Sandra P. Welch

Synergistic interactions of endogenous opioids and cannabinoid systems.

Cannabinoids and opioids are distinct drug classes historically used in combination to treat pain. Delta(9)-THC, an active constituent in marijuana, releases endogenous dynorphin A and leucine enkephalin in the production of analgesia. The endocannabinoid, anandamide (AEA), fails to release dynorphin A. The synthetic cannabinoid, CP55,940, releases dynorphin B. Neither AEA nor CP55,940 enhances morphine analgesia. The CB1 antagonist, SR141716A, differentially blocks Delta(9)-THC versus AEA. Tolerance to Delta(9)-THC, but not AEA, involves a decrease in the release of dynorphin A. Our preclinical studies indicate that Delta(9)-THC and morphine can be useful in low dose combination as an analgesic. Such is not observed with AEA or CP55,940. We hypothesize the existence of a new CB receptor differentially linked to endogenous opioid systems based upon data showing the stereoselectivity of endogenous opioid release. Such a receptor, due to the release of endogenous opioids, may have significant impact upon the clinical development of cannabinoid/opioid combinations for the treatment of a variety of types of pain in humans.

Dynorphin B and spinal analgesia: induction of antinociception by the cannabinoids CP55,940, Δ9-THC and anandamide

The endogenous opioid dynorphin B was evaluated for its role in cannabinoid-induced antinociception. Previous work in our laboratory has shown that the synthetic, bicyclic cannabinoid, CP55,940, induces the release of dynorphin B whilst the naturally occurring cannabinoid, Delta(9)-tetrahydrocannabinol (Delta(9)-THC), releases dynorphin A. The dynorphins contribute in part to the antinociceptive effects of both cannabinoids at the level of the spinal cord. The present study compares dynorphin B released from perfused rat spinal cord in response to acute administration of anandamide (AEA), Delta(9)-THC and CP55,940 at two time points, 10 min and 30 min post administration, and attempts to correlate such release with antinociceptive effects of the drugs. Dynorphin B was collected from spinal perfusates of rats pretreated with Delta(9)-THC, CP55,940 or AEA. The supernatant was lyophilized and the concentrations of dynorphin B were measured via radioimmunoassay. At a peak time of antinociception (10 min), CP55,940 and Delta(9)-THC induced significant two-fold increases in the release of dynorphin B. AEA did not significantly release dynorphin B. Upon a 30-min pretreatment with the drugs, no significant dynorphin B release was observed, although antinociceptive effects persisted for CP55,940 and Delta(9)-THC. Previous work indicates that Delta(9)-THC releases dynorphin A while AEA releases no dynorphin A. This study confirms that although all three test drugs produced significant antinociception at 10 min, the endocannabinoid, AEA, does not induce antinociception via dynorphin release. Thus, our data indicate a distinct mechanism which underlies AEA-induced antinociception.

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.

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

Cannabinoids and opioids produce antinociceptive synergy. Cannabinoids such as Δ-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.

Prolonged reversal of morphine tolerance with no reversal of dependence by protein kinase C inhibitors.

The phosphatidylinositol (PI) cascade plays a pivotal role in mediating behavioral tolerance to the antinociceptive effects of morphine. Earlier we reported that antinociceptive tolerance was completely reversed 30 min after the administration of inhibitors of each step in the PI cascade. The aim of this study was to determine whether injection of a single dose of protein kinase C (PKC) inhibitor would elicit a prolonged reversal of morphine tolerance for up to 24 h. Three days after implantation of placebo- or 75-mg morphine pellets, mice received intracerebroventricular (i.c.v.) injections of vehicle or PKC inhibitor drug. Morphine challenge doses were then administered 4, 8 and 24 h later to test for tolerance reversal. In non-tolerant mice, Gö-7874 and sangivamycin had no effect on the potency of morphine. However, Gö-7874 and sangivamycin significantly reversed morphine tolerance at 4, 8 and 24 h. In addition, the role of PKC in morphine physical dependence was determined. Gö-7874 and sangivamycin by themselves did not precipitate spontaneous morphine withdrawal. Therefore, experiments were conducted to determine whether the PKC inhibitors would block naloxone-precipitated withdrawal. However, neither a 30-min nor a 24-h pretreatment with Gö-7874 or sangivamycin blocked naloxone withdrawal. Our results along with other publications indicate that PKC is a pivotal kinase essential for maintaining animals in an opioid tolerant state. Finally, the use of persistent PKC inhibitors that lasted for 24 h demonstrated that the neuronal systems in these animals did not adapt by increasing the activity of other protein kinase cascades to re-establish morphine tolerance.

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 [35S]GTPγS autoradiography using the S1P1‐selective agonist SEW2871 and the S1P1/3‐selective antagonist VPC44116 show that in several regions a majority of this activity is mediated by S1P1 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 CB1 receptors. At least one of the central effects of S1P, thermal antinociception, is also at least partly S1P1 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 S1P1 receptor contributes to S1P‐mediated antinociception.

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.

Alterations in L-type calcium channels in the brain and spinal cord of acutely treated and morphine-tolerant mice

Opiate tolerance has been associated with changes in neuronal intracellular calcium levels. In vivo studies have indicated the involvement of dihydropyridine (DHP)-sensitive (L-type) voltage-gated calcium channels in the morphine-tolerant state. In this study, we assessed how the morphine-tolerant state would affect the antinociception produced by intrathecal administration of two agents (Bay K 8644 and thapsigargin) that increase intracellular calcium. Morphine-tolerant mice displayed a 7-fold greater ED50 for Bay K 8644 antinociception than placebo-treated animals; no difference was found in the dose-response curve for thapsigargin. To further explore the role of the L-type channel in morphine tolerance development, we performed radiolabeled nitrendipine binding studies in the spinal cord and brain regions associated with pain modulation from acutely treated, tolerant, and control mice. Our treatment protocol produced a 4-fold shift in the ED50 for s.c. morphine antinociception, as determined by the tail-flick procedure. Binding site number and affinity were determined using Scatchard analysis for the following timepoints: 20 min and 60 min after s.c. injection of morphine or vehicle, as well as after 4 days of chronic administration, and after naloxone-precipitated withdrawal. Although some changes were observed in the affinity of nitrendipine for its receptor, a significant change in these measures was found only following naloxone administration, which produced increases in Bmax in both placebo- and morphine-treated mice.

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.