This study was conducted to assess the utility of unbound brain EC50 (EC50,u) as a measure of in vivo potency for centrally active drugs. Seven mu-opioid agonists (alfentanil, fentanyl, loperamide, ...methadone, meperidine, morphine, and sufentanil) were selected as model central nervous system drugs because they elicit a readily measurable central effect (antinociception) and their clinical pharmacokinetics/pharmacodynamics are well understood. Mice received an equipotent subcutaneous dose of one of the model opioids. The time course of antinociception and the serum and brain concentrations were determined. A pharmacokinetic/pharmacodynamic model was used to estimate relevant parameters. In vitro measures of opioid binding affinity (Ki) and functional activity EC50 for agonist stimulated guanosine 5'-O-(3-35Sthio)triphosphate binding and relevant clinical parameters were obtained to construct in vitro-to-preclinical and preclinical-to-clinical correlations. The strongest in vitro-to-in vivo correlation was observed between Ki and unbound brain EC50,u (r2 approximately 0.8). A strong correlation between mouse serum and human plasma EC50 was observed (r2 = 0.949); the correlation was improved when corrected for protein binding (r2 = 0.995). Clinical equipotent i.v. dose was only moderately related to Ki. However, estimates of ED50 and EC50 (total serum, unbound serum, total brain, and unbound brain) were significant predictors of clinical equipotent i.v. dose; the best correlation was observed for brain EC50,u (r2 = 0.982). For each opioid, brain equilibration half-life in mice was almost identical to the plasma effect-site equilibration half-life measured clinically. These results indicate that the mouse is a good model for opioid human brain disposition and clinical pharmacology and that superior in vitro-to-preclinical and preclinical-to-clinical correlations can be achieved with relevant unbound concentrations.
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Long-term cannabinoid administration produces region-dependent CB 1 receptor desensitization and down-regulation. This study examined the time course for normalization of CB 1 receptors and G-protein ...activation using 3 H-labeled N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboximide hydrochloride (SR141716A) and guanosine 5â²- O -(3- 35 Sthio)triphosphate ( 35 SGTPγS binding), respectively, in hippocampus and striatum/globus pallidus (GP). Mice were treated with escalating doses
of Î 9 -tetrahydrocannabinol (Î 9 -THC) or R (+)-2,3-dihydro-5-methyl-3-(morpholinyl)methylpyrrolo-1,2,3- de -1,4-benzoxazinyl-(1-naphthalenyl)methanone mesylate (WIN55,212-2) for 15 days, and tissue was collected 1, 3, 7, or 14
days after final injection. 3 HSR141716A and WIN55,212-2-stimulated 35 SGTPγS binding were decreased in both regions 1 day after treatment. WIN55,212-2-stimulated G-protein activation in striatum/GP
returned to control level at 3 days after cessation of treatment with either drug but did not return to control level in hippocampus
until 14 days. CB 1 receptor binding did not recover to control levels until day 7 or 14 after treatment in striatum/GP and hippocampus, respectively.
The mechanism of CB 1 binding site down-regulation was investigated after long-term Î 9 -THC treatment. Analysis of CB 1 receptor mRNA in hippocampus and striatum/GP showed that transcriptional regulation could not explain prolonged recovery
rates from CB 1 receptor down-regulation. In contrast, CB 1 receptor protein, as determined by immunoblot analysis, matched the down-regulation and recovery rates of CB 1 receptor binding sites relatively closely. These data demonstrate that cannabinoid-induced decreases in CB 1 receptor function persist for relatively long time periods after cessation of long-term drug treatment and that CB 1 receptor signaling recovers more quickly in striatum/GP than hippocampus. Moreover, down-regulation of CB 1 receptor binding sites does not seem to result mainly from transcriptional regulation, suggesting that adaptive regulation
of CB 1 receptors in brain primarily occurs at the protein level.
Low‐efficacy mu‐opioid receptor (MOR) agonists represent promising therapeutics, but existing compounds (e.g., buprenorphine, nalbuphine) span a limited range of low MOR efficacies and have poor MOR ...selectivity. Accordingly, new and selective low‐efficacy MOR agonists are of interest. A novel set of chiral C9‐substituted phenylmorphans has been reported to display improved MOR selectivity and a range of high‐to‐low MOR efficacies under other conditions; however, a full opioid receptor binding profile for these drugs has not been described. Additionally, studies in mice will be useful for preclinical characterization of these novel compounds, but the pharmacology of these drugs in mice has also not been examined. Accordingly, the present study characterized the binding selectivity and in vitro efficacy of these compounds using assays of opioid receptor binding and ligand‐stimulated 35SGTPɣS binding. Additionally, locomotor effects were evaluated as a first step for in vivo behavioral assessment in mice. The high‐efficacy MOR agonist and clinically effective antidepressant tianeptine was included as a comparator. In binding studies, all phenylmorphans showed improved MOR selectivity relative to existing lower‐efficacy MOR agonists. In the ligand‐stimulated 35SGTPɣS binding assay, seven phenylmorphans had graded levels of sub‐buprenorphine MOR efficacy. In locomotor studies, the compounds again showed graded efficacy with a rapid onset and ≥1 h duration of effects, evidence for MOR mediation, and minor sex differences. Tianeptine functioned as a high‐efficacy MOR agonist. Overall, these in vitro and in vivo studies support the characterization of these compounds as MOR‐selective ligands with graded MOR efficacy and utility for further behavioral studies in mice.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
The CB 1 cannabinoid receptor is a G-protein coupled receptor that has important physiological roles in synaptic plasticity, analgesia,
appetite, and neuroprotection. We report the discovery of two ...structurally related CB 1 cannabinoid receptor interacting proteins (CRIP1a and CRIP1b) that bind to the distal C-terminal tail of CB 1 . CRIP1a and CRIP1b are generated by alternative splicing of a gene located on chromosome 2 in humans, and orthologs of CRIP1a
occur throughout the vertebrates, whereas CRIP1b seems to be unique to primates. CRIP1a coimmunoprecipitates with CB 1 receptors derived from rat brain homogenates, indicating that CRIP1a and CB 1 interact in vivo. Furthermore, in superior cervical ganglion neurons coinjected with CB 1 and CRIP1a or CRIP1b cDNA, CRIP1a, but not CRIP1b, suppresses CB 1 -mediated tonic inhibition of voltage-gated Ca 2+ channels. Discovery of CRIP1a provides the basis for a new avenue of research on mechanisms of CB 1 regulation in the nervous system and may lead to development of novel drugs to treat disorders where modulation of CB 1 activity has therapeutic potential (e.g., chronic pain, obesity, and epilepsy).
Evidence has shown that downstream signaling by mu opioid receptor (MOR) agonists that recruit β-arrestin2 may lead to the development of tolerance. Also, it has been suggested that opioid receptor ...desensitization and cyclic AMP overshoot contributes to the development of tolerance and occurrence of withdrawal, respectively. Therefore, studies were conducted with 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3-carboxamido)morphinan (NAQ), a MOR selective partial agonist discovered in our laboratory, to characterize its effect on β-arrestin2 recruitment and precipitation of a cyclic AMP overshoot. DAMGO, a MOR full agonist dose-dependently increased β-arrestin2 association with the MOR, whereas NAQ did not. Moreover, NAQ displayed significant, concentration-dependent antagonism of DAMGO-induced β-arrestin2 recruitment. After prolonged morphine treatment of mMOR-CHO cells, there was a significant overshoot of cAMP upon exposure to naloxone, but not NAQ. Moreover, prolonged incubation of mMOR-CHO cells with NAQ did not result in desensitization nor downregulation of the MOR. In functional studies comparing NAQ with nalbuphine in the cAMP inhibition, Ca2+ flux and 35SGTPγS binding assays, NAQ did not show agonism in the Ca2+ flux assay but showed partial agonism in the cAMP and 35SGTPγS assays. Also, NAQ significantly antagonized DAMGO-induced intracellular Ca2+ increase. In conclusion, NAQ is a low efficacy MOR modulator that lacks β-arrestin2 recruitment function and does not induce cellular hallmarks of MOR adaptation and fails to precipitate a cellular manifestation of withdrawal in cells pretreated with morphine. These characteristics are desirable if NAQ is pursued for opioid abuse treatment development.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Sphingosine-1-phosphate (S1P) and cannabinoid receptors are G-protein-coupled receptors that mediate the effects of S1P and
endocannabinoids, respectively. Cannabinoid receptors also mediate the ...effects of Î 9 -tetrahydrocannabinol, the primary psychoactive ingredient in marijuana, whereas S1P receptors contribute to the immunosuppressant
effects of 2-amino-2-(2-4-octylphenylethyl)-1,3-propanediol (FTY720). FTY720 is a sphingosine analog that can prevent renal
graft rejections and suppress a variety of autoimmune disorders in animal models and clinical trials. We now report that both
FTY720 and sphingosine interact with CB 1 but not CB 2 cannabinoid receptors. FTY720 and sphingosine inhibited the binding of the CB 1 -selective antagonist 3 H N- (piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide ( 3 HSR141716A) and the cannabinoid agonist 3 H(â)- cis -3-2-hydroxy-4-(1,1-dimethylheptyl)phenyl- trans -4-(3-hydroxypropyl)cyclohexanol ( 3 HCP55,940) in a concentration-dependent manner in both CB 1 -expressing cell lines and mouse cerebellum. However, these compounds did not significantly alter 3 HCP55,940 binding to CB 2 receptors. In G-protein activation assays, FTY720 and sphingosine inhibited the maximal stimulation of guanosine 5â²- O -(3- 35 Sthio)triphosphate binding by the cannabinoid agonist R -(+)-2,3-dihydro-5-methyl-3-(morpholinyl)methylpyrrolo1,2,3- de -1,4-benzoxazinyl-(1-naphthalenyl)methanone mesylate (WIN55,212-2) in a concentration-dependent manner, and this antagonist
effect was not mimicked by S1P. FTY720 and sphingosine also inhibited activation of extracellular signal-regulated kinases
1 and 2 and Akt by WIN55,212-2 in intact Chinese hamster ovary (CHO) cells expressing CB 1 receptors and attenuated WIN55,212-2-stimulated internalization of a fluorescence-tagged CB 1 receptor in CHO cells. Moreover, both FTY720 and sphingosine produced rightward shifts in the concentration-effect curves
of cannabinoid agonists for G-protein activation, indicating that they act as competitive CB 1 antagonists. These results suggest that the CB 1 receptor could be a novel target of FTY720 and that sphingosine could be an endogenous CB 1 antagonist.
The main pharmacological effects of marijuana, as well as synthetic and endogenous cannabinoids, are mediated through G‐protein‐coupled receptors (GPCRs), including CB
1
and CB
2
receptors. The CB
1
...receptor is the major cannabinoid receptor in the central nervous system and has gained increasing interest as a target for drug discovery for treatment of nausea, cachexia, obesity, pain, spasticity, neurodegenerative diseases and mood and substance abuse disorders. Evidence has accumulated to suggest that CB
1
receptors, like other GPCRs, interact with and are regulated by several other proteins beyond the established role of heterotrimeric G‐proteins. These proteins, which include the GPCR kinases, β‐arrestins, GPCR‐associated sorting proteins, factor associated with neutral sphingomyelinase, other GPCRs (heterodimerization) and the novel cannabinoid receptor‐interacting proteins: CRIP
1a/b
, are thought to play important roles in the regulation of intracellular trafficking, desensitization, down‐regulation, signal transduction and constitutive activity of CB
1
receptors. This review examines CB
1
receptor‐interacting proteins, including heterotrimeric G‐proteins, but with particular emphasis on non‐G‐protein entities, that might comprise the CB
1
receptosomal complex. The evidence for direct interaction with CB
1
receptors and potential functional roles of these interacting proteins is discussed, as are future directions and challenges in this field with an emphasis on the possibility of eventually targeting these proteins for drug discovery.
This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit
http://dx.doi.org/10.1111/j.1476‐5381.2010.00831.x
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Inhibition of the metabolism of the endocannabinoids, anandamide (AEA) and 2-arachidonyl glycerol (2-AG), by their primary metabolic enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol ...lipase (MAGL), respectively, has the potential to increase understanding of the physiological functions of the endocannabinoid system. To date, selective inhibitors of FAAH, but not MAGL, have been developed. The purpose of this study was to determine the selectivity and efficacy of N-arachidonyl maleimide (NAM), a putative MAGL inhibitor, for modulation of the effects of 2-AG. Our results showed that NAM unmasked 2-AG activity in a tetrad of in vivo tests sensitive to the effects of cannabinoids in mice. The efficacy of 2-AG (and AEA) to produce hypothermia was reduced compared with Delta(9)-tetrahydrocannabinol; however, 2-AG differed from AEA by its lower efficacy for catalepsy. All tetrad effects were partially CB(1) receptor-mediated because they were attenuated (but not eliminated) by SR141716A N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide HCl and in CB(1)(-/-) mice. In vitro, NAM increased endogenous levels of 2-AG in the brain. Furthermore, NAM raised the potency of 2-AG, but not AEA, in agonist-stimulated guanosine 5'-O-(3-(35)Sthio)triphosphate binding assay, a measure of G-protein activation. These results suggest that NAM is an MAGL inhibitor with in vivo and in vitro efficacy. NAM and other MAGL inhibitors are valuable tools to elucidate the biological functions of 2-AG and to examine the consequences of dysregulation of this endocannabinoid. In addition, NAM's unmasking of 2-AG effects that are only partially reversed by SR141716A offers support for the existence of non-CB(1), non-CB(2) cannabinoid receptors.
The main pharmacological effects of marijuana, as well as synthetic and endogenous cannabinoids, are mediated through G‐protein‐coupled receptors (GPCRs), including CB1 and CB2 receptors. The CB1 ...receptor is the major cannabinoid receptor in the central nervous system and has gained increasing interest as a target for drug discovery for treatment of nausea, cachexia, obesity, pain, spasticity, neurodegenerative diseases and mood and substance abuse disorders. Evidence has accumulated to suggest that CB1 receptors, like other GPCRs, interact with and are regulated by several other proteins beyond the established role of heterotrimeric G‐proteins. These proteins, which include the GPCR kinases, β‐arrestins, GPCR‐associated sorting proteins, factor associated with neutral sphingomyelinase, other GPCRs (heterodimerization) and the novel cannabinoid receptor‐interacting proteins: CRIP1a/b, are thought to play important roles in the regulation of intracellular trafficking, desensitization, down‐regulation, signal transduction and constitutive activity of CB1 receptors. This review examines CB1 receptor‐interacting proteins, including heterotrimeric G‐proteins, but with particular emphasis on non‐G‐protein entities, that might comprise the CB1 receptosomal complex. The evidence for direct interaction with CB1 receptors and potential functional roles of these interacting proteins is discussed, as are future directions and challenges in this field with an emphasis on the possibility of eventually targeting these proteins for drug discovery.
This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476‐5381.2010.00831.x
Full text
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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