Pharmacological Characterization of a Novel ENaCα siRNA (GSK2225745) With Potential for the Treatment of Cystic Fibrosis.

Lung pathology in cystic fibrosis is linked to dehydration of the airways epithelial surface which in part results from inappropriately raised sodium reabsorption through the epithelial sodium channel (ENaC). To identify a small-interfering RNA (siRNA) which selectively inhibits ENaC expression, chemically modified 21-mer siRNAs targeting human ENaCα were designed and screened. GSK2225745, was identified as a potent inhibitor of ENaCα mRNA (EC(50) (half maximal effective concentration) = 0.4 nmol/l, maximum knockdown = 85%) and protein levels in A549 cells. Engagement of the RNA interference (RNAi) pathway was confirmed using 5' RACE. Further profiling was carried out in therapeutically relevant human primary cells. In bronchial epithelial cells, GSK2225745 elicited potent suppression of ENaCα mRNA (EC(50) = 1.6 nmol/l, maximum knockdown = 82%). In human nasal epithelial cells, GSK2225745 also produced potent and long-lasting (≥72 hours) suppression of ENaCα mRNA levels which was associated with significant inhibition of ENaC function (69% inhibition of amiloride-sensitive current in cells treated with GSK2225745 at 10 nmol/l). GSK2225745 showed no evidence for potential to stimulate toll-like receptor (TLR)3, 7 or 8. In vivo, topical delivery of GSK2225745 in a lipid nanoparticle formulation to the airways of mice resulted in significant inhibition of the expression of ENaCα in the lungs. In conclusion, GSK2225745 is a potent inhibitor of ENaCα expression and warrants further evaluation as a potential novel inhaled therapeutic for cystic fibrosis.Molecular Therapy - Nucleic Acids (2013) 2, e65; doi:10.1038/mtna.2012.57; published online 15 January 2013.

Combining ion pairing agents for enhanced analysis of oligonucleotide therapeutics by reversed phase-ion pairing ultra performance liquid chromatography (UPLC).

The burgeoning field of oligonucleotide therapeutics is based upon synthetically derived biopolymers comprised of relatively simple RNA and DNA building blocks. Significant gains in knowledge around mechanisms of action (RNA interference, RNA splicing, etc.) and oligonucleotide design (ASO, siRNA, DsiRNA, miRNA, locked nucleic acid, etc.) have been the main drivers of recent investment for this field [1,2]. As therapeutics, there is currently great interest in oligonucleotides due to the reduced time required to achieve lead molecules and to their potential for treating previously untractable diseases. One of the more challenging areas for the field of oligonucleotide therapeutics is the development of high-quality analysis schemes for the determination of purity in drug substance and product. This, in part, is due to the fact that the synthesis of oligonucleotides results in a significant number of closely related impurities that are not easily removed during purification [1]. As a result, these macromolecules (4000-8000 MW on average, depending on chain length) and their soup of closely related impurities are typically not well resolved from one another via conventional chromatographic approaches. One of the more common chromatographic techniques used for oligonucleotide analysis is reversed phase-ion pairing liquid chromatography (RP-IP). Our research led us to the discovery that the use of multiple ion pairing agents combined in the mobile phase can improve the overall chromatographic resolution and peak shape of the oligonucleotide analytes over the use of a single ion pairing agent alone, resulting in enhanced purity analysis and the opportunity to identify related impurities with greater certainty. In addition, the use of combined ion pairing agents allowed for the development of a "universal" method which has provided superior chromatography for several different oligonucleotide compounds and their related impurities regardless of differences in nucleotide sequence. The RP-IP UPLC method conditions are ESI-MS compatible and have allowed for the mass identification of five positional isomeric impurities chromatographically resolved and present at less than 1% of the nominal parent peak area.

Chemistry at the 2' position of constituent nucleotides controls degradation pathways of highly modified oligonucleotide molecules.

A forced degradation study of a proprietary short interfering RNA (siRNA) molecule most of whose constituent nucleotides have been modified at the 2' position was conducted to assess degradation pathways and stability liabilities. The siRNA was subjected to various conditions as a solid and in solution followed by analysis with reverse-phase ultra-performance liquid chromatography-mass spectrometry. Positional isomers of degradants gave rise to multiple chromatographic peaks with identical masses. In some instances, the exact location of a modification was elucidated, but in most cases although the identity of the nucleotide affected was proposed with a high degree of confidence, its position within the oligonucleotide sequence was not determined. Reaction mechanisms were proposed for all observed major degradants based on reverse-phase ultra-performance liquid chromatography-mass spectrometry data generated in this laboratory and a search of literature sources. This work demonstrates that the chemistry at the 2' position of constituent nucleotides controls degradation pathways of highly modified siRNA molecules under various conditions and that classes of degradants can be predicted with a fair amount of confidence. A table of mass differences is presented that can be used as an aid to making partial structural assignments in oligonucleotide molecules containing similarly modified nucleotides.

Using a nanoelectrospray-differential mobility spectrometer-mass spectrometer system for the analysis of oligosaccharides with solvent selected control over ESI aggregate ion formation.

Differential mobility spectrometry (DMS), also commonly referred to as high field asymmetric waveform ion mobility spectrometry (FAIMS) is a rapidly advancing technology for gas-phase ion separation. The interfacing of DMS with mass spectrometry (MS) offers potential advantages over the use of mass spectrometry alone. Such advantages include improvements to mass spectral signal/noise, orthogonal/complementary ion separation to mass spectrometry, enhanced ion and complexation structural analysis, and the potential for rapid analyte quantitation. In this report, we demonstrate the successful use of our nanoESI-DMS-MS system, with a methanol drift gas modifier, for the separation of oligosaccharides. The tendency for ESI to form oligosaccharide aggregate ions and the negative impact this has on nanoESI-DMS-MS oligosaccharide analysis is described. In addition, we demonstrate the importance of sample solvent selection for controlling nanoESI oligosaccharide aggregate ion formation and its effect on glycan ionization and DMS separation. The successful use of a tetrachloroethane/methanol solvent solution to reduce ESI oligosaccharide aggregate ion formation while efficiently forming a dominant MH(+) molecular ion is presented. By reducing aggregate ion formation in favor of a dominant MH(+) ion, DMS selectivity and specificity is improved. In addition to DMS, we would expect the reduction in aggregate ion complexity to be beneficial to the analysis of oligosaccharides for other post-ESI separation techniques such as mass spectrometry and ion mobility. The solvent selected control over MH(+) molecular ion formation, offered by the use of the tetrachloroethane/methanol solvent, also holds promise for enhancing MS/MS structural characterization analysis of glycans.

Rapid separation and quantitative analysis of peptides using a new nanoelectrospray- differential mobility spectrometer-mass spectrometer system.

Differential mobility spectrometry (DMS) (see Buryakov, I. A.; Krylov, E. V.; Nazarov, E. G.; Rasulev, U. Kh. Int. J. Mass Spectrom. Ion Processes 1993, 128, 143-148), also commonly referred to as high-field asymmetric waveform ion mobility spectrometry (FAIMS) (see Purves, R. W.; Guevremont, R.; Day, S.; Pipich, C. W.; Matyjaszcyk, M. S. Rev. Sci. Instrum. 1998, 69, 4094-4105), is a rapidly advancing technology for gas-phase ion separation. The interfacing of DMS with mass spectrometry (MS) offers potential advantages over the use of mass spectrometry alone. Such advantages include improvements to mass spectral signal-to-noise, orthogonal/complementary ion separation to mass spectrometry, enhanced ion and complexation structural analysis, and the potential for rapid analyte quantitation. In this report, we investigate the use of our nanoESI-DMS-MS system to demonstrate differential mobility separation of peptides. The formation of higher order peptide aggregate ions (ion complexes) via electrospray ionization and the negative impact this has on DMS peptide separation are examined. The successful use of differential mobility drift gas modifiers (dopants) to reduce aggregate ion size and improve DMS peptide ion separation is presented. Following optimization of DMS peptide separation conditions, we examined next the feasibility of a new analytical platform which uses direct sample infusion with nanoESI-DMS-MS for ultrarapid analyte quantitation. Quantitation of a selected peptide from a semicomplex peptide mixture is presented. Initial feasibility results with this new approach demonstrate good accuracy and reproducibility, as well as an absolute mass sensitivity of 6.8 amol and a minimum dynamic range of 2500 for the peptide of interest. This report offers a first look at utilizing nanoESI-DMS-MS to create an ultrarapid (under 5 s) quantitative analysis platform and its potential in the high-throughput arena. Each ion separation technique, DMS and MS, offers orthogonal ion separation to one another, enhancing the overall specificity for this quantitative approach.

Characterization of gas-phase molecular interactions on differential mobility ion behavior utilizing an electrospray ionization-differential mobility-mass spectrometer system.

Differential mobility spectrometry (DMS) is a rapidly advancing technology for gas-phase ion separation. The interfacing of DMS with mass spectrometry (MS) offers potential advantages over the use of mass spectrometry alone. Such advantages include improvements to mass spectral signal/noise ratios, orthogonal/complementary ion separation to mass spectrometry, enhanced ion and complexation structural analysis, and potential for rapid analyte quantitation. The introduction of a new ESI-DMS-MS system and its utilization to aid in the understanding of DMS separation theory is described. A current contribution to DMS separation theory is one of an association/dissociation process between ions/molecules in the gas phase during the differential mobility separation. A model study was designed to investigate the molecular dynamics and chemical factors influencing the theorized association/dissociation process, and the mechanisms by which these gas-phase interactions affect an ion's DM behavior. Five piperidine analogues were selected as model analytes, and three alcohol drift gas dopants/modifiers were used to interrogate the analyte ions in the gas phase. Two proposed DMS separation mechanisms, introduced as Core and Façade, corresponding to strong and weak attractions between ions/molecules in the gas phase, are detailed. The proposed mechanisms provide explanation for the observed changes in analyte separation by the various drift gas modifiers. Molecular modeling of the proposed mechanisms provides supportive data and demonstrates the potential for predictive optimization of analyte separation based on drift gas modifier effects.