PRELIMINARY STUDIES ON INCREASED NALOXONE POTENCY AND ITS RELATIONSHIP TO MORPHINE TOLERANCE AND DEPENDENCE IN MICE

1. Morphine pretreatment (8 0 mg/kg s.c.) induced no overt tolerance to its antinociceptive effect in mice 4 h later, but enhanced the antagonistic potency of naloxone. 2. A molecular sieve morphine pellet implanted for 24 h induced measurable tolerance, but the relative potency of naloxone was not significantly different from that observed after single-dose morphine pretreatment. The development of tolerance and increased naloxone potency do not, therefore, run parallel. 3. Naloxone-precipitated withdrawal symptoms were observed after single-dose morphine and after pellet implantation. However, molecular sieve morphine pellet implantation induced a higher degree of dependence as compared with single dose morphine pretreatment. 4. These results indicate that the rate of development of increased naloxone potency and of morphine tolerance and dependence do not run parallel. This implies that caution must be exercised in regarding increased naloxone potency as a sensitive indicator of the initiation and development of tolerance and dependence to morphine.     

Interaction between the inhibitory effects of morphine and clonidine on intestinal transit in mice

Both clonidine and morphine dose-dependently inhibited intestinal transit in mice. This inhibitory effect of clonidine was antagonized by prior administration of yohimbine but not by naloxone, while morphine's effect was antagonized by both yohimbine and naloxone. Morphine pretreatment did not induce any apparent tolerance to the effect of clonidine and morphine tested 4 h later. However, yohimbine became more potent in antagonizing the effect of clonidine while naloxone remained ineffective. Morphine pretreatment enhanced the antagonistic effectiveness of both yohimbine and naloxone against morphine. Clonidine pretreatment at a dose that did not induce any tolerance slightly enhanced the antagonistic effects of yohimbine and naloxone against morphine. Yohimbine also became more effective against clonidine but naloxone remained ineffective. At higher doses of clonidine pretreatment tolerance to the effects of morphine and clonidine were observed. These results suggest that both alpha 2-adrenoceptors and opioid receptors are involved in the inhibitory action of morphine on intestinal transit, while clonidine mainly acts through the alpha 2-adrenoceptors. In addition, there is a close interaction between the alpha 2-adrenoceptors and opioid receptors in the intestine.     

The effect of morphine pretreatment on hypothermia induced by morphine in mice.

Morphine caused an apparently dose-dependent hypothermia in mice. Co-administration of naloxone antagonised this effect. Pretreatment with a single dose of morphine induced detectable tolerance to the hypothermic effect of a second dose of morphine given 3 h later and naloxone was more effective in antagonising the hypothermic effect of morphine in morphine-pretreated mice than in saline-pretreated animals. The present study has shown that morphine pretreatment can augment the antagonistic effect of naloxone towards the hypothermic action of morphine.     

THE EFFECT OF ADRENALECTOMY ON THE DEVELOPMENT OF MORPHINE TOLERANCE AND PHYSICAL DEPENDENCE IN MICE

A molecular sieve morphine pellet implanted for 24 h induced measurable tolerance and physical dependence in mice. 2 Adrenalectomy sensitized the animals to the antinociceptive effect of morphine. However, the degree of tolerance induced by morphine pellet implantation was not significantly affected. 3 Quantitative assessment of naloxone-precipitated withdrawal symptoms showed that adrenalectomy slightly enhanced the development of physical dependence. 4 These results indicate that adrenalectomy has no effect on the rate of development of morphine tolerance but may be involved in the development of physical dependence.     

Central and peripheral inhibitory effects of morphine on intestinal transit in mice

In the present study, intestinal motility was measured by the transit of charcoal meal through the small intestine in mice. Morphine, given subcutaneously, caused a dose-dependent slowing of the rate of intestinal transit. This inhibitory effect of morphine was antagonised by prior subcutaneous administration of naloxone hydrochloride or its quaternary analog naloxone methylbromide. However, on a weight basis, naloxone methylbromide was only about half as potent as naloxone hydrochloride. Morphine given centrally, either intracerebroventricularly or intracisternally, caused a dose-related inhibition of intestinal transit, the intracerebroventricular route appearing to be more effective. The effects of centrally administered morphine were antagonised by prior subcutaneous administration of naloxone hydrochloride but not by naloxone methylbromide. The narcotic antagonists administered centrally were effective in suppressing the inhibitory effect of subcutaneously administered morphine. When given intracerebroventricularly naloxone methylbromide was as effective as naloxone hydrochloride, while by intracisternal route, it was about half as potent as naloxone hydrochloride. These results provide evidence that peripherally administered morphine inhibits intestinal transit by both central and peripheral mechanisms. Of the central routes of administration studied, it appears that the sites around the lateral ventricles play a more significant role in the intestinal inhibitory actions of morphine.     

THE POSSIBLE INVOLVEMENT OF ADRENOCEPTORS IN THE INTESTINAL EFFECT OF MORPHINE IN MICE

The inhibitory effect of morphine on intestine was observed by following the intestinal transit of a charcoal meal. This inhibitory effect of morphine was antagonized by naloxone. In addition, the inhibitory effect of morphine was also suppressed by prior administration of yohimbine and phentolamine. However, prazosin, a selective alpha 1-adrenoceptor antagonist, had no effect on the inhibitory effect of morphine on intestinal transit. Furthermore, prior administration of propranolol did not alter this effect of morphine. These adrenoceptor antagonists by themselves, at the doses used, had no effect on the rate of intestinal transit of a charcoal meal in mice. These results suggest that alpha 2-adrenoceptors may be involved in the intestinal effect of morphine while alpha 1- and beta-adrenoceptors do not appear to play any significant role in this aspect of morphine action.     

The effects of naltrexone on the development of physical dependence on morphine

A single i.p. injection of naltrexone (20 mg/kg) partially inhibited the development of physical dependence upon morphine in mice rendered dependent on morphine by implantation of a pellet containing 75 mg of morphine free base for three days. This was evidenced by an increase in the dose of naloxone (ED50) required to precipitate withdrawal jumping response. The increase in naloxone ED50 was much more pronounced when naltrexone was given prior to and during the course of pellet implantation. Inhibition was also observed when naltrexone was administered one day after the morphine pellet implantation, i.e., after some dependence had already developed. Naltrexone administration prior to and during the development of dependence also inhibited, but only partially, the loss of body weight and hypothermic response observed during abrupt withdrawal of morphine in morphine-dependent mice. The inhibitory effect of naltrexone on morphine dependence development was not associated with changes in brain morphine concentration.     

Some relations between tolerance and physical dependence to morphine in mice

Tolerance to morphine analgesia and precipatated physical dependence were studied in mice under different conditions. There was a gradual loss of tolerance during the continous absorption of morphine from a pellet. Tolerance was decreased by nalorphine during morphine absorption. An attenuated physical dependence was observed two or four days after a single dose of morphine. In animals previously treated with pellets of morphine, single doses of morphine induced less tolerance than in mice that had never been implanted with pellets; in both cases cycloheximide prevented development of tolerance. Tolerance persisted for more than 20 days after absorption of the morphine pellet. These results reinforce the hypothesis that tolerance and physical dependence are produced by a similar mechanism and that an inhibitory process of tolerance exists.     

The effect of chronic administration of caffeine on morphine-induced analgesia, tolerance and dependence in mice

Morphine-induced analgesia, and the development of morphine-induced tolerance and dependence was determined in mice which had drunk caffeinated water (1 mg/ml) for 14 days or in mice which had received (-)-N6-(phenylisopropyl)-adenosine (PIA) 1 mg/kg i.p. for 14 days. Analgesia was assessed by the tail flick assay. The development of dependence was assessed by determining the ED50 of naloxone to precipitate withdrawal jumping (3 h after 100 mg/kg morphine pretreatment or 72 h after s.c. implantation of a morphine 75 mg pellet) and by determining the extent of naloxone-precipitated hypothermia in morphine-implanted animals. In mice chronically administered caffeine, the ED50 for morphine-induced analgesia was significantly decreased while the naloxone ED50 for withdrawal jumping increased by 2-fold after both types of morphine pretreatment. In control animals (tap water for 14 days), doses of 1 and 10 mg/kg of naloxone caused significant hypothermia in morphine-implanted animals. Doses of naloxone up to 100 mg/kg did not cause significant hypothermia in morphine-implanted animals which had received chronic caffeine. The development of tolerance was determined by computing the morphine potency ratio for the tail flick assay (tolerant ED50/control ED50). In mice chronically administered caffeine, the potency ratio was decreased significantly in morphine-implanted animals when compared to control. Morphine-induced analgesia, tolerance and dependence was not changed significantly in animals chronically administered PIA. Neither the distribution of morphine to the brain nor the opioid receptor binding parameters for [3H]etorphine and [3H]naltrexone were altered in mice chronically administered caffeine or PIA.(ABSTRACT TRUNCATED AT 250 WORDS)     

A dual action of valproic acid upon morphine analgesia and morphine withdrawal

Effects of valproic acid administration on morphine analgesia and on morphine tolerance and dependence were investigated in mice. Valproate increased the reaction time to thermal stimulation in naive animals. This effect was additive with morphine when valproate was administered shortly before the analgesic. However, an antagonism was observed if a 4-hour period elapsed between valproate and morphine administration. When administered to mice receiving a sustained release preparation of morphine, valproate antagonized the development of tolerance to morphine. Valproate elicited a dual action on the abstinence signs observed after naloxone administration in morphine-treated mice. The effect consisted in a reduction of abstinence behavior if the anticonvulsant was administered a few minutes before naloxone; the same treatment increased the severity of the abstinence behavior when valproate was injected 1 h before the precipitating dose of naloxone. In this latter schedule, concomitant administration of gamma-vinyl-GABA failed to reduce the severity of the convulsions observed during the abstinence syndrome. These results suggest that valproate is metabolized to a compound responsible for decreased analgesia and intensified withdrawal signs.     

Differential tolerance to the intestinal inhibitory effect of opiates in mice

The occurrence of unidirectional noncross-tolerance to heroin and etorphine induced antinociception in morphine tolerant mice prompted this study to determine whether this phenomenon occurs for opiate induced inhibition of intestinal transit. Tolerance to the intestinal inhibitory effect (charcoal meal test) of s.c. morphine, etorphine and heroin was reflected by an increase in the ED50 value and in most cases, a flattening of the dose response curve in morphine and etorphine pelleted mice (tested with pellet in place). A similar response was noted following intracerebroventricular administration of these agents in morphine pelleted mice. More tolerance developed to the relatively hydrophilic compound morphine than to the lipophilic compounds etorphine and heroin. Factors related to hydrophilicity and lipophilicity may be involved in the development of tolerance to opiates in the intestine although unidirectional non cross-tolerance did not occur.     

The effects of cholinergic compounds on the development of morphine tolerance,dependence and increased naloxone potency in mice

The effects of chronic treatment with morphine and cholinergic compounds on the development of morphine tolerance, physical dependence and increased naloxone potency were studied. Using the abdominal constriction method, it was shown that morphine tolerance was apparent after s.c. administration of morphine 20.0 mg/kg three times a day for four days. It was found that, in animals which showed a low degree of morphine tolerance, the naloxone potency was similar to that determined in mice which had been pretreated with only a single dose of morphine which causes no measurable tolerance. Thus, the development of increased naloxone potency and tolerance to morphine do not parallel each other. In addition, while atropine inhibited, and anti-cholinesterase drugs enhanced, the development of increased naloxone potency caused by morphine treatment they had no or little effect on the development of morphine tolerance. Futhermore, chronic treatment with cholinergic agonists reduced, while muscarinic antagonist enhanced, the development of physical dependendence on morphine as assessed by withdrawal jumping and body weight loss. It is concluded that the increased potency of naloxone in antagonising the antinociceptive effect of morphine can be dissociated from the development of tolerance to, and physical dependen on, morphine in mice.     

Increase in plasma cyclic AMP levels elicited by naloxone in morphine-dependent male mice

Naloxone increased the levels of plasma cyclic AMP in morphine-dependent male mice in which a morphine pellet had been implanted for 72 hr, but not in the nondependent mice. The effect of naloxone on the cyclic AMP levels disappeared 24 hr after the removal of the morphine pellet. Pretreatment with propranolol, hexamethonium or adrenalectomy, but not with atropine or phentolamine inhibited the effect of naloxone. These results suggest that naloxone increases the level of plasma cyclic AMP by releasing catecholamines from the adrenal medulla, thereby activating the adenylate cyclase in the tissues through the stimulation of β-adrenergic receptors. A moderate increase in plasma cyclic AMP was also seen in morphine-dependent mice 1 hr after the removal of the morphine pellet. It is proposed that the increase in the levels of plasma cyclic AMP elicited by naloxone-precipitated or abrupt withdrawal is one of the withdrawal symptoms and probably can be used as a screening method for assessing the degree of physical dependence in laboratory animals.     

The possible involvement of alpha-adrenoceptors in the intestinal effect of nalbuphine in mice

In the present study, intestinal motility was measured by the transit of charcoal meal through the small intestine in mice. Nalbuphine, given subcutaneously, caused a dose-dependent inhibition of the intestinal transit in mice. This inhibitory effect of nalbuphine was antagonized by prior s.c. administration of naloxone and MR2266. In addition, the inhibitory effect of nalbuphine was also suppressed by prior administration of yohimbine, an alpha 2-adrenoceptor antagonist, and phentolamine, an antagonist for both alpha 1- and alpha 2-adrenoceptors. Unlike morphine, the intestinal inhibitory effect of nalbuphine was also antagonized by prazosin, a selective alpha 1-adrenoceptor antagonist. However, prior administration of propranolol did not alter this effect of nalbuphine. These adrenoceptor antagonists by themselves, at the doses used, had no effect on the rate of intestinal transit of a charcoal meal in mice. These results suggest that both alpha 1- and alpha 2-adrenoceptors may be involved in the intestinal effect of nalbuphine while beta-adrenoceptors do not appear to play any significant role in this aspect of nalbuphine's action.     

A novel combination of opiates and endothelin antagonists to manage pain without any tolerance development

Several neurotransmitter mechanisms have been proposed as playing a role in the development of morphine tolerance. We provide evidence for the first time that endothelin antagonists can restore morphine analgesia in morphine-tolerant rats and prevent the development of tolerance to morphine. Studies were carried out in rats and mice treated with implanted placebo or implanted morphine pellet. The maximal tail-flick latency in morphine pellet + vehicle-treated rats (7.54 seconds) was significantly lower when compared with placebo pellet + vehicle-treated rats (10 seconds), indicating that tolerance developed to the analgesic effect of morphine. BQ123 potentiated tail-flick latency by 30.0% in placebo-tolerant rats and 94.5% in morphine-tolerant rats compared with respective controls. BMS182874 potentiated tail-flick latency by 30.2% in placebo-tolerant rats and 66.7% in morphine-tolerant rats. The enhanced analgesic effect of morphine after treatment with endothelin antagonists could be blocked by naloxone, indicating an opiate-mediated effect; but naloxone binding to brain membranes was not affected by BQ123. Guanosine triphosphate binding was stimulated by morphine and endothelin-1 in non-tolerant mice and not in morphine-tolerant mice; however, guanosine triphosphate binding was stimulated by BQ123 in morphine-tolerant mice and was unaffected in non-tolerant mice. These results suggest that uncoupling of G-protein occurs in morphine tolerance and endothelin antagonist restores the coupling of G-protein to its receptors. A combination use of endothelin antagonist and opiates could provide a novel approach in improving analgesia and eliminating tolerance.     

An improved implantation pellet for rapid induction of morphine dependence in mice.

A new type of morphine implantation pellet for the rapid induction of physical dependence in mice can be prepared by absorbing 7 mg morphine sulphate onto molecular sieves Type 4A (BDH). The small cylindrical pellets can be implanted subcutaneously without trauma and the need for anaesthesia, and are easily removed at any time from the animals. The peak of physical dependence is reached 24 h after implantation, and mortality is negligible. Withdrawal symptoms can be precipitated by intraperitoneal injection of naloxone, without removal of the pellet, and up to 70% of a group of mice show the characteristic urge to jump off a raised platform. This type of pellet has definite advantages over some other sustained-release preparations used in studies on morphine addiction in small animals.     

Facilitation of morphine-induced tolerance and physical dependence by prolyl-leucyl-glycinamide

The pretreatment of mice with 30 mg/kg morphine s.c. did not alter the analgesic effect of morphine in mice pretreated with saline but decreased the analgesic effect of morphine in mice pretreated with prolyl-leucyl-glycinamide (PLG). Tolerance was evaluated by the effect of PLG on morphine-induced enhancement of naloxone potency which is a measure of the capacity of naloxone to antagonize morphine-induced analgesia and is postulated to be an indicator of tolerance development. The naloxone potency of PLG-treated mice was 2-fold greater than that of control mice. PLG did not alter the whole brain levels of morphine, nor did it alter the naloxone potency in mice which were not pretreated with 30 mg/kg morphine. In mice treated with 100 mg/kg morphine or implanted with 50 mg morphine pellets for 24 or 72 h, the amount of naloxone required to induce jumping was not altered by PLG. However, PLG treatment did increase the hypothermia and body weight loss seen after naloxone-induced withdrawal. Administration of PLG to morphine-dependent mice 1 h prior to naloxone did not modify the resultant hypothermia or body weight loss. These results indicate that PLG facilitated the development of morphine tolerance and dependence.     

Influence of 5,6-dihydroxytryptamine on morphine tolerance and physical dependence

The intracerebral administration of 5,6-dihydroxytryptamine (5,6-DHT) in the mouse inhibited the development of tolerance to and physical dependence on morphine induced by morphine pellet implantation. Reduction in tolerance development by 5,6-DHT was evidenced by the decreased amount of morphine necessary to produce analgesia and reduction in dependence development by the increase in the amount of naloxone necessary to induce precipitated withdrawal jumping in comparison with morphine-implanted animals receiving saline. Further evidence that 5,6-DHT reduced dependence development on morphine was evidenced by the fact that 5,6-DHT decreased the loss in body weight which occurred after abrupt morphine withdrawal. At the dose of 5,6-DHT used in this study (60 μg of the creatinine sulfate dihydrate 24 hr prior to morphine pellet implantation), the 5-HT level in the brain 4 days later was 75% of that of the control group while catecholamine levels remain unchanged. These studies substantiate the suggestion from this laboratory that central serotonergic system may be associated in the development of morphine tolerance and dependence.     

On the mechanism of tolerance to morphine-induced Straub tail reaction in mice

The effect of 5-HT and opioid receptor antagonists on morphine-induced Straub tail was studied in mice. Straub tail behavior was induced by subcutaneous administration of different doses (20, 30, and 40 mg/kg) of morphine hydrochloride to mice. The effect of morphine was dose-dependent. Maximum response was obtained with 40 mg/kg of the drug. The response induced by morphine (20 and 40 mg/kg) was decreased by different doses of intraperitoneal injection of naloxone (1 and 2 mg/kg) or methysergide, mianserin, and ritanserin (1 and 2 mg/kg). The effect of morphine (40 mg/kg) was also reduced by intracerebroventricular injection of naloxone (0.4-0.8 microg/animal) or mianserin (2 and 4 microg/animal). Different groups of mice received one daily dose (50 mg/kg sc) of morphine sulfate for 3 days to develop tolerance to morphine. The Straub tail reaction induced by morphine hydrochloride (40 mg/kg) was tested on the fourth day. Naloxone injection (1 and 2 mg/kg ip) on Day 3 (1 h after morphine sulfate injection) or on Day 4 (1 h before test dose of morphine hydrochloride), decreased tolerance induced to morphine. Methysergide, mianserin, or ritanserin (intraperitoneal) on Days 2 and 3 (1 h after morphine sulfate injection) or on Day 4 (1 h before test dose of morphine hydrochloride), also decreased tolerance induced to morphine. Intracerebroventricular injection of either naloxone or mianserin also reduced tolerance to morphine. It is concluded that 5-HT(2) and opioid receptor mechanisms are involved in morphine-induced Straub tail reaction and tolerance induced to morphine also may be mediated through these receptors.     

Repeated administration of nicotine attenuates the development of morphine tolerance and dependence in mice

Clinical use of morphine in pain management is a controversial issue. Both nicotine and morphine are widely abused. So, investigating the interaction between nicotinic and opioid receptors is of great interest to both basic mechanistic and clinical view. We investigated the influence of repeated administration of nicotine on the development of morphine tolerance and dependence. Adult male albino mice were rendered dependent on morphine by subcutaneous (s.c.) injections three times daily for 3 days. Repeated intraperitoneal (i.p.) injection of nicotine (0.001-2 mg/kg) or saline (1 ml/kg) was performed 15 min prior to each morphine injection. Maximal possible effect (MPE%) of morphine (50 mg/kg; s.c.) was used on the fourth day as an index for the development of tolerance. Likewise, to assess the occurrence of dependence in drug-treated mice, naloxone (5 mg/kg; i.p.) was injected 2 h after the last dose of morphine. Repeated nicotine administration significantly attenuated the development of tolerance in a dose-dependent manner whereas it significantly decreased withdrawal jumping behavior in a biphasic profile (V-shape) manner. Furthermore, the central nicotinic receptor antagonist mecamylamine (0.01-0.1 mg/kg; i.p.) neither the peripheral nicotinic receptor antagonist hexamethonium (0.01 and 0.1 mg/kg; i.p.) nor the muscarinic receptor antagonist atropine (2.5-10 mg/kg; i.p.), dose-dependently antagonized both the inhibition of withdrawal jumping as well as increase in MPE% which was produced by repeated nicotine administration (0.1 mg/kg; i.p.). On the other hand, 3 days of solely nicotine treatment resulted in significant jumping behavior precipitated by naloxone after single morphine injection on the test day. The data suggests that the inhibitory effect of nicotine on the morphine tolerance and dependence is mediated by central nicotinic receptors and there is a cross-dependence between nicotine and morphine.