To determine the feasibility of using fluorophotometry to assess the PK of AF488-ranibizumab administered intravitreally, an initial study was performed as illustrated in
Figure 1A. The total amount of administered ranibizumab remained constant between treatment groups while the level of AF488 dye varied. This allowed initial investigation of the dynamic range of the instrument while keeping the total protein load constant. An example of the raw fluorophotometry depth scans over the course of 14 days is shown in
Figure 2A. The broad vitreous peak between approximately 0 to 20 mm was observed over the entire time period and for all AF488 levels (
Fig. 2A,
Supplementary Fig. S2). Two methods were employed to translate the raw fluorescence signal to the concentration of ranibizumab. The first was the peak method that utilized the maximum peak measured between 0 and 20 mm (
Figs. 2B,
3). The second method utilized the total area under the curve (
AUC) between 0 and 20 mm (
Fig. 2B,
Supplementary Fig. S3). As shown in
Figure 3A and
Supplementary Figure S4A, the fluorescence signal measured using the fluorophotometer showed a first order loss over the course of 14 days for all fluorescence levels evaluated. Signal normalized to fluorescence and to an AF488-ranibizumab standard curve (
Figs. 3B,
3C,
Supplementary Figs. S4B, S4C) also displayed first-order kinetics. Normalized ranibizumab concentrations were used to calculate the PK parameters (
Table 1) for individual animals for both the peak and
AUC methods. Animal-to-animal variability was low for all PK parameters as evidenced by the standard deviations in
Table 1. Using the peak method, the calculated vitreous clearance (
CL) of ranibizumab was 0.25 ± 0.02 mL/day, the volume of distribution at steady state (
Vss) was 1.1 ± 0.13 mL, the half-life (
t1/2) was 3.2 ± 0.25 days, and the
AUC was 1944 ± 114 μg·day/mL. Using the
AUC method, the calculated vitreous
CL of ranibizumab was 0.33 ± 0.06 mL/day, the
Vss was 1.5 ± 0.32 mL, the
t1/2 was 3.3 ± 0.12 days, and the
AUC was 1540 ± 372 μg·day/mL. As shown in
Table 1, differences in
CL,
Vss, and
AUC values were statistically significant (
P value < 0.05) between the peak method and
AUC method, whereas
t1/2 values were not. However, all values calculated were within the ranges observed historically, suggesting that both the peak and
AUC methods are viable approaches for normalizing the fluorescence signal obtained with fluorophotometry. For subsequent analysis, only the peak method was used with fluorophotometry.