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Jeffery J Prusakiewicz, Carol A Rasmussen, Christian Hafele, Justine Oswald, Craig B Struble; Non-Invasive Quantitation of a Near-Infrared Fluorescence Dye Following Intravitreal Administration To Non-human Primates Using The Heidelberg Spectralis HRA+OCT. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2109.
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The pharmacokinetics of ophthalmic biotherapeutics can be difficult to directly determine in vitreous humor, and may require terminal samples in preclinical studies. Because of the high transparency of living ocular tissues to near-infrared light, the temporal changes in vitreous concentrations of a near-infrared fluorescent (NIRF) probe can be monitored in situ following an intravitreal dose. The concentration of NIRF probe was calculated from a standard curve using a scanning laser ophthalmoscope and verified following ex vivo sampling of vitreous humor with a fluorescent plate reader.
A NIRF probe (IRDye800CW) was administered by intravitreal injection into cynomologous monkey eyes. Method optimization was performed by monitoring the NIRF intensity with various combinations of parameters using the Heidelberg Spectralis HRA+OCT in ICGA mode. Scans of the vitreous were taken immediately postdose and over the next day. Intensity gray scale values were converted to concentrations using standard curves. Upon imaging completion, animals were euthanized and vitreous humor samples were collected for in vitro analysis. Concentrations were assessed under a variety of parameters to define those that were the most accurate.
Prior to dosing little background fluorescence was observed in any of the imaging modes. Minimal matrix effects were observed for vitreous humor, but 10% difference was observed in radial location intensity when comparing the pixel density in different regions of the image from dosed primate eyes. It was verified that calibration curves at 50% and 80% intensities were sufficient to cover over 3 orders of magnitude (1 nM-3.2 μM) in gray scale pixel density. Focusing at 8 diopters from the retina gave the most consistent imaging results.
This study demonstrates the feasibility and accuracy of determining the concentration of intraocular NIRF probes by noninvasive imaging methods. These methods may be extended to the quantitation of NIRF-labeled biotherapeutics, and can afford serial measurements from the same animal, resulting in an overall reduction in animal usage. In addition, the Heidelberg Spectralis HRA+OCT has multiple modes of detection with the ability to simultaneously quantitate co-administered molecules when evaluating combination therapies in the preclinical and clinical studies.
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