Aripiprazole Prolongs Morphine Antinociception Effect and Disrupts Acute Morphine Tolerance

Aripiprazole is an atypical antipsychotic drug mainly characterized by partial agonist activity at dopamine D2 and serotonin-1A receptors with minimal side effects. Based on typical antipsychotic pharmacological activity, including antinociception effect and disruption opioid anti-nociceptive tolerance, Aripiprazole activity and its interaction with morphine on nociception was evaluated by tail flick and hot plate assay in the mouse. In experiment 1, mice received aripiprazole (5, 10 and 20 mg/kg IP), saline (1 ml/kg, IP) and morphine (5 mg/kg, IP) 30 minutes prior to the test. The tail flick and hot-plate methods were used for pain evaluation. In order to assess the effect of aripiprazole on morphine antinociception in experiment 2, it was administered 30 min after morphine injection and then the test was assessed. Also, in experiment 3, the effect of aripiprazole (10 and 20 mg/kg IP), on acute morphine tolerance was studied. Comparisons between the groups were carried out using the Analysis of Variance (ANOVA), and post hoc Tukey’s test. P<0.05 was considered statistically significant. The results revealed that aripiprazole, at doses that had no affected themselves (10–20 mg/kg), were significantly (P<0.001) effective on prolonging the morphine antinociceptive effect by tail flick test in mice. Also Aripiprazole (20mg/kg) significantly increased the duration of morphine antinociception effect by hot plate test, but it did not significantly influence the morphine antinociception time course at 5 and 10 mg/kg of drug. Pretreatment with aripiprazole (20 mg/kg i.p.) prevented the acute morphine tolerance in hotplate test. These results also suggest that aripiprazole might have therapeutic value in combination to morphine as an adjuvant analgesic. It was also shown that partial agonist properties of D2 and 5-HT1A as well as antagonist properties of 5-HT2A in aripiprazole likely account for the potentiation of morphine antinociception.


INTRODUCTION
The role of antipsychotic drugs as adjuvant analgesics has been studied in human and animals, but the existing data are contradictory 1,2 .Aripiprazole is a unique atypical antipsychotic that seems to act as a partial agonist at 5-HT 1A and D 2 receptors of dopamine and also as an antagonist at 5-HT 2A receptors [3][4][5][6] .However, like most central nervous system drugs, the actual mechanism of its action is not entirely understood.Aripiprazole has a low tendency for extrapyramidal side effects.It causes minimal sedation or weight gain and produces no elevation in serum prolactin levels or cardiovascular side-effects 7 .Dopamine receptor's antagonists such as haloperidol are used to stand such adverse effects of opioids as hallucination and delirium 8 ; though, most of these drugs have other side effects such as extrapyramidal 9 .A previous study reported that morphine-induced hyper-locomotion, reward and dopamine release in the nucleus accumbens were suppressed by aripiprazole pretreatment.In this regard, co-administration morphine with aripiprazole might be valuable for decreasing the severity of morphine-induced dopamine-related side effects 10 .On the other hand, different antipsychotic drugs did not seem to have similar effects on morphine antinociception.Haloperidol has been reported to potentiate the morphine antinociception effect in rats 11 , but aripiprazole did not appear to have any effect on morphine antinociception activity in mice 12 .Dopamine and opioid systems interactions have been studied extensively 13,14 .The critical role of dopamine in descending inhibition pathway has also been demonstrated.In clinical studies, abnormalities in dopaminergic neurotransmission have been objectively shown in painful clinical conditions such as burning mouth syndrome, restless legs syndrome and fibromyalgia 15 .In addition, convincing evidence from animal studies showed that spinal cord and different brain nuclei dopaminergic system are involved in nociception.For example, it was reported that amphetamine-and morphine-induced analgesia are involved in increasing dopamine levels in nucleus accumbens 16 .Increasing the dopamine release in nucleus accumbens has an antinociceptive effect which is mediated through D1 and D2 receptors 17 .In addition to its stimulusinduced antinociception, dopamine may also inhibit nociception in the mesolimbic/mesocortical circuits tonically, because the lesion of dopaminergic neurons of Ventral Tegmental Area (VTA) results in hyperalgesic responses 18 .In VTA, Dopaminergic neurons are particularly involved in both endogenous and morphine-induced antinociception.It has been indicated that D1 and D2 dopamine receptors in NAc and D1 receptors in VTA are involved in developing the sensitization to morphine in rats 19 .Recently, Reisi et al. revealed that D1 and D2 antagonist receptors microinjection into NAc and D1 antagonist receptor into VTA can prevent the morphine antinociceptive effects in the tail flick test.

Objectives
Against this background, this study was conducted to evaluate whether aripiprazole could affect on morphine antinociception in experimental animals.

Animals
In the present study, male albino mice (20-25 g) were housed in groups of six to eight and were allowed free access to food and water.All experiments were conducted between 10 am and 3 pm with 12 hours of regular light/dark cycle and constant temperature (22±1°C) according with the guidelines of Pharmaceutical Sciences Branch of Islamic Azad University for animal care and use.

Experiments
Tail-flick and hot plate tests were carried on separate animals groups.The first experiment was conducted to observe whether aripiprazole (2, 5, 10 and 20 mg/kg IP) could change the basal nociception.The animals were injected with aripiprazole or saline and placed in the Tail flick apparatus and/or hot plate device.
In order to determine the effective doses of morphinek the second experiment was conducted in which the animals received 5 mg/kg morphine (with strong antinociceptive and minor sedation effect) and were tested in the Hotplate and Tail flick.To assess the effect of D2 receptor partial agonist on the anti-nociceptive response of µ receptor agonist (morphine), aripiprazole (10 and 20 mg/Kg IP) was then administered 30 min after receiving 5 mg/kg IP morphine.They were immediately placed in the analgesia meter.

Tail-flick
The tail-flick latency (TFL) was recorded by the tail-flick apparatus (Borj Sanat, Iran).Reaction time between the onset of heat stimulus and movement of the tail away from the noxious stimulus was determined by an automatic sensor as TFL.
To avoid tissue injuryk cut-off point was set in 9.0 and 10.0 (S).To estimate the animals' sensitivity to nociceptive stimulusk the individual TFL was considered as a pain threshold before and after (15, 30, 60 and 180 min) drug treatment.

Hot-Plate
Animals were placed on a thermostatically controlled hot-plate (Borj Sanat, Iran), set at 55 ± 0.5 °C.The time between placement animals on platform of hot-plate apparatus and shaking or licking of the paws or jumping was recorded as latency of pain response.In order to avoid the animals paw injury, cut-off time set to 15 seconds.Latency response was recorded before treatment and at 15, 30, 60 and 180 min after drugs/vehicles administration.

Statistical Analysis
Data were expressed as mean ± SEM.The effect of antinociception was measured and the mean latencies in all animal groups were subjected to one-way ANOVA followed by protected Tukey's test for multiple comparisons.P<0.05 were considered to be statistically significant.

Tail flick test
In the first experiment, the aripiprazole effect on pain threshold was assessed in the tail flick test.The results for TFLs revealed that there are no significant differences in TFLs at any time intervals among the vehicle and aripiprazole (5, 10 and 20 mg/kg) dose groups.Though, aripiprazole (2 mg/kg) would provide a significant reduction TFLs in compared to vehicle group at 30 and 60min after treatment (p<0.05,p<0.01 respectively).Furthermore, no significant drug effect was observed, indicating that aripiprazole did not alter pain threshold in the higher doses administered in the current study.
The second experiment was designed to analyze the aripiprazole effects on the morphine antinociception time course.The results (Fig. 2) revealed that antipsychotic doses (10, 20) were significantly effective in prolonging the action of morphine (P<0.001).

Hot-Plate Test
Aripiprazole alone at 5, 10 and 20 mg/ kg had no effect on the hot plate response latency (fig.3).
The second experiment was designed to analyze the aripiprazole effects on morphine antinociception time course, morphine produced significant antinociception, which peaked at 30 min and lasted for 60 min (P<0.05).Morphine plus aripiprazole at 20mg/kg significantly (P<0.05)increased the duration of morphine antinociception effect for 180 min, but it did not significantly influence the morphine antinociception time course at 5 and 10 mg/kg doses (Fig. 4).
Aripiprazole (20mg/kg) significantly increased the duration of morphine antinociception effect, but it did not significantly influence the

Prevention of acute morphine tolerance by aripiprazole
We investigated aripiprazole effect on the development of morphine tolerance in acute model of opioid tolerance.Mice received an induction dose of morphine (100 mg/kg S.C.) and were exhibit significantly reduced antinociception for 4 h later (2.6% MPE versus 96.4% MPE in saline-pretreated mice, p<0.0001) by a test dose of morphine (10 mg/kg S.C.), indicative of the development of acute morphine tolerance (Fig. 5).In mice pretreated with aripiprazole (20 mg/kg I.P.) 30 min before the induction dose of morphine, morphineantinociception keep on largely intact (91.95%, respectively; not significantly different from control).While at the lower dose used, aripiprazole (10 mg/ kg I.P.) was unable to prevent morphine tolerance.These results confirmed that aripiprazole blocked the development of morphine tolerance in higher dose.

DISCUSSION
The purpose of this study was to evaluate the effect of aripiprazole, an antipsychotic drug, on the morphine antinociception effect in two pain assessment models in mice.It was demonstrated that aripiprazole at doses that had no affected themselves (10-20 mg/kg) prolonged the morphine antinociception effect.Also in this study, we revealed that aripiprazole disrupted morphine antinociceptive tolerance.
Interestingly, it was obser ved that aripiprazole (10 and 20 mg/kg IP) increased the action time of morphine antinociception in the tail flick test.Also there was an immediate decrease in pain threshold after the administration of aripiprazole (2 mg/kg IP) in this test.
In hotplate test, aripiprazole (20 mg/kg IP) significantly prolonged the morphine antinociception effect.Though, the lower dose of aripiprazole (5 and 10 mg/kg) did not significantly change the morphine antinociception time course.Also in this study, we demonstrated that aripiprazole (20 mg/kg IP) prevented morphine antinociceptive tolerance in hot plate model.
There is little evidence regarding the possible interaction in antinociceptive effect of morphine and aripiprazole.An experimental study showed that aripiprazole did not affect the antinociceptive action of morphine.Aripiprazole prevented the stimulant action of morphine, without interfering with basal motor activity 12 .Also, it was reported that aripiprazole did not induce place preference or aversion by itself; however, it inhibited both the development and expression of morphineinduced CPP 12 .Previous studies have revealed that aripiprazole pretreatment reduced the reinstatement of CPP induced by morphine, but had no effect on the expression of morphine-induced CPP or locomotor activity 20 .Moreover, former studies showed that aripiprazole did not reduce the spontaneous locomotion and had no marked sedation in the dose study in mice and rats 21,22 .
One reason for unpredictable reports on analgesic interaction of aripiprazole and morphine might be the use of different methods for testing the analgesia and dose of aripiprazole.This discrepancy could be attributed to the used dose of aripiprazole.In this study, the antinociceptive potentiation effect of aripiprazole was observed in higher doses.
Previous studies on other antipsychotics such as haloperidol indicate that their interaction with morphine on the antinociception action is dose-dependent and may also differ among animal species.It was reported that haloperidol (0.1-1 mg/ kg i.p.) by itself neither produce any antinociception effect nor alter morphine antinociception in mice 23 .However, another study showed that haloperidol could potentiate the antinociception of morphine in rats, possibly by acting as a Ã-receptor antagonist 11 .
The interaction between dopamine and opioid systems is well documented 14 .Significant evidence suggests that dopamine activity affects the opioid system by modulating opiate peptide transcripts 24 , synthesis 25 , release and biotransformation 26 .In contrast, opioids modify the dopamine system by several mechanisms, such as dopamine synthesis 27 , release 28,29 , biotransformation 30 , and activity of dopaminergic neurons 31,32 .Moreover, behavioral evidence suggests that changes in dopaminergic and/or opioidergic systems are involved in the behavioral sensitization to morphine.Since morphine increases both dopamine synthesis and release in the dopaminergic system via activation of ¼-opioid receptors, it is likely that morphine sensitization might be caused by a similar mechanism 33 .It has been stated that there is a functional relationship among morphine and dopaminergic system [34][35][36] .Morphine locomotion 37 may be mediated by dopaminergic system.In addition, dopaminergic system has also been implicated in antinociceptive effect and expression of morphine withdrawal signs 38 .It has been proposed that sulpiride, a D 2 receptor antagonist, decreased the response to morphine (6 and 9 mg/kg) in the formalin test, whereas SCH 23390 did not influence the morphine antinociception 39 .In contrast, Ozdemir et al., demonstrated that eticlopride, D 2 antagonist significantly increased the morphine analgesic effect 40 .Considering the animal studies focusing on pain behavior, clinical data and genetic associations, a common suggestion is that dopamine is antinociceptive by D 2 receptors.(13).Animal studies have directed that the administration of D 2 D D 3 receptor agonists in the striatum suppresses pain-related responses, whereas D 2 D D 3 receptor antagonists in the striatum increase the pain [41][42][43] .Subsequently aripiprazole has a unique pharmacological profile that includes partial agonism at D 2 receptors with actions on both postsynaptic D 2 /D 3 receptors and presynaptic dopamine auto-receptors with varying degrees of efficacy.Additionally, aripiprazole acts as a partial agonist at 5HT 1A receptors 44 and an antagonist at 5HT 2A receptors (4, 45).Considering the abovementioned studies, the partial activation of D 2 receptors can be considered as the main mechanism through which this compound stimulates the effects of morphine and alters dopaminergic receptor functions in pain pathway.
The main finding in the present study is that aripiprazole (at doses used here) significantly prolonged morphine antinociception effect and disrupted morphine antinociceptive tolerance in mice.Although the detailed mechanism by which aripiprazole affect the morphine antinociception is yet unclear, the partial activation of D 2 receptors can be considered as the main mechanism aripiprazole on prolongation of morphine antinociception effect.However, further pharmacological research are needed to elucidate the actual mechanism of aripiprazole on modulating morphine-induced antinociception in animal models of pain.