INTRODUCTION:

Opioids such as morphine have been used for thousands of years for the treatment of pain. It is considered as the most powerful drugs to relieve pain. However, the development of tolerance, dependence and severe withdrawal symptoms limits their long-term use after repeated opioid administration. These phenomenon is caused by the adaptation that occurred to counteract prolonged opioid receptor activation [1-3] Adaptive changes have been observed within the central nervous system at difference organization level. It involved from alteration in receptor and cellular level to functional modifications of different neuronal networks [4-5].

The mu-opioid receptor (MOR) is key to morphine’s action, and there are several lines of evidence on the strong relationship between MOR activation and tolerance, dependence and withdrawal. Abnormal prolong activation of MOR by opiates causes multiple cellular adaptation in downstream. Previous studies reported that endocytosis of the MOR can prevent the morphine tolerance and dependence subsequently block the onset of withdrawal [6].

It has been suggested that other’s type opioid that has high ability to promote MOR endocytosis could reduce tolerance as compared to morphine [7-11]. Other than that, series of studies proposed that enhancing morphine-induced MOR endocytosis significantly reduce the development of tolerance and/or dependence in both in vitro and in vivo [9, 12-17].

Methadone is a synthetic opioid medication that is administered orally in the treatment of opioid dependence. Methadone quickly achieves steady-state plasma levels after repeated administration [18]. Its effects are qualitatively similar to morphine and other opiates hence it is widely being used in methadone maintenance therapy [19]. Couple of studies reported that adequate therapeutic doses of methadone could reduce morphine craving and subsequently block the onset of withdrawal [20,21].

It has long been known that physiological concentrations of sodium facilitate and promote the activation of MOR [22]. Parallel to this mechanism, we proposed that Zamzam water, which contain highly abundant of sodium might have an effect on the regulation of MOR. Zamzam water is natural water that come from well located in Makkah and few meters east of Kabaah. The well is 35 meters deep. A series of studies have shown that Zamzam water contain high concentration of mineral compared to other normal mineral water [23,24]. It has been found that Zamzam water has curative effect in the management of opioid dependence due to its highly mineral contain [25].

The aim of this study was to investigate the effects of methadone, Zamzam water and co-treatment methadone and Zamzam water to alleviate the withdrawal symptoms among morphine dependent rats.

MATERIALS AND METHODS:

ANIMALS:

Healthy male Sprague-Dawley rats weighing between 180–220 gram were housed and kept at 25–30^°C in the Animal Center, Faculty of Medicine, Universiti Sultan Zainal Abidin, UniSZA. Animals were divided into five groups (n = 10) in a temperature-controlled room. They were maintained under standard laboratory conditions with natural dark and light cycle. They were allowed free access to food and tap water ad libitum. Animals were acclimatized for at least one week to adapt to the laboratory prior to test. All animal experimental procedures were approved (Approval no: BNU/EC/01/2011) by the Ethics Committee of Universiti Sultan Zainal Abidin (UniSZA). At the end of the treatment duration, rats were sacrificed by decapitation and their brains were dissected rapidly for Ventral Tegmental Area (VTA) regions. All samples were placed on dry ice and stored at -80^°C until the days of assay.

Drug:

Morphine sulphate (10mg/ml) and methadone hydrochloride (5mg/ml) were purchased from Merck (Germany). Morphine sulfate was dissolved in normal saline solution and was injected to the rats intraperitoneally. Meanwhile for treatment, methadone syrup was dissolved in the distilled water and Zamzam water, respectively and then consumed by morphine dependent rat orally.

Induction of Morphine Dependence:

In first day, rats were made dependent by repeated intraperitoneal injections of 10 mg/kg morphine at 08.00 AM and 19.00 PM. The morphine doses was increased daily by 2 mg/kg increments per day until a maximum of 68 mg/kg twice daily for 30 days was achieved [26]. (Ghowsi and Yousofvand, 2015).

Experimental Groups:

Rats were randomly divided into five experimental groups, each comprising 10 rats as follows; negative control group, received distilled water orally for 30 consecutive days. No morphine was injected to this group. In positive control group, rats were received variable doses of morphine sulfate solution for 30 consecutive days. Morphine was stop after 30 days and no treatment was given in this group during treatment period. Next, in the methadone treatment group, rats received variable doses of morphine sulfate solution for 30 consecutive days. Subsequently, the rats were given methadone orally as a treatment with increasing dose by 1 mg/100 mL per day until a maximum of 2.5 mg/100 mL for 30 days was achieved. In Zamzam water treatment group, the rats were received variable doses of morphine sulfate solution for 30 consecutive days. Then, the rats were supplemented with Zamzam water orally as a treatment for 30 consecutive days after day one of withdrawal. Last group, co-treatment methadone and Zamzam water group, rats were received variable doses of morphine sulfate solution for 30 consecutive days. Afterwards, the rats were orally given with methadone mixed with Zamzam water as a treatment with increasing dose by 1 mg/100 mL per day until a maximum of 2.5 mg/100 mL for 30 days was achieved.

Spontaneous Animal Withdrawal Behaviour Observation:

The animals were placed individually into plexiglas cages at 10:00 a.m. on days one, seven, fourteen, twenty-one, thirty after the withdrawal of morphine for 5 minutes for acclimation. Then, signs of spontaneous withdrawal like diarrhea, wet dog shake, body writhing, jumping and teeth chattering were observed for 30 minutes period. Scores were averaged for each behavior test.

RESULT AND DISCUSSION:

As shown in figure 1, co-treatment of methadone and Zamzam water was significantly reduced the number of diarrhea in rats undergoing morphine withdrawal. Tukey’s Post-test analysis revealed a statistically significant (P<0.05) reduction in the number of diarrhea in a group of co-treatment methadone and Zamzam as compared to others treatment group after 14 days of treatment.

Figure 1 Effects of methadone, Zamzam water and co-treatment methadone with Zamzam water on diarrhea in rats undergoing spontaneous morphine withdrawal.

Figure 2 shows the number of wet dog shake in the five groups of experiments. In this study, all treatment group, methadone alone, Zamzam water alone and co-treatment methadone with Zamzam water together show significantly reduced on wet dog shake compared with non-treatment group on the last day of morphine withdrawal (Day 30) (P < 0.05) (Figure 1).

Figure 2 Effects of methadone, Zamzam water and co-treatment methadone with Zamzam water on wet dog shake in rats undergoing spontaneous morphine withdrawal.

As shown in figure 3, Tukey’s Post-test analysis revealed that morphine-dependent rats treated with methadone alone, Zamzam water alone and co-treatment methadone and Zamzam water were significantly reduced (P<0.05) in the number of jumping compared to untreated group.

Figure 3 Effects of methadone, Zamzam water and co-treatment methadone with Zamzam water on jumping behaviour in rats undergoing spontaneous morphine withdrawal.

As shown in figure 4, only rats that treated with methadone and co-treated methadone and Zamzam water showed a significant reduction (P<0.05) in the number of teeth chattering when compared to positive control group.

Figure 4 Effects of methadone, Zamzam water and co-treatment methadone with Zamzam water on teeth chattering in rats undergoing spontaneous morphine withdrawal.

Post-test analysis revealed there was not a significant reduction (p>0.05) in the number of body writhing in rats treated with methadone alone, Zamzam water alone and co-treatment methadone and Zamzam water compared to positive control group (figure 5).

Figure 5 Effects of methadone, Zamzam water and co-treatment methadone with Zamzam water on body writhing in rats undergoing spontaneous morphine withdrawal.

The result of the current study showed the treated groups including methadone, Zamzam water and co-treated of both methadone and Zamzam water exhibited significant reduction in the number of diarrhea, wet dog shake and jumping (p<0.05) compared to the non-treatment group. Another noteworthy fact was that regarding the diarrhea and wet dog shake manifestation, the treatment group that was treated with both methadone and Zamzam water showed a more promising result than the group being treated with methadone alone. This combination treatment Zamzam water and methadone show a significant reduction as compared to methadone treatment (p<0.05). This result probably due to synergistic effects exhibited by Zamzam water when combine together with methadone.

Watery diarrhea is a common manifestation of withdrawal from opioid in dependent rats [27] and it is considered as one of the Narcotic bowel syndrome (NBS) [28] and opioid induced bowel dysfunction [29]. Until now, researcher still debate on the mechanism of opioid induced diarrhea although some researcher believed that the increased production of free radical induced by nitric oxide synthase (NOS) is main causes of diarrhea in morphine-dependent rats [30]. Some studies have shown that free radical scavengers such as vitamin C derivative and a vitamin E derivative potently suppressed several withdrawal signs like body weight loss and diarrhea induced by morphine, indicate that the role of antioxidant agent in alleviate the morphine withdrawal in rat [27, 31]. Furthermore, fullerene, a strong free radical scavenger, almost completely suppressed naloxone-precipitated withdrawal symptoms including diarrhea [27, 31].

In the current study, 30ml of Zamzam water drinking orally by rat for 30 days reduce the number of diarrhea after chronic morphine administration. We postulated that Zamzam water have capability to reduce the number of diarrhea in morphine dependent rats by eliminating free radical. Alkaline nature of the Zamzam water has been reported and it makes it as beneficial water especially in term of a potential antioxidant agent [32]. Other than its alkaline nature, its composition in terms of minerals and trace elements may also play a vital role as a potential antioxidant agent in boosting of antioxidant based mechanisms [33].

Wet dog shake behaviour is commonly used in animal model for qualifying serotoninergic activity in the brain [34,35]. It is reported that the level of serotonin depleted during morphine withdrawal which is responsible for the occurrence of wet dog shake behaviour in rats [36-38]. These mechanisms may explain the rout by which combination of methadone and Zamzam water led to the reduction of wet dog shake. We postulated that the reduction of wet dog shake amount after treated with Zamzam water or co-treatment methadone and Zamzam water is due to the presence of lithium inside the Zamzam water. Series of studies have shown that lithium has been proven to exerts an inhibitory effect on the cAMP production [39-41]. Furthermore, lithium was reported increase the level of serotonin in rat [42, 43] and 5-HT release from rat brain slices [44-45] which can restore the depleted serotonin level during morphine withdrawal.

It is also interesting to note that in Zamzam water treatment group show a significant decrease in the number of jumping compared to non-treatment group (p<0.05). It is well-known that jumping behaviour is a common morphine withdrawal sign in rat. By definition, jumping is considered as an attempt to escape from the test chamber [46]. According to Nishikawa et al. [47], stress and anxiety are the factor behind this phenomenon, which is a result of drug-induced maladaptation in noradrenergic and dopaminergic systems in rat brain. It is interesting to note that in Zamzam water treatment group show a significant decrease in the number of jumping compared to non-treatment group and this is might be a novel report. Zamzam water reduce the number of jumping which shows that it has a highly significant inhibitory effect on anxiety and stress. This may be due to the fact that Zamzam water is known as antidepressant agents, which can further contribute in the decrease in the number of jumping [48].

CONCLUSION:

In conclusion, we have demonstrated that combination of methadone and Zamzam water reduce/prevent the development of morphine dependence and this is speculated to be mediated by up regulated of MOR after chronic morphine administration. This is owing to the higher mineral content in Zamzam water especially sodium that promote and facilitate the activation and endocytosis process of MOR.

ACKNOWLEDGEMENT:

This study was supported by Niche Research Grant Scheme (NRGS) from the Ministry of Education (MOE), Malaysia RR057-1.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 28.04.2017 Modified on 05.09.2017

Research J. Pharm. and Tech 2017; 10(11): 3783-3788.

DOI: 10.5958/0974-360X.2017.00686.2