Intravitreal Pharmacokinetics of a Near-Infrared Fluorescence-Labeled Biotherapeutic Determined In Situ Using Confocal Scanning Laser Ophthalmoscopy

摘要

The pharmacokinetics (PK) of ophthalmics in the different compartments of the human eye are difficult to determine. Because of the high transparency of living tissue to near-infrared (NIR) light, the temporal changes in vitreous concentrations of a biomolecule labelled with a NIR fluorescent probe may be measured by in situ monitoring with a scanning laser ophthalmoscope (SLO), allowing the PK of the biotherapeutic to be determined at the site of administration by non-invasive techniques. In this study, a surrogate biotherapeutic, in the form of a humanized IgG (CVX-2000), was labelled with the NIR probe IRDye 800CW to yield CVX-4164. Rabbits were injected intravitreally with CVX-4164 (or 800 CW alone, or CVX 2000) and NIR fluorescence intensity measured in the central plane of the vitreous humour using a SLO. Fluorescence intensities were converted to concentrations using standard curves. Little background fluorescence was observed and the minimum concentration of CVX-4164 detected was 10 nM. Vitreal concentrations of CVX-4164 determined in situ declined over time, with C_(max) ≈1 muM and t_(1/2) ≈145 hours (112 mug dose). The t_(1/2) of CVX-4164 was three times greater than that of IRDye 800CW alone, whereas the vitreal clearance and volume of distribution of the native dye were 2000- and 550-fold greater than the conjugate. CVX-4164 concentrations determined in situ were 2.6 to 4.4 times higher than those determined by NIR fluorescence or ELISA in ex vivo, homogenized vitreous humour samples, respectively. This may reflect the greater spatial resolution of in situ imaging compared to the dispersion of CVX-4164 throughout the homogenized vitreous. Moreover, vitreal concentrations determined in situ were ≈3 orders of magnitude greater than plasma concentrations of CVX-4164 determined by ELISA, and showed a different kinetic profile. In conclusion, repeated monitoring of the intraocular concentrations of biotherapeutics labelled with NIR fluorescent probes in situ over relatively long periods of time (240 hours) may allow the PK of the agent to be determined in the intact eye at the site of administration with minimal disruption of the target tissues.