RT-qPCR Methods to Support Pharmacokinetics and Drug Mechanism of Action to Advance Development of RNAi Therapeutics.

The goal of this study was to develop a reverse transcription quantitative polymerase chain reaction (RT-qPCR) method for the accurate quantification of chemically modified small interfering RNA (siRNA) including but not restricted to thermally destabilizing modifications such as glycol nucleic acid (GNA). RT-qPCR was found to be superior to mass spectrometry-based siRNA detection in terms of sensitivity and throughput. However, mass spectrometry is still the preferred method when specific metabolite detection is required and is also insensitive to siRNA chemical modifications such as GNA. The RT-qPCR approach can be optimized to take chemical modifications into account and works robustly in different matrices without optimization, unlike mass spectrometry. RT-qPCR and mass spectrometry both have their strengths and weaknesses for the detection of siRNA and must be used appropriately depending on the questions at hand. Considerations such as desired throughput, assay sensitivity, and metabolite identification must be weighed when choosing which methodology to apply.

Evaluation of electrophoretic mobility shift assay as a method to determine plasma protein binding of siRNA.

Aim: The electrophoretic mobility shift assay (EMSA) was evaluated as an alternative to ultrafiltration (UF) to assess plasma protein binding (PPB) of small interfering RNAs (siRNA) and antisense oligonucleotides (ASO). Results & methodology: EMSA analysis showed that PPB depended on siRNA and plasma concentration. Conversely, when analyzed by ultrafiltration, siRNA bound the filtration device nonspecifically and PPB remained >98% across physiologically relevant siRNA concentrations. Using EMSA, siRNA exhibited charge-based interactions with plasma proteins, while ASO remained highly bound to plasma proteins or albumin in the presence of 500 mM salt. Conclusion: PPB characteristics of siRNA and ASO can be distinguished using EMSA. Characterization of siRNA PPB by EMSA enhances our knowledge of siRNA absorption, distribution, metabolism and excretion and advanced development of RNA interference therapeutics.

In Vitro Drug-Drug Interaction Evaluation of GalNAc Conjugated siRNAs Against CYP450 Enzymes and Transporters.

Small interfering RNAs (siRNAs) represent a new class of medicines that are smaller (∼16,000 Da) than biologic therapeutics (>150,000 Da) but much larger than small molecules (<900 Da). Current regulatory guidance on drug-drug interactions (DDIs) from the European Medicines Agency, Food and Drug Administration, and Pharmaceutical and Medical Devices Agency provides no recommendations for oligonucleotide therapeutics including siRNAs; therefore, small molecule guidance documents have historically been applied. Over ∼10 years, in vitro DDI investigations with siRNAs conjugated to a triantennary N-acetylgalactosamine [(GalNAc)-siRNA] ligand have been conducted during nonclinical drug development to elucidate the potential clinical DDI liability. GalNAc siRNAs were evaluated as substrates, inhibitors, or inducers of major cytochrome P450s (P450s) and as substrates and inhibitors of transporters. Aggregate analysis of these data demonstrates a low potential for DDI against P450s. Zero of five, 10, and seven are inducers, time-dependent inhibitors, or substrates, respectively, and nine of 12 do not inhibit any P450 isoform evaluated. Three GalNAc siRNAs inhibited CYP2C8 at supratherapeutic concentrations, and one mildly inhibited CYP2B6. The lowest K i value of 28 µM is >3000-fold above the therapeutic clinical C max at steady state, and importantly no clinical inhibition was projected. Of four GalNAc siRNAs tested none were substrates for transporters and one caused inhibition of P-glycoprotein, calculated not to be clinically relevant. The pharmacological basis for DDIs, including consideration of the target and/or off-target profiles for GalNAc siRNAs, should be made as part of the overall DDI risk assessment. If modulation of the target protein does not interfere with P450s or transporters, then in vitro or clinical investigations into the DDI potential of the GalNAc siRNAs are not warranted. SIGNIFICANCE STATEMENT: Recommendations for evaluating DDI potential of small molecule drugs are well established; however, guidance for novel modalities, particularly oligonucleotide-based therapeutics are lacking. Given the paucity of published data in this field, in vitro DDI investigations are often conducted. The aggregate analysis of GalNAc-siRNA data reviewed herein demonstrates that, like new biological entities, these oligonucleotide-based therapeutic drugs are unlikely to result in DDIs; therefore, it is recommended that the need for in vitro or clinical investigations similarly be determined on a case-by-case basis. Given the mechanism of siRNA action, special consideration should be made in cases where there may be a pharmacological basis for DDIs.