Association of cannabinoid receptor modulation with normal and abnormal skeletal remodelling: A systematic review and meta-analysis of in vitro, in vivo and human studies.

To address the inconsistent findings from studies that used different models to explore the role of classical cannabinoid type 1 (CB1) and 2 (CB2) receptors in skeletal remodelling, we searched Medline, Web of Science and Embase for relevant studies from inception to June 23, 2020. We identified 38 in vitro, 34 in vivo and 9 human studies. A meta-analysis of in vitro studies showed that exposure to the inverse-agonists AM251 (mean difference [MD]:-26.75, 95% confidence interval [CI]:-45.36,-8.14, p = 0.005), AM630 (standardised[std.] MD:-3.11, CI:-5.26,-0.97, p = 0.004; SR144528, std.MD:-4.88, CI -7.58,-2.18, p = 0.0004) and CBD (std.MD:-1.39, CI -2.64,-0.14, p = 0.03) is associated with reduced osteoclastogenesis, whereas the endocannabinoid 2-AG (std.MD:2.00, CI:0.11-3.89, p = 0.04) and CB2-selective agonist HU308 (MD:19.38, CI:11.75-27.01, p < 0.00001) were stimulatory. HU308 also enhanced osteoblast differentiation (std.MD:2.22, CI:0.95-3.50, p = 0.0006) and activity (std.MD:2.97, CI:1.22-4.71, p = 0.0008). In models of bone loss, CB1/2 deficiency enhanced peak bone volume (std.MD:3.70, CI:1.77-5.63, p = 0.0002) but reduced bone formation (std.MD:-0.54, CI:-0.90,-0.17, p = 0.004) in female mice. In male rats, CB1/2 deficiency (std.MD:2.31, CI:0.30-4.33, p = 0.02) and AM251 or CBD treatments (std.MD:2.19, CI:0.46-3.93, p = 0.01) enhanced bone volume. CB1/2 deficiency (std.MD:9.78, CI:4.96-14.61, p < 0.0001) and AM251 or AM630 treatments (std.MD:28.19, CI:19.13-37.25, p < 0.0001) were associated with osteoprotection. The CB2-selective agonists JWH133 and 4Q3C enhanced bone volume in arthritic rodents (std.MD:14.45, CI:2.08-26.81, p = 0.02). In human, CB2 SNPs (AA:rs2501431, MD:-0.28, CI:-0.55,-0.01, p = 0.04; CC:rs2501432, MD:-0.29, CI:-0.56,-0.02, p = 0.03) were associated with reduced bone mineral density, however the association of Marijuana use remains unclear. Thus, CB1/2 modulation is associated with altered bone metabolism, however findings are confounded by low study number and heterogenicity of models.

JZL184, A Monoacylglycerol Lipase Inhibitor, Induces Bone Loss in a Multiple Myeloma Model of Immunocompetent Mice.

Multiple myeloma (MM) patients develop osteolysis characterised by excessive osteoclastic bone destruction and lack of osteoblast bone formation. Pharmacological manipulation of monoacylglycerol lipase (MAGL), an enzyme responsible for the degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG), reduced skeletal tumour burden and osteolysis associated with osteosarcoma and advanced breast and prostate cancers. MM and hematopoietic, immune and bone marrow cells express high levels of type 2 cannabinoid receptor and osteoblasts secrete 2-AG. However, the effects of MAGL manipulation on MM have not been investigated. Here, we report that treatment of pre-osteoclasts with non-cytotoxic concentrations of JZL184, a verified MAGL inhibitor, enhanced MM- and RANKL-induced osteoclast formation and size in vitro. Exposure of osteoblasts to JZL184 in the presence of MM cell-derived factors reduced osteoblast growth but had no effect on the ability of these cells to mature or form bone nodules. In vivo, administration of JZL184 induced a modest, yet significant, bone loss at both trabecular and cortical compartments of long bones of immunocompetent mice inoculated with the syngeneic 5TGM1-GFP MM cells. Notably, JZL184 failed to inhibit the in vitro growth of a panel of mouse and human MM cell lines, or reduce tumour burden in mice. Thus, MAGL inhibitors such as JZL184 can exacerbate MM-induced bone loss.

Pharmacological Inhibition of NFκB Reduces Prostate Cancer Related Osteoclastogenesis In Vitro and Osteolysis Ex Vivo.

NFκB is implicated in cancer and bone remodelling, and we have recently reported that the verified NFκB inhibitor Parthenolide (PTN) reduced osteolysis and skeletal tumour growth in models of metastatic breast cancer. Here, we took advantage of in vitro and ex vivo bone cell and organ cultures to study the effects of PTN on the ability of prostate cancer cells and their derived factors to regulate bone cell activity and osteolysis. PTN inhibited the in vitro growth of a panel of human, mouse and rat prostate cancer cells in a concentration-dependent manner with a varying degree of potency. In prostate cancer cell-osteoclast co-cultures, the rat Mat-Ly-Lu, but not human PC3 or mouse RM1-BT, enhanced RANKL stimulated osteoclast formation and PTN reduced these effects without affecting prostate cancer cell viability. In the absence of cancer cells, PTN reduced the support of Mat-Ly-Lu conditioned medium for the adhesion and spreading of osteoclast precursors, and survival of mature osteoclasts. Pre-exposure of osteoblasts to PTN prior to the addition of conditioned medium from Mat-Ly-Lu cells suppressed their ability to support the formation of osteoclasts by inhibition of RANKL/OPG ratio. PTN enhanced the ability of Mat-Ly-Lu derived factors to increase calvarial osteoblast differentiation and growth. Ex vivo, PTN enhanced bone volume in calvaria organ-Mat-Ly-Lu cell co-culture, without affecting Mat-Ly-Lu viability or apoptosis. Mechanistic studies in osteoclasts and osteoblasts confirmed that PTN inhibit NFκB activation related to derived factors from Mat-Ly-Lu cells. Collectively, these findings suggest that pharmacological inhibition of the skeletal NFκB signalling pathway reduces prostate cancer related osteolysis, but further studies in the therapeutic implications of NFκB inhibition in cells of the osteoblastic lineage are needed.

Raman spectroscopy as a predictive tool for monitoring osteoporosis therapy in a rat model of postmenopausal osteoporosis.

Pharmacological therapy of osteoporosis reduces bone loss and risk of fracture in patients. Modulation of bone mineral density cannot explain all effects. Other aspects of bone quality affecting fragility and ways to monitor them need to be better understood. Keratinous tissue acts as surrogate marker for bone protein deterioration caused by oestrogen deficiency in rats. Ovariectomised rats were treated with alendronate (ALN), parathyroid hormone (PTH) or estrogen (E2). MicroCT assessed macro structural changes. Raman spectroscopy assessed biochemical changes. Micro CT confirmed that all treatments prevented ovariectomy-induced macro structural bone loss in rats. PTH induced macro structural changes unrelated to ovariectomy. Raman analysis revealed ALN and PTH partially protect against molecular level changes to bone collagen (80% protection) and mineral (50% protection) phases. E2 failed to prevent biochemical change. The treatments induced alterations unassociated with the ovariectomy; increased beta sheet with E2, globular alpha helices with PTH and fibrous alpha helices with both ALN and PTH. ALN is closest to maintaining physiological status of the animals, while PTH (comparable protective effect) induces side effects. E2 is unable to prevent molecular level changes associated with ovariectomy. Raman spectroscopy can act as predictive tool for monitoring pharmacological therapy of osteoporosis in rodents. Keratinous tissue is a useful surrogate marker for the protein related impact of these therapies.The results demonstrate utility of surrogates where a clear systemic causation connects the surrogate to the target tissue. It demonstrates the need to assess broader biomolecular impact of interventions to examine side effects.

Raman spectroscopy predicts the link between claw keratin and bone collagen structure in a rodent model of oestrogen deficiency.

Osteoporosis is a common disease characterised by reduced bone mass and an increased risk of fragility fractures. Low bone mineral density is known to significantly increase the risk of osteoporotic fractures, however, the majority of non-traumatic fractures occur in individuals with a bone mineral density too high to be classified as osteoporotic. Therefore, there is an urgent need to investigate aspects of bone health, other than bone mass, that can predict the risk of fracture. Here, we successfully predicted association between bone collagen and nail keratin in relation to bone loss due to oestrogen deficiency using Raman spectroscopy. Raman signal signature successfully discriminated between ovariectomised rats and their sham controls with a high degree of accuracy for the bone (sensitivity 89%, specificity 91%) and claw tissue (sensitivity 89%, specificity 82%). When tested in an independent set of claw samples the classifier gave 92% sensitivity and 85% specificity. Comparison of the spectral changes occurring in the bone tissue with the changes occurring in the keratin showed a number of common features that could be attributed to common changes in the structure of bone collagen and claw keratin. This study established that systemic oestrogen deficiency mediates parallel structural changes in both the claw (primarily keratin) and bone proteins (primarily collagen). This strengthens the hypothesis that nail keratin can act as a surrogate marker of bone protein status where systemic processes induce changes.

Pharmacological Inhibition of the Skeletal IKKß Reduces Breast Cancer-Induced Osteolysis.

IKKß has previously been implicated in breast cancer bone metastasis and bone remodelling. However, the contribution of IKKß expressed by bone cells of the tumour microenvironment to breast cancer-induced osteolysis has yet to be investigated. Here, we studied the effects of the verified selective IKKß inhibitors IKKßIII or IKKßV on osteoclast formation and osteoblast differentiation in vitro and in vivo, human and mouse breast cancer cells' support for osteoclast formation and signalling in vitro and osteolysis ex vivo and in immunocompetent mice after supracalvarial injection of human MDA-MB-231 conditioned medium or intra-cardiac injection of syngeneic 4T1 breast cancer cells. Pre-treatment with IKKßIII or IKKßV prior to exposure to tumour-derived factors from human and mouse breast cancer cell lines protected against breast cancer-induced osteolysis in two independent immunocompetent mouse models of osteolysis and the ex vivo calvarial bone organ system. Detailed functional and mechanistic studies showed that direct inhibition of IKKß kinase activity in osteoblasts and osteoclasts was associated with significant reduction of osteoclast formation, enhanced osteoclast apoptosis and reduced the ability of osteoblasts to support osteoclastogenesis in vitro. When combined with previous findings that suggest NFκB inhibition reduces breast cancer tumorigenesis and metastasis our present findings have an important clinical implication on raising the possibility that IKKß inhibitors, as bone anabolics, osteoclast inhibitors as well as anti-metastatic agents, may have advantages over anti-osteoclasts agents in the treatment of both skeletal and non-skeletal complications associated with metastatic breast cancer.

Emerging therapeutic targets in cancer induced bone disease: A focus on the peripheral type 2 cannabinoid receptor.

Skeletal complications are a common cause of morbidity in patients with primary bone cancer and bone metastases. The type 2 cannabinoid (Cnr2) receptor is implicated in cancer, bone metabolism and pain perception. Emerging data have uncovered the role of Cnr2 in the regulation of tumour-bone cell interactions and suggest that agents that target Cnr2 in the skeleton have potential efficacy in the reduction of skeletal complications associated with cancer. This review aims to provide an overview of findings relating to the role of Cnr2 receptor in the regulation of skeletal tumour growth, osteolysis and bone pain, and highlights the many unanswered questions and unmet needs. This review argues that development and testing of peripherally-acting, tumour-, Cnr2-selective ligands in preclinical models of metastatic cancer will pave the way for future research that will advance our knowledge about the basic mechanism(s) by which the endocannabinoid system regulate cancer metastasis, stimulate the development of a safer cannabis-based therapy for the treatment of cancer and provide policy makers with powerful tools to assess the science and therapeutic potential of cannabinoid-based therapy. Thus, offering the prospect of identifying selective Cnr2 ligands, as novel, alternative to cannabis herbal extracts for the treatment of advanced cancer patients.

Bone Cell-autonomous Contribution of Type 2 Cannabinoid Receptor to Breast Cancer-induced Osteolysis.

The cannabinoid type 2 receptor (CB2) has previously been implicated as a regulator of tumor growth, bone remodeling, and bone pain. However, very little is known about the role of the skeletal CB2 receptor in the regulation of osteoblasts and osteoclasts changes associated with breast cancer. Here we found that the CB2-selective agonists HU308 and JWH133 reduced the viability of a variety of parental and bone-tropic human and mouse breast cancer cells at high micromolar concentrations. Under conditions in which these ligands are used at the nanomolar range, HU308 and JWH133 enhanced human and mouse breast cancer cell-induced osteoclastogenesis and exacerbated osteolysis, and these effects were attenuated in cultures obtained from CB2-deficient mice or in the presence of a CB2 receptor blocker. HU308 and JWH133 had no effects on osteoblast growth or differentiation in the presence of conditioned medium from breast cancer cells, but under these circumstances both agents enhanced parathyroid hormone-induced osteoblast differentiation and the ability to support osteoclast formation. Mechanistic studies in osteoclast precursors and osteoblasts showed that JWH133 and HU308 induced PI3K/AKT activity in a CB2-dependent manner, and these effects were enhanced in the presence of osteolytic and osteoblastic factors such as RANKL (receptor activator of NFκB ligand) and parathyroid hormone. When combined with published work, these findings suggest that breast cancer and bone cells exhibit differential responses to treatment with CB2 ligands depending upon cell type and concentration used. We, therefore, conclude that both CB2-selective activation and antagonism have potential efficacy in cancer-associated bone disease, but further studies are warranted and ongoing.

Genetic background modifies the effects of type 2 cannabinoid receptor deficiency on bone mass and bone turnover.

Cannabinoid receptors and their ligands play significant roles in regulating bone metabolism. Previous studies of type 1 cannabinoid receptor-deficient mice have shown that genetic background influences the skeletal phenotype. Here, we investigated the effects of genetic background on the skeletal phenotype of mice with type 2 cannabinoid receptor deficiency (Cnr2 (-/-)). We studied Cnr2 (-/-) mice on a CD1 background and compared the findings with those previously reported in Cnr2 (-/-) C57BL/6 mice. Young female Cnr2 (-/-) CD1 mice had low bone turnover and high trabecular bone mass compared with wild-type (WT), contrasting with the situation in Cnr2 (-/-) C57BL/6 mice where trabecular bone mass has been reported to be similar to WT. The Cnr2 (-/-) CD1 mice lost more trabecular bone at the tibia with age than WT due to reduced bone formation, and at 12 months there was no difference in trabecular bone volume between genotypes. This differs from the phenotype previously reported in C57BL/6 Cnr2 (-/-) mice, where bone turnover is increased and bone mass reduced with age. There were no substantial differences in skeletal phenotype between Cnr2 (-/-) and WT in male mice. Cortical bone phenotype was similar in Cnr2 (-/-) and WT mice of both genders. Deficiency of Cnr2 has site- and gender-specific effects on the skeleton, mainly affecting trabecular bone, which are influenced by genetic differences between mouse strains. Further evaluation of the pathways responsible might yield new insights into the mechanisms by which cannabinoid receptors regulate bone metabolism.

Modulation of strain-specific differences in gene expression by cannabinoid type 2 receptor deficiency.

Previous studies have shown that the skeletal consequences of cannabinoid receptor deficiency differ in different strains of mice. In order to explore the mechanisms responsible, we analysed global gene expression in bone from wild-type CD1 mice and littermates with targeted inactivation of the type 2 cannabinoid receptor (Cnr2 (-/-)) and compared the results with those obtained from a similar analysis of wild-type and Cnr2 (-/-) C57BL/6 mice. Trabecular bone volume was increased in Cnr2 (-/-) CD1 mice compared with wild-type littermates but decreased in Cnr2 (-/-) C57BL/6 mice. Microarray analysis identified 354 genes in which substantial differences in gene expression (>1.5-fold) were observed that were specifically affected by Cnr2 deficiency. Bioinformatic analysis of data from wild-type mice of each strain revealed Cnr2-dependent differences in expression of genes clustering within the gene ontology (GO) terms immune response (p < 0.0001), positive regulation of response to stimulus (p < 0.0001), nucleotide binding (p = 0.002), and ribonucleotide binding (p = 0.003). Bioinformatic analysis of data from Cnr2 (-/-) mice of each strain revealed associations between GO terms corresponding to the extracellular region (p = 0.002), the cell surface (p = 0.02), antigen binding (p = 0.03), external side of plasma membrane (p = 0.04), and regulation of the force of heart contraction (p = 0.04). We conclude that Cnr2 deficiency affects expression of a large number of genes in different strains of mice, and that these differences are likely to be responsible in part for the differences in skeletal phenotype that we and others have observed in mice with defective cannabinoid receptor signalling in different genetic backgrounds.

Classical cannabinoid receptors as target in cancer-induced bone pain: a systematic review, meta-analysis and bioinformatics validation.

To test the hypothesis that genetic and pharmacological modulation of the classical cannabinoid type 1 (CB1) and 2 (CB2) receptors attenuate cancer-induced bone pain, we searched Medline, Web of Science and Scopus for relevant skeletal and non-skeletal cancer studies from inception to July 28, 2022. We identified 29 animal and 35 human studies. In mice, a meta-analysis of pooled studies showed that treatment of osteolysis-bearing males with the endocannabinoids AEA and 2-AG (mean difference [MD] - 24.83, 95% confidence interval [95%CI] - 34.89, - 14.76, p < 0.00001) or the synthetic cannabinoid (CB) agonists ACPA, WIN55,212-2, CP55,940 (CB1/2-non-selective) and AM1241 (CB2-selective) (MD - 28.73, 95%CI - 45.43, - 12.02, p = 0.0008) are associated with significant reduction in paw withdrawal frequency. Consistently, the synthetic agonists AM1241 and JWH015 (CB2-selective) increased paw withdrawal threshold (MD 0.89, 95%CI 0.79, 0.99, p < 0.00001), and ACEA (CB1-selective), AM1241 and JWH015 (CB2-selective) reduced spontaneous flinches (MD - 4.85, 95%CI - 6.74, - 2.96, p < 0. 00001) in osteolysis-bearing male mice. In rats, significant increase in paw withdrawal threshold is associated with the administration of ACEA and WIN55,212-2 (CB1/2-non-selective), JWH015 and AM1241 (CB2-selective) in osteolysis-bearing females (MD 8.18, 95%CI 6.14, 10.21, p < 0.00001), and treatment with AM1241 (CB2-selective) increased paw withdrawal thermal latency in males (mean difference [MD]: 3.94, 95%CI 2.13, 5.75, p < 0.0001), confirming the analgesic capabilities of CB1/2 ligands in rodents. In human, treatment of cancer patients with medical cannabis (standardized MD - 0.19, 95%CI - 0.35, - 0.02, p = 0.03) and the plant-derived delta-9-THC (20 mg) (MD 3.29, CI 2.24, 4.33, p < 0.00001) or its synthetic derivative NIB (4 mg) (MD 2.55, 95%CI 1.58, 3.51, p < 0.00001) are associated with reduction in pain intensity. Bioinformatics validation of KEGG, GO and MPO pathway, function and process enrichment analysis of mouse, rat and human data revealed that CB1 and CB2 receptors are enriched in a cocktail of nociceptive and sensory perception, inflammatory, immune-modulatory, and cancer pathways. Thus, we cautiously conclude that pharmacological modulators of CB1/2 receptors show promise in the treatment of cancer-induced bone pain, however further assessment of their effects on bone pain in genetically engineered animal models and cancer patients is warranted.

TRAF6 as a potential target in advanced breast cancer: a systematic review, meta-analysis, and bioinformatics validation.

TRAF6 has emerged as a key regulator of breast cancer (BCa). However, the TRAF family constitutes of seven members that exhibit distinct and overlapping functions. To explore which TRAF represents a potential druggable target for BCa treatment, we searched Medline, Web of Science and Scopus for relevant studies from inception to June 27, 2021. We identified 14 in vitro, 11 in vivo and 4 human articles. A meta-analysis of pharmacological studies showed that in vitro inhibition of TRAF2/4 (mean difference (MD): - 57.49, 95% CI: - 66.95, - 48.02, P < 0.00001) or TRAF6 (standard(Std.)MD: - 4.01, 95% CI: - 5.75, - 2.27, P < 0.00001) is associated with reduction in BCa cell migration. Consistently, inhibition of TRAF2/4 (MD: - 51.08, 95% CI: - 64.23, - 37.94, P < 0.00001) and TRAF6 (Std.MD: - 2.80, 95% CI: - 4.26, - 1.34, P = 0.0002) is associated with reduced BCa cell invasion, whereas TRAF2/4 inhibition (MD: - 40.54, 95% CI: - 52.83, - 28.26, P < 0.00001) is associated with reduced BCa cell adhesion. Interestingly, only inhibition of TRAF6 (MD: - 21.46, 95% CI: - 30.40, - 12.51, P < 0.00001) is associated with reduced cell growth. In animal models of BCa, administration of pharmacological inhibitors of TRAF2/4 (Std.MD: - 3.36, 95% CI: - 4.53, - 2.18, P < 0.00001) or TRAF6 (Std.MD: - 4.15, 95% CI: - 6.06, - 2.24, P < 0.0001) in mice is associated with reduction in tumour burden. In contrast, TRAF6 inhibitors (MD: - 2.42, 95% CI: - 3.70, - 1.14, P = 0.0002) reduced BCa metastasis. In BCa patients, high expression of TRAF6 (Hazard Ratio: 1.01, CI: 1.01, 1.01, P < 0.00001) is associated with poor survival rate. Bioinformatics validation of clinical and pathway and process enrichment analysis in BCa patients confirmed that gain/amplification of TRAF6 is associated with secondary BCa in bone (P = 0.0079), and poor survival rate (P < 0.05). Overall, TRAF6 inhibitors show promise in the treatment of metastatic BCa. However, low study number and scarcity of evidence from animal and human studies may limit the translation of present findings into clinical practice.

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.

Anti-inflammatory, but not osteoprotective, effect of the TRAF6/CD40 inhibitor 6877002 in rodent models of local and systemic osteolysis.

NFκB plays a key role in inflammation and skeletal disorders. Previously, we reported that pharmacological inhibition of NFκB at the level of TRAF6 suppressed RANKL, CD40L and IL1ß-induced osteoclastogenesis and attenuated cancer-induced bone disease. TNFα is also known to regulate TRAF6/NFκB signalling, however the anti-inflammatory and osteoprotective effects associated with inhibition of the TNFα/TRAF6/NFκB axis have not been investigated. Here, we show that in vitro and ex vivo exposure to the verified small-molecule inhibitor of TRAF6, 6877002 prevented TNFα-induced NFκB activation, osteoclastogenesis and calvarial osteolysis, but it had no effects on TNFα-induced apoptosis or growth inhibition in osteoblasts. Additionally, 6877002 disrupted T-cells support for osteoclast formation and synoviocyte motility, without affecting the viability of osteoblasts in the presence of T-cells derived factors. Using the collagen-induced arthritis model, we show that oral and intraperitoneal administration of 6877002 in mice reduced joint inflammation and arthritis score. Unexpectedly, no difference in trabecular and cortical bone parameters were detected between vehicle and 6877002 treated mice, indicating lack of osteoprotection by 6877002 in the arthritis model described. Using two independent rodent models of osteolysis, we confirmed that 6877002 had no effect on trabecular and cortical bone loss in both osteoporotic rats or RANKL- treated mice. In contrast, the classic anti-osteolytic alendronate offered complete osteoprotection in RANKL- treated mice. In conclusion, TRAF6 inhibitors may be of value in the management of the inflammatory component of bone disorders, but may not offer protection against local or systemic bone loss, unless combined with anti-resorptive therapy such as bisphosphonates.

Small molecule inhibitors of IkappaB kinase signaling inhibit osteoclast formation in vitro and prevent ovariectomy-induced bone loss in vivo.

The NFκB pathway plays a critical role in the regulation of osteoclast activity, and activation of the pathway is dependent on IκB kinase (IKK), which phosphorylates IκB, targeting it for proteasomal degradation. Pharmacological inhibitors of IKK exhibit anti-inflammatory properties and prevent bone erosions in models of inflammatory arthritis. However, the effects of these agents on osteoblast function and ovariectomy-induced bone loss remain unknown. Here we examined the effects of the IKK inhibitors celastrol, BMS-345541, and parthenolide on bone cell function in vitro and ovariectomy-induced bone loss in vivo. All three compounds inhibited RANKL-induced signaling in osteoclasts, caused osteoclast apoptosis, and inhibited osteoclast formation. Although parthenolide and BMS-345541 had no inhibitory effects on osteoblast function, celastrol prevented IL1ß-induced TAK1 activation and inhibited osteoblast growth, differentiation, and bone nodule formation. The selective IKK inhibitors parthenolide and BMS-345541 prevented ovariectomy-induced bone loss by inhibiting osteoclastic bone resorption. We conclude that pharmacological inhibitors of IKK inhibit several critical signaling pathways in osteoclasts necessary for cell survival, formation, and activity in vitro and bone loss in vivo. Accordingly, IKK inhibitors may be of value in the prevention and treatment of bone diseases characterized by increased bone loss such as postmenopausal osteoporosis.

Cannabinoids and bone: friend or foe?

The endocannabinoid system is recognized to play an important role in regulating a variety of physiological processes, including appetite control and energy balance, pain perception, and immune responses. The endocannabinoid system has also recently been implicated in the regulation of bone metabolism. Endogenously produced cannabinoids are hydrophobic molecules derived from hydrolysis of membrane phospholipids. These substances, along with plant-derived and synthetic cannabinoids, interact with the type 1 (CB(1)) and 2 (CB(2)) cannabinoid receptors and the GPR55 receptor to regulate cellular function through a variety of signaling pathways. Endocannabinoids are produced in bone, but the mechanisms that regulate their production are unclear. Skeletal phenotyping of mice with targeted inactivation of cannabinoid receptors and pharmacological studies have shown that cannabinoids play a key role in the regulation of bone metabolism. Mice with CB(1) deficiency have high peak bone mass as a result of an osteoclast defect but develop age-related osteoporosis as a result of impaired bone formation and accumulation of bone marrow fat. Mice with CB(2) deficiency have relatively normal peak bone mass but develop age-related osteoporosis as a result of increased bone turnover with uncoupling of bone resorption from bone formation. Mice with GPR55 deficiency have increased bone mass as a result of a defect in the resorptive activity of osteoclasts, but bone formation is unaffected. Cannabinoids are also produced within synovial tissues, and preclinical studies have shown that cannabinoid receptor ligands are effective in the treatment of inflammatory arthritis. These data indicate that cannabinoid receptors and the enzymes responsible for ligand synthesis and breakdown play important roles in bone remodeling and in the pathogenesis of joint disease.

The biphenyl-carboxylate derivative ABD328 is a novel orally active antiresorptive agent.

We previously described a novel series of biphenyl carboxylic acid derivatives which have potent antiresorptive effects in vitro and in vivo and do not affect osteoblast function. However, none of the previous compounds showed oral activity, probably because they were esters, which would be expected to be metabolized very rapidly. Here, we tested whether derivatives where the ester link was replaced by a ketone link were orally active. Compounds were tested in murine osteoclast and osteoblast cultures and in the mouse ovariectomy (OVX) model of osteoporosis. The ketones were at least as potent at inhibiting osteoclast formation and RANKL signaling in vitro as the esters and did not inhibit osteoblast differentiation or function. The basic ketone-linked compound ABD68 was only partially able to inhibit OVX-induced bone loss at an oral dose of 20 mg/kg daily. Substitutions on the phenyl rings increased the potency of the compounds in vitro and may prevent metabolism of the compounds in vivo. The most promising derivative, ABD328, completely prevented OVX-induced bone loss when administered by intraperitoneal injection at 3 mg/kg daily. Furthermore, ABD328 was also able to fully prevent OVX-induced bone loss when given orally at 10 mg/kg daily. The results indicate that biphenyl carboxylates like ABD328 are oral candidate drugs for the treatment of diseases characterized by increased bone resorption, such as postmenopausal osteoporosis.

Combined administration of a small-molecule inhibitor of TRAF6 and Docetaxel reduces breast cancer skeletal metastasis and osteolysis.

Tumour necrosis factor receptor-associated factor 6 (TRAF6) has been implicated in breast cancer and osteoclastic bone destruction. Here, we report that 6877002, a verified small-molecule inhibitor of TRAF6, reduced metastasis, osteolysis and osteoclastogenesis in models of osteotropic human and mouse breast cancer. First, we observed that TRAF6 is highly expressed in osteotropic breast cancer cells and its level of expression was higher in patients with bone metastasis. Pre-exposure of osteoclasts and osteoblasts to non-cytotoxic concentrations of 6877002 inhibited cytokine-induced NFκB activation and osteoclastogenesis, and reduced the ability of osteotropic human MDA-MB-231 and mouse 4T1 breast cancer cells to support bone cell activity. 6877002 inhibited human MDA-MB-231-induced osteolysis in the mouse calvaria organ system, and reduced soft tissue and bone metastases in immuno-competent mice following intra-cardiac injection of mouse 4T1-Luc2 cells. Of clinical relevance, combined administration of 6877002 with Docetaxel reduced metastasis and inhibited osteolytic bone damage in mice bearing 4T1-Luc2 cells. Thus, TRAF6 inhibitors such as 6877002 - alone or in combination with conventional chemotherapy - show promise for the treatment of metastatic breast cancer.

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.