INTRODUCTION:
Piper betel leaves are consumed after food along with betel nut as a traditional practice in South Asia and the Indian subcontinent1. Piper betel leaves in particular were demonstrated to have many health benefits. Betel leaves were known to have antioxidant2, antiulcer3,4, antidiabetic5, cardioprotective6, anthelmitic7, anticancer8, 9 and antifungal activity10. However, its consumption with betel nut is known to cause habit formation and lead ill effects on health. It is known to cause oral cancers11. Despite its well-known carcinogenic potential, it is the fourth known, self-administered psychotropic substance after tobacco, alcohol and caffeine. The principal neuroactive alkaloid in betel nut is arecoline which is a non-selective agonist at both muscarinic and nicotinic cholinergic receptors. At the muscarinic receptors, it causes many central and peripheral nervous system effects but they do not have addiction potential.
Its activity on nicotinic acetylcholine receptor (nAChR) α4β2 accounts for its addictive property12. The active ingredient in tobacco is nicotine which is responsible for habit-forming behavior. Nicotine acts on the nicotinic acetylcholine receptor (nAChR). Stimulation of mesolimbic dopamine neurons by nicotine was associated with addiction behaviour observed in tobacco smoking. Similarly, betel nut which contains arecoline acts as an agonist at nAChR, was found to be associated with habit-forming with betel nut despite its ill effects on health. Since, there is a common underlying mechanism with both of them, we propose that usage of drugs for tobacco cessation, could also be effective against betel nut addiction.
Studies done for smoking cessation therapies have found that the drugs acting as partial agonists at nicotinic receptors leads to cessation of smoking by releasing dopamine sufficient to offset withdrawal symptoms and to reduce satisfaction. The α4β2 nACh receptor partial agonist varenicline was approved by the U.S. Food and Drug Administration for use in smoking cessation13.
Conditioned Place Preference (CPP) is a form of Pavlovian conditioning in which an environmental cue associates with the motivational effects of abusing drugs. This technique popularly used to study the dependency effects of abused drugs. Hence, in our study we would like to determine the beneficial effect of varenicline in betel nut dependence, because of the involvement of common nicotinic acetylcholine receptor α4β2 path in addiction caused by tobacco and betel nut14.
AIMS AND OBJECTIVES:
To study the effects of varenicline on betel nut dependence in Albino Wistar rats.
MATERIALS AND METHODS:
Animal selection:
Healthy inbred rats were procured from animal house of the institute. Forty male albino rats of wistar strain weighing 150-250 grams were used in the present study. They were housed under temperature of (23±2°C), humidity of (50±5 %) and 10-14 hours of light and dark cycles. Each animal was housed in separate, labeled polypropylene cages with sterile husk bedding, food pellets and water dispenser. Water and food pellets were provided ad libitum. Rats were randomized to 5 groups of 8 rats each and experiment conducted from 8 AM to 5 PM daily. The study was conducted in accordance to CPCSEA guidelines and after approval of Institutional animal ethics committee (IEC no: IAEC/KMC/16/2017).
Drugs:
Varenicline was purchased from the hospital pharmacy (Cipla, Mumbai manufactured).
Betel nuts were purchased from the local Udupi market and authenticated by a botanist.
Betel nut preparation:
Whole Areca catechu nuts were dried, weighed and pulverized in a grinder. The powder was added to Ringer's solution (pH 7.2) at 200mg per ml. This mixture was stirred for 10 minutes at room temperature and filtered using a filter paper12.
Drugs were prepared freshly every day and administered accordingly. The dosage was selected based on the earlier studies. Betel nut extract was given orally at a dose of 5mg/kg15 and varenicline was given subcutaneously at doses of 0.5mg/kg, 1mg/kg and 2 mg/kg16.
Saline was prepared in the laboratory freshly from NaCl of analytical grade.
Apparatus:
The test apparatus for CPP model consisted of three compartments: two large compartments measuring 20 × 35cm each and separated by minor start compartment measuring 10 x 10cm. The central start compartment has removable guillotine doors separating each of the large compartments. One of its walls was painted white with a
grid floor and the other wall was painted black with a hole floor. The central area connects the two chambers and serves as a start compartment17.
Table 1: Treatment Groups
|
Group |
Drug |
No. of Rats |
|
1 |
Saline + saline |
8 |
|
2 |
Saline + betel nut |
8 |
|
3 |
Varenicline 0.5mg/kg + betel nut 5mg/kg |
8 |
|
4 |
Varenicline 1mg/kg + betel nut 5mg/kg |
8 |
|
5 |
Varenicline 2mg/kg + betel nut 5mg/kg |
8 |
Saline 1ml/kg administered PO; betel nut 5mg/kg PO; Varenicline administered at 0.5mg/kg, 1mg/kg and 2 mg/kg for groups 3, 4 and 5 respectively by subcutaneous route (SC)
The body weight of all the rats were measured at the beginning and at the end of the study.
Conditioned Place Preference (CPP) model:
Experimental Procedure:
Session durations: Habituation = 15 min, Conditioning = 20 min, Post-conditioning = 20 min
Pre- conditioning (Habituation):
Day 1 (Monday) – Habituation for 15 min: Habituation precedes the conditioning phase in which rats will be allowed to adapt to the environment to prevent any effects of experimental novelty. Each animal was placed separately in the start chamber with opened guillotine doors to allow free access to whole experimental apparatus for 15 min. The total time spent by the rat in each of the large compartments was measured to find the baseline preference.
Rats with > 75% baseline preference for one chamber were excluded from further study. The chamber that rat spends more time at baseline was paired with saline and the other was paired with test drug. In the present experiment, we observed that all the rats spent more time in black chamber, hence saline was paired to it and the test drug (betel extract and varenicline) was paired to white chamber.
Conditioning (14 trial conditioning):
During this phase, rats were conditioned for each of the drug paired- chambers for 14 days. Conditioning sessions began from day 2 and were conducted in the morning and afternoon till day 18. During the session guillotine doors were closed and rats were paired in their respective chambers for 20 minutes. Sessions were conducted daily at the same time but the timing of administration of saline and the test drug were interchanged every day for each rat to eliminate the influence of biological clock on drug dependence behavior of animals. Betel nut extract was administered at 5mg/kg orally. Control group received saline before pairing to either of the chambers. Group 2 rats received only saline before pairing to black chamber and oral betel nut 30 minutes before being conditioned to white chamber. Groups 3,4,5 animals were pretreated with varenicline at the dose of 0.5mg/kg, 1mg/kg and 2 mg/kg by subcutaneous route (SC) route respectively and 30 minutes later, betel nut extract was given before conditioning to the white chamber. A constant time gap of six hours was maintained between the morning and afternoon sessions throughout the experiment. If the time spent in the chamber which was paired with the betel nut extract was more than the time spent in chamber paired with saline, then conditioned place preference was said to be established for the rats17.
Table 2: Conditioning schedule of rats with paired-chambers
|
|
8AM |
2PM |
|
Day 2 (Tuesday) |
Saline trial |
Drug trial |
|
Day 3 (Wednesday) |
Drug trial |
Saline trial |
|
Day 4 (Thursday) |
Saline trial |
Drug trial |
|
Day 5 (Friday) |
Drug trial |
Saline trial |
|
Day 6, 7 (Saturday, Sunday) – drug and saline free period. |
||
|
Day 8 (Monday) |
Saline trial |
Drug trial |
|
Day 9 (Tuesday) |
Drug trial |
Saline trial |
|
Day 10(Wednesday) |
Saline trial |
Drug trial |
|
Day 11 (Thursday) |
Drug trial |
Saline trial |
|
Day 12 (Friday) |
Saline trial |
Drug trial |
|
Day 13 (Saturday-Sunday) - drug and saline free period. |
||
|
Day 14 (Monday) |
Drug trial |
Saline trial |
|
Day 15 (Tuesday) |
Saline trial |
Drug trial |
|
Day 16 (Wednesday) |
Drug trial |
Saline trial |
|
Day 17 (Thursday) |
Saline trial |
Drug trial |
|
Day 18 (Friday) |
Drug trial |
Saline trial |
Conditioning period -20 minutes for each rat with saline and drugs in the morning and afternoon5
Post Conditioning:
On Day 19, treatment in all the groups stopped and each animal was placed in the neutral chamber, door was opened and allowed access to both the chambers freely. The time spent in each chamber was documented for 20 minutes. Meantime spent in each chamber estimated in different groups and CPP score is estimated. The CPP score is the time difference in seconds between post-conditioning and preconditioning in the drug-associated compartment.
RESULTS:
Table 3: Bodyweight changes in all the groups before and at the end of the treatment.
|
Group |
Basal weight |
End Weight |
P |
|
Control |
197.5±8.4 |
234±8.5 |
0.001 |
|
BN |
171±3.7 |
181±3.5 |
0.001 |
|
BN+VN 0.5 |
199±6.4 |
217±5.6 |
0.001 |
|
BN+VN 1 |
199±5 |
230±7 |
0.001 |
|
BN+VN 2 |
198±3.7 |
219±3.6 |
0.001 |
Values are expressed as mean ± SEM. P≤0.05 was considered significant.
Body weight changes are shown in Table 3. There was significant increase in body weight in all the groups compared to their baseline weight.
 |
Figure 1: Effects of varenicline on betel nut extract induced place preference.
Values expressed as mean ± SEM; ** P<0.01 vs. betel nut-conditioned rats;* P<0.01 vs. saline-conditioned rats; $ P<0.05 Vs. Group 3
The CPP score was significantly increased in betel nut group compared to all the other groups. Administration of varenicline reduced the CPP score compared to betel nut group. Betel nut administration during conditioning induced a clear place preference on the test day. Varenicline administered at all the 3 doses was effective in ameliorating acquisition behavior of betelnut (P<0.05; Fig. 1). The reduction was maximum at varenicline dose of 2 mg/kg and there was significant reduction compared to dose 0.5mg/kg.
Statistical analysis:
Statistical analysis was done by using SPSS version 20. Data was represented as mean with standard error mean for continuous data. ANOVA test with post-hoc Tukey test was done for analysis. P value of ≤0.05 was considered statistically significant.
DISCUSSION:
Our experiment showed increase in weight in betel nut and varenicline groups and demonstrated the dose dependent decrease in CPP score in varenicline treated groups. There were inconsistent reports on the effects of betel nut on body weight depending on species. Studies in rats reports either no effect on body weight or impaired weight gain18, 19. On the contrary, in humans its use was found to increase body weight and cause central obesity20. Previous reports suggests that betel nut inhibits GABA receptor which increases appetite and results in weight gain and fat accumulation21. Earlier study on varenicline in rats had reported a reduction in body weight in rats22. In our study, rats treated with varenicline had increased body weight. However, our study differs from the above study in the duration of treatment and drugs used, as varenicline was used concomitantly with betel nut.
In the present experiment we have used CPP model, an established model for studying drug seeking behavior. This is for the first-time, betel nut dependence was studied and the efficacy of varenicline in ameliorating betel nut dependence was evaluated. Varenicline was studied by a similar model for its efficacy in blocking the nicotine acquisition17. The current study demonstrated the beneficial actions of varenicline and its potential for use in betel nut dependence. Varenicline administration with the arecanut reduced the time spent in the drug paired chamber at all the doses. The benefits may be due to its action on nicotinic receptors. Areca nut contains a neuroalkaloid, arecoline which is known to produce addictive effects by its agonistic activity on nicotinic acetylcholine receptor (nAChR) α4β212. Varenicline by its partial agonistic activity could impair the rewarding effects of arecanut. The rewarding effect is believed to be due to activation of mesolimbic dopamine system due to nicotinic action. This drug is proven and approved drug for its similar mechanism in smoking cessation therapy13.
LIMITATION:
We couldn’t demonstrate the underlying mechanisms for the benefits of the drug. However, this study is the first to demonstrate the potential of varenicline as a cessation therapy in betel nut dependence. Further, clinical studies are required before its clinical application.
CONCLUSION:
Varenicline was found to be effective in cessation of betel nut seeking behavior in rat model.
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Received on 04.03.2022 Modified on 12.08.2022
Accepted on 21.12.2022 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(6):2791-2794.
DOI: 10.52711/0974-360X.2023.00459