(M1330-10-63) Effect of pH on Metabolite Profiling and Identification of GalNAc Conjugated siRNA in In Vitro Metabolic System

Purpose: The metabolites of GalNAc conjugated siRNA in different species were compared in in vitro incubation systems under different pH values, which is to investigate the PH influence for the metabolism of GalNAc conjugated siRNA, and then help to find suitable experiment conditions to evaluate the in vitro metabolism of siRNA.

Methods: SiRNA-01 was incubated in mouse, rat, dog, monkey and human liver S9 fractions for 24 hours at 37 ℃ with the buffer of pH = 5.0, 6.0 and 8.0, respectively. SiRNA-01 was incubated in mouse, rat, and monkey liver homogenate for 24 hours at 37 ℃ with the buffer of pH = 5.0, 6.0 and 8.0, respectively. An LC-UV-MS/MS method was successfully developed with Waters ACQUITY UPLC-PDA coupled with HRMS (Q-Exactive Plus) in full MS/ddMS² mode for analysis of siRNA-01. The sample treatment process was involved in special liquid-liquid extraction or solid phase extraction.

Results: In the incubation system with pH = 5.0, the major metabolite of antisense strand (AS) was AS 3’ n-1, with relative abundance of 2.64%, 1.80%, 2.30%, 4.30% and 6.18% in mouse, rat, dog, monkey and human, respectively. The relative abundance of intact AS was more than 93.5% in all five species. For sense strand (SS), the loss of one, two and three GalNAc groups were the major metabolites. And the sum of their relative abundance was 90.91%, 95.20%, 63.18%, 74.56% and 43.92% in mouse, rat, dog, monkey and human, respectively. The truncations of GalNAc groups were the most prominent metabolites yielding between 98.6 to 100% of total identified metabolites related to SS. In the incubation system with pH = 6.0, the major metabolites of AS were AS 3’ n-1, and AS 5’ n-5. The relative abundance of AS 3’ n-1 in mouse, rat, dog, monkey and human was 18.42%, 8.99%, 15.67%, 17.37% and 34.77%, respectively. The AS 5’ n-5 metabolite accounted 22.93% in rat, and less than 4.5% in other four species. The relative abundance of intact AS was between 54.31% and 78.79% in all five species. For SS, the loss of one, two and three GalNAc groups were also the major metabolites. Their total relative abundance was 28.73%, 57.17%, 39.18%, 56.51% and 18.36% in mouse, rat, dog, monkey and human, respectively. Their proportion was obviously lower than that of pH = 5. In the incubation system with pH = 8.0, the major metabolites of AS were AS 3’ n-1, AS 5’ n-5 with and without 3’ n-1. The relative abundance of AS 3’ n-1 in mouse, rat, dog, monkey and human was 17.06%, 6.36%, 24.63%, 16.92% and 28.42%, respectively. The AS 5’ n-5 accounted 24.10% in monkey, less than 2.5% in other four species. The AS 5’ n-5 with 3’ n-1 accounted 15.17% in monkey, and less than 1% in other four species. For SS, the intact SS was the dominated component, with relative abundance of 98.63% to 100% in five species. No SS related metabolites were detected in rat. Liver homogenate - In the incubation system with pH = 5.0, only a small amount of AS metabolized. Total three to five AS related metabolites were detected in mouse, rat and monkey. The intact AS accounted between 96.42% and 97.13% in mouse, rat and monkey. For SS, top three metabolites (loss of GalNAc groups) were detected with total relative abundance of 66.45%, 80.1% and 49.69% in mouse, rat and monkey, respectively. In the incubation system with pH = 6.0, total 12 to 14 AS related metabolites were detected in mouse, rat and monkey. The top metabolite AS 3’ n-1 accounted 46.65%, 30.96% and 30.46% in mouse, rat and monkey, respectively. For SS, the top three metabolites (loss of GalNAc groups) were detected with total relative abundance of 48.58%, 84.65% and 57.62% in mouse, rat and monkey, respectively. Small amount of SS 3’ n-2, n-3, n-4, n-5 and n-6 metabolites were detected in rat. In the incubation system with pH = 8.0, total 6 to 7 AS related metabolites were detected in mouse, rat and monkey. The intact AS accounted between 94.01% and 95.61% in mouse, rat and monkey. The intact SS accounted more than 99%.

Conclusion: In the incubation system with pH = 6.0, both AS and SS of siRNA-01 had comprehensive metabolism in liver S9 and liver homogenate. Under this incubation condition, we can investigate the metabolism of both AS and SS of siRNA-01 at the same time, which is close to their metabolism in vivo, and demonstrated this in vitro metabolic system can help to predict the in vivo metabolism. As for the effect of PH values to the metabolism of other siRNAs and whether PH=6 is appropriate, more studies are under evaluation. Herein research provides a reference and some clues.

Figure 1. Major Metabolites in Different PH of Incubation Systems

Figure 2. AS&SS metabolites under different PH in in vitro system of rat liver S9 vs. rat in vivo liver

Table 1. Relative Abundance Summary of The Metabolites of siRNA-01 in Liver S9 Fractions and Liver Homogenate Under different PH Incubation systems