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Anita Nikolova Penkova, Juan Carlos Martinez, Mark Humayun, Abegail Tadle, Ana Galesic, Alejandra Calle, Mark Thompson, Matthew Pratt, Satwindar Singh Sadhal; BEVACIZUMAB DIFFUSION COEFFICIENT IN VIVO MEASUREMENT OF RABBIT VITREOUS HUMOR WITH FLUORESCEIN LABELING. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6103.
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© ARVO (1962-2015); The Authors (2016-present)
The primary purpose of this work is to measure the diffusion coefficient of the macromolecule bevacizumab in the live rabbit vitreous humor using fluorescein labeling for visualization.
The method consists of injecting into rabbit vitreous a solution of bevacizumab that was successfully labeled with Alexa Flour 488 dye (6.74µM concentration). An anesthetized dutch-belted live rabbit was used for the experiment. The labeled drug solution was injected into the vitreous with a 30-gauge needle via pars plana (1.5 mm away from the limbus) of an anesthetized rabbit at a slow rate of 2µl/min over a period of 10 minutes using a syringe pump. The drug diffusion was then tracked using HRA+OCT Spectralis (Heidelberg Engineering, Inc; Germany) at different time points, approximately 15 minutes apart in a 3-hour time period.
The fluorescence intensity data were analyzed to produce colormap contours. Using a predictive model, these contours are also obtained by an analytical solution of the diffusion equation. The theoretical prediction and the experimental contours were matched while floating the unknown diffusion coefficient (see ). The best fit yields a value for the diffusion coefficient which was found to be approximately 4x10-7 cm2/s.
While Gd-labeled molecules with MRI have provided very accurate measurements of the respective diffusion coefficients, fluorescein labeling provides an alternative when the former presents challenges. With bevacizumab, this alternative worked out very successfully, and fluorescein labeling provided images that have been analyzed to obtain the in vivo concentration distribution of this drug in the rabbit vitreous. A theoretical model based on a spherical bolus of the drug and its quantitative comparison with experimental data provided the value of the unknown diffusion coefficient. The is somewhat higher than the value for Gd-IgG (160 kDa) for the ex vivo bovine vitreous but about the same order of magnitude (2.1x10-7 cm2/s). While the current results provide information towards developing a comprehensive model for fluid flow and transport in the eye, experimental and analytical refinements in the approach are being explored to provide a higher degree of experimental accuracy.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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