Since the first siRNA drug was approved in 2018, oligonucleotide therapeutics have increased as an emerging drug modality. Oligonucleotides are short single or double-stranded fragments of DNA or RNA molecules that have a wide range of potential applications. However, due to its novelty and complexity, oligonucleotides have more technical challenges and unknown consequences for drug developers compared to many more conventional drugs. As such, oligonucleotides require pharmacokinetic evaluation systems that differ from traditional testing methods. WuXi AppTec’s Drug Metabolism and Pharmacokinetics (DMPK) Service Department’s extensive experience with oligonucleotide studies – combined with guidelines issued by various drug regulatory authorities on oligonucleotide development and studies in frontier literatures – has enabled us to build a set of ADME evaluation systems for oligonucleotides that significantly shorten development cycles.
Investigate multiple special routes of administration in pharmacokinetic studies.
Determine plasma protein binding rates through customized experimental procedures.
Conduct studies on tissue distribution through non-radiolabeled or radiolabeled oligonucleotides.
Investigate drug distribution with a quantitative whole-body autoradiography (QWBA) study.
Evaluate drug metabolism through various in vitro metabolic models.
Evaluate metabolic transformation through in vivo and in vitro metabolite identification.
Investigate excretion pathways through in vivo excretion experiments.
Well-established DDI evaluation based on drug metabolic enzymes and transporters.
Diverse types: The different chemical modifications, sequences, or delivery systems of oligonucleotides may have their respective specificities, thus requiring different pharmacokinetic evaluation methods.
Complex sample pretreatment: It is difficult to establish a proper mass spectrometry analysis method due to its poor stability, matrix effect, ion inhibition, and metabolite interference.
Multivalent in mass spectrometry: Both oligonucleotides and their metabolites tend to be multivalent in mass spectrometry, and there are various metabolites in the tissue matrix, which makes it difficult to identify metabolites.
Non-final regulatory guidelines: Neither FDA nor ICH has formulated final specific guidelines for the preclinical pharmacokinetics study of oligonucleotides.
An appropriate in vitro metabolic system needs to be selected in the screening stage.
Appropriate bioanalytical methods are critical for PK studies of oligonucleotides.
Metabolite identification facilitates early detection of active metabolites.
Exposure to the drug in target tissues rather than plasma exposure is more relevant to drug efficacy.
DMPK study capabilities for multiple types of oligonucleotides such as ASO, siRNA, microRNA, and aptamer.
Comprehensive bioanalysis (mass spectrometry, qPCR, fluorescent probes, etc.), biomarker analysis (target mRNA, target protein, or downstream biomarkers), in vitro and in vivo metabolite identification, and radiolabeled non-clinical ADME and clinical AME study capabilities.
Diverse in vitro metabolic study models.
Special administration routes, such as intrathecal injection and intravitreal injection.
Liver biopsy for large animals to support PK study of oligonucleotides.
Study on tissue distribution of oligonucleotides using QWBA technology.
There are various types of oligonucleotides, and their pharmacokinetic properties vary depending on mechanism, delivery system, and chemical modification. In the preclinical development stage, study strategies should be customized according to the specific characteristics of the oligonucleotide to accelerate its development. For in vitro ADME study, it is necessary to select an appropriate in vitro metabolic model based on the structural characteristics of the drug; for in vivo PK study, different administration methods can be used to investigate the PK properties. An appropriate detection method should be developed in accordance with the properties of the oligonucleotide.
Committed to Your ProgramWe have a specialized and dedicated service model. Each client will be connected to a dedicated study director who will provide comprehensive management services for the pharmacokinetic project from drug discovery to the clinical phase.
Extensive Experience and Custom-DesignedWe have more than four years of oligonucleotide study experience. We provide customized designs of pharmacokinetic study strategies for our customers' new molecules, together with rapid optimization and timely adjustment based on flexible study concepts.
Cross-Department Cooperation and High EfficiencyOur team works closely with the chemistry and biology departments internally to promote the smooth operation of the project, reduce the wait time, and ensure data security.
Comprehensive Capabilities and Diverse Bioanalytical MethodsWith a professional oligonucleotide study team and comprehensive instruments and equipment, WuXi AppTec's DMPK team can conduct extensive oligonucleotide studies and analysis, capable of delivering high-quality in vitro and in vivo data – all to accelerate the drug development process.
Take the preclinical PK study of Inclisiran (Novartis Leqvio®) as an example. In December 2020, the European Union granted market approval for Inclisiran. It is an innovative gene-based therapy, the world’s first small interfering nucleic acid (siRNA) in cholesterol-lowering field, targeting the liver proprotein convertase subtilisin/kexin type 9 (PCSK9) protein for the treatment of hypercholesterolemia or mixed dyslipidemia.
Inclisiran has undergone multiple chemical modifications, such as replacement of the phosphodiester bond with thiophosphate ester bond, and ribose modification by 2'-OMe (2'-O-methyl) or 2'-F(fluorine), which covalently links to three GalNAc molecules at the 3’-end of sense strand.
Based on relevant published literatures, the contents of the preclinical pharmacokinetic study of Inclisiran are summarized as follows:
1. In vitro ADME studies: Electrophoretic mobility shift assay (EMSA) on plasma protein binding rate, serum and liver S9 stability, CYP enzyme inhibition and induction, and drug transporter substrate and inhibition, etc.
2. In vivo PK studies: Studies on the pharmacokinetic characteristics in rats and monkeys under subcutaneous or intravenous administration conditions. In some studies, multiple samples were taken from the same animal through liver biopsy to analyze the drug concentrations in the liver. 14C labeled Inclisiran was used to investigate the tissue distribution and excretion pathway in rats and monkeys; the sampling periods lasted up to 98 days (rats) and 42 days (cynomolgus monkeys) by utilizing QWBA technology.
3. Metabolite identification studies: Thorough identification of metabolites was conducted. In vitro evaluation mainly included the metabolite identification of Inclisiran in human, mouse, rat and monkey serum and liver S9. In vivo evaluation included the metabolite identification of 14C labeled Inclisiran in the plasma, urine, bile (rat only), feces, livers, kidneys and injection sites (rat only) of rats and cynomolgus monkeys after administration; the metabolites in the plasma and liver tissues of rats and cynomolgus monkeys after the administration of Inclisiran were also identified.
 Yu, S. S., Hu, X. M., Wang, H. X., Wang, Y. & Wang, Q. L. An overview of nonclinical study and evaluation of therapeutic single-stranded oligonucleotides drugs. Chinese J. New Drugs 27, 1122‒1129 (2018).
 Guidance, D. Clinical Pharmacology Considerations for the Development of Oligonucleotide Therapeutics. Guidance for Industry. (2022)
 https://www.ema.europa.eu/en/documents/assessment-report/leqvio-epar-public-assessment-report_en.pdf. (2020)