Development of a targeted carrier for Rapamycin in a murine model of Sjögren’s syndrome

Abstract

Purpose : Sjögren’s syndrome (SS) is a systemic autoimmune disease affecting lacrimal glands (LG) and salivary glands. There is currently no therapy that targets inflammation in affected tissue. Elastin-Like Polypeptides (ELPs) are protein polymers that are thermally-responsive, easy to engineer, and of low immunogenicity, making them ideal candidates for drug carriers. Rapamycin (Rapa), an immunosuppressant, shows promising immunomodulatory effects on autoimmune dacryoadenitis in male NOD mice, a model for SS-associated dacryoadenitis. Since ICAM-1 is elevated in LG of these mice, we developed an ICAM-1-targeted ELP carrier to increase delivery of Rapa to the LG.

Methods : An ICAM-1-binding peptide was fused to the untargeted (VPGAG)₁₉₂-FKBP fusion protein (AF) and termed IBPAF. The biophysical properties, Rapa binding, and ICAM-1 binding ability of IBPAF and AF purified by inverse transition cycling were determined. Plasma concentrations of fluorescently-labeled ELPs versus time curve were obtained to analyze their pharmacokinetic properties. Biodistribution of fluorescently-labeled ELPs were compared by IVIS and fluorescence intensity in tissue lysates in 14 wk male NOD mice at 2 h, 8 h and 24 h following i.v. injection.

Results : Both AF and IBPAF were purified to >90% homogeneity and formed monodisperse nanoparticles with radius of ~7nm. Both AF and IBPAF efficiently bound Rapa with K_d ~ 5 nM. IBPAF showed significantly higher (4-fold, p<0.05) binding to TNF-α stimulated bENd.3 cells which overexpress ICAM-1. Pharmacokinetics studies in male NOD mice showed no significant differences between AF and IBPAF with respect to t_1/2 (8.9 ± 3.2 vs 5.5 ± 2.9 h, respectively), CL (0.179 ± 0.015 vs 0.168 ± 0.026 mL/h, respectively), V_d (2.3 ± 0.4 vs 1.3 ± 0.7 mL, respectively) and AUC (183.8 ± 15.6 vs 196.2 ± 34.3 μM*h, respectively)(mean ± SD, n=5). Both IVIS imaging and analysis of ELP fluorescence in tissue lysates showed that IBPAF had significantly higher LG accumulation in NOD mice when compared with AF (p<0.05, n=5) within 2 h of injection. No significant differences were detected in any other organs between IBPAF and AF at any other time points.

Conclusions : We developed an ICAM-1 targeted protein carrier for Rapa that specifically binds to ICAM-1 in vitro and accumulates in the LG of diseased NOD mice.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Schematic illustration of therapeutic mechanism of ICAM-1 targeted ELP carrier for Rapa