The Endocannabinoid System Alleviates Pain in a Murine Model of Cancer-Induced Bone Pain.

Metastatic breast cancer is prevalent worldwide, and one of the most common sites of metastasis is long bones. Of patients with disease, the major symptom is pain, yet current medications fail to adequately result in analgesic efficacy and present major undesirable adverse effects. In our study, we investigate the potential of a novel monoacylglycerol lipase (MAGL) inhibitor, MJN110, in a murine model of cancer-induced bone pain. Literature has previously demonstrated that MAGL inhibitors function to increase the endogenous concentrations of 2-arachydonylglycerol, which then activates CB1 and CB2 receptors to inhibit inflammation and pain. We demonstrate that administration of MJN110 significantly and dose dependently alleviates spontaneous pain behavior during acute administration compared with vehicle control. In addition, MJN110 maintains its efficacy in a chronic-dosing paradigm over the course of 7 days without signs of receptor sensitization. In vitro analysis of MJN110 demonstrated a dose-dependent and significant decrease in cell viability and proliferation of 66.1 breast adenocarcinoma cells to a greater extent than KML29, an alternate MAGL inhibitor, or the CB2 agonist JWH015. Chronic administration of the compound did not appear to affect tumor burden, as evidenced by radiograph or histologic analysis. Together, these data support the application for MJN110 as a novel therapeutic for cancer-induced bone pain. SIGNIFICANCE STATEMENT: Current standard of care for metastatic breast cancer pain is opioid-based therapies with adjunctive chemotherapy, which have highly addictive and other deleterious side effects. The need for effective, non-opioid-based therapies is essential, and harnessing the endogenous cannabinoid system is proving to be a new target to treat various types of pain conditions. We present a novel drug targeting the endogenous cannabinoid system that is effective at reducing pain in a mouse model of metastatic breast cancer to bone.

Genetic and pharmacological antagonism of NK1 receptor prevents opiate abuse potential.

Development of an efficacious, non-addicting analgesic has been challenging. Discovery of novel mechanisms underlying addiction may present a solution. Here we target the neurokinin system, which is involved in both pain and addiction. Morphine exerts its rewarding actions, at least in part, by inhibiting GABAergic input onto substance P (SP) neurons in the ventral tegmental area (VTA), subsequently increasing SP release onto dopaminergic neurons. Genome editing of the neurokinin 1 receptor (NK1R) in the VTA renders morphine non-rewarding. Complementing our genetic approach, we demonstrate utility of a bivalent pharmacophore with dual activity as a µ/δ opioid agonist and NK1R antagonist in inhibiting nociception in an animal model of acute pain while lacking any positive reinforcement. These data indicate that dual targeting of the dopaminergic reward circuitry and pain pathways with a multifunctional opioid agonist-NK1R antagonist may be an efficacious strategy in developing future analgesics that lack abuse potential.

Cancer-induced bone pain: Mechanisms and models.

Cancerous cells can originate in a number of different tissues such as prostate, breast and lung, but often go undetected and are non-painful. Many types of cancers have a propensity to metastasize to the bone microenvironment first. Tumor burden within the bone causes excruciating breakthrough pain with properties of ongoing pain that is inadequately managed with current analgesics. Part of this failure is due to the poor understanding of the etiology of cancer pain. Animal models of cancer-induced bone pain (CIBP) have revealed that the neurochemistry of cancer has features distinctive from other chronic pain states. For example, preclinical models of metastatic cancer often result in the positive modulation of neurotrophins, such as NGF and BDNF, that can lead to nociceptive sensitization. Preclinical cancer models also demonstrate nociceptive neuronal expression of acid-sensing receptors, such as ASIC1 and TRPV1, which respond to cancer-induced acidity within the bone. CIBP is correlated with a significant increase in pro-inflammatory mediators acting peripherally and centrally, contributing to neuronal hypersensitive states. Finally, cancer cells generate high levels of oxidative molecules that are thought to increase extracellular glutamate concentrations, thus activating primary afferent neurons. Knowledge of the unique neuro-molecular profile of cancer pain will ultimately lead to the development of novel and superior therapeutics for CIBP.

Building a better analgesic: multifunctional compounds that address injury-induced pathology to enhance analgesic efficacy while eliminating unwanted side effects.

The most highly abused prescription drugs are opioids used for the treatment of pain. Physician-reported drug-seeking behavior has resulted in a significant health concern among doctors trying to adequately treat pain while limiting the misuse or diversion of pain medications. In addition to abuse liability, opioid use is associated with unwanted side effects that complicate pain management, including opioid-induced emesis and constipation. This has resulted in restricting long-term doses of opioids and inadequate treatment of both acute and chronic debilitating pain, demonstrating a compelling need for novel analgesics. Recent reports indicate that adaptations in endogenous substance P/neurokinin-1 receptor (NK1) are induced by chronic pain and sustained opioid exposure, and these changes may contribute to processes responsible for opioid abuse liability, emesis, and analgesic tolerance. Here, we describe a multifunctional mu-/delta-opioid agonist/NK1 antagonist compound [Tyr-d-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bn(CF3)2 (TY027)] that has a preclinical profile of excellent antinociceptive efficacy, low abuse liability, and no opioid-related emesis or constipation. In rodent models of acute and neuropathic pain, TY027 demonstrates analgesic efficacy following central or systemic administration with a plasma half-life of more than 4 hours and central nervous system penetration. These data demonstrate that an innovative opioid designed to contest the pathology created by chronic pain and sustained opioids results in antinociceptive efficacy in rodent models, with significantly fewer side effects than morphine. Such rationally designed, multitargeted compounds are a promising therapeutic approach in treating patients who suffer from acute and chronic pain.

Intrathecal PKA-selective siRNA treatment blocks sustained morphine-mediated pain sensitization and antinociceptive tolerance in rats.

Sustained morphine treatment has been shown to produce paradoxical pain sensitization (opioid-induced hyperalgesia) and also causes increase in spinal pain neurotransmitter, such as calcitonin gene related peptide (CGRP), concentration in experimental animals. Studies have also shown that cyclic adenosine-monophosphate (cAMP)-dependent protein kinase (PKA) plays a major role in the regulation of presynaptic neurotransmitter (such as CGRP and substance P) synthesis and release. We have previously shown that in cultured primary sensory dorsal root ganglion (DRG) neurons sustained in vitro opioid agonist treatment upregulates cAMP levels (adenylyl cyclase (AC) superactivation) and augments basal and capsaicin evoked CGRP release in a PKA dependent manner. In the present study, we investigated the in vivo role of PKA in sustained morphine-mediated pain sensitization. Our data indicate that selective knock-down of spinal PKA activity by intrathecal (i.th.) pretreatment of rats with a PKA-selective small interference RNA (siRNA) mixture significantly attenuates sustained morphine-mediated augmentation of spinal CGRP immunoreactivity, thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. The present findings indicate that sustained morphine-mediated activation of spinal cAMP/PKA-dependent signaling may play an important role in opioid induced hyperalgesia.

Spinal or systemic TY005, a peptidic opioid agonist/neurokinin 1 antagonist, attenuates pain with reduced tolerance.

BACKGROUND AND PURPOSE: The use of opioids in treating pain is limited due to significant side effects including somnolence, constipation, analgesic tolerance, addiction and respiratory depression. Pre-clinical studies have shown that neurokinin 1 (NK(1) ) receptor antagonists block opioid-induced antinociceptive tolerance and may inhibit opioid-induced rewarding behaviours. Here, we have characterized a bifunctional peptide with both opioid agonist and NK(1) antagonist pharmacophores in a rodent model of neuropathic pain. EXPERIMENTAL APPROACH: Rats were evaluated for behavioural responses to both tactile and thermal stimuli in either an uninjured, sham- or nerve-injured state. TY005 (Tyr-DAla-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bn(CF(3) )(2) ) was delivered spinally or systemically to assess the antinociceptive effects after acute exposure. Motor skills were evaluated using the rotarod test to determine potential sedative effects. Spinal TY005 was given chronically to sham- or nerve-injured animals to determine the development of tolerance. KEY RESULTS: Bolus injections of TY005 produced dose-dependent antinociception in non-injured animals and alleviated nerve injury-induced thermal and tactile hypersensitivities (i.e. antihyperalgesia) more effectively than morphine. Sedative effects were not evident from the rotarod test at doses that were antihyperalgesic, nor at doses threefold higher. Repeated administration of TY005 did not lead to the development of antihyperalgesic tolerance or alter sensory thresholds. CONCLUSIONS AND IMPLICATIONS: Collectively, the data suggest that opioid agonist/NK(1) antagonist bifunctional peptides represent a promising novel approach to the management of chronic pain without the development of tolerance, reducing the need for escalation of doses and unwanted side effects associated with opiates alone.

Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1-selective siRNA.

BACKGROUND AND PURPOSE: Long-term morphine treatment enhances pain neurotransmitter [such as calcitonin gene-related peptide (CGRP)] levels in the spinal cord. It has been suggested previously that increased spinal CGRP may contribute to sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance. Previous in vitro studies from our group indicated that Raf-1 kinase-mediated adenylyl cyclase superactivation played a crucial role in sustained morphine-mediated augmentation of basal and evoked CGRP release from cultured primary sensory neurons. The present study was aimed to evaluate the physiological significance of this molecular mechanism in vivo, in rats. EXPERIMENTAL APPROACH: Rats were intrathecally (i.th) injected with a Raf-1-selective small interfering RNA (siRNA) mixture for 3 days and were subsequently infused with saline or morphine, s.c. for 7 days. Thermal and mechanical sensory thresholds of the animals were assessed by daily behavioural tests. After final behavioural testing (day 6), spinal cords were isolated from each animal group and spinal CGRP and Raf-1 protein levels were measured using elisa and immunohistochemistry. KEY RESULTS: Selective knockdown of spinal Raf-1 protein levels by i.th Raf-1-selective siRNA pretreatment significantly attenuated sustained morphine-mediated up-regulation of CGRP immunoreactivity in the spinal cord of rats and prevented the development of thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. CONCLUSIONS AND IMPLICATIONS: Raf-1 played a significant role in sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance in vivo. These findings suggest novel pharmacological approaches to improve the long-term utility of opioids in the treatment of chronic pain.

Preclinical assessment of candidate analgesic drugs: recent advances and future challenges.

In analgesic drug development, preclinical procedures are widely used to assess drug effects on pain-related behaviors. These procedures share two principal components: 1) a manipulation intended to produce a pain-like state in the experimental subject and 2) measurement of behaviors presumably indicative of that pain state. Drugs can then be evaluated for their ability to attenuate pain-related behaviors. In the simplest procedures, the pain state is produced by delivery of an acute noxious stimulus (e.g., a warm thermal stimulus), and the primary dependent measures focus on withdrawal responses or other nocifensive behaviors that increase in rate, frequency, or intensity in response to the noxious stimulus. This approach has been refined in two ways. First, new methods have been developed to induce more clinically relevant pain states. In particular, pain requiring clinical intervention is often associated with inflammation or neuropathy, and novel procedures have emerged to model these conditions and their ability to produce hypersensitive pain states, such as allodynia and hyperalgesia. Second, studies are incorporating a broader array of pain-related behaviors as dependent measures. For example, pain not only stimulates nocifensive behaviors but also suppresses many adaptive behaviors, such as feeding or locomotion. Measures of pain-suppressed behaviors can provide new insights into the behavioral consequences of pain and the effects of candidate analgesics. In addition, functional magnetic resonance imaging has emerged as a noninvasive tool for investigating changes in neural activity associated with pain and analgesia. Integration of these complementary approaches may improve the predictive validity of analgesic drug development.

Antinociceptive pharmacology of N-[[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl) sulfonyl]methylamino]ethoxy]-N-methylacetamide, fumarate (LF22-0542), a novel nonpeptidic bradykinin B1 receptor antagonist.

The antinociceptive pharmacology of N-[[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl) sulfonyl]methylamino]ethoxy]-N-methylacetamide fumarate (LF22-0542), a novel nonpeptidic B1 antagonist, was characterized. LF22-0542 showed high affinity for human and mouse B1 receptors with virtually no affinity for the human B2 receptor; a selectivity index of at least 4000 times was obtained when LF22-0542 was profiled throughout binding or cell biology assays on 64 other G-protein-coupled receptor, 10 ion channels, and seven enzymes. LF22-0542 was a competitive B1 receptor antagonist and elicited significant antinociceptive actions in the mouse acetic acid-induced writhing assay, as well as in the second phases of formalin-induced nociception in mice and in both the first and second phases of the formalin response in rats. LF22-0542 was active after s.c. but not p.o. administration. In B1 receptor knockout (KO) mice, acetic acid and formalin responses were significantly reduced and LF22-0542 had no additional effects in these animals. LF22-0542 alleviated thermal hypersensitivity in both acute (carrageenan) and persistent inflammatory (complete Freund's adjuvant) pain models in rats. LF22-0542 produced a full reversal of experimental neuropathic thermal hypersensitivity but was inactive in reversing nerve injury-induced tactile hypersensitivity in rats. In agreement with this observation, B1 KO mice subjected to peripheral nerve injury did not show thermal hypersensitivity but developed nerve injury-induced tactile hypersensitivity normally. The data demonstrate the antihyperalgesic actions of a selective systemically administered B1 receptor antagonist and suggest the utility of this class of agents for the treatment of inflammatory pain states and for some aspects of neuropathic pain.

Melanocortinergic control of penile erection.

Melanocortin receptors in the forebrain and spinal cord can be activated by endogenous or synthetic ligands to induce penile erection in rats and human subjects. To better understand how melanocortin circuits play a role in sex behavior, we review the contribution of melanocortin receptors and/or neurons in the hypothalamus, hindbrain, spinal cord and peripheral nerves to erectile function. New information regarding neuropeptides that mediate penile erection has extended our understanding of the central control of sex behavior, and melanocortin agonists may provide alternatives to existing treatment for highly prevalent problems including erectile dysfunction.

Glial cell line-derived neurotrophic factor normalizes neurochemical changes in injured dorsal root ganglion neurons and prevents the expression of experimental neuropathic pain.

Glial cell line-derived neurotrophic factor (GDNF) is necessary for the development of sensory neurons, and appears to be critical for the survival of dorsal root ganglion (DRG) cells that bind the lectin IB4. Intrathecal infusion of GDNF has been shown to prevent and reverse the behavioral expression of experimental neuropathic pain arising from injury to spinal nerves. This effect of GDNF has been attributed to a blockade of the expression of the voltage gated, tetrodotoxin-sensitive sodium channel subtype, Na(V)1.3, in the injured DRG. Here we report that GDNF given intrathecally via osmotic-pump to nerve-injured rats (L5/L6 spinal nerve ligation) prevented the changes in a variety of neurochemical markers in the DRG upon injury. They include a loss of binding of IB4, downregulation of the purinergic receptor P2X(3), upregulation of galanin and neuropeptide Y immunoreactivity in large diameter DRG cells, and expression of the transcription factor ATF3. GDNF infusion concomitantly prevented the development of spinal nerve ligation-induced tactile hypersensitivity and thermal hyperalgesia. These observations suggest that high dose, exogenous GDNF has a broad neuroprotective role in injured primary afferent. The receptor(s) that mediates these effects of GDNF is not known. GDNF's ability to block neuropathic pain states is not likely to be specific to Na(V)1.3 expression.

Enhanced evoked excitatory transmitter release in experimental neuropathy requires descending facilitation.

Nerve injury-induced afferent discharge is thought to elicit spinal sensitization and consequent abnormal pain. Experimental neuropathic pain, however, also depends on central changes, including descending facilitation arising from the rostral ventromedial medulla (RVM) and upregulation of spinal dynorphin. A possible intersection of these influences at the spinal level was explored by measuring evoked, excitatory transmitter release in tissues taken from nerve-injured animals with or without previous manipulation of descending modulatory systems. Spinal nerve ligation (SNL) produced expected tactile and thermal hyperesthesias. Capsaicin-evoked calcitonin gene-related peptide (CGRP) release was markedly enhanced in lumbar spinal tissue from SNL rats when compared with sham-operated controls. Enhanced, evoked CGRP release from SNL rats was blocked by anti-dynorphin A(1-13) antiserum; this treatment did not alter evoked release in tissues from sham-operated rats. Dorsolateral funiculus lesion (DLF) or destruction of RVM neurons expressing mu-opioid receptors with dermorphin-saporin, blocked tactile and thermal hypersensitivity, as well as SNL-induced upregulation of spinal dynorphin. Spinal tissues from these DLF-lesioned or dermorphin-saporin-treated SNL rats did not exhibit enhanced capsaicin-evoked CGRP-IR release. These data demonstrate exaggerated release of excitatory transmitter from primary afferents after injury to peripheral nerves, supporting the likely importance of increased afferent input as a driving force of neuropathic pain. The data also show that modulatory influences of descending facilitation are required for enhanced evoked transmitter release after nerve injury. Thus, convergence of descending modulation, spinal plasticity, and afferent drive in the nerve-injured state reveals a mechanism by which some aspects of nerve injury-induced hyperesthesias may occur.

Design of novel peptide ligands which have opioid agonist activity and CCK antagonist activity for the treatment of pain.

Disease states such as neuropathic pain offer special challenges in drug design due to the system changes which accompany these diseases. In this manuscript we provide an example of a new approach to drug design in which we have modified a potent and selective peptide ligand for the CCK-2 receptor to a peptide which has potent agonist binding affinity and bioactivity at delta and mu opioid receptors, and simultaneous antagonist activity at CCK receptors. De novo design based on the concept of overlapping pharmacophores was a central hypothesis of this design, and led to compounds such as H-Tyr-DPhe-Gly-DTrp-NMeNle-Asp-Phe-NH(2) (i.e., RSA 601) which have the designed properties.

Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2 induces penile erection via brain and spinal melanocortin receptors.

Penile erection induced by alpha-melanocyte-stimulating hormone and melanocortin receptors (MC-R) in areas of the spinal cord and periphery has not been demonstrated. To elucidate sites of the proerectile action of melanocortin peptides, in awake male rats we administered the MC-R agonist Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH(2) (MT-II) i.c.v., intrathecal (i.th.) and i.v. and scored penile erection and yawning. Injection of the MC-R antagonist Ac-Nle-c[Asp-His-DNal(2')-Arg-Trp-Lys]-NH(2) (SHU-9119) i.c.v. or i.th. in combination with i.th. MT-II differentiated spinal from supraspinal effects. To exclude a site of action in the penis, we recorded intracavernous pressure responses to intracavernosal injection of MT-II in the anesthetized rat.I.c.v., i.th., and i.v. MT-II induced penile erections in a dose-dependent fashion. Yawning was observed with i.c.v. and i.v. MT-II, while spinal injection did not produce this behavior. Intrathecal delivery of MT-II to the lumbosacral spinal cord was more efficacious in inducing erections than i.c.v. or i.v. administration; SHU-9119 blocked the erectile responses to i.th. MT-II when injected i.th. but not i.c.v. Intracavernosal MT-II neither increased intracavernous pressure nor augmented neurostimulated erectile responses. We confirmed the central proerectile activity of MT-II and demonstrated that in addition to a site of action in the brain, the distal spinal cord contains melanocortin receptors that can initiate penile erection independent of higher centers. These results provide new insight into the central melanocortinergic pathways that mediate penile erection and may allow for more efficacious melanotropin-based therapy for erectile dysfunction.

Dynorphin-independent spinal cannabinoid antinociception.

Spinal antinociception produced by delta 9-tetrahydro-cannabinol (Delta(9)-THC) and other cannabinoid agonists has been suggested to be mediated by the release of dynorphin acting at the kappa opioid receptor. Alternatively, as cannabinoid receptors are distributed appropriately in the pain transmission pathway, cannabinoid agonists might act directly at the spinal level to inhibit nociception, without requiring dynorphin release. Here, these possibilities were explored using mice with a deletion of the gene encoding prodynorphin. Antinociceptive dose-response curves were constructed for spinal Delta(9)-THC and WIN 55,212-2 in prodynorphin knock-out mice and in wild-type littermates. WIN 55,212-2 and Delta(9)-THC were equipotent in the wild-type and prodynorphin knock-out mice. Spinal pretreatment with a kappa opioid receptor antagonist, nor-binaltorphimine (nor-BNI), did not alter the dose-response curves for either WIN 55,212-2 or Delta(9)-THC in prodynorphin knock-out and wild-type mice. However, the same dose of nor-BNI used blocked U50,488H-induced antinociception in both wild-type and prodynorphin knock-out mice, confirming kappa opioid receptor activity. Pretreatment with SR141716A, a cannabinoid receptor antagonist blocked the antinociceptive actions of both WIN 55,212-2 and Delta(9)-THC. These data support the conclusion that antinociception produced by spinal cannabinoids are likely to be mediated directly through activation of cannabinoid receptors without the requirement for dynorphin release or activation of kappa opioid receptors.

Distinct potassium channels on pain-sensing neurons.

Differential expression of ion channels contributes functional diversity to sensory neuron signaling. We find nerve injury induced by the Chung model of neuropathic pain leads to striking reductions in voltage-gated K(+) (Kv) channel subunit expression in dorsal root ganglia (DRG) neurons, suggesting a potential molecular mechanism for hyperexcitability of injured nerves. Moreover, specific classes of DRG neurons express distinct Kv channel subunit combinations. Importantly, Kv1.4 is the sole Kv1 alpha subunit expressed in smaller diameter neurons, suggesting that homomeric Kv1.4 channels predominate in A delta and C fibers arising from these cells. These neurons are presumably nociceptors, because they also express the VR-1 capsaicin receptor, calcitonin gene-related peptide, and/or Na(+) channel SNS/PN3/Nav1.8. In contrast, larger diameter neurons associated with mechanoreception and proprioception express high levels of Kv1.1 and Kv1.2 without Kv1.4 or other Kv1 alpha subunits, suggesting that heteromers of these subunits predominate on large, myelinated afferent axons that extend from these cells.

Inhibition of neuropathic pain by selective ablation of brainstem medullary cells expressing the mu-opioid receptor.

Neurons in the rostroventromedial medulla (RVM) project to spinal loci where the neurons inhibit or facilitate pain transmission. Abnormal activity of facilitatory processes may thus represent a mechanism of chronic pain. This possibility and the phenotype of RVM cells that might underlie experimental neuropathic pain were investigated. Cells expressing mu-opioid receptors were targeted with a single microinjection of saporin conjugated to the mu-opioid agonist dermorphin; unconjugated saporin and dermorphin were used as controls. RVM dermorphin-saporin, but not dermorphin or saporin, significantly decreased cells expressing mu-opioid receptor transcript. RVM dermorphin, saporin, or dermorphin-saporin did not change baseline hindpaw sensitivity to non-noxious or noxious stimuli. Spinal nerve ligation (SNL) injury in rats pretreated with RVM dermorphin-saporin failed to elicit the expected increase in sensitivity to non-noxious mechanical or noxious thermal stimuli applied to the paw. RVM dermorphin or saporin did not alter SNL-induced experimental pain, and no pretreatment affected the responses of sham-operated groups. This protective effect of dermorphin-saporin against SNL-induced pain was blocked by beta-funaltrexamine, a selective mu-opioid receptor antagonist, indicating specific interaction of dermorphin-saporin with the mu-opioid receptor. RVM microinjection of dermorphin-saporin, but not of dermorphin or saporin, in animals previously undergoing SNL showed a time-related reversal of the SNL-induced experimental pain to preinjury baseline levels. Thus, loss of RVM mu receptor-expressing cells both prevents and reverses experimental neuropathic pain. The data support the hypothesis that inappropriate tonic-descending facilitation may underlie some chronic pain states and offer new possibilities for the design of therapeutic strategies.

The cannabinoid agonist WIN55,212-2 suppresses opioid-induced emesis in ferrets.

BACKGROUND: Cannabinoid receptor agonists reverse nausea and vomiting produced by chemotherapy and radiation therapy in animals and humans but have not been tested against opioid-induced emesis. This study tests the hypothesis that cannabinoid receptor agonists will prevent opioid-induced vomiting. METHODS: Twelve male ferrets were used. They weighed 1.2-1.6 kg at the beginning and 1.8-2.3 kg at the end of the experiments. All drugs were injected subcutaneously. WIN55,212-2, a mixed CB1-CB2 cannabinoid receptor agonist, was administered 25 min before morphine. Retches and vomits were counted at 5-min intervals for 30 min after morphine injection. RESULTS: Retching and vomiting responses increased with increasing morphine doses up to 1.0 mg/kg, above which the responses decreased. Previous administration of naloxone prevented morphine-induced retching and vomiting. WIN55,212-2 dose-dependently reduced retching and vomiting. The ED50 was 0.05 mg/kg for retches and 0.03 mg/kg for vomits. At 0.13 mg/kg, retching decreased by 76% and vomiting by 92%. AM251, a CB1 receptor-selective antagonist, blocked the antiemetic actions of WIN55,212-2, but AM630, a CB2 receptor-selective antagonist, did not. CONCLUSIONS: These results demonstrate that WIN55,212-2 prevents opioid-induced vomiting and suggest that the antiemetic activity of WIN55,212-2 occurs at CB1 receptors. This is consistent with findings that CB1 receptors are the predominant cannabinoid receptors in the central nervous system and that antiemetic effects of cannabinoids appear to be centrally mediated.