Differential Tolerance to FTY720-Induced Antinociception in Acute Thermal and Nerve Injury Mouse Pain Models: Role of Sphingosine-1-Phosphate Receptor Adaptation.

The immunomodulatory prodrug 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720), which acts as an agonist for sphingosine-1-phosphate (S1P) receptors (S1PR) when phosphorylated, is proposed as a novel pain therapeutic. In this study, we assessed FTY720-mediated antinociception in the radiant heat tail-flick test and in the chronic constriction injury (CCI) model of neuropathic pain in mice. FTY720 produced antinociception and antiallodynia, respectively, and these effects were dose-dependent and mimicked by the S1PR1-selective agonist CYM-5442. Repeated administration of FTY720 for 1 week produced tolerance to acute thermal antinociception, but not to antiallodynia in the CCI model. S1PR-stimulated [35S]GTPγS autoradiography revealed apparent desensitization of G protein activation by S1P or the S1PR1 agonist 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole (SEW-2871) throughout the brain. Similar results were seen in spinal cord membranes, whereby the Emax value of S1PR-stimulated [35S]GTPγS binding was greatly reduced in repeated FTY720-treated mice. These results suggest that S1PR1 is a primary target of FTY720 in alleviating both acute thermal nociception and chronic neuropathic nociception. Furthermore, the finding that tolerance develops to antinociception in the tail-flick test but not in chronic neuropathic pain suggests a differential mechanism of FTY720 action between these models. The observation that repeated FTY720 administration led to desensitized S1PR1 signaling throughout the central nervous system suggests the possibility that S1PR1 activation drives the acute thermal antinociceptive effects, whereas S1PR1 desensitization mediates the following: 1) tolerance to thermal antinociceptive actions of FTY720 and 2) the persistent antiallodynic effects of FTY720 in neuropathic pain by producing functional antagonism of pronociceptive S1PR1 signaling.

Sphingosine lysolipids in the CNS: endogenous cannabinoid antagonists or a parallel pain modulatory system?

A significant number of patients experience chronic pain and the intractable side effects of currently prescribed pain medications. Recent evidence indicates important pain-modulatory roles for two classes of G-protein-coupled receptors that are activated by endogenous lipid ligands, the endocannabinoid (eCB) and sphingosine-1-phosphate (S1P) receptors, which are widely expressed in both the immune and nervous systems. In the central nervous system (CNS), CB1 cannabinoid and S1P1 receptors are most abundantly expressed and exhibit overlapping anatomical distributions and similar signaling mechanisms. The eCB system has emerged as a potential target for treatment of chronic pain, but comparatively little is known about the roles of S1P in pain regulation. Both eCB and S1P systems modulate pain perception via the central and peripheral nervous systems. In most paradigms studied, the eCB system mainly inhibits pain perception. In contrast, S1P acting peripherally at S1P1 and S1P3 receptors can enhance sensitivity to various pain stimuli or elicit spontaneous pain. However, S1P acting at S1P1 receptors and possibly other targets in the CNS can attenuate sensitivity to various pain stimuli. Interestingly, other endogenous sphingolipid derivatives might play a role in central pain sensitization. Moreover, these sphingolipids can also act as CB1 cannabinoid receptor antagonists, but the physiological relevance of this interaction is unknown. Overall, both eCB and sphingolipid systems offer promising targets for the treatment of chronic pain. This review compares and contrasts the eCB and S1P systems with a focus on their roles in pain modulation, and considers possible points of interaction between these systems.

Sphingosine-1-phosphate receptors as emerging targets for treatment of pain.

Lysolipids are important mediators of cellular communication in multiple physiological processes. Sphingosine-1-phosphate (S1P) is a major lysolipid in many organs, including the central nervous system (CNS). This commentary discusses recent findings on the role of S1P in regulating pain perception, and highlights advances and challenges in the field. S1P interacts with multiple cellular targets, including G-protein-coupled receptors. Known S1P receptors include five types, four of which are expressed in the CNS (S1P(1,2,3,5)) where they are localized on neurons and glia. S1P receptor-mediated G-protein activation has been demonstrated throughout the CNS, including regions that regulate nociception. S1P receptors couple to multiple G-proteins to produce various intracellular responses, and can mediate both excitatory and inhibitory neuromodulation, depending on the receptor type and cellular context. Both antinociceptive and pro-nociceptive effects of S1P have been reported, and both actions can involve S1P(1) receptors. Current evidence suggests that antinociception is mediated by CNS neurons, whereas pro-nociception is mediated by primary afferent neurons or immune cells in the periphery, or CNS glia. Nonetheless, peripheral administration of the S1P(1,3,4,5) agonist pro-drug, FTY720, produces antinociception. FTY720 is approved to treat multiple sclerosis, and produces potent anti-inflammatory effects, which suggests potential utility for painful autoimmune diseases. Furthermore, evidence suggests that the S1P system interacts with other pain-modulatory systems, such as endogenous cannabinoid and opioid systems, and putative novel sphingolipid targets in the CNS. These findings suggest that drugs targeting the S1P system could be developed as novel analgesics, either as monotherapy or potential adjuncts to established analgesics.

Morphine potentiates neurodegenerative effects of HIV-1 Tat through actions at µ-opioid receptor-expressing glia.

Individuals infected with human immunodeficiency virus-1 who abuse opiates can have a higher incidence of virus-associated neuropathology. Human immunodeficiency virus does not infect neurons, but viral proteins such as transactivator of transcription and glycoprotein 120, originating from infected glia, are neurotoxic. Moreover, functional changes in glial cells that enhance inflammation and reduce trophic support are increasingly implicated in human immunodeficiency virus neuropathology. In previous studies, co-exposure with morphine enhanced transactivator of transcription neurotoxicity towards cultured striatal neurons. Since those cultures contained µ-opioid receptor-expressing astroglia and microglia, and since glia are the principal site of infection in the central nervous system, we hypothesized that morphine synergy might be glially mediated. A 60 hour, repeated measures paradigm and multiple co-culture models were used to investigate the cellular basis for opiate-enhanced human immunodeficiency virus neurotoxicity. Morphine co-exposure significantly enhanced transactivator of transcription-induced neuron death when glia were present. Synergistic effects of morphine on transactivator of transcription neurotoxicity were greatest with neuron-glia contact, but also occurred to a lesser extent with glial conditioned medium. Importantly, synergy was lost if glia, but not neurons, lacked µ-opioid receptors, indicating that opiate interactions with human immunodeficiency virus converge at the level of µ-opioid receptor-expressing glia. Morphine enhanced transactivator of transcription-induced inflammatory effectors released by glia, elevating reactive oxygen species, increasing 3-nitrotyrosine production by microglia, and reducing the ability of glia to buffer glutamate. But neuron survival was reduced even more with glial contact than with exposure to conditioned medium, suggesting that noxious elements associated with cell contact augment the toxicity due to soluble factors. Similar morphine-transactivator of transcription synergy was also observed in studies with the clade C sequence of HIV-1 transactivator of transcription, which did not cause neuron death unless morphine was present. Several paradoxical observations related to opiate effects were noted when µ-opioid receptors were specifically ablated from either glia or neurons. This suggests that µ-opioid receptor loss in isolated cell types can fundamentally distort cell-to-cell signalling, revealing opponent processes that may exist in individual cell types. Our findings show the critical role of glia in orchestrating neurotoxic interactions of morphine and transactivator of transcription, and support the emerging concept that combined exposure to opiates and human immunodeficiency virus drives enhanced pathology within the central nervous system.

Sphingosine-1-phosphate receptors mediate neuromodulatory functions in the CNS.

Sphingosine-1-phosphate (S1P) is a ubiquitous, lipophilic cellular mediator that acts in part by activation of G-protein-coupled receptor. Modulation of S1P signaling is an emerging pharmacotherapeutic target for immunomodulatory drugs. Although multiple S1P receptor types exist in the CNS, little is known about their function. Here, we report that S1P stimulated G-protein activity in the CNS, and results from [(35)S]GTPgammaS autoradiography using the S1P(1)-selective agonist SEW2871 and the S1P(1/3)-selective antagonist VPC44116 show that in several regions a majority of this activity is mediated by S1P(1) receptors. S1P receptor activation inhibited glutamatergic neurotransmission as determined by electrophysiological recordings in cortical neurons in vitro, and this effect was mimicked by SEW2871 and inhibited by VPC44116. Moreover, central administration of S1P produced in vivo effects resembling the actions of cannabinoids, including thermal antinociception, hypothermia, catalepsy and hypolocomotion, but these actions were independent of CB(1) receptors. At least one of the central effects of S1P, thermal antinociception, is also at least partly S1P(1) receptor mediated because it was produced by SEW2871 and attenuated by VPC44116. These results indicate that CNS S1P receptors are part of a physiologically relevant and widespread neuromodulatory system, and that the S1P(1) receptor contributes to S1P-mediated antinociception.

Interaction of the cannabinoid and opioid systems in the modulation of nociception.

Cannabinoids and opioids produce antinociceptive synergy. Cannabinoids such as Delta-9-tetrahydrocannabinol (THC) release endogenous opioids and endocannabinoids such as anandamide (AEA) also alter endogenous opioid tone. Opioids and cannabinoids bind distinct receptors that co-localize in areas of the brain involved with the processing of pain signals. Therefore, it is logical to look at interactions of these two systems in the modulation of both acute and chronic pain. These drugs are often co-abused. In addition, the lack of continued effectiveness of opioids due to tolerance development limits the use of such drugs. The cost to society and patients in terms of dollars, loss of productivity, as well as quality of life, is staggering. This review summarizes the data indicating that with cannabinoid/opioid therapy one may be able to produce long-term antinociceptive effects at doses devoid of substantial side effects, while preventing the neuronal biochemical changes that accompany tolerance. The clinical utility of modulators of the endocannabinoid system as a potential mimic for THC-like drugs in analgesia and tolerance-sparing effects of opioids is a critical future direction also addressed in the review.

Modulation of opioids via protection of anandamide degradation by fatty acid amide hydrolase.

Lack of involvement of the opioid system with the endocannabinoid, arachidonylethanolamide (anandamide) was possibly due to hydrolysis by fatty acid amide hydrolase (FAAH). Cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597) is an inhibitor of FAAH, increases brain anandamide levels and enhances anandamide-induced antinociception in male ICR mice (25-30 g). The combination of URB597 (10 mg/kg, i.p.) and anandamide (40 mg/kg, i.p.) produced maximal antinociception in the mouse tail-flick test [68.7+/-16.8 percent maximum possible effect (%MPE)], versus either substance alone (27.3+/-7.9%MPE and 4.6+/-2.3%MPE, respectively) and is significantly blocked (p<0.05) by the cannabinoid CB(1) receptor antagonist, SR141716A (rimonabant), the kappa opioid receptor-selective antagonist, nor-Binaltorphimine (10 microg i.t.; 12.7+/-4.0%MPE) and the mu opioid receptor antagonist, naloxone (1 mg/kg, s.c.; 6.0+/-3.8%MPE), but not by the delta opioid receptor-selective antagonist, naltrindole (2 mg/kg, s.c.; 29.7+/-8.2%MPE) or the cannabinoid CB(2) receptor antagonist, SR144528. In addition, nor-BNI (10 microg i.t) administration to FAAH(-/-) knockout mice produced a nociceptive response. The URB597/anandamide combination was not active in the CB(1)(-/-) knockout mice, but retained activity in the MOR(-/-) knockout mice. The sub-active combination of (URB597 10 mg/kg, i.p/anandamide 10 mg/kg, i.p.; 15.5+/-4.3%MPE) shifted the dose response curve of morphine to the left (morphine alone ED(50)=4.6 mg/kg [3.7-5.6] versus morphine/URB597/anandamide (ED(50)=2.5 mg/kg [1.9-3.4]). These data are the first demonstration that anandamide, if protected from degradation, acts via the CB(1) receptor to interact with kappa opioid receptor systems in opioid analgesia.

Decreased basal endogenous opioid levels in diabetic rodents: effects on morphine and delta-9-tetrahydrocannabinoid-induced antinociception.

We have previously demonstrated synergy between morphine and Delta(9)-tetrahydrocannabinol (Delta(9)-THC) in the expression of antinociception in acute pain models and in arthritic models of chronic pain. Our data has been extended to include acute pain in both diabetic mice and rats. In diabetic mice, Delta(9)-THC p.o. was more active in the tail-flick test in the diabetic mouse than in the non-diabetic mouse. Morphine (s.c.) was less potent in diabetic than in non-diabetic mice [6.1 (5.1-7.2) versus 3.2 (2.4-4.1) mg/kg, respectively], an effect previously extensively documented in pre-clinical and clinical testing. In addition, the combination of Delta(9)-THC with morphine produced a greater-than-additive relief of acute pain in mice. In the rat, the induction of the diabetic state decreased the antinociceptive effect of morphine, an effect temporally related to a decreased release of specific endogenous opioids. Conversely, Delta(9)-THC retained the ability to release endogenous opioids in diabetic rats and maintained significant antinociception. Extrapolation of such studies to the clinical setting may indicate the potential for use of Delta(9)-THC-like drugs in the treatment of diabetic neuropathic pain, alone or in combination with very low doses of opioids.

Chronic morphine treatment decreases the Cav1.3 subunit of the L-type calcium channel.

Voltage-gated L- and N-type calcium channels (VOCs) are implicated in the activity of morphine, but their contribution to the expression of opioid tolerance remains uncertain. L- and N-type VOCs are heteropentamers of alpha(1), alpha(2)delta, beta, and gamma subunits. The alpha(1) subunit forms both the ion pore and the binding site for ligands. The Ca(v)1.2 and Ca(v)1.3 are the neuronal dihydropyridine (DHP)-sensitive L-type channel subunit types. The Ca(v)2.2 subunit is found in omega conotoxin GVIA-sensitive N-type calcium channels. Ca(v)1.2 VOC gating properties are phosphorylation-dependent with many kinases implicated. We hypothesized that changes in channel subunit structure or phosphorylation state, induced by chronic opioid exposure, may in part explain changes in calcium regulation observed both in vivo and in vitro. Antibodies, specific for the Ca(v)1.2, Ca(v)1.3, and Ca(v)2.2 subunits of VOCs were employed with Western immunoassays to access whether chronic morphine treatment had an effect on receptor protein levels. The L-type channel Ca(v)1.3 protein, but not the Ca(v)1.2 protein or phosphorylation state, significantly decreased upon chronic morphine treatment. The Ca(v)2.2 subunit protein of the N-type channel of VOCs remained unchanged. The Ca(v)1.3 subunit modification may represent one of many potential adaptive changes in tolerance to morphine-induced changes in intracellular calcium.

The role of gonadal hormones on opioid receptor protein density in arthritic rats.

The purpose of this study was to evaluate the effects of the gonadal hormones on the opioid receptor protein levels of Freund's adjuvant-treated (arthritic) male and female Lewis rats. Following a paw pressure nociception assay, the midbrain and spinal cord tissues were collected for comparison of mu, delta, and kappa opioid receptor protein levels. The effects of Freund's adjuvant-induced hyperalgesia resulted in significantly decreased nociception thresholds in both males and females, compared to vehicle treated animals in the paw pressure test. It was hypothesized that the presence or lack thereof of gonadal hormones would alter nociception, an effect temporally correlated with a change in opioid receptor protein expression. Nociceptive thresholds were altered by arthritis in both sexes, but not further altered by gonadal changes in males. A small, but significant increase in threshold was shown in ovariectomized females. In spite of the small gonadal-induced changes in the nociceptive threshold sensitivity to pressure, significant changes in the plasticity of the opioid system were observed. There was a significant increase in kappa opioid receptor protein levels in the spinal cord of arthritic ovariectomized females. Mu opioid receptor and kappa opioid receptor protein levels in the spinal cord tissue of non-arthritic male rats were significantly higher than in arthritic rats, a difference eliminated by gonadectomy. Gonadectomy produced similar results in the mu opioid receptor protein level in the male midbrain tissue as well. Sex differences were observed in both the mu and kappa opioid receptor protein levels. The spinal cord tissue of male rats, regardless of the presence of gonads or arthritis displayed significantly greater levels of mu opioid receptor protein levels than females. The removal of gonadal hormones appears to have opposite effects in males and females in terms of opioid receptor proteins, but not nociception as quantified by the paw pressure test. The role of changes in the plasticity of the opioid systems in response to arthritis or gonadal hormones remains to be elucidated.

Low dose combination of morphine and delta9-tetrahydrocannabinol circumvents antinociceptive tolerance and apparent desensitization of receptors.

Morphine and delta9-tetrahydrocannabinol (THC) produce antinociception via mu opioid and cannabinoid CB1 receptors, respectively, located in central nervous system (CNS) regions including periaqueductal gray and spinal cord. Chronic treatment with morphine or THC produces antinociceptive tolerance and cellular adaptations that include receptor desensitization. Previous studies have shown that administration of combined sub-analgesic doses of THC+morphine produced antinociception in the absence of tolerance. The present study assessed receptor-mediated G-protein activity in spinal cord and periaqueductal gray following chronic administration of THC, morphine or low dose combination. Rats received morphine (escalating doses from 1 to 6x75 mg s.c. pellets or s.c. injection of 100 to 200 mg/kg twice daily), THC (4 mg/kg i.p. twice daily) or low dose combination (0.75 mg/kg each morphine (s.c) and THC (i.p.) twice daily) for 6.5 days. Antinociception was measured in one cohort of rats using the paw pressure test, and a second cohort was assessed for agonist-stimulated [35S]GTPgammaS binding. Chronic administration of morphine or THC produced antinociceptive tolerance to the respective drugs, whereas combination treatment did not produce tolerance. Administration of THC attenuated cannabinoid CB1 receptor-stimulated G-protein activity in both periaqueductal gray and spinal cord, and administration of morphine decreased mu opioid receptor-stimulated [35S]GTPgammaS binding in spinal cord or periaqueductal gray, depending on route of administration. In contrast, combination treatment did not alter cannabinoid CB1 receptor- or mu opioid receptor-stimulated G-protein activity in either region. These results demonstrate that low dose THC-morphine combination treatment produces antinociception in the absence of tolerance or attenuation of receptor activity.

The antinociceptive effect of Delta9-tetrahydrocannabinol in the arthritic rat involves the CB(2) cannabinoid receptor.

Cannabinoid CB(2) receptors have been implicated in antinociception in animal models of both acute and chronic pain. We evaluated the role both cannabinoid CB(1) and CB(2) receptors in mechanonociception in non-arthritic and arthritic rats. The antinociceptive effect of Delta(9)-tetrahydrocannabinol (Delta(9)THC) was determined in rats following administration of the cannabinoid CB(1) receptor-selective antagonist, SR141716A, the cannabinoid CB(2) receptor-selective antagonist, SR144528, or vehicle. Male Sprague-Dawley rats were rendered arthritic using Freund's complete adjuvant and tested for mechanical hyperalgesia in the paw-pressure test. Arthritic rats had a baseline paw-pressure of 83 +/- 3.6 g versus a paw-pressure of 177 +/- 6.42 g in non-arthritic rats. SR144528 or SR141716A (various doses mg/kg; i.p.) or 1:1:18 (ethanol:emulphor:saline) vehicle were injected 1 h prior to Delta(9)THC (4 mg/kg; i.p) or 1:1:18 vehicle and antinociception determined 30min post Delta(9)THC. AD(50)'s for both antagonists were calculated with 95% confidence limits. In addition, midbrain and spinal cord were removed for determination of cannabinoid CB(1) and CB(2) receptor protein density in the rats. SR144528 significantly attenuated the antinociceptive effect of Delta(9)THC in the arthritic rats [AD(50) = 3.3 (2.7-4) mg/kg], but not in the non-arthritic rats at a dose of 10/mg/kg. SR141716A significantly attenuated Delta(9)THC-induced antinociception in both the non-arthritic [AD(50) = 1.4 (0.8-2) mg/kg] and arthritic rat [AD(50) = 2.6 (1.8-3.1) mg/kg]. SR141716A or SR144528 alone did not result in a hyperalgesic effect as compared to vehicle. Our results indicate that the cannabinoid CB(2) receptor plays a critical role in cannabinoid-mediated antinociception, particularly in models of chronic inflammatory pain.

Synergy between delta9-tetrahydrocannabinol and morphine in the arthritic rat.

We have shown in past isobolographic studies that a small amount of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) can enhance morphine antinociception in mice. However, previous studies of the Delta(9)-THC/morphine interaction were performed using normal mice or rats and evaluated acute thermal antinociception. Less is known about cannabinoid and opioid interactions involved in mechanical nociception and in chronic inflammatory pain models, such as Freund's complete adjuvant-induced arthritic model. One fixed-ratio combination was chosen for testing the interaction between Delta(9)-THC and morphine in the Freund's adjuvant-induced arthritic model. This combination represented a 1:1 ratio of the drugs and thus consisted of equieffective doses ranging from 0.1 to 5 mg/kg Delta(9)-THC and from 0.1 to 5 mg/kg morphine. The combination ED(50) value for the fixed ratios (total dose) in relation to the ED(50) value of the drugs alone was determined. The isobolographic analysis indicated a synergistic interaction between Delta(9)-THC and morphine in both the non-arthritic and the arthritic rats. Since Freund's adjuvant-induced alteration in endogenous opioid tone has been previously shown, our data indicate that such changes did not preclude the use of Delta(9)-THC and morphine in combination. As with acute preclinical pain models in which the Delta(9)-THC/morphine combination results in less tolerance development, the implication of the study for chronic pain conditions is discussed.

Non-cannabinoid CB1, non-cannabinoid CB2 antinociceptive effects of several novel compounds in the PPQ stretch test in mice.

The analgesic and anti-hyperalgesic effects of cannabinoid- and vanilloid-like compounds, plus the fatty acid amide hydrolase (FAAH) inhibitor Cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597), and acetaminophen, were evaluated in the phenyl-p-quinone (PPQ) pain model, using different routes of administration in combination with opioid and cannabinoid receptor antagonists. All the compounds tested produced analgesic effects. Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and (R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide ((R)-methanandamide) were active by three routes of administration: i.p., s.c. and, p.o. Delta(9)-THC produced ED(50)s of 2.2 mg/kg (0.3-15.6) i.p., 9 mg/kg (4.3-18.9) s.c., and 6.4 mg/kg (5.5-7.6) p.o. Similarly, (R)-methanandamide yielded ED(50)s of 2.9 mg/kg (1-8) i.p., 11 mg/kg (7-17) s.c., and 11 mg/kg (0.9-134) p.o. N-vanillyl-arachidonyl-amide (arvanil) was active by two routes, producing ED(50)s of 4.7 mg/kg (3.0-7.4) s.c. and 0.06 mg/kg (0.02-0.2) i.p. Palmitoylethanolamide, URB597, and acetaminophen were active i.p., resulting in ED(50)s of 3.7 mg/kg (3.2-4.2), 22.9 mg/kg (11.1-47.2), and 160 mg/kg (63-405), respectively. None of the cannabinoid or opioid receptor antagonists tested blocked the compounds evaluated, with two exceptions: the antinociceptive effects of Delta(9)-THC and URB597 were completely blocked by SR141716A, a cannabinoid CB(1) receptor antagonist. Western immunoassays performed using three opioid receptor antibodies, a cannabinoid CB(1) receptor antibody and a transient receptor potential vanilloid type 1(TRPV(1)) receptor antibody, yielded no change in receptor protein levels after short-term arvanil, (R)-methanandamide or Delta(9)-THC administration. These data suggest that all the compounds tested, except Delta(9)-THC and URB597, produced analgesia via a non-cannabinoid CB(1), non-cannabinoid CB(2) pain pathway not yet identified.

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.

Enhancement of transdermal fentanyl and buprenorphine antinociception by transdermal delta9-tetrahydrocannabinol.

Previous studies have demonstrated that delta9-tetrahydrocannabinol (THC) enhances the antinociceptive potency of many opioids administered by a variety of different routes of administration. We hypothesized that THC would enhance fentanyl or buprenorphine analgesia via the transdermal route of administration. THC was first demonstrated to enhance opioid antinociception when both drugs were administered parenterally in a hairless guinea pig model using the pin prick test. A low dose of THC (50 mg/kg, i.p.) produced no antinociception. However, THC enhanced the potency of s.c. fentanyl by 6.7-fold, and s.c. buprenorphine in a non-parallel fashion. For the transdermal studies, THC, fentanyl or buprenorphine was applied by pipette to the skin of the dorsum between the fore- and hind-flanks and covered with individual Tegederm patches. THC (400 mg/kg) produced no antinociception. However, THC enhanced fentanyl's potency by 3.7-fold at 2-h, and 5.8-fold at 4-h. Buprenophine's potency was increased 8.2-fold at 2-h and 7.2-fold at 4-h when co-administered with THC. These results indicate that the enhancement of transdermal opioids by THC could lead to the design of an effective combination analgesic patch.

Mu and delta opioid-stimulated [35S]GTP gamma S binding in brain and spinal cord of polyarthritic rats.

Polyarthritis induced by inoculation with complete Freund's adjuvant alters opioid peptides, but does not affect opioid receptor binding. This study was conducted to measure mu and delta opioid receptor-stimulated G-protein activity in brain and spinal cord of rats 19 days after injection of complete Freund's adjuvant or vehicle. Mu and delta opioid-stimulated [35S]GTPgammaS binding measured autoradiographically in caudate-putamen, medial thalamus and periaqueductal gray was unchanged in polyarthritic rats. Delta opioid-stimulated [35S]GTPgammaS binding was significantly decreased in the spinal cord of polyarthritic rats, whereas mu opioid-stimulated activity was unchanged. These data reveal that the functional activity of delta opioid receptors in the spinal cord is altered in polyarthritis.

Reversal of delta 9-tetrahydrocannabinol-induced tolerance by specific kinase inhibitors.

Tolerance develops to the pharmacological effects of Delta9-tetrahydrocannabinoid (THC) following repetitive administration. Adaptations in signaling pathways involved in tolerance to THC-induced behaviors are not understood. The objective of our study was the evaluation of kinase involvement in the expression of tolerance to the above four THC-induced behaviors. Kinase inhibitors that specifically inhibit cyclic AMP-dependent protein kinase (PKA), cyclic GMP-dependent protein kinase (PKG), calmodulin-dependent protein kinase (PKC) and src tyrosine kinase were tested for reversal of tolerance to THC's effects. PKG and PKC inhibitors did not reverse tolerance in any behavioral measure. Src tyrosine kinase inhibition reversed tolerance to only the hypoactive effects of THC. PKA inhibition reversed tolerance to all measures, although the doses of inhibitor and time-course of inhibition varied among behaviors. Thus, our data suggest that PKA activity plays a major role in THC-induced tolerance, and that THC produces its multiple effects through different signaling pathways.

The antinociceptive effect of Delta9-tetrahydrocannabinol in the arthritic rat.

Our study addressed the hypothesis that spinal release of endogenous opioids underlies Delta9-tetrahydrocannabinol (Delta9-THC)-induced antinociception in Freund's adjuvant-induced arthritic and nonarthritic rats. The paw-pressure test was used to assess the antinociceptive effects of Delta9-THC versus those of morphine, and opioid and cannabinoid receptor-selective antagonists were used to characterize the involved receptors. Cerebrospinal fluid was collected after Delta9-THC injection (i.p.) for the measurement of endogenous opioid peptides. Our results indicate that morphine or Delta9-THC is equally potent and efficacious in both nonarthritic and arthritic rats. Delta9-THC-induced antinociception is attenuated by the kappa opioid receptor antagonist, nor-binaltorphimine, in arthritic rats only. Delta9-THC induces increased immunoreactive dynorphin A (idyn A) levels in nonarthritic rats while decreasing idyn A in arthritic rats. We hypothesize that the elevated idyn A level in arthritic rats contributes to hyperalgesia by interaction with N-methyl-D-aspartate receptors, and that Delta9-THC induces antinociception by decreasing idyn A release.

Modulation of oral morphine antinociceptive tolerance and naloxone-precipitated withdrawal signs by oral Delta 9-tetrahydrocannabinol.

Previous studies have demonstrated a functional interaction between cannabinoid and opioid systems in the development and expression of morphine tolerance and dependence. In these experiments, we examined the effect of a low oral dose of Delta 9-tetrahydrocannabinol (Delta 9-THC) on the development of oral morphine tolerance and the expression of naloxone-precipitated morphine withdrawal signs of jumping and diarrhea in ICR mice. Chronic treatment with high-dose oral morphine produced a 3.12-fold antinociceptive tolerance. Tolerance to morphine was prevented in groups receiving a daily cotreatment with a nonanalgetic dose (20 mg/kg p.o.) of Delta 9-THC, except when challenged with a very high dose of morphine. The chronic coadministration of low-dose Delta 9-THC also reduced naloxone-precipitated (1 mg/kg s.c.) platform jumping by 50% but did not reduce diarrhea. In separate experiments, mice treated chronically with high-dose morphine p.o. were not cross-tolerant to Delta 9-THC; in fact, these morphine-tolerant mice were more sensitive to the acute antinociceptive effects of Delta 9-THC. Delta 9-THC (20 mg/kg p.o.) also reduced naloxone-precipitated jumping but not diarrhea when administered acutely to morphine-tolerant mice. These results represent the first evidence that oral morphine tolerance and dependence can be circumvented by coadministration of a nonanalgetic dose of Delta 9-THC p.o. In summary, cotreatment with a combination of morphine and Delta 9-THC may prove clinically beneficial in that long-term morphine efficacy is maintained.