Pharmacokinetics, Metabolism, and Biodistribution of a Liver‐Targeted siRNA Therapeutic in Preclinical Species

Almost twenty years ago, small interfering RNA (siRNA) was first demonstrated as a tool to silence gene expression in vitro. Today siRNA is part of an emerging drug class of RNA interference‐based therapeutics undergoing development against formerly undruggable targets. siRNA is a double‐stranded molecule typically 21–23 nucleotides in length. Upon uptake into the cell, the siRNA duplex is loaded into a multi‐protein RNA‐induced silencing complex (RISC). One strand, referred to as the sense strand, of the siRNA duplex is degraded while the remaining strand, the antisense strand, guides RISC to a target mRNA sequence for cleavage, subsequently decreasing the expression of the corresponding protein. The primary objective of this work was to determine an extensive pharmacokinetic profile of a liver‐targeting siRNA construct in preclinical species. To aid in measuring low levels of siRNA drug, an ultrasensitive hybridization‐ligation enzyme‐linked immunosorbent assay was developed utilizing locked nucleic acid probes for the quantitation of the sense and antisense strands across multiple biological matrices. Following a single subcutaneous injection of siRNA (0.1, 1 or 10 mg/kg) in non‐human primates (NHP), we observed peak serum concentrations of sense and antisense strands to occur as soon as 1–2 hours post‐dose from which circulating siRNA levels then declined, but were detected out to 7 days post‐dose. In addition to this work, we aimed to gain a better understanding of siRNA tissue distribution, specifically to the liver and kidneys, as well as the metabolism of the molecule that may occur in vivo potentially compromising drug efficacy. Interestingly, we observed a similar accumulation of both sense and antisense strands in the liver, whereas, the sense strand reached concentrations three orders of magnitude greater than the antisense strand in the kidneys, suggesting denaturation of the siRNA duplex. We also confirmed siRNA loading of RISC via immunoprecipitation from the liver homogenate and observed comparable loading levels of both sense and antisense strands. Further, these liver homogenates were then analyzed for metabolite formation using high‐resolution liquid chromatography‐tandem mass spectrometry. Metabolite profiling was performed on the antisense strand of the siRNA duplex as this is the pharmacologically active component of the molecule. We were excited to observe that the full‐length antisense strand was the predominant analyte detected (71.3% of total drug and metabolites) in liver homogenate followed by six metabolites of lesser abundance that resulted from single nucleotide cleaving at the 3′ end, n‐1 (21.5%), n‐2 (1%), n‐3 (0.6%), n‐4 (1.5%), n‐5 (2.8%) and n‐6 (1.5%). Concurrent to this work, efforts are underway to develop a fluorescent‐based in situ hybridization to investigate intra‐ and extra‐hepatic distribution of the siRNA construct at the cellular level. Through the described studies, we have successfully achieved a more complete understanding of the absorption, distribution, metabolism and excretion properties of an siRNA therapeutic in preclinical species which will inform clinical translation. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .