Cannabinoid CB1 and CB2 receptor ligand specificity and the development of CB2-selective agonists

Cannabinoids in current use such as nabilone activate both CB1 and CB2 receptors. Selective CB2 activation may provide some of the therapeutic effects of cannabinoids, such as their immuno-modulatory properties, without the psychoactive effects of CB1 activation. Therefore, cannabinoid CB2 receptors represent an attractive target for drug development. However, selective and potent CB2 agonists remain in development. CB1 and CB2 differ considerably in their amino acid sequence and tertiary structures. Therefore, clinical development of potent and selective CB2 agonists is probable. Mutational and ligand binding studies, functional mapping, and computer modelling have revealed key residues and domains in cannabinoid receptors that are involved in agonist and antagonist binding to CB1 and CB2. In addition, CB2 has undergone more rapid evolution, and results for ligand binding and efficacy cannot be automatically extrapolated from rat or mouse CB2 to human. Furthermore, loss of CB1 affinity is a crucial property for CB2-selective ligands, and although rat CB1 is 97% homologous with human CB1, critical differences do exist, with potential for further exploitation in drug design. In this paper we briefly review previous cannabinoid receptor models and mutation/binding studies. We also review binding affinity ratios with respect to CB1 and CB2. We then employ our own models to illustrate key cannabinoid receptor residues and binding subdomains that are involved in these differences in binding affinities and discuss how these might be exploited in the development of CB2 specific ligands. Published reports for species specific binding affinities for CB2 are scarce, and we argue that this needs to be corrected prior to the progression of CB2 agonists from pre-clinical to clinical research.     

The role of the cannabinoid CB2 receptor in pain transmission and therapeutic potential of small molecule CB2 receptor agonists

This review gives a brief overview of the expression patterns, molecular pharmacology and physiological role of the cannabinoid 2 receptor (CB2) in pain. Particular emphasis is given to the therapeutic utility of CB2 receptor agonists. Through studies utilizing selective CB2 receptor agonists, non-selective cannabinoid agonists in conjunction with selective CB1 and CB2 receptor antagonists, or CB2 receptor knockout mice, it is now clear that this receptor plays a critical role in nociception. To this end, CB2 receptors have been shown to modulate acute pain, chronic inflammatory pain, post-surgical pain, cancer pain and pain associated with nerve injury. Here we review these studies and the compounds that were utilized. We hypothesize the mechanism of action by which the CB2 receptor could be involved in these processes. Finally we summarize the most recent novel chemical scaffolds that are being investigated towards advancing selective CB2 receptor agonists into the clinic. Many new pharmacological agents have been identified by high throughput screening and small molecule lead discovery and optimization in the past 10 years. It is anticipated that at least some of these agents may ultimately constitute effective new pain therapeutics that lack the side effects associated with traditional cannabinoid ligands.     

Cannabidiol displays unexpectedly high potency as an antagonist of CB1 and CB2 receptor agonists in vitro

Background and purpose:     

Neuroprotective potential of CB1 receptor agonists in an in vitro model of Huntington's disease

Background and purpose:

The therapeutic potential of cannabinoids in Huntington''s disease (HD) has been investigated by several groups with complex and sometimes contrasting results. We sought to examine key points of intersection between cannabinoid receptor 1 (CB₁) signalling, survival and the formation of mutant huntingtin aggregates in HD.

Experimental approach:

Using a simplified pheochromocytoma (PC12) cell model of HD expressing exon 1 of wild-type or mutant huntingtin, we assayed cell death and aggregate formation using high-throughput cytotoxicity and image-based assays respectively.

Key results:

CB₁ activation by HU210 conferred a small but significant level of protection against mutant huntingtin-induced cell death. Pertussis toxin uncoupled HU210 from the inhibition of cAMP, preventing rescue of cell death. Phosphorylation of extracellular signal-regulated kinase (ERK) was also critical to CB₁-mediated rescue. Conversely, treatments that elevated cAMP exacerbated mutant huntingtin-induced cell death. Despite opposing effects on HD cell survival, both HU210 and compounds that elevated cAMP increased the formation of mutant huntingtin aggregates. The increase in aggregation by HU210 was insensitive to Pertussis toxin and UO126, suggesting a G-protein alpha subtype s (G_s)-linked mechanism.

Conclusions and implications:

We suggest that the CB₁ receptor, through G-protein alpha subtype i/o (G_i/o)-linked, ERK-dependent signal transduction, is a therapeutic target in HD. However the protective potential of CB₁ may be limited by promiscuous coupling to G_s, the stimulation of cAMP formation and increased aggregate formation. This may underpin the poor therapeutic efficacy of cannabinoids in more complex model systems and suggest that therapies that are selective for the G_i/o, ERK pathway may be of most benefit in HD.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     

Cannabinoid CB(1) receptor upregulation in a rat model of chronic neuropathic pain

Although cannabinoids are known to be more effective analgesics against chronic rather than acute pain, the mechanism underlying this phenomenon is still unclear. We report now that contralateral thalamic cannabinoid CB(1) receptors are upregulated after unilateral axotomy of the tibial branch of the sciatic nerve, a rat model of chronic neuropathic pain, and hypothesize that cannabinoid CB(1) receptor upregulation contributes to the increased analgesic efficacy of cannabinoids in chronic pain conditions.     

CB1 cannabinoid receptors promote oxidative/nitrosative stress, inflammation and cell death in a murine nephropathy model

Background and purpose:

Accumulating recent evidence suggests that cannabinoid-1 (CB₁) receptor activation may promote inflammation and cell death and its pharmacological inhibition is associated with anti-inflammatory and tissue-protective effects in various preclinical disease models, as well as in humans.

Experimental approach:

In this study, using molecular biology and biochemistry methods, we have investigated the effects of genetic deletion or pharmacological inhibition of CB₁ receptors on inflammation, oxidative/nitrosative stress and cell death pathways associated with a clinically relevant model of nephropathy, induced by an important chemotherapeutic drug cisplatin.

Results:

Cisplatin significantly increased endocannabinoid anandamide content, activation of p38 and JNK mitogen-activated protein kinases (MAPKs), apoptotic and poly (ADP-ribose)polymerase-dependent cell death, enhanced inflammation (leucocyte infiltration, tumour necrosis factor-α and interleukin-1β) and promoted oxidative/nitrosative stress [increased expressions of superoxide-generating enzymes (NOX2(gp91phox), NOX4), inducible nitric oxide synthase and tissue 4-hydroxynonenal and nitrotyrosine levels] in the kidneys of mice, accompanied by marked histopathological damage and impaired renal function (elevated creatinine and serum blood urea nitrogen) 3 days following its administration. Both genetic deletion and pharmacological inhibition of CB₁ receptors with AM281 or SR141716 markedly attenuated the cisplatin-induced renal dysfunction and interrelated oxidative/nitrosative stress, p38 and JNK MAPK activation, cell death and inflammatory response in the kidney.

Conclusions and implications:

The endocannabinoid system through CB₁ receptors promotes cisplatin-induced tissue injury by amplifying MAPK activation, cell death and interrelated inflammation and oxidative/nitrosative stress. These results also suggest that inhibition of CB₁ receptors may exert beneficial effects in renal (and most likely other) diseases associated with enhanced inflammation, oxidative/nitrosative stress and cell death.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     

Cannabinoid receptor 2 (CB2) agonists and antagonists: a patent update

Introduction: Modulation of the CB₂ receptor is an interesting approach for pain and inflammation, arthritis, addictions, neuroprotection, and cancer, among other possible therapeutic applications, and is devoid of central side effects.

Areas covered: This review highlights the novel scaffolds for CB₂ ligands and the diverse therapeutic applications for CB₂ modulators disclosed in patents published since 2012.

Expert opinion: Structural diversity of CB₂ modulator scaffolds characterized the patent literature. Several CB₂ agonists reached clinical Phase II for pain management and inflammation. Other therapeutic applications need to be explored such as neuroprotection and/or neurodegeneration.     

Biology and therapeutic potential of cannabinoid CB2 receptor inverse agonists

Evidence has emerged suggesting a role for the cannabinoid CB2 receptor in immune cell motility. This provides a rationale for a novel and generalized immunoregulatory role for cannabinoid CB2 receptor-specific compounds. In support of this possibility, we will review the biology of a class of cannabinoid CB2 receptor-specific inverse agonist, the triaryl bis-sulfones. We will show that one candidate, Sch.414319, is potent and selective for the cannabinoid CB2 receptor, based on profiling studies using biochemical assays for 45 enzymes and 80 G-protein coupled receptors and ion channels. We will describe initial mechanistic studies using this optimized triaryl bis-sulfone, showing that the compound exerts a broad effect on cellular protein phosphorylations in human monocytes. This profile includes the down regulation of a required phosphorylation of the monocyte-specific actin bundling protein L-plastin. We suggest that this observation may provide a mechanism for the observed activity of Sch.414319 in vivo. Our continued analysis of the in vivo efficacy of this compound in diverse disease models shows that Sch.414319 is a potent modulator of immune cell mobility in vivo, can modulate bone damage in antigen-induced mono-articular arthritis in the rat, and is uniquely potent at blocking experimental autoimmune encephalomyelitis in the rat.     

Alterations in behavioral flexibility by cannabinoid CB1 receptor agonists and antagonists

Rationale Cannabinoid CB₁ receptors are expressed in the prefrontal cortex, but their role in mediating executive functions such as behavioral flexibility is unclear.Objective The present study examined the effect of pharmacological activation or blockade of the cannabinoid CB₁ receptors on behavioral flexibility using a strategy set-shifting task conducted on a cross maze.Materials and methods In experiment 1, rats initially were trained to turn left or right while ignoring the visual cue to obtain a food; on the second test day, rats had to inhibit the previously learned rule and approach the cue to obtain the food. In experiment 2, the order of discrimination training was reversed.Results Administration of the cannabinoid CB₁ receptor agonist HU-210 before the set-shift on day 2 elicited dose-dependent effects on performance. A 20-μg/kg dose of HU-210 increased perseverative errors, whereas the effects of a lower, 5-μg/kg dose caused differential effects depending on whether rats were required to shift from a response to a visual-cue discrimination strategy or vice versa. Conversely, administration of a 2-mg/kg, but not a 5-mg/kg dose of the CB₁ receptor antagonist AM251 reduced perseverative errors.Conclusions These data demonstrate a biphasic and dose-sensitive role for the cannabinoid system in behavioral flexibility, which in turn may have clinical implications for the role of the endocannabinoid system in psychiatric disorders where behavioral flexibility is compromised.     

Cannabinoid CB2 receptor agonist activity in the hindpaw incision model of postoperative pain

The identification of peripherally expressed CB2 receptors and reports that the selective activation of cannabinoid CB2 receptors produces antinociception without traditional cannabinergic side effects suggests that selective cannabinoid CB2 receptor agonists might be useful in the management of pain. In a rat hindpaw incision model, we examined the antiallodynic activity of the selective cannabinoid CB2 receptor agonists AM1241 (3-30 mg/kg i.p.), GW405833 (3-30 mg/kg i.p.), and HU-308 (0.3-30 mg/kg i.p.). The rank order for efficacy in the hindpaw incision model following a dose of 10 mg/kg, i.p. was AM1241 > GW405833 = HU-308, and the selective cannabinoid CB2 receptor antagonist, SR144528, reversed the antiallodynic effect of HU-308. Together, these data suggest that selective cannabinoid CB2 receptor agonists might represent a new class of postoperative analgesics.     

Involvement of central cannabinoid CB2 receptor in reducing mechanical allodynia in a mouse model of neuropathic pain

We sought to examine the involvement of central cannabinoid CB2 receptor activation in modulating mechanical allodynia in a mouse model of neuropathic pain. JWH133 was demonstrated to be a selective cannabinoid CB2 receptor agonist in mice, reducing forskolin-stimulated cAMP production in CHO cells expressing mouse cannabinoid CB2 and cannabinoid CB1 receptors with EC50 values of 63 nM and 2500 nM, respectively. Intrathecal administration of JWH133 (50 and 100 nmol/mouse) significantly reversed partial sciatic nerve ligation-induced mechanical allodynia in mice at 0.5 h after administration. In contrast, systemic (intraperitoneal) or local (injected to the dorsal surface of the hindpaw) administration of JWH133 (100 nmol/mouse) was ineffective. Furthermore, the analgesic effects of intrathecal JWH133 (100 nmol/mouse) were absent in cannabinoid CB2 receptor knockout mice. These results suggest that the activation of central, but not peripheral, cannabinoid CB2 receptors play an important role in reducing mechanical allodynia in a mouse model of neuropathic pain.     

Synthesis and SAR studies of 2-oxoquinoline derivatives as CB2 receptor inverse agonists

The highly CB2 selective cannabinoid receptor inverse agonist, 7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxylic acid N-benzo[1,3]dioxol-5-ylmethyl)amide (JTE-907; 9b), served as the lead compound for investigating the structure-activity relationships of its analogues and in the search for more potent and effective CB2 receptor inverse agonists. A series of aromatic amides of 7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxylic acid 6 was synthesized, and the CB2 receptor activities of the compounds were determined by a [35S]GTPgammaS-binding assay using membranes of CHO cells stably transfected with the human CB2 receptor. As a result, all the compounds were defined as full CB2 receptor inverse agonists, and additionally, except for two 3,4-dihydroxyphenylalkylamides, they were found to be equally potent as SR144528.     

Synthesis and in vitro evaluation of novel 2-oxo-1,2-dihydroquinoline CB2 receptor inverse agonists

Aliphatic amides of 7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxylic acid 6 were synthesized and evaluated for their CB2 and CB1 receptor activities by a [35S]GTPgammaS membrane-binding assay. Compounds 7-18 produced CB2 inverse agonist activities with a variety of potencies (IC50: 1 nm-1 microm). By contrast, no considerable antagonism of CB1 receptor was observed by 1-10 microm concentrations indicating that synthesized compounds were CB2 receptor selective.     

Analgesic and antiinflammatory effects of cannabinoid receptor agonists in a rat model of neuropathic pain

Cannabinoid receptor (CB) agonists are known to attenuate allodynia in a range of pain models, but their long-term effects and their mechanisms of action are controversial. The present study compares the antiallodynic effects of long-term treatment with a mixed CB1/CB2 (WIN55,212-2) and a selective CB2 (GW405833) cannabinoid receptor agonist and correlates these effects with their influences on spinal cord (SC) glial activation. The substances were applied daily in a rat neuropathic pain model. Tactile allodynia was assessed, and the development of gliosis was illustrated with immunohistochemical methods. Both substances reduced mechanical allodynia. Their analgesic effect was accompanied by a significant reduction in reactive gliosis and cathepsins (CAT) X and S expression. A daily injection of either substance for 8 days was sufficient to induce a sustained antiallodynic effect, which persisted up to 6 days after the last injection. The re-appearance of mechanical allodynia after this period was associated with a breakout of a strong gliotic response in the lumbar SC. Our results emphasize the therapeutic efficacy of cannabinoid receptor agonists and their inhibitory effects on the formation of gliosis.     

Differentiation between low- and high-efficacy CB1 receptor agonists using a drug discrimination protocol for rats

Rationale

The “subjective high” from marijuana ingestion is likely due to Δ⁹-tetrahydrocannabinol (THC) activating the central cannabinoid receptor type 1 (CB₁R) of the endocannabinoid signaling system. THC is a weak partial agonist according to in vitro assays, yet THC mimics the behavioral effects induced by more efficacious cannabinergics. This distinction may be important for understanding similarities and differences in the dose–effect spectra produced by marijuana/THC and designer cannabimimetics (“synthetic marijuana”).

Objective

We evaluated if drug discrimination is able to functionally detect/differentiate between a full, high-efficacy CB₁R agonist [(±)AM5983] and the low-efficacy agonist THC in vivo.

Materials and methods

Rats were trained to discriminate between four different doses of AM5983 (0.10 to 0.56 mg/kg), and vehicle and dose generalization curves were determined for both ligands at all four training doses of AM5983. The high-efficacy WIN55,212-2 and the lower-efficacy (R)-(+)-methanandamide were examined at some AM5983 training conditions. Antagonism tests involved rimonabant and WIN55,212-2 and AM5983. The separate (S)- and (R)-isomers of (±)AM5983 were tested at one AM5983 training dose (0.30 mg/kg). The in vitro cyclic adenosine monophosphate (cAMP) assay examined AM5983 and the known CB₁R agonist CP55,940.

Results

Dose generalization ed₅₀ values increased as a function of the training dose of AM5983, but more so for the partial agonists. The order of potency was (R)-isomer > (±)AM5983 > (S)-isomer and AM5983 > WIN55,212-2 ≥ THC > (R)-(+)-methanandamide. Surmountable antagonism of AM5983 and WIN55,212-2 occurred with rimonabant. The cAMP assay confirmed the cannabinergic nature of AM5983 and CP55,940.

Conclusions

Drug discrimination using different training doses of a high-efficacy, full CB₁R agonist differentiated between low- and high-efficacy CB₁R agonists.     

Virtual screening of novel CB2 ligands using a comparative model of the human cannabinoid CB2 receptor

To identify novel selective CB2 lead compounds, a comparative model of the CB2 receptor was constructed using the high-resolution bovine rhodopsin X-ray structure as a template. The CB2 model was utilized both in building the database queries and in filtering the hit compounds by a docking and scoring method. In G-protein activation assays, 1-isoquinolyl[3-(trifluoromethyl)phenyl]methanone (40, NRB 04079) was found to act as a selective agonist at the human CB2 receptor.     

SR141716A诱导大鼠产生与CB1受体激动剂相反的行为模式(英文)

AIM: To examine the acute actions of the CB1cannabinoid receptor antagonist SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carbowamide] on typical behavioralpattern of psychoactive cannabinoids in rats.METHODS:At different time after injection the tail-flick response latency,the rectal temperature,thelocomotor activity,and the immobility on a ring as wellas the numbers of rears,self-grooming episodes(lasting 5 s),and fecal pellets were measured.RESULTS:Achte administration of SR141716A(3 mg/kg ip) induced a significant increase inhorizontal locomotor activity assayed by an activitymeter,in stereotypic activity(such as rearing and self-     

Expression of the murine CB2 cannabinoid receptor using a recombinant Semliki Forest virus

A recombinant Semliki Forest virus (SFV) RNA construct, SFV1-mCB(2) RNA, was employed for the high-level expression of the murine CB(2) (mCB(2)) cannabinoid receptor in baby hamster kidney cells. Biosynthetic radiolabel incorporation studies in concert with urea-sodium dodecylsulfate-polyacrylamide gel electrophoresis (urea-SDS-PAGE) and western immunoblotting revealed that two major proteins of approximately 26 and 40kDa were produced by the construct. The 40kDa product, but not the 26kDa product, was glycosylated as determined by 2-deoxy-D-glucose incorporation and peptide-N-glycosidase F digestion analysis. Assessment of [3H]CP55940 ([3H]-(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) binding data for membranes of cells transfected with SFV1-mCB(2) RNA indicated a K(d) of 0.35+/-0.04nM and a B(max) of 24.4+/-2.7pmol/mg. A rank order of binding affinities for cannabinoids, which paralleled that reported for native mCB(2) receptors, was observed. The CB(2) receptor-specific antagonist SR144528 (N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) blocked binding of [3H]CP55940, while the CB(1) receptor-specific antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] had a minimal effect. These results indicate that the recombinant receptor expressed from SFV1-mCB(2) RNA exhibits properties, including ligand binding features, that are consistent with those for the native mCB(2) receptor. However, the presence of both 26 and 40kDa receptor species is consistent with alternative translation from two AUG start sites using the SFV1-mCB(2) RNA expression system.     

Morphine analgesia suppresses tumor growth and metastasis in a mouse model of cancer pain produced by orthotopic tumor inoculation

The present study was conducted to clarify whether relief from cancer pain by morphine would suppress tumor growth and metastasis. When given orthotopic inoculation of B16-BL6 melanoma cells into the hind paw, C57BL/6 mice showed moderate and marked hyperalgesia on days 7-10 and from day 14 post-inoculation, respectively. The volume of inoculated hind paw was increased exponentially as a function of time from day 8 post-inoculation, a phenomena being due to melanoma growth. Lung metastasis was apparent after day 12 post-inoculation. On day 16 post-inoculation, the hyperalgesia was completely inhibited by subcutaneous injection of morphine hydrochloride (5 and 10 mg/kg). The tumor growth and lung metastasis were markedly inhibited by repeated administration of morphine (5 and 10 mg/kg daily for 6 days) and also by the neurectomy of sciatic nerve innervating the inoculated region. The results suggest that relief from cancer pain by morphine inhibits tumor growth and metastasis.     

SR141716A诱导大鼠产生与CB1受体激动剂相反的行为模式

AIM: To examine the acute actions of the CB1 cannabinoid receptor antagonist SR141716A [N-piperidino-5-(4-chlorophenyl)- 1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide] on typical behavioral pattern of psychoactive cannabinoids in rats. METHODS: At different time after injection the tail-flick response latency, the rectal temperature, the locomotor activity, and the immobility on a ring as well as the numbers of rears, self-grooming episodes (lasting 5 s), and fecal pellets were measured. RESULTS: Acute administration of SR141716A (3 mg/kg i.p.) induced a significant increase in horizontal locomotor activity assayed by an activity meter, in stereotypic activity (such as rearing and self-grooming) and in defecation, and a decrease in nociceptive threshold recorded as tail-flick latency. This dose had no effect on ring immobility and did not change the body temperature. CONCLUSION: These results demonstrate that this cannabinoid antagonist itself was inducing behavior opposite to that of CB1 receptor agonists.