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. In the absence of metabolite standards for quantitation, we apply an ionization efficiency correction across a panel of siRNA molecules and find that it improves in vitro-in vivo correlation in a transgenic mouse model. Finally, we lay out a case for why Michaelis-Menten kinetics will likely be inadequate for describing Ago2-mediated metabolic activation kinetics, and propose several alternative models that can be solved numerically and applied to quantitated kinetic data when it becomes available.

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. Nevertheless, many siRNAs of different sequences and chemical modification patterns must still be evaluated before arriving at a final candidate. One of the major difficulties in streamlining this workflow is the well-known phenomenon that the in vitro data obtained from oligonucleotides transfected into cells are not directly predictive of their in vivo activity. Consequently, all oligonucleotides with some degree of in vitro activity are typically screened in vivo before final lead selection. Here, we demonstrate that the stability of liver-targeting GalNAc-conjugated siRNAs in a mouse liver homogenate shows an acceptable correlation to their in vivo target knockdown efficacy. Therefore, we suggest the incorporation of an in vitro liver homogenate stability assay during the lead optimization process for siRNAs. The addition of this assay to a flow scheme may decrease the need for animal studies, and it could bring cost savings and increase efficiency in siRNA drug development.

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. We developed a liquid chromatography-mass spectrometry method for the identification and semi-quantitation of the metabolites of eluforsen and then applied the method for in vitro and in vivo metabolism studies. Solid-phase extraction was used following proteinase K digestion for sample preparation. Chain-shortened metabolites of eluforsen by 3' exonuclease were observed in mouse liver in an in vitro incubation system and by either 3' exonuclease or 5' exonuclease in liver and lung samples from an in vivo mouse and monkey study. This study provides approaches for further metabolite characterization of 2'-ribose-modified phosphorothioate oligonucleotides in in vitro and in vivo studies to support the development of oligonucleotide therapeutics.

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

Understanding small interfering RNA (siRNA) fraction unbound (f u) 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 N-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 f u 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 f u in biologic matrices including human, nonhuman primate, rat, and mouse plasma, and human liver homogenate. To enhance understanding of the siRNA-plasma interaction landscape, we examined the effects of various common oligonucleotide therapeutic modifications to the ribose and helix backbone on siRNA f u in plasma (f u,plasma) and found that chemical modifications can alter plasma protein binding by at least 20%. Finally, to gain insight into which specific plasma proteins bind to siRNA, we developed a qualitative screen to identify binding "hits" across a panel of select purified human plasma proteins.

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. Described herein, a Locked Nucleic Acid (LNA) Hybridization-Ligation ECL ELISA was designed for ultra-sensitive quantification of both sense and antisense strands of siRNA independent of structural modifica-tions. This assay was applied to measure siRNA in serum and tissue homogenate in preclinical species. We observed rapid clearance of siRNA from the systemic circulation which contrasted the prolonged accumulation within the tissue. The assay was also able to distinguish and quantify free siRNA from RNA-induced silencing complex (RISC) and Argonaute 2 (Ago2) associated with therapeutic siRNA. We utilized an orthogonal method, LC-MS, to investigate 3' exonuclease activity toward the antisense strand metabolism. Taken together, we have demonstrated that the LNA Hybridization-Ligation ECL ELISA is arobust analytical method with direct application to measuring the exposure of siRNA therapeutics seamlessly across biological matrices.

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. In this study, we report the first mass spectrometry-based quantitation of miR-451, a circulatory microRNA. Using a highly selective sample preparation method with an average recovery of 83.6% and a novel mobile phase chemistry, we were able to reach an LOQ of 0.5 ng/mL. Because of such high sensitivity, we could detect and quantify the endogenous miR-451 from both human and rat plasma. Considering the increased precision of LC-MS compared to other methods, these results usher in a new era of miRNA biomarker discovery and validation.

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. Interestingly, optimal ion-pairing agents should be selected on a case by case basis as their choice is strongly influenced by the sequence of the oligonucleotide under investigation. Despite imposing major practical difficulties to analytical method development, such a highly variable system that responds very strongly to the nuances of the electrospray composition provides an excellent opportunity for a fundamental study of the electrospray ionization process. Our investigations using this system quantitatively revealed the major factors that influenced the ESI ionization efficiency of oligonucleotides. Parameters such as boiling point, proton affinity, partition coefficient, water solubility, and Henry's law constants for the ion-pairing reagents and the hydrophobic thymine content of the oligonucleotides were found to be the most significant contributors. Identification of these parameters also allowed for the development of a statistical predictive algorithm that can assist with the choice of an optimum IP agent for each particular oligonucleotide sequence. We believe that research in the field of oligonucleotide bioanalysis will significantly benefit from this algorithm (included in Supplementary Material) as it advocates for the use of lesser-known but more suitable ion-pair alternatives to TEA for many oligonucleotide sequences. Graphical Abstract ᅟ.

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. Although HFIP is a very good choice to be used alongside less hydrophobic IP agents, other fluorinated alcohols such as 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFMIP) can significantly outperform HFIP when used with more hydrophobic IP agents. We also found that more acidic fluorinated alcohols assist with the transfer of oligonucleotides with secondary structure (e.g., folded strands and hairpins) into the gas phase. Graphical Abstract ᅟ.

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. In the current study we compared the mass spectral response of several compounds with a range of physiochemical properties in aCSF (essentially a mixture of salts and buffers), diluted plasma (ranging from 1:5 to 1:200) and real CSF to find the best surrogate for CSF in LC/MS/MS methods. METHODS: A number of analytes from polar to non-polar, high protein binding to low protein binding, employing different sample preparation methods, were prepared in diluted plasma, actual CSF or aCSF and tested using LC/MS/MS. The analytes included cotinine and its metabolites, quetiapine, norquetiapine, chlorpromazine, efavirenz and lamivudine. The similarity of MS responses from these compounds in aCSF and diluted plasma to CSF was assessed by comparing the slopes of the calibration curves generated from using linear regression modeling. RESULTS: For all compounds, the lowest percent difference in response ratio (0 to 17%) was observed from 1:200 diluted plasma. Our results indicated that, irrespective of the inherent physiochemical properties of the analytes or the method of sample preparation, 1:200 diluted plasma performed as the best surrogate for CSF in LC/MS/MS methods. CONCLUSIONS: The percent difference in response ratio has been established to demonstrate how different compounds behave between CSF, aCSF and dilute plasma. Although among the compounds tested some of them showed a very similar MS response in actual and aCSF, there were analytes that demonstrated significant differences in ESI-MS signal when sprayed from these two matrices. However, even in such cases, 1:200 diluted plasma generated results with no significant difference from CSF. Therefore, we recommend that in order to develop robust and dependable bioanalytical LC/MS methods from CSF samples, it is more appropriate to prepare calibration curves and quality control samples in diluted plasma instead of aCSF. Copyright © 2016 John Wiley & Sons, Ltd.

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. Detection of the fluorescence derivatives of the analytes as well as the IS allowed for lower detection limits in order to support biological applications in cell culture models. The linearity, sensitivity, specificity, accuracy and precision of the method were evaluated and met the requirements specified by the US Food and Drug Administration. The calibration curves proved to be linear and the method was validated over the range from 1.0-4000 nM for both cells and the media where complete recovery of the analytes was also achieved.

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., hybridization and PCR) are not fully equipped to deal with the experimental needs arising from these new discoveries. More importantly, as the intracellular capacity of oligonucleotides is being harnessed for biomedical applications, alternative bioanalytical techniques become indispensable in order to comply with ever-increasing regulatory requirements. Owing to its ability to detect oligonucleotides independent of their sequence, LC-MS is emerging as the analytical method of choice for oligonucleotides. In this article, the current applications of LC-MS in the analysis of oligonucleotides, with an emphasis on RNA therapeutics and biomarkers, will be examined. In addition, the theoretical framework of oligonucleotide ESI is carefully inspected with the purpose of identifying the contributing factors to MS signal intensity.