Global Transcriptomic Profiling Using Small Volumes of Whole Blood: A Cost-Effective Method for Translational Genomic Biomarker Identification in Small Animals

Blood is an ideal tissue for the identification of novel genomic biomarkers for toxicity or efficacy. However, using blood for transcriptomic profiling presents significant technical challenges due to the transcriptomic changes induced by ex vivo handling and the interference of highly abundant globin mRNA. Most whole blood RNA stabilization and isolation methods also require significant volumes of blood, limiting their effective use in small animal species, such as rodents. To overcome these challenges, a QIAzol-based RNA stabilization and isolation method (QSI) was developed to isolate sufficient amounts of high quality total RNA from 25 to 500 μL of rat whole blood. The method was compared to the standard PAXgene Blood RNA System using blood collected from rats exposed to saline or lipopolysaccharide (LPS). The QSI method yielded an average of 54 ng total RNA per μL of rat whole blood with an average RNA Integrity Number (RIN) of 9, a performance comparable with the standard PAXgene method. Total RNA samples were further processed using the NuGEN Ovation Whole Blood Solution system and cDNA was hybridized to Affymetrix Rat Genome 230 2.0 Arrays. The microarray QC parameters using RNA isolated with the QSI method were within the acceptable range for microarray analysis. The transcriptomic profiles were highly correlated with those using RNA isolated with the PAXgene method and were consistent with expected LPS-induced inflammatory responses. The present study demonstrated that the QSI method coupled with NuGEN Ovation Whole Blood Solution system is cost-effective and particularly suitable for transcriptomic profiling of minimal volumes of whole blood, typical of those obtained with small animal species

Substituted Indazoles as Na_v1.7 Blockers for the Treatment of Pain

The genetic validation for the role of the Na_v1.7 voltage-gated ion channel in pain signaling pathways makes it an appealing target for the potential development of new pain drugs. The utility of nonselective Na_v blockers is often limited due to adverse cardiovascular and CNS side effects. We sought more selective Na_v1.7 blockers with oral activity, improved selectivity, and good druglike properties. The work described herein focused on a series of 3- and 4-substituted indazoles. SAR studies of 3-substituted indazoles yielded analog <b>7</b> which demonstrated good in vitro and in vivo activity but poor rat pharmacokinetics. Optimization of 4-substituted indazoles yielded two compounds, <b>27</b> and <b>48</b>, that exhibited good in vitro and in vivo activity with improved rat pharmacokinetic profiles. Both <b>27</b> and <b>48</b> demonstrated robust activity in the acute rat monoiodoacetate-induced osteoarthritis model of pain, and subchronic dosing of <b>48</b> showed a shift to a lower EC₅₀ over 7 days

Substituted Indazoles as Na_v1.7 Blockers for the Treatment of Pain

The genetic validation for the role of the Na_v1.7 voltage-gated ion channel in pain signaling pathways makes it an appealing target for the potential development of new pain drugs. The utility of nonselective Na_v blockers is often limited due to adverse cardiovascular and CNS side effects. We sought more selective Na_v1.7 blockers with oral activity, improved selectivity, and good druglike properties. The work described herein focused on a series of 3- and 4-substituted indazoles. SAR studies of 3-substituted indazoles yielded analog <b>7</b> which demonstrated good in vitro and in vivo activity but poor rat pharmacokinetics. Optimization of 4-substituted indazoles yielded two compounds, <b>27</b> and <b>48</b>, that exhibited good in vitro and in vivo activity with improved rat pharmacokinetic profiles. Both <b>27</b> and <b>48</b> demonstrated robust activity in the acute rat monoiodoacetate-induced osteoarthritis model of pain, and subchronic dosing of <b>48</b> showed a shift to a lower EC₅₀ over 7 days