Selective Kappa-opioid Agonist Research Articles

Electrophysiological effects of kappa-opioid analgesic, RU-1205, using machine learning methods

The study is focused to the investigation of a new kappa-opioid agonist RU-1205, which exhibits an analgesic effect without causing dysphoric or aversive actions. It is assumed that this effects may be due to its functional selectivity, or the presence of an additional mechanism of action that involves blocking p38 mitogen-activated protein kinase (MAPK).The aim of the study was an experimental identification of RU-1205 mechanisms of action associated with the inhibition of MAPK p38 and functional selectivity for kappa opioid receptors.Materials and methods. The LFP activity was recorded in the male rats weighing 260–280 g (n=62) and implanted with chronic cortical and deep electrodes, after the intracerebroventricular administration of the well-studied reference substances: the selective kappa-opioid agonist U-50488 100 μg; the MAPK p38 blocker SB203580 1 μg; and the investigational compound RU-1205 at 350 μg. The weighted phase lag index (WPLI) was calculated. Subsequently, machine learning methods were employed to reduce the dimensionality and extract connectivity features using the principal component analysis method, then a signal classification was performed (models based on Gaussian processes). Using the local patch-clamp technique in the “whole-cell” configuration, the spike activity of pyramidal neurons in the basolateral amygdala was studied. Neurons were identified by their accommodation properties. After local perfusion of the test compounds, 3 dose-response curves were obtained for: (1) U-50488 at concentrations ranging from 0.001 to 10 μM; (2) combinations of U-50488 (0.001–10 μM) and RU-1205 (10 μM); and (3) the combinations of U-50488 (0.01–10 μM) and RU-1205 (100 μM).Results. The developed models made it possible to classify the compound RU-1205 as a “non-inhibitor” of MAPK p38 with a high probability. The results obtained were confirmed in patch-clamp experiments on acute brain slices where it was demonstrated that U-50488 statistically significantly increases the spike activity of pyramidal neurons of the basolateral amygdala (p <0.05), and RU-1205 interacts with U-50488, competitively suppressing its effect on the spike activity of neurons.Conclusion. The findings suggest that compound RU-1205 displays properties consistent with a functional kappa agonist activity and does not have a significant effect on MAPK p38. The study demonstrates the possibility of integrating electrophysiological measurements and advanced data analysis methods for a deep understanding of drug action and underscores the potential for further research in this area.

Effect of adjuvant drugs on the analgesic activity of opioid morphine analgesics and compound RU-1205

Introduction: Adjuvant medications can be used to increase the analgesic effect of opioid analgesics, reduce the manifestation of side effects, and also for premedication. This paper provides information on the effect of clonidine, haloperidol, metocloparmide, diazepam, midazolam on opioid analgesics: - morphine and the selective kappa-opioid agonist compound RU-1205.
 Materials and methods: A probable interaction between RU-1205, morphine and adjuvant drugs in pain behaviors was carried out on the model of somatogenic pain. 95 male mice received either RU-1205 (5 mg/kg, i.p.) and morphine (1 mg/kg, i.p.) separately or in combination with haloperidol (0.45 mg/kg, i.p.); midazolam (0.3 mg/kg, i.p.); diazepam (1 mg/kg, i.p.); metoclopramide (5 mg/kg, i.p.), and clonidine (1 mg/kg, i.p.). The analgesic effect was assessed by tail flick test. Registration of the latent period of the reaction was carried out 30, 60 and 90 minutes after the adjuvant drug administration.
 Results: When studying the interaction with morphine, it was found that clonidine, haloperidol and metoclopramide enhanced the effects; diazepam offset them, and midazolam had no affect on the analgesic properties. In the course of the studies, RU-1205 showed an increase in analgesic activity when combined with clonidine, a slight increase with midazolam, and a decrease when co-administered with diazepam. Haloperidol had no influence on the effect of RU-1205, while metoclopramide both potentiated and reduced the analgesic effect.
 Discussion: Pharmacodynamic and pharmacokinetic interactions of RU-1205 with an α2AR agonist, benzodiazepine receptor agonists, D2P antagonist, and σ-receptor blocker were established.
 Conclusion: The presented data make it possible to more accurately formulate ideas about the localization and action mechanism of the kappa-agonist of opioid receptors, the compound RU-1205.

Evaluation of Antinociceptive Effects of Chitosan-Coated Liposomes Entrapping the Selective Kappa Opioid Receptor Agonist U50,488 in Mice.

Background and Objectives: The selective kappa opioid receptor agonist U50,488 was reported to have analgesic, cough suppressant, diuretic and other beneficial properties. The aim of our study was to analyze the effects of some original chitosan-coated liposomes entrapping U50,488 in somatic and visceral nociceptive sensitivity in mice. Materials and Methods: The influence on the somatic pain was assessed using a tail flick test by counting the tail reactivity to thermal noxious stimulation. The nociceptive visceral estimation was performed using the writhing test in order to evaluate the behavioral manifestations occurring as a reaction to the chemical noxious peritoneal irritation with 0.6% acetic acid (10 mL/kbw). The animals were treated orally, at the same time, with a single dose of: distilled water 0.1 mL/10 gbw; 50 mg/kbw U50,488; 50 mg/kbw U50,488 entrapped in chitosan-coated liposomes, according to the group they were randomly assigned. Results: The use of chitosan-coated liposomesas carriers for U50,488 induced antinociceptive effects that began to manifest after 2 h, andwere prolonged but with a lower intensity than those caused by the free selective kappa opioid in both tests. Conclusion: In this experimental model, the oral administration of nanovesicles containing the selective kappa opioid agonist U50,488 determined a prolonged analgesic outcome in the tail flick test, as well as in the writhing test.

Ligand/kappa-opioid receptor interactions: Insights from the X-ray crystal structure

During the past five years, the three-dimensional structures of 14 different G-protein coupled receptors (GPCRs) have been resolved by X-ray crystallography. The most recently published structures, those of the opioid receptors (ORs), are remarkably important in pain modulation, drug addiction, and mood disorders. These structures, confirmed previously proposed key interactions conferring potency and antagonistic properties, including the well–known interaction with Asp138, conserved in all aminergic GPCRs. In addition, crystallization of the opioid receptors highlighted the potential function of the ECL2 and ICL2 loops. We have previously reported a set of potent and selective kappa opioid receptor peptide agonists, of which ff(D-nle)r-NH2 is among the most potent and selective ones. These peptides were identified from the deconvolution of a 6,250,000 tetrapeptide combinatorial library. A derivative of this set is currently the subject of a phase 2 clinical trial in the United States. In this work, we describe comparative molecular modeling studies of kappa-OR peptide agonists with the co-crystallized antagonist, JDTic, and also report structure–activity relationships of 23 tetrapeptides. The overall binding and contact interactions are sound and interactions known to favor selectivity and potency were observed. Additional modeling studies will reveal conformational changes that the kappa-OR undergoes upon binding to these peptide agonists.

LC-MS/MS quantification of salvinorin A from biological fluids

A facile method for quantifying the concentration of the powerful and widely available hallucinogen salvinorin A (a selective kappa opioid agonist) from non-human primate cerebrospinal fluid (CSF) and human plasma has been developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive electrospray ionization (ESI) mode. With CSF solid phase extraction can be avoided completely by simply diluting each sample to 10 % (v/v) acetonitrile, 1 % (v/v) formic acid and injecting under high aqueous conditions for analyte focusing. Extensive plasma sample preparation was investigated including protein precipitation, SPE column selection, and plasma particulate removal. Human plasma samples were centrifuged at 21,000 × gravity for 4 minutes to obtain clear particulate-free plasma, from which 300 μl was spiked with internal standard and loaded onto a C18 SPE column with a 100 mg mL-1 loading capacity. Guard columns (C18, hand packed 1 mm × 20 mm) were exchanged after backpressure increased above 4600psi, about 250 injections. A shallow acetonitrile/water gradient was used, 29 to 33% CH3CN over 8 minutes to elute salvinorin A. Reduction of chemical noise was achieved using tandem mass spectrometry with multiple reaction monitoring while sensitivity increases were observed using a 50 μL injection volume onto a small bore analytical column (C18, 1 mm ID × 50 mm) thus increasing peak concentration. Limits of quantification were found to be 0.0125 ng mL-1 (CSF) and 0.05 ng mL-1 (plasma) with interday precision and accuracy below 1.7 % and 9.42 % (CSF) and 3.47 % and 12.37 % (plasma) respectively. This method was used to determine the concentration of salvinorin A from an in vivo Rhesus monkey study and a trial of healthy human research participants, using behaviorally active doses.

The effects of the selective kappa-opioid agonist MR 2034 on the guinea-pig ileum.

The effects of the selective kappa-opioid agonist MR 2034 on the guinea-pig ileum were compared with those produced by the mu-agonist morphine. MR 2034 induced acute tolerance and inhibited electrically evoked contractions to the same extent as morphine. The slope of the concentration-effect curve to MR 2034 was not significantly different from that of morphine. However, MR 2034 and morphine may act at their respective receptors since naloxone was 6.57 times more effective at inhibiting responses to morphine than MR 2034. MR 2034, like morphine, induced acute dependence as revealed by naloxone-induced contractions of tissues that had been incubated with MR 2034 for 5 h. The functional properties of kappa-opoid receptors are the same as mu-receptors in the guinea-pig ileum.

Comparison of the discriminative stimulus effects of salvinorin A and its derivatives to U69,593 and U50,488 in rats

Research interests regarding the psychopharmacology of salvinorin A have been motivated by the recreational use and widespread media focus on the hallucinogenic plant, Salvia divinorum. Additionally, kappa opioid (KOP) receptor ligands may have therapeutic potential in the treatment of some neuropsychiatric conditions, including drug dependence and mood disorders. Salvinorin A is a selective KOP agonist, but only a few studies have explored the discriminative stimulus effects of this compound. This study compared the discriminative stimulus effects of salvinorin A and two synthetic derivatives of salvinorin B to the KOP agonists, U69,593 and U50,488. Sixteen male Sprague-Dawley rats trained to discriminate U69,593 (0.13 mg/kg, s.c., N = 8) or U50,488 (3.0 mg/kg, i.p., N = 8) under a fixed-ratio 20 schedule of food reinforcement were administered substitution tests with salvinorin A (0.125-3.0 mg/kg, i.p.). The animals trained to discriminate U69,593 were also administered substitution tests with salvinorin B ethoxymethyl ether (0.005-0.10 mg/kg, i.p.) and salvinorin B methoxymethyl ether (0.03-0.10 mg/kg, i.p.). Another eight rats were trained to discriminate 2.0 mg/kg salvinorin A and tested with U69,593 (0.04-0.32 mg/kg) and U50,488 (0.4-3.2 mg/kg). Salvinorin A and both synthetic derivatives of salvinorin B substituted completely for U69,593. Additionally, cross-generalization was observed between salvinorin A and both KOP agonists. These findings support previous reports indicating that the discriminative stimulus effects of salvinorin A are mediated by kappa receptors. Future studies may assist in the development and screening of salvinorin A analogs for potential pharmacotherapy.

Crucial role of peripheral κ‐opioid receptors in a model of periodontal disease in rats

Periodontal disease is a chronic inflammatory condition of the tooth supporting tissues, the periodontium. Opioids have been shown to account for the relief of various chronic and acute inflammatory conditions. The aim of the present study was to investigate the participation of peripheral opioid receptors in development of periodontal disease. Morphine and selective agonists and antagonists of opioid receptors were used in an experimental model of ligature-induced periodontal disease in rats. To evaluate the development of disease, the loss of fiber attachment, alveolar bone and number of cells in periodontal tissues were assessed. Measurements of these indicators were obtained by morphometric analysis of histological sections of periodontal-diseased tissues stained with hematoxylin and eosin. Local administration of either morphine or a selective kappa-opioid agonist for three consecutive days from the onset of periodontal disease reduced the loss of periodontal tissues, without changing the number of leukocytes in inflamed periodontium. Nor-binaltorphimine, a selective kappa-antagonist, reversed the beneficial effects of both morphine and the compound U-50,488 in this model. The use of either an agonist or an antagonist of delta-opioid receptors, however, did not affect disease progression. Our results showed that the beneficial effect of opioids in periodontal disease depended mainly on the activation of specific kappa-opioid receptors located in the periphery. Activation of such receptors could be considered in the management of periodontal disease, since it would not present the classical central side-effects associated with opioid use.

Total Synthesis of the Hallucinogenic Neoclerodane Diterpenoid Salvinorin A

Total synthesis of salvinorin A (1), a neoclerodane diterpenoid having the most potent hallucinogenic activity and a selective kappa-opioid agonist, was completed in 20 steps starting from enantiomerically pure hydroxy-Wieland-Miescher ketone 5.

Opposing effects of intra-nucleus accumbens mu and kappa opioid agonists on sensory specific satiety

Mu opioid (MOP) agonists acting in the nucleus accumbens (NAcc) robustly enhance consumption of palatable foods. In addition, the effect on consumption of palatable foods produced by MOP agonists acting in the NAcc depends on both recent flavor exposure and the availability of a choice between different-flavored foods. In contrast, kappa opioid (KOP) agonists have variable effects on feeding and KOP agonists have MOP opposing behavioral actions when microinjected at several brain sites. We previously demonstrated that NAcc MOP agonists reverse the devaluation (satiety) effect of pre-feeding for a given flavor; in fact, NAcc MOP agonists selectively increase consumption of a recently sampled food. In contrast, in the present study, we found that the selective KOP agonist U50488 injected into the NAcc of rats reduced consumption of a recently sampled flavor while increasing consumption of the flavor that was not pre-fed. Intra-NAcc U50488 did not affect overall consumption or flavor preference in the absence of pre-feeding. The present data, in conjunction with our previous findings, highlight the robust and opposing role of NAcc MOP and KOP opioid receptors in palatability-based food choice and consumption and raise the possibility that an endogenous KOP agonist acting in the NAcc contributes to the phenomenon of sensory specific satiety.

ICER and c‐Fos expression in the hindbrain following central administration of U‐50488 (U‐50), a selective kappa opioid agonist

Central kappa opioid administration produces a marked increase in renal sympathetic nerve activity with a concurrent water diuresis in conscious rats. Because kappa opioid agonists produce their neuronal inhibitory effects in part via the inhibition of cAMP through Gi/o, they may also change the expression of endogenous transcriptions factors involved in suppression of cAMP regulated transcription factors. Thus, inducible cAMP early repressor (ICER) staining may serve as a neuronal marker for the activation of 2nd messenger systems that suppress cAMP regulated genes. The present study examines the effects of intracerebroventricular (ICV) U-50 (10 μg/5μl) on ICER and c-Fos staining in several hindbrain regions that express kappa binding sites and are also involved in sympathetic outflow. Alternate sets of brainstem sections were processed for ICER using polyclonal anti-ICER antibody raised in rabbit and c-Fos using a commercially available antibody. Sections were also double labeled with DBH to anatomically define these regions. ICV U-50 produced significant increases in ICER staining in the medial (VEH, 8 ± 4; U50 24 ± 4; P < .001) and anterior (VEH, 3 ± 3; U50 12 ± 3; P < .05) nucleus of the solitary tract, area postrema (VEH, 2.1± 1.9; U-50 41 ± 14; P < .001), and locus coeruleus (VEH, 1.3± .7; U-50 23± 10; P < .001). In contrast to ICER, c-Fos expression only decreased in the area postrema without any significant changes in the other hindbrain regions. Together, central kappa opioid receptor mediated responses in these hindbrain regions may contribute to the central effects of U-50. Supported by NIH HL62579 & DK57822.

Depressive-Like Effects of the κ-Opioid Receptor Agonist Salvinorin A on Behavior and Neurochemistry in Rats

Endogenous opioids seem to play a critical role in the regulation of mood states. For example, there is accumulating evidence that stimulation of kappa-opioid receptors, upon which the endogenous opioid dynorphin acts, can produce depressive-like behaviors in laboratory animals. Here we examined whether systemic administration of salvinorin A (SalvA), a potent and highly selective kappa-opioid agonist, would produce depressive-like effects in the forced swim test (FST) and intracranial self-stimulation (ICSS) test, which are behavioral models often used to study depression in rats. We extracted, isolated, and purified SalvA from Salvia divinorum plant leaves and examined its effects on behavior in the FST and ICSS test across a range of doses (0.125-2.0 mg/kg) after systemic (intraperitoneal) administration. SalvA dose dependently increased immobility in the FST, an effect opposite to that of standard antidepressant drugs. Doses of SalvA that produced these effects in the FST did not affect locomotor activity in an open field. Furthermore, SalvA dose dependently elevated ICSS thresholds, an effect similar to that produced by treatments that cause depressive symptoms in humans. At a dose that caused the depressive-like effects in both the FST and ICSS assays, SalvA decreased extracellular concentrations of dopamine (DA) within the nucleus accumbens (NAc), a critical component of brain reward circuitry, without affecting extracellular concentrations of serotonin (5-HT). These data provide additional support for the hypothesis that stimulation of brain kappa-opioid receptors triggers depressive-like signs in rats and raise the possibility that decreases in extracellular concentrations of DA within the NAc contribute to these effects.

Differential time course of effects of kappa-opioid agonist treatment on dynorphin A levels and kappa-opioid receptor density.

The effects of kappa-opioid agonist treatment on kappa-opioid receptor density and on dynorphin A levels in the rat brain were studied. Rats were treated with the selective kappa-opioid agonist U-69593 or vehicle for 5 d. Dynorphin A levels and kappa-opioid receptor binding were measured on day 8 (3 d after the last injection) or 22 (17 d after the last injection). On day 8, kappa-opioid receptor density was increased in the hypothalamus of rats treated with U-69593; there were no changes in the frontal cortex or caudate putamen. In contrast, there was an increase in dynorphin A levels in the frontal cortex and no changes in hypothalamus and caudate putamen in response to U-69593. On day 22, Bmax was decreased in frontal cortex and caudate putamen of U-69593-treated rats, whereas dynorphin A levels were increased in the caudate putamen and in the frontal cortex. These findings suggest that kappa-opioid receptor agonist treatment has long-term, continually changing effects on the kappa-opioid system.

Effect of acute and chronic administration of U50,488, a kappa opioid receptor agonist, in 6-OHDA-lesioned rats chronically treated with levodopa

To evaluate the possible involvement of kappa opioid receptor-mediated mechanisms in levodopa-induced motor fluctuations, we have investigated the effects of U50,488, a selective kappa opioid agonist, on levodopa-induced motor alterations in rats with unilateral 6-OHDA lesion. Acute and chronic administration of U50,488 has been studied to evaluate the possible reversion or prevention of these levodopa effects. In a first set of experiments, rats were treated with levodopa (25 mg/kg with benserazide, twice daily, ip) for 22 days and, on Day 23 U50,488 (0.5, 1, or 3 mg/kg, ip) was administered immediately before levodopa. In a second set of experiments, rats were treated daily for 22 days with levodopa and U50,488 (1 or 3 mg/kg/day, ip). The duration of the rotational behavior induced by chronic levodopa decreased after 22 days (P < 0.05). Acute administration of U50,488 on Day 23 reversed this effect when low doses were administered (P < 0.05). Chronic U50,488 administration did not prevent the shortening in response duration induced by levodopa. Our results demonstrate that the kappa opioid receptor agonist U50,488 reverses but does not prevents levodopa-induced motor alterations in parkinsonian rats. These results suggest a role for kappa opioid receptor-mediated mechanisms in the pathophysiology of levodopa-induced motor response complications. These findings suggest that the stimulation of kappa opioid receptors might confer clinical benefit to parkinsonian patients under levodopa therapy suffering from motor complication syndrome.

Regulation of dynorphin gene expression by kappa-opioid agonist treatment.

The effects of K-opioid agonist treatment on prodynorphin mRNA expression in the rat brain were studied. Rats were treated with the selective kappa-opioid agonist U-69593 or vehicle for 5 days and prodynorphin mRNA was measured on day 8 (3 days after the last injection) or 22 (17 days after the last injection). On day 8 prodynorphin mRNA was increased in the hypothalamus and decreased in the striatum, frontal cortex, and hippocampus of rats treated with U-69593. On day 22, prodynorphin mRNA was increased in the hypothalamus, frontal cortex and striatum of U-69593 treated rats. These findings suggests that kappa-opioid receptor agonist treatment has long-term, continually changing effects on prodynorphin mRNA expression.

Effects of gonadal steroid hormone treatments on opioid antinociception in ovariectomized rhesus monkeys.

Gonadal steroid hormones altered opioid antinociception under some conditions in rodents, and we reported previously that chronic estradiol enhanced kappa but not mu opioid antinociception in ovariectomized rhesus monkeys. Sex differences have also been observed in the antinociceptive effects of opioid agonists. These findings suggest that gonadal hormones may modulate opioid antinociception. To extend our previous studies of estradiol by examining the effects of progesterone alone, estradiol in combination with progesterone, and testosterone alone on opioid antinociception in ovariectomized rhesus monkeys. Opioid effects were studied during chronic treatment with vehicle (sesame oil) or with progesterone alone (P; 0.32 mg/kg per day), a combination of progesterone+estradiol (P+E; 0.32 mg/kg per day P + 0.002 mg/kg per day E), or testosterone alone (T; 0.32 mg/kg per day). Opioid antinociception in a warm-water tail-withdrawal procedure was examined with the selective kappa opioid agonist U50,488, the selective mu agonist morphine, and the two mixed-action opioids butorphanol and nalbuphine. The steroid treatment regimens produced physiological levels of progesterone and estradiol similar to peak levels observed during the luteal phase of the menstrual cycle and physiological levels of testosterone similar to those observed in intact males. Treatment with P, P+E, or T did not alter baseline thermal nociception. P+E significantly increased the potency of U50,488 at 50 degrees C but not at 54 degrees C. Gonadal hormone treatments had little or no effect on antinociception produced by morphine, butorphanol, or nalbuphine at either temperature. These findings further suggest that chronic treatment with physiological levels of gonadal hormones may modulate the antinociceptive effects of U50,488 in ovariectomized rhesus monkeys.

Kappa opioid agonists alter dopamine markers and cocaine-stimulated locomotor activity.

To better understand the influence of kappa-opioid agonists on the effects of cocaine, rats were treated with daily injections of the selective kappa-opioid agonist U-69593 or bremazocine. In combination with 10 mg/kg cocaine, both compounds, at a dose of 0.32 mg/kg, greatly diminished locomotor activity, and these effects were maintained over a period of 5 days. In addition, the response to a challenge injection of 10 mg/kg cocaine several days after the end of kappa-Opioid agonist treatment with or without cocaine was markedly reduced. When naltrexone was given in combination with U-69593, it blocked the reduction in cocaine-induced locomotor activity after U-69593 treatment alone. However, a single injection of either kappa-opioid agonist alone had no effect on cocaine-induced locomotion several days later (i.e. no long-term effects), suggesting that multiple injections of the kappa-opioid agonist are needed to reduce the locomotor activating effects of cocaine other than acutely. In addition, treatment with the kappa-opioid agonist U-69593 (0.32 mg/kg) over a 5-day period decreased dopamine transporter densities in the caudate putamen, and this was also blocked by co-administration of naltrexone.

Sex Differences in Brainstem Neural Activation after Injury to the TMJ Region

The basis for a higher prevalence of painful temporomandibular disorders (TMD) among women than men is not known. The present study used Fos-like immunoreactivity (Fos-LI) to quantify the pattern and magnitude of neural activation within the trigeminal brainstem complex of male and female rats caused by acute inflammatory injury to the temporomandibular joint (TMJ) region. Also, Fos-LI was assessed in animals given morphine, a preferential mu opioid receptor agonist, or U50,488H, a selective kappa opioid agonist, prior to TMJ injury to determine if opioid modulation of neural activation was similar in males and females. The general pattern of Fos-LI after TMJ injury was similar in males and females. This pattern was characterized by a high density of Fos-positive neurons in the dorsal paratrigeminal nucleus (dPa5), subnucleus interpolaris/caudalis transition region (Vi/Vc-vl), and in the superficial laminae at the subnucleus caudalis/upper cervical spinal cord (Vc/C2) junction ipsilateral to TMJ injury. In contrast to other regions the number of Fos-positive neurons produced at the Vc/C2 junction was proportional to the concentration of mustard oil injected into the TMJ region. In addition, proestrus females produced higher levels of Fos-LI at the Vc/C2 junction than diestrus females or males. Morphine caused a greater dose-related reduction in Fos-LI at the dPa5 and Vc/C2 junction in males than females. By contrast, U50,488H caused a dose-related reduction in Fos-LI only at the Vc/C2 junction of proestrus females. These results support the hypothesis that the Vc/C2 junction region plays a critical role in the integration of pain signals originating from the TMJ region and may underlie sex differences in sensory processing related to TMJ pain.

Effect of opioid active therapeutics on the ascending reflex pathway in the rat ileum

For a long time therapeutic agents that interact with opioid receptors have been used in antidiarrheal therapy. The action of the opioid active substances on motility and transit have already been characterized; however, their effects on myenteric reflexes and their possible luminal action have not yet been investigated. Loperamide, fedotozine and β-casomorphin-4, as well as the casomorphin-analogue β-CM-4027, are, or have been, suggested as therapeutic agents and were studied in the isolated rat ileum for their effect on the ascending reflex pathway. β-CM-4027 > fedotozine > loperamide > β-casomorphin-4 caused a concentration-dependent inhibition of the ascending contractile reflex response with an IC50of 1.4×10–7M, 1.5×10–6M, 4.1×10–6M and 4.5×10–6M respectively. At the same time as the oral contractile reflex response was inhibited, all four opioid agonists (CM-4027 > β-casomorphin-4 > fedotozine > loperamide) increased the latency of the reflex response. Both effects were blocked by naloxone, indicating the involvement of opioid receptors. These results demonstrate that opioid-active drugs and substances modify the peristaltic reflex by reducing the efficacy of the reflex response and modulating the timing of the reflex pathway. In a second series of experiments, luminal application of opioid-active drugs was compared with serosal application. β-casomorphine-4 caused a concentration-dependent inhibition of the oral reflex response with an IC50of 3×10–3M which was 750 times higher than after serosal application. In contrast, a stable and highly selective kappa opioid agonist (U-50,488), which caused potent inhibition upon serosal application (IC50: 2.3×10–7M), showed no inhibitory effect after luminal application up to a concentration of 10–2M. Thus casomorphins could have a local effect on the gut wall with no need for systemic absorption. This might be used for a possible therapeutic application.

Reduction of stress-induced analgesia but not of exogenous opioid effects in mice lacking CB1 receptors.

CB1 cannabinoid receptors are widely distributed in the central nervous system where they mediate most of the cannabinoid-induced responses. Here we have evaluated the interactions between the CB1 cannabinoid receptors and the endogenous opioid system by assaying a number of well-characterized opioid responses, e.g. antinociception and stress-mediated effects, on mutant mice in which the CB1 receptor gene was invalidated. The spontaneous responses to various nociceptive stimuli (thermal, mechanical and visceral pain) were not changed in mutant CB1 mice. Furthermore, the absence of the CB1 cannabinoid receptor did not modify the antinociceptive effects induced by different opioid agonists: morphine (preferential mu opioid agonist), D-Pen2-D-Pen5-enkephalin (DPDPE) and deltorphin II (selective delta opioid agonists), and U-50,488H (selective kappa opioid agonist) in the hot-plate and tail-immersion tests. In contrast, the stress-induced opioid mediated responses were modified in CB1 mutants. Indeed, these mutants did not exhibit antinociception following a forced swim in water at 34 degrees C and presented a decrease in the immobility induced by the previous exposure to electric footshock. However, the antinociception induced by a forced swim in water at 10 degrees C was preserved in CB1 mutants. These results indicate that CB1 receptors are not involved in the antinociceptive responses to exogenous opioids, but that a physiological interaction between the opioid and cannabinoid systems is necessary to allow the development of opioid-mediated responses to stress.