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A.M. Laties, C. Mitchell; Method for the Assessment of Ocular Distribution of Pharmacological Agents . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3531.
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Purpose: We describe a method for the precise and accurate definition of ocular penetration of a given agent. The method provides: protection of tissue integrity and prevents diffusion or translocation. Methods: Preservation of tissue integrity is accomplished by quick freezing in isopentane cooled in liquid nitrogen to a temperature of –105° C. Thereafter freeze drying at a temperature of –35° C retains tissue integrity and at the same time prevent diffusion. In effect, low temperature sublimation of water from the ice interface can be compared to agent fixation in that diffusible substances do not move to a perceptible degree. Results: For the eye, the end result is a dried globe of perfect form that permits several alternatives for localization and quantification of drug: 1) dried eyes can be dissected under a controlled, low humidity to obtain discrete samples of specific regions from individual ocular structures. Dry, diffusion–free, small tissue samples from retina at various locations can be obtained. In addition, after weighing the tissue sample, drug can be extracted for analysis. 2) If, instead, a radiolabeled compound was applied, multiple samples from eyes, each dosed for a specific duration can be individually placed in a suitable medium and counts per minute recorded. Each set of samples is equivalent to a stop–action assay at a given timepoint. As one example, greater than a 20 fold difference in activity is found in the rabbit lens between anterior cortex and central nucleus one hour after topical administration of radiolabeled epinephrine. 3) Alternatively, tissue samples can be embedded in non–hydrophilic embedding media to permit visualization of fluorescent probes. When this is done and a highly diffusible, small molecular weight probe such as fluorescein is administered intravenously, the validity of the method is immediately apparent: brilliant yellow fluorescence sharp–edged and confined within retinal blood vessels stands in stark contrast to the blue autofluorescence of adjacent tissue. Conclusions: With this method the ocular distribution to the eye of drugs administered either topically or systemically can be accurately localized. #
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