Christina M. Barrett

Estrogen Receptor-β-selective Ligands Alleviate High-fat Diet- and Ovariectomy-induced Obesity in Mice

Obesity is an epidemic problem affecting millions of people in the Western hemisphere and costs the United States economy more than

Design, Synthesis, and Biological Evaluation of Stable Colchicine Binding Site Tubulin Inhibitors as Potential Anticancer Agents

200 billion annually. Currently, there are no effective treatments to combat obesity. Recent studies have implicated the constitutive activity of estrogen receptor (ER) β as an important regulator of metabolic diseases. However, the potential of ER-β-selective ligands to offset obesity is not clear. We evaluated the pharmacological effect of ER-β-selective ligands (β-LGNDs) in animal models of high-fat diet- and ovariectomy-induced obesity. Ligand binding, transactivation, and uterotrophic studies with β-LGNDs demonstrated selectivity for ER-β over ER-α. Animals fed a high-fat diet showed a significant increase in body weight, and this weight gain was attenuated by β-LGNDs. High-fat diet-mediated increases in serum cholesterol, leptin, glucose, and fat accumulation in organs were also reduced by β-LGNDs. In addition, MRI scanning indicated that β-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 β-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 β-LGNDs were due to indirect peroxisome proliferator-activated receptor γ antagonistic actions requiring the ligand binding domain of ER-β and through abrogation of the ability of PGC-1 to coactivate peroxisome proliferator-activated receptor γ. In conclusion, these studies indicate that ligand-activated ER-β is a potential therapeutic target to combat obesity and obesity-related metabolic diseases.

Discovery and Preclinical Characterization of Novel Small Molecule TRK and ROS1 Tyrosine Kinase Inhibitors for the Treatment of Cancer and Inflammation

To block the metabolically labile sites of novel tubulin inhibitors targeting the colchicine binding site based on SMART, ABI, and PAT templates, we have designed, synthesized, and biologically tested three focused sets of new derivatives with modifications at the carbonyl linker, the para-position in the C ring of SMART template, and modification of A ring of the PAT template. Structure–activity relationships of these compounds led to the identification of new benzimidazole and imidazo[4,5-c]pyridine-fused ring templates, represented by compounds 4 and 7, respectively, which showed enhanced antitumor activity and substantially improved the metabolic stability in liver microsomes compared to SMART. MOM group replaced TMP C ring and generated a potent analogue 15, which showed comparable potency to the parent SMART compound. Further modification of PAT template yielded another potent analogue 33 with 5-indolyl substituent at A ring.

Role and pharmacologic significance of cytochrome P-450 2D6 in oxidative metabolism of toremifene and tamoxifen†‡

Receptor tyrosine kinases (RTKs), in response to their growth factor ligands, phosphorylate and activate downstream signals important for physiological development and pathological transformation. Increased expression, activating mutations and rearrangement fusions of RTKs lead to cancer, inflammation, pain, neurodegenerative diseases, and other disorders. Activation or over-expression of ALK, ROS1, TRK (A, B, and C), and RET are associated with oncogenic phenotypes of their respective tissues, making them attractive therapeutic targets. Cancer cDNA array studies demonstrated over-expression of TRK-A and ROS1 in a variety of cancers, compared to their respective normal tissue controls. We synthesized a library of small molecules that inhibit the above indicated RTKs with picomolar to nanomolar potency. The lead molecule GTx-186 inhibited RTK-dependent cancer cell and tumor growth. In vitro and in vivo growth of TRK-A-dependent IMR-32 neuroblastoma cells and ROS1-overexpressing NIH3T3 cells were inhibited by GTx-186. GTx-186 also inhibited inflammatory signals mediated by NFκB, AP-1, and TRK-A and potently reduced atopic dermatitis and air-pouch inflammation in mice and rats. Moreover, GTx-186 effectively inhibited ALK phosphorylation and ALK-dependent cancer cell growth. Collectively, the RTK inhibitor GTx-186 has a unique kinase profile with potential to treat cancer, inflammation, and neuropathic pain.

β-LGND2, an ERβ selective agonist, inhibits pathologic retinal neovascularization

We investigated the in vitro metabolism and estrogenic and antiestrogenic activity of toremifene (TOR), tamoxifen (TAM) and their metabolites to better understand the potential effects of cytochrome P‐450 2D6 (CYP2D6) status on the activity of these drugs in women with breast cancer. The plasma concentrations of TOR and its N‐desmethyl (NDM) and 4‐hydroxy (4‐OH) metabolites during steady‐state dosing with TOR were also determined. Unlike TOR, TAM and its NDM metabolite were extensively oxidized to 4‐OH TAM and 4‐OH‐NDM TAM by CYP2D6, and the rate of metabolism was affected by CYP2D6 status. 4‐OH‐NDM TOR concentrations were not measurable at steady state in plasma of subjects taking 80 mg of TOR. Molecular modeling provided insight into the lack of 4‐hydroxylation of TOR by CYP2D6. The 4‐OH and 4‐OH‐NDM metabolites of TOR and TAM bound to estrogen receptor (ER) subtypes with fourfold to 30‐fold greater affinity were 35‐ to 187‐fold more efficient at antagonizing ER transactivation and had antiestrogenic potency that was up to 360‐fold greater than their parent drugs. Our findings suggest that variations in CYP2D6 metabolic capacity may cause significant differences in plasma concentrations of active TAM metabolites (i.e., 4‐OH TAM and 4‐OH‐NDM TAM) and contribute to variable pharmacologic activity. Unlike TAM, the clinical benefits in subjects taking TOR to treat metastatic breast cancer would not likely be subject to allelic variation in CYP2D6 status or affected by coadministration of CYP2D6‐inhibiting medications.

GTx-822, an ERβ-Selective Agonist, Protects Retinal Pigment Epithelium (ARPE-19) from Oxidative Stress by Activating MAPK and PI3-K Pathways

PURPOSE The goal of our study was to evaluate the in vitro and in vivo anti-angiogenic effects of ERβ selective agonist, β-LGND2, using human retinal microvascular endothelial cell (HRMVEC) cultures and a mouse model for oxygen-induced retinopathy (OIR). METHODS The selectivity of β-LGND2 was determined using binding and transactivation assays. The effects of β-LGND2 on pathologic neovascularization were evaluated in OIR mice by histology and retinal mounts stained with isolectin B4 to quantify aberrant angiogenesis. Gene expression and protein levels were evaluated using Q-PCR, angiogenesis protein array, and Western blotting. A cell death detection ELISA kit was used to evaluate HRMVECs following hypoxic and hyperoxic conditions. In vitro angiogenesis was evaluated by growth factor-induced proliferation, tube formation, and cell migration assays. RESULTS β-LGND2-treated OIR mice had a reduced number of neovascular tufts compared to vehicle-treated animals and a significant amount of normal blood vessel maturation similar to normoxia controls. β-LGND2 inhibited in vitro hypoxia- or hyperoxia-induced cell death and the formation of endothelial tubular structures in an ERβ-specific mechanism. However, β-LGND2 did not inhibit significantly growth factor-induced HRMVEC proliferation and migration. Gene and protein studies revealed that OIR mice treated with β-LGND2 had lower levels of pro-angiogenic factors, like VEGF and HIF1α. CONCLUSIONS β-LGND2 inhibited in vitro and in vivo pathologic neovascularization in the retina in an ERβ-specific mechanism. These results show that β-LGND2, a non-steroidal ERβ selective agonist, could be a useful therapeutic for ocular diseases involving aberrant angiogenesis, like ROP, wet-AMD, and diabetic retinopathy.

Preclinical characterization of a novel diphenyl benzamide selective ERα agonist for hormone therapy in prostate cancer

PURPOSE The goal of this study was to determine whether an estrogen receptor-β (ERβ)-selective agonist (GTx-822; GTx, Inc., Memphis, TN) could prevent hydrogen peroxide (H(2)O(2))-induced oxidative stress in ARPE-19 cells and to elucidate the molecular pathways involved in this protection. METHODS The selectivity of GTx-822 for ERβ was determined by receptor-binding assay (RBA) and transactivation assay. Cultured ARPE-19 cells were subjected to oxidative stress with t-butyl hydroxide (t-BH) or hydrogen peroxide (H(2)O(2)) in the presence and absence of GTx-822. Reactive oxygen species (ROS) was measured by using H(2)DCFDA fluorescence. Apoptosis was evaluated by cell death ELISA. Mitochondrial membrane potential was measured with the JC-1 assay. Gene expression and protein expression and activation were quantitated with qRT-PCR and Western blot analysis. Phospho-protein arrays elucidated the activation of protein kinases. RESULTS The RBA and transactivation assay revealed that GTx-822 is an ERβ-selective agonist (K(i) = 0.53 nM). GTx-822 prevented oxidative stress in ARPE-19 cells. It preserved mitochondrial function and prevented cellular apoptosis. Pretreatment with GTx-822 increased ERβ gene and protein expression during oxidative stress. Upregulation of the phase II antioxidant genes GPx-2 and HO-1 was also seen in an ERβ-dependent mechanism. GTx-822 pretreatment induced phosphorylation of ERK1/2, PI3-K, and Bad. CONCLUSIONS This is the first report to show that GTx-822, an ERβ agonist, can protect ARPE-19 cells from the cellular apoptosis induced by oxidative stress. GTx-822 mediated cytoprotection was mediated through induction of both genomic and nongenomic pathways. The results of this study open new avenues for the use of a selective ERβ agonist in treatment of ocular diseases like AMD where oxidative stress plays a major role in disease pathogenesis.

Abstract 2788: GTx-230: an orally available novel tubulin inhibitor that disrupts tumor vasculature and displays single-agent antitumor efficacy in multidrug-resistant prostate cancer with less neurotoxicity

Androgen deprivation therapy (ADT) is the mainstay of treatment for advanced prostate cancer. ADT improves overall and disease-free survival rates, but long-term therapy is associated with severe side effects of androgen and estrogen depletion including hot flashes, weight gain, depression, and osteoporosis. Effective hormone reduction can be achieved without estrogen deficiency-related side effects by using therapy with estrogenic compounds. However, cardiovascular complications induced by estrogens coupled with the availability of LHRH agonists led to discontinuation of estrogen use for primary androgen deprivation therapy in the 1980s. New treatments for prostate cancer that improve patient outcomes without the serious estrogen deficiency-related toxicities associated with ADT using LHRH analogs are needed. Herein we describe a novel nonsteroidal selective estrogen receptor-α agonist designed for first-line therapy of advanced prostate cancer that in animal models induces medical castration and minimizes many of the estrogen deficiency-related side effects of ADT. The present studies show that orally administered GTx-758 reversibly suppressed testosterone to castrate levels and subsequently reduced prostate volume and circulating prostate-specific antigen in relevant preclinical models without inducing hot flashes, bone loss, thrombophilia, hypercoagulation, or increasing fat mass.

A novel bis-indole destabilizes microtubules and displays potent in vitro and in vivo antitumor activity in prostate cancer

A novel indole antimitotic compound, GTx-230, was identified in cell-based screening assays that binds to the colchicine site in tubulin and destabilizes microtubules. In this study, the pharmacokinetics, antitumor efficacy, neurotoxicity, and vascular disrupting ability of GTx-230 were evaluated. GTx-230 inhibited the in vitro growth of a number of human cancer cell lines including DU-145, LNCaP, PC-3, and PPC-1 prostate cancer cells with low nanomolar IC50 values. Following a 10 mg/kg intravenous (i.v.) dose to mice, the plasma clearance (CL), volume of distribution at steady state (Vdss), and terminal half-life (T1/2) of GTx-230 were 19 ml/min/kg, 2.9 l/kg, and 1.7 h, respectively. Oral bioavailability of GTx-230 was 36% in mice. In vivo, GTx-230 (6.7 mg/kg, qd, p.o.) was effective with near 100% tumor growth inhibition in xenograft models using PC-3/TXR cells over-expressing P-glycoprotein. In contrast, docetaxel, a known antimitotic agent, was not effective in the PC-3/TXR xenograft model. In vivo studies of mouse performance on an accelerating rotarod and hotplate showed that GTx-230 did not induce any symptoms of peripheral neuropathy. In addition, we evaluated the effect of GTx-230 on in vitro vascular permeability using a transwell system with confluent HUVEC monolayers. GTx-230 resulted in increased permeability after 1 h incubation and showed potency similar to CA4. In summary, the potent in vitro and in vivo antitumor activities of GTx-230 suggest that it may represent a new antimitotic agent for management of solid malignancies, particularly for patients with drug resistant cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2788. doi:1538-7445.AM2012-2788