and B (p < 0.001) compared to the control
group. The compound PCP did not produce any
significant change in OAE compared to the control group.
Effect of PCP and Its Derivatives
(S, M, P, B, L, F)
on Locomotor Activity of Mice
The results are shown in Figure 3. One-way
ANOVA revealed a significant change of CAE
between treatment groups [F (7,74) = 5.246, p <
310
R. Hajikhani et al.
B
A
Fig. 2. Effects of the i.p. injection of PCP (10 mg/kg) and its derivatives (S, M, P, B, L, F; 10 mg/kg) on anxiety-like
behavior of mice assessed by measuring open arm time (OAT; Fig. 2A) and open arm entry (OAE; Fig. 2B) in an elevated-plus maze test. Each bar indicated the mean ± SEM of 10 treatment mice. “** P < 0.01, *** p < 0.001 compared
with the control saline-injected mice (CTL)”
Ryc. 2. Działanie iniekcji i.p. PCP (10 mg/kg) i jej pochodnych (S, M, P, B, L, M, 10 mg/kg) na zachowania lękowe
myszy ocenione przez pomiar czasu przebywania na otwartym ramieniu (OAT; ryc. 2A) i liczenie wejść na otwarte
ramiona (OAE; ryc. 2B) w uniesionym labiryncie krzyżowym. Każdy słupek oznacza średnią ± SEM 10 badanych
myszy. „** P < 0,01, *** p < 0,001 w porównaniu z grupą kontrolną myszy, którym podawano sól fizjologiczną (CTL)”
0.0001; Figure 3A]. A significant increase of CAE
was observed in mice treated with compounds P (p
< 0.001), and B (p < 0.05) compared to the control group. One-way ANOVA also revealed a significant change in velocity of mouse movement
between treatment groups [F (7,74) = 5.778, p <
0.0001; Figure 3B]. A significant increase in OAE
was observed in mice treated with compounds
P (p < 0.001), B (p < 0.05), and S (p < 0.001) compared to the control group. No significant CAE or
velocity changes of the mouse movements were
detected in the treatment groups compared to the
control group.
A
Discussion
Recent developments in the neurobiology of
anxiety have highlighted the neurotransmitter
glutamate as an important factor in anxiety and
anxious behaviors. The effects of glutamate on
anxiety behaviors are mediated through different
combinations of ionotropic and metabotropic glutamate receptors and potentially different sub-unit
combinations [22]. Also, it seems that all NMDA
glutamatergic system, nicotinic acetylcholine and
serotonin (5-HT) receptors can impose an effect
on the modulation of anxiolytic behaviors caused
Fig. 3. Effects of PCP (10 mg/kg) and its derivatives (S, M, P, B, L, F; 10 mg/kg) on locomotor activities of the
mice assessed by measuring the number of closed arm entries (CAE; Fig. 3A) and the velocity (Fig. 3B) of animal
movement in an elevated-plus maze test. Each bar represents the mean ± SEM of 10 treatment mice.“** P < 0.01, ***
p < 0.001 compared with the control saline-injected mice (CTL)”
B
Ryc. 3. Działanie iniekcji i.p. PCP (10 mg/kg) i jej pochodnych (S, M, P, B, L, M, 10 mg/kg) na aktywność ruchową
myszy ocenione przez liczenie wejść na zamknięte ramiona (CAE; ryc. 3A) i pomiar prędkości poruszania się zwierząt
(ryc. 3B) po uniesionym labiryncie krzyżowym. Każdy słupek oznacza średnią ± SEM 10 badanych myszy.
„** P < 0,01, *** p < 0,001 w porównaniu z grupą kontrolną myszy, którym podawano sól fizjologiczną (CTL)”
Phencyclidine Derivatives and Anxiety-like Behavior
by phencyclidine and its derivatives [23–29]. PCP
(I) and some of its substitutive derivatives (S, M,
P, B, L, F) making some changes in phenyl (consisting of high electron donating and dipole moments of methyl and methoxy groups), piperidine
(consisting of high polarity hydroxyl) or alternative morpholine ring (Fig. 1) were applied to evaluate the anti-anxiety behaviors of the mice. The
results showed that the addition of methoxy and
hydroxyl groups with high electron donating and
hydrophilic properties in M, and only the methoxy group in L, were more effective in reducing
anxiety-related behavior in mice with less side effects relative to other derivatives (PCP, S, P, B, F).
311
The addition of the methyl group was effective not
only in compound F but also when the piperidinyl
group was added (e.g. compounds S and P), similar to altering the piperidine by morpholine ring in
compound B. Therefore, it seems such changes on
the PCP molecule could facilitate an affinity to the
mentioned receptors and it causes an increase of
anxiolytic behaviors by the above-mentioned analogues compared to PCP and the vehicle (control).
Based on the results in the elevated-plus maze
test, it was concluded that, whereas compounds
M and L could produce specific anti-anxiety activities with less side effects, compound M was more
effective.
Acknowledgements
The authors would like to express their gratitude to Dr. Ahmad Jahan Latibari and Dr. Natasha Qale Pourdana,
the CamTESOL certified editor, for their patience in proofreading the initial drafts of this article.
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Address for correspondence:
Ramin Hajikhani
Department of Physiology
Faculty of Science
Islamic Azad University
Karaj branch
P.O. Box: 31485-313
Karaj
Iran
Conflict of interest: None declared
Received: 28.08.2011
Revised: 10.10.2011
Accepted: 29.03.2012