(T1230-01-03) Absorption, Distribution, Metabolism, and Excretion of ION775, a Hepatocyte-Targeting siRNA Designed to Silence APOC3 Expression, Evaluated After Single and Multiple SC Dose(s) in Mice and Monkeys

Purpose: The purpose was to evaluate the absorption, distribution, metabolism, and excretion (ADME) of ION775 in CD-1 mice and cynomolgus monkeys. ION775 is a hepatocyte-targeting small interfering RNA (siRNA) designed to silence APOC3 expression currently being investigated in a Phase 1 study in healthy volunteers with elevated triglycerides (≥150 mg/dL and ≤500 mg/dL).

Methods: ION775 was evaluated in a pharmacokinetic (PK) study following a single subcutaneous (SC) dose in mice (3 or 30 mg/kg) and monkeys (5 mg/kg), with plasma, tissue, urine and fecal samples collected at specific timepoints/intervals in the post-dose period. ION775 was also evaluated in a 13-week Toxicology study following once monthly (q4w) SC dose administrations in mice (30, 100, or 300 mg/kg), and monkeys (5, 25, or 100 mg/kg), with plasma and/or tissues collected at specific timepoints over the course of the study. An electrophoretic mobility shift assay (EMSA) was used to evaluate the extent of whole plasma protein binding (PPB) of ION775 in vitro. Concentrations of ION775 in plasma, tissue, urine, and feces were determined using ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) methods for detection of the antisense strand which is considered representative of the duplex (Berman et al. 2023).  In general, concentrations of plasma and tissue were similar (within 2-fold) between males and females in both species, and thus were summarized as gender combined.

Results: ION775 PPB was similar in mice and monkeys with ≥93.77% bound at the lowest concentration evaluated (2.00 μg/mL). Following SC administration, ION775 was rapidly absorbed to systemic circulation with Cmax observed at 0.5 hours for mice and between 2 to 8 hours for monkeys (TABLE 1). ION775 concentrations were dose dependent with greater than dose proportional increases observed in plasma peak (Cmax) & total (AUC_0-24h) exposure in both species across the respective dose ranges evaluated. Similar kinetics were observed between Day 1 (1^st dose) and Day 85 (4^th dose) indicating a lack of accumulation in plasma exposure measures following repeat dose administration (q4w) in monkeys (TABLE 1). Plasma peak (Cmax) and/or total (AUC_0=24h) exposure of ION775 following SC administration, was several-fold lower in mice compared to monkeys at comparable mg/kg dose levels (TABLE 1), consistent with the more rapid clearance in rodents typically observed for this class of compounds (Nair et al. 2017). After peaking, ION775 plasma concentrations rapidly declined over approximately the first 24 hours with a relatively short apparent elimination half-life ranging from 0.589 to 1.24 hours in mice and 2.47 to 8.19 hours in monkeys (TABLE 1). The initial rapid distribution phase was followed by a much slower elimination phase, with an apparent plasma terminal elimination half-life of 65.3 days determined in the high dose monkeys (100 mg/kg) following 4 doses of ION775 (TABLE 1). The apparent elimination half-life may be underestimated in the lower dose groups due to limited sensitivity of the bioanalytical method. Rapid clearance from plasma corresponded to extensive uptake into systemic tissues, predominantly liver, the target site of drug action, with substantially lower distribution to the kidneys (TABLE 2). Tissue exposure was dose dependent but increased greater than dose proportionally in the kidney and less than dose proportionally in the liver, likely reflecting saturation of ASGPR mediated hepatocyte uptake of ION775. Following cessation of treatment during the 3-month recovery period in mice (300 mg/kg) and monkeys (100 mg/kg), tissue concentrations decreased significantly with less than 2% remaining in kidney for both species, and less than 4% remaining in liver for mice (TABLE 2). Just over 40% of ION775 remained in monkey liver with an estimated half-life of 71.6 days which is consistent with the apparent terminal elimination half-life of 65.3 days in monkey plasma. While no circulating metabolites were detected in mouse or monkey plasma, the GalNAc3-moiety appears to be rapidly hydrolyzed intracellularly once taken up into tissues, after which duplex siRNA was metabolized slowly via exo- and endonucleases resulting in shortened oligonucleotide strands of varying lengths over time. Urinary excretion of ION775 accounted for only a small percentage of the administered dose, with 7.89 and 5.06% excreted over the 48-hour collection interval in mice (3 mg/kg) and monkeys (5 mg/kg), respectively. Renal clearance was more than 30-fold lower in mice and 25-fold lower in monkeys, than plasma clearance over the first 24 hours, which is consistent with tissue uptake being the primary mechanism of plasma clearance rather than urinary excretion. Similarly, the excretion of ION775 in feces accounted for a very small percentage of the administered dose with < 0.100% excreted over the 48-hour collection period for both species.

Conclusion: Overall, the ADME properties observed for ION775 were consistent between mice and monkeys and were generally similar to those observed for other GalNAc3-conjugated siRNAs in this chemical class (McDougall et al. 2022). Furthermore, the observed liver half-life of 71.6 days in monkeys supports less frequent dosing and continued development in the clinic.

References: Berman CL, Antonsson M, Batkai S, … Wu JT. (2023) OSWG Recommended Approaches to the Nonclinical Pharmacokinetic (ADME) Characterization of Therapeutic Oligonucleotides. Nucleic Acid Therapeutics, 33(5):286 – 305.
McDougall, R., Ramsden, D., Agarwal, S., …Wu, J.-T. (2022). The Nonclinical Disposition and Pharmacokinetic/Pharmacodynamic Properties of N -Acetylgalactosamine–Conjugated Small Interfering RNA Are Highly Predictable and Build Confidence in Translation to Human. Drug Metabolism and Disposition, 50(6), 781–797.
Nair, J. K., Attarwala, H., Sehgal, A., … Maier, M. A. (2017). Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc–siRNA conjugates. Nucleic Acids Research, 45(19), 10969–10977.

Acknowledgements: The author, Gina McMullen, is an Ionis Pharmaceuticals employee and holds stock in the company. This project is sponsored by Ionis Pharmaceuticals. The author, Rosie Yu, is an Ionis Pharmaceuticals employee and holds stock in the company. This project is sponsored by Ionis Pharmaceuticals. The author, Asa Wahlander, is an Ionis Pharmaceuticals employee and holds stock in the company. This project is sponsored by Ionis Pharmaceuticals. The author, Noah Post, is an Ionis Pharmaceuticals employee and holds stock in the company. This project is sponsored by Ionis Pharmaceuticals. The author, Jeremy Dixon, is an Ionis Pharmaceuticals employee and holds stock in the company. This project is sponsored by Ionis Pharmaceuticals. The author, TP Prakash, is an Ionis Pharmaceuticals employee and holds stock in the company. This project is sponsored by Ionis Pharmaceuticals. The author, Adam Mullick, is an Ionis Pharmaceuticals employee and holds stock in the company. This project is sponsored by Ionis Pharmaceuticals. The author, Xiang Gao, is an Ionis Pharmaceuticals employee and holds stock in the company. This project is sponsored by Ionis Pharmaceuticals.

Plasma PK Summary Table of ION775 following SC Dose Administration in Mice and Monkeys (Gender Combined)

Summary Table of ION775 Tissue Exposure in Mice and Monkeys
(Gender Combined) after Multiple SC Dose Administrations