T2DM progresses from compensated insulin resistance to beta cell failure resulting in uncompensated hyperglycemia, a process replicated in the Zucker diabetic fatty (ZDF) rat. Our earlier findings (Nature Medicine 19:1132, 2013) implicate pro-inflammatory macrophages infiltrating pancreatic islets in a paracrine mechanism of beta cell loss, and identify macrophage-expressed CB1 receptors as a therapeutic target in T2DM. In a follow up study, we have addressed the question whether CB1 receptors have an obligatory role in the development of T2DM, by generating CB1 receptor knockout rats on a ZDF genetic background, using the zinc finger technology. ZDF-Cnr1 rats retain the inactivating mutation of the leptin receptor present in the parent ZDF strain, and also lack CB1 receptors globally. Unlike their ZDF littermates that develop T2DM, ZDF-Cnr1 rats remain normoglycemic due to preservation of pancreatic beta cells and beta-cell function. They are also protected from the nephropathy and dyslipidemia affecting their ZDF littermates, which were reported earlier (PNAS 111:E5420-28, 2014). Adoptive transfer of ZDF-Cnr1 bone marrow to ZDF rats also protects them from the development of hyperglycemia and T2DM, but not from the nephropathy or dyslipidemia. These findings confirm that obligatory role of CB1 receptors on bone marrow-derived macrophages in the beta-cell loss of diabetic ZDF rats, but indicates that role of CB1 receptors in other cell types in the associated nephropathy and dyslipidemia. This work has been written up and will be submitted for publication. We have developed novel, dual-target compounds for the treatment of various pathologies associated with fibrosis. Liver fibrosis, a consequence of chronic liver injury and a waystation to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via CB1R induce pro-fibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted duse to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies. We have introduced a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in the liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis, induced by CCl4 or bile duct ligation, the hybrid CNB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor W1400, without causing CB1R occupancy in the CNS and the related anxiety-like behaviors. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated fibrotic pathways, including increased PDGF/PDGF receptor1 and alphaVbeta6 integrin signaling, as judged by its ability to inhibit these pathways in CB1R-/- but not in nos2-/- mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus dual-target peripheral CB1R/iNOS inhibitors have therapeutic potential in liver fibrosis. This work has been published in JCI Insight (2016). The hybrid CB1R/iNOS inhibitor described above has also been tested in different forms of pulmonary fibrosis. Pulmonary fibrosis (PF) is a life-threatening disease with poor prognosis and in need of novel treatment strategies, as currently available single target therapies have yielded modest success. For complex and multi-factorial diseases such as PF, targeting multiple pathways may improve therapeutic efficacy. We discovered that the endocannabinoid/CB1R system is overactive in the lung of patients with idiopathic PF and mice with bleomycin-induced PF where it contributes to disease progression, marking CB1R as a novel therapeutic target. Moreover, we demonstrate that engaging the secondary target iNOS by a peripherally restricted, orally bioavailable hybrid CB1R/iNOS inhibitor improves anti-fibrotic efficacy over inhibition of CB1R alone, and minimizes CNS side effects caused by brain-penetrant CB1R antagonists. This work has been written up and submitted for publication.
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