Imaging Targeted-Agent Binding In Vivo with Two Probes

An approach to quantitatively image targeted-agent binding rate in vivo is demonstrated with dual-probe injection of both targeted and nontargeted fluorescent dyes. Images of a binding rate constant are created that reveal lower than expected uptake of epidermal growth factor in an orthotopic xenograft pancreas tumor (2.3×10−5 s−1), as compared to the normal pancreas (3.4×10−5 s−1). This approach allows noninvasive assessment of tumor receptor targeting in vivo to determine the expected contrast, spatial localization, and efficacy in therapeutic agent delivery. Targeting therapeutic drugs to tumors based on their overexpression of cellular receptors is widely researched and has important clinical success.1, 2 Yet there are essentially no good tools to assess the in vivo receptor expression contrast between tumor as compared to normal surrounding tissue.3, 4 In tumors with very high molecular signaling such as in the pancreas,4, 5 it is not obvious when a particular receptor is actually up-regulated as compared to the surrounding normal tissue versus upregulated without biopsy. Imaging of receptor status in vivo is problematic, because the majority of any targeted agent in vivo is often not cell-associated yet. Thus, any single image simply provides a measure of the whole tissue concentration rather than the bound concentration. Delivery from the vascular supply to tumor cells requires transvascular leakage, followed by diffusion through the interstitial space, and binding to the targeted receptor followed by possible internalization.6 As such, imaging concentration values in vivo usually do not provide information about binding,7 since most of the agent is in the interstitial space. In this work, we demonstrate a new methodology for quantitative imaging of effective binding rate in vivo, using the difference in fluorescence signal between a targeted and untargeted agent. We use this to demonstrate that a tumor known to have high EGFR expression in vitro 5 actually has lower EGF activity than the surrounding normal pancreas in vivo. Most contrast agent imaging has been interpreted with a simple pharmacokinetic model that is designed with as few compartments and rate constants as possible to not overinterpret the data. A three compartment model [Fig. 1 ] can be used effectively to model targeted agent delivery in the tumor, which includes compartments for 1. the concentration of drug in the plasma within the vasculature, 2. the concentration in the interstitial space of the tissue, and 3. the cellular-associated fraction of drug.7 The dominant fast rates in this model are transvascular delivery of contrast agent through rate constantK12 role= presentation \u3eK12 , and then cell-associating rate constant due to binding and uptake, K23 role= presentation \u3eK23 . The dominant clearance from the plasma is given by excretion mechanisms, such as those in the liver and kidneys, through rate constant Ke role= presentation \u3eKe . Then the slowest rates tend to be those involved in backflow from the interstitial space to the vasculature K21 role= presentation \u3eK21 , and from the cell-associated space to the interstitial space K32 role= presentation \u3eK32 . Each of these is shown in the illustration of the model in Fig. 1

A Critical Examination of Subgroup Analyses: The National Acute Spinal Cord Injury Studies and Beyond

The use of high-dose methylprednisolone for acute spinal cord injury continues to be a topic of debate. This controversy largely stems from fundamental issues in statistical interpretation of trial data, most notably subgroup analyses. The purpose of this review is to discuss important examples of improper subgroup analysis and encourage better practices in future research