Plasma kallikrein, a member of the kallikrein-kinin system, catalyzes the release of the bioactive peptide bradykinin, which induces inflammation, vasodilation, vessel permeability, and pain. Preclinical evidence implicates the activity of plasma kallikrein in diabetic retinopathy, which is a leading cause of visual loss in patients suffering from diabetes mellitus. Employing a technology based on phage-display combined with chemical cyclization, we have identified highly selective bicyclic peptide inhibitors with nano- and picomolar potencies toward plasma kallikrein. Stability in biological matrices was either intrinsic to the peptide or engineered via the introduction of non-natural amino acids and nonpeptidic bonds. The peptides prevented bradykinin release in vitro, and in vivo efficacy was demonstrated in both a rat paw edema model and in rodent models of diabetes-induced retinal permeability. With a highly extended half-life of ∼40 h in rabbit eyes following intravitreal administration, the bicyclic peptides are promising novel agents for the treatment of diabetic retinopathy and diabetic macular edema.

The Fab-fragment of 6B4, a murine monoclonal antibody targeting the human platelet glycoprotein (GP) Ibalpha and blocking the binding of von Willebrand factor (VWF), is a powerful antithrombotic. In baboons, this was without side effects such as bleeding or thrombocytopenia. Recently, we developed a fully recombinant and humanized version of 6B4-Fab-fragment, h6B4-Fab, which maintains its inhibitory capacities in vitro and ex vivo after injection in baboons. We here investigated the antithrombotic properties, the effect on bleeding time and blood loss and initial pharmacokinetics of h6B4-Fab in baboons. The antithrombotic effect of h6B4-Fab on acute platelet-mediated thrombosis was studied in baboons where thrombus formation is induced at an injured and stenosed site of the femoral artery, allowing for cyclic flow reductions (CFRs) which are measured on an extracorporeal femoral arteriovenous shunt. Injection of 0.5 mg/kg h6B4-Fab significantly reduced the CFRs by 80%, whereas two extra injections, resulting in cumulative doses of 1.5 and 2.5 mg/kg, completely inhibited the CFRs. Platelet receptor occupancy, plasma concentrations and effects ex vivo were consistent with what was previously observed. Finally, minimal effects on bleeding time and blood loss, no spontaneous bleeding and no thrombocytopenia were observed. We therefore conclude that h6B4-Fab maintains the antithrombotic capacities of the murine 6B4-Fab, without causing side effects and therefore can be used for further development.

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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.