Short-term tolerance to morphine: effects of diazepam, phenobarbital, and amphetamine

Short-term tolerance to morphine, which can be demonstrated in as little as 3 hours after a single administration of the opiate, was examined in animals chronically pretreated with diazepam, phenobarbital, or amphetamine. Tail-flick latency in mice and changes in plasma corticosterone in rats were the parameters tested in these experiments. Rats primed with either saline or morphine, 10 mg/kg, were injected 3 hours subsequently with morphine, 5 mg/kg. Those primed with saline showed the characteristic plasma corticosterone elevation following morphine, when serial blood samples were examined, whereas those previously treated with morphine did not. Mice were primed with saline or either of two doses of morphine, 30 or 100 mg/kg, 3.5 hours prior to estimation of tail-flick latency and ED50 determinations. Mice primed with either dose of morphine had significantly higher ED50's than those primed with saline. Chronic treatment with diazepam or amphetamine in either species did not significantly alter short-term tolerance development by either parameter. However, with phenobarbital pretreatment, the plasma corticosterone response was attenuated and short-term tolerance to morphine's analgesic effects did not occur. Further studies in morphine-pelleted mice showed that analgesic tolerance occurred similarly in all groups. This suggests that barbiturates may delay the process.     

Apparent pA2 value of naltrexone is not changed in rats following continuous exposure to morphine or naloxone.

Chronic opioid antagonist administration increases opioid binding sites and potentiates behavioral responses to morphine. Conversely, chronic opioid agonist administration attenuates behavioral responses to morphine, though this is not necessarily accompanied by a parallel loss of binding sites. We examined the possibility that the in vivo affinity of the mu receptors might be altered as a consequence of the continuous administration of either naloxone or morphine. Rats were implanted sc with naloxone- or morphine-filled osmotic pumps; control animals were implanted with sham pumps. One week later, 24 hr after removing the osmotic pumps, cumulative dose-response curves for fentanyl analgesia were generated in the presence of 0.0, 0.03, 0.1, or 0.3 mg/kg naltrexone, using a tail-flick procedure. The analgesic ED50 (with 95% C. L.) of fentanyl in sham implanted animals, following saline pretreatment was 0.027 mg/kg (0.019, 0.039). The potency of fentanyl was decreased in rats infused with morphine, ED50 = 0.051 mg/kg (0.028, 0.093), and increased in rats that received naloxone, ED50 = 0.018 mg/kg (0.015, 0.022). The mean apparent pA2 value for naltrexone (with 95% C.L.) in the control group was 7.7 (7.5, 7.9). No differences were detected in animals that had received either naloxone or morphine for 7 days, pA2 = 7.8 (7.5, 8.1) and 7.4 (7.3, 7.6), respectively. Our results indicate that there is no change in the apparent affinity of the mu-receptor following continuous exposure to either an opioid agonist or antagonist, at a time when the analgesic potency of the agonist is decreased or increased, respectively.     

Effects of fluoxetine and LY 367265 on tolerance to the analgesic effect of morphine in rats

Morphine is widely used to treat chronic pain, however its utility is hindered by the development of tolerance to its analgesic effects. The aim of this study was to investigate effects of fluoxetine, a specific serotonin (5-HT) reuptake inhibitor, and LY 367265, an inhibitor of the 5-HT transporter and 5-HT2A receptor antagonist, on tolerance induced to the analgesic effect of morphine in rats. The study was carried out on male Wistar Albino rats (weighing 170-190 g). To constitute morphine tolerance, animals received morphine (50 mg/kg; s.c.) once daily for 3 days. After last dose of morphine, injected on day 4, morphine tolerance was evaluated. The analgesic effects of fluoxetine (10 mg/ kg; i.p.), LY 367265 (3 mg/kg; i.p.) and morphine were considered at 30-min intervals by tail-flick and hot-plate tests. The results showed that fluoxetine and LY 367265 significantly attenuated the development and expression of morphine tolerance. The maximal antinociceptive effects were obtained 30 min after administration of fluoxetine and 60 min after administration of LY 367265. In conclusion, we observed that co-injection of morphine with fluoxetine and LY 367265 increased the analgesic effects of morphine and delayed development of tolerance to morphine analgesia.     

Modification of antiallodynic and antinociceptive effects of morphine by peripheral and central action of fluoxetine in a neuropathic mice model

We have previously reported that serotonin concentration was reduced in the brain of mice with neuropathic pain and that it may be related to reduction of morphine analgesic effects. To further prove this pharmacological action, we applied fluoxetine, a selective serotonin reuptake inhibitor, to determine whether it suppressed neuropathic pain and examined how its different administration routes would affect antinociceptive and antiallodynic effects of morphine in diabetic (DM) and sciatic nerve ligation (SL) mice, as models of neuropathic pain. Antiallodynia and antinociceptive effect of drugs were measured by using von Frey filament and tail pinch tests, respectively. Fluoxetine given alone, intracerebroventicularly (i.c.v., 15 microg/mouse) or intraperitoneally (i.p., 5 and 10 mg/kg) did not produce any effect in either model. However, fluoxetine given i.p. enhanced both antiallodynic and antinociceptive effects of morphine. Administration of fluoxetine i.c.v., slightly enhanced only the antiallodynic effect of morphine in SL mice. Ketanserine, a serotonin 2A receptor antagonist (i.p., 1 mg/kg) and naloxone, an opioid receptor antagonist (i.p., 3 mg/kg), blocked the combined antinociceptive effect of fluoxetine and morphine. Our data show that fluoxetine itself lacks antinociceptive properties in the two neuropathy models, but it enhances the analgesic effect of morphine in the periphery and suggests that co-administration of morphine with fluoxetine may have therapeutic potential in treatment of neuropathic pain.     

Analgesia induced by intrathecal injection of dynorphin B in the rat

A dose-dependent analgesic effect of intrathecally injected dynorphin B was observed in rats using the tail flick as nociceptive test. Intrathecal injection of 20 nmol of dynorphin B increased the tail flick latency by 90 +/- 23%, an effect that lasted about 90 min. For the same degree of analgesia, dynorphin B was 50% more potent than morphine on a molar basis. The analgesic effect of this dose of dynorphin B was partially blocked by 10 mg/kg, but not by 1 mg/kg, of subcutaneous naloxone, showing a relative resistance to naloxone reversal as compared with morphine analgesia. The analgesia produced by dynorphin B was unchanged in morphine-tolerant rats but was significantly decreased in rats tolerant to ethylketazocine. These results suggest that dynorphin B produces its potent analgesic effect by activation of kappa rather than mu opioid receptors in the rat spinal cord.     

Measuring naloxone antagonism of discriminative opioid stimulus

The numerous studies of opioids as discriminative stimuli, beginning in 1971, have shown specificity, similarity of several opioids, differences in potency (fentanyl greater than heroin greater methadone greater than morphine), and antagonism by naloxone and naltrexone. The discriminative opioid stimulus is differentiated from those of other classes of drugs, such as sedatives and anxiolytics. Greater potency of the opioid stimulus has been found in rats after subcutaneous (s.c.) than intraperitoneal administration. The discriminative opioid stimulus and its antagonism by naloxone or naltrexone have been demonstrated in rats, squirrel monkeys, gerbils, and pigeons. A few studies have quantified the competitive agonist-antagonist interaction at the receptor by calculating the pA2, which reflects the dose of the antagonist that requires doubling the agonist dose to obtain the original agonist response. The pA2 for naloxone is the same in groups of rats trained to discriminate different doses of morphine (1, 2, or 4 mg/kg s.c.) from saline. Higher pA2 values in tests after fentanyl and methadone than after heroin and morphine in rats trained to discriminate fentanyl (0.04 mg/kg s.c.) from saline reflect greater susceptibility of the synthetic than the natural exogenous opioids to antagonism by naloxone. Different pA2 values are usually interpreted as indicating differences among populations of receptors.     

Antinociceptive effect of 7-hydroxymitragynine in mice: Discovery of an orally active opioid analgesic from the Thai medicinal herb Mitragyna speciosa

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa. We previously reported the morphine-like action of mitragynine and its related compounds in the in vitro assays. In the present study, we investigated the opioid effects of 7-hydroxymitragynine, which is isolated as its novel constituent, on contraction of isolated ileum, binding of the specific ligands to opioid receptors and nociceptive stimuli in mice. In guinea-pig ileum, 7-hydroxymitragynine inhibited electrically induced contraction through the opioid receptors. Receptor-binding assays revealed that 7-hydroxymitragynine has a higher affinity for micro-opioid receptors relative to the other opioid receptors. Administration of 7-hydroxymitragynine (2.5-10 mg/kg, s.c.) induced dose-dependent antinociceptive effects in tail-flick and hot-plate tests in mice. Its effect was more potent than that of morphine in both tests. When orally administered, 7-hydroxymitragynine (5-10 mg/kg) showed potent antinociceptive activities in tail-flick and hot-plate tests. In contrast, only weak antinociception was observed in the case of oral administration of morphine at a dose of 20 mg/kg. It was found that 7-hydroxymitragynine is a novel opioid agonist that is structurally different from the other opioid agonists, and has potent analgesic activity when orally administered.     

Morphine-Induced Apoptosis in the Ventral Tegmental Area and Hippocampus After the Development but not Extinction of Reward-Related Behaviors in Rats

Some data suggest that morphine induces apoptosis in neurons, while other evidences show that morphine could have protective effects against cell death. In this study, we suggested that there is a parallel role of morphine in reward circuitry and apoptosis processing. Therefore, we investigated the effect of morphine on modifications of apoptotic factors in the ventral tegmental area (VTA) and hippocampus (HPC) which are involved in the reward circuitry after the acquisition and extinction periods of conditioned place preference (CPP). In behavioral experiments, different doses of morphine (0.5, 5, and 10 mg/kg) and saline were examined in the CPP paradigm. Conditioning score and locomotor activity were recorded by Ethovision software after acquisition on the post-conditioning day, and days 4 and 8 of extinction periods. In order to investigate the molecular mechanisms in each group, we then dissected the brains and measured the expression of apoptotic factors in the VTA and HPC by western blotting analysis. All of the morphine-treated groups showed an increase of apoptotic factors in these regions during acquisition but not in extinction period. In the HPC, morphine significantly increased the ratio of Bax/Bcl-2, caspases-3, and PARP by the lowest dose (0.5 mg/kg), but, in the VTA, a considerable increase was seen in the dose of 5 mg/kg; promotion of apoptotic factors in the HPC and VTA insinuates that morphine can affect the molecular mechanisms that interfere with apoptosis through different receptors. Our findings suggest that a specific opioid receptor involves in modification of apoptotic factors expression in these areas. It seems that the reduction of cell death in response to high dose of morphine in the VTA and HPC may be due to activation of low affinity opioid receptors which are involved in neuroprotective features of morphine.     

Nicotine improves morphine-induced impairment of memory: possible involvement of N-methyl-D-aspartate receptors in the nucleus accumbens

The possible involvement of N-methyl-D-aspartate (NMDA) receptors in the nucleus accumbens (NAc) in nicotine's effect on impairment of memory by morphine was investigated. A passive avoidance task was used for memory assessment in male Wistar rats. Subcutaneous (s.c.) administration of morphine (5 and 10 mg/kg) after training impaired memory performance in the animals when tested 24 h later. Pretest administration of the same doses of morphine reversed impairment of memory because of post-training administration of the opioid. Moreover, administration of nicotine (0.2 and 0.4 mg/kg, s.c.) before the test prevented impairment of memory by morphine (5 mg/kg) given after training. Impairment of memory performance in the animals because of post-training administration of morphine (5 mg/kg) was also prevented by pretest administration of a noncompetitive NMDA receptor antagonist, MK-801 (0.75 and 1 microg/rat). Interestingly, an ineffective dose of MK-801 (0.5 microg/rat) in combination with low doses (0.075 and 0.1 mg/kg) of nicotine, which had no effects alone, synergistically improved memory performance impaired by morphine given after training. On the other hand, pretest administration of NMDA (0.1 and 0.5 microg/rat), which had no effect alone, in combination with an effective dose (0.4 mg/kg, s.c.) of nicotine prevented the improving effect of nicotine on memory impaired by pretreatment morphine. The results suggest a possible role for NMDA receptors of the NAc in the improving effect of nicotine on the morphine-induced amnesia.     

Reversal of morphine, methadone and heroin induced effects in mice by naloxone methiodide

Opioid overdose, which is commonly associated with opioid induced respiratory depression, is a problem with both therapeutic and illicit opioid use. While the central mechanisms involved in the effects of opioids are well described, it has also been suggested that a peripheral component may contribute to the effects observed. This study aimed to further characterise the effects of the peripherally acting naloxone methiodide on the respiratory, analgesic and withdrawal effects produced by various opioid agonists. A comparison of the respiratory and analgesic effects of morphine, methadone and heroin in male Swiss-Albino mice was conducted and respiratory depressive ED(80) doses of each opioid determined. These doses (morphine 9 mg/kg i.p., methadone 7 mg/kg i.p., and heroin 17 mg/kg i.p.) were then used to show that both naloxone (3 mg/kg i.p.) and naloxone methiodide (30-100 mg/kg i.p.) could reverse the respiratory and analgesic effects of these opioid agonists, but only naloxone precipitated withdrawal. Further investigation in female C57BL/6J mice using barometric plethysmography found that both opioid antagonists could reverse methadone induced decreases in respiratory rate and increases in tidal volume. Its effects do not appear to be strain or sex dependent. It was concluded that naloxone methiodide can reverse the respiratory and analgesic actions of a variety of opioid agonists, without inducing opioid withdrawal.     

Teratogenic and postnatal developmental studies of morphine in Sprague-Dawley rats

The teratogenic and postnatal developmental effects of morphine exposure during pregnancy were studied in Sprague-Dawley rats in three separate experiments using chronically implanted osmotic minipumps in order to avoid respiratory depression. In the first experiment, the teratogenic effects of three different morphine dosages were studied: a low dose (10 mg/kg/day), an intermediate dose (35 mg/kg/day), and a high dose (70 mg/kg/day). On day 5 of gestation, osmotic minipumps that deliver their contents at a constant rate for 15 days were implanted subcutaneously on the back of the rats. On day 20 of gestation, cesarean sections were performed, reproductive indices were determined, and fetuses were examined externally and then preserved for subsequent visceral and skeletal examinations. The pregnancy rate was significantly reduced at the intermediate and high doses to 57% and 6%, respectively (control, 83%). No teratogenic effects were observed at any dosage, but growth retardation was present in the intermediate-dose group. In the second experiment, postnatal survival of the offspring of dams treated with either normal saline, morphine (35 mg/kg/day), or the synthetic opioid, fentanyl (500 micrograms/kg/day) were studied. Offspring of morphine-treated dams had a significantly higher mortality rate, which peaked at 56% within 2 days. No effect was seen after fentanyl treatment. In the third experiment, pups of morphine-treated dams were cross-fostered by saline-treated dams; the postnatal mortality in offspring of morphine-treated dams remained high (62%). Our results indicate that doses of morphine up to 35 mg/kg/day delivered by osmotic minipumps are not teratogenic in rats but cause other adverse fetal effects that result in increased postnatal mortality.     

Comparative effects of Pro-Leu-Gly-NH2 and cyclo(Leu-Gly) administered orally on the development of tolerance to the analgesic effect of morphine in the rat

Comparative effects of Pro-Leu-Gly-NH2 (MIF) and cyclo(Leu-Gly) (CLG) administered orally at different stages of chronic morphine treatment on the development of tolerance to the analgesic effect of morphine in the rat were determined. Male Sprague-Dawley rats were implanted with either 6 placebo or morphine pellets during a 7-day period. Implantation of morphine pellets resulted in the development of a high degree of tolerance as evidenced by a decrease in the analgesic response to morphine. Administration of CLG (8 and 16 mg/kg/day) on day 5, 6 and 7 of implantation inhibited the development of tolerance to morphine but 4 and 32 mg/kg doses had no effect. Further, CLG (2 mg/kg/day for 7 days) inhibited the development of tolerance but higher doses (4 and 8 mg/kg) had no effect. MIF (26 and 52 mg/kg) administered orally on the last three days of the implantation schedule inhibited the development of tolerance to morphine. MIF (6.5 mg/kg/day for 7 days) inhibited the development of tolerance but the higher doses had no effect. Concurrent administration of MIF (6.5 mg/kg) and CLG (2 mg/kg) for seven days failed to inhibit the development of tolerance. A single dose of MIF or CLG administered a day before the assessment of tolerance did not affect the morphine tolerance. Thus, even after a significant degree of tolerance to morphine had developed, neuropeptides like MIF and CLG given orally, in appropriate doses, can inhibit development of tolerance to morphine and restore the analgesic effect of morphine.     

Naloxone-induced enhancement of carbon dioxide stimulated respiration

In unanesthetized rats, naloxone (5 mg/kg, s.c.) produced an increase in both respiratory frequency and tidal volume as compared to saline administered animals. Maximal respiratory stimulation was observed within 5 minutes after naloxone injection and duration of the response was greater than 30 minutes. Exposure to different atmospheres of carbon dioxide potentiated the increase in ventilation in a step-wise manner as the carbon dioxide concentration was increased. Pretreatment with low doses of morphine sulfate (2 mg/kg daily for 2 days) or naloxone HCl (5 mg/kg daily for 5 days) enhanced respiratory stimulation induced by naloxone. It was concluded that naloxone increases the sensitivity of central ventilatory response to carbon dioxide as a result of displacement of endogenous endorphins from central opioid receptors.     

The effect of pentobarbital on the antinociceptive action of morphine in morphine-tolerant and non-tolerant rats

The interaction of sodium pentobarbital with morphine sulfate in both morphine-tolerant and non-tolerant rats was investigated using the tail-compression test for analgesia. Male Sprague-Dawley rats (300–350 g) were given pentobarbital (4, 8, or 16 mg/kg) 5 min before morphine (2, 4, 6, or 8 mg/kg). Control animals received two saline injections, or pentobarbital plus saline, or saline plus morphine. All injections were subcutaneous. Prior to the first injection, a baseline nociceptive threshold was determined for each rat by applying a modified micrometer to its tail and increasing the pressure until a squeak was elicited. Test readings were taken every half-hour for 2 hr beginning 30 min after the second injection. For the chronic studies, animals were first made tolerant to morphine by the administration of the narcotic twice a day for 3 days, increasing the dose from 10 to 50 mg/kg/injection. Identical testing procedures were then followed with these rats except that the test dose of morphine given on day 4 was in the range 8–128 mg/kg. It was found that Na pentobarbital, in the subanesthetic doses used, had neither antinociceptive nor hyperalgesic properties. Furthermore, the barbiturate had no effect on the antinociceptive action of morphine in either morphine-tolerant or non-tolerant rats.     

Reduction of the sevoflurane minimum alveolar concentration induced by methadone, tramadol, butorphanol and morphine in rats

This study aimed to estimate the reduction in the minimum alveolar concentration (MAC) of sevoflurane induced by low and high doses of methadone (5 and 10 mg/kg), tramadol (25 and 50 mg/kg), butorphanol (5 and 10 mg/kg) or morphine (5 and 10 mg/kg) in the rat. A control group received normal saline. Sixty-three adult male Sprague-Dawley rats were anaesthetized with sevoflurane (n = 7 per group). Sevoflurane MAC was then determined before and after intraperitoneal administration of the opioids or saline. The duration of the sevoflurane MAC reduction and basic cardiovascular and respiratory measurements were also recorded. The baseline MAC was 2.5 (0.3) vol%. Methadone, tramadol and morphine reduced the sevoflurane MAC (low dose: 31 ± 10, 38 ± 15 and 30 ± 13% respectively; high dose: 100 ± 0, 83 ± 17 and 77 ± 25%, respectively) in a dose-dependent manner. The low and high doses of butorphanol reduced the sevoflurane MAC to a similar extent (33 ± 7 and 31 ± 4%, low and high doses, respectively). Two rats developed apnoea following administration of high-dose butorphanol and methadone. These anaesthetic-sparing effects are clinically relevant and may reduce the adverse effects associated with higher doses of inhalational anaesthetics.     

Short-term tolerance to morphine: Effects of indomethacin

Short-term tolerance to opiates has been demonstrated in as little as three hours after priming with a single dose of morphine in naive animals. Tail-flick latency in mice and changes in plasma corticosterone in rats were the indicators tested in these experiments. Rats primed with either saline or morphine, 10 mg/kg, were injected 3 hrs. subsequently with morphine, 5 mg/kg. Those primed with saline showed the characteristic plasma corticosterone elevation following morphine, when serial blood samples were examined, whereas those previously treated with morphine did not. Mice were primed with saline or either of two doses of morphine, 30 or 100 mg/kg, 3.5 hrs. prior to estimation of tail-flick latency and ED 50 determinations. Mice primed with either dose of morphine had significantly higher ED50's than those primed with saline. The effects of indomethacin, 5 or 10 mg/kg, were examined on both systems. Rats and mice were pretreated with indomethacin at 2.25 or 3 hrs., respectively, before morphine-priming. In all cases, indomethacin did not produce alterations in responses previously observed in correspondently treated controls.     

Adrenalectomy potentiates the morphinebut not cocaine-induced place preference in rats

The conditioned place preference paradigm is commonly used to study the reinforcing properties of various drugs. In the present study, the effect of adrenalectomy (ADX) on the morphine-induced place preference was examined in rats. Morphine produced a significant preference for the drug-associated place in sham-operated (sham) and ADX rats. In sham rats, only the highest dose of morphine (8 mg/kg, i.p.) produced a significant preference, while in ADX rats, lower doses of morphine (1 and 2 mg/kg, i.p.) produced a significant preference for the drug-associated place. Furthermore, the morphine-induced place preference was blocked by the dopamine D₁ antagonist SCH23390 in both sham and ADX rats. On the other hand, the cocaineinduced place preference was not affected by ADX. In the present study, we found that ADX potentiates the reinforcing effect induced by morphine, but not that induced by cocaine, which suggests that the enhancement by ADX may be due to a change in opioid receptors, morphine metabolism and/or some other cause, but not to a change in dopamine receptors.     

"Paradoxical" analgesia and aggravated morphine dependence induced by opioid antagonists

Chronic treatment with naloxone (Nx) or naltrexone (Ntx) induces paradoxical analgesia. In the present study, the effects of chronic treatment with opioid receptor antagonists, such as nor-binaltorphimine (nor-BNI) for kappa and naltrindole (NTI) for delta receptors, on analgesic response using the hot plate test and on morphine physical dependence in rats were examined. The hot plate latency was significantly increased by pretreatment with Nx (5 mg/kg, s.c.), nor-BNI (20 mg/kg, i.p.) or NTI (20 mg/kg, i.p.) for 5 days. After chronic pretreatment with these antagonists, the rats were treated with morphine-admixed food (0.5 mg/g of food) for 3 days. Chronic pretreatment with Nx and NTI significantly increased Nx precipitated body weight loss in morphine dependent rats, while chronic pretreatment with nor-BNI produced small increase. These results indicate that chronic treatment with nor-BNI or NTI as well as with Nx induces obviously paradoxical analgesia, and that chronic blockade of mu or delta may enhance the development of physical dependence on morphine.     

Acute analgesic effect of loperamide as compared to morphine after intrathecal administration in rat

Loperamide, a mu opioid receptor agonist, which is commonly used as an antidiarrhoeal agent has been reported to possess analgesic activity after intrathecal administration. However, the exact analgesic profile, i.e., onset, duration and intensity of analgesia in relation to morphine is not fully known. In the present study, the acute analgesic effect of loperamide (5 microg) was compared with that of morphine (5 microg) and morphine + loperamide (5 microg of each) using the tail flick method after intrathecal administration. Naloxone (5 mg/kg) reversibility of the analgesic effect was also studied. The analgesic response of loperamide was significantly higher than morphine. Even after 22 hr, maximum possible effect was greater than 49%. Naloxone partially antagonized the analgesic effect of loperamide. This suggested that loperamide may be acting through blockade of Ca2+ channels besides activating mu opioid receptors. Loperamide may prove to be a better substitute for morphine as spinal analgesic.