RNA interference (RNAi) is an exciting new tool to effect acute in vivo knockdown of genes for pharmacological target validation. Testing the application of this technology to metabolic disease targets, three RNAi delivery methods were compared in two frequently utilized preclinical models of obesity and diabetes, the diet-induced obese (DIO) and B6.V-Lep/J (ob/ob) mouse. Intraperitoneal (i.p.) and high pressure hydrodynamic intravenous (i.v.) administration of naked siRNA, and low pressure i.v. administration of shRNA-expressing adenovirus were assessed for both safety and gene knockdown efficacy using constructs targeting cJun N-terminal kinase 1 (JNK1). Hydrodynamic delivery of siRNA lowered liver JNK1 protein levels 40% in DIO mice, but was accompanied by iatrogenic liver damage. The ob/ob model proved even more intolerant of this technique, with hydrodynamic delivery resulting in severe liver damage and death of most animals. While well-tolerated, i.p. injections of siRNA in DIO mice did not result in any knockdown or phenotypic changes in the mice. On the other hand, i.v. injected adenovirus expressing shRNA potently reduced expression of JNK1 in vivo by 95% without liver toxicity. In conclusion, i.p. and hydrodynamic injections of siRNA were ineffective and/or inappropriate for in vivo gene targeting in DIO and ob/ob mice, while adenovirus-mediated delivery of shRNA provided a relatively benign and effective method for exploring liver target silencing.
396 Objectives ABT-806 is a humanized antibody targeting a unique epitope on EGFR only available for binding when EGFR is overexpressed, activated, or when the EGFRvIII mutation is present. This property allows for a higher tumor to normal tissue contrast and thus enables ABT-806 for payload delivery. We have therefore developed 111In-labeled ABT-806 (ABT-806i) for SPECT imaging of EGFR positive tumors. Methods The ABT-806 antibody was conjugated with either DTPA or CHX-A’-DTPA and used to generate 111In-labeled ABT-806 (ABT-806i) which was characterized to evaluate its radiochemical features and its relative tumor-specific binding properties using nanoSPECT/CT image quantification. Results Selective tumor targeting of 111In-labeled ABT-806 (ABT-806i) was demonstrated in both wild-type EGFR overexpressing tumor xenografts (11 ± 2.1 %ID/cc) and EGFRvIII positive tumor xenografts, 21.5 ± 4.3%ID/cc in flank and 75.1 ± 11.3 %ID/cc as orthotopic glioma. The specific tumor uptake was inhibited gradually by co-dosing increasing concentrations of unlabeled ABT-806 antibody, from which a tumor occupancy curve was generated to estimate the dose for 50% tumor saturation as 13 mg/kg in flank xenografts. ABT-806 conjugated to CHX-A’-DTPA was GMP manufactured as a formulated kit. The labeling efficiency with sterile 111In was consistently higher than 95% for the instant preparation. The immunoreactive fraction was 0.8 ± 0.06 and the product is stable for 5 days and the prepared ABT-806i retained the favorable in vivo biodistribution and tumor targeting features. Conclusions These features render ABT-806i a promising imaging agent currently under clinical validation for characterization of tumor EGFR status. Research Support Abbott Labs
Gel permeation methods have been commonly used to screen combinatorial libraries synthesized on a solid support. We report here three screens of combinatorial libraries using gel permeation assays. These include a simple enzymatic assay to identify inhibitors of the influenza enzyme neuraminidase, and two more complex assays designed to screen for inhibitors of the interleukin-8 (IL-8)-IL-8 receptor and the urokinase-urokinase receptor interactions, respectively. The IL-8 ligand-receptor assay makes use of IL-8 receptor-expressing cells attached to a membrane, thus enabling washing steps as part of the assay. The urokinase ligand-receptor assay employs an enzyme-linked immunosorbent assay-type format, previously thought to be amenable only to well-based assays. The results of these three screens are reported here, including the discovery of a novel series of acyclic inhibitors of neuraminidase. The development of complex assays in a gel permeation format allows for the routine screening of combinatorially as well as noncombinatorially made compound collections against virtually any kind of target, and is being widely used in our high throughput screening operations.
The objective of this study was to determine the utility of 99mTc-3P-Arg-Gly-Asp (RGD2) single photon emission computed tomography (SPECT)/computed tomography (CT) for noninvasive monitoring of integrin αvβ3-expression response to antiangiogenic treatment with linifanib. Linifanib or vehicle therapy was carried out in female athymic nu/nu mice bearing U87MG glioma (high αvβ3 expression) or PC-3 prostate (low αvβ3 expression) tumors at 12.5 mg/kg twice daily. The average tumor volume was 180 ± 90 mm3 the day prior to baseline SPECT/CT. Longitudinal 99mTc-3P-RGD2 SPECT/CT imaging was performed at baseline (–1 day) and days 1, 4, 11, and 18. Tumors were harvested at all imaging time points for histopathological analysis with H&E and immunohistochemistry. A significant difference in tumor volumes between vehicle- and linifanib-treated groups was observed after 4 days of linifanib therapy in the U87MG model. The percent injected dose (%ID) tumor uptake of 99mTc-3P-RGD2 peaked in the vehicle-treated group at day 11, while the %ID/cm3 tumor uptake decreased slowly over the whole study period. During the first 2 days of linifanib treatment, a rapid decrease in both %ID/cm3 tumor uptake and tumor/muscle ratios of 99mTc-3P-RGD2 was observed, followed by a slow decrease until day 18. No decrease in tumor uptake of 99mTc-3P-RGD2 or tumor volume was observed for either treatment group in the PC-3 model. Changes in tumor vasculature were confirmed by histopathological H&E analysis and immunohistochemistry. Longitudinal imaging using 99mTc-3P-RGD2 SPECT/CT may be a useful tool for monitoring the downstream biologic effects of linifanib therapy.
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