Case Study 12: Roadmap to Quantifying Ago2-Mediated siRNA Metabolic Activation Kinetics.

Therapeutic siRNA is a prodrug that requires Ago2-mediated site-specific hydrolysis of the sense strand before RNA interference can occur. Although this metabolic activation step was first described 15 years ago, the kinetics of this reaction, and its relationship to in vivo siRNA efficacy, remains unexplored in the literature. To provide a roadmap to address these gaps, we describe a liquid chromatography-mass spectrometry method to monitor formation of the cleaved sense-strand metabolites in a reconstituted system.

Introducing an In Vitro Liver Stability Assay Capable of Predicting the In Vivo Pharmacodynamic Efficacy of siRNAs for IVIVC.

There has been a renewed interest in therapeutic small interfering RNAs (siRNAs) over the past few years. This is particularly the result of successful and efficient delivery of N-acetylgalactosamine (GalNAc)-conjugated siRNAs to the liver. In general, the lead selection process for siRNA drugs is faster and more straightforward than traditional small molecules.

Metabolite Profiling of the Antisense Oligonucleotide Eluforsen Using Liquid Chromatography-Mass Spectrometry.

Eluforsen (previously known as QR-010) is a 33-mer 2'-O-methyl modified phosphorothioate antisense oligonucleotide targeting the F508del mutation in the gene encoding CFTR protein of cystic fibrosis patients. In this study, eluforsen was incubated with endo- and exonucleases and mouse liver homogenates to elucidate its in vitro metabolism. Mice and monkeys were used to determine in vivo liver and lung metabolism of eluforsen following inhalation.

Plasma and Liver Protein Binding of -Acetylgalactosamine-Conjugated Small Interfering RNA.

Understanding small interfering RNA (siRNA) fraction unbound ( ) in relevant physiologic compartments is critical for establishing pharmacokinetic-pharmacodynamic relationships for this emerging modality. In our attempts to isolate the equilibrium free fraction of -acetylgalactosamine-conjugated siRNA using classic small-molecule in vitro techniques, we found that the hydrodynamic radius was critical in determining the size exclusion limit requirements for isolation, largely validating the siRNA "rigid rod" hypothesis. With this knowledge, we developed an orthogonally validated 50 kDa molecular-mass cutoff ultrafiltration assay to quantify in biologic matrices including human, nonhuman primate, rat, and mouse plasma, and human liver homogenate.

Application of Locked Nucleic Acid Oligonucleotides for siRNA Preclinical Bioanalytics.

Despite the exquisite potential of siRNA as a therapeutic, the mechanism(s) responsible for the robust indirect exposure-response relationships have not been fully elucidated. To understand the siRNA properties linked to potent activity, requires the disposition of siRNA to be characterized. A technical challenge in the characterization is the detection and quantitation of siRNA from biological samples.

Direct identification of microribonucleic acid miR-451 from plasma using liquid chromatography mass spectrometry.

In recent years, small endogenous RNAs have come to the forefront of both basic and translational research. For example, many studies have pointed to the potential role of microRNAs (miRNAs) as disease biomarkers. However, precise quantitative methods for the analysis of miRNAs are still lacking.

Assessing the Interplay between the Physicochemical Parameters of Ion-Pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides.

Alkylamines are widely used as ion-pairing agents during LC-MS of oligonucleotides. In addition to a better chromatographic separation, they also assist with the desorption of oligonucleotide ions into the gas phase, cause charge state reduction, and decrease cation adduction. However, the choice of such ion-pairing agents has considerable influence on the MS signal intensity of oligonucleotides as they can also cause significant ion suppression.

The Role of Fluorinated Alcohols as Mobile Phase Modifiers for LC-MS Analysis of Oligonucleotides.

Hexafluoroisopropanol (HFIP) has been widely used as an acidic modifier for mobile phases for liquid chromatography-mass spectrometry (LC-MS) analysis of oligonucleotides ever since the first report of its use for this purpose. This is not surprising, considering the exceptional performance of HFIP compared with carboxylic acids, which cause significant MS signal suppression in electrospray ionization. However, we have found that other fluorinated alcohols can also be utilized for mobile phase preparation and the choice of optimal fluorinated alcohol is determined by the ion-pairing (IP) agent.

Development of a surrogate matrix for cerebral spinal fluid for liquid chromatography/mass spectrometry based analytical methods.

Rationale: In recent years, several liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods have been reported for the quantitative determination of drugs and metabolites in cerebral spinal fluid (CSF). Artificial CSF (aCSF) is often used as a surrogate for preparing calibration curves and quality control samples in these methods. However, aCSF does not accurately represent the composition of real CSF because it is missing all of the proteins and lipids, which may alter the electrospray ionization (ESI) response when performing LC/MS/MS analyses.

Ion pair liquid chromatography method for the determination of thiamine (vitamin B1) homeostasis.

A new method for reversed phase HPLC determination of thiamine and its major in vivo phosphorylation products, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP), was developed using tetrabutylammonium hydroxide as the ion-pairing agent. The separation was performed on a Phenomenex Kinetex EVO C18 column with a gradient of a phosphate-buffered aqueous solution of the ion-pair reagent and methanol. The duty cycle for the assay was 13 min and pyrithiamine was successfully used as the internal standard for the first time in a thiamine HPLC measurement protocol.

LC-MS of oligonucleotides: applications in biomedical research.

Recent findings have elucidated numerous novel biological functions for oligonucleotides. Current standard methods for the study of oligonucleotides (i.e.