Nonclinical absorption, distribution, metabolism, excretion and biomarkers properties of a modified N-acetylgalactosamine–conjugated small-interfering RNA for hypercholesteremia

DTX-007 is a double-stranded small-interfering RNA that decreases the expression of the proprotein convertase subtilisin/kexin type 9 protein through downregulation of its mRNA by conjugating with 3 N-acetylgalactosamine moieties to improve its hepatic distribution when administered via subcutaneous injection for the treatment of hypercholesterolemia. The nonclinical pharmacokinetics and the absorption, distribution, metabolism, and excretion characteristics of DTX-007 were evaluated in both in vivo and in vitro systems. Plasma protein binding exhibited concentration dependence across all examined species, approximating 99% at clinically relevant concentrations in humans. DTX-007 was absorbed after subcutaneous injection, with a mean half-life of around 0.9 hours in rats and 7.4 hours in monkeys following a dosage of 3 mg/kg. Our analysis indicates that the plasma pharmacokinetics for area under the concentration-time curve and Cmax of DTX-007 are approximately dose proportional and confirmed a linear relationship indicating approximately linear pharmacokinetics. DTX-007 is primarily transported to the liver by asialoglycoprotein receptor-mediated absorption, with a significantly prolonged half-life (t1/2) of 87.5 hours in the liver. The tissue to plasma concentration ratio of DTX-007-AS was greatest in the liver (4589) and kidney (536). DTX-007 was metabolized by nucleases rather than cytochrome P450 isozymes across many species, with no metabolites peculiar to humans. The primary metabolites in human and monkey were AS-3'N-1 (0.19%) and AS-5'N-2 (0.96%), respectively. Renal and fecal excretion constituted minor pathways for the removal of DTX-007. In the 1 mg/kg dosage group, proprotein convertase subtilisin/kexin type 9 protein levels were slightly diminished by 15 days postadministration (63%). Subcutaneous injections of cynomolgus monkeys at doses of 1, 3, and 10 mg/kg led to a substantial decrease in serum low-density lipoprotein cholesterol levels (28%-59%). It demonstrated similar pharmacokinetics profile in both species, indicating significant higher bioavailability than the approved small-interfering RNA drug and long-term effect on pharmacodynamic markers. SIGNIFICANCE STATEMENT: This study describes the ADME of GalNAc-RNA drug in rats and monkeys in vivo and across human and animal matrices in vitro. These studies disclose the excellent ADME profile with higher bioavailability than the approved siRNA drug and support the interpretation of toxicology studies, helping characterize the disposition of drug in humans and support the clinical use. It also highlights adequate exposure of drug to the target organ liver and long-term effect on pharmacodynamic markers after single dose.