sFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life In Vivo

Abstract

Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.

Polysaccharides in Ocular Drug Delivery

“…The half-lives were about ten times longer for the HA conjugates than for the unconjugated sFlt-1. Conjugation of sFlt-1 with HA did not change the pharmacological activity of this protein drug [170,171]. This study nicely demonstrated the potential use of polymer conjugation in intravitreal drug delivery.…”

“…HA has been used to reduce clearance and increase the residence time of drugs in the vitreous humor [136]. Altiok et al [170,171] conjugated anti-VEGF drug (soluble VEGF receptor; sFlt-1) to HA (MW 300 kDa, 650 kDa, and 1 MDa) to provide sustained release and prolonged retention in the vitreous [170]. The half-lives were about ten times longer for the HA conjugates than for the unconjugated sFlt-1.…”

Hyaluronic Acid–Antibody Fragment Bioconjugates for Extended Ocular Pharmacokinetics

“…24,25 Because the fate of both pathways is systemic clearance, PEGylation poses a potential challenge for ocular long-acting delivery in that achieving a sufficiently extended ocular half-life will cause a concomitant increase in systemic exposure and may result in liver accumulation if PEG molecules larger than 3−5 nm R H are used. 26 The use of HA as a polymer scaffold, as has been previously suggested for vitreal half-life extension, 27 may ameliorate some of the concerns with PEGylation. Indeed, we demonstrate that HA-rabFab conjugates with R H up to 29 nm were found to clear slowly from the vitreous humor without indication of significantly increased systemic exposure.…”

Synthesis and Characterization of Novel Succinyl Chitosan-Dexamethasone Conjugates for Potential Intravitreal Dexamethasone Delivery

“…Covalent conjugation with macromolecular compounds (especially non-toxic, biocompatible, and biodegradable natural polymers) can increase the size of a drug molecule, thereby increasing its residence time in the vitreous humor [27,28]. Altiok et al [29,30] conjugated an anti-vascular endothelial growth factor (anti-VEGF) drug (sFlt-1) with polyanionic hyaluronic acid to decrease sFlt-1 clearance and increase drug retention time in the vitreous. This resulted in a tenfold increase in the drug half-life with no change in pharmacological activity.…”