Comparative Metabolism of a GalNAc-Conjugated siRNA, Inclisiran, Among Various In Vitro Systems and Correlations with In Vivo Metabolism in Rats

N-acetylgalactosamine-conjugated small interfering RNAs (siRNAs) have emerged as a promising new class of therapeutics, demonstrating potential across various disease areas. Metabolic stability is a critical factor in the development of siRNA therapies, necessitating the selection of suitable in vitro metabolism models for comprehensive studies. However, many existing in vitro models often fail to accurately predict in vivo metabolism. Therefore, our objective was to systematically identify the optimal models and conditions that can simulate in vivo metabolism. Inclisiran, a representative N-acetylgalactosamine-conjugated siRNA, was chosen as the model compound for this investigation. A thorough comparison of in vitro metabolism was conducted to identify optimal models and corresponding conditions that could predict inclisiran's metabolic profiles in rat plasma and liver in vivo. The models tested included serum, plasma, plated hepatocytes, liver homogenate, S9 fractions, tritosomes, lysosomes, and cytosol, reflecting inclisiran's trajectory from injection to RNA-induced silencing complex in hepatocytes. The results indicated that both rat serum and plasma obtained after anticoagulation with heparin are suitable models to mimic the nucleolytic metabolism of inclisiran in in vivo blood. For hepatic metabolism, liver homogenate in phosphate buffer (pH 6.0), liver tritosomes in acetate buffer (pH 5.0), and plated hepatocytes were suitable models. Liver homogenate and cytosol in Tris-HCl buffer (pH 7.4) were effective for studying nuclease-mediated metabolism of inclisiran's antisense strand under neutral conditions, though they did not fully simulate in vivo metabolism of the sense strand. These findings underscore the importance of selecting appropriate in vitro models and conditions to accurately simulate in vivo metabolism of inclisiran. SIGNIFICANCE STATEMENT: This research offers profound insights into the metabolic pathways and characteristics of inclisiran across various in vitro models and in vivo plasma and liver, enhancing our understanding of inclisiran's metabolism. It establishes a critical foundation for selecting appropriate in vitro systems for future studies of other N-acetylgalactosamine-conjugated small interfering RNAs, aiding in the optimization and development of oligonucleotide therapeutics, and thereby advancing the field of RNA-based drug development.