Pharmacologic and Pharmacokinetic Assessment of Anti-TGFβ2 Aptamers in Rabbit Plasma and Aqueous Humor

Abstract

The aim of the study is to determine the bioactivity and effects of PEGylation on the pharmacokinetics in rabbit aqueous humor and plasma of an aptamer directed against TGFbeta2. Pharmacological activity of anti-TGFbeta2 aptamer in rabbit ocular fluid was demonstrated using a mink lung epithelial cell proliferation assay. For pharmacokinetic analyses, concentrations of aptamers in plasma and aqueous humor were determined over time following bilateral subconjunctival administration to Dutch-belted rabbits using a hybridization-based pseudo-enzyme-linked immunosorbent assay (ELISA) assay. Anti-TGFbeta2 aptamer (ARC81) binds to human TGFbeta2 with a K(D) of approximately 5 nM and inhibits the activity of human TGFbeta2 in vitro in a cell-based assay with an IC(50) of approximately 100 nM. ARC81 blocks endogenously derived TGFbeta2 in rabbit aqueous humor in vitro with an IC(50) of approximately 200 nM and an IC(90) of approximately 1 microM. In vivo in rabbit, ARC81 [no polyethylene glycol (PEG)] entered systemic circulation rapidly (t(max) = 1 h in plasma) relative to aptamer conjugates ARC117 (20 kDa PEG) and ARC119 (40 kDa PEG), which showed prolonged residence in the subconjunctival space and aqueous compartment (t(max) = 6 and 12 h, respectively, in plasma). Both 20- and 40-kDa aptamer conjugates reached maximal concentrations (C(max)) in aqueous humor of 23-30 nM and remained at or above 1 nM for as long as 12 h. Pharmacologically active levels of anti-TGFbeta2 aptamers can be sustained in the ocular fluid and local tissue environment over a 12-h period after single administration. Daily subconjunctival administration of PEGylated anti-TGFbeta2 aptamers should allow further pharmacological evaluation of these agents in a rabbit conjunctival scarring model. Perioperative administration, via subconjunctival injection, may prove to be an effective means to deliver therapeutic quantities of TGFbeta2 aptamer conjugates in trabeculectomy procedures.