Oligonucleotide pharmacokinetic/drug metabolism

The research and development of oligonucleotide analytes as pharmaceuticals for the treatment of a wide variety of diseases has been ongoing in academia and in the pharmaceutical industry for more than 30 years. Over the past 3 decades, the field has expanded considerably to include antisense oligonucleotides (ASOs), aptamers, small interfering RNAs (siRNAs), microRNAs (miRNAs), splicing-switched oligonucleotides (SSOs), and, more recently, CRISPR/ Cas9, among others [1].

Considerable advances in chemical modifications of oligonucleotides have also increased target-binding affinity and exonuclease resistance to increase half-life in vivo [2–8]. Furthermore, advances in the delivery of oligonucleotides to their target organs, either through targeted delivery (i.e., intrathecal, intranasal, intravitreal, etc.) [9–15] or through drug delivery mechanisms (i.e. , conjugation with GalNAc, lipid nanoparticles, etc. )

[2,9,16 – 22], when combined with advances in chemical modifications [23], has increased potency, thus minimizing the dosing schedule, as well as the amount of drug needed to be effective.

There has been renewed interest in oligonucleotides given the recent approvals of Spinraza® (Ionis/Biogen) for the treatment of spinal muscular atrophy [24] and Eteplirsen (Sarepta) for the treatment of Duchenne muscular dystrophy [25]. The last 5 years have seen an increase in the number of pharmaceutical companies whose focus is on the development of oligonucleotide-based compounds.

Furthermore, the importance of pharmaceuticals based on oligonucleotides is evident, since most of the big pharmaceutical companies have oligonucleotides in their current projects, either through partnerships or independent development. The appeal is considerable given that oligonucleotides can be used to target a wide range of disease states including, but not limited to: cancer, viral infections, and rare diseases (including genetic diseases), as well as numerous targets previously considered non-susceptible. treatment with traditional therapies.

Oligonucleotide Bioanalysis Platforms

Given the wide range of oligonucleotide-based compounds and the advances made in chemical modifications and drug delivery, the appropriate assay platform must be selected for pharmacokinetic (PK) profiling, drug metabolism, and drug metabolism assessment. anatomical distribution of compounds.

There are a number of assay formats that can be followed for PK analysis, with chromatographic and enzyme-linked immunosorbent assays (ELISAs) being the preferred ones. Liquid chromatography assays such as UPLC-UV, LC-FL, LC-MS/MS, and LC-HRAM are typically highly selective, can distinguish between full-length oligonucleotides and their metabolites, and have a wide dynamic range, but generally require complex sample preparation and have lower sample throughput.

An alternative to chromatography-based methods are hybridization ELISA methods. Briefly, the oligonucleotide analyte in the sample is hybridized with complementary capture and, if necessary, detection probes. Special care must be taken when designing capture and detection probes to ensure adequate affinity for the oligonucleotide analyte and to minimize the potential for self-hybridization. The assay is completed with the addition of detection reagent and substrate.

There are three hybridization ELISA formats that are commonly used: 1) ligation hybridization ELISA; 2) Nuclease-based hybridization ELISA; and 3) double probe hybridization ELISA. The Immunochemistry Department at PPD Laboratories Bioanalytical Laboratory (PPD BioA Richmond, VA, USA) has been performing hybridization ELISA assays since 2001 to support the testing of PK/DM samples for non-clinical PK/DM shipments, similar to GLP and clinical. PPD BioA ICD has developed, transferred and validated methods using all three hybridization ELISA formats.