Biodistribution and Targeting of Anti-5T4 Antibody-Drug Conjugate Using Fluorescence Molecular Tomography.

Understanding a drug's whole-body biodistribution and tumor targeting can provide important information regarding efficacy, safety, and dosing parameters. Current methods to evaluate biodistribution include in vivo imaging technologies like positron electron tomography and single-photon emission computed tomography or ex vivo quantitation of drug concentrations in tissues using autoradiography and standard biochemical assays. These methods use radioactive compounds or are cumbersome and do not give whole-body information. Here, for the first time, we show the utility of fluorescence molecular tomography (FMT) imaging to determine the biodistribution and targeting of an antibody-drug conjugate (ADC). An anti-5T4-antibody (5T4-Ab) and 5T4-ADC were conjugated with a near-infrared (NIR) fluorophore VivoTag 680XL (VT680). Both conjugated compounds were stable as determined by SEC-HPLC and plasma stability studies. Flow cytometry and fluorescence microscopy studies showed that VT680-conjugated 5T4-ADC specifically bound 5T4-expressing cells in vitro and also exhibited a similar cytotoxicity profile as the unconjugated 5T4-ADC. In vivo biodistribution and tumor targeting in an H1975 subcutaneous xenograft model demonstrated no significant differences between accumulation of VT680-conjugated 5T4-Ab or 5T4-ADC in either normal tissues or tumor. In addition, quantitation of heart signal from FMT imaging showed good correlation with the plasma pharmacokinetic profile suggesting that it (heart FMT imaging) may be a surrogate for plasma drug clearance. These results demonstrate that conjugation of VT680 to 5T4-Ab or 5T4-ADC does not change the behavior of native biologic, and FMT imaging can be a useful tool to understand biodistribution and tumor-targeting kinetics of antibodies, ADCs, and other biologics. Mol Cancer Ther; 15(10); 2530-40. ©2016 AACR.

Axitinib inhibits retinal and choroidal neovascularization in in vitro and in vivo models.

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.

Axitinib and crizotinib combination therapy inhibits bone loss in a mouse model of castration resistant prostate cancer.

BACKGROUND: Castration 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. METHODS: VCaP-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. RESULTS: Subcutaneous 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. CONCLUSION: Our 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.

Patient-derived xenografts reveal limits to PI3K/mTOR- and MEK-mediated inhibition of bladder cancer.

BACKGROUND: Metastatic bladder cancer is a serious condition with a 5-year survival rate of approximately 14 %, a rate that has remained unchanged for almost three decades. Thus, there is a profound need to identify the driving mutations for these aggressive tumors to better determine appropriate treatments. Mutational analyses of clinical samples suggest that mutations in either the phosphoinositide-3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) or RAS/MEK/ERK pathways drive bladder cancer progression, although it remains to be tested whether the inhibition of either (or both) of these pathways can arrest PI3K/mTOR- or Ras-driven proliferation. METHODS: Herein, we used several bladder cancer cell lines to determine drug sensitivity according to genetic background and also studied mouse models of engrafted UM-UC-3 cells and patient-derived xenografts (PDXs) to test PI3K/mTOR and MEK inhibition in vivo. RESULTS: Inhibition of these pathways utilizing PF-04691502, a PI3K and mTOR inhibitor, and PD-0325901, a MEK inhibitor, slowed the tumor growth of PDX models of bladder cancer. The growth inhibitory effect of combination therapy was similar to that of the clinical maximum dose of cisplatin; mechanistically, this appeared to predominantly occur via drug-induced cytostatic growth inhibition as well as diminished vascular endothelial growth factor secretion in the tumor models. Kinase arrays of tumors harvested after treatment demonstrated activated p53 and Axl as well as STAT1 and STAT3. CONCLUSION: 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.

Contrast agents for quantitative microCT of lung tumors in mice.

The identification and quantitative evaluation of lung tumors in mouse models is challenging and an unmet need in preclinical arena. In this study, we developed a noninvasive contrast-enhanced microCT (µCT) method to longitudinally evaluate and quantitate lung tumors in mice. Commercially available µCT contrast agents were compared to determine the optimal agent for visualization of thoracic blood vessels and lung tumors in naïve mice and in non-small-cell lung cancer models. Compared with the saline control, iopamidol and iodinated lipid agents provided only marginal increases in contrast resolution. The inorganic nanoparticulate agent provided the best contrast and visualization of thoracic vascular structures; the density contrast was highest at 15 min after injection and was stable for more than 4 h. Differential contrast of the tumors, vascular structures, and thoracic air space by the nanoparticulate agent enabled identification of tumor margins and accurate quantification. µCT data correlated closely with traditional histologic measurements (Pearson correlation coefficient, 0.995). Treatment of ELM4-ALK mice with crizotinib yielded 65% reduction in tumor size and thus demonstrated the utility of quantitative µCT in longitudinal preclinical trials. Overall and among the 3 agents we tested, the inorganic nanoparticulate product was the best commercially available contrast agent for visualization of thoracic blood vessels and lung tumors. Contrast-enhanced µCT imaging is an excellent noninvasive method for longitudinal evaluation during preclinical lung tumor studies.

Combination of a MEK inhibitor at sub-MTD with a PI3K/mTOR inhibitor significantly suppresses growth of lung adenocarcinoma tumors in Kras(G12D-LSL) mice.

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.

ß-LGND2, an ERß selective agonist, inhibits pathologic retinal neovascularization.

PURPOSE: The goal of our study was to evaluate the in vitro and in vivo anti-angiogenic effects of ERß selective agonist, ß-LGND2, using human retinal microvascular endothelial cell (HRMVEC) cultures and a mouse model for oxygen-induced retinopathy (OIR). METHODS: The selectivity of ß-LGND2 was determined using binding and transactivation assays. The effects of ß-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: ß-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. ß-LGND2 inhibited in vitro hypoxia- or hyperoxia-induced cell death and the formation of endothelial tubular structures in an ERß-specific mechanism. However, ß-LGND2 did not inhibit significantly growth factor-induced HRMVEC proliferation and migration. Gene and protein studies revealed that OIR mice treated with ß-LGND2 had lower levels of pro-angiogenic factors, like VEGF and HIF1α. CONCLUSIONS: ß-LGND2 inhibited in vitro and in vivo pathologic neovascularization in the retina in an ERß-specific mechanism. These results show that ß-LGND2, a non-steroidal ERß selective agonist, could be a useful therapeutic for ocular diseases involving aberrant angiogenesis, like ROP, wet-AMD, and diabetic retinopathy.

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

GTx-822, an ER{beta}-selective agonist, protects retinal pigment epithelium (ARPE-19) from oxidative stress by activating MAPK and PI3-K pathways.

PURPOSE: The goal of this study was to determine whether an estrogen receptor-ß (ERß)-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 ERß 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 ERß-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 ERß gene and protein expression during oxidative stress. Upregulation of the phase II antioxidant genes GPx-2 and HO-1 was also seen in an ERß-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 ERß 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 ERß agonist in treatment of ocular diseases like AMD where oxidative stress plays a major role in disease pathogenesis.

17-ß estradiol protects ARPE-19 cells from oxidative stress through estrogen receptor-ß.

PURPOSE: To elucidate the mechanism of 17-ß estradiol (17ß-E(2))-mediated protection of retinal pigment epithelium (RPE) from oxidative stress. METHODS: Cultured ARPE-19 cells were subjected to oxidative stress with t-butyl hydroxide or hydrogen peroxide in the presence or absence of 17ß-E(2). Reactive oxygen species (ROS) were measured using H(2)DCFDA fluorescence. Apoptosis was evaluated by cell-death ELISA kit and Hoechst-3486 staining. Mitochondrial membrane potential was measured using the JC-1 assay. Cellular localization of estrogen receptor (ER) was evaluated by confocal microscopy. Gene expression and protein expression was quantified using qRT-PCR and western blotting. Superoxide dismutase and ATP levels were measured using commercial kits. RESULTS: ARPE-19 cells expressed significant amounts of ERα and ERß. Pretreatment with 17ß-E2 protected ARPE-19 cells from oxidative stress and apoptosis. 17ß-E(2) reduced the ROS levels and mitochondrial depolarization. The 17ß-E(2)-mediated cytoprotection was inhibited by ER antagonists ICI (ERα and ERß) and THC (ERß) but not by tamoxifen (ERα). Knockdown of ERß expression by siRNA abolished the protective effects of 17ß-E(2). Further, qRT-PCR analysis revealed that 17ß-E(2) pretreatment upregulated the expression of ERß and phase II cellular antioxidant genes. CONCLUSIONS: These results indicate that 17ß-E(2) protects ARPE-19 cells from oxidative stress through an ERß-dependent mechanism. 17ß-E(2)-mediated cytoprotection occurred through the preservation of mitochondrial function, reduction of ROS production, and induction of cellular antioxidant genes.