Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense.

Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.

Case studies addressing human pharmacokinetic uncertainty using a combination of pharmacokinetic simulation and alternative first in human paradigms.

PF-184298 ((S)-2,3-dichloro-N-isobutyl-N-pyrrolidin-3-ylbenzamide) and PF-4776548 ((3-(4-fluoro-2-methoxy-benzyl)-7-hydroxy-8,9-dihydro-3H,7H-pyrrolo[2,3-c][1,7]naphthyridin-6-one)) are novel compounds which were selected to progress to human studies. Discordant human pharmacokinetic predictions arose from pre-clinical in vivo studies in rat and dog, and from human in vitro studies, resulting in a clearance prediction range of 3 to >20 mL min⁻¹ kg⁻¹ for PF-184298, and 5 to >20 mL min⁻¹ kg⁻¹ for PF-4776548. A package of work to investigate the discordance for PF-184298 is described. Although ultimately complementary to the human pharmacokinetic data in characterising the disposition of PF-184298 in humans, these data did not provide any further confidence in pharmacokinetic prediction. A fit for purpose human pharmacokinetic study was conducted for each compound, with an oral pharmacologically active dose for PF-184298, and an intravenous and oral microdose for PF-4776548. This provided a relatively low cost, clear decision making approach, resulting in the termination of PF-4776548 and further progression of PF-184298. A retrospective analysis of the data showed that, if the tools had been available at the time, the pharmacokinetics of PF-184298 in human could have been predicted from a population based simulation tool in combination with physicochemical properties and in vitro human intrinsic clearance.

Selection, optimization, and pharmacokinetic properties of a novel, potent antiviral locked nucleic acid-based antisense oligomer targeting hepatitis C virus internal ribosome entry site.

We have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC(50)]) and cytotoxic (50% cytotoxic concentration [CC(50)]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC(50) = 4 nM) and low cytotoxicity (CC(50) >880 nM) targeting the 25- to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25- to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25- to 40-nt region have no antiviral effect, revealing important regulatory features of the 25- to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required for in vitro antiviral activity.

Part 3: Design and synthesis of proline-derived α2δ ligands.

A potent series of substituted (2S,4S)-benzylproline α(2)δ ligands have been designed from the readily available starting material (2S,4R)-hydroxy-L-proline. The ligands have improved pharmacokinetic profile over the (4S)-phenoxyproline derivatives described previously and have potential for development as oral agents for the treatment of neuropathic pain. Compound 16 has been progressed to clinical development.

Part 2: Design, synthesis and evaluation of hydroxyproline-derived α2δ ligands.

Conformational constraint has been used to design a potent series of α(2)δ ligands derived from the readily available starting material (2S,4R)-hydroxy-l-proline. The ligands have improved physicochemistry and potency compared to their linear counterparts (described in our earlier publication) and the lead compound has been progressed to clinical development.

Retention-directed and selectivity controlled chromatographic resolution: Rapid post-hoc analysis of DMPK samples to achieve high-throughput LC-MS separation.

High quality chromatographic separation underpins robustness in LC-MS, frequently the analytical method of choice for pharmaceutical drug discovery work. The potential improvements in chromatographic selectivity afforded by serial column coupling (SCC), provide a useful means to enhance the resolution of complex samples. In this work, we present a revised high-throughput form of SCC, in which just two individual mixed phase columns were coupled together and combined with a gradient-optimised, retention-directed ultra-high pressure method to achieve rapid separations, with no further method optimisation necessary. The overall performance was evaluated from an open access DMPK analytical working environment perspective; where in anticipation of bioanalytical or metabolite identification chromatography challenges, or with the knowledge that stronger resolution was required for in-vitro sample analysis, the methodology could be immediately implemented by the analyst. Retention-directed selection of a shallow SCC gradient method was successful in separating peaks throughout the chromatographic window, resulting in a runtime still congruent to high-throughput analyses (3.5 min). In-vitro assay sample interferences were resolved 44-72% of the time, and the overall resolving power for isomeric separations significantly improved against single column comparisons (1.7-fold mean RS improvement). Over a sustained period of time in our laboratory, SCC methods have been used for metabolite identification and bioanalytical samples, where both convenience and effectiveness in solving analytical challenges has been consistently demonstrated. Examples that highlight SCC chromatography, and a guided discussion of the main high-throughput considerations, are included. The technique offers wide applicability, and we would recommend it as a toolbox consideration to the laboratory analyst.

Sensitive quantitation of reboxetine enantiomers in rat plasma and brain, using an optimised reverse phase chiral LC-MS/MS method.

A sensitive liquid chromatography-mass spectrometry (LC-MS) method has been developed for stereoselective determination of reboxetine in rat plasma and brain homogenate (LLOQ, 50 pg/ml). The method optimised ionisation efficiency with an electro-ionspray source, by adjusting the composite flow conditions (rate, pH, organic content) from column eluent and post-column organic modifier. LC conditions utilized a chiral AGP column (5 microm) with 12.5 mM ammonium carbonate buffer adjusted with formic acid (pH 6.7) and included a wash step (0.05% acetic acid in water) to maintain assay robustness and chromatographic performance. The total method cycle time was 23 min. Imprecision (R.S.D.) was below 10% and inaccuracy (% error) below 7% for both enantiomers in plasma and brain homogenate, over a 2000-fold dynamic range (0.05-100 ng/ml). An automated liquid-liquid extraction technique was used (borate buffer, pH 10/tert-butyl methyl ether) and the matrix type used produced no difference in the assay performance. The method was successfully applied to determine the pharmacokinetic profiles of S,S- and R,R-reboxetine in rats, following subcutaneous administration of racemate drug.

Development of a micro-turbulent flow chromatography focus mode method for drug quantitation in discovery bioanalysis.

An online turbulent flow chromatography method coupled to tandem mass spectrometry (TFC-MS/MS) has been developed within our bioanalytical group, suited to the analysis of mid to late stage discovery compounds. A dual column configuration utilising isocratic focusing of the analyte upon the analytical column maintained an excellent peak shape for a large proportion of compounds encountered and enabled consistent quantitation to sub-nanogram concentrations (<15 pg on column). Furthermore, the low sample injection volume coupled with rapid column washing using basic and acidic mobile phases, has proved advantageous in removing sample carryover and also the overall exposure to biological material; favourable for good system robustness. All the data discussed were generated with a method cycle time of 5 min providing accurate quantitation (acceptance criteria based upon FDA method validation guidelines) with multiple analytes and biological matrices.

Bioanalysis of Pseudomonas aeruginosa alkyl quinolone signalling molecules in infected mouse tissue using LC-MS/MS; and its application to a pharmacodynamic evaluation of MvfR inhibition.

Alkyl quinolone molecules 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) are important quorum sensing signals, which play a mediatory role in the pathogenesis of acute and chronic Pseudomonas aeruginosa infection. A targeted approach inhibiting the bacterial 'multiple virulence factor regulon' (MvfR) protein complex, offers the possibility to block the synthesis of MvfR-dependant signal molecules. Here, a high throughput bioanalytical method was developed using LC-MS/MS detection for the selective determination of HHQ and PQS in mouse tissue homogenate, over a sensitive range of 1-5000 and 10-5000pg/mL, respectively. Chromatographic peak distortion of the iron chelator PQS was overcome with the applied use of a bidentate chelator mobile phase additive 2-Picolinic acid at 0.2mM concentration, giving an improved separation and response for the analyte, whilst maintaining overall MS system robustness. Following thigh infection with P. aeruginosa strain 2-PA14 in mice, the concentration and time course of HHQ and PQS (4-hydroxy-2-alkyl-quinolone (HAQ) biomarkers) residing in the biophase were evaluated, and exhibited a low level combined with a substantial inter-individual variability. Quantifiable levels could be obtained from approximately 15h post infection, to the study termination at 21-22h. A dose dependant reduction in HAQ tissue concentrations at selected time points were obtained following MvfR inhibitor administration versus drug vehicle (p<0.01, Kruskal-Wallis-one way ANOVA) and meta -analyses of several studies enabled an inhibitory concentration (IC50) of 80nM free drug to be determined. However, due to the experimental limitations a defined time profile for in-vivo HAQ production could not be characterised. Microsomal stability measurements demonstrated a rapid metabolic clearance of both alkyl quinolone biomarkers in the bacterial host, with a hepatic extraction ratio greater than 0.96 (the measurable assay limit). High clearance underpinned the low concentrations present in the well-perfused thigh tissue. Along with method development and validation details, this paper considers the kinetics of in-vivo HAQ bio-synthesis during Pseudomonas infection; and risks of biomarker over-estimation from samples which contain an exogenous population of bacteria.

Quantitation of locked nucleic acid antisense oligonucleotides in mouse tissue using a liquid-liquid extraction LC-MS/MS analytical approach.

BACKGROUND: A significant challenge of oligonucleotide bioanalysis is the selective extraction from complex tissue samples, where the molecules that distribute into the intracellular space are extensively protein bound and sit amongst a high concentration of endogenous nucleic acid material. Published analytical methodology currently purports extensive sample preparation requirements that include cell lysis steps, homogenization and dual cleanup with liquid-liquid extraction and solid-phase extraction, prior to injection. RESULTS: We have developed a simple liquid-liquid extraction approach to rapidly isolate antisense oligonucleotides from biological tissues with high recovery and combined these preparative steps with a robust monolithic column LC-MS/MS setup. The platform showed improved chromatographic resolution and detection sensitivity over standard reversed-phase columns and required a low sample volume. CONCLUSION: The high-throughput method was sufficient to accurately quantify multiple antisense oligonucleotides in mouse tissue and plasma down to low ng/g and ng/ml levels, respectively, for pharmacokinetic determination, and exhibited a high degree of specificity.