Bioactivity and circulation time of PEGylated NELL-1 in mice and the potential for osteoporosis therapy

Abstract

Osteoporosis is a progressive bone disease due to low osteoblast activity and/or high osteoclast activity. NELL-1 is a potential therapy for osteoporosis because it specifically increases osteoblast differentiation. However, similar to other protein drugs, the bioavailability of NELL-1 may be limited by its in vivo half-life and rapid clearance from body. The purpose of the present study is to prolong NELL-1 circulation time in vivo by PEGylation with three monomeric PEG sizes (5, 20, 40 kDa). While linear PEG 5k yielded the most efficient PEGylation and the most thermally stable conjugate, linear PEG 20k resulted in the conjugate with the highest Mw and longest in vivo circulation. Compared to non-modified NELL-1, all three PEGylated conjugates showed enhanced thermal stability and each prolonged the in vivo circulation time significantly. Furthermore, PEGylated NELL-1 retained its osteoblastic activity without any appreciable cytotoxicity. These findings motivate further studies to evaluate the efficacy of PEGylated NELL-1 on the prevention and treatment of osteoporosis.

Pharmacokinetics and osteogenic potential of PEGylated NELL-1 in vivo after systemic administration

“…and PEGylated NELL-1 were labeled with fluorescein isothiocyanate (FITC) according to our previous paper [34]. FITC (Sigma Aldrich, MO) and either NELL-PEG or NELL-1 (4 mg/mL) were reacted at a 50:1 molar ratio in a 0.1 M sodium carbonate-bicarbonate buffer (pH 9.0) for 3 h at room temperature under magnetic stirring at 250 rpm.…”

“…Then, the mice were intravenously injected with 100 μl of PBS/PEG or NELL-PEG via the lateral tail veins at a q4d or q7d dosing interval over a 4-week experimental period. The type of PEG (linear 5 KDa) for NELL-PEG and the optimal doses for PBS/PEG (1.52 mg/kg) and NELL-PEG (1.25 mg/kg) were determined according to our previous studies [29,34].…”

“…PEGylation of a protein not only prolongs its half-life, but also reduces its injection frequency by consequence of extending circulation time in vivo. Seeking to reduce the injection frequency of NELL-1, we previously used PEGylation technology to enhance the systemic pharmacokinetics of NELL-1 [34]. Three different PEGylated types of NELL-1 (5K-linear, 20K-linear, and 40K-branched) were tested, each demonstrating an increased thermostability and a maintained bioactivity in vitro in mouse osteoblast cells and human adipose-derived perivascular stem cells (hPSCs) compared to naked NELL-1 [34].…”

“…Seeking to reduce the injection frequency of NELL-1, we previously used PEGylation technology to enhance the systemic pharmacokinetics of NELL-1 [34]. Three different PEGylated types of NELL-1 (5K-linear, 20K-linear, and 40K-branched) were tested, each demonstrating an increased thermostability and a maintained bioactivity in vitro in mouse osteoblast cells and human adipose-derived perivascular stem cells (hPSCs) compared to naked NELL-1 [34]. Importantly, the levels of PEGylated NELL-1 were found to remain significantly higher compared to naked NELL-1 at 24 hours in vivo.…”

“…Previously, we tested three different PEGylation types of NELL-1 (NELL-PEG-5k, NELL-PEG-20k and NELL-PEG-40k) and determined NELL-PEG-5k to be the most optimal type of PEGylation for this in vivo study [34]. Thus, in the present study, we examined the osteogenic potential of systemically administered PEGylated NELL-1 (NELL-PEG-5k) compared to naked NELL-1 and a carrier control.…”