In Situ Determination of Intravitreal Concentrations of a Biotherapeutic Labeled with a Near-Infrared Fluorescent Ligand Using Confocal Scanning Laser Ophthalmoscopy

Abstract

Purpose: : The pharmacokinetics (PK) of ophthalmic biotherapeutics are difficult to directly determine in human vitreous humor. Because of the high transparency of living tissue to near-infrared (NIR) light, the temporal changes in vitreous concentrations of a biomolecule labeled with a NIR fluorescent probe (NIRF) were monitored in situ using a scanning laser ophthalmoscope (SLO).

Methods: : A humanized IgG was labeled with the NIRF probe IRDye-800 CW (CVX-4164). NZW rabbits were intravitreally administered CVX-4164 and NIRF intensity measured in the center of the vitreous humor using a SLO. Scans were repeatedly taken from 1 to 240 hours after administration. Intensity gray scale values were converted to concentrations using standard curves. PK data were analyzed by non-compartmental and compartmental analysis using WinNonlin. The care and use of the animals in this study were conducted in accordance with the guidelines of the NRC USA and the Canadian Council on Animal Care.

Results: : There was little background fluorescence and the minimum detectable concentration of CVX-4164 was < 10 nM. Vitreal CVX-4164 concentrations determined in situ declined according to a single compartment, 1^st order kinetics model, with C_max ≈1 µM, and t_1/2 = 145 hr, consistent with the rabbit vitreal t_1/2 of other mAbs (eg, bevacizumab) as measured by ELISA. Vitreal CVX-4164 concentrations were 2.6 to 4.4 times higher than those determined by ex vivo NIRF or ELISA in homogenized vitreous humor, reflecting the superior spatial resolution of in situ imaging. Vitreal CVX-4164 concentrations determined in situ were >3 orders of magnitude greater than plasma concentrations determined by ELISA, and showed a different PK profile.

Conclusions: : This study demonstrates the feasibility of determining the PK of intraocular biotherapeutics labeled with NIRF probes by in situ monitoring. This concept may be extended to the imaging of NIRF-labeled biologicals with either SLO and/or optical coherence tomography for monitoring biomarkers in the cornea and retina, or for diagnosing ocular diseases.

Keywords: imaging/image analysis: non-clinical • clinical research methodology • vitreous