In-vivo pharmacological evaluation of the CB1-receptor allosteric modulator Org-27569
Abstract
Several allosteric modulators (AMs) of the CB1 receptor have been characterized in vitro, including Org27569, which enhances CB1-specific binding of [3H]CP55,940, but behaves as an insurmountable CB1-receptor antagonist in several biochemical assays. Although a growing body of research has investigated the molecular actions of this unusual AM, it is unknown whether these actions translate to the whole animal. The purpose of the present study was to determine whether Org27569 would produce effects in well-established mouse behavioral assays sensitive to CB1 orthosteric agonists and antagonists. Similar to the orthosteric CB1 antagonist/inverse agonist rimonabant, Org27569 reduced food intake; however, this anorectic effect occurred independently of the CB1 receptor. Org27569 did not elicit CB1-mediated effects alone and lacked efficacy in altering antinociceptive, cataleptic, and hypothermic actions of the orthosteric agonists anandamide, CP55,940, and Δ9-tetrahydrocannabinol. Moreover, it did not alter the discriminative stimulus effects of anandamide in FAAH-deficient mice or Δ9-tetrahydrocannabinol in wild-type mice in the drug discrimination paradigm. These findings question the utility of Org27569 as a ‘gold standard’ CB1 AM and underscore the need for the development of CB1AMs with pharmacology that translates from the molecular level to the whole animal.
Introduction
The discovery of molecules acting as CB1-receptor allosteric modulators (AMs) has fueled interest in developing novel therapeutic agents that elicit minimal cannabimimetic side effects associated with orthosteric CB1agonists and antagonists (Price et al., 2005; Horswill et al., 2007; Navarro et al., 2009; Bauer et al., 2012; Pamplona et al., 2012). Org27569, the most widely investigated CB1 AM, possesses an unusual pharmacological profile. It increases the binding affinity of CP55,940, a potent and efficacious orthosteric CB1 agonist, but abates receptor responses in functional assays, including CP55,940-stimulated GTPγS binding, contractions of mouse vas deferens, agonist-induced inhibition of cAMP production, and β-arrestin recruitment assays (Price et al., 2005; Ahn et al., 2012; Baillie et al., 2013). Other recent work has revealed that β-arrestin plays a role in CB1-biased signaling produced by Org27569 (Ahn et al., 2013). These paradoxical effects of Org27569 (i.e. enhanced orthosteric agonist binding, but antagonism of signaling events) have been proposed as being mediated by increased rates of CB1 desensitization that concomitantly cause cAMP levels and hyperpolarization states to return to baseline more rapidly than in the absence of the modulator (Cawston et al., 2013).
Although Org27569 represents the most extensively studied CB1 AM reported in the scientific literature, this work is based solely on in-vitro studies. If CB1 AMs are to be pursued as potential therapeutic agents, it will be important to ensure that prototypical compounds under investigation are active in the whole animal (Ross, 2007). Thus, the primary objective of the present study was to determine whether Org27569 functions as a CB1 AM in mice. On the basis of its dampening effect on CB1-receptor signaling, we initially assessed whether it would reduce food consumption, a hallmark of CB1 blockade (Di Marzo and Matias, 2005). Previously, PSNCBAM-1, which is structurally and pharmacologically similar to Org27569, was shown to reduce food consumption in rats, although CB1 involvement was not assessed (Horswill et al., 2007). Thus, in the present study CB1 (+/+) and (−/−) mice were used to infer whether any observed anorectic effects were CB1dependent. In addition, we tested Org27569 in well-established animal models sensitive to the cannabimimetic effects of CB1 orthosteric agonists, alone and in combination with CP55,940 or anandamide (AEA), which have been used in its in-vitro characterization, as well as with Δ9-tetrahydrocannabinol (THC).
Methods
Subjects
Male and female CB1 (+/+) and (−/−) mice were used in feeding studies, and male C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine, USA) or FAAH (−/−) mice were used in drug discrimination experiments. Male ICR mice (Harlan, Indianapolis, Indiana, USA) and C57BL/6 J mice were used in other experiments. All animal protocols were approved by the VCU IACUC and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (Resources IoLA, 2011).
Effects of Org27569 on feeding
Following a 1-week acclimation period, CB1 (+/+) and (−/−) mice were food-deprived, given an intraperitoneal injection of Org27569 (30mg/kg), rimonabant (10mg/kg; positive control), or vehicle at 23 h, and placed in a plastic cage with access to water. A premeasured amount (2.3–2.6 g) of sweet cereal (Froot Loops; Kellogg NA Co., Battle Creek, Michigan, USA) or standard chow (Teklad, Madison, Wisconsin, USA) was placed in the test cage from 24 to 26h. All mice received each treatment condition in a counterbalanced design, with at least 96 h between test days.
Cannabimimetic pharmacological effects of Org27569 in combination with orthosteric agonists
Cumulative dose–response relationships were assessed for AEA, CP55,940, and THC.
Drug discrimination
FAAH (−/−) and C57BL/6J mice were respectively trained to discriminate AEA (6.0mg/kg) and THC (5.6mg/kg) from vehicle using a fixed ratio 10 (FR10) schedule in nose-poke operant conditioning chambers (MED Associates, St Albans, Vermont, USA), as previously described (Ignatowska-Jankowska et al., 2013). Bioserv 14mg Dustless Precision Pellets (product # F05684; Bioserv, Frenchtown, New Jersey, USA) served as the reinforcer. During test sessions, responses in either aperture delivered reinforcement under an FR10 schedule.
Org27569 quantification in blood and brain
Org27569 was administered either intraperitoneally (30mg/kg) or intracerebroventricularly (100 μg) and levels of the drug were quantified, respectively, in the blood and brain, or brain, according to established methods (Kinsey et al., 2009), using the HPLC/MS/MS method with an Applied Bio systems (Carlsbad, CA, USA) 3200 Q trap with a turbo V source for TurboIonSpray attached to a Shimadzu SCL HPLC system (Kyoto, Japan). Chromatographic separation was performed using a Discovery HS C18, 2.1×150mm, 3 μm column (Supelco, St. Louis, MO, USA).
Drugs
Org27569 (University of Aberdeen, Scotland) and CP55,940, THC, AEA, PF-3845 (FAAH inhibitor that prevents AEA degradation), and rimonabant (CB1 antagonist/inverse agonist; NIDA, Bethesda, Maryland, USA) were dissolved in ethanol (Pharmco Products Inc., Brookfield, Connecticut, USA), Emulphor-620 (Rhodia, Cranbury, New Jersey, USA), and saline, in a ratio of 1 : 1 : 18. Org27569 was administered systemically in a volume of 20μl/g, and for intracerebroventricular injection it was dissolved in 100 % dimethyl sulfoxide and administered in a volume of 5μl. The injection volume for all other systemically administered compounds was 10 μl/g.
Data analysis
All data are reported as mean±SEM and were analyzed using analysis of variance. Dunnett’s test was used for the dose–response experiments and the Bonferroni–Dunn test was used for other post-hoc comparisons. Analyses were considered significant at P values less than 0.05.
Results
Org27569 (30mg/kg) significantly reduced consumption of standard chow (data not shown) and sweet cereal in both CB1 (−/−) and (+/+) mice [genotype by drug treatment interaction: F(2,60)=11.1, P<0.001; Fig. 1a]. In contrast, rimonabant (positive control) reduced intake in CB1 (+ /+) mice only. Org27569 did not alter the discriminative stimulus effects of or substitute for AEA (Fig. 1b and c) or THC (5.6mg/kg intraperitoneally; data not shown) in FAAH (−/−) mice and C57BL/6J mice, respectively. Moreover, Org27569 alone did not produce antinociception, catalepsy, or hypothermia. Further, Org27569 did not reduce the cataleptic, antinociceptive, or hypothermic effects produced by AEA in PF-3845-treated mice or FAAH (−/−) mice (data not shown). In fact, in FAAH (−/−) mice, Org27569 produced small but significant increases in the potency of AEA (potency ratio with 95 % confidence interval) to elicit catalepsy [1.3 (1.1–1.7)] and antinociception [1.9 (1.3–2.8)], but not hypothermia [1.1 (0.9–1.4)], compared with vehicle-pretreated FAAH (−/−) mice (data not shown). Org27569 (30mg/kg intraperitoneally) was detected in the brain (4.09±0.39 μg/mg) and blood (3.71±1.77mg/ml) 1 h after intraperitoneal administration. In an effort to assess the impact of further increasing brain levels of this compound, Org27569 (100 μg) was administered intracerebroventricularly. Despite achieving increased brain levels of Org27569 (i.e. 13.4±2.5 and 10.56±1.8 μg/mg at 0.75 and 1.75 h after injection, respectively), this compound did not alter the antinociceptive, cataleptic, or hypothermic dose–response relationships of intraperitoneally administered CP55,940 (0.1, 0.3, and 1mg/kg; Fig. 2). Finally, Org27569 (30 mg/kg, intraperitoneal) did not affect these pharmacological endpoints produced by intraperitoneally administered THC (3, 10, 30, and 100 mg/kg; data not shown).
Discussion
This study represents the first systematic evaluation of the CB1 AM Org27569 in well-established in-vivo assays sensitive to CB1 orthosteric agonists and antagonists. Org27569 produced hypophagic effects in both CB1 (−/−) and (+/+) mice. This finding is in contrast to that for rimonabant, which reduced food consumption only in CB1 (+/+) mice. These results indicate that the CB1 receptor is dispensable for Org27569-induced hypophagia. Org27569 did not elicit relevant behavioral or cannabimimetic effects when administered alone or in combination with three distinct CB1orthosteric agonists. Specifically, it did not substitute for either AEA or THC and did not modify the discriminative stimulus effects of either agonist in the drug discrimination paradigm. Moreover, Org27569 did not alter the cataleptic, antinociceptive, or hypothermic effects of THC in wild-type mice or AEA in PF-3845-treated FAAH (+/+) mice, although it slightly enhanced AEA-induced antinociception and catalepsy in FAAH (−/−) mice. Although pharmacokinetic issues represent a potential hurdle when studying drugs in the whole animal, it is noteworthy that 1 h after intraperitoneal administration, Org27569 was detected at equivalent concentrations in the brain and blood. Moreover, direct administration of Org27569 to the brain yielded a 2–3-fold increase in its levels in the brain; however, it still did not alter the cataleptic, antinociceptive, or hypothermic effects of systemically administered CP55,940.
The findings that Org27569 did not elicit CB1-mediated effects alone and lacked efficacy in altering the actions of orthosteric agonists in established murine behavioral assays sensitive to cannabimimetic activity indicate a lack of translation between its interesting in-vitro actions at the CB1 receptor and its effects in the whole animal. These results question the utility of Org27569 as a tool to elucidate CB1 allosteric site(s), if these compounds are to be pursued as potential pharmacotherapies. The development of new CB1 AMs with a pharmacology that extends from the molecular level to the whole animal is needed.
Acknowledgments
This research has been supported by the National Institute on Drug Abuse grants DA009789, DA017259, DA027113, DA026449, and 2T32DA007027. The authors thank Scott O’Neil for his assistance with these studies.
References
- Ahn KH, Mahmoud MM, Kendall DA. Allosteric modulator ORG27569 induces a CB1 cannabinoid receptor high affinity agonist binding state, receptor internalization and Gi-independent ERK1/2 activation. J Biol Chem. 2012;287:12070–12082.[PMC free article] [PubMed]
- Ahn KH, Mahmoud MM, Shim J-Y, Kendall DA. Distinct roles of β-arrestin 1 and β-arrestin 2 in Org27569-induced biased signaling and internalization of the cannabinoid receptor 1 (CB1) J Biol Chem. 2013;288:9790–9800. [PMC free article] [PubMed]
- Baillie GL, Horswill JG, Anavi-Goffer S, Reggio PH, Bolognini D, Abood ME, et al. CB1 receptor allosteric modulators display both agonist and signaling pathway specificity. Mol Pharmacol. 2013;83:322–338. [PMC free article] [PubMed]
- Bauer M, Chicca A, Tamborrini M, Eisen D, Lerner R, Lutz B, et al. Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors. J Biol Chem. 2012;287:36944–36967.[PMC free article] [PubMed]
- Cawston EE, Redmond WJ, Breen C, Grimsey N, Connor M, Glass M. Real-time characterisation of cannabinoid receptor 1 (CB1) allosteric modulators reveals novel mechanism of action. Br J Pharmacol. 2013;170:893–907. [PMC free article] [PubMed]
- Di Marzo V, Matias I. Endocannabinoid control of food intake and energy balance. Nat Neurosci. 2005;8:585–589. [PubMed]
- Falenski KW, Thorpe AJ, Schlosburg JE, Cravatt BF, Abdullah RA, Smith TH, et al. FAAH−/− mice display differential tolerance, dependence, and cannabinoid receptor adaptation after Δ9-tetrahydrocannabinol and anandamide administration. Neuropsychopharmacology. 2010;35:1775–1787. [PMC free article][PubMed]
- Horswill J, Bali U, Shaaban S, Keily J, Jeevaratnam P, Babbs A, et al. PSNCBAM-1, a novel allosteric antagonist at cannabinoid CB1 receptors with hypophagic effects in rats. Br J Pharmacol. 2007;152:805–814. [PMC free article] [PubMed]
- Ignatowska-Jankowska B, Ghosh S, Crowe M, Kinsey S, Niphakis M, Abdullah R, et al. In vivo characterization of the highly selective monoacylglycerol lipase inhibitor KML29: antinociceptive activity without cannabimimetic side effects. Br J Pharmacol. 2013 doi: 10.1111/bph.12298. Epub ahead of print. [PMC free article][PubMed] [Cross Ref]
- Kinsey SG, Long JZ, O’Neal ST, Abdullah RA, Poklis JL, Boger DL, et al. Blockade of endocannabinoid-degrading enzymes attenuates neuropathic pain. J Pharmacol Exp Ther. 2009;330:902–910. [PMC free article] [PubMed]
- Navarro HA, Howard JL, Pollard GT, Carroll F. Positive allosteric modulation of the human cannabinoid (CB1) receptor by RTI-371, a selective inhibitor of the dopamine transporter. Br J Pharmacol. 2009;156:1178–1184. [PMC free article] [PubMed]
- Pamplona FA, Ferreira J, de Lima OM, Duarte FS, Bento AF, Forner S, et al. Anti-inflammatory lipoxin A4 is an endogenous allosteric enhancer of CB1 cannabinoid receptor. Proc Natl Acad Sci USA. 2012;109:21134–21139. [PMC free article] [PubMed]
- Price MR, Baillie GL, Thomas A, Stevenson LA, Easson M, Goodwin R, et al. Allosteric modulation of the cannabinoid CB1 receptor. Mol Pharmacol. 2005;68:1484–1495. [PubMed]
- Resources IoLA. Guide for the care and use of laboratory animals.Washington, DC: Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council; 2011.
- Ross RA. Allosterism and cannabinoid CB1 receptors: the shape of things to come. Trends Pharmacol Sci. 2007;28:567–572. [PubMed]
