The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.

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2-Methoxyestradiol (2-ME), an endogenous metabolite of estradiol with no affinity for estrogen receptors, is a potent anticarcinogenic agent (in phase II clinical trials) and mediates the inhibitory effects of estradiol on smooth muscle cell (SMC) growth. Here we studied the intracellular mechanisms by which 2-ME inhibits SMC growth and whether 2-ME prevents injury-induced neointima formation. 2-ME concentrations that inhibit proliferation of cycling human aortic SMCs by >or=50% blocked cell-cycle progression in G(0)/G(1) and in G(2)/M phase, as determined by flow cytometry. Consistent with the cell-cycle effects, at a molecular level (Western blots), 2-ME inhibited cyclin D(1) and cyclin B(1) expression; cyclin-dependent kinase (cdk)-1 and cdk-2 activity; and retinoblastoma protein (pRb), extracellular signal-regulated kinase (ERK) 1/2, and Akt phosphorylation. 2-ME also upregulated the Cdk inhibitor p27 and interfered with tubulin polymerization. Moreover, 2-ME augmented COX-2 expression, suggesting that it may also inhibit SMC growth via prostaglandin formation. In rats, treatment with 2-ME abrogated injury-induced neointima formation; decreased proliferating SMCs; downregulated expression of proliferating-cell nuclear antigen (PCNA), c-myc, cyclin D(1), cyclin B(1), phosphorylated Akt, phosphorylated ERK1/2, p21, and pRb; inhibited cdk-1 and cdk-4 activity; and upregulated expression of cyclooxygenase (COX)-2 and p27. Caspase-3 cleavage assay and fluorescence-activated cell-sorting (FACS) analysis showed no evidence of apoptosis in 2-ME-treated SMCs, and TUNEL staining in carotid segments showed no evidence of 2-ME-induced apoptosis in vivo. The antimitotic effects of 2-ME on SMCs are mediated by the inhibition of key cell-cycle regulatory proteins and effects on tubulin polymerization and COX-2 upregulation. These effects of 2-ME most likely contribute to the antivasoocclusive actions of this endogenous compound.

Abstract-Estrogen receptors (ERs) are considered to mediate the ability of 17␤-estradiol (estradiol) to reduce injury-induced proliferation of vascular smooth muscle cells (VSMCs), leading to vascular lesions. However, the finding that estradiol attenuates formation of vascular lesions in response to vascular injury in knockout mice that lack either ER-␣ or ER-␤ challenges this concept. Our hypothesis is that the local metabolism of estradiol to methoxyestradiols, metabolites of estradiol with little affinity for ERs, mediates the ER-independent antimitogenic effects of estradiol on VSMCs. In human VSMCs, 2-methoxyestradiol and 2-hydroxyestradiol were more potent than was estradiol in inhibiting DNA synthesis ( 3 [H]-thymidine incorporation), collagen synthesis ( 3 [H]-proline incorporation), cell proliferation (cell number), and cell migration (movement of cells across a polycarbonate membrane). The inhibitory effects of estradiol on VSMCs were enhanced by cytochrome-P450 (CYP450) inducers 3-methylcholanthrene and phenobarbital. Moreover, the inhibitory effects of estradiol were blocked in the presence of the CYP450 inhibitor 1-aminobenzotriazole and the catechol-O-methyltransferase inhibitors quercetin and OR486. Both OR486 and quercetin blocked the conversion of 2-hydroxyestradiol to 2-methoxyestradiol; moreover, they blocked the antimitogenic effects of 2-hydroxyestradiol but not of 2-methoxyestradiol. The ER antagonist ICI182780 blocked the inhibitor effects of estradiol on VSMCs, but only at concentrations (Ͼ50 mol/L) that also inhibit the metabolism of estradiol to hydroxyestradiols (precursors of methoxyestradiols). In conclusion, the inhibitory effects of locally applied estradiol on human VSMCs are mediated via a novel ER-independent mechanism involving estradiol metabolism. These findings imply that vascular estradiol metabolism may be an important determinant of the cardiovascular protective effects of estradiol and that nonfeminizing estradiol metabolites may confer cardiovascular protection regardless of gender.

our data indicate that chymase inhibition can inhibit pro-atherogenic and plaque destabilizing effects which are associated with perivascular mast cell activation. Our study thus identifies pharmacological chymase inhibition as a potential therapeutic modality for atherosclerotic plaque stabilization.

Background/Aims: A critical involvement of the endocannabinoid/cannabinoid receptor system in diabetes and its complications has been recognized. Experimental evidence suggested that activation of the cannabinoid receptor type 2 (CB₂), which is expressed in the kidney by podocytes and inflammatory cells, had a protective role in early streptozotocin-induced type 1 diabetes in mice. No experimental evidence is so far available on the effects of CB₂ agonists in type 2 diabetes. In this study, we investigated the effects of a CB₂ agonist given at a phase of overt disease on renal functional and structural changes in BTBR ob/ob mice, a model of type 2 diabetic nephropathy. Methods: BTBR ob/ob mice received, from 10 to 21 weeks of age, vehicle, the selective CB₂ agonist HU910, or lisinopril used as standard therapy for comparison. BTBR wild-type mice served as controls. Results: Treatment with CB₂ agonist reduced progressive albuminuria of BTBR ob/ob mice to a similar extent as ACE inhibitor. The antiproteinuric effect of CB₂ agonist was associated with the amelioration of the defective nephrin expression in podocytes of diabetic mice. CB₂ agonist limited mesangial matrix expansion, fibronectin accumulation and sclerosis. Glomerular infiltration of Mac-2-positive monocytes/machrophages was attenuated by CB₂ agonist, at least in part due to the drug's ability to reduce MCP-1 chemotactic signals. Renoprotective effects of CB₂ were similar to those achieved by ACE inhibitor. Conclusion: These results suggest that CB₂ agonism is a potential option to be added to the available therapeutic armamentarium for type 2 diabetic nephropathy.

Abstract-Determinants of restenosis after angioplasty include constrictive remodeling and intimal hyperplasia. Both processes require extensive matrix turnover, so matrix metalloproteinases (MMPs) have become potential targets of antirestenosis therapies. We studied the effects of RO113-2908, a broad-spectrum MMP inhibitor (MMPI), on the response to iliac artery angioplasty and stenting in atherosclerotic cynomolgus monkeys. Lumen diameter (LD) was measured angiographically, and artery wall geometry was assessed after perfusion-fixation at 4 weeks. Angiogenesis was measured in subcutaneous polyvinyl alcohol disks. Key Words: restenosis Ⅲ constrictive remodeling Ⅲ metalloproteinases Ⅲ angioplasty Ⅲ stents E ach year, Ϸ3 million patients undergo reconstruction of atherosclerotic arteries, and many subsequently develop recurrent stenoses at the sites of repair. Restenosis after angioplasty or stenting is due primarily to constrictive artery wall remodeling or intimal hyperplasia, respectively. These processes involve extensive reorganization of extracellular matrix to facilitate changes in artery wall geometry and accumulation of new intimal mass.Matrix degradation is tightly regulated in the normal artery wall through a balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors (eg, TIMPs). Atherosclerosis shifts the balance toward degradation, as MMPs are produced by accumulating macrophages and activated smooth muscle cells (SMCs). 1-3 Angioplasty further increases MMP production, 4 -7 and matrix turnover and maturation continue for months after angioplasty in all layers of the arterial wall, likely contributing to SMC proliferation, migration, and remodeling. 7-9 These observations have led to speculation that inhibiting MMP activity could limit restenosis. However, experiments with MMP inhibitors to prevent intimal hyperplasia in animal models have shown mixed results, and effects on remodeling have not been well characterized. 5,6,10 -13 Whether this approach can improve the response to angioplasty or stenting in human beings with advanced atherosclerosis is yet to be defined.To extend this approach to a preclinical model, we treated atherosclerotic cynomolgus monkeys with a broad-spectrum MMPI for 4 weeks after angioplasty and stent-angioplasty to determine the impact on restenosis. Treatment provided continuous, systemic MMP inhibitory activity and inhibited angiogenesis, but artery wall remodeling and intimal hyperplasia were not improved. Additional preclinical research is needed to define the spectrum of matrix-degrading proteases critical in constrictive remodeling and intimal hyperplasia of atherosclerotic arteries after angioplasty. Methods Animal ModelThirty-one adult male monkeys (Macaca fascicularis; mean age, 13.5 years) consumed an atherogenic diet (0.28 mg cholesterol/kcal) for 2.5Ϯ0.1 years to induce advanced iliac artery lesions. 8,14 -17 Animals were then randomized into MMPI (nϭ16) or control (nϭ15) groups based on diet duration and the ratio of total to HDL choleste...

The endocannabinoid system, and in particular the cannabinoid type 2 receptor (CB2R), raised the interest of many medicinal chemistry programs for its therapeutic relevance in several (patho)physiologic processes. However, the physico-chemical properties of tool compounds for CB2R (e.g., the radioligand [H]CP55,940) are not optimal, despite the research efforts in developing effective drugs to target this system. At the same time, the importance of drug-target binding kinetics is growing since the kinetic binding profile of a ligand may provide important insights for the resulting in vivo efficacy. In this context we synthesized and characterized [H]RO6957022, a highly selective CB2R inverse agonist, as a radiolabeled tool compound. In equilibrium and kinetic binding experiments [H]RO6957022 showed high affinity for human CB2R with fast association () and moderate dissociation () kinetics. To demonstrate the robustness of [H]RO6957022 binding, affinity studies were carried out for a wide range of CB2R reference ligands, spanning the range of full, partial, and inverse agonists. Finally, we used [H]RO6957022 to study the kinetic binding profiles (i.e., and values) of selected synthetic and endogenous (i.e., 2-arachidonoylglycerol, anandamide, and noladin ether) CB2R ligands by competition association experiments. All tested ligands, and in particular the endocannabinoids, displayed distinct kinetic profiles, shedding more light on their mechanism of action and the importance of association rates in the determination of CB2R affinity. Altogether, this study shows that the use of a novel tool compound, i.e., [H]RO6957022, can support the development of novel ligands with a repertoire of kinetic binding profiles for CB2R.