Differential displacement of opioids from plasma protein binding sites by di-isopropylfluorophosphate in the mouse

Di-isopropylfluorophosphate (DFP) displaced the opioid drug, alfentanil, from plasma proteins in-vivo and in-vitro in mice but was without effect on the structurally related compound, fentanyl. This action probably accounts for the enhanced entry of alfentanil into the brain of DFP-treated animals and its enhanced antinociceptive activity.     

Di-isopropylfluorophosphate induced antinociception and its interactions with opioid drugs in the rat

The irreversible anticholinesterase, di-isopropylfluorophosphate (DFP), was shown to be antinociceptive in the paw pressure test in rats only at doses where animals exhibited marked signs of anticholinesterase poisoning. At sub-antinociceptive doses DFP potentiated the opioid drugs fentanyl and alfentanil but failed to alter morphine antinociception. These interactions differ from our previous studies using the hot plate test in mice and suggest that opioid/cholinergic interactions are species and test dependent.     

P-glycoprotein ATPase activating effect of opioid analgesics and their P-glycoprotein-dependent antinociception in mice

It is well known that opioid analgesics exert central antinociceptive actions. However, in vivo and in vitro studies have shown that some opioid analgesics given systemically have limited access to the central nervous system because of the blood-brain barrier (BBB). P-glycoprotein (P-gp), an ATP-dependent drug efflux transporter, is one component of the BBB. In this report, we assessed the antinociceptive effect of morphine, fentanyl, and meperidine in P-gp deficient (mdr1a KO) mice, and compared these effects with those in wild type (WT) mice. The antinociceptive effects of morphine and fentanyl in mdr1a KO mice were significantly greater than those in WT mice. However, there was no clear difference in the antinociceptive effects of meperidine in the two genotypes. In addition, we determined the effect of opioid analgesics on P-gp ATPase activity, which is requisite for drug transport, using mouse brain capillary endothelial cells. In our observations, morphine and fentanyl, but not meperidine, significantly increased P-gp ATPase activity, and the drugs' concentration-response curves were bell-shaped, reaching a peak at a concentration of 1 muM. These results suggest that P-gp ATPase activity may be, at least in part, involved in the antinociceptive potencies of those opioid analgesics that are substrates for P-gp.     

G-protein receptor kinase 3 (GRK3) influences opioid analgesic tolerance but not opioid withdrawal

1. Tolerance to opioids frequently follows repeated drug administration and affects the clinical utility of these analgesics. Studies in simple cellular systems have demonstrated that prolonged activation of opioid receptors produces homologous receptor desensitization by G-protein receptor kinase mediated receptor phosphorylation and subsequent beta-arrestin binding. To define the role of this regulatory mechanism in the control of the electrophysiological and behavioral responses to opioids, we used mice having a targeted disruption of the G-protein receptor kinase 3 (GRK3) gene. 2. Mice lacking GRK3 did not differ from wild-type littermates neither in their response latencies to noxious stimuli on the hot-plate test nor in their acute antinociceptive responses to fentanyl or morphine. 3. Tolerance to the electrophysiological response to the opioid fentanyl, measured in vitro in the hippocampus, was blocked by GRK3 deletion. In addition, tolerance to the antinociceptive effects of fentanyl was significantly reduced in GRK3 knockouts compared to wild-type littermate controls. 4. Tolerance to the antinociceptive effects of morphine was not affected by GRK3 deletion although morphine tolerance in hippocampal slices from GRK3 knockout mice was significantly inhibited. Tolerance developed more slowly in vitro to morphine than fentanyl supporting previous work in in vitro systems showing a correlation between agonist efficacy and GRK3-mediated desensitization. 5. The results of these studies suggest that GRK3-mediated mechanisms are important components of both electrophysiologic and behavioral opioid tolerance. Fentanyl, a high efficacy opioid, more effectively produced GRK3-dependent effects than morphine, a low efficacy agonist.     

Analysis of the opioid–opioid combinations according to the nociceptive stimulus in mice

The purpose of the present study was to characterize the antinociceptive effects of tramadol, fentanyl and morphine, when two of them were systemically combined in a 1:1 potency ratio, in the hot plate, the acetic acid writhing, and the formalin tests in mice. Interaction indexes and isobolographic analysis were used to assess the type of interaction. Fentanyl was the most potent drug, followed by morphine and tramadol, with the exception in the phase I of formalin test. Synergistic interactions were obtained when tramadol was combined with fentanyl or with morphine in the writhing and formalin tests. But, in the hot plate only additive interactions were obtained. Changes were induced on the type of interaction depending on the level of effect of opioid–opioid combinations. Moreover, co-administration of fentanyl with morphine showed additivity, regardless of the type of stimulus. Standard rotarod test analysis confirmed intact motor coordination. The present findings suggest that the type of interaction between opioids is not only related to the nature of nociceptive stimulus but also to non-opioid analgesic pathways.     

Variable modulation of opioid brain uptake by P-glycoprotein in mice

The efflux transporter P-glycoprotein (P-gp) is an important component of the blood-brain barrier (BBB) that limits accumulation of many compounds in brain. Some opioids have been shown to interact with P-gp in vitro and in vivo. Genetic or chemical disruption of P-gp has been shown to enhance the antinociceptive and/or toxic effects of some opioids, although the extent of this phenomenon has yet to be understood. The purpose of this study was to assess quantitatively the influence of mdr1a P-gp on initial brain uptake of chemically diverse opioids in mice. The brain uptake of opioids selective for the mu (fentanyl, loperamide, meperidine, methadone, and morphine), delta (deltorphin II, DPDPE, naltrindole, SNC 121) and kappa (bremazocine and U-69593) receptor subtypes was determined in P-gp-competent (wild-type) and P-gp-deficient [mdr1a(-/-)] mice with an in situ brain perfusion model. BBB permeability of the opioids varied by several orders of magnitude in both mouse strains. The difference in brain uptake between P-gp-competent and P-gp-deficient mice ranged from no detectable effect (meperidine) to >/=8-fold increase in uptake (DPDPE, loperamide, and SNC 121). In addition, loperamide efflux at the BBB was inhibited by quinidine. These results demonstrate that P-gp modulation of opioid brain uptake varies substantially within this class of compounds, regardless of receptor subtype. P-gp-mediated efflux of opioids at the BBB may influence the onset, magnitude, and duration of analgesic response. The variable influence of P-gp on opioid brain distribution may be an important issue in the context of pharmacologic pain control and drug interactions.     

Potentiation of morphine analgesia by adjuvant activation of 5-HT7 receptors

Spinal blockade of 5-HT7 receptors has been reported to inhibit the antinociceptive effect of opioids. In this study, we found that subcutaneous administration of the selective 5-HT7 receptor agonist E-55888 (10 mg/kg) or the antagonist SB-258719 (5 mg/kg) exerted no effect on the tail-flick test in mice. However, E-55888, but not SB-258719, increased (2.6-fold) the analgesic potency of oral morphine. The potentiating effect exerted by E-55888 was prevented by SB-258719. A pharmacokinetic interaction was discarded as morphine plasma and brain concentrations were not significantly modified when co-administered with E-55888. These results reinforce the involvement of 5-HT7 receptors in opioid analgesia and point to a potential use of 5-HT7 receptor agonists as adjuvants of opioid analgesia.     

Effect of mu- and kappa-opioid agonists on the electroconvulsive seizure threshold in mice and antagonism by naloxone and MR 2266

The effects of mu-agonists (morphine, fentanyl) and kappa-agonists (U-50,488, U-69,593, bremazocine, nalbuphine, tifluadom) on the electroconvulsive threshold were studied in mice. The threshold could be significantly elevated by all drugs tested in a dose range that was in the order of magnitude of the antinociceptive ED50. Mice tolerant to the antielectroshock effect of morphine still reacted to U-69,593. The antagonism of the anticonvulsant effect by the mu-antagonist naloxone and the kappa-antagonist MR 2266 was receptor-specific only with fentanyl and U-50,488. The other opioid agonists were either antagonized by both drugs (morphine, U-69,593, bremazocine, nalbuphine) or even by the opposite antagonist (tifluadom). A synergistic effect of mu- and kappa-stimulation is assumed for the mediation of the antielectroshock effect of opioid drugs, but drugs with high affinity and intrinsic activity at one receptor type (fentanyl, U-50,488) are obviously able to bring about their antielectroshock effect through the one respective opioid binding site.     

Di-isopropyl phosphofluoridate-induced antinociception: possible role of endogenous opioids

Di-isopropyl phosphofluoridate (DFP, 0.1--1.5 mg/kg, s.c.) produced antinociception in rats as measured by the hot plate test. Naloxone reduced DFP-induced antinociception but did not affect the attenuated locomotor activity or hypothermia produced by DFP. Animals rendered tolerant to the antinociceptive action of morphine failed to exhibit cross tolerance to the antinociceptive action of DFP. Morphine- and DFP-induced antinociceptive states were antagonized by MR 2266 and GPA 1843, the (-)-isomers of 5,9 alpha-Diethyl-2-(3-furylmethyl)-2'-hydroxy-6, 7-benzomorphan and -2-allyl-2'-hydroxy-9 beta-methyl-5-phenyl-6, 7-benzomorphan hydrochloride, respectively; the corresponding (+)-isomers, MR 2267 and GPA 1847, did not antagonize the antinociceptive state produced by DFP or morphine. These results suggest that DFP-induced antinociception may be mediated via the release of endogenous opioids; however, this could occur at sites different from those concerned with morphine tolerance.     

Effect of μ- and -Opioid Agonists on the Electroconvulsive Seizure Threshold in Mice and Antagonism by Naloxone and MR 2266

Abstract: The effects of μ-agonists (morphine, fentanyl) and x-agonists (U-50,488, U-69,593, bremazocine, nalbuphine, tifluadom) on the electroconvulsive threshold were studied in mice. The threshold could be significantly elevated by all drugs tested in a dose range that was in the order of magnitude of the antinociceptive ED50. Mice tolerant to the antielectroshock effect of morphine still reacted to U-69,593. The antagonism of the anticonvulsant effect by the μ-antagonist naloxone and the x-antagonist MR 2266 was receptor-specific only with fentanyl and U-50,488. The other opioid agonists were either antagonized by both drugs (morphine, U-69,593, bremazocine, nalbuphine) or even by the opposite antagonist (tifluadom). A synergistic effect of μ- and x-stimulation is assumed for the mediation of the antielectroshock effect of opioid drugs, but drugs with high affinity and intrinsic activity at one receptor type (fentanyl, U-50,488) are obviously able to bring about their antielectroshock effect through the one respective opioid binding site.     

Effect of a selective GABA(B) receptor agonist baclofen on the mu-opioid receptor agonist-induced antinociceptive, emetic and rewarding effects

The management of excessive adverse effects of opioids is a major clinical problem. The present study was undertaken to investigate the effect of a selective γ-aminobutyric acid (GABA)_B receptor agonist baclofen on the μ-opioid receptor agonist-induced antinociceptive, emetic and rewarding effects. Either morphine or fentanyl produced a dose-dependent antinociceptive effect in both ferrets using Randall-Selitto test and mice using tail-flick test. Under these conditions, pretreatment of baclofen produced an additive antinociception induced by morphine or fentanyl. Furthermore, the augmentation of antinociception induced by systemic administration of baclofen with morphine or fentanyl was completely abolished by either i.c.v. or i.t. pretreatment with the selective GABA_B receptor antagonist CGP 35348 in mice. We next investigated the emetic response induced by μ-opioid receptor agonist in ferrets. Morphine at lower doses than that used for antinociceptive assay produced both retching and vomiting, whereas fentanyl failed to produce the retching and vomiting in ferrets. Here we reported for the first time that baclofen significantly suppressed the retching and vomiting induced by morphine, indicating the involvement of GABA_B receptor in emetic control pathway. Furthermore, baclofen also inhibited place preference elicited morphine or fentanyl in rats. Taken together, these results suggest that co-administration of baclofen with μ-opioid receptor agonist produced a potentiation of antinociceptive effect, whereas an untoward effect was completely blocked.     

Dextromethorphan and ketamine potentiate the antinociceptive effects of mu- but not delta- or kappa-opioid agonists in a mouse model of acute pain

Animal and clinical studies have reported potentiation of opioid antinociception by NMDA receptor antagonists such as ketamine and dextromethorphan. The aim of this study was to compare these clinically available NMDA antagonists in combination with classical morphine, mu-selective fentanyl-like opioids, the delta-opioid agonist SNC80 and the kappa-opioid agonist U50,488H. Using a mouse hot-plate test, dose-response relationships were first determined for all compounds individually and then for opioids co-administered with fixed doses of ketamine or dextromethorphan. All compounds were administered intraperitoneally ED(50) values were calculated from the proportion of animals failing to exhibit any response within a fixed cut-off criterion of 30 s. To varying degrees, all compounds produced increases in response latencies over time. Dextromethorphan produced lower ED(50) values for morphine, fentanyl and sufentanil but exerted no effect on the potency of SNC80 or U50,488H. Similarly, ketamine potentiated the antinociceptive potency of morphine, fentanyl and sufentanil but not SNC80 or U50,488H. In summary, these results support the use of mu-opioid agonists in combination with NMDA antagonists, but suggest that there may be no advantage in combining dextromethorphan or ketamine with delta- or kappa-opioids in the management of acute pain.     

Antinociceptive effects of morphine, fentanyl, tramadol and their combination, in morphine-tolerant mice

The development of morphine-tolerance after chronic administration, reduces analgesic efficacy and is a significant clinical problem in some patients; may be managed clinically by increasing the doses of morphine and/or the administration of a second mu-opioid agonist. In morphine-tolerant mice, we investigated the presence of an interaction when two opioids are administered simultaneously. We determined the antinociceptive effects of morphine (M), fentanyl (FEN), and tramadol (TRM) individually and combined in a 1:1 proportion, based on their potency. Nociceptive thresholds were evaluated in CD1 mice using the hot plate test. Morphine tolerance was induced by the subcutaneous implantation of a 75 mg morphine pellet, whereas control animals received a placebo pellet; the experiments were performed three days later. In both (placebo and morphine pellets), dose-response curves for M, FEN and TRM, individually and combined were obtained, and the doses that produced 50% inhibition (ED₅₀) were determined. Sustained exposure to morphine induced a significant decrease in antinociceptive potency to acute M or FEN administration (tolerance), which was of a lesser magnitude after acute TRM; in these experiments the analysis of the interaction between chronic morphine and each opioid, demonstrated functional antagonism. The simultaneous administration of two opioids in morphine-tolerant mice, demonstrated antagonism for the M:FEN combination, whereas the effects of TRM combined with M or FEN, remained additive. The results suggest that during morphine-tolerance, TRM could be a useful drug to induce effective analgesia when combined with FEN or M.     

Electrophysiological characterization of 14-benzoyltalatisamine, a selective blocker of the delayed rectifier K+ channel found in virtual screening

The objective of this study was to investigate spinal and supraspinal antinociceptive effects of a new synthetic compound, (±)-cis-(6-ethyl-tetrahydropyran-2-yl)-formic acid (tetrahydropyran derivative). Its activity was compared with those from morphine. In peripheral models of inflammation and hyperalgesia, tetrahydropyran derivative significantly reduced nociceptive effect induced by acetic acid or formalin in mice. Tetrahydropyran derivative developed antinociceptive effect on the tail-flick and hot-plate tests with a long-acting curve maintaining the effect for 4 h longer than morphine.

The opioid receptor antagonist naloxone totally reverted tetrahydropyran derivative effects on both models. Morphine as well as tetrahydropyran derivative induced tolerance and sedation in mice. However, tetrahydropyran derivative-induced tolerance had its onset retarded and the sedative activity was lower when compared to that induced by morphine. These results indicate that this new substance develops an antinociceptive activity and may be used in the future as a substitute for traditional opioids.     

Spinal antinociceptive actions and naloxone reversibility of intravenous mu- and kappa-opioids in spinalized rats: potency mismatch with values reported for spinal administration

1. The relative spinal effectiveness of mu- and kappa-opioids has been assessed by their intravenous potencies on nociceptive responses (heat and/or pinch) of single motoneurones recorded in alpha-chloralose anaesthetized, spinalized rats. 2. The depressant actions of both mu- and kappa-opioids were reversed by low intravenous doses of naloxone (10 to 100 micrograms kg-1). When tested at a dose of 1 microgram kg-1 i.v., naloxone antagonized the effects of the mu-agonist morphine but had no effect on the kappa-opioid U-50,488. This provides further support for the theory that the actions of mu- and kappa-ligands were mediated at different subclasses of opioid receptor but highlights the difficulties in using antagonists with poor receptor selectivity to differentiate between mu- and kappa-receptor-mediated effects in vivo. 3. The molar potency rations of fentanyl: morphine:U-50,488: tifluadom for thermal and mechanical nociceptive responses were 620: 1.0:0.74:5.7 and 520:1.0:0.56:7.7 respectively. These potency ratios, as well as the absolute potencies, agree well with those reported in several behavioural studies in which systemic administration of agonists was used in non-thermal tests. 4. The agonist potency values obtained in this study contrast with those reported for local spinal administration. By this route, the potency of lipophilic opioids (e.g. fentanyl, U-50,488 and tifluadom) relative to hydrophilic opioids (e.g. morphine) is much reduced, implying that activity of intrathecally administered opioids is more dependent on the physico-chemical properties of the agonists used than on the relative abundance in the spinal cord of functional opioid receptors of the mu- and kappa-subtypes. This conclusion indicates that the results with locally applied opioids should not be used to assess spinal opioid receptor function.     

Analgesic activity and pharmacological characterization of N-[1-phenylpyrazol-3-yl]-N-[1-(2-phenethyl)-4-piperidyl] propenamide, a new opioid agonist acting peripherally

We previously reported the synthesis of three new opioid agonists as well as their in vitro and in vivo activity [Girón, R., Abalo, R., Goicoechea, C., Martín, M.I., Callado, L.F., Cano, C., Goya, P., Jagerovic, N. 2002. Synthesis and opioid activity of new fentanyl analogs. Life Sci. 71, 1023-1034]. One of them, N-[1-phenylpyrazol-3-yl]-N-[1-(2-phenethyl)-4-piperidyl)] propenamide (IQMF-4), showed an interesting antinociceptive activity. Intraperitoneally (i.p.) administered, it was as effective as fentanyl or morphine, being less potent than fentanyl but more so than morphine. The aim of the present work was to evaluate its antinociceptive effect by different routes of administration, using the hot plate test, and to investigate possible side effects, such as tolerance and withdrawal, in vitro, using the myenteric plexus-longitudinal muscle strip preparation from guinea pig ileum, and in vivo, using the hot plate test. IQMF-4 was more potent than morphine when administered per os (p.o.), but less potent when administered intracerebroventricularly (i.c.v.). By both routes, fentanyl is more potent that IQMF-4. When IQMF-4 was administered i.p., naloxone methiodide, a peripherally acting antagonist, was able to completely block its antinociceptive effect, whereas, after i.c.v. administration, the blockade was only partial. An interesting feature of the new compound is that it induces tolerance in vitro but not in vivo. Moreover, though in vitro withdrawal was not different from fentanyl or morphine, in vivo withdrawal symptoms were significantly less frequent in mice treated with IQMF-4 than in those treated with morphine or fentanyl. Although more assays are required, these results show that IQMF-4 appears to be a potent analgesic compound with an interesting peripheral component, and reduced ability to induce dependence.     

Differences in tolerance to anti-hyperalgesic effects between chronic treatment with morphine and fentanyl under a state of pain.

The present study was undertaken to investigate the possible change in anti-hyperalgesic effect following repeated treatment with morphine or fentanyl using the dose to improve the thermal hyperalgesia under an inflammatory pain-like state. The anti-hyperalgesic effect induced by fentanyl in complete Freund's adjuvant (CFA)-pretreated mice rapidly disappeared during the consecutive administration of fentanyl, whereas morphine preserved its potency of anti-hyperalgesic effect. In addition, repeated treatment with fentanyl, but not morphine, resulted in the increase in levels of phosphorylated-mciro-opioid receptor (MOR) associated with the enhanced inactivation of protein phosphatase 2A and the reduction in Rab4-dependent MOR resensitization. Next, we investigated the specific involvement of the opioid receptor types and MOR subtypes in analgesic properties of morphine and fentanyl in the mouse spinal cord. In the competitive displacement binding assay with [1H]DAMGO, no significant difference in the binding affinity to MOR between morphine and fentanyl was noted in membranes obtained from the mouse spinal cord. Furthermore, there was no significant difference between morphine and fentanyl in either antinociceptive effect or G-protein activation in mice partially lacking MOR-1B, which shows a greater resistance to agonist-induced desensitization than for other MOR subtypes. These findings point out the possibility that the chronic treatment with fentanyl may cause the different modulation from chronic treatment with morphine on either the internalization or resensitization of MORs in the spinal cord under a pain-like state. The present data provide the first evidence for the mechanism underlying the development of tolerance to fentanyl-induced anti-hyperalgesic effect under chronic pain.     

Time course of the enhancement and restoration of the analgesic efficacy of codeine and morphine by delta9-tetrahydrocannabinol

Delta9-tetrahydrocannabinol (delta9-THC) synergizes with morphine and codeine by releasing endogenous opioids. These studies determined 1) the duration of enhancement of morphine and codeine by delta9-THC, 2) the effect of (delta9-THC on the time course of fully efficacious doses of the opioids, 3) restoration of efficacy of morphine and codeine by delta9-THC, and 4) duration of restoration. Sub-active combination doses of delta9-THC/morphine or delta9-THC/codeine are equivalent in duration of action and efficacy to high-dose opioids alone. Delta9-THC (20 mg/kg p.o.) significantly restores the antinociceptive effects of both high-dose morphine and codeine (100 and 200 mg/kg p.o., respectively) at later time points at which morphine or codeine was no longer active (360- and 120-min post-administration, respectively). Thus, the cannabinoid/opioid combination might be useful in therapeutics to enhance opioid activity, as well as to restore the efficacy of opioids.     

Receptor-selective antagonism of opioid antinociception in female versus male rats

This study was conducted to determine whether sex differences in opioid antinociception may be explained by sex differences in opioid receptor activation. The time course, dose-effect and selectivity of antagonists that have been previously shown to be relatively mu (beta-funaltrexamine, beta-FNA), kappa (norbinaltorphimine, norBNI), or delta (naltrindole, NTI) receptor selective in male animals were compared in female and male Sprague-Dawley rats using a 52 degrees C hotplate test. In both sexes, beta-FNA (10 or 20 microg intracerebroventricularly [i.c.v.]) dose-dependently blocked the antinociceptive effects of fentanyl (0.056 mg/kg subcutaneously); antagonism was observed 24 h after beta-FNA, and diminished within 7-14 days. In both sexes, norBNI (1 or 10 microg i.c.v.) dose-dependently blocked the antinociceptive effects of U69,593 (1.0 mg/kg subcutaneously); antagonism was maximal by 1-3 days post-norBNI and lasted longer than 56 days. NTI (1 or 10 microg i.c.v.) dose-dependently blocked the antinociceptive effects of [D-Pen2, D-Pen5]enkephalin (DPDPE, 100 nmol i.c.v.) in both sexes; however, the duration of action of NTI was shorter in females than in males. The antinociceptive effects of the mu receptor-preferring agonists fentanyl, morphine and buprenorphine were significantly and dose-dependently antagonized by beta-FNA, but not by norBNI or NTI, in both sexes. Beta-FNA antagonism was significantly greater in females compared with males given morphine, but not fentanyl or buprenorphine. The antinociceptive effects of the kappa receptor-preferring agonists U69,593 and U50,488 were significantly and dose-dependently antagonized by norBNI; U50,488 but not U69,593 was also antagonized to a lesser extent by NTI and beta-FNA, in both sexes. The antinociceptive effect of the delta receptor-preferring agonist SNC 80 was significantly antagonized by NTI, but not by norBNI or beta-FNA, in both sexes. The sex difference in beta-FNA antagonism of morphine suggests that there may be sex differences in functional mu opioid receptor reserve or signal transduction; however, the lack of consistency across all mu agonists weakens this hypothesis. Overall, the opioids tested had very similar receptor selectivity in male and female subjects.     

Synergistic antinociceptive actions and tolerance development produced by morphine-fentanyl coadministration: correlation with μ-opioid receptor internalization

It has been described that coadministration of opioids with low doses of other analgesics can reduce adverse effects and increase antinociception, but combinations of two μ-opioid receptor agonists have been poorly explored. The objective of this work was threefold: 1) to evaluate the antinociceptive combination of i.c.v. morphine and fentanyl at different doses; 2) to compare the antinociception produced by acute or repeated administration of an effective morphine dose (1 μg) alone, or combined with a low fentanyl dose (1 ng); and 3) to correlate these effects with μ-opioid receptor internalization in periaqueductal gray matter and locus coeruleus. Antinociception was evaluated by the tail-flick test and receptor internalization was analyzed by confocal microscopy in Wistar rats. Drug interactions were examined by administering combinations of opioids in 1:3, 1:1 and 3:1 ratios of their respective ED(50) fractions. For tolerance and internalization studies, animals were i.c.v. injected only once (acute treatment) or twice a day until five administrations were completed. Our results show that morphine and fentanyl have synergistic effects. The combination of 1 ng fentanyl with 1 μg morphine increases the magnitude and duration of antinociception not only after a single injection, but also after five administrations when tolerance develops to morphine alone. Increased and long-lasting antinociception correlates positively with increased β-arrestin 2 activity and μ-opioid receptor internalization in periaqueductal gray matter and locus coeruleus. These results suggest that combined administration of morphine and fentanyl increases long-lasting antinociception and β-arrestin 2 signaling contributes to the combination effects.