PEGylation of a proprotein convertase peptide inhibitor for vaginal route of drug delivery: In vitro bioactivity, stability and in vivo pharmacokinetics

Uterine proprotein convertase (PC) 6 is critical for embryo implantation in mice and women. It is also one of the PC family members that play a vital role in HIV infectivity. We hypothesized that inhibiting PC6 in the female reproductive tract (vagina, cervix and uterus), may protect women from both pregnancy and HIV infection. One key requirement to prove this concept in an animal model is a vaginally deliverable PC6 inhibitor. Nona-d-arginine (Poly R) is a potent peptide PC inhibitor and is able to inhibit HIV in cell culture. We modified Poly R by PEGylation with different strategies and determined their biochemical properties in vitro and in vivo. PEGylation at the C-terminus, regardless of the PEG size (30 kDa or 1239 Da) did not compromise the inhibitory potency of Poly R. In contrast, PEGylation at both termini (1239 Da) dramatically reduced its inhibitory activity. Poly R and C-PEGylated Poly Rs also showed equal potency in inhibiting a PC6-dependent cellular process critical for embryo implantation. Poly R and the equipotent C-PEGylated Poly Rs were further tested for their serum stability in vitro and pharmacokinetics in vivo following vaginal administration in mice. All Poly Rs were equally stable in mouse serum in vitro for 24 h; C-PEGylated Poly Rs showed enhanced vaginal absorption and penetration across the vaginal mucosa/epithelium. This is the first report that C-terminal PEGylation significantly enhances the therapeutic properties of Poly R for vaginal drug delivery. Our findings also provide important insights into future design of Poly R derivatives.

► We PEGylated a peptide inhibitor of proprotein convertase, nona-d-arginine (Poly R).
► Poly R tolerates PEGylation at C-terminus regardless PEG size 1239 Da or 30 kDa.
► C-PEGylated Poly Rs showed enhanced vaginal absorption and mucosal penetration.
► First report a peptide with improved properties for vaginal delivery after PEGylation.
► Findings provide important insights into future design of Poly R derivatives.