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Obesity is an epidemic problem affecting millions of people in the Western hemisphere and costs the United States economy more than $200 billion annually. Currently, there are no effective treatments to combat obesity. Recent studies have implicated the constitutive activity of estrogen receptor (ER) ␤ as an important regulator of metabolic diseases. However, the potential of ER-␤-selective ligands to offset obesity is not clear. We evaluated the pharmacological effect of ER-␤-selective ligands (␤-LGNDs) in animal models of high-fat diet-and ovariectomyinduced obesity. Ligand binding, transactivation, and uterotrophic studies with ␤-LGNDs demonstrated selectivity for ER-␤ over ER-␣. Animals fed a high-fat diet showed a significant increase in body weight, and this weight gain was attenuated by ␤-LGNDs. High-fat diet-mediated increases in serum cholesterol, leptin, glucose, and fat accumulation in organs were also reduced by ␤-LGNDs. In addition, MRI scanning indicated that ␤-LGNDs altered body composition by reducing fat mass and increasing lean body mass. Organ weights and gene expression analyses demonstrated that adipose tissue is the center of action for ␤-LGNDs, and the reduction in body weight is likely due to increased energy expenditure. In vitro and in vivo mechanistic studies indicated that the anti-obesity effects of ␤-LGNDs were due to indirect peroxisome proliferator-activated receptor ␥ antagonistic actions requiring the ligand binding domain of ER-␤ and through abrogation of the ability of PGC-1 to coactivate peroxisome proliferator-activated receptor ␥. In conclusion, these studies indicate that ligand-activated ER-␤ is a potential therapeutic target to combat obesity and obesity-related metabolic diseases.Obesity is an epidemic disease affecting over 400 million people globally (1). Two-thirds of adults and children in the United States are either overweight or obese, making it a serious health risk and economic burden to society (2). Obesity is not a standalone disease, as its emergence leads to various complications, including type 2 diabetes mellitus (T2DM), 3 hypertension, atherosclerosis, and other cardiovascular diseases, osteoporosis, and clinical depression (3, 4). The United States Food and Drug Administration required an anti-obesity drug to reduce the body weight by 5% and/or better results than placebo in 12 months, indicating that even a marginal reduction in body weight will cause a significant improvement in the welfare of these patients (5). Despite the exponentially growing global obesity pharmaceutical market, only two Food and Drug Administration-approved drugs are available for this indication: 1) amphetamines and sibutramine that act on the hypothalamus to control appetite stimulation in the central nervous system, and 2) Orlistat, which is a lipase inhibitor that blocks gastrointestinal absorption of fat and decreases energy uptake (6). Despite mediocre performance, these drugs are commonly associated with side effects such as tachycardia, hypertension, fecal incontinence, ...

These results indicate that 17β-E(2) protects ARPE-19 cells from oxidative stress through an ERβ-dependent mechanism. 17β-E(2)-mediated cytoprotection occurred through the preservation of mitochondrial function, reduction of ROS production, and induction of cellular antioxidant genes.

Taken together, these results indicate that clinically relevant doses of PF-04691502 and PD-0325901 can suppress bladder tumor growth in PDX models, thus offering additional potential treatment options by a precision medicine approach.

Non-invasive imaging using radiolabels is a common technique used to study the biodistribution of biologics. Due to the limited shelf-life of radiolabels and the requirements of specialized labs, non-invasive optical imaging is an attractive alternative for preclinical studies. Previously, we demonstrated the utility of fluorescence molecular tomography (FMT) an optical imaging modality in evaluating the biodistribution of antibody-drug conjugates. As FMT is a relatively new technology, few fluorophores have been validated for in vivo imaging. The goal of this study was to characterize and determine the utility of near-infrared (NIR) fluorophores for biodistribution studies using interleukin-13 receptor subunit alpha-2 antibody (IL13Rα2-Ab). Eight fluorophores (ex/em: 630/800 nm) with an N-hydroxysuccinimide (NHS) linker were evaluated for Ab conjugation. The resulting antibody-fluorophore (Ab-F) conjugates were evaluated in vitro for degree of conjugation, stability and target-binding, followed by in vivo/ex vivo FMT imaging to determine biodistribution in a xenograft model. The Ab-F conjugates (except Ab-DyLight800) showed good in vitro stability and antigen binding. All Ab-F conjugates (except for Ab-BOD630) resulted in a quantifiable signal in vivo and had similar biodistribution profiles, with peak tumor accumulation between 6 and 24 h post-injection. In vivo/ex vivo FMT imaging showed 17-34% ID/g Ab uptake by the tumor at 96 h. Overall, this is the first study to characterize the biodistribution of an Ab using eight NIR fluorophores. Our results show that 3-dimensional optical imaging is a valuable technology to understand biodistribution and targeting, but a careful selection of the fluorophore for each Ab is warranted.

Age-related Macular Degeneration (AMD) is the leading cause of visual impairment and blindness in the elderly in developed countries. Neovascular/exudative (wet) AMD is the aggressive form of AMD and can involve choroidal neovascularization and vascular leakage. Anti-vascular endothelial growth factor (anti-VEGF) medications have significantly improved treatment of wet-AMD. However, only approximately 40% of patients obtain full benefit from anti-VEGF therapy and the medications are given by intravitreal injection. Axitinib, a small molecule multi-receptor tyrosine kinase inhibitor used for the treatment of advanced renal cell carcinoma, is taken orally and inhibits VEGF activity by blocking VEGF receptors. Axitinib also has the advantage of blocking platelet derived growth factor (PDGF) receptors which play a role in neovascularization. Using in vitro human retinal microvascular endothelial cells (HRMVECs), human brain vascular pericytes (HBVRs), 3D co-culture vessel sprout assay, and in vivo laser induced rat choroidal neovascularization (CNV) models, the effect of axitinib on neovascularization was evaluated. Axitinib inhibited neovascularization better than anti-VEGF and/or anti-hPDGF-B mAb in the in vitro models demonstrating that combined inhibition of both VEGF and PDGF pathways may be synergistic in treating wet-AMD. Additionally, axitinib showed good efficacy at a low dose (0.875 mg/day) in laser-induced CNV model in rats. In conclusion our data shows that axitinib, an inhibitor of VEGF and PDGF-B pathways may be useful in ameliorating wet-AMD therapy.

The role of PI3K and MAPK pathways in tumor initiation and progression is well established; hence, several inhibitors of these pathways are currently in different stages of clinical trials. Recent studies identified a PI3K/mTOR (PF-04691502) and a MEK inhibitor (PD-0325901) with strong potency and efficacy in different cell lines and tumor models. PD-0325901, however, showed adverse effects when administered at or above MTD (maximum tolerated dose) in the clinic. Here, we show in preclinical models that PD-0325901 at doses well below MTD (sub-MTD 1.5 mg/kg SID) is still a potent compound as single agent or in combination with PF-04691502. We first observed that PD-0325901 at 1.5 mg/kg SID and in combination with PF-04691502 (7.5 mg/kg; SID) significantly inhibited growth of H460 (carry Kras and PIK3CA mutations) orthotopic lung tumors. Additionally, we tested efficacy of PD-0325901 in Kras(G12D-LSL) conditional GEMMs (genetically engineered mouse models) which are a valuable tool in translational research to study tumor progression. Intranasal delivery of adenoviruses expressing Cre recombinase (Adeno-Cre) resulted in expression of mutant Kras leading to development of tumor lesions in lungs including adenomatous hyperplasia, large adenoma, and adenocarcinoma. Similar to H460 tumors, PD-0325901 as single agent or in combination with PF-04691502 significantly inhibited growth of tumor lesions in lungs in Kras(G12D-LSL) mice when treatment started at adenocarcinoma stage (at 14 weeks post-Adeno-Cre inhalation). In addition, immunohistochemistry showed inhibition of pS6 (phosphorylated ribosomal S6) in the treated animals particularly in the combination group providing a proof of mechanism for tumor growth inhibition. Finally, m-CT imaging in live Kras(G12D-LSL) mice showed reduction of tumor burdens in PD-0325901-treated animals at sub-MTD dose. In conclusion, our data suggest that PD-0325901 at doses below MTD is still a potent compound capable of tumor growth inhibition where Kras and/or PI3K are drivers of tumor growth and progression.

BackgroundCastration resistant prostate cancer (CRPC) is a leading cause of cancer-related deaths in men. The primary cause of mortality and morbidity in patients is bone metastases and remodeling resulting in osteoblastic and osteolytic lesions. Recently, cabozantinib, a multi-kinase inhibitor (VEGFR2 and c-MET inhibitor), was shown to have efficacy on bone lesions in patients. In this study we tested multi-kinase inhibitors: axitinib (VEGFR inhibitor) and crizotinib (c-MET inhibitor) in a combination trial in mice models.MethodsVCaP-Luc cells were grown as subcutaneous implants in intact and castrated NOD-SCID-gamma (NSG) mice to confirm the androgen dependency. For bone metastasis model two cohorts of NSG mice (castrated and intact) received orthotopic injection of VCaP-Luc cells into the bone marrow cavity of left tibia. Mice were monitored weekly for tumor growth using bioluminescence imaging. Animals were randomized into 4 groups based on the tumor bioluminescence signal: vehicle, crizotinib alone, axitinib alone, crizotinib and axitinib in combination. Animals were imaged weekly by in vivo 2-D X-ray imaging to monitor bone remodeling. At the end of the study animals were euthanized and both tibias were extracted for ex vivo high-resolution 3-D micro-computed tomography (μCT) imaging.ResultsSubcutaneous model showed that androgen stimulation may be helpful but not essential for the growth of VCaP-Luc cells. VCaP-Luc cells grown intra-tibially in intact animals caused extensive remodeling of bone with mixed osteoblastic (bone formation) and osteolytic (bone matrix dissolution) lesions. The osteoblastic lesions were predominant and at times extended beyond the tibial shaft into the surrounding tissue. In contrast, only osteolytic lesions were prominent throughout the study in castrated animals. Treatment with crizotinib alone reduced the osteolytic lesions in castrated animals. Axitinib alone reduced the osteoblastic lesions in the intact animals. Combination therapy with axitinib and crizotinib remarkably inhibited the tibial remodeling by VCaP-Luc cells which resulted in a significant reduction of both osteoblastic and osteolytic lesions.ConclusionOur data show that combined inhibition of c-MET and VEGFR can be beneficial for treatment of metastatic bone disease in CRPC and that the drugs act on two different stages of the disease.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-742) contains supplementary material, which is available to authorized users.