Andrew Healey

Whole-body section fluorescence imaging--a novel method for tissue distribution studies of fluorescent substances.

The present study demonstrates the usefulness of whole body section fluorescence imaging, a novel technique used in optical imaging drug discovery. This method is in principle an analog of whole body autoradiography, except that fluorescence is measured instead of radioactivity. The method was shown to have a linear concentration-response relationship over a 1000-fold concentration range. Densitometric image analysis allowed semiquantitative studies of drug disposition and selective tumor retention of an optical imaging drug candidate.

A targeted molecular probe for colorectal cancer imaging

Colorectal cancer is a major cause of cancer death. Morbidity, mortality and healthcare costs can be reduced if the disease can be detected at an early stage. Screening is a viable approach as there is a clear link to risk factors such as age. We have developed a fluorescent contrast agent for use during colonoscopy. The agent is administered intravenously and is targeted to an early stage molecular marker for colorectal cancer. The agent consists of a targeting section comprising a peptide, and a fluorescent reporter molecule. Clinical imaging of the agent is to be performed with a far red fluorescence imaging channel (635 nm excitation/660-700 nm emission) as an adjunct to white light colonoscopy. Preclinical proof of mechanism results are presented. The compound has a Kd of ~3nM. Two human xenograft tumour models were used. Tumour cells were implanted and grown subcutaneously in nude mice. Imaging using a fluorescence reflectance imaging system and quantitative biodistribution studies were performed. Substances tested include the targeted agent, and a scrambled sequence of the peptide (no binding) used as a negative control. Competition studies were also performed by co-administration of 180 times excess unlabelled peptide. Positive imaging contrast was shown in the tumours, with a clear relationship to expression levels (confirmed with quantitative biodistribution data). There was a significant difference between the positive and negative control substances, and a significant reduction in contrast in the competition experiment.

Imaging efficacy of a targeted imaging agent for fluorescence endoscopy

Colorectal cancer is a major cause of cancer death. A significant unmet clinical need exists in the area of screening for earlier and more accurate diagnosis and treatment. We have identified a fluorescence imaging agent targeted to an early stage molecular marker for colorectal cancer. The agent is administered intravenously and imaged in a far red imaging channel as an adjunct to white light endoscopy. There is experimental evidence of preclinical proof of mechanism for the agent. In order to assess potential clinical efficacy, imaging was performed with a prototype fluorescence endoscope system designed to produce clinically relevant images. A clinical laparoscope system was modified for fluorescence imaging. The system was optimised for sensitivity. Images were recorded at settings matching those expected with a clinical endoscope implementation (at video frame rate operation). The animal model was comprised of a HCT-15 xenograft tumour expressing the target at concentration levels expected in early stage colorectal cancer. Tumours were grown subcutaneously. The imaging agent was administered intravenously at a dose of 50nmol/kg body weight. The animals were killed 2 hours post administration and prepared for imaging. A 3-4mm diameter, 1.6mm thick slice of viable tumour was placed over the opened colon and imaged with the laparoscope system. A receiver operator characteristic analysis was applied to imaging results. An area under the curve of 0.98 and a sensitivity of 87% [73, 96] and specificity of 100% [93, 100] were obtained.

Molecular targeting as a contrast agent mechanism for fluorescence endoscopy

Several mechanisms of action can be employed for a molecular imaging contrast agent for use with endoscopy. Targeting of cell surface molecules that are up regulated at an early disease stage, with a fluorescent labelled vector is one attractive approach. However, it suffers from the inherent limitation that the concentration of agent available is fundamentally limited by the concentration of receptor molecules available. Simple models indicate that for successful imaging with a targeting approach, the imaging system should be able to adequately image concentrations in the nanomolar region. Such low reporter molecule concentrations have implications for the choice of contrast agent. Target tissue size and location, the tissue native fluorescence contribution, the brightness of the reporter molecule, and photobleaching thresholds are all factors which contribute to the choice of reporter. For endoscopic imaging of millimetre sized target tissue volumes close to the surface Cy5TM (650-700nm) wavelengths are preferable to Cy3TM (550-600nm) and Cy7TM (750-800nm). We have constructed a system optimised for sensitivity by tailoring light delivery, collection, filtering and detection, in order to address the fundamental technical performance limits for endoscopic applications. It is demonstrated through imaging system calibration, phantom based measurement and animal imaging data that low nanomolar concentrations of Cy5 based fluorescent contrast agent in millimetre sized superficial lesions are adequately imaged with a clinically relevant endoscope system in real time. It is concluded that targeting is a technically viable approach for endoscopic applications.

Photon transport models for predictive assessment of imageability

Accurate calculation of internal fluence excited in tissue from an optical source can be used for predicting the performance of fluorescent contrast agents for clinical applications. Solutions of excitation fluence for a steady-state Monte Carlo model and a finite element implementation of the 3d diffusion equation have been compared up to depths of 20mm from a point source located on top of a homogeneous cylindrical phantom for a range of reduced scattering-to-absorption ratios. Differences between the fluence calculated by Monte Carlo and diffusion model is found to be dependent on the transport mean free path (mfp), size of the phantom in relation to the penetration depth, distance from the source and mesh resolution. The differences are small at depths ~ mfp and peak at depths ~2mfp. The differences should ideally reduce to zero at large depths but the magnitude of the differences tend to increase due to the finite boundary in the diffusion model. As an example, for a mfp = 0.817mm similar in magnitude to mesh resolution, diffusion fluence at 1mm, 2mm, 10mm and 14mm is 76%, 59%, 66% and 63% respectively of Monte Carlo fluence. For large mfps characteristic of non- diffusive regimes, diffusion model overestimates fluence at distances less than one mfp. This work demonstrates that mean free path and mesh resolution are the critical parameters that distinguish the performance of Monte Carlo and diffusion models to define error margins that could be utilized for predictive assessment of imageability of fluorescent agents using the diffusion model.