|Year : 2013 | Volume
| Issue : 4 | Page : 383-387
Neuro-pharmacological evaluation of structurally novel 5-hydroxytryptamine type 3 receptor antagonist, N-n-propyl-3-ethoxyquinoxaline-2-carboxamide (6n) for its anxiolytic potential
Shvetank Bhatt, Radhakrishnan Mahesh, Ankur Jindal, Thangaraj Devadoss
Department of Pharmacy, Birla Institute of Technology and Science, Pilani, Rajasthan, India
|Date of Submission||26-Apr-2013|
|Date of Acceptance||29-Jul-2013|
|Date of Web Publication||15-Oct-2013|
Department of Pharmacy, Birla Institute of Technology and Science, Pilani - 333 031, Rajasthan
Source of Support: UGC., Conflict of Interest: None
| Abstract|| |
Aim: The present study was designed to investigate the anxiolytic activity of "N-n-propyl-3-ethoxyquinoxaline-2-carboxamide (6n)," a novel 5-hydroxytryptamine Type 3 (5-HT 3 ) receptor antagonist in experimental mouse models of anxiety. Materials and Methods: The anxiolytic activity of "6n" (1 and 2 mg/kg, intraperitoneally (i.p.)) was evaluated in mice by using a battery of behavioral tests of anxiety such as elevated plus maze (EPM), light/dark (L and D) box, hole board (HB) and open field test (OFT) with diazepam (2 mg/kg, i.p.) as standard anxiolytic. All the tested doses of "6n" did not affect the base line locomotion. Results: The new chemical entity "6n" (1 and 2 mg/kg, i.p.) and diazepam (2 mg/kg, i.p.) significantly increased the percentage of time spent and number of entries in open arm in the EPM test. In the L and D test compound "6n" (1 and 2 mg/kg, i.p.) and diazepam (2 mg/kg, i.p.) significantly increased the total time spent in light compartment as well as the number of transitions from one compartment to other. While "6n" (2 mg/kg, i.p.), diazepam (2 mg/kg) showed a significant increase in number of square crossed and "6n" at (1 mg/kg, i.p.) did not affect the number of square crossed significantly. Compound "6n" (1 and 2 mg/kg, i.p.) and diazepam (2 mg/kg, i.p.) also significantly increased number of head dips and number of square crossed in HB test whereas significantly decreased the head dipping latency as compared with vehicle control group. In addition, "6n" (1 and 2 mg diazepam (2 mg/kg, i.p.) significantly increased the ambulation scores and number of rearing in OFT. Conclusion: In conclusion, these findings indicated that compound "6n" exhibited an anxiolytic-like effect in animal models of anxiety.
Keywords: Anxiety, elevated plus maze, hole board, 5-hydroxytryptamine type 3 receptor antagonists, open field test
|How to cite this article:|
Bhatt S, Mahesh R, Jindal A, Devadoss T. Neuro-pharmacological evaluation of structurally novel 5-hydroxytryptamine type 3 receptor antagonist, N-n-propyl-3-ethoxyquinoxaline-2-carboxamide (6n) for its anxiolytic potential. Int J Nutr Pharmacol Neurol Dis 2013;3:383-7
|How to cite this URL:|
Bhatt S, Mahesh R, Jindal A, Devadoss T. Neuro-pharmacological evaluation of structurally novel 5-hydroxytryptamine type 3 receptor antagonist, N-n-propyl-3-ethoxyquinoxaline-2-carboxamide (6n) for its anxiolytic potential. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2019 Nov 21];3:383-7. Available from: http://www.ijnpnd.com/text.asp?2013/3/4/383/119855
| Introduction|| |
Anxiety and related disorders such as generalized anxiety, panic and obsessive compulsive disorder, phobias or post-traumatic stress disorder are the most common mental illness and a major cause of disability and most frequently occurring psychiatric disorder across the globally.  Emotional challenges in anxiety tend to exacerbate physical illness and which, in turn, are likely to create a problem of health-related quality-of-life.  Despite a steady increase in the development of anxiolytic drugs, the prevalence of the disorder remains stable that could be attributed to the unclear neurobiological understanding of pathophysiology or the inconsistent efficacy of current pharmacological treatment. 
Animal studies have shown that positive modulation of the gamma-aminobutyric acid Type A receptor by benzodiazepines results in anxiolytic activity. Apart from few chemical remedies such as benzodiazepines and selective serotonin reuptake inhibitors (SSRIs) not much treatment options are available to cure generalized anxiety disorders. , However, the realization that benzodiazepines have a narrow safety margin and sexual side-effects related to SSRI have prompted many researchers to screen new potential compounds in the hope of identifying the drugs with better anxiolytic potential with a novel mechanistic pathway and fair safety margin.
Serotonin is the major neurotransmitter involved in the anxiety. Until now seven superfamilies of serotonin receptors are identified in that serotonin 5-HT 3 receptors are pentameric ligand gated ion channels with fast synaptic potential belonging to the superfamily of Cys-loop receptors. 5-HT 3 receptors are expressed in the central nervous system in regions involved in the vomiting reflex, processing of pain, the reward system, cognition, depression and anxiety control. ,
The involvement of 5-HT 3 receptors in depression and anxiety is complemented by studies of 5-HT 3 knockout mice, which revealed the regulation of 5-HT 3 (3A subtype) in anxiety-related behaviors.  Serotonergic neurotransmission in the prefrontal cortex plays a key role in regulating emotion and cognition under normal and pathological conditions. Increased availability of 5-HT on 5-HT 2 and 5-HT 3 receptors increases anxiety  and is probably at least in part responsible for anxiogenic-like effects of antidepressants while 5-HT 3 -receptor blockade has anxiolytic effects.  The proposed mechanism for anti-depressant effect of "N-n-propyl-3- ethoxyquinoxaline -2-carboxamide (6n)" is postsynaptic 5-HT 3 receptor antagonism in serotonergic neurons can facilitate specific binding of 5-HT to other postsynaptic receptors such as 5-HT 1B , 5-HT 2A and 5-HT 2C , thereby aiding in serotonergic transmission.  Both hippocampal and accumbens 5-HT 3 receptors seem to contribute to the anxiolytic-like effects of 5-HT 3 -receptor antagonists. It also appears that this effect of 5-HT 3 -receptor antagonists is related to their action on postsynaptic 5-HT 3 receptors within the nucleus accumbens and depends upon the functional state of the 5-HT innervation ascending from the raphe nuclei. 
5-HT 3 receptor antagonists have been tested on anxiety and depressive syndromes associated to other diseases. The well-known 5-HT 3 receptor antagonist, ondansetron (OND) reduced depressive symptoms in patients with chronic hepatitis C,  with alcoholism  and in bulimic patients. 
Similarly, in fibromyalgic patients, tropisetron improved anxiety and depressive scores. 
Despite increased interest among the clinical neurosciences, information regarding the antidepressant activity of serotonin Type 3 modulators is still lacking. Thus, selecting the test sensitive to antidepressant drugs, the present study was designed to investigate the antidepressant potential of "6n" in rodent models of anxiety.
In the present study, compound "6n" which exhibited an optimum log P (2.52) and pA 2 (7.6) value greater than the standard 5-HT 3 receptor antagonist, OND pA 2 (6.9) was selected from a series of compounds synthesized in our laboratory for the preliminary anxiolytic screening in the standard rodent models of anxiety. 
| Materials and Methods|| |
Behavioral based experiments were carried out using male Swiss albino mice (20-25 g), procured from Chaudhary Charan Singh Haryana Agricultural University, Hisar, India. Animals were kept in polypropylene boxes under standard laboratory conditions (temperature 23 ± 2°C and room humidity 60 ± 10%), maintained on 12:12 h light/dark (L and D) cycle. Standard diet and filtered water were given ad libitum. All the experiments were carried out between 09.00 a.m. and 02.00 p.m. in accordance with the Institutional Animal Ethics Committee of Birla Institute of Technology and Science (BITS), Pilani, India (Protocol No. IAEC/RES/14/04).
Drugs and treatments
Diazepam was purchased from Cipla Ltd. India. Compound "6n" and diazepam were prepared freshly before use in distilled water. "6n" (1 and 2 mg/kg, intraperitoneally (i.p.)) and diazepam (2 mg/kg, i.p.) were administered i.p. to respective groups, 30 min prior to testing in each test. Dose of the compound "6n" was selected based on the previous studies conducted in our laboratory. 
Elevated plus maze
The Elevated plus maze (EPM) test was first evaluated for rats  and later adapted for mice.  In brief, the apparatus consisted of a wooden maze with two enclosed arm (30 cm ° 5 cm ° 15 cm) and two open arms (30 cm ° 5 cm ° 0.25 cm) that extended from a central platform (5 cm ° 5 cm) to form a plus sign. The plus-maze apparatus was elevated to a height of 45 cm and placed inside a sound-attenuated room. The trial was started by placing a mouse on the central platform of the maze facing its head toward an open arm. The behavioral performances recorded during a 5 min test period were; percentage open arm entries and percentage time spent in open arm. Entry into an arm was considered valid only when all four paws of the mouse were inside that arm.  The apparatus was thoroughly cleaned with 70% ethanol after each trial.
L and D aversion test
The L and D apparatus comprised of a box divided into two separate compartments, occupying two-thirds and one-third of the total size, respectively. The larger compartment (light compartment) was illuminated by a 60-W bulb, while the smaller (dark compartment) was entirely black and enclosed under a dark cover. The L and D compartments were separated by a partition with a tunnel to allow passage from one compartment to the other.  At the beginning of the test, the mouse was placed individually at the center of the light compartment facing towards the tunnel and was allowed to explore the entire apparatus for 5 min. The behavioral parameters such as total time spent in the light compartment, number of transitions between the L and D compartments and number of square crossed in each compartment were measured. The apparatus was thoroughly cleaned with 70% ethanol after each trial.
Hole board test
The HB apparatus consisted of a grey Plexiglas platform (40 cm ° 40 cm) raised to a height of 15 cm from the floor of a gray wooden box (40 cm ° 40 cm ° 40 cm). The grey Plexiglas platform consisted of 16 equivalent square compartments (12 peripheral and 4 central), each featuring a central circular hole (3 cm diameter). Test session was started by placing each animal in the center of the HB and allowed to freely explore on the apparatus for 5 min. The behavioral performances such as the number of head dipping, number of square crossed and latency to the first head dipping were measured. ,
Open field test
The apparatus consisted of a wooden box (60 cm ° 60 cm ° 30 cm) with the floor divided into 16 squares (15 cm ° 15 cm) by black parallel and intersecting lines. The apparatus was illuminated with 60 W bulb suspended 100 cm above. At the beginning of the test, the mouse was placed individually at the center of the square arena. The ambulation scores (number of square crossed) and rearing number (standing upright on the hind legs) were measured. After each individual test session the floor was thoroughly cleaned with 70% ethanol. 
All values were expressed as mean ± standard error of the mean. The data obtained from various groups were statistically analyzed using one-way analysis of variance followed by the post-hoc Dunnett's test in Graph pad prism 3. The P < 0.05 was considered to be statistically significant.
| Results|| |
Acute treatment with "6n" (1 and 2 mg/kg, i.p.) and diazepam (2 mg/kg, i.p.) significantly increased the percentage of both open arm entries and time spent in open arm at P < 0.05 as compared to vehicle control group [Table 1].
L and D test
Compound "6n" (1 and 2 mg/kg, i.p.) and diazepam (2 mg/kg, i.p.) treatment significantly at P < 0.05 increased the number of entries from one compartment to other as well as increase the total time spent in lit area. Moreover, "6n" (2 mg/kg, i.p.) and diazepam (2 mg/kg, i.p.) increased number of square crossed in each compartment while lower dose of "6n" (1 mg/kg, i.p.) did not produce a significant effect at P < 0.05 in number of square crossed [Table 2].
Compound "6n" (1 and 2 mg/kg, i.p.) and diazepam (2 mg/kg, i.p.) treatment significantly at P < 0.05 increased the number of head dips, number of square crossed and decreased the head dipping latency [Table 3].
Compound "6n" (1 and 2 mg/kg, i.p.) and diazepam (2 mg/kg, i.p.) treatment significantly increased the ambulation score and decreased number of rearing at P < 0.05 as compared with vehicle treatment group [Table 4].
| Discussion|| |
The serious side-effects associated with classical anti-anxiety drugs namely rebound insomnia, sedation, withdrawal and tolerance with benzodiazepine, barbiturates and alcohol; sexual dysfunction with antihistaminics and tricyclic antidepressants have limited their use in patients. , Hence search for alternative treatments of anxiety disorders have been evolving rapidly. In the present study, the anxiolytic effects of "6n" was evaluated in mouse models of anxiety such as the EPM, L and D test, hole-board test and OFT.  The results of the present study verified the designed hypothesis that 5-HT 3 receptor antagonists plays an important role in the pathogenesis of anxiety disorder via increasing the availability of serotonin at postsynaptic receptors. Although, it is uncertain that any single animal model captures all of the components of the complex expression of anxiety, thus a battery of tests have been used to evaluate the potential anxiolytic effect of "6n" in the present study.
The EPM test is most popular animal test for evaluation of anxiolytic compounds used in behavioral pharmacology. The EPM test used to evaluate the psychomotor performance and emotional aspects of rodents. EPM is considered as one of the well-established model for unconditioned anxiety i.e., height, unprotected opening, novelty to detect anxiolytic/anxiogenic-like activity by investigating aspects of physiological and pharmacological behavior. In the EPM test percentage increase number of entries and time spent into the open arms arm the most obvious index/reliable indicators of decreased anxiety or indicating the anxiolytic-like activity of a compound while anxiogenic substances have the opposite effect.  In our study, treatment with "6n" produced anxiolytic-like effects in the EPM test as evidenced by increased percentages of both open arm entries and time spent in open arms test.  In addition, diazepam used as reference anxiolytic also showed the potential anxiolytic effects in EPM.
The L and D test is another widely used animal model for screening anxiolytic or anxiogenic drugs  by utilizing the animal's natural preference for dark spaces. The L and D paradigm is ethological approach based on natural aversion of mice to brightly lit place and it is sensitive to drugs affect anxiety. Anxiolytics reduce the natural aversion to light and increase the time spent in light area and number of transition from one compartment to other showed anxiolytic potential of compound.  In the present study, we found that "6n" treatment significantly increased the time spent in lit compartment, number of square crossed as well as the number of transitions. Some studies have reported that an anxiolytic drug increased the transitions between the two compartments. ,
The anxiolytic-like effects of "6n" were further confirmed using the HB test. Recently, hole-board test has been popular as a model of anxiety and offers a simple method for measuring the behavioral response of rodents to an unfamiliar environment.  The head dipping behavior of a rodent in HB is sensitive to change in an emotional state of the animal.  The present study results revealed that "6n" treatment significantly increased the number of head dipping and decreased the latency of head dipping reflecting the anxiolytic activity of the compound. This effect is in agreement with previous studies, which suggest that increase in the head dipping number and square crossed and decrease latency of head dipping reflected the anxiolytic like activity of a compound. 
The OFT is also widely used for the screening of anxiolytic/anxiogenic drugs. This test is used to evaluate the animals emotional state normal aversion of a rodent to the brightly lit area produces the anxiety and fear, which is characterized by alteration in the behavioral parameters of animal in open field. Thus, the animal removed from their acclimatized cage and placed in environment express anxiety and fear by showing alteration in some or all parameters. Previous reports suggested that anxiolytic compound have a tendency to reduce the fearful behaviors of rodents in open field.  "6n" treatment increased the ambulation scores in OFT indicating the anxiolytic effect of "6n."
In summary, the results of the present study suggest the anxiolytic activity of "6n" in animal models of anxiety. However, further studies are required in order to better evaluate the possible mechanisms, underlying the anxiolytic-like effects of "6n."
| Acknowledgment|| |
We thank the University Grants Commission, India, BITS, Pilani, India and Sophisticated Analytical Instrument Facilities, Panjab University, Chandigarh, India for providing financial support, laboratory and analytical facilities, respectively.
| References|| |
|1.||Cross-national comparisons of the prevalences and correlates of mental disorders. WHO International Consortium in Psychiatric Epidemiology. Bull World Health Organ 2000;78:413-26. |
|2.||Al-Maskari MY, Petrini K, Al-Zakwani I, Al-Adawi SH, Dorvlo AS, Al-Adawi S. Mood dysfunction and health-related quality of life among type 2 diabetic patients in Oman: Preliminary study. Int J Nutr Pharmacol Neurol Dis 2011;1:56-63. |
|3.||Rickels K, Garcia-Espana F, Mandos LA, Case GW. Physician withdrawal checklist (PWC-20). J Clin Psychopharmacol 2008;28:447-51. |
|4.||Banga PV, Patil CY, Deshmukh GA, Chandaliya KC, Baig MS, Doifode SM. Biosynthesis, mechanism of action, and clinical importance of neuroactive steroids: Pearls from literature. Int J Nutr Pharmacol Neurol Dis 2013;3:77-86. |
|5.||Grundmann O, Nakajima J, Seo S, Butterweck V. Anti-anxiety effects of apocynum venetum L. in the elevated plus maze test. J Ethnopharmacol 2007;110:406-11. |
|6.||Haus U, Späth M, Färber L. Spectrum of use and tolerability of 5-HT3 receptor antagonists. Scand J Rheumatol Suppl 2004;119:12-8. |
|7.||Mahesh R, Perumal RV, Pandi PV. Cancer chemotherapy-induced nausea and vomiting: Role of mediators, development of drugs and treatment methods. Pharmazie 2005;60:83-96. |
|8.||Kelley SP, Bratt AM, Hodge CW. Targeted gene deletion of the 5-HT3A receptor subunit produces an anxiolytic phenotype in mice. Eur J Pharmacol 2003;461:19-25. |
|9.||Bell R, Gilmore, PE. Effects of the 5-HT3 receptor agonist SR 57227A and antagonist Y-25130 on the behaviour of rats in the elevated zero-maze model of anxiety. J Psychopharmacol 2003;17:A30. |
|10.||Faerber L, Drechsler S, Ladenburger S, Gschaidmeier H, Fischer W. The neuronal 5-HT3 receptor network after 20 years of research - Evolving concepts in management of pain and inflammation. Eur J Pharmacol 2007;560:1-8. |
|11.||Rajkumar R, Mahesh R. The auspicious role of the 5-HT3 receptor in depression: A probable neuronal target? J Psychopharmacol 2010;24:455-69. |
|12.||Piche T, Vanbiervliet G, Cherikh F, Antoun Z, Huet PM, Gelsi E, et al. Effect of ondansetron, a 5-HT3 receptor antagonist, on fatigue in chronic hepatitis C: A randomised, double blind, placebo controlled study. Gut 2005;54:1169-73. |
|13.||Johnson BA, Ait-Daoud N, Ma JZ, Wang Y. Ondansetron reduces mood disturbance among biologically predisposed, alcohol-dependent individuals. Alcohol Clin Exp Res 2003;27:1773-9. |
|14.||Faris PL, Eckert ED, Kim SW, Meller WH, Pardo JV, Goodale RL, et al. Evidence for a vagal pathophysiology for bulimia nervosa and the accompanying depressive symptoms. J Affect Disord 2006;92:79-90. |
|15.||Haus U, Varga B, Stratz T, Späth M, Müller W. Oral treatment of fibromyalgia with tropisetron given over 28 days: Influence on functional and vegetative symptoms, psychometric parameters and pain. Scand J Rheumatol Suppl 2000;113:55-8. |
|16.||Mahesh R, Bhatt S, Devadoss T, Jindal A, Gautam B, Pandey D. Antidepressant potential of 5-HT3 receptor antagonist, N-n-propyl-3-ethoxyquinoxaline-2-carboxamide (6n). J Young Pharm 2012;4:235-44. |
|17.||Adeyemi OO, Yemitan OK, Taiwo AE. Neurosedative and muscle-relaxant activities of ethyl acetate extract of Baphia nitida AFZEL. J Ethnopharmacol 2006;106:312-6. |
|18.||Yadav AV, Kawale LA, Nade VS. Effect of Morus alba L. (mulberry) leaves on anxiety in mice. Indian J Pharmacol 2008;40:32-6. |
|19.||Mi XJ, Chen SW, Wang WJ, Wang R, Zhang YJ, Li WJ, et al. Anxiolytic-like effect of paeonol in mice. Pharmacol Biochem Behav 2005;81:683-7. |
|20.||Saitoh A, Hirose N, Yamada M, Yamada M, Nozaki C, Oka T, et al. Changes in emotional behavior of mice in the hole-board test after olfactory bulbectomy. J Pharmacol Sci 2006;102:377-86. |
|21.||Rebai O, Djebli NE. Chronic exposure to aluminum chloride in mice: Exploratory behaviors and spatial learning. Adv Biol Res 2008;2:26-33. |
|22.||Sonavane GS, Sarveiya VP, Kasture VS, Kasture SB. Anxiogenic activity of myristica fragrans seeds. Pharmacol Biochem Behav 2002;71:239-44. |
|23.||Roy-Byrne PP. The GABA-benzodiazepine receptor complex: Structure, function, and role in anxiety. J Clin Psychiatry 2005;66 Suppl 2:14-20. |
|24.||Zohar J, Westenberg HG. Anxiety disorders: A review of tricyclic antidepressants and selective serotonin reuptake inhibitors. Acta Psychiatr Scand Suppl 2000;403:39-49. |
|25.||Nic Dhonnchadha BA, Bourin M, Hascoët M. Anxiolytic-like effects of 5-HT2 ligands on three mouse models of anxiety. Behav Brain Res 2003;140:203-14. |
|26.||Jain NN, Ohal CC, Shroff SK, Bhutada RH, Somani RS, Kasture VS, et al. Clitoria ternatea and the CNS. Pharmacol Biochem Behav 2003;75:529-36. |
|27.||Bhagwat V, Chowta MN, Shoeb A, Maskeri R, Venkatesh V, Rai A. Evaluation of anxiolytic activity of vanillin in wistar albino rats. Int J Nutr Pharmacol Neurol Dis 2013;3:96-101. |
|28.||Bhatt S, Mahesh R, Devadoss T, Jindal AK. Anxiolytic-like effect of (4-benzylpiperazin-1-yl)(3-methoxyquinoxalin-2-yl) methanone (6g) in experimental mouse models of anxiety. Indian J Pharmacol 2013;45:248-51. |
[Table 1], [Table 2], [Table 3], [Table 4]