Discovery and implementation of transcriptional biomarkers of synthetic LXR agonists in peripheral blood cells

<p>Abstract</p> <p>Background</p> <p>LXRs (Liver X Receptor α and β) are nuclear receptors that act as ligand-activated transcription factors. LXR activation causes upregulation of genes involved in reverse cholesterol transport (RCT), including ABCA1 and ABCG1 transporters, in macrophage and intestine. Anti-atherosclerotic effects of synthetic LXR agonists in murine models suggest clinical utility for such compounds.</p> <p>Objective</p> <p>Blood markers of LXR agonist exposure/activity were sought to support clinical development of novel synthetic LXR modulators.</p> <p>Methods</p> <p>Transcript levels of LXR target genes ABCA1 and ABCG1 were measured using quantitative reverse transcriptase/polymerase chain reaction assays (qRT-PCR) in peripheral blood from mice and rats (following a single oral dose) and monkeys (following 7 daily oral doses) of synthetic LXR agonists. LXRα, LXRβ, ABCA1, and ABCG1 mRNA were measured by qRT-PCR in human peripheral blood mononuclear cells (PBMC), monocytes, T- and B-cells treated <it>ex vivo </it>with WAY-252623 (LXR-623), and protein levels in human PBMC were measured by Western blotting. ABCA1/G1 transcript levels in whole-blood RNA were measured using analytically validated assays in human subjects participating in a Phase 1 SAD (Single Ascending Dose) clinical study of LXR-623.</p> <p>Results</p> <p>A single oral dose of LXR agonists induced ABCA1 and ABCG1 transcription in rodent peripheral blood in a dose- and time-dependent manner. Induction of gene expression in rat peripheral blood correlated with spleen expression, suggesting LXR gene regulation in blood has the potential to function as a marker of tissue gene regulation. Transcriptional response to LXR agonist was confirmed in primates, where peripheral blood ABCA1 and ABCG1 levels increased in a dose-dependent manner following oral treatment with LXR-623. Human PBMC, monocytes, T- and B cells all expressed both LXRα and LXRβ, and all cell types significantly increased ABCA1 and ABCG1 expression upon <it>ex vivo </it>LXR-623 treatment. Peripheral blood from a representative human subject receiving a single oral dose of LXR-623 showed significant time-dependent increases in ABCA1 and ABCG1 transcription.</p> <p>Conclusion</p> <p>Peripheral blood cells express LXRα and LXRβ, and respond to LXR agonist treatment by time- and dose-dependently inducing LXR target genes. Transcript levels of LXR target genes in peripheral blood are relevant and useful biological indicators for clinical development of synthetic LXR modulators.</p

Clinical and molecular characterisation of KCNT1-related severe early onset epilepsy

Objective: To characterise the phenotypic spectrum, molecular genetic findings and functional consequences of pathogenic variants in early onset KCNT1-epilepsy. Methods: We identified a cohort of 31 patients with epilepsy of infancy with migrating focal seizures (EIMFS) and screened for variants in KCNT1 using direct Sanger sequencing, a multiple gene next generation sequencing panel and whole exome sequencing. Additional patients with non-EIMFS early onset epilepsy in whom we identified KCNT1 variants on local diagnostic multiple gene panel testing were also included. Where possible, we performed homology modelling to predict putative effects of variants on protein structure and function. We undertook electrophysiological assessment of mutant KCNT1 channels in a Xenopus oocyte model system. Results: We identified pathogenic variants in KCNT1 in 12 patients, four of which are novel. Most variants occurred de novo. Ten had a clinical diagnosis of EIMFS and the other two presented with early onset severe nocturnal frontal lobe seizures. Three patients had a trial of quinidine with good clinical response in one. Computational modelling analysis implicates abnormal pore function (F346L) and impaired tetramer formation (F502V) as putative disease mechanisms. All evaluated KCNT1 variants resulted in marked gain-of-function, with significantly increased channel amplitude and variable blockade by quinidine. Conclusions: Gain-of-function KCNT1 pathogenic variants cause a spectrum of severe focal epilepsies with onset in early infancy. Currently, genotype-phenotype correlations are unclear, though clinical outcome is poor for the majority of cases. Further elucidation of disease mechanisms may facilitate the development of targeted treatments, much needed for this pharmacoresistant genetic epilepsy

Direct Delivery of a Cytotoxic Anticancer Agent into the Metastatic Lymph Node Using Nano/Microbubbles and Ultrasound

<div><p>Direct injection of an anticancer agent into a metastatic lymph node (LN) has not been used as a standard treatment because evidence concerning the efficacy of local administration of a drug into a metastatic LN has not been established. Here we show that the combination of intralymphatic drug delivery with nano/microbubbles (NMBs) and ultrasound has the potential to improve the chemotherapeutic effect. We delivered <i>cis</i>-diamminedichloroplatinum (II) (CDDP) into breast carcinoma cells <i>in vitro</i> and found that apoptotic processes were involved in the antitumor action. Next, we investigated the antitumor effect of intralymphatic chemotherapy with NMBs and ultrasound in an experimental model of LN metastasis using MXH10/Mo-<i>lpr</i>/<i>lpr</i> mice exhibiting lymphadenopathy. The combination of intralymphatic chemotherapy with NMBs and ultrasound has the potential to improve the delivery of CDDP into target LNs without damage to the surrounding normal tissues. The present study indicates that intralymphatic drug delivery with NMBs and ultrasound will potentially be of great benefit in the clinical setting.</p></div

Histopathology analysis.

<p>(A) Representative sections stained with HE are shown for (Aa) control, (Ab) US + ALs, (Ac) CDDP + ALs, and (Ad) CDDP + ALs + US groups. Panels (Ae) and (Af) show higher magnification views of the regions highlighted in panels (Aa) (white dotted square) and (Ad) (black dotted square), respectively. CD31-immunostained sections are shown for the control (Ag) and CDDP + ALs + US (Ah) groups. Black dotted lines indicate carcinomatous lesions and white arrows indicate necrotic lesions. Black triangles indicate the border between normal tissue and necrotic areas. In the control and US + ALs groups, widespread cancerous lesions can be seen (Aa, b), while smaller lesions were evident in sections from the CDDP + ALs group (Ac). In sections from the CDDP + ALs + US group, carcinomatous lesions could not be detected, but necrotic lesions were observed (Ad, f). In the control group, CD31-positive vasculature was distributed over the entire lesion (Ag), whereas in the CDDP + ALs + US group CD31-positive vasculature was rarely observed in necrotic lesions (Ah). (B) Quantification of macrovessel and microvessel densities of tumor-bearing SiLNs. No significant differences in macrovessel and microvessel densities were detected between any of the experimental groups.</p

Evaluation of the treatment efficacy of delivery of CDDP with ALs and US.

<p>(A) <i>In vivo</i> bioluminescence imaging on day 10. (B) Longitudinal analysis of normalized luciferase activity (<i>n</i> = 4 for each group). Values at different time points were normalized to those on day 3. Black arrows indicate the days on which treatment was administered (days 3, 4 and 7). On day 10, normalized luciferase activity in the CDDP + ALs + US group was significantly lower than that in the control group (<i>P</i> < 0.05) and showed a trend towards being lower than that in the US + ALs or CDDP + ALs group. Mean ± SEM values are shown. *<i>P</i> < 0.05.</p

Blood vessel extraction images of tumor-bearing SiLNs.

<p>(A) Representative vascular extraction images for each group, taken from the widest cross-section of the tumor-bearing SiLN. The white circles indicate the boundaries of each SiLN. In the control, US + ALs and CDDP + ALs groups, no obvious changes in vessel structures were observed between days 0 and 9. In the CDDP + ALs + US group, the proportion of the tumor-bearing SiLN colored green in the vascular extraction image was lower on day 9 than on day 0. (B) Normalized blood vessel densities of tumor-bearing SiLNs (<i>n</i> = 4 for each group). Values at different time points were normalized to those on day 0. There was a trend for normalized blood vessel density on day 9 to be lower in the CDDP + ALs + US group than in the other 3 groups, although there were no significant differences between any of the groups. Mean ± SEM values are shown. Scale bars represent 2 mm.</p