Oligonucleotides (oligos), including small interference RNAs (siRNAs) and antisense oligonucleotides (ASOs), represent a novel class of therapeutic agents capable of precisely targeting and regulating gene expression. This precision allows the potential treatment of various diseases, from genetic disorders to cancers, by silencing harmful genes or correcting genetic abnormalities.
Oligos’ mechanism of action involves binding to mRNA molecules, preventing them from being translated into proteins, or altering splicing patterns, providing a highly targeted approach to treating diseases. In addition, oligos’ efficacy is closely linked to their stability in plasma. Stability influences their pharmacokinetic profiles—i.e., absorption, distribution, metabolism, and excretion (ADME)—which are critical factors in determining therapeutic effectiveness and safety.
A key challenge in oligonucleotide drug development is ensuring that these molecules remain stable in the bloodstream long enough to reach their targets and have their intended effect. Therefore, developing robust assays to evaluate the plasma stability of oligonucleotides accurately is essential for advancing these promising therapeutic agents through drug discovery and development, optimizing their design for enhanced stability and improving patient outcomes.
A new oligo assay
WuXi AppTec scientists designed a robust and reproducible in vitro assay to accurately evaluate oligos’ stability in plasma. The assay aims to mimic the in vivo conditions these therapeutic agents encounter in the bloodstream, providing crucial data on their degradation rates and stability profiles. By accurately assessing these parameters, the assay will enable researchers to predict oligos’ pharmacokinetic behavior and guide their design and optimization for therapeutic use.
A secondary objective of the study is to investigate the effect of anticoagulants, plasma stored condition, and the choice of plasma or serum in oligo metabolism. Anticoagulants prevent blood from clotting; some can even influence the activity of nucleases that degrade oligos, potentially affecting the assay’s accuracy. Similarly, the plasma’s condition—i.e., whether it is frozen or fresh—may impact the metabolic stability of these molecules.
Understanding these effects is crucial to ensure that the newly developed assay provides reliable and consistent results, facilitating the development of oligo therapeutics with enhanced efficacy and safety profiles.
The study’s methodology & primary findings
The study meticulously selected a range of oligos, including siRNAs and ASOs, to ensure a comprehensive evaluation of plasma stability. These oligos were chosen based on their therapeutic potential and diversity in sequence and structure, aiming to represent a broad spectrum of the molecules currently under investigation for drug development purposes.
The methodology involved preparing plasma samples using different anticoagulants to assess their effects on oligo stability. The study also explored the effects of frozen versus fresh plasma, and serum or plasma to determine how the factors might affect oligos’ metabolic stability.
The study used liquid chromatography-tandem-mass spectrometry (LC-MS/MS), a highly sensitive and specific analytical technique to assess oligo stability. This method enabled the precise measurement of oligo degradation products, providing detailed insights into these molecules’ stability and metabolic pathways in plasma. The LC-MS/MS technique was critical for generating reliable data that could inform the development of more stable and effective oligo-based therapeutics. The study’s three primary findings include:
- Effect of different anticoagulants on oligo stability
The study found significant differences in oligo stability depending on the type of anticoagulant used in plasma preparation. Heparin was identified as having a minimal impact on oligo stability compared to other anticoagulants, suggesting it is the preferred choice for plasma stability assays. - Analysis of the freezing treatment effect on enzymatic activity
The effect of freezing and thawing on the enzymatic activity within plasma was analyzed, showing that these treatments do not significantly alter the enzymes’ activity that metabolizes oligos. This finding suggests that oligo stability assays can be conducted on plasma samples subjected to standard freezing and thawing processes without compromising the assay’s integrity. - Impact of plasma or serum on oligonucleotide metabolism
The research investigates the differences in the metabolism of oligonucleotide in plasma or serum and showing that these matrices do not influence the metabolism. This finding suggests that both plasma and serum are suitable for oligonucleotide metabolism research.
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Figure 1. Comparison of the remaining data in rat plasma for Patisiran and WuXi AppTec Control 01 in (A) frozen plasma, with EDTA-K2 and heparin as anticoagulants (B) fresh and frozen heparin anticoagulated plasma (C) frozen plasma and frozen serum.
The significance of the study results
The study results illustrate the critical role of anticoagulant usage, plasma handling conditions, and the choice of plasma or serum in assessing oligonucleotide stability. These factors are pivotal in developing a reliable plasma stability assay for accurate pharmacokinetic profiling of oligo-based therapeutics.
By identifying the conditions under which oligos remain stable or are degraded in the bloodstream, researchers can design more effective and durable therapies. This allows them to optimize dosing regimens and improve the success rate of preclinical and clinical evaluations of new drug candidates.
Comparisons with prior research highlight this study’s significance, particularly in its systematic evaluation of anticoagulants and handling conditions. This approach addresses gaps in existing methodologies, offering a more flexible framework for oligo stability assessment. Continued exploration into these drugs is crucial, focusing on understanding the interaction between oligos and alternative biological matrices. Further research could aim to elucidate the mechanisms of degradation in different environments and develop enhanced stabilization strategies to extend the half-life and efficacy of oligo-based therapeutics.
The implications of these results are far-reaching for oligos’ pharmacokinetics and metabolism profiles. By providing a clearer understanding of how these molecules behave in plasma, the study helps optimize oligo designs for improved stability, efficacy, and safety. This ultimately enhances drug development and therapeutic outcomes for these molecules.
Future work could explore its use in personalized medicine, predicting patient-specific responses based on plasma stability profiles and guiding dosage and treatment regimens for oligonucleotide therapeutics.
A final word
The study successfully developed a reliable in vitro plasma stability assay for oligonucleotides, highlighting the importance of anticoagulant usage, plasma/serum choice, and plasma handling in assessing oligo stability. It also demonstrated that Heparin is a more suitable anticoagulant and that freezing treatments do not significantly impact enzymatic activity. It is recommended that this plasma stability assay be integrated into the early stages of drug development to guide the selection and optimization of oligo candidates. This assay can significantly contribute to designing oligo-based drugs efficiently and with improved pharmacokinetic profiles, supporting the advancement of safe and effective therapeutics.
As a global company with operations across Asia, Europe, and North America, WuXi AppTec provides a broad portfolio of R&D and manufacturing services that enable the global pharmaceutical and life sciences industry to advance discoveries and deliver groundbreaking treatments to patients. Through its unique business models, WuXi AppTec’s integrated, end-to-end services include chemistry drug CRDMO (Contract Research, Development and Manufacturing Organization), biology discovery, preclinical testing and clinical research services, advanced therapies CTDMO (Contract Testing, Development and Manufacturing Organization), helping customers improve the productivity of advancing healthcare products through cost-effective and efficient solutions. WuXi AppTec received an AA ESG rating from MSCI for the third consecutive year in 2023 and its open-access platform is enabling more than 6,000 customers from over 30 countries to improve the health of those in need – and to realize the vision that “every drug can be made, and every disease can be treated.”
