Anti-anxiety effect of a novel 5-HT₃ receptor antagonist N-(benzo[d]thiazol-2-yl)-3-ethoxyquinoxalin-2-carboxamide (6k) using battery tests for anxiety in mice.

OBJECTIVE: To investigate the anti-anxiety activity of "6k", a novel 5-hydroxytryptamine type 3 (5-HT₃) receptor antagonist in in mice.
MATERIALS AND METHODS: Anti-anxiety activity of "6k" (1, 2, and 4 mg/kg, intraperitoneally (i.p.)) was evaluated in mice by behavioral tests such as elevated plus maze (EPM), open field test (OFT), light-dark box (L&D), and hole board test (HBT). Diazepam (2 mg/kg, i.p.) served as reference standard.
RESULTS: "6k" significantly (P < 0.05) increased the time and entries in open arm in EPM as compared to vehicle control group. Further, "6k" significantly (P < 0.05) increased the central and peripheral ambulation along with rearings and time in central area; whereas, reduced the fecal pellets in OFT as compared to vehicle control group. There was significant (P < 0.05) reduction in the latency to enter dark chamber; whereas, increased number of crossings and time in light chamber in L&D aversion test by treatment with "6k" as compared to vehicle control group. In HBT, "6k" significantly (P < 0.05) increased the number of head dipping and squares crossed; whereas, reduced the latency for first head dip and number of fecal pellets as compared to vehicle control group.
CONCLUSION: A novel 5-HT3 receptor antagonist has anti-anxiety action.

Introduction

Anxiety disorder is a most common psychological behavioral disorder globally.[1] It leaves negative impact on the quality of life with life time prevalence about 15% and spontaneous remission about 14%.[2] This probably raise questions over the efficacy of the current therapeutic treatment or lack of understanding the neurobiological pathophysiology of anxiety disorder.[3]

Since, last 3 decades, benzodiazepines (BZD) have been the drug of choice for anxiety treatment due to their effectiveness and relative safety. But, continued use of BZD leads to side effects like tolerance, withdrawal effects, and psychological and physical dependence.[4] Hence, better alternative therapeutics with wide safety window against anxiety is a hunting need in psychological research in the welfare of social and public health.

In these concern, 5-hydroxytryptamine (5-HT3) receptor antagonists represent an attractive alternative to BZD because they do not produce sedation, motor impairment, or amnesia, nor do they have addictive properties or produce significant withdrawal symptoms after discontinuation of chronic administration in animal studies.[5] The role of serotonin in anxiety disorder has been studied previously and it is well accepted that serotonin plays a crucial role in the pathophysiology of anxiety.[6] 5-HT3 receptors because of its selective ligands, is identified and characterized in the brain tissues.[7] It has been reported earlier that 5-HT3 receptors are present in the brain areas involved in control of vomiting reflex, emotion, cognition, pain, depression, and anxiety.[8] Specific studies on selectivity of 5-HT3 receptors in knockout animals reflected that, 5-HT3-A receptors are involved in depression and anxiety-like disorders.[9]

Moreover, 5-HT3 receptors antagonists like ondansetron, granisetron, zacopride, and tropisetron has been reported for some promising results in animal models of anxiety.[10]

Clinical reports of 5-HT3 antagonist have shown some promising results. Preliminary clinical studies of ondansetron are being reported for reducing the symptoms of obsessive-compulsive disorder (OCD).[11] Another 5-HT3 receptor antagonist tropisetron was found to be effective in the treatment of generalized anxiety disorder.[12] The post-synaptic 5-HT3 receptors in the hippocampus and nucleus accumbens and the functional state of serotonin in the ascending raphe nuclei seems to be involved in the anxiolytic effect.[12] Also, it is earlier reported that, 5-HT3 receptor antagonists blocks the postsynaptic receptors thereby, facilitating the serotonergic transmission by increasing the synaptic level of serotonin which binds to other 5-HT receptors especially, 5-HT1B and 5-HT2C.[13]

Hence, the present study was designed to investigate the anti-anxiety activity in a battery tests of anxiety in mice, of a novel 5-HT3 receptor antagonist N-(benzo[d]thiazol-2-yl)-3-ethoxyquinoxalin-2-carboxamide “6k” having optimum log P-value (4.56) and pA2 value (6.8) which is comparable to the standard ondansetron (pA2 6.9), performed on guinea pig ileum against 2-methyl-5-HT (5-HT3 agonist).[14]

Materials and Methods

Animals

Male Swiss albino mice (20-25 g) were obtained from Hisar Agricultural University, Hisar, India (Reg. No. 417/01/a/ CPCSEA). The animals were housed under standard laboratory conditions (temperature 23 ± 2°C and room humidity 60 ± 10%) and maintained on 12:12 h light-dark cycle. Standard diet and filtered water was provided ad libitum. Animals were acclimatized to laboratory conditions 24 h before the commencement of experiment. Each group consisted of six animals. All the experiments were carried out between 0900 and 1500 h. The experimental protocol was approved by Institutional Animal Ethics Committee (IAEC) of Birla Institute of Technology and Science, Pilani, India (Protocol No. IAEC/RES/16/6).

Drugs and Treatments

“6k” was synthesized by medicinal chemistry group of the BITS-Pilani Institute. Diazepam was procured from Cipla Ltd. India. “6k” and diazepam were freshly prepared on the day of experiment in distilled water. “6k” (1, 2, and 4 mg/kg, intraperitoneally (i.p.)) and diazepam (2 mg/kg, i.p.) were injected to the animals in respective groups 30 min before commencement of each assay of the battery tests.

Chemistry of “6k”

The chemistry of compound “6k” was followed according to Mahesh et al.[14]

Elevated Plus Maze (EPM)

The EPM was performed by the method mentioned elsewhere.[15] Briefly, it consisted of two open and two closed arms (all arms: 20 × 4 × 12 cm) made of wooden blocks elevated at a height of 25 cm from floor, which was lighten with 60 W bulb at a height of 100 cm. Each mouse was placed in the central square (5 cm × 5 cm) facing an open arm and allowed to explore the maze for 5 min of test period. The parameters measured were % time spent in open arms and % entries in open arms. Entry was considered valid only when all four paws of animals were in that particular arm. The maze was cleaned with 70% (v/v) alcohol in between two test sessions to get rid of residual odor.

Open Field Test (OFT)

The open field exploration was performed as described previously.[16] The apparatus consisted of a circular (90 cm diameter) arena with 75 cm high aluminum walls and white floor equally divided into 10 cm squares. A 60 W light bulb was positioned 90 cm above the base of the arena, which was the only source of illumination in the testing room. During the test, number of central and peripheral ambulation score and number of rearing episodes and fecal pellets were recorded for 5 min. Each mouse was transferred, 1 h before to the testing room using the home cage. After each test, the apparatus was sprayed with 70% (v/v) alcohol and wiped thoroughly to eliminate the residual odor. The test was performed in a temperature-, noise-, and light-controlled room.

Light-Dark Box (L&D) Aversion Test

The method developed by Crawley and Goodwin (1980),[17] based on the natural aversion of animal for brightly lit places was adopted. Briefly, in a two compartment box, one dark and one brightly lit, the time spent in the light compartment and the crossings between the light and dark compartment was recorded for a test time of 5 min. After each test, the apparatus was sprayed with 70% v/v alcohol and wiped thoroughly to eliminate the residual odor. Testing was performed in a temperature-, noise-, and light-controlled room.

Hole Board Test (HBT)

The HBT was performed according to Nolan and Parkes (1973),[18] with slight modifications. Briefly, the hole board apparatus consisted of a gray plexiglas chamber (40 cm × 40 cm) raised to a height of 15 cm from the floor of a wooden gray box (40 cm × 40 cm × 40 cm) having 16 equidistant holes, each of 3 cm in diameter distributed on the floor. Animals were individually placed in the center of the hole board and allowed to freely explore the apparatus for 5 min. The latency for first head dip, numbers of squares crossed, the number of head-dipping, and fecal contents were recorded. A head dip was scored if both eyes disappeared into the hole. After each test, the apparatus was sprayed with 70% v/v alcohol and wiped thoroughly to eliminate the residual odor.

Statistical Analysis

Data were analyzed using Graph Pad PRISM software version 2.01 (GraphPad Software, La Jolla, USA). One specific group of mice was assigned to one specific drug treatment condition. All the values are expressed as mean ± standard error of the mean (SEM). The significance of differences between groups for behavioral and biochemical assays were analyzed using one-way analysis of variance (ANOVA) followed by post-hoc Dunnett's test. For statistical analysis P < 0.05 was considered statistically significant.

Results

EPM

Table 1 showed that treatment with “6k” (1, 2, and 4 mg/kg, i.p.) and diazepam (2 mg/kg, i.p.) there was significant (P < 0.05) dose-dependent increase in number of entries and time spent in open arm as compared to vehicle control group.

Table 1

Comparison of effect of “6k” and diazepam on mice behavior in elevated plus maze test

OFT

Table 2 showed increase in the central and pehripheral ambutaions along with the time in central and number of rearing in dose-dependent manner with “6k” (1, 2, and 4 mg/kg, i.p.) and diazepam (2 mg/kg, i.p.) significantly (P < 0.05) as compared to vehicle control group.

Table 2

Comparison of effect of “6k” and diazepam on mice behavior in open field test

L&D Aversion Test

“6k” (1, 2, and 4 mg/kg, i.p.) dose dependently and diazepam (2 mg/kg, i.p.) significantly (P < 0.05) increased the number of transitions and time in light chamber; whereas, reduced the latency to enter dark chamber as compared to vehicle control group as mentioned in Table 3.

Table 3

Comparision of effect of “6k” and diazepam on mice behavior in light-dark aversion test

HBT

“6k” (1, 2, and 4 mg/kg, i.p.) dose dependently and diazepam (2 mg/kg, i.p.) significantly (P < 0.05) increased the number of head dips and square crossings, whereas reduced the latency for first head dip and number of fecal pellets as compared to vehicle control group as shown in Table 4.

Table 4

Comparison of effect of “6k” and diazepam on mice behavior in hole board test

Discussion

The present study investigated the anti-anxiety activity of “6k” with battery tests in mice The results clearly indicate the proposed hypothesis of important role of 5-HT3 receptor antagonist in the pathogenesis of anxiety disorder by increasing the serotonergic transmission at postsynaptic receptors.[13] A battery tests were employed for evaluating anti-anxiety effect of “6k” to avoid the false positive data from a single test to understand the complexity of anxiety disorder.

The EPM test at preclinical level is one of the most widely used method for screening anxiolytics,[19] which has high validity with respect to etiology as it uses natural stimuli such as fear of a new, brightly-lit open space, and the fear of balancing on a relatively narrow raised platform. EMP test is mostly used to evaluate the psychomotor and emotional aspects in rodents which correlate with unconditioned anxiety. Moreover, it is known that anti-anxiety agents increase the frequency of entries and time spent in open arms in EPM.[20] Accordingly, in the present study, “6k” increased the percent entries and time spent in open arms, which is an indicator of anti-anxiety potential of “6k”.

In OFT, another paradigm of battery tests for anxiety, in which anxious behavior is triggered by two factors: Individual testing and agoraphobia. Individual testing refers to the isolation of animal from the social group, while agoraphobia refers to the fear to the large environment provided as the open field.[16] Higher levels of anxiety should mainly lead to decrease in the number of squares visited in center and number of squares visited in periphery. “6k” significantly raised the central as well as peripheral ambulation in OFT which shows the increase in horizontal performance. Further, “6k” increased the number of rearings indicating vertical exploratory effect of the compound. Thus, the horizontal and vertical exploratory effect is increased by “6k”. Moreover, the time spent in central area is increased on “6k” dosing. Overall, “6k” showed anxiolytic potential in OFT.

L&D test is another widely used test for screening anti-anxiety agents. It is based on the innate aversion of rodents to brightly illuminated areas and on the spontaneous exploratory behavior of rodents in response to mild stressors, that is, a novel environment and light.[21] It is reported that an animal spending more time in light chamber along with increased transition number is considered to show anxiolytic activity.[21] In the present study, “6k” increased the latency to enter the dark chamber, time spent in light chamber, and number of transitions (that refers to the entry of the animal from one chamber to another). The decreased latency to enter in the dark chamber when initially kept in brightly illuminated light chamber indicates the anti-anxiety effect of the “6k” as the onset of anxiety of the animal is decreased. Further, the increased time spent in light chamber indicated less anxiety behavior and increased transitions showed increased exploratory behavior of the animal treated with “6k”. Thus, “6k” produced anxiolytic effect in L & D model.

The HBT provides a simple method for measuring the response of an animal to an unfamiliar environment and is widely used to assess emotionality, anxiety, and/or responses to stress in animals.[22] It has been shown that head dipping behavior reflects the sensitivity towards the changes in emotional state of the animal and provides information that a fearless state in animals may be reflected by the raised head dipping behavior.[18] “6k” increased the number of head dipping along with the number of crossing which showed an anti-anxiety activity of “6k” in HBT paradigm.

The mechanism of action of 5-HT3 receptor antagonist “6k” for anti-anxiety potential is not exactly cleared. Some of the earlier reports suggest that, 5-HT3 receptor antagonists like “6k” antagonizes the post-synaptic 5-HT3 receptors and increases the serotonergic neurotransmission. These increased serotonin acts through allosteric modulation of serotonergic system on the other 5-HT receptors mainly 5-HT1 and 5-HT2, thereby producing the anti-anxiety effect.[2324]

Conclusively, the present study aimed at investigating the anxiolytic potential of “6k” in animal models, namely EPM, OFT, L&D test, and HBT with standardized behavioral parameters. Based on the results obtained, it is cleared that the exposure to the battery test for anxiety, produced anxiety behavior in animals which was significantly attenuated by “6k” indicating anti-anxiety effect of “6k”. Future studies focused on the molecular mechanisms will shed lights on the anti-anxiety effect of “6k”.