|Year : 2012 | Volume
| Issue : 4 | Page : 235-244
Antidepressant potential of 5-HT 3 receptor antagonist, N-n- propyl-3-ethoxyquinoxaline-2-carboxamide (6n)
R Mahesh, S Bhatt, T Devadoss, AK Jindal, BK Gautam, DK Pandey
Department of Pharmacy, FD-III, Birla Institute of Technology and Science, Pilani, Rajasthan, India
|Date of Web Publication||6-Dec-2012|
Department of Pharmacy, FD-III, Birla Institute of Technology and Science, Pilani, Rajasthan
| Abstract|| |
The present study was designed to evaluate the antidepressant potential of 5-HT3 receptor antagonist N-n-propyl-3-ethoxyquinoxaline-2-carboxamide ( 6n). The compound ' 6n' with optimum log P and pA 2 value identified from a series of compounds synthesized in our laboratory was subjected to forced Swim Test (FST) (1, 2, and 4 mg/kg, i.p) and Tail Suspension Test (TST) (1, 2, and 4 mg/kg, i.p.). The compound ' 6n' significantly reduced the duration of immobility in mice without affecting the baseline locomotion. Moreover, ' 6n' (2 mg/kg, i.p.) potentiated the 5-hydroxytryptophan (5-HTP)-induced head twitch responses in mice and ' 6n' at tested dose (1 and 2 mg/kg, i.p.) reversed the reserpine-induced hypothermia in rats. In interaction studies of ' 6n' with various standard drugs/ligands using FST, ' 6n' (1 mg/kg, i.p.) potentiated the antidepressant effect of venlafaxine (4 and 8 mg/kg, i.p.) and fluoxetine (10 and 20 mg/kg, i.p.). Additionally, ' 6n' (1 and 2 mg/kg, i.p.) influenced the effect of harmane (5 mg/ kg, i.p.) as well as reversed the effect of parthenolide (1 mg/kg, i.p.) by reducing the duration of immobility in FST. Furthermore, ' 6n' (1 mg/kg, i.p.) potentiated the effect of bupropion (10 and 20 mg/kg, i.p.) in TST. Chronic ' 6n' (1 and 2 mg/kg, i.p.) treatment attenuated the behavioral abnormalities in olfactory bulbectomized rats. In conclusion, these various findings reiterated the antidepressant-like effects of ' 6n' in behavioral models of depression.
Keywords: 5-HT 3 receptor antagonists, forced swim test, head twitch, reserpine, tail suspension test
|How to cite this article:|
Mahesh R, Bhatt S, Devadoss T, Jindal A K, Gautam B K, Pandey D K. Antidepressant potential of 5-HT 3 receptor antagonist, N-n- propyl-3-ethoxyquinoxaline-2-carboxamide (6n). J Young Pharmacists 2012;4:235-44
|How to cite this URL:|
Mahesh R, Bhatt S, Devadoss T, Jindal A K, Gautam B K, Pandey D K. Antidepressant potential of 5-HT 3 receptor antagonist, N-n- propyl-3-ethoxyquinoxaline-2-carboxamide (6n). J Young Pharmacists [serial online] 2012 [cited 2013 Apr 19];4:235-44. Available from: /text.asp?2012/4/4/235/104367
| Introduction|| |
Depression is a chronic, recurring, and potentially life- threatening illness that affects up to 20% of the population across the globe. ,,, It is one of the top ten causes of morbidity and mortality worldwide, based on a survey by the World Health Organization. Despite a steady increase in the number of antidepressants over the years, the prevalence of the disorder remains stable which may be due to unclear pathophysiology or the inconsistent efficacy of currently available antidepressants with undesirable side effects. However, there is a direct correlation between the catecholaminergic neuronal systems and depression. Serotonin is the major neurotransmitter involved in the depression. Till now, seven superfamilies of serotonin receptors are identified; in that, serotonin type 3 (5-HT 3 ) receptors are pentameric ligand-gated ion channels belonging to the superfamily of Cys-loop receptors. 5-HT 3 receptors are known to be 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 abundance of 5-HT 3 receptors in the chemoreceptor triggering zone has qualified them as primary targets for anti-emetic agents. Selective 5-HT 3 receptor antagonists, such as; ondansetron (OND) and tropisetron are now recognized as drugs of choice in managing cancer chemotherapy- induced and postoperative nausea and vomiting. , The motivating outcomes from preliminary behavioral tests on 5-HT 3 receptor antagonists, their good safety profile, and the complementary effectual regional distribution of 5-HT 3 receptors in the central nervous system have urged further research to establish their potential usage in a range of central nervous system disorders. Studies on human beings using selective 5-HT 3 receptor antagonists discovered heterogeneous distribution throughout the brain within the brainstem, e.g., nucleus tractus solitarius, area postrema, and spinal trigeminal nucleus as well as the forebrain, e.g., hippocampus, amygdala, nucleus accumbens, putamen, caudate. ,
The role of 5-HT 3 receptors in anxiety is confirmed by studies of 5-HT 3A knockout mice which revealed that 5-HT 3A receptor subtypes regulate depression- and anxiety-related behaviors.  Evidence for the relevance of 5-HT 3 receptor antagonists in the treatment of depression stems from clinical trials in which patients suffering from complex disorders such as fibromyalgia and bulimia showed improvement of the comorbid depression. ,
According to proposed hypothesis by Rajkumar and Mahesh (2010), 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.  Thus, the paucity of evidence on the direct influence of 5-HT 3 receptor antagonist treatment on rodent depression-like behavior triggered the present study with objectives of which are (i) to screen the molecule '6n', preclinically in various behavioral and mechanistic rodent models of depression (ii) to conduct drug interaction studies with conventional antidepressants/ligands in order to propose the plausible mechanism underlying the above effect.
Using the three-component pharmacophore mode,  a series of 5-HT 3 receptor antagonists were designed, synthesized, and screened for their 5-HT 3 antagonist potential [Table 1]. The compounds were tested for their ability to inhibit the 5-HT 3 receptor in isolated guinea pig ileum, and the pA 2values were determined against 2-methyl- 5-hydroxytryptamine with OND as the reference drug. ,
|Table 1: Log P and pA2 values of series of 3-ethoxyquinoxali-2-carboxamides|
Click here to view
Preclinical screening of new chemical entity (NCE) in depression have been utilized vigorously to evaluate the novel compounds.  These tests neglect the aspect of face validity but have a strong predictive validity to aid in the identification of efficient antidepressant molecules.  Hence, a battery of behavioral tests were adopted for the study which included acute models like Forced Swim Test (FST),  Tail Suspension Test (TST), , mechanistic models like 5-hydroxytryptophan (5-HTP)-induced head twitch response in mice, and reserpine-induced hypothermia (RIH).  Evaluation of chronic effect of the compound was studied on olfactory bulbectomized (OBX) rats , to provide significant information on antidepressant activity of 6n, which was identified for this study based on pA 2 and log P values.
In the present study, compound N-n-propyl-3-ethoxyquinoxaline-2-carboxamide (6n) which exhibited an optimum log P and pA 2 values (pA 2- 7.6) greater than the standard 5-HT 3 receptor antagonist, OND (pA2 -6.9), , was selected for the preliminary antidepressant screening in the standard rodent models of depression as mentioned above.
| Materials and Methods|| |
Albino mice (25 ± 2 g), Wistar rats (250 ± 20 g), and Dunkin Hartley guinea pigs (370 ± 20 g) were obtained from Hissar Agriculture University, Haryana, India. All procedures were in adherence to protocol approved by Institutional Animal Ethics Committee (IAEC) of Birla Institute of Technology and Science, Pilani, India (Protocol No. IAEC/RES/14/04, dated 07.09.11). The animals were kept for at least one week before the experiments, at optimum temperature (23 ± 2°C) and humidity-controlled (50-60%) animal rooms under a 12:12-h light/dark cycle (light on 6:00-18:00 h) with free access to food and water ad libitum. Behavioral studies were carried out during the light phase (9.00 a.m. - 2.00 p.m.). The animals were used only once for each experiment.
Chemistry of 6n
The target compound 6n (N-n-propyl-3-ethoxyquinoxaline-2-carboxamide) was synthesized by coupling the 3-ethoxyquinoxalin-2-carboxylic acid with n-propylamine in the presence of coupling agents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC•HCl) and 1-hydroxybenzotriazole (HOBt) under nitrogen atmosphere. The key intermediate, 3-ethoxyquinoxalin-2- carboxylic acid, was synthesized from the starting material o-phenylenediamine in a sequence of reactions, involving cyclic condensation with diethyl ketomalonate, chlorination with phosphorous oxychloride followed by saponification then nucleophilic displacement with sodium ethoxide, the synthetic route of 6n is depicted in [Figure 1].
|Figure 1: Synthesis of 6n (N-n-propyl-3-ethoxyquinoxaline-2-carboxamide), Reagents and conditions: (a) Diethyl ketomalonate, ethanol, reflux, 6 h., 60%; (b) POCl 3 , DMF, reflux, 30 min. 80%; (c) Na 2 CO 3, reflux, 6 h. (d) dil. HCl, 94%; (e) NaOCH 2 CH 3, ethanol, MW, 6 min., dil. HCl, 85%; (f) EDC·HCl, HOBt, THF, N 2, 0°C– rt, 1h.; (g) propylamine, rt, 6h|
Click here to view
The basic pharmacophore of 5-HT 3 receptor antagonists is shown in [Figure 2].
Drugs and chemicals
Fluoxetine (FLX), paroxetine (PAR), venlafaxine (VLA), and bupropion (BUP) were obtained as gift samples from Cipla Pharmaceuticals and IPCA Laboratories Private Limited, India, respectively. Escitalopram (ESC) was obtained as generous gift samples from Glenmark Pharmaceuticals. Harmane was purchased from Tocris Bioscience, U.K. The drugs for anesthesia, namely ketamine and xylazine, were purchased from Reidel Neon Labs, Indian Immunologicals (Mumbai, India). The drugs were freshly prepared in distilled water and administered per os (p.o.) or intra-peritoneally (i.p.) (as specified) in a constant volume of 10 ml/kg. For interaction studies, the antidepressants/ligands and standards were administered i.p., 45 and 30 minutes, respectively, before testing in FST and TST as per the protocol adopted, earlier in our laboratory. The drugs were administered p.o. once a day for 14 days in the chronic treatment schedule.
5-HT 3 receptor antagonistic activity
The compounds '6n' was tested for its ability to inhibit the 5-HT 3 receptor in isolated guinea-pig ileum, and the pA2 value was determined against 2-methyl-5- hydroxytryptamine. , In order to assess the 5-HT 3 receptor antagonistic activity, guinea pigs were sacrificed under mild anesthesia. The abdomen was cut open and a length of ileum excised about 2 cm from the ileo-caecal junction; the longitudinal muscle-myenteric plexus, 3-4 cm in length, was prepared and mounted as cited in the literature. The tissue was equilibrated for 30 minutes under a resting tension of 500 mg with constant aeration in a 40 ml organ bath containing Tyrode solution, maintained at 37°C.
Non-cumulative concentrations of 2-methyl-5-HT were added with a 15-minute dosing cycle (to prevent desensitization) and left in contact with the tissue until the maximal contraction had developed. To study the antagonist effect of the test compounds on the response evoked by 2-methyl-5-HT, the compounds were added to the organ bath and left in contact with the tissue for at least 10 minutes prior to the addition of 2-methyl-5-HT. The contractions were recorded using a T-305 Force transducer coupled to a Student's physiograph (Bio Devices, Ambala, India). Antagonism was expressed in the form of pA2 values, which were graphically determined. The pA2 values of the test compound were compared with the standard antagonist, OND.
Forced swim test
The FST was carried out as described elsewhere,  with slight modifications. Mice were dropped individually into a plexi-glass cylinder (height: 30 cm, diameter: 22.5 cm) filled with water to a depth of 15 cm and maintained at 23-25°C. After an initial vigorous activity (2 minutes), the mice acquired an immobile posture in this test, which was characterized by motionless floating in the water, making only those movements necessary to keep the head above the water. The duration of immobility which reflects the state of depression was recorded during the last 4 minutes of the 6-minute test. The mice were subjected to 15-minute training under similar conditions, 24 hours before the test.
Tail suspension test
Mice were individually suspended by the tail to a horizontal bar (distance from floor was 50 cm) using scotch tape (distance from tip of tail was approximately 1 cm). Typically, mice exhibited several escape-oriented behavior interspersed with temporally increasing bouts of immobility. , The duration of immobility (in seconds) during the 6-minute test session was recorded.
Spontaneous locomotor activity
The spontaneous locomotor activity was assessed using an actophotometer.  The animals were individually placed in a square arena (30 cm × 30 cm) with walls painted black and fitted with photocells just above the floor level. The photocells were checked before the beginning of the experiment. After an initial two minute familiarization period, the digital locomotor scores were recorded for the next 10 minutes in a dimly lit room. The arena was cleaned with dilute alcohol and dried between trails.
5-hydroxytryptophan-induced head twitch response
The method mentioned elsewhere  was adopted with substantial modifications. The vehicle/drug treated mice were injected with pargyline (75 mg/kg i.p) and 5-HTP (5 mg/kg), 30 and 15 minutes prior to drug administration, respectively, and gently placed in separate, clear plexi-glass cages (12 ×12 × 10 cm). The total number of head twitches (characterized by abrupt lateral movements) episodes was recorded for the next 15 minutes.
Interaction studies in forced swim test and tail suspension test
The animals were treated either with vehicle or with one of the following test compounds namely, FLX (10 and 20 mg/kg, i.p.), a serotonin re-uptake inhibitor, VLA (4 and 8 mg/kg, i.p.), a serotonin norepinephrine (NE) reuptake, inhibitor harmane (5 mg/kg, i.p.), a monoamine oxidase inhibitor, parthenolide (1 mg/kg i.p.), a serotonin release inhibitor, Interaction of BUP (10 and 20 mg/kg, i.p), a NE and dopamine re-uptake inhibitor was carried out in TST since it is more sensitive than that of FST. , All the standard drug doses were adopted from the previous work and standard responses recorded. ,
Reserpine-induced hypothermia in rats
Rats were gently hand restrained and the glycerol lubricated digital thermometer probe was inserted into the rectum. The rectal temperature of the rats treated with reserpine (1 mg/kg, i.p.) was recorded at 30, 60, 90, and 120 minutes after the drug administration. The difference in the rectal temperature between the baseline and 60 th minute values were recorded. On the day preceding to experimentation, the rectal temperature of the rats were assessed in a similar manner in order to habituate the animals to the experimental procedures. 
Bilateral olfactory bulbectomy (OBX) was carried out as described earlier. , In brief, the rat was anesthetized with a combination of ketamine (75 mg/kg, i.p.) and xylazine (5 mg/kg, i.p.). The head was fixed to a stereotaxic frame (Inco, India) and the skull exposed by a midline incision. Burr holes (2 mm in diameter) were drilled 8 mm anterior to bregma and 2 mm on either side of the midline at a point corresponding to the posterior margin of the orbit of the eye. The dura was punctured and the olfactory bulbs were removed by suction. Hemostatic sponge was used to prevent excessive bleeding and to fill the dead space. The scalp was then sutured and the wound was dabbed with antiseptic solution. Sham surgery was carried out in the same way (including the piercing of the dura matter) except that olfactory bulbs were left intact. Sulprim (each ml containing 200 and 40 mg of sulfadiazine and trimethoprim, respectively), was administered (0.2 ml/300 g, i.m.) once a day for the first 3 days for both OBX and sham-operated rats. The animals were individually housed for the first 3 days following surgery and thereafter in groups of two (one sham and one OBX rat) until the end of the study. The OBX and sham rats were allowed 14-day rehabilitation period during which they were handled by the same experimenter to prevent aggression which would have developed, otherwise. The surgery, rehabilitation, treatment, and behavioral screening of OBX/sham rats were carried out based on the customized schedule previously reported. 
Open field test behavior
OBX and sham control rats were subjected to the Open field test (OFT) on the 29 th day after surgery. The open field exploration was conducted as described elsewhere,  with substantial modifications. The apparatus consisted of a circular (90 cm diameter) arena with 75 cm high aluminium-wated walls and 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. Each animal was individually placed in the center of the OFT apparatus and the following parameters such as ambulation scores (number of squares crossed), number of rearing episodes (when the rat stands upright on its hind paws), and number of fecal pellets were noted for 5 minutes by a trained observer, unaware of the specific treatments (observer blind). After each test, the apparatus was sprayed with dilute alcohol and wiped thoroughly. 
Data are expressed as the mean ± SD. The single treatment studies were analyzed using a one-way analysis of variance followed by a post-hoc Dunnett' s test and Tukey' s test (FST and TST). The interaction studies were analyzed using a two-way analysis of variance followed by a post-hoc Bonferroni test. The level of statistical significance was fixed at P < 0.05.
| Results|| |
Based on the pA2 and log P values, compound 6n was taken for extensive behavioral pharmacological testing from amongst the series of compounds.
In FST, the acute treatment with 6n (1, 2, and 4 mg/ kg, i.p.) significantly decreased the duration of immobility as compared with vehicle treatment [Figure 3]. Likewise, a significant decrease in duration of immobility was evident at 6n (1, 2, and 4 mg/kg) in the TST [Figure 4]. The positive control, ESC (10 mg/kg) and BUP (20 mg/kg), also significantly reduced the duration of immobility in FST and TST, respectively, as compared with the control group. None of the tested doses influenced the locomotion of mice in actophotometer [Figure 5]. Depletion of brain serotonin induced by reserpine affects the central nervous system as demonstrated by RIH. Administration of reserpine (1 mg/kg i.p) elicited a pronounced decrease in core body temperature of rats. This effect was significantly (P < 0.05) reversed by 6n, ESC (10 mg/kg) treatments [Figure 6].
|Figure 3: Effect of 6n on duration of immobility of mice FST. The columns represent mean duration of immobility in seconds (s) and error bars indicate S.D. n = 8 per group. *P< 0.05 compared with vehicle treated group. #P< 0.05, ESC compared with 1 mg/kg treatment group. ESC = Escitalopram|
Click here to view
|Figure 4: Effect of 6n on duration of immobility of mice TST. The columns represent mean duration of immobility in seconds (s) and error bars indicate S.D. n = 8 per group. *P< 0.05 compared with vehicle treated group. #P< 0.05, BUP compared with all treatment group. $P< 0.05, 2 mg/kg compared with 1 mg/kg treated group. @P< 0.05, 4 mg/kg compared with 1 mg/kg treated group. BUP = Bupropion|
Click here to view
|Figure 5: Effect of 6n on spontaneous locomotor activity of mice. The columns represent mean locomotor scores recorded in a 10-minute observation period. The error bars indicate S.D., n = 8 per group|
Click here to view
|Figure 6: Effect of 6n on reserpine-induced hypothermia in rats. The columns represent mean decrease in temperature (°C) and error bars indicate S.D. n = 8 per group. *P< 0.05 compared with vehicle-treated group. ESC = Escitalopram|
Click here to view
5-HTP-induced head twitches were performed to confirm the involvement of serotonergic pathway. The combination of 5-HTP (5 mg/kg, i.p.) and pargyline (75 mg/kg, i.p.) induced the characteristic head twitch response. 6n (2 mg/ kg, i.p.) and FLX (20 mg/kg, i.p.) significantly potentiated the 5-HTP/pargyline-induced head twitches, respectively [Figure 7].
|Figure 7: Effect of 6n and fluoxetine (FLX) on 5-hydroxytryptophan (5 mg/kg) + pargyline (75 mg/kg)-induced head twitch response in mice. The columns represent mean number of head twitches and error bars indicate S.D. n = 8 per group. *P< 0.05 compared with vehicle-treated group. FLX = Fluoxetine|
Click here to view
The peak dose of 6n (1 and 2 mg/kg, i.p.) that induced a significant (P < 0.05) decrease in the duration of immobility was selected for the interaction studies. The reference compounds were tested individually to determine their effects in FST and TST. 6n (1 and 2 mg/ kg, i.p.) significantly (P < 0.05) affected the duration of immobility throughout the interaction study as compared with control.
For a conclusive evaluation of antidepressant potential of 5-HT 3 receptor antagonists, interaction studies with SSRIs were carried out. Animals pretreated with 6n (1 mg/kg, i.p.) was found to enhance antidepressant-like effects of FLX (10 and 20 mg/kg, i.p.) and VLA (4 and 8 mg/ kg, i.p.) as shown in [Figure 8] and [Figure 9], respectively. Pre-treatment with 6n (1 and 2 mg/kg, i.p.) enhanced the antidepressant-like effects of harmane (5 mg/kg, i.p.) [Figure 10] indicating the facilitatory effects of 6n on the serotonergic neurotransmission. 6n significantly reversed the depressant- like effect of parthenolide (1 mg/kg, i.p.) as shown in [Figure 11]. Further, 6n (1 mg/kg) significantly enhanced the antidepressant activity of BUP (10 and 20 mg/kg) in mice TST [Figure 12].
|Figure 8: Effect of 6n (1 mg/kg) on antidepressant activity of fluoxetine (10 and 20 mg/kg) in mice FST. The columns represent mean duration of immobility (s) and error bars indicate S.D., n = 8 per group. *P< 0.05 compared with vehicle-treated group. #P< 0.05 compared with fluoxetine (FLX) treated group. $P< 0.05 compared with 6n treated group|
Click here to view
|Figure 9: Effect of 6n (1 mg/kg) on antidepressant activity of venlafaxine (VLA) in mice FST. The columns represent mean duration of immobility (s) and error bars indicate S.D. n = 8 per group. *P< 0.05 compared with vehicle-treated group. #P< 0.05 compared with venlafaxine (4 and 8 mg/ kg) treated group. $P< 0.05 compared with 6n - treated group|
Click here to view
|Figure 10: Effect of 6n (1 and 2 mg/kg) on antidepressant activity of Harmane (5 mg/kg) in mice FST. The columns represent mean duration of immobility in seconds (s) and error bars indicate S.D. n = 8 per group. *P< 0.05 vs. vehicle treated Group. #P< 0.05 compared with 6n treated group. $P< 0.05 compared vs harmane (5 mg/kg)-treated group|
Click here to view
|Figure 11: Effect of 6n (1 and 2 mg/kg) on depressant activity of parthenolide (PTN) in mice FST. The columns represent mean duration of immobility in seconds (s) and error bars indicate S.D. n = 8 per group. *P< 0.05 vs vehicle-treated group. #P< 0.05 compared with PTN (1 mg/kg)-treated group|
Click here to view
|Figure 12: Effect of 6n (1 mg/kg) on antidepressant activity of bupropion (BUP) in mice TST. The columns represent mean duration of immobility in seconds (s) and error bars indicate S.D., n = 8 per group. *P< 0.05 vs vehicle-treated group. #P< 0.05 compared with bupropion (10 and 20 mg/kg) treated group. $P< 0.05 compared with 6n-treated group|
Click here to view
The effects of 6n on the behavior of OBX/sham rats were analyzed in different circumstances as shown in [Table 2]. Chronic (14 days, p.o) treatment with 6n (1 and 2 mg/kg) significantly (P < 0.05) reduced the number of ambulation, rearing, and fecal pellets in OBX rats as compared with the vehicle-treated OBX rats. Moreover, OND (1 mg/kg, i.p.) and PAR (10 mg/kg) also significantly reduced the number of ambulation, rearing, and fecal pellets as compared with control group.
|Table 2: Effects of paroxetine (10 mg/kg, po) and 6n (1 and 2 mg/kg, po) open field behavior (ambulation/ Rearing/Fecal pellet) in OBX/sham rats|
Click here to view
| Discussion|| |
Preclinical studies led to the consensus that 5-HT 3 receptor antagonists have antidepressant effects by blocking limbic hyperactivity response.  The results of this behavioral investigation divulge the antidepressant-like effects of 6n,Since 5-HT 3 receptors are expressed in nucleus tractussolitarius, area postrema, and spinal trigeminal nucleus as well as the forebrain, e.g., hippocampus, amygdala, nucleus accumbens, putamen, caudate indicate their role in depression and anxiety , as the areas mentioned affected in these disorders. The FST takes advantage of the observation that rodents, following initial escape-oriented movements in an inescapable situation (in a cylinder filled with water), rapidly adopt a characteristic immobile posture (indicative of despair). It is also a useful tool in the better understanding of the role of specific monoamines and receptor subtypes implicated in depressive states. The TST is similar and the antidepressant-like activity of a compound is determined by a decrease in the duration of immobility during the FST and TST. Both of these models of depression are widely used to screen NCEs for their antidepressant potential. , These tests are quite sensitive and relatively specific to all classes of antidepressants. In the present study, 6n significantly decreased the duration of immobility in mice FST and TST. The decreased duration of immobility in mice FST and TST reflects the antidepressant-like activity of a compound. ,
Further, to investigate the plausible mechanism of 6n RIH, and 5-HTP induced head twitch was done. Reserpine being a non-specific monoamine depleting agent, acts by blocking the monoamine transport into synaptic vesicle. The depletion of brain biogenic amines affect the central nervous system characterized by hypothermia.  The decrease in body temperature induced by reserpine was reported to be antagonized by antidepressants.  6n and ESC significantly prevented the hypothermic effect of reserpine-exhibiting antidepressant-like effects in this sensitive model. 5-HTP being the immediate precursor of 5-HT, significantly increases the serotonergic transmission inducing a characteristic head twitch response in mice. 6n increased the head twitches in presence of pargyline, a MAO inhibitor. In this regard, the antidepressant-like effect of 6n appears to be modulated by an increase in monoamines, particularly 5-HT concentrations in the synapse. 
Effect of 6n on aforementioned models gives us certain clue regarding the probable mechanism of 6n which could be mainly through modulation of serotonin in synaptic cleft; further interaction studies were performed to strengthen the mechanistic hypothesis of the molecule.
Interaction studies with ligands/conventional antidepressants in FST and TST were carried out not only to predict the probable mechanism (possible receptor targets) of antidepressant-like effects of 6n, but also to pharmacologically validate the 6n - induced antidepressant-like behavior in the above mentioned predictive tests. For a conclusive evaluation of antidepressant potential of 5-HT 3 receptor antagonists, interaction studies with standard ligands/conventional antidepressants were carried out.  6n (1 mg/kg, i.p.) significantly enhanced the antidepressant-like action of FLX (10 and 20 mg/ kg, i.p.). VLA, an SNRI in a lower dose range (4-8 mg/kg), mainly influences the serotonergic system, whereas at higher doses the involvement of the NE system predominate. , 6n (1 mg/ kg, i.p.) significantly enhanced the antidepressant- like action of VLA (4 and 8 mg/kg, i.p.) as compared with alone treatment group.
Harmane, a β-carboline alkaloid, increases the monoamine levels by inhibiting the enzyme monoamine oxidase-A and B.  6n (1 and 2 mg/kg, i.p.)decreased the duration of immobility and influenced the effect of harmane (5 mg/kg, i.p.) in FST. Parthenolide, a serotonin release inhibitor  that produces depressant-like effects,  was tested with 6n to explore the serotonergic influence of 6n. A depressant-like effect induced by parthenolide was considered as a model to identify antidepressants acting through serotonergic mechanisms.  6n (1 and 2 mg/ kg, i.p.) reversed the depressogenic effect of parthenolide (1 mg/kg, i.p.), possibly through enhancing serotonin release. 6n (1 mg/kg, i.p.) potentiated the effect of BUP (10 and 20 mg/kg, i.p.), possibly by interacting with the dopaminergic system, following the dopamine hypothesis of depression. , Though the exact mechanism is not clear, it could be due to the release of dopamine through inhibition of 5-HT 3 receptors. 
Olfactory bulbectomy has been proposed to be an agitated hyposerotonergic model of depression  and used to explore the antidepressant potential of novel agents.  OBX rats exhibited a specific, abnormal behavioral pattern in the open field test characterized by increased ambulation, rearing, and fecal pellets. This abnormal behavior is reversed by antidepressants.  The increased locomotor/ exploratory behavior of OBX rat in open field test may be due to exposure to a novel environment.  Moreover, other possible reason for hyperactivity of OBX rats in OFT may be due to decrease in activity in competing behavior or delay/failure to habituate to novel environment. 
The present neurobehavioral investigation showed antidepressant-like effects of 6n, a novel 5-HT 3 antagonist, in animal models of depression. The precise mechanism by which 6n produced antidepressant-like effect is not clear. However, on the basis of results obtained in various mechanistic and interaction studies like potentiation of head twitch responses and reversal of RIH, it is suggested that 6n exhibited antidepressant-like effect by increasing the concentration of monoamine neurotransmitter in the synapse. Increase in serotonin level through blockade of 5-HT 3 receptor could possibly explain the overall antidepressant-like effect of 6n. Reversal of parthenolide-induced depression-like behavior indicated the antidepressant-like action of 6n via serotonergic modulation.
| Conclusion|| |
Compound 6n showed antidepressant-like effects in rodents' models of depression, without affecting the locomotion of rodents at tested doses. The interaction studies with several standard ligands/antidepressants indicated that compound 6n expressed the antidepressant- like effect by enhancing the level of monoamine in synapse, probably through 5-HT 3 receptor antagonism. These results correlated the beneficial effects of 5-HT 3 receptor antagonist in depression, particularly comorbid disorders. 6n potentiate the effects of antidepressants, which indicated that the combination of antidepressant with 5-HT 3 receptor antagonist can potentially accelerate the onset of antidepressant action. Further studies on 6n are warranted to investigate the molecular effect of 6n and safety of the molecule.
| Acknowledgments|| |
We thank the University Grants Commission (UGC), India, Birla Institute of Technology and Science (BITS), Pilani, India, and SAIF, Panjab University, Chandigarh, India, for providing financial support, laboratory and analytical facilities, respectively. We also express our gratitude to IPCA, Cipla, Natco, and Glenmark Laboratories, India, for providing drug sample in short notice.
| References|| |
|1.||Manji HK, Drevets WC, Charney DS. The cellular neurobiology of depression. Nat Med 2001;7:541-7. |
|2.||Nestler EJ, Barrot M, Dileone RJ, Eisch AJ, Gold SJ, Monteggia L. M. Neurobiology of depression. Neuron 2002;34:13-25. |
|3.||Gillespie CF, Nemerof CB. Hypercortisolemia and depression.Psychosom Med 2005;67:S26-8. |
|4.||Charne DS. Psychobiological mechanisms of resilience and vulnerability: Implications for successful adaptation to extreme stress. Am J Psychiatry 2004;161:195-216. |
|5.||Haus U, Spath M, Farber L. Spectrum of use and tolerability of 5-HT3 receptor Antagonists. Scand J Rheumatol 2004;119:12-8. |
|6.||Mahesh R, Perumal RV, Pandi PV. Cancer chemotherapyinduced nausea and vomiting: Role of mediators, development of drugs and treatment methods. Pharmazie 2005;60:83-96. |
|7.||Barnes JM, Barnes NM, Costal B, Ironside JW, Naylor RJ. Identification and characterisation of 5-hydroxytryptamine 3 recognition sites in human brain tissue. J Neurochem 1989;53:1787-93. |
|8.||Abi-Dargham A, Laruelle M, Wong DT, Robertson DW, Weinberger DR, Kleinman JE. Pharmacological and regional characterization of [3H] Y278584 binding sites in human brain. J Neurochem 1993;60:730-7. |
|9.||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. |
|10.||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. |
|11.||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. |
|12.||Rajkumar R, Mahesh R. The auspicious role of the 5-HT3 receptor in depression: A probable neuronal target? J Psychopharmacol 2010;24:455-69. |
|13.||Hibert MF, Hoffmann R, Miller RC, Carr AA. Conformation-activity relationship study of 5-HT3 receptor antagonists and a definition of a model for this receptor. J Med Chem 1990;33:1594-600. |
|14.||Mahesh R, Perumal RV, Pandi PV. Microwave assisted synthesis of 2-(4- substituted piperazin-1-yl)-1, 8- naphthyridine-3-carbonitrile as a new class of serotonin 5-HT 3 receptor antagonists. Bioorg Med Chem Lett 2004;14:5179-81. |
|15.||Bourin M. Is it possible to predict the activity of a new antidepressant in animals with simple psychopharmacological tests? Fundam Clin Pharmacol 1990;4:49-64. |
|16.||Lucki I. The forced swimming test as a model for core and component behavioral effects of antidepressant drugs. Behav Pharmacol 1997;8:523-32. |
|17.||Porsolt RD, Bertin A, Jalfre M. Behavioral despair in mice: A primary screening test for antidepressants. Arch Int Pharmacodyn Ther 1977;229:327-36. |
|18.||Steru L, Chermat R, Thierry B, Simon P. The tail suspension test: A new method for screening antidepressant drugs. Psychopharmacology (Berl) 1985;85:367-70. |
|19.||Thierry B, Steru L, Simon P, Porsolt RD. The tail suspension test: Ethical considerations. Psychopharmacology (Berl) 1986;90:284-5. |
|20.||Devadoss T, Pandey DK, Mahesh R, Yadav SK. Effect of acute and chronic treatment with QCF-3 (4-benzylpiperazin-1-yl) (quinoxalin-2- yl) methanone, a novel 5- HT 3 reeptor antagonist, in animal models of depression. Pharmacol Rep 2010;62:245-57. |
|21.||Kelly JP, Wyrnn AS, Leonard BE. The olfactory bulbectomized rat as a model of depression: An an update. Pharmacol Ther 1997;74:299-316. |
|22.||Mahesh R, Bhatt S, Pandey DK, Gautam B. Antidepressant like effect of pimozide in acute and chronic animal models of depression. Ind J Pharm Educ Res 2011;45:46-53. |
|23.||Paton WD, Zar AM. The origin of acetylcholine released from guinea-pig intestine and longitudinal muscle strips. J Physiol 1968;194:13-33. |
|24.||MacKay D. How should values of pA2 and affinity constants for pharmacological competitive antagonists be estimated? J Pharm Pharmacol 1978;30:312-3. |
|25.||Boissier JR, Simon P. Action de la caféine sur la motilité spontanée de la souris. Arch Int Pharmacodyn Ther 1965;158:212-21. |
|26.||Martin P, Massol J, Soubrie P, Puech AJ. Effects of triiodothyronine (T 3 ) on the potentiation by antidepressants of L-5-hydroxytryptophan-induced head-twitches in mice. Prog Neuropsychopharmacol Biol Psychiatry 1989;13:735-48. |
|27.||Ripoll N, David DJ, Dailly E, Hascoët M, Bourin M. Effects in various mice strains in the tail suspension test. Behav Brain Res 2003;143:193-200. |
|28.||Bourin M, Chenu F, Ripoll N, David DJP. A proposal of decision tree to screen putative antidepressants using forced swim and tail suspension tests . Behav Brain Res 2005;164:266-9. |
|29.||Englert LF, Ho BT, Taylor D. The effects of. (-)-delta9-tetrahydrocannabinol on reserpine- induced hypothermia in rats. Br J Pharmacol 1973;49:243-52. |
|30.||Simpson KH, Murphy P, Colthup PV, Whelan P. Concentration of ondansetron in cerebrospinal fluid following oral dosing in volunteers. Psychopharmacology (Berl) 1992;109:497-8. |
|31.||Wolf H. Preclinical and clinical pharmacology of the 5-HT3 receptor antagonists. Scand J Rheumatol Suppl 2000;113:37-45. |
|32.||Marles RJ, Kaminski J, Arnason JT, Pazos-Sanou L, Heptinstall S, Fischer NH, et al. A bioassay for inhibition of serotonin release from bovine platelets. J Nat Prod 1992;55:1044-56. |
|33.||Sheehan M, Wong HR, Hake PW, Malhotra V, O'Connor M, Zingarelli B, et al. Parthenolide, an inhibitor of the nuclear factor-B pathway, ameliorates cardiovascular derangement and outcome in endotoxic shock in rodents. Mol Pharmacol 2002;61:953-63. |
|34.||Van Hoomissen J, Kunrath J, Dentlinger R, Lafrenz A, Krause M, Azar A. Cognitive and locomotor/exploratory behavior after chronic exercise in the olfactory bulbectomy model of depression. Behav Brain Res 2011;222:106- 16. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12]
[Table 1], [Table 2]