Iain R. Greig

Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors

Accelerated osteoclastic bone resorption has a central role in the pathogenesis of osteoporosis and other bone diseases. Identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and to the development of new treatments. Here we show that mice with inactivation of cannabinoid type 1 (CB1) receptors have increased bone mass and are protected from ovariectomy-induced bone loss. Pharmacological antagonists of CB1 and CB2 receptors prevented ovariectomy-induced bone loss in vivo and caused osteoclast inhibition in vitro by promoting osteoclast apoptosis and inhibiting production of several osteoclast survival factors. These studies show that the CB1 receptor has a role in the regulation of bone mass and ovariectomy-induced bone loss and that CB1- and CB2-selective cannabinoid receptor antagonists are a new class of osteoclast inhibitors that may be of value in the treatment of osteoporosis and other bone diseases.

Modulation of l-α-Lysophosphatidylinositol/GPR55 Mitogen-activated Protein Kinase (MAPK) Signaling by Cannabinoids

Background: The endogenous l-α-lysophosphatidylinositol activates GPR55. Results: Structural analogues of SR141716A act both as agonists alone and as inhibitors of l-α-lysophosphatidylinositol. Certain CB2 receptor agonists also modulate GPR55 activity. Conclusion: Certain cannabinoids can both activate GPR55 and attenuate l-α-lysophosphatidylinositol-mediated phosphorylated ERK1/2 activation. This has mechanistic implications for the antinociceptive effects of certain CB2 agonists. Significance: Cannabinoid ligands have complex interactions with the l-α-lysophosphatidylinositol/GPR55 signaling system. GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.

MODULATION OF L-α-LYSOPHOSPHATIDYLINOSITOL /GPR55 MAP KINASE SIGNALLING BY CANNABINOIDS

Background: The endogenous l-α-lysophosphatidylinositol activates GPR55. Results: Structural analogues of SR141716A act both as agonists alone and as inhibitors of l-α-lysophosphatidylinositol. Certain CB2 receptor agonists also modulate GPR55 activity. Conclusion: Certain cannabinoids can both activate GPR55 and attenuate l-α-lysophosphatidylinositol-mediated phosphorylated ERK1/2 activation. This has mechanistic implications for the antinociceptive effects of certain CB2 agonists. Significance: Cannabinoid ligands have complex interactions with the l-α-lysophosphatidylinositol/GPR55 signaling system. GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.

In-vivo pharmacological evaluation of the CB1-receptor allosteric modulator Org-27569

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 &Dgr;9-tetrahydrocannabinol. Moreover, it did not alter the discriminative stimulus effects of anandamide in FAAH-deficient mice or &Dgr;9-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 CB1 AMs with pharmacology that translates from the molecular level to the whole animal.

Identification of Biphenylcarboxylic Acid Derivatives as a Novel Class of Bone Resorption Inhibitors

A novel class of biphenylcarboxylic acid derivatives are described that inhibit osteoclastic bone resorption in vitro by promoting osteoclast apoptosis and that prevent ovariectomy‐induced bone loss in vivo. The compounds act by a novel mechanism that seems to be distinct from existing antiresorptive drugs.

ABD56 causes osteoclast apoptosis by inhibiting the NFκB and ERK pathways

We have previously shown that the biphenylcarboxylic acid butanediol ester (ABD56) inhibits osteoclast formation and activity in vitro and in vivo. However, the mechanism of action of this compound is unknown. ABD56 inhibited osteoclast formation and caused osteoclast apoptosis, but had no effects on osteoblasts or macrophages. As the NFkappaB and MAPK pathways are essential for osteoclast formation and survival, we studied the effects of ABD56 on these pathways. ABD56 caused phosphorylation of p38, JNK and nuclear translocation of c-jun in osteoclasts. ABD56-induced apoptosis was prevented by the caspase inhibitor zVAD-fmk but was not prevented by the p38- or JNK-inhibitors. ABD56 completely abolished RANKL-induced IkappaB and ERK1/2 phosphorylation. Increasing the amount of RANKL partially rescued ABD56-induced apoptosis, indicating that the apoptosis is most probably due to the inhibition of survival signals such as ERK and NFkappaB, rather than activation of the p38 or Jnk MAPK pathways.

Identification of novel biphenyl carboxylic acid derivatives as novel antiresorptive agents that do not impair parathyroid hormone-induced bone formation.

Bisphosphonates are widely used in the treatment of osteoporosis, but they inhibit bone formation and blunt the anabolic effect of PTH. Here we describe a novel series of compounds that have potent antiresorptive effects in vitro and in vivo that do not adversely affect osteoblast function. The effects of the compounds on osteoclast formation and survival were studied on mouse osteoclasts generated from bone marrow macrophages and on osteoblast function using primary mouse calvarial osteoblast cultures and bone nodule cultures. Studies were performed in vivo using sham-operated or ovariectomized mice. The most potent compound tested was ABD350, a halogen-substituted derivative of the parent compound ABD56 in which the labile ester bond was replaced by a reduced ketone link, with IC50 osteoclast formation at a concentration of 1.3 microm. All compounds inhibited receptor activator of nuclear factor-kappaB ligand-induced inhibitor of nuclear factor kappaB phosphorylation and caused osteoclast apoptosis but no inhibitory effects on osteoblast function were observed at concentrations of up to 20 microm. ABD350 prevented ovariectomy-induced bone loss when given ip (5 mg/kg.d), whereas ABD56 was only partially effective at this dose. In contrast to the bisphosphonate alendronate, ABD350 had no inhibitory effect on PTH-induced bone formation in ovariectomized mice. In conclusion, the biphenyl carboxylic acid derivatives like ABD350 represent a new class of antiresorptive drugs that inhibit osteoclast activity but have no significant inhibitory effects on osteoblast activity in vitro or PTH-induced bone formation in vivo.

The synthesis and evaluation of o-phenylenediamine derivatives as fluorescent probes for nitric oxide detection

A series of molecular probes for the determination of nitric oxide (NO) have been prepared. Each probe consists of an anthracene, coumarin or acridine fluorophore coupled to an electron rich o-phenylenediamine group. The o-phenylenediamine group can be substituted with methyl or methoxy groups to enhance its electron rich nature. The fluorophore fluorescence is quenched by photoelectron transfer (PET) from the aromatic amine to the lowest unoccupied orbital of the excited state fluorophore. Reaction with nitrosating species converts the o-phenylenediamine group into an electron deficient benzotriazole derivative. This group has a higher oxidation potential and does not quench the fluorophore fluorescence by photoelectron transfer so that these products are highly fluorescent. Some benzotriazole derivatives were made preparatively by alternative synthetic routes. The formation of fluorescent probes was evaluated by treatment of the precursors with nitrous fumes and S-nitroso-N-acetylpenicillamine (SNAP).

Development of triarylsulfonamides as novel anti-inflammatory agents

Triaylsulfonamides were identified as novel anti-inflammatory agents, acting by inhibition of RANKL and TNFα signaling. Structure-activity studies led to the identification of compounds with in vitro potencies of <100 nM against J774 macrophages and osteoclasts, but with little activity against osteoblasts or hepatocytes (IC(50) >50 μM). A representative compound (4k, ABD455) was able to completely prevent inflammation in vivo in a prevention model and was highly effective at controlling inflammation in a treatment model.

Identification of small molecule inhibitors of RANKL and TNF signalling as anti-inflammatory and antiresorptive agents in mice

Introduction Inflammatory joint diseases such as rheumatoid arthritis are associated with local bone erosions and systemic bone loss, mediated by increased osteoclastic activity. The receptor activator of nuclear factor (NF) κB ligand (RANKL) plays a key role in mediating inflammation-induced bone loss, whereas tumour necrosis factor (TNF) plays a central role in the inflammatory process. Here we tested whether a recently identified class of small molecule inhibitors of RANKL signalling (ABD compounds) also affect TNF signalling and whether these compounds inhibit inflammation in an animal model of rheumatoid arthritis. Methods The inhibitory effects of the ABD compounds on TNF-induced signalling were tested in mouse macrophage cultures by western blotting and in an NFκB luciferase-reporter cell line. The anti-inflammatory effects of the compounds were tested in the mouse collagen-induced arthritis model of rheumatoid arthritis. Results The ABD compounds ABD328 and ABD345 both inhibited TNF-induced activation of the NFκB pathway and the extracellular signal-regulated kinase (ERK) and Jun kinase (JNK) mitogen activated protein kinases (MAPKs). When tested in the mouse collagen-induced arthritis model of rheumatoid arthritis, the compounds suppressed inflammatory arthritis, inhibited joint destruction and prevented systemic bone loss. Furthermore, one of the compounds (ABD328) showed oral activity. Conclusions Here we describe a novel class of small molecule compounds that inhibit both RANKL- and TNF-induced NFκB and MAPK signalling in osteoclasts and macrophages, and inflammation and bone destruction in a mouse model of rheumatoid arthritis. These novel compounds therefore represent a promising new class of treatments for inflammatory diseases, such as rheumatoid arthritis.