Assessment of the Drug Interaction Potential of Unconjugated and GalNAc3-Conjugated 2'-MOE-ASOs

Abstract

Antisense oligonucleotides are metabolized by nucleases and drug interactions with small drug molecules at either the cytochrome P450 (CYP) enzyme or transporter levels have not been observed to date. Herein, a comprehensive in vitro assessment of the drug-drug interaction (DDI) potential was carried out with four 2'-O-(2-methoxyethyl)-modified antisense oligonucleotides (2'-MOE-ASOs), including a single triantennary N-acetyl galactosamine (GalNAc₃)-conjugated ASO. Several investigations to describe the DDI potential of a 2'-MOE-ASO conjugated to a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors are explored. The inhibition on CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 and induction on CYP1A2, CYP2B6, and CYP3A4 were investigated in cryopreserved hepatocytes using up to 100 μM of each ASO. No significant inhibition (half maximal inhibitory concentration [IC₅₀] > 100 μM) or induction was observed based on either enzymatic phenotype or mRNA levels. In addition, transporter interaction studies were conducted with nine major transporters per recommendations from regulatory guidances and included three hepatic uptake transporters, organic cation transporter 1 (OCT1), organic anion transporting polypeptide 1B1 (OATP1B1), and OATP1B3; three renal uptake transporters, organic anion transporter 1 (OAT1), OAT3, and OCT2; and three efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and bile salt export pump (BSEP). None of the four ASOs (10 μM) were substrates of any of the nine transporters, with uptake <2-fold compared to controls, and efflux ratios were below 2.0 for BCRP and P-gp. Additionally, neither of the four ASOs showed meaningful inhibition on any of the nine transporters tested, with the mean percent inhibition ranging from -38.3% to 24.2% with 100 μM ASO. Based on these findings, the unconjugated and GalNAc₃-conjugated 2'-MOE-ASOs would have no or minimal DDI with small drug molecules via any major CYP enzyme or drug transporters at clinically relevant exposures.

Keywords: antisense oligonucleotides; drug interaction; metabolism; pharmacokinetics; transport.

Graphical abstract

Figure 1

CYP Inhibition Studies (A and B) CYP1A2, CYP2B6, CYP2C8, CYP2C9, and CYP2C19 (A) and CYP2D6, CYP2E1, CYP3A4 (midazolam 1′-hydroxylation), and CYP3A4 (testosterone 6β-hydroxylation) (B) enzyme activity by ISIS 304801, 396443, 420915, 681257, positive control, and vehicle. Data for ASOs represent the mean fold inhibition of triplicate analysis ± corresponding SD. Human hepatocytes were pre-incubated with CYP-inhibitor-positive control or ASO solutions were followed by the addition of CYP isoenzyme-specific substrates for 30 min using a 45-min incubation time.

Figure 2

Cell Uptake Summary Uptake of ISIS 304801, ISIS 396443, ISIS 420915, and ISIS 681257 in human hepatocytes. (A and B) Concentration ratio in pellet normalized to supernatant amount at time 0 (A) and percent of supernatant normalized to time 0 (B). Incubation amount refers to μM for ISIS 304801, 420915, and 681257 and μg/mL for ISIS 396443, respectively. Samples, curves, QCs, and internal standard were processed by liquid-liquid extraction and solid phase extraction prior to analysis by LC-UV/MS. Two calibration curves were used, with the linear range for LC-MS and LC-UV/Vis as 0.064–641.03 μM and 32.05–4,807.69 μM, respectively.

Figure 3

In Vitro Uptake Mediated by SLC and ABC Transporters For all transporters except BSEP, the cell line was MDCK. BSEP was studied in membrane vesicles prepared from Sf9 cells. Transporting ratio is the signal to noise for SLC transporters, as defined by the ratio of substrate transported in transporter-expressed cells/non-expressed cells. The concentration of each ASO, including ISIS 304801, ISIS 396443, ISIS 420915, and ISIS 681257 was studied at 10 μM. Positive control substrates for each transporter included 2 μM p-aminohippurate (OAT1), 10 μM p-aminohippurate (OAT3), 2 μM MPP⁺ (OCT1), 10 μM metformin (OCT2), 2 μM estradiol-17-β-D-glucuronide (OATP1B1), and 2 μM CCK-8 (OATP1B3). Efflux ratio is the P_app in the B to A direction divided by the P_app in the A to B direction. Accumulation ratio is the signal to noise for the BSEP transporter, as defined by the vesicular accumulation (ATP)/vesicular accumulation (AMP). The concentration of each ASO, including ISIS 304801, ISIS 396443, ISIS 420915, and ISIS 681257, was studied at 10 μM. Positive control substrates for each transporter included 2 μM prazosin (BCRP), 100 nM quinidine (P-gp), and 1 μM taurocholate (BSEP). Data represent the mean and SD of triplicate samples.

Figure 4

Inhibition of SLC and ABC Transporter-Mediated Probe Substrate Transport (A–E) Inhibition of positive controls (A), ISIS 304801 (B), ISIS 396443 (C), ISIS 420915 (D), and ISIS 681257 (E). For all transporters except BSEP, the cell line was MDCK. BSEP was studied in membrane vesicles prepared from Sf9 cells. The concentration of reference inhibitor probenecid was 100 μM (OAT1) (OAT3), quinidine was 1,000 μM (OCT1) (OCT2), rifampicin was 100 μM (OATP1B1) (OATP1B3) or 300 μM (BSEP), Ko143 was 1 μM (BCRP), and elacridar was 3 μM (P-gp). The concentration of each ASO was studied at 100 μM. Probe substrates for each transporter included 2 μM p-aminohippurate (OAT1), 10 μM p-aminohippurate (OAT3), 2 μM MPP⁺ (OCT1), 10 μM metformin (OCT2), 2 μM estradiol-17-β-D-glucuronide (OATP1B1), 2 μM CCK-8 (OATP1B3), 2 μM prazosin (BCRP), 100 nM quinidine (P-gp), and 1 μM taurocholate (BSEP). Data represent the mean and SD of triplicate samples. Dashed line = 100%. A highly statistically significant inhibition was obtained for all positive controls (p < 0.001).