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The purpose of the present study was to evaluate the nature of the antinociceptive interaction among dexketoprofen (DEX), a mixed inhibitor of the cyclo-oxygenases, and tramadol (TRAM), a weak opioid with monoaminergic activity that inhibits norepinephrine and serotonin re-uptake. We assessed antinociception in the acetic acid writhing test, the tail flick and the formalin (FT) tests, and gastrointestinal transit (GIT) after the administration of a charcoal meal. The analysis of the interaction was carried out using isobolograms and interaction indexes or the fixed-dose method GIT. The administration of DEX or TRAM individually induced dose-dependent antinociception in all the algesiometric tests. In the three tests, TRAM was between 5.2 (FT, phase I) and 35 times (FT, Phase II) more potent than DEX. When testing combinations at different potency ratios (1 : 1, 1 : 3, 3 : 1), we could demonstrate synergy in all algesiometric tests, only when drugs were combined in a 1 : 1 proportion. Interestingly, the proportion of the drugs in the combination could change the type of interaction from synergy to antagonism. On the inhibition of GIT, a dose-related inhibition was established for TRAM, but not for DEX. Using a fixed-dose protocol, we could demonstrate antagonism between DEX and TRAM on the inhibition of GIT. The results of the present study suggest that a combination of DEX and TRAM in a 1 : 1 proportion could be adequate to use in future clinical trials in humans.

Background: Morphine and clonidine show synergy or antagonism inhibiting gastrointestinal transit depending on their proportion and level of effect. Their interaction during morphine tolerance and intestinal inflammation were assessed., Methods: Gastrointestinal transit in mice was evaluated with charcoal and antitransit effects expressed as percent mean values +/- SEM. Tolerance was induced with a morphine pellet (75 mg) implanted for 72 h, and inflammation with intragastric croton oil. Dose-response curves for morphine and clonidine alone and combined at a 1:1 potency ratio were obtained, and doses producing a 50% and 60% inhibition were calculated (ED50, ED60). Interaction was established by isobolograms, interaction indexes, and analysis of variance., Results: In naive and tolerant mice, the combination induced linear dose-response curves up to the ED60 and then reached a plateau. In naive mice, ED50 values were as follows: morphine 1.52 +/- 0.15 mg/kg, clonidine 0.09 +/- 0.008 mg/kg, and combined 0.506 +/- 0.084 mg/kg (0.478 +/- 0.08 mg/kg morphine plus 0.028 +/- 0.004 mg/kg clonidine). During tolerance, ED50 values were as follows: morphine 9.73 +/- 0.8 mg/kg, clonidine 0.09 +/- 0.007 mg/kg, combination 0.131 +/- 0.09 mg/kg (morphine 0. 13 +/- 0.09 mg/kg plus clonidine 0.0013 +/- 0.0005 mg/kg). In both groups, the interaction was synergistic up to the ED60 and antagonistic thereafter; synergy was enhanced during tolerance. During inflammation, ED50 values were as follows: morphine 0.17 +/- 0.04 mg/kg, clonidine 0.015 +/- 0.006 mg/kg, combined 0.62 +/- 0.04 mg/kg (morphine 0.568 +/- 0.04 mg/kg plus clonidine 0.052 +/- 0.004 mg/kg); thus, potencies of morphine and clonidine increased 9.3 and 7.1 times, while the combination remained unaltered. Moreover, inflammation transformed synergy into antagonism., Conclusions: The interaction between morphine and clonidine was significantly altered during tolerance and inflammation. During tolerance, synergy was present up to 60% effect and then became antagonistic. Inflammation converted synergy to antagonism. A common pathway in signal transduction could partially explain the results.

1. We have studied the effects of mu and delta opioids on intestinal function (permeability, PER; gastrointestinal transit, GIT), and their antagonism after the intracerebroventricular (i.c.v.) administration of specific antibodies (ABs) or antisense oligodeoxynucleotides (ODN) to mu-receptors (OR). Central versus peripheral site/s of action of subcutaneous (s.c.) mu-opioids, were also assessed. 2. Male Swiss CD-1 mice were used. GIT was measured with charcoal and PER by the passage of 51Cr-EDTA from blood to lumen. 3. Morphine and fentanyl (i.c.v. and s.c.) inhibited GIT and PER in a dose-related manner; they were more potent by i.c.v. route, both on GIT and PER (70 and 17 times for morphine and fentanyl). They also had a greater effect on GIT than PER (4.3 and 1.6 times). DPDPE had a lower potency than mu-agonists in all experiments, and no dose-response could be obtained after s.c. administration on GIT. 4. Pretreatment with i.c.v. ABs (24 h) or antisense ODN (5 days), decreased the effects (GIT and PER) of i.c.v. morphine and fentanyl, while those of DPDPE remained unchanged. The ABs did not alter the peripheral effects of mu-opioids. 5. The results show that (i.c.v. or s.c.) mu opioids produce dose-related inhibitions of PER and GIT, being more potent by the i.c.v. route. Delta-opioids had a greater effect on PER than GIT, while the opposite occurred for mu-agonists. Pretreatment with ABs or ODN to mu-OR, blocked the central effects of mu (but not delta) agonists on GIT and PER.

The study describes a model of chronic intestinal inflammation in mice. Inflammation was induced by the administration of one dose of croton oil (CO) (acute CO) or two doses (chronic CO) of intragastric CO, whereas controls received saline (SS); GI transit was measured with charcoal. Chronic CO induced intestinal inflammation substantiated by optical microscopy, weight loss (20%) and a 25% increase in GI transit. The ED50 values in SS animals were 1.67 +/- 0.13 mg/kg for morphine and 0.038 +/- 0.006 mg/kg for fentanyl; chronic CO significantly decreased the ED50 values to 0.16 +/- 0.03 mg/kg (morphine) and 0.006 +/- 0.0005 mg/kg (fentanyl). Thus the potency of morphine increased 10.4 times and that of fentanyl 6.3 times. The effects of enkephalin, but not those of U-50488H, were also significantly enhanced during chronic CO. The antitransit effects of p.o. loperamide increased 11.7 times during chronic CO. All effects were reversed by specific antagonists. The fraction of the active opioid receptor that mediates the antitransit effects of morphine was evaluated using beta-funaltrexamine. In chronic CO, the doses of beta-funaltrexamine needed to antagonize 1 mg/kg of morphine were significantly higher than in SS and acute CO, and the ED50/KA ratio was 20 times lower. These results suggest an increase in the active concentration of mu-opioid receptors during chronic inflammation.

1. The aim of investigation was to establish and compare the reversibility of tolerance to the antitransit effects of morphine by three different procedures: (a) acute inflammation of the gut, (b) lorglumide a cholecystokininA (CCKA) receptor antagonist, or (c) MK-801, an N-methyl-D-aspartate (NMDA) receptor ion channel blocker. The type of interaction between morphine and lorglumide or MK-801 on the inhibition of gastrointestinal transit (GIT) in naive animals was also evaluated. 2. Male Swiss CD-1 mice were implanted with 75 mg of morphine base or placebo pellets. Gastrointestinal transit was assessed with a charcoal meal and results expressed as % inhibition of GIT. Inflammation was induced by the intragastric (p.o.) administration of croton oil (CO), while controls received castor oil (CA) or saline (SS). Morphine was administered by subcutaneous (s.c.) or intracerebroventricular (i.c.v.) injection, to naive and tolerant animals treated with CO, CA or SS. Dose-response curves for s.c. morphine were also performed in naive and tolerant mice receiving 5.2 or 7.4 nmol (s.c.) lorglumide or MK-801, respectively. 3. The ED50 values for inhibition of GIT by s.c. morphine were: 45.9 +/- 2.7 and 250.1 +/- 3.1 nmol in naive and tolerant animals, respectively, demonstrating a five fold decrease in the potency of morphine. In naive animals, inflammation (CO) decreased the ED50 of morphine three times (14.4 +/- 2.2 nmol). However, no tolerance to s.c. morphine (ED50 16.4 +/- 2.6 nmol) was manifested during intestinal inflammation. After i.c.v. administration, a similar degree of tolerance to morphine was observed (4.8 fold decrease in potency). Intestinal inflammation had no effect on the ED50 values of i.c.v. morphine in naive and tolerant animals, showing that reversal of tolerance is related to local mechanism/s. Mean values for intestinal pH were 6.9 +/- 0.04 and 6.2 +/- 0.04 in SS and CO treated mice, respectively. In addition, morphine was 74 times more potent by the i.c.v. than by the s.c. route (naive-SS). 4. Morphine and lorglumide interacted synergistically in naive animals; in addition, the administration of lorglumide reversed tolerance to s.c. morphine. No interaction (additivity) was observed in naive animals when morphine and MK-801 were administered in combination. However, the drug completely reversed tolerance to the antitransit effects of morphine. 5. The present investigation shows that acute inflammation of the gut reverses tolerance to the antitransit effects of s.c. morphine by a peripheral mechanism. Qualitatively similar results were obtained after the administration of lorglumide or MK-801. Our results suggest that a local decrease in pH could play an important role during inflammation, while antagonism of endogenous compensatory systems would explain the reversal of tolerance induced by lorglumide or MK-801.

1. The peripheral effects of alpha(2)-adrenoceptor agonists were investigated in a model of intestinal inflammation induced by intragastric administration of croton oil (CO). Our hypothesis was that inflammation would 'sensitize' adrenoceptors in peripheral and/or central terminals of myenteric and submucous plexus neurones, and enhance systemic effects of alpha(2)-adrenoceptor agonists. 2. Male swiss CD-1 mice, received intragastrically CO (0.05 ml), castor oil (CA, 0.1 ml) or saline (SS) 3 h before the study: gastrointestinal transit (GIT) was evaluated 20 min afterwards with a charcoal meal. The presence of inflammation was assessed by electron microscopy. 3. The intragastric administration of CA or CO caused an increase in GIT and weight loss, but only CO induced an inflammatory response. Both clonidine (imidazoline1/alpha(2)-agonist) and UK-14304 (alpha(2)-agonist) produced dose-related inhibitions of GIT in all groups. During inflammatory diarrhoea (CO), potencies of systemic (s.c.) clonidine and UK-14304 were significantly increased 3.5 and 2.1 times, respectively, while potencies remained unaltered in the presence of diarrhoea without inflammation (CA). The effects were reversed by administration (s.c.) of receptor-specific adrenoceptor antagonists, but not by naloxone. 4. Clonidine was 8.3 (SS) and 2.8 (CO) times more potent when administered intracerebroventricularly (i.c.v.), than when administered s.c. Inflammation of the gut did not alter the potency of i.c.v. clonidine, demonstrating that enhanced effects of s.c. clonidine are mediated by peripheral receptors. During inflammation, i.c.v. efaroxan did not antagonize low doses of s.c. clonidine (ED20 and ED50S), but partially reversed ED80S, further supporting the peripheral effects of the agonists in CO treated animals. 5. The results demonstrate that inflammation of the gut enhances the potency of alpha(2)-adrenoceptor agonists by a peripheral mechanism. The results also suggest that the inflammatory response induces an up-regulation or sensitization of alpha(2)-adrenoceptors and/or imidazoline receptors.

Background: Combinations of drugs are frequently used to achieve effective analgesia while minimizing side effects. Although the analgesic effects of morphine and clonidine seem to be synergistic, few studies have investigated other effects. Their role in inhibiting gastrointestinal transit was evaluated using different methods of analysis., Methods: Percentage inhibition of transit induced by morphine, clonidine, or their combination was measured in mice that had been given an intragastric charcoal meal. Dose-response curves were obtained for each drug individually; for morphine:clonidine at 1:3, 1:1, and 1:0.33 ratios; and for morphine in the presence of 0.0138 mg/kg clonidine. The interaction was evaluated by isobolograms, combination indexes, and fixed-dose analysis., Results: Each drug and their combinations inhibited transit in a dose-related manner. Combinations of morphine and clonidine produced interaction that depended on the ratio and level of response. The interaction analyzed by isobolograms and combination indexes showed that combinations in 1:1 and 1:3 proportions were synergistic at the median effective doses or less and were antagonistic at larger doses. Fixed-dose analysis using different ratios showed similar results. The effects of the combination (median effective dose at 1:1 ratio) were antagonized by efaroxan but not by naloxone, suggesting a predominant role of alpha-2-mediated effects., Conclusions: Investigations into drug interactions should include several levels of response and combinations at different ratios. Isobolographic analysis permits the statistical evaluation of results without making assumptions about mechanisms of action of the drugs or their interactions. In this study, the combination of morphine and clonidine should produce synergy, antagonism, or no interaction depending on the relative doses and the level of effect.

We have investigated the antitransit effects of free and liposomal morphine in a model of intestinal inflammation. Mice received saline or croton oil orally, 3 h prior to evaluation, and gastrointestinal transit was measured 20 min afterwards. Peak/duration of effects, potency (ED50) and antagonism by naloxone and naloxone methiodide were evaluated. Peak effects occurred 30 and 40 min after administration of morphine and liposomal morphine, respectively. Encapsulated morphine had a more pronounced and prolonged effect than morphine. Comparison of the ED50S demonstrated that the potency of liposomal morphine was 3.5 times higher than that of morphine during inflammation; in addition, inflammation increased the potency of morphine and liposomal morphine, 3 and 9.2 times, respectively. The effects of morphine and liposomal morphine in croton oil-treated mice were reversed by naloxone and naloxone methiodide. The results show that during inflammation, the potency and duration of the antitransit effects of morphine are significantly enhanced by encapsulation.

The aim of the study was to determine the effects of intestinal inflammation induced by croton oil on the antitransit action of systemically administered receptor-specific opioids. Our hypothesis was that inflammation would "sensitize" opioid receptors in peripheral and/or central terminals of myenteric and submucous plexus neurons and enhance the effects of exogenously administered opioids. Diarrhea was induced by p.o. administration of croton oil and was demonstrated by weight loss and increased gastrointestinal transit. Histologically, an increased number of clear vesicles in the cytoplasm of jejunal epithelial cells and enlarged spaces filled with fine granular material in the extravascular compartment were observed. Subcutaneous morphine and fentanyl produced dose-related inhibitions of gastrointestinal transit in saline-treated controls with ED50 values of 1.24 +/- 0.06 and 0.036 +/- 0.010 mg/kg, respectively. In animals with diarrhea, dose-response curves were parallel and shifted to the left with a significant decrease in ED50 values of 2.95 times for morphine and 1.89 for fentanyl. The effects of the delta agonist Tyr-D-Pen-Gly-Phe-D-Pen, but those of U50,488H [trans-3,4-dichloro-N-methyl-N-(2-(1-pyrrolydynil)cyclohexyl) benzeneazetamine] also were increased significantly during diarrhea associated with inflammation. Naloxone (0.1 mg/kg), MR-2266 [(-)-a-5,9-diethyl-2'-hydroxy-2-(3-furylmethyl)-6,7-benzomorphan] (3 mg/kg) and naltrindole (1 mg/kg) antagonized the effects of the receptor-specific opioid agonists used in the study. Our results show that the potency of s.c. mu and delta opioids is increased during inflammation of the gut and that the effect is mediated by the same type of opioid receptors present in the noninflamed tissue. These results support the view that a sensitization of opioid receptors occurs during acute inflammation of the gut.

The effects of kappa-opiate agonists on gastrointestinal motility was assessed in Swiss Webster, C57BL/6, BALB/c and DBA/2 strains of mice. The kappa-agonists, PD 117302, U-69593 and U-50488H (3 mg kg-1), were injected subcutaneously and the distance travelled by a charcoal meal in the gastrointestinal tract was measured. All kappa-agonists induced significant inhibition of charcoal meal transit; however, there were significant strain differences in the antitransit effect. It is concluded that kappa-receptors are involved in the inhibition of gastrointestinal transit and that the negative data reported in the literature may be due to a genotype-dependent effect of kappa-agonists.

1. The effect of alpha 2-adrenoceptor agonists on gastrointestinal motility was assessed in normoglycaemic and streptozotocin-diabetic mice. 2. The alpha 2-adrenoceptor agonists used were: clonidine (0.1, 0.3 and 1 mg kg-1, azepexole (10, 20 and 40 mg kg-1), tizanidine (1, 3 and 10 mg kg-1) and ST-91 (10, 20 and 30 mg kg-1). 3. Acute hyperglycaemia was induced by D-(+)-glucose (5 g kg-1) and chronic hyperglycaemia by streptozotocin (200 mg kg-1) injection. 4. The gut motility was quantitated using the charcoal meal test. 5. The results indicate that in normoglycaemic and acutely hyperglycaemic mice, all of the alpha 2-adrenoceptor agonists used produced significant inhibition of meal transit. 6. However, in streptozotocin-diabetic mice, the anti-transit effect of alpha 2-adrenoceptor agonists was attenuated. 7. Since streptozotocin-induced diabetes but not acute hyperglycaemia was associated with the attenuation of anti-transit effect, elevated blood sugar is not the mechanism for the observed effect. 8. As with groups treated with clonidine, azepexole or tizanidine, the anti-transit effect of a peripherally acting alpha 2-adrenoceptor agonist, ST-91, was attenuated in streptozotocin-diabetic mice. This suggests the involvement of peripheral mechanism(s) in attenuating the anti-transit effect of alpha 2-adrenoceptor agonists. 9. These results identify the need for critical evaluation of the role and efficacy of alpha 2-adrenoceptor agonists in the therapeutic management of diabetic diarrhoea.

Using the charcoal meal test, the effects of morphine (3, 5 and 7 mg/kg) on gastrointestinal transit was assessed in normoglycemic as well as in acute and chronic hyperglycemic mice. Acute hyperglycemia was induced by intraperitoneal injection of glucose (5.04 g/kg), while chronic hyperglycemia was induced by streptozotocin injection (200 mg/kg i.p., 7-8 days before). Acute hyperglycemia augmented the inhibitory effect of morphine on gut transit, however, chronic hyperglycemia failed to modify the effects of morphine. The results indicate that hyperglycemia per se may not be the primary mechanism for the altered sensitivity to morphine in experimental models of diabetes.

Systemic administration of highly selective kappa opiate agonists, U-50488H and U-69593 (3, 10 and 30 mg/kg i.p.) produced significant inhibition of the gastrointestinal transit in mice as assessed by charcoal meal test. In contrast, the (+) stereoisomer of U-50488H, U-53455E did not inhibit the gastrointestinal motility. Furthermore, the kappa-selective antagonist, Mr 2266 (3 mg/kg) when administered along with the agonists, reversed the effects of the agonists. These results suggest that stereospecific kappa opiate receptors are involved in inhibition of gut motility in mice.

The effects of highly selective kappa-opiate agonists were assessed on the gastrointestinal motility in normoglycaemic and hyperglycaemic conditions in mice. Chronic hyperglycaemia was induced by streptozocin injection (200 mg kg-1 i.p.), 7-8 days before the experiment. Acute hyperglycaemia was induced by glucose injection (5 g kg-1 i.p.) at the time of opiate administration. The kappa-opiate agonists, U-50488H and U-69593 (1, 3 and 10 mg kg-1) were injected (i.p.) just before the charcoal meal. The animals were killed 45 min later and the distance travelled by the test meal was measured. In the normoglycaemic mice, both kappa-agonists significantly (P less than 0.05) inhibited the meal transit and this effect was significantly (P less than 0.05) augmented in acute hyperglycaemic animals. However, in chronic hyperglycaemic animals U-50488H failed to inhibit the charcoal meal transit, while U-69593 produced anti-transit effect comparable to that observed in normoglycaemic mice. These results demonstrate that kappa-opiate agonists produce anti-transit effects in mice that these effects are enhanced during acute hyperglycaemia. The disparity of anti-transit effects of kappa-opiate agonists in acute vs chronic hyperglycaemia supports the hypothesis that elevated glucose levels are not the primary mechanism for the altered response to opiates observed in the experimental models of diabetes.