ABSTRACT
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 â¼40 h in rabbit eyes following intravitreal administration, the bicyclic peptides are promising novel agents for the treatment of diabetic retinopathy and diabetic macular edema.
Subject(s)
Bridged Bicyclo Compounds/chemical synthesis , Bridged Bicyclo Compounds/pharmacology , Diabetes Complications/drug therapy , Diabetic Retinopathy/drug therapy , Macular Edema/drug therapy , Macular Edema/etiology , Plasma Kallikrein/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Animals , Bradykinin/metabolism , Edema/drug therapy , Eye/metabolism , Foot/pathology , Half-Life , Intravitreal Injections , Male , Mice , Mice, Inbred C57BL , Permeability , Protease Inhibitors/administration & dosage , Rabbits , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Substrate Specificity , Vitreous Body/chemistry , Vitreous Body/metabolismABSTRACT
Using capillary electrophoresis coupled to laser-induced fluorescence (HPCE-LIF), it was possible to profile N-linked oligosaccharides from EPO, including species containing sialic acid, during the course of batch cultures performed either in serum-free or serum-containing medium. Although an unusual high heterogeneity of the N-linked oligosaccharides was observed by both SDS-PAGE and HPCE analysis, the patterns of mEPO glycans after desialylation by mild acid hydrolysis were found to be quite constant over the course of the cultures either with or without serum supplementation. In contrast, when the protein was analyzed by HPCE without acidic desialylation, fingerprints of N-linked oligosaccharides changed with time in serum-containing conditions. This phenomenon appeared to be mainly due to the desialylation of mEPO as a result of a sialidase activity released upon cell lysis. These results demonstrate that though a higher EPO titer was obtained in serum supplemented conditions, sialylation of EPO was severely affected by the presence of serum in the culture medium.