Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys

Abstract

Triantennary N-acetyl galactosamine (GalNAc3) is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc3 via a trishexylamino (THA)-C6 cluster significantly enhances antisense oligonucleotide (ASO) potency. Herein, the biotransformation, disposition, and elimination of the THA cluster of ION-681257, a GalNAc3-conjugated ASO currently in clinical development, are investigated in rats and monkey. Rats were administered a single subcutaneous dose of (3)H-radiolabeled ((3)H placed in THA) or nonradiolabeled ION-681257. Mass balance included radiometric profiling and metabolite fractionation with characterization by mass spectrometry. GalNAc3-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc3 conjugate at the 5'-end of the ASO was rapidly metabolized and excreted with 25.67 ± 1.635% and 71.66 ± 4.17% of radioactivity recovered in urine and feces within 48 hours postdose. Unchanged drug, short-mer ASOs, and linker metabolites were detected in urine. Collectively, 14 novel linker associated metabolites were discovered including oxidation at each branching arm, initially by monooxidation at the β-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining β-arms. Metabolites in bile and feces were identical to urine except for oxidized linear and cyclic linker metabolites. Enzymatic reaction phenotyping confirmed involvement of N-acetyl-β-glucosaminidase, deoxyribonuclease II, alkaline phosphatase, and alcohol + aldehyde dehydrogenases on the complex metabolism pathway for THA supplementing in vivo findings. Lastly, excreta from monkeys treated with ION-681257 revealed the identical series as observed in rat. In summary, our findings provide an improved understanding of GalNAc3-conjugated-ASO metabolism pathways which facilitate similar development programs.

Figure 1

Chemical structure of ³H-ION 681257 labeled on the THA linker. T* denotes the tritium radiolabel position.

Figure 2

Percent of dose eliminated in excreta and found in tissue. (a) Mass percentages of cumulative radioactivity and total dose eliminated in Sprague Dawley rats following single s.c. administration of 4.3 mg/kg of ³H-ION 681257 labeled on the THA Linker at 114.4 µCi/kg (n = 2 rats, M). Values are listed as the percentage of normalized dose excreted in the urine, feces, and combined. (b) Percent of dose in major and minor tissues at 24 and 168 hours.

Figure 3

Representative HPLC radiochromatograms of (a) dosing solution, (b) rat urine, (c) rat bile, and (d) rat feces. Conditions of the mobile phase evaluated include (black trace) 5 mmol/l ammonium acetate pH 8.0 and (blue trace) 0.1% aqueous formic acid pH 2.7. All samples were diluted to 100,000 DPM per 20 µl injection. DPM, disintegrations per minute; M#, identified metabolite listed in Table 1; P, parent; S, short-mer; U, unidentified.

Figure 4

Representative in vivo metabolism of the major GalNAc₃-associated metabolites identified in rat feces. MS/MS product ion spectra (a–h) represent metabolites (M5–M12) and are listed with x-axes representing m/z values, with the y-axes as relative abundance (counts).

Figure 5

Representative HPLC–MS chromatograms depicting in-vitro cleavage of GalNAc sugars from conjugated oligonucleotide (ION-681257) using β-N-acetylglucosaminidase and DNase II. Reaction conditions include (a) 100 µmol/l ION-681257 without β-Nag was used as a negative control, and (b) 100 µmol/l with 122 U/ml of β-Nag. Reactions were incubated for 30 minutes at 37 °C. The mass spectra illustrate rapid cleavage of all sugars from the intact drug substance. (c) Neat standards corresponding to −0 sugar (black trace), −1 sugar (red trace), and −3 sugars (blue trace), respectively, were diluted in water to 0.32 mg/ml and incubated with 0.12 mg/ml of DNase II at 37 °C for 24 hours.

Figure 6

Representative HPLC–MS chromatograms depicting in-vitro metabolism of GalNAc₃-conjugated oligonucleotide (ION-681257) using DNase II treatment followed by alkaline phosphatase, alcohol, and aldehyde dehydrogenases. (a) Combined samples including the −3 sugar standard absent enzyme as a control depicted in the total ion chromatogram. (b) Mass spectra of control samples following DNase II treatment, but absent alkaline phosphatase revealed the presence of linear metabolite (M4). (c, d) Mass spectra of the −3 sugar standard treated by DNase II for 18 hours and incubated with alkaline phosphatase for 2 hours depicting (M8) in high abundance in addition to residual cyclized linker (M5). ALH and ALDH treatment using (M8) standard alone (e–h). (e) Total ion chromatogram of (M8) treated with alcohol dehydrogenase, and (f) corresponding mass spectra. (g) Total ion chromatogram of (M8) treated with alcohol dehydrogenase followed by aldehyde dehydrogenase and corresponding mass spectra (h). Both mono and di-oxidations are observed.

Figure 7

Proposed major metabolism pathways of GalNAc₃-conjugated antisense oligonucleotides. Metabolites were detected in rat urine (blue, single asterisk), bile and feces (red, two asterisks), and/or combined (violet, three asterisks). The proposed biotransformation pathway is a result of characterization by LC–MS, including identity of precursor mass, product ions, retention times, and radiometric detection. An in vitro approach using enzymatic incubations was also used to confirm metabolite formation. From the scheme, it is observed that the full mass conjugated parent is rapidly metabolized into predominately oxidized species.