Evaluation of GalNAc-siRNA Conjugate Activity in Pre-clinical Animal Models with Reduced Asialoglycoprotein Receptor Expression.

The hepatocyte-specific asialoglycoprotein receptor (ASGPR) is an ideal candidate for targeted drug delivery to the liver due to its high capacity for substrate clearance from circulation together with its well-conserved expression and function across species. The development of GalNAc-siRNA conjugates, in which a synthetic triantennary N-acetylgalactosamine-based ligand is conjugated to chemically modified siRNA, has enabled efficient, ASGPR-mediated delivery to hepatocytes. To investigate the potential impact of variations in receptor expression on the efficiency of GalNAc-siRNA conjugate delivery, we evaluated the pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates in multiple pre-clinical models with reduced receptor expression. Despite greater than 50% reduction in ASGPR levels, GalNAc conjugate activity was retained, suggesting that the remaining receptor capacity was sufficient to mediate efficient uptake of potent GalNAc-siRNAs at pharmacologically relevant dose levels. Collectively, our data support a broad application of the GalNAc-siRNA technology for hepatic targeting, including disease states where ASGPR expression may be reduced.

Comparative in silico-in vivo evaluation of ASGP-R ligands for hepatic targeting of curcumin Gantrez nanoparticles.

The present study aims to design hepatic targeted curcumin (CUR) nanoparticles using Gantrez (GZ) as a polymer. Three carbohydrate-based hepatocyte asialoglycoprotein receptor (ASGP-R) ligands were selected for the study, namely kappa carrageenan (KC), arabinogalactan (AG), and pullulan (P). AG and KC are galactose based while P is a glucose-based polymer. CUR-GZ nanoparticles were prepared by nanoprecipitation and anchored with the ligands by nonspecific adsorption onto preformed nanoparticles. The change in zeta potential values confirmed adsorption of the ligands. Docking simulation was evaluated as a tool to predict ligand ASGP-R interactions, using grid-based ligand docking with energies (Glide). Monomers and dimers were used as representative units of polymer for docking analysis. The binding of ASGP-R was validated using D-galactose as monomer. The interaction of the ligands with the receptor was evaluated based on Glide scores and E model values, both for monomers and dimers. The data of the docking study based on Glide scores and E model values suggested higher affinity of AG and P to the ASGP-R, compared to KC. At 1 h, following intravenous administration of the nanoparticles to rats, the in vivo hepatic accumulation in the order CUR-GZAG > CUR-GZKC > CUR-GZP correlated with the docking data based on Glide scores. However, at the end of 6 h, pullulan exhibited maximum hepatic accumulation and arabinogalactan minimum accumulation (p < 0.05). Nevertheless, as predicted by docking analysis, arabinogalactan and pullulan revealed maximum hepatic accumulation. Docking analysis using dimers as representative stereochemical units of polymers provides a good indication of ligand receptor affinity. Docking analysis provides a useful tool for the preliminary screening of ligands for hepatic targeting.

The position of cysteine relative to the transmembrane domain is critical for palmitoylation of H1, the major subunit of the human asialoglycoprotein receptor.

The mammalian hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic recycling receptor that mediates the internalization of desialylated glycoproteins and their delivery to lysosomes where they are degraded. The human ASGP-R is a hetero-oligomeric complex composed of two subunits designated H1 and H2. Both subunits are palmitoylated at the cytoplasmic Cys residues near their transmembrane domains (TMD). The cytoplasmic Cys(36) in H1 is located at a position that is five amino acids from the transmembrane junction. Because the sequences of subunits in all mammalian ASGP-R species are highly conserved especially at the region near the palmitoylated Cys, we sought to identify a recognition signal for the palmitoylation of H1. Various types of H1 mutants were created by site-directed or deletion mutagenesis including alteration of the amino acids surrounding Cys(36), replacing portions of the TMD with that of a different protein and partial deletion of the cytoplasmic domain as well as transposing the palmitoylated Cys to positions further away from the TMD. Mutant H1 cDNAs were transiently expressed in COS-7 cells, and the H1 proteins were analyzed after metabolic labeling with [(3)H]palmitate. The results indicate that neither the native amino acid sequence surrounding Cys(36) nor the majority of the cytoplasmic domain sequence is critical for palmitoylation. Palmitoylation was also not dependent on the native TMD of H1. In contrast, the attachment of palmitate was abolished if the Cys residue was transposed to a position that was 30 amino acids away from the transmembrane border. We conclude that the spacing of a Cys residue relative to the TMD in the primary protein sequence of H1 is the major determinant for successful palmitoylation.