Ocriplasmin, a 249-amino acid recombinant C-terminal fragment of human plasmin, has the potential to degrade, within the eye, the protein scaffold that links the vitreous to the retina. This may be beneficial to the treatment of a number of important ophthalmic indications, such as symptomatic vitreomacular adhesion. We demonstrate here that ocriplasmin used at therapeutically-relevant concentrations is inactivated in porcine vitreous through autolytic degradation. Autolytic cleavage occurs at a limited number of sites, primarily K156-E157, K166-V167 and R177-V178, which, as predicted, contain a positively-charged arginine or lysine residue at the P1 position. Our data also suggest that autolytic degradation requires at least local or partial unfolding of the protein.

Ocriplasmin, a truncated form of plasmin, is commercialized in the USA and in Europe under the trade name Jetrea(®), and indicated for the treatment of symptomatic vitreomacular adhesion and vitreomacular traction including when associated with macular hole ≤400 µm, respectively. We have shown in a previous study that ocriplasmin undergoes autolytic degradation when injected in eye vitreous, which leads to its rapid inactivation. In order to investigate this process further, we have introduced in ocriplasmin a variety of amino acid substitutions within or in the immediate vicinity of the three major autolytic cleavage sites. We demonstrate here that autolytic inactivation of ocriplasmin is a sequential process where initial cleavage occurs primarily between residues 156 and 157. Reduction or even blocking of autolysis can be achieved by mutating a limited number of key residues. In this study, we also report the identification of a series of ocriplasmin variants with improved resistance to autolysis and unimpaired catalytic activity. Such variants represent useful tools for the exploration of therapeutic approaches aiming at non-surgical resolution of vitreomacular adhesion.

PurposeRetinal cells participate in the transport and clearance of therapeutics. In this study, we used in vitro retinal cell models to investigate the uptake and transport of ocriplasmin, a protease used for the treatment of vitreomacular traction.MethodsCultures of primary porcine Müller and human ARPE‐19 cells were incubated with Alexa488‐labeled ocriplasmin or its inactive form for up to 3 h. Spatial distribution and colocalization with vesicle transport proteins were assessed at several time points.ResultsOcriplasmin was rapidly detected in Müller and RPE cells. Uptake was observed as cytoplasmic foci and confirmed by confocal microscopy. In contrast, enzymatically inactive ocriplasmin was taken up at a significantly slower rate. Given its focal cytoplasmic distribution, we investigated whether ocriplasmin was present in cellular transport organelles. In Müller cells, ocriplasmin colocalized partly with Rab5‐positive early endosomes and Rab7‐positive lysosomes but with very few Rab11‐positive recycling endosomes. In RPE cells, ocriplasmin also colocalized in part with early endosomes and lysosomes, but to a larger extent with recycling endosomes.ConclusionsTaken together, our data indicate that ocriplasmin can be taken up by Müller and RPE cells and that this uptake depends in part on its enzymatic activity. Ocriplasmin was found in transport vesicles, indicating active transport mainly through the degradation pathway in Müller cells whereas in RPE cells, outward transport is preferred. It is described that RPE cells transport anti‐VEGFs which emphasizes their role in retinal drug clearance. Our study suggests that retinal cells might participate in ocriplasmin drug clearance. Further in vivo studies will need to assess the ocriplasmin transport in the retina as well as the impact of uptake on ocriplasmin activity.