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Amy Shelton, Susan Crowell, Hanine Anezinos, Danielle Mandikian, Phillip Chu; Utility of quantitative autoradiography for drug pharmacology and ocular tissue distribution in New Zealand White Rabbits. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6104.
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© ARVO (1962-2015); The Authors (2016-present)
Radiolabeling of test articles is a common method to assess drug concentration and biodistribution to ocular compartments. The purpose of this study is to determine if quantitative autoradiography can be used on whole cranium sections of New Zealand White rabbits to measure drug concentration and assess tissue distribution including retinal penetration of an I-125 labeled drug product.
Dose AdministrationA 50µL ITV injection was administered to the inferior temporal location of each eye.Solid Scintillation CountingFor comparison to autoradiography, ocular matrices were dissected and homogenized before being analyzed in duplicate for radioactivity by SSC.Quantitative Autoradiography Sections were collected in the transverse plane on adhesive tape. After drying, a representative section from each level of interest was mounted and wrapped with Mylar film.Autoradiographic standards were sampled to create a calibrated standard curve. LLOQ was based on the lowest readable concentration. Tissue concentrations were interpolated from each standard curve as nanocuries/g and then converted to ng equivalents/g.Mass SpectometryIsotopically-labeled material was spiked into each tissue sample and was extracted from the using acetonitrile precipitation. The supernatant was run on LC-MS (Orbitrap Elite) and drug concentration quantitated by comparing AUC to the internal standard.
Ocular tissues were harvested and counted to determine the total drug concentration post ITV injection, including the aqueous humor, vitreous humor and retina. Aqueous and vitreous concentrations were reliable and corroborated by mass spectometry. Isolation methods for retina were found to be insufficient, and unlikely to represent true retinal concentrations. Autoradiography was then used to inform drug distribution within ocular compartments, but lacked fine resolution to determine concentration or drug penetration into the thin retinal layers.
Radioanalysis of isolated tissue is a valuable method to assess drug concentration in whole ocular tissue samples, such as the aqueous and vitreous humors. Autoradiography, although unable to determine retinal concentration, should be considered as a companion analysis to SSC or MS, to assess heterogeneity of test article distribution to, and within, ocular compartments.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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