Dr. Yan Pan- Associate Director, DMPK Department, WuXi AppTec
Small interfering RNA (siRNA) therapeutics have emerged as a trans formative class of drugs, offering targeted gene silencing capabilities that hold promise for treating various diseases. Since the landmark approval of the first siRNA drug Patisiran in 2018, the therapeutic siRNA landscape has expanded significantly, with multiple drugs, including Givosiran, Lumasiran, Inclisiran, and Vutrisiran, now approved in both the US and EU markets. Understanding these drugs’ absorption, distribution, metabolism, and excretion (ADME) characteristics is critical for optimizing their development and clinical application of siRNA therapeutics.
Structural modifications (e.g., 2’-O-methyl, phosphorothioate) and delivery strategies (e.g., lipid nanoparticles, GalNAc conjugation) will continue to shape the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of siRNA drugs. Lessons from these pioneering therapies are now guiding the next generation of RNA-based therapeutics, expanding their reach beyond hepatic targets.
ADME in Early siRNA Drug Development
In vitro ADME studies are essential to early-stage drug discovery, falling under the broader umbrella of drug metabolism and pharmacokinetics (DMPK). These assays help scientists gather important absorption, distribution, metabolism, and excretion data. In vitro testing for siRNA drugs also informs structural optimization, delivery method selection, and initial dosing strategies.
These assays allow researchers to assess drug permeability (e.g., using Caco-2 or PAMPA models), transporter interactions (e.g., OATP or MDR1 assays), and metabolic stability (e.g., liver S9 fractions or microsomes). The ability to predict in vivo PK, including potential drug-drug interactions (DDIs), accelerates lead optimization and de-risks preclinical development.
Absorption: Overcoming Delivery Challenges
Delivering siRNA therapeutics presents unique challenges due to their size, polarity, and susceptibility to degradation. Unlike small molecules, siRNAs are poorly absorbed through the gastrointestinal tract, necessitating alternative administration routes to achieve therapeutic concentrations.
Intravenous vs. Subcutaneous Administration
Patisiran utilizes lipid nanoparticle (LNP) encapsulation, with particles approximately 60–100 nm in diameter, facilitating intravenous (IV) administration.¹ This delivery system enhances liver targeting, as the LNPs are taken up by hepatocytes via apolipoprotein E-mediated endocytosis, leading to higher hepatic exposure compared to subcutaneous (SC) administration.²
In contrast, Givosiran, Lumasiran, Inclisiran, and Vutrisiran employ N-acetylgalactosamine (GalNAc) conjugation, enabling SC administration. The GalNAc moiety specifically binds the asialoglycoprotein receptor (ASGPR) on hepatocytes, enabling efficient receptor-mediated endocytosis.³ SC administration provides a sustained-release effect, allowing gradual uptake by hepatocytes and reducing the risk of ASGPR saturation that may occur with high-concentration IV dosing.4
Pharmacokinetic Profiles
Following SC administration, these siRNA drugs exhibit a ttwo-compartmentPK PK model, with plasma concentration-time profiles showing an initial distribution phase (α phase) followed by a slower elimination phase (β phase). Systemic exposure, measured by Cmax and AUC, generally increases proportionally with dose, and no significant sex differences have been observed in PK parameters.
For siRNA therapeutics, conventional permeability models (e.g., PAMPA) are less relevant due to their macromolecular nature. Instead, absorption studies typically focus on:
- Cellular uptake assays using hepatocyte models (primary or immortalized)
- ASGPR binding/uptake studies for GalNAc-conjugated siRNAs
- Endosomal escape efficiency measurements
Distribution: Hepatic Targeting of siRNA Therapeutics
The liver is the primary target organ for clinically approved siRNA drugs due to its unique physiological and molecular characteristics, including high blood perfusion (30% of cardiac output), fenestrated sinusoidal endothelium (100–150 nm pores) that facilitates macromolecule extravasation, and abundant expression of ASGPR on hepatocytes (~500,000 receptors/ cell), which enables efficient uptake of GalNAc-conjugated siRNAs.
In vivo studies, including quantitative whole-body autoradiography (QWBA) and tissue collection analyses, have demonstrated that siRNA drugs predominantly accumulate in the liver. For instance, QWBA studies of radiolabeled siRNAs have confirmed significant hepatic uptake, with minimal distribution to other tissues 5,6
Plasma protein binding (PPB) significantly influences the pharmacokinetics of oligonucleotide therapeutics, affecting both volume of distribution and clearance rates. PPB assays - such as equilibrium dialysis and ultrafiltration - show that Inclisiran exhibits approximately 87% binding, while Lumasiran shows 77–85%. These values reflect the complex binding behavior characteristic of oligonucleotides, attributable to their unique physicochemical properties (high molecular weight, polyanionic nature) and propensity for nonspecific interactions with multiple plasma protein classes.
Challenges in PPB assays include assay sensitivity, nonspecific binding to assay apparatus, and interspecific variability in plasma protein composition. Translational extrapolation from in vitro to in vivo remains a key hurdle, requiring advanced modeling and interpretation.
Metabolism: Nuclease-Mediated Degradation of siRNA Therapeutics
Unlike small molecule drugs, which are primarily metabolized by cytochrome P450 enzymes, siRNA therapeutics are mainly degraded by endonucleases and exonucleases present in the liver and systemic tissues .7 This degradation process results in shorter oligonucleotide fragments, which are subsequently eliminated from the body. The chemical modifications incorporated into siRNA molecules, such as 2’-O-methyl and phosphorothioate linkages, enhance their stability against nuclease activity, prolonging their half-life and therapeutic effects.
In vitro metabolism studies typically involve liver S9 fractions, hepatocytes, lysosomes, and tritium preparations to assess metabolic stability and identify potential metabolites. These data are crucial for understanding species-specific metabolism and informing in vivo study design.
Excretion: Renal & Hepatic Pathways
The elimination of siRNA therapeutics involves both renal and hepatic pathways, with the balance between these routes influenced by the drug’s structure and delivery mechanism.
Renal Excretion
Due to their hydrophilic nature and negative charge, siRNA drugs are primarily excreted via the kidneys. Studies have shown that Vutrisiran is eliminated through renal pathways, with approximately 15–25% of the administered dose recovered unchanged in urine. However, most of the drug is cleared through hepatic uptake and subsequent metabolism.
Impact of Renal Impairment
Inclisiran has explicitly been studied in patients with varying degrees of renal impairment. Results indicate that while Cmax and AUC values increase in patients with renal dysfunction, these changes do not necessitate dose adjustments, as the therapeutic efficacy remains unaffected. Population PK analyses suggest that mild to moderate renal impairment does not significantly impact drug exposure or efficacy for the other siRNA drugs.8
Drug-Drug Interaction: Minimal CYP450 Involvement
siRNA therapeutics typically demonstrate a low potential for DDIs, primarily because they are not substrates, inhibitors, or inducers of cytochrome P450 enzymes or major drug transporters.
Givosiran presents a notable exception, as it targets delta-aminolevulinic acid synthase 1 (ALAS1), a rate-limiting enzyme in the heme biosynthesis pathway. By suppressing ALAS1 expression, Givosiran may indirectly modulate the activity of heme-dependent enzymes, including certain CYP450 isoforms. Clinical studies have observed increased exposure to substrates of CYP1A2, CYP2C9, CYP2C19, and CYP3A4 in patients treated with Givosiran, suggesting a potential for DDI in this context.6
Standard in vitro screening panels (CYP inhibition, Ki determination, and time-dependent inhibition assays) help evaluate interaction risks during development. Although most siRNA drugs exhibit minimal DDI liability, thorough preclinical assessment remains a regulatory requirement to ensure therapeutic safety.
Strategic ADME Optimization & Automation in ADME Testing
Early understanding of ADME properties plays a pivotal role in optimizing siRNA therapeutics. Early ADME screening helps identify candidates with favorable PK profiles, reduces late-stage attrition, and informs critical preclinical and clinical development decisions.
These insights guide delivery methods, dose selection, and safety assessment. When combined with in vivo studies, they enable robust in vitro-in vivo correlations (IVIVC). Though challenges such as nonspecific binding, inter-individual variability, and in vitro-in vivo extrapolation persist, emerging advancements in assay design and modeling techniques help address these limitations.
Implementation of automation in in vitro ADME testing offers significant advantages for siRNA drug development. Automated liquid handling systems enhance assay reproducibility, boost throughput, and minimize human error. These systems enable rapid sample processing and consistent reagent dispensing, which are essential for generating high-quality, reliable data.
High-throughput automation enables screening multiple candidates across different conditions, streamlining the decision-making process during lead optimization. With real-time data capture and reduced variability, automation expedites progression from early discovery through IND submission.
Concluding Remarks on ADME Profiles of Successful siRNA Drugs
The ADME profiles of clinically approved siRNA therapeutics highlight the importance of tailored delivery strategies, metabolic stability, and comprehensive pharmacokinetic evaluations in the development of this drug class. Advancements in chemical modifications and delivery technologies have enabled the successful translation of siRNA drugs from bench to bedside, opening new avenues for treating previously intractable diseases.
As RNA-based therapeutics continue to advance, a thorough understanding of the ADME characteristics will remain critical for guiding the design and optimization of future siRNA drugs, ultimately ensuring their safety, efficacy, and regulatory success.
References
1. Chen S, Tam YYC, Lin PJC, et al. Development of lipid nanoparticle formulations of siRNA for hepatocyte gene silencing following subcutaneous administration. J Control Release. 2014;196:106-112.
2. Kozauer N. Cross-Discipline Team Leader Review: Patisiran NDA. Alnylam Pharmaceuticals; 2016.
3. Springer AD, Dowdy SF. GalNAc-siRNA conjugates: Leading the way for delivery of RNAi therapeutics. Nucleic Acid Ther. 2018;28(3):109-118.
4. Witzigmann D, Kulkarni JA, Leung J, et al. Variable asialoglycoprotein receptor 1 expression in liver disease: Implications for therapeutic intervention. Hepatol Res. 2016;46(7):686-696.
5. European Medicines Agency. European Public Assessment Report: Onpattro (patisiran). EMA; 2018.
6. Alnylam Pharmaceuticals. Givosiran NDA Multi-Discipline Review. FDA; 2019.
7. Andersson P, Den Besten C. Preclinical and clinical drug-metabolism, pharmacokinetics and safety of therapeutic oligonucleotides. In: Drug Metabolism in Drug Design and Development: Basic Concepts and Practice. RSC Drug Discovery Series. 2019:474-531.
8. Jing X, Arya V, Reynolds KS, Rogers H. Clinical pharmacology of RNAi-based therapeutics: A summary based on FDA-approved small-interfering RNAs. Drug Metab Dispos. 2022. doi:10.1124/dmd.122.001107
Dr. Yan Pan is a group leader of the DMPK Study Director Team at WuXi AppTec’s Shanghai office. He has more than 15 years of experience in drug metabolism and pharmacokinetics and holds a Ph.D. in neurobiology with a specialization in neuropharmacology from Fudan University.