Effect of acute and chronic treatment with QCF-3 (4-benzylpiperazin-1-yl) (quinoxalin-2-yl)
methanone, a novel 5-HT 3 receptor antagonist, in animal models of depression
Thangaraj Devadoss1, Dilip K. Pandey1, Radhakrishnan Mahesh1, Shushil K. Yadav2
Pharmacy Group, Central Animal Facility, FD-III, Birla Institute of Technology & Science, Pilani, Rajasthan-333031, India
Correspondence: Dilip K. Pandey, e-mail: pandeysdl1408@gmail.com
Abstract:
The serotonin type 3 (5-HT3) receptor is unique among the seven recognized serotonin receptor “families”. The existence serotonin type 3 receptor (5-HT3) in neuro-anatomical regions stimulated the research interest for novel therapeutic targets such as anxiety, depression, nociception and cognitive function. In the current study, (4-benzylpiperazin-1-yl) (quinoxalin-2-yl) methanone (QCF-3), a novel 5-HT3receptor antagonist, with an optimal log P (the logarithm of the ratio of the concentrations of the un-ionized solute in the solvents is called log P) and significant pA2 value (is a negative logarithm of the molar concentration of antagonist re- quired to reduce the effect of multiple dose agonist to that of single dose) was screened for its anti-depressant potential using rodent behavioral models of depression. Psycho-pharmacological investigations involved acute and chronic treatment (14 days) with QCF-3 and assessment of behavior during the forced swim test (FST) and tail suspension test (TST) in mice and olfactory bulbecto- mised rats. A dose response study in mice revealed an initial anti-depressant-like effect of QCF-3 (0.5–4 mg/kg, ip) in the FST and TST. Interaction studies showed that QCF-3 (1 and 2 mg/kg) significantly enhanced the antidepressant action of fluoxetine and bupropion in the FST and TST, respectively. QCF-3 (1 and 2 mg/kg) potentiated the 5-hydroxytryptophan (5-HTP) induced head twitches response in mice and reversed reserpine-induced hypothermia in rats. Further, OBX rats exhibited behavioral anomalies in the open field and hyper-emotionality tests that were attenuated by chronic QCF-3 treatment. In conclusion, this behavioral study de- scribes an antidepressant-like effect of QCF-3 in rodent behavioral models of depression.
Key words:
antidepressant-like effect, 5-HT!antagonist, forced swim test, olfactory bulbectomy
Abbreviations: FST – forced swim test, 5-HT – serotonin, 5-HT!– serotonin type 3 receptor, 5-HTP – 5-hydroxytryptophan, mCPP – m-chlorophenylpiperazine, OBX – olfactory bulbec- tomized, 8-OH-DPAT – 8-hydroxy-N,N-dipropyl-2-amino te- tralin, TST – tail suspension test
Introduction
Serotonergic neurons have regional distribution in brain areas, which implicated the range of neurologi-
cal phenomenon and there has been much interest in the therapeutic potential of serotonin type 3 (5-HT3) re- ceptor antagonists for anti-psychotic, anti-nociceptive and other neurological and psychiatric disorders. Pro- gressive research studying the neurobiology of de- pression has focused on the serotonergic neuro- transmitter system [11, 38] as potential target for the anti-depressants [50]. Other reported antidepressant mechanisms include changes in the density and sensi- tivity of 5-HT1 and 5-HT2 receptors as a result of chronic treatment with antidepressants. The 5-HT3 has been identified in the central nervous system [43],
and antagonists such as ondansetron, granisetron, do- lasetron and tropisetron are routinely used for the treatment of radiotherapy- and chemotherapy-induced nausea and vomiting in patients with cancer [20, 33].
Furthermore, 5-HT3 receptors antagonists are ef- fective in neuropsychiatric disorders, including bulimia and fibromyalgia [60]. In the last decade, these molecules have been mainly screened for neuropsychopharmacological uses in various pre-clinical and clinical studies [19, 64]. The possible involvement of 5-HT3receptors in neuropsychological disorders was suggested by Greenshaw [14]; furthermore Redrobe and Bourin [49] demonstrated that 5-HT3receptors play a partial role in the effectiveness of antidepressants during the FST. The involvement of 5-HT3receptors in the neu- ral pathways of depression has led to research investi- gating these receptors as antidepressant drug targets [8]. The 5-HT3 receptors are ligand-gated ion chan- nels that mediate the release of a number of neuro- transmitters [65], and 5-HT3 receptor antagonists freely pass the blood-brain barrier, which make com- pounds targeting these receptors as perfect therapeutic candidates for neuro-behavioral studies [14]. Depres- sion affects 15% of the population worldwide [17]
and is associated with both cancer and cancer chemo- therapy [36, 44]. Other condition comorbid with de- pression include anxiety [23], obsessive compulsive disorder [23], dementia [21], neuropathic pain [37]
and Parkinson’s disease [10]. Several antidepressants are commercially available for clinical use. However, many patients do not respond to conventional anti- depressant treatments, and the beneficial therapeutic effects appear after several weeks [18, 41]. Although there are several molecular targets available for the development of new antidepressant drugs, most of the current treatments for depression directly or indirectly affect the monoaminergic system [57].
Using the three-component pharmacophore model [17] for the 5-HT3 receptor antagonists as a guide, a series of compounds have been designed, synthesized and screened for their 5-HT3antagonist potential (Tab.
1). The compounds were tested for their ability to in- hibit the 5-HT3 receptor in isolated guinea pig ileum, and the pA2values were determined against 2-methyl-5- hydroxytryptamine [32, 33].
Animal models of depression have been frequently used to screen novel compounds [6] and were origi- nally designed as screening tests to assess the efficacy of antidepressant drugs. These tests are strong predic- tors of efficient antidepressant compounds [29].
Tab. 1. The pA and log P values for novel quinoxalin-2-carboxa- mides
Basic structure
Compd.a Rb pA2c logpd
QCF-1 5.9 1.1
QCF-2 7.3 2.84
QCF-3* 7.8 2.49
QCF-4 5.0 2.71
QCF-5
4.5 2.71
QCF-6
4.5 3.76
QCF-7
7.0 2.71
QCF-8
5.9 0.77
QCF-9
6.7 1.10
QCF-10
7.3 2.32
* (4-Benzylpiperazin-1-yl)(quinoxalin-2-yl) methanone was screened for its antidepressant potential in the current study.=A series of com- pounds synthesized in the laboratory and coded as QCF-1 to QCF-10.>Group attached to the carbonyl group of the basic struc- ture.?The pA values are calculated against 2-methyl-5-hydroxy- tryptamine.@The log p values are calculated using Chem Draw Ultra 11 (Cambridge Software)
N N
N N
N N
N
N OCH3
N N
H3CO
N N
CF N HN
HN N
HN N HN NH
Therefore, a behavioral test battery, including the FST [46] and TST [4, 59], was used for the preliminary an- tidepressant evaluation of the test compound. Further, interaction studies with various serotonergic modula- tors that exhibit an antidepressant effect, including 8- OH-DPAT, a 5-HT1A receptor agonist [31], and mCPP, a non selective 5-HT2Aagonist [47, 48], were conducted to determine the mechanism of the test compound. Parthenolide, a serotonin release inhibitor [35] that produces depressant-like effects [43], was tested with QCF-3 to explore the serotonergic influ- ence of QCF-3. Bupropion, a norepinephrine and do- pamine reuptake inhibitor [2], interacted with QCF-3 during the TST, which suggests that QCF-3 also has a dopaminergic effect. The 5-hydroxytryptophan (5- HTP)-induced head twitch response in mice, the reserpine-induced hypothermia test and an olfactory bulbectomy in rats were used to determine the antidepressant-like effects of the novel 5-HT3 anta- gonist in rodents. In the present study, the antidepressant potential of QCF-3 was evaluated in validated animal models of depression (FST, TST and olfactory bulbec- tomy).
Materials and Methods
Animals
Male Swiss albino mice, 25–35 g, and Wistar albino rats, 250–300 g, were obtained from Hissar Agricul- tural University (Hissar, Haryana, India) and maintained under standard lighting (lights on: 07.00–19.00 h), tem- perature (23 ± 2°C), and room humidity (60 ± 10%) conditions. The rodents were housed in standard poly- carbonate cages and provided with free access to food (standard pellet chow) and filtered water. Experimen- tal sessions began after the rodents had been quaran- tined for 20 days. The animals were used only once for each experiment and were strictly acclimated to the experimental room for 1 h before testing. Experi- ments on animals were approved by the Institutional Animal Ethics Committee of Birla Institute of Tech- nology & Science, Pilani, India (Protocol No. IAEC/
RES/04/01, dated 13.08.08).
Drugs
Fluoxetine and paroxetine were obtained from Sun Pharmaceuticals and IPCA Laboratories, (India), re- spectively. m-Chlorophenylpiperazine (mCPP) was obtained from Lancaster chemicals, (UK). Parthe- nolide and 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) were purchased from Tocris, (UK).
Bupropion hydrochloride was a gift from Ranbaxy Research Laboratories, (India). Pargyline and 5- hydroxytryptophan were purchased from Sigma Chemicals, (USA). Reserpine was purchased from Sisco Research Laboratories, (India). All drugs were freshly prepared in distilled water and administered per-orally (po) and intraperitoneally (ip). Dose-re- sponse studies were carried out using the mouse spon- taneous locomotor activity test, FST and TST to de- termine the QCF-3 doses that significantly exhibited antidepressant-like activity without affecting the baseline locomotion. QCF-3 (0.5–4 mg/kg) was ad- ministered to mice 30 min before starting the sponta- neous locomotor activity test, FST, and TST. The QCF-3 dose that had the maximum effect in prelimi- nary study was selected for the interaction and chronic studies.
Interaction studies
For the interaction studies, QCF-3 and 5-HT ligands were administered ip 30 min before testing, and mCPP and parthenolide were administered ip 45 min before testing, as described by Redrobe and Bourin [50] and Bourin et al. [5]. The acute dose-response profiles of QCF-3 were studied in both the FST and TST. Interaction studies with QCF-3 and the conven- tional antidepressants and mCPP were conducted with the FST.
Confirmatory studies
Confirmatory studies included the assessment of the effects of QCF-3 (1–2 mg/kg) on the 5-HTP-induced head twitch response in mice, the reserpine-induced hypothermia (1 mg/kg, ip) test in rats and the behav- ior response during the modified open-field and hyper-emotionality paradigm in OBX rats. For the 5- HTP-induced head twitch response, mice were treated with pargyline and 5-HTP (75 + 5 mg/kg) 30 and 45 min after QCF (1–2 mg/kg) administration, respectively.
For the olfactory bulbectomy experiment, which is
a model of chronic depression, rats received QCF-3 (1 and 2 mg/kg), paroxetine (10 mg/kg) or vehicle once a day for 14 days (15th to 28th day) after a post- surgical rehabilitation period of 14 days. Administra- tion of all drugs and behavioral testing were done be- tween 10.00–14.00 h. On the 29th day, OBX rats were subjected to an open field test followed by a hyper- emotionality test on the 30th day (Tab. 2). Behavioral assays were performed by trained experimenters who were blind to the treatment.
Chemistry
The title compound, (4-benzylpiperazin-1-yl) (quino- xalin-2-yl)methanone (QCF-3), was synthesized in three steps, involving oxidative cyclization of o-phe- nylenediamine with D-fructose to afford the 2- tetrahydroxybutylquinoxaline, which was oxidized to carboxylic acid (2-quinoxaline carboxylic acid) ac- cording to the procedures described by Harms [16].
The resultant carboxylic acid was stirred with dry tet- rahydrofuran, 1-(3-diethylaminopropyl)-3-ethyl car- bodiimide hydrochloride and 1-hydroxybenzotriazole under a nitrogen atmosphere in an ice bath for 45 min to yield the active ester (above reaction mass). An N- benzylpiperazine was added to the active ester and stirred at room temperature for 6 h. The reaction mass was concentrated under vacuum, and the resultant residue was diluted with ethyl acetate and washed with 10% aqueous sodium bicarbonate and water. The aqueous layer was back extracted with ethyl acetate, and the combined organic layers were dried over an- hydrous sodium sulfate and concentrated under vac- uum to yield the target product. The product was con- verted into a hydrochloride salt by treating the prod- uct with ethanolic hydrochloric acid.
BEHAVIORAL ASSAYS
Spontaneous locomotor activity
Spontaneous locomotor activity was assessed using the actophotometer [3], which contains a square arena (30 × 30 cm) with walls that are fitted with photocells just above the floor level. The photocells were checked before the beginning of the experiment. The drug/vehicle treated mice (n = 8) were then individu- ally placed in the arena. After a two minute acclima- tion period, the digital locomotor scores were re- corded for the next 10 min.
Forced swim test
The FST described by Porsolt et al. [46] was slightly modified [34]. In brief, each mouse was placed indi- vidually in a glass cylinder (diameter 22.5 cm, height 30 cm) that was filled to the 15 cm mark with water.
The floor of the cylinder was demarcated into four equal quadrants. The mice were forced to swim for 15 min on the pre-test day. Mice were then allowed to return to their home cage. After 24 h from the pre-test day, each mouse (vehicle/drug treated) was placed into the water and forced to swim for 6 min. The dura- tion of immobility and number of quadrants crossed during the last 4 min was measured. The mouse was considered to be immobile when it stopped struggling and passively moved to remain floating and keep its head above water. Water was changed between trials and temperature was maintained at 22 ± 2°C.
Tail suspension test
Behavioral despair was induced by a TST according to the procedure of Steru et al. [59]. Mice were sus- pended individually from a horizontal bar 50 cm above
Tab. 2. Surgery and treatment schedule to assess the effect of QCF-3 on olfactory bulbectomized rats
Day 0 0th – 1st day 1st – 14th day 15th – 28th day 29th – 30th day
Behavioral assessments
29th 30th
Surgery Recovery from surgery (continuous care)
Rehabilitation period (daily handling and
observation)
Drug/vehicle treatment (once a daypo administration for
14 days)
Modified open field exploration
Hyper-emotionality
the tabletop using adhesive tape. The point of attach- ment on the tail was 1 cm from the tip. The duration of immobility during the 6-min observation period was recorded. Mice were considered immobile only when they were completely motionless. The parame- ter recorded was the number of seconds spent immo- bile.
5-Hydroxytryptophan-induced head twitch response
The method has been mentioned elsewhere [36], and it was adopted with slight modifications. Male mice that were pre-treated with QCF-3 were treated with par- gyline hydrochloride (75 mg/kg) and 5-HTP (5 mg/kg) 30 min and 15 min before the observations began, re- spectively. Fifteen minutes after 5-HTP administra- tion, the number of head twitches exhibited by the mice in a 15 min was recorded as the head twitch score. The head twitch response was characterized by abrupt lateral movements, which may or may not be accompanied by body twitches and hind limb retrac- tions.
Reserpine-induced hypothermia
Male Wistar rats were treated with reserpine (1 mg/kg, ip) 30 min after po administration of QCF-3. The ani- mals were gently restrained by hand while inserting the probe rectally with lubrication. On the day preced- ing the experiments, the rats were individually iden- tified, weighed and subjected to a temperature reading in order to habituate the animals to the experimental procedures. The effects of QCF-3 (1–2 mg/kg) on reserpine-induced hypothermia (measured with digi- tal thermometer) were recorded 30 min before admin- istering reserpine and 30, 60, 90 and 120 min after.
Hypothermia was measured by calculating the tem- perature difference between 120 min and 0 min.
OLFACTORY BULBECTOMY Surgery
A bilateral olfactory bulbectomy was performed as described previously [34, 48] with a slight modifica- tion. The rats were anaesthetized with xylazine (5 mg/
kg) and ketamine (75 mg/kg, ip). The head of the rat was fixed in a stereotaxic frame, and the skull was ex- posed by a midline sagittal incision. Burr holes (2 mm
in diameter) were drilled 8 mm anterior to the bregma and 2 mm on either side of the midline at a point cor- responding to the posterior margin of the orbit of the eye. The olfactory bulbs were removed by suction, the holes were filled with hemostatic sponge to control excessive bleeding, and the scalp was sutured. To pre- vent post-surgical infection, the rats were given a Sul- prim injection (each ml containing 200 mg of sulfadi- azine and 40 mg of trimethoprim) intramuscularly (0.2 mL/300 g) once a day for 3 days. Sham-operated rats went through the same procedure, including piercing of the duramater, with their bulbs left intact.
Open field exploration
The OBX and sham rats were subjected to an open field test 29 days after surgery and 15 days after start- ing the chronic drug/vehicle treatment. The open field exploration was conducted as described [23, 48]. The apparatus consisted of a circular (90 cm diameter) arena with 75-cm high aluminum walls, and a 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 test- ing room. Each rat was individually placed in the cen- ter of the open field apparatus and the ambulation scores (number of squares crossed), rearing and fecal pellet were counted for 5 min.
Hyper-emotionality
Hyper-emotionality was measured using a modifica- tion of the procedure described by Brady and Nauta [7] and Shibata et al. [54]. Hyper-emotionality of rats was measured by scoring responses to the following stimuli: (1) Startle response: was induced by a stream of air (using a 10-ml syringe) directed at the dorsum, (2) Struggle response: was induced by handling with a gloved hand, (3) Fight response: was induced by pinching the tail with forceps, and (4) Bite response:
was induced by presenting a rod 4–5 cm in front of the snout. A trained researcher performed these opera- tions. These responses were graded as follows: 0, none; 1, slight; 2, moderate; 3, marked; or 4, extreme.
All animals in each group were observed in 1 day.
The score for each animal was determined within 5 min of the observed response. The observers were blind with respect to the drug treatment.
Statistics
Data were expressed as the mean ± SEM. The single treatment studies were analyzed using a one-way analysis of variance followed by a Dunnett post-hoc
test. The interaction studies were analyzed using a two-way analysis of variance followed by a Tukey post-hoc test. The level of statistical significance was fixed at p < 0.05.
Results
Locomotor scores
Figure 1 displays the effects of QCF-3 on locomotor activity in mice. Lower doses of QCF-3 (0.5–4 mg/kg, ip) had no influence on locomotor activity when com- pared to the control group, whereas a higher QCF-3 (8 mg/kg) dose significantly (p < 0.05) decreased the locomotor activity compared to the control group.
Forced swim and tail suspension tests
These tests for depressive-like behavior measure the duration of immobility, which reflects behavioral despair.
Post-hoc analysis revealed that QCF-3 (1–4 mg/kg) in- duced a significant [F (5,42) = 24.73, p < 0.05] reduc- tion in immobility time and a significant increase in swimming behavior in mice during the FST [F (5,42)
= 26.44, p < 0.05] as compared to the control group (Fig. 2A–B). The positive control, fluoxetine (10 mg/kg), also induced a significant change in immobility and swimming behavior (p < 0.05) as compared to control group. In the TST, QCF–3 (1–4 mg/kg) treatment sig-
Fig. 1. Effect of QCF-3 on spontaneous locomotor activity in mice.
The columns represent the mean locomotor scores recorded in a 10- min observation period. The error bars indicate the SEM, n = 8 per group
Fig. 2. Effect of QCF-3 and fluoxetine on the duration of immobility in mice during the FST. The columns represent the mean (A) duration of immobility (s) and (B) swimming episodes. The error bars indicate the SEM, * p < 0.05 when compared with the vehicle-treated group;
n = 8 per group
Fig. 3. Effect of QCF-3 and bupropion on duration of immobility in mice during the TST. The columns represent the mean duration of im- mobility (s) and error bars indicate the SEM, * p < 0.05 when com- pared with the vehicle-treated group; n = 8 per group
nificantly [F (5,42) = 43.64, p < 0.05] decreased the duration of immobility as compared to the control group (Fig. 3).
Interaction studies
The peak dose of QCF-3 (1–2 mg/kg) that induced a significant (p < 0.05) decrease in the duration of im- mobility and a significant (p < 0.05) increase in swim- ming episodes was selected for the interaction studies.
During the FST, mCPP (1 mg/kg) significantly in- creased the duration of immobility [F (5,42) = 6.90, p < 0.05] and decreased the swimming episodes [F (5,42) = 21.51, p < 0.05] in mice as compared to the control group (Fig. 4A–B). QCF-3 failed to reverse the mCPP-i4251nduced immobility. During the FST, 8-OH-DPAT (1 mg/kg, ip) showed a significant
antidepressant-like activity in mice. Pre-treatment with QCF-3 had no significant effect on the duration of immobility [F (5,42) = 38.64, p < 0.05] and the swimming episodes [F (5,42) = 16.39, p < 0.05] as compared to the effects induced by 8-OH-DPAT alone (Fig. 5A–B). The antidepressant-like effects of QCF-3 were weaker than that of fluoxetine (10 mg/kg, ip).
Pre-treatment with QCF-3 significantly (p < 0.05) en- hanced the antidepressant action of fluoxetine by de- creasing the duration of immobility [F (5, 42) = 38.64, p < 0.05] and increasing the swimming episodes [F (5,42) = 22.91, p < 0.05], as shown in Figure 6A–B.
Parthenolide significantly induced a depressant-like effect in mice during the FST, which was character- ized by an increase in the duration of immobility [F (5,32) = 42.44, p < 0.05] and a decrease in swimming behavior [F (5, 32) = 86.09, p < 0.05]; QCF-3 signifi- cantly reversed the depressant-like effect of parthe- nolide (Fig. 7A–B). Co-administration of QCF-3 (1–2
Fig. 4. Effect of QCF-3 (1–2 mg/kg, ip) on the mCPP-induced (1 mg/kg,ip) increase in (A) the duration of immobility (s) and (B) swimming episodes in mice during the FST. The columns represent the mean of the values. Error bars indicate the SEM, * p < 0.05 when compared to the control group, (n = 8 per group)
Fig. 5. Effect of QCF-3 (1–2 mg/kg, ip) pre-treatment on the antidepressant effects of 8-OH-DPAT (1 mg/kg,ip) in mice during the FST. Columns represent the mean of the (A) duration of immobility and (B) swimming episodes. Error bars represent the SEM, * p < 0.05 when compared to the control group, n = 8 per group
mg/kg) and bupropion significantly [F (5,42) = 57.72 p < 0.05] augmented the antidepressant activity of bupropion in mice during the TST when compared to bupropion alone (Fig. 8).
5-HTP-induced head twitch response
The co-administration of pargyline and 5-HTP (75 + 5 mg/kg) induced the characteristic head twitch re- sponse. Pre-treatment with QCF-3 (1–2 mg/kg) and fluoxetine significantly [F (3,28) = 31.75, p < 0.05]
potentiated the head twitch response as compared to a combination of pargyline and 5-HTP alone (Fig. 9).
Reserpine-induced hypothermia
Administration of reserpine (1 mg/kg, ip) to rats elic- ited a pronounced decrease [F (4,25) = 69.42, p <
Fig. 6. Effect of QCF-3 (1–2 mg/kg,ip) pre-treatment on the antide- pressant effects of fluoxetine (10 mg/kg,ip) in mice during the FST.
Columns represent the mean of the (A) duration of immobility and (B) swimming episode. Error bars represent the SEM, * p < 0.05 when compared to the control group,p < 0.05 when compared to the fluoxetine-treated group and * p < 0.05 when compared to the QCF- 3-treated group, n = 8 per group
Fig. 7. Effect of QCF-3 (1–2 mg/kg,ip) pre-treatment on the depres- sant effect of parthenolide (1 mg/kg,ip) in mice during the FST. Col- umns represent the mean of the (A) duration of immobility and (B) swimming episodes. Error bars represent the SEM, * p < 0.05 when compared to the control group,p < 0.05 when compared to the parthenolide-treated group, n = 8 per group
Fig. 8. Effect of QCF-3 (1–-2 mg/kg,ip) pre-treatment on the antide- pressant effect of bupropion (20 mg/kg) in mice during the TST. Col- umns represent the mean of the duration of immobility. Error bars rep- resent the SEM, * p < 0.05 when compared to the control group,
p < 0.05 when compared to the bupropion-treated group and p <
0.05 when compared to the QCF-3-treated group, n = 8 per group
0.05] in core body temperature. This effect was sig- nificantly (p < 0.05) attenuated by QCF-3. Similarly, fluoxetine reversed the hypothermic effect of re- serpine (Fig. 10).
OLFACTORY BULBECTOMY IN RATS Open field test
The effect of different treatment combinations on the behavior of the OBX/sham rats was investigated (Tab.
3). Removal of the olfactory bulbs from rats produced a characteristic hyperactivity during the open field test compared to sham rats. Chronic (14 days) treat-
ment with QCF-3 significantly (p < 0.05) reduced the amount of ambulation [F (7,40) = 16.59, p < 0.05], rearing [F (7,40) = 31.50, p < 0.05] and fecal pellets [F (7,40) = 4.88, p < 0.05] in the OBX rats as com- pared to the vehicle-treated OBX rats. QCF-3 (1–2 mg/kg) exhibited antidepressant-like effects, while paroxetine was the most effective antidepressant among all treatments.
Hyper-emotionality
Figure 11 displays the mean scores for hyper- emotionality in the experimental groups, sham and OBX rats. OBX rats showed significant (p < 0.05)
Tab. 3. Effect of QCF-3 and paroxetine on the behavior of OBX rats in the modified open field test
Groups Dose (mg/kg) Ambulation Rearing Fecal Pellets
Sham control 0 91.17 ± 6.88 10.00 ± 1.24 2.81 ± 0.49
Sham + paroxetine 10 99.67 ± 9.31 8.67 ± 0.97 2.00 ± 0.70
Sham + QCF-3 1 103.00 ± 8.87 9.33 ± 1.05 2.33 ± 0.71
Sham + QCF-3 2 102.17 ± 7.34 8.33 ± 1.86 2.00 ± 0.76
OBX control 0 212.5 ± 14.59* 36.33 ± 2.89* 6.83 ± 0.70*
OBX + paroxetine 10 113.11 ± 9.76# 11.83 ± 1.78# 2.33 ± 0.49#
OBX + QCF-3 1 153.50 ± 9.59# 23.17 ± 3.15# 3.67 ± 0.99#
OBX + QCF-3 2 124.67 ± 14.29# 15.67 ± 2.49# 2.67 ± 0.95#
The values are expressed as the mean ± SEM. The drug/vehicle treatments were administered once a day for 14 days. * p < 0.05 when com- pared to the sham control,p < 0.05 when compared to the vehicle-treated OBX group, n = 6 per group
Fig. 9. The effect of QCF-3 on the 5-HTP/pargyline-induced head twitches in mice. The columns represent the mean number of head twitches during a 15 min . All the animals were administered 5-HTP (5 mg/kg,ip) and pargyline (75 mg/kg, ip). Error bars represent the SEM, * p < 0.05 when compared to the control group, n = 8 per group
Fig. 10. Effects of fluoxetine (10 mg/kg,po) and QCF-3 (1–2 mg/kg, po) treatment on reserpine-induced hypothermia in rats. The col- umns represent the mean decrease in rectal temperature (°C). Error bars represent the SEM, * p < 0.05 when compared to the 2nd h value of vehicle treatment, n = 6 per group
hyper-emotional behaviors, which included evalua- tion of the bite, startle, struggle, and fight response, when compared to sham rats. The hyper-emotional behavior exhibited by the OBX rats was significantly [F (7,40) = 39.13, p < 0.05] reduced by chronic treat- ment with QCF-3.
Discussion
The present behavioral investigation with various ani- mal models of depression revealed the antidep- ressant-like effects of a novel 5-HT3 antagonist, QCF-3. According to the Hibert et al. [17] pharma- cophore model, a compound should show better 5-HT3 receptor antagonism, and it should possess three necessary elements, namely an aromatic ring, a carbonyl linking moiety and a basic nitrogen center in a specific spatial arrangement. Most of the reported 5-HT3receptor antagonists satisfy these criteria. The synthesized compounds (Tab. 1) comply with the pharmacophore model. When assessing the antide- pressant potential of a drug, an increase in baseline lo- comotion can lead to a false positive result [46].
QCF-3 exhibited antidepressant-like effects in the FST and TST without affecting baseline locomotion.
The antidepressant-like activity of a compound is determined by a decrease in the duration of immobil- ity during the FST and TST. Both of these models of depression are widely used to screen new chemical entities for their antidepressant potential [46, 59].
These tests are quite sensitive and relatively specific to all classes of antidepressants. In the present study, QCF-3 significantly decreased the duration of immo- bility in mice during the FST and TST. A reduction in the duration of immobility is a measure of the antide- pressant properties of a drug [46]. The antidepressant- like effect of QCF-3 does not seem to be associated with any motor effects because it did not show a sig- nificant change in locomotion in mice. Testing for in- teractions with selective serotonin reuptake inhibitors is necessary to assess the antidepressant potential of 5-HT3receptor antagonists [9, 50]. QCF-3 (1–2 mg/kg) significantly enhanced the antidepressant-like action of fluoxetine. There is an antidepressant-like effect of 8-OH-DPAT (partial 5-HT1A agonist) at a dose of 1 mg/kg [28, 40, 47] in mice during the FST. Pre- treatment with QCF-3 had no influence on the antide- pressant activity of 8-OH-DPAT, which suggests that the stimulation of the 5-HT1 autoreceptor by 8-OH- DPAT [51] led to saturation of the mobility effects. To determine the functional profile of QCF-3 on the 5-HT2receptor, an interaction study with QCF-3 and mCPP, a non-selective 5-HT2 receptor agonist [48], was conducted. QCF-3 failed to reverse the depres- sant-like action of mCPP, which eliminates a possible effect of QCF-3 on the 5-HT2receptor. A depressant- like effect induced by parthenolide was considered as a model to identify antidepressants acting through se- rotonergic mechanisms [43]. QCF-3 significantly re- versed the depressant-like effect of parthenolide, pos- sibly through serotonin release. To investigate if QCF-3 interacts with the dopaminergic system, fol- lowing the dopamine hypothesis of depression [49], an interaction study with QCF-3 and bupropion was conducted in mice using the TST. Due to species- dependent variation, bupropion failed to exhibit anti- depressant effects in the FST with Swiss albino mice [4]. Hence, the interaction study with the TST was ex- pected to shed some light on the influence of the dopa- minergic system. In the present study, QCF-3 pre- treatment augmented the antidepressant effect of bupropion in the TST, which suggests that the dopamin- ergic system is involved in the antidepressant-like effect of QCF-3. Though the exact mechanism is unclear, it
Fig. 11. Effects of paroxetine (10 mg/kg,po) and QCF-3 (1 and 2 mg/kg,po) on the hyper-emotionality behavior in OBX /sham rats. All drug and vehicle solutions were administered once a day (po) for 14 days. Results are exprerssed as the mean of the hyper-emotionality scores. Error bars represent the SEM, * p < 0.05 when compared to the sham operated rats, p < 0.05 when compared to the vehicle-treated OBX rats (n = 6 per group)
could involve the release of dopamine through inhibi- tion of the 5-HT3receptor.
One of the pharmacological mechanisms of antide- pressants is the enhancement of synaptic concentra- tions of monoamines, particularly serotonin. 5-HTP is the immediate precursor of serotonin, and administra- tion of 5-HTP was reported to increase serotonergic transmission and induce a characteristic head twitch response in mice [41, 43, 53]. In the present study, pargyline- and 5-HTP-induced head twitch responses were significantly potentiated by fluoxetine and QCF-3 pre-treatment. In this regard, the antidepres- sant-like effect of QCF-3 appears to be modulated by an increase in serotonin concentrations at the synapse [43].
Reserpine is monoamine depleting agent that blocks the transport of monoamines into synaptic vesicles. The depletion of brain serotonin affects the central nervous system and is characterized by hypo- thermia and ptosis [12]. The decrease in body tem- perature induced by reserpine is antagonized by anti- depressants [12]. QCF-3 and fluoxetine significantly prevented the hypothermic effect of reserpine, which indicates antidepressant effect of QCF-3 in this sensi- tive model.
Olfactory bulbectomy has been proposed to be an agitated hypo-serotonergic model of depression [30]
and is used to explore the antidepressant potential of novel agents [5]. OBX rats exhibited a specific, ab- normal behavioral pattern in the open field test [23, 57] characterized by increased ambulation, rearing and fecal pellets [56], and this abnormal behavior is reversed by antidepressants [62]. In the open field test, QCF-3 and paroxetine significantly reversed the hyperactivity exhibited by OBX rats.
Hyper-emotionality is manifested by violent at- tacks or flight reactions in response to previously neu- tral or innocuous stimuli. In the current study, expos- ing OBX rats to noxious stimuli elicited this hyper- emotional behavior [27, 60, 64]. Thus, it is possible that OBX-induced hyper-emotional behavior resem- bles psychomotor agitation, which is a diagnostic cri- terion for depression [14,]. This hyper-emotionality of OBX rats is useful for evaluating antidepressant activ- ity [26, 54, 55, 63]. Chronic treatment with QCF-3 significantly reduced the hyper-emotional behavior exhibited by OBX rats.
The present neuro-behavioral studies showed an antidepressant-like effect of QCF-3, a 5-HT3antago- nist, in animal models of depression. The precise
mechanism by which QCF-3 produced an antidepres- sant-like effect is not understood. However, a poten- tiation of the head twitch response and reversal of reserpine-induced hypothermia suggests that QCF-3 produced an antidepressant-like effect by increasing the concentration of a neurotransmitter [52]. An in- crease in the level of serotonin through blockade of the 5-HT3 receptor [52] could possibly explain the antidepressant-like effect of QCF-3 [15]. Reversal of the parthenolide-induced depression-like behavior in- dicates that serotonin is involved in the antidepressant like action of QCF-3. The location of the 5-HT3 re- ceptor in a neuro-anatomical region might be regulat- ing the 5-HT transmission indirectly [25].
Based on the results, we suggest that QCF-3, a novel 5-HT3 antagonist, could be a potential candidate for the management of depression. A major drawback of all marketed antidepressants, regardless of their mechanism of action, is a delayed onset (2–4 weeks) of therapeutic efficacy. Combining these drugs with a 5-HT3 antagonist could potentially accelerate the onset of the antidepressant effect. Hopefully, future studies will provide a better understanding of the pro- miscuous nature of the novel 5-HT3 antagonists, which will lead to agents that are useful for control- ling cancer and chemotherapy-induced depression. To characterize the effect of QCF-3 at the cellular level, future studies with molecular techniques are neces- sary. The development of more specific ligands may also allow for a more directed approach, with their long-term effectiveness in depression
Acknowledgment:
This work was supported by the University Grants commission, India.
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Received:
February 10, 2009; in revised form: Novmber 18, 2009.
