Development and characterization of a tumor specific contrast agent for in vivo imaging of therapeutic response

Derivatives of the tubulin binding agent colchicine have been utilized as vascular-disrupting agents (VDA’s) as they selectively target immature tumor endothelial cells, inducing thrombosis and vessel occlusion, resulting in extensive necrosis within 24h of administration. This study has investigated the use of a novel gadolinium labeled colchicine derivative (Gd.DOTA.colchicinic acid) as a contrast medium for magnetic resonance imaging (MRI) to assess the effects of VDA’s in vivo.Gd.DOTA.colchicinic acid and the non-gadolinium DOTA.colchicinic acid was synthesized and provided by Imperial College Genetic Therapies Group, Imperial College London. Human ovarian OVCAR3 carcinoma cells were either plated for in vitro experiments or grown as subcutaneous tumors for in vivo evaluation by MRI. In vitro cell studies consisted of cell death assays 24 hours post-incubation of either DOTA.colchicinic acid or colchicine (1nM - 1mM) recorded by histological examination and FACS analysis. For in vivo studies tumor bearing mice were imaged using a muliti sliced spin echo MRI sequence covering the whole abdomen to evaluate biodistribution as well intratumoural response, prior to, 2, 12 and 24 hours post administration of 200mg/kg Gd.DOTA.colchicinic acid, 200mg/kg DOTA.colchicinic acid, or water as a control. After the final scan tumors and selective organs were excised, frozen and sections where taken for Heamotoxylin and Eosin staining.The results showed that in vitro the DOTA.colchicinic acid maintained efficiency at higher concentrations enabling administration of a larger dose than that of unlabeled colchicine, further facilitating its function as a contrast agent. In vivo MRI within 2 hours of administration of 200mg/kg Gd.DOTA.colchicinic acid showed a slight reduction in T1 compared to that of base line, with a 40% reduction in T1 at 12 hours post injection, which was sustained for 24 hours. Tumor images showed specific morphological changes within the tumor over the 24 hour time course, which maybe due to entrapment within the necrotic spaces after vascular occlusion. Histology confirmed that all tumors treated with a colchicinic acid tagged molecule caused large central necrosis within the tumor at the 24 hour time point. The liver and kidney also exhibited reductions in T1 over the time course of the study. However, resulting histology confirmed no significant differences between treated and non-treated morphology in either organ.This project has established that Gd.DOTA.colchicinic acid as a functional contrast agent that can bind tubulin leading to tumor vascular shutdown as well as shortening T1 in vivo. Gd.DOTA.colchicinic acid maintains efficiency at high concentrations enabling administration of a larger dose of Gd.DOTA.colchicinic acid, further facilitating its function as a contrast agent and can be utilized to image vascular therapy of VDA’s in vivo.