Peptides and oligonucleotides are two of the most promising classes of drugs being developed today. Their extraordinary potential means sponsors and developers around the world are investing significant time and resources to make progress with these modalities.
With such competition, quick, safe development becomes crucial. Integrated DMPK, bioanalysis, and toxicology can significantly accelerate progress.
Before designing a proper integrated toxicology approach, it’s important to understand where to look for toxicity. The primary drivers of toxicity in peptides are exaggerated pharmacology, immunogenicity, and local or injection site reactions. For oligos, chemistry-driven protein binding and class effects, tissue accumulation, hybridization-dependent off-target effects, and sequence-specific contributions within a chemical platform are the major causes of toxicity.
1. Establish Specific Preclinical Safety Strategies
Preclinical safety strategies should be set according to the modality rather than applied generically. Establishing a plan early will ensure toxicology progresses much smoother.
For peptides, preclinical safety strategies should focus on:
- Immunogenicity: ADA monitoring in all repeat-dose studies; in silico epitope prediction; NHP preferred when feasible; recovery groups are important due to potentially delayed immune effects.
- Local tolerance: Detailed injection-site histopathology (SC/IM common routes).
- Secondary pharmacology: Focused receptor/enzyme panel rather than broad off-target screens.
- Species selection: Pharmacologic relevance and immune system similarity.
- Chronic toxicity: Emphasize kidney (renal clearance) and immunogenicity-related findings.
For oligonucleotides, researchers need to examine the following carefully:
- Hybridization-dependent off-targets: In silico (BLAST) + confirmatory in vitro assays.
- Class effects: Complement activation, platelet counts, liver enzymes, kidney biomarkers.
- Tissue accumulation: Focused histopathology of liver, kidney, spleen, lymph nodes.
- Safety pharmacology: Core battery in relevant species; off-target assessment even without hybridization.
- Surrogates: Species-specific sequences when clinical candidate is inactive.
- Delivery systems: Separate evaluation of GalNAc, LNPs, or other vehicles.
2. Carefully Evaluate Exposure Considerations
Researchers need to keep in mind the essential exposure considerations that can affect how toxicology data is interpreted for peptides and oligos.
For peptides, researchers should take into account the necessity of frequent sampling early post-dose, monitor urine recovery and kidney exposure, appreciate that injection-site depot effects can prolong exposure, assess safety margins based on both systematic exposure and PD markers, consider that high-dose may be limited by solubility or local irritation, and remember that anti-drug antibodies (ADA) can alter clearance in later phases of repeat dose studies.
There are also several exposure considerations for oligonucleotides. Plasma half-life is often short, while tissue half-life is long, peak plasma levels may not reflect tissue exposure or PD duration, both plasma and key tissue levels must be reported to properly understand distribution, tissue exposure can be more useful than plasma exposure for judging safety margins, accumulation with repeat dosing is expected and must be quantified, and for conjugated oligos, researchers must characterize conjugate stability and free oligo.
3. Conduct a Thorough Biodistribution Examination
Biodistribution is an essential consideration for preclinical testing of oligos and peptides. It helps researchers connect pharmacological effects and toxicities to tissue exposure, rather than relying on the administered dose or plasma concentration.
In peptides, biodistribution informs species selection and kidney findings, and researchers need to take the following into account:
- They have limited distribution compared to small molecules
- Common high-exposure sites are the kidney, liver, and the injection site.
- Urinary excretion of intact peptide or fragments is often significant.
- Local retention at SC injection site can drive local toxicity
- Most peptides have limited brain penetration.
Biodistribution of oligonucleotides informs the duration of recovery groups, the need for chronic studies, and the potential for off-target organ toxicity. Again, there are key factors to consider:
- Strong accumulation in highly perfused and reticuloendothelial system organs.
- The liver and kidney usually dominate biodistribution, but the spleen and lymph nodes are also important.
- Chemical modifications and conjugates dramatically alter distribution.
- Long tissue residence can lead to delayed or cumulative toxicity.
- Hybridization assays enable sequence-specific quantification in tissues.
4. Ensure Regulatory Compliance
Regulatory guidelines should be followed from the very beginning of development. For peptide development, ICH M3(R2) applies, with ICH S6(R1) principles for larger or modified peptides. For oligonucleotides, the FDA Draft Guidance "Nonclinical Safety Assessment of Oligonucleotide-Based Therapeutics and the Clinical Pharmacology guidance from June 2024 should be consulted. ICH S13, covering the nonclinical safety evaluation of oligonucleotide-based therapeutics, is also emerging.
Common regulatory expectations across both modalities include justification of species and any surrogate molecules used, robust exposure and biodistribution data, and a weight-of-evidence approach to genotoxicity and carcinogenicity. Early discussions with regulators can be crucial for novel chemistries or delivery systems.
5. Build a Comprehensive Integrated Study Design
An integrated study design allows toxicology, exposure, biodistribution, pharmacology, and bioanalysis data to be interpreted together, rather than in isolation. This can improve the speed and quality of development.
Developers looking to take advantage of these benefits should ensure they use dose-range finding to guide definitive repeat-dose studies and select high doses using exposure and pharmacological effect, supported by PK/PD data and relevant ICH guidance. Integrating key safety assessments early is vital, including class-specific biomarkers, safety pharmacology, biodistribution and toxicokinetics.
Researchers must also ensure they address modality-specific risks, including immunogenicity and local tolerance for peptides and off-target hybridization for oligonucleotides. Finally, they also need to include recovery groups to assess whether adverse effects persist or reverse as tissue exposure declines.
A Final Word
Peptides and oligonucleotides will continue to dominate development priorities, so sponsors and developers must become familiar with the challenges and opportunities these modalities present in preclinical research.
By taking an integrated approach and optimizing that route through the tips listed above, developers can ensure they make the most of this wave of innovation and bring safe and secure drugs to market. For any sponsors or developers seeking to ensure their toxicology studies are properly prepared, executed, and analyzed, working with an experienced lab partner can make a huge difference.
