CNS Adverse Effects: From Functional Observation Battery/Irwin Tests to Electrophysiology.

This chapter describes various approaches for the preclinical assessment of drug-induced central nervous system (CNS) adverse effects. Traditionally, methods to evaluate CNS effects have consisted of observing and scoring behavioral responses of animals after drug is administered. Among several behavioral testing paradigms, the Irwin and the functional observational battery (FOB) are the most commonly used assays for the assessment of CNS effects. The Irwin and FOB are considered good first-tier assays to satisfy the ICH S7A guidance for the preclinical evaluation of new chemical entities (NCE) intended for humans. However, experts have expressed concern about the subjectivity and lack of quantitation that is derived from behavioral testing. More importantly, it is difficult to gain insight into potential mechanisms of toxicity by assessing behavioral outcomes. As a complement to behavioral testing, we propose using electrophysiology-based assays, both in vivo and in vitro, such as electroencephalograms and brain slice field-potential recordings. To better illustrate these approaches, we discuss the implementation of electrophysiology-based techniques in drug-induced assessment of seizure risk, sleep disruption, and cognitive impairment.

Resistance to everolimus driven by epigenetic regulation of MYC in ER+ breast cancers.

Acquired resistance to PI3K/mTOR/Akt pathway inhibitors is often associated with compensatory feedback loops involving the activation of oncogenes. Here, we have generated everolimus resistance in ER+ breast cancer cells and in long-term estrogen deprived (LTED) models that mimic progression on anti-estrogens. This allowed us to uncover MYC as a driver of mTOR inhibitor resistance. We demonstrate that both everolimus resistance and acute treatment of everolimus can lead to the upregulation of MYC mRNA, protein expression and, consequently, the enrichment of MYC signatures as revealed by RNA sequencing data. Depletion of MYC resulted in resensitization to everolimus, confirming its functional importance in this setting. Furthermore, ChIP assays demonstrate that MYC upregulation in the everolimus resistant lines is mediated by increased association of the BRD4 transcription factor with the MYC gene. Finally, JQ1, a BRD4 inhibitor combined with everolimus exhibited increased tumor growth inhibition in 3D Matrigel models and an in vivo xenograft model. These data suggest that MYC plays an important role in mediating resistance to everolimus in ER+ and ER+/LTED models. Furthermore, given the regulation ofMYCby BRD4 in this setting, these data have implications for increased therapeutic potential of combining epigenetic agents with mTOR inhibitors to effectively downregulate otherwise difficult to target transcription factors such as MYC.

Early prediction of polymyxin-induced nephrotoxicity with next-generation urinary kidney injury biomarkers.

Despite six decades of clinical experience with the polymyxin class of antibiotics, their dose-limiting nephrotoxicity remains difficult to predict due to a paucity of sensitive biomarkers. Here, we evaluate the performance of standard of care and next-generation biomarkers of renal injury in the detection and monitoring of polymyxin-induced acute kidney injury in male Han Wistar rats using colistin (polymyxin E) and a polymyxin B (PMB) derivative with reduced nephrotoxicity, PMB nonapeptide (PMBN). This study provides the first histopathological and biomarker analysis of PMBN, an important test of the hypothesis that fatty acid modifications and charge reductions in polymyxins can reduce their nephrotoxicity. The results indicate that alterations in a panel of urinary kidney injury biomarkers can be used to monitor histopathological injury, with Kim-1 and α-GST emerging as the most sensitive biomarkers outperforming clinical standards of care, serum or plasma creatinine and blood urea nitrogen. To enable the prediction of polymyxin-induced nephrotoxicity, an in vitro cytotoxicity assay was employed using human proximal tubule epithelial cells (HK-2). Cytotoxicity data in these HK-2 cells correlated with the renal toxicity detected via safety biomarker data and histopathological evaluation, suggesting that in vitro and in vivo methods can be incorporated within a screening cascade to prioritize polymyxin class analogs with more favorable renal toxicity profiles.

A multi-site comparison of in vivo safety pharmacology studies conducted to support ICH S7A & B regulatory submissions.

INTRODUCTION: Parts A and B of the ICH S7 guidelines on safety pharmacology describe the in vivo studies that must be conducted prior to first time in man administration of any new pharmaceutical. ICH S7A requires a consideration of the sensitivity and reproducibility of the test systems used. This could encompass maintaining a dataset of historical pre-dose values, power analyses, as well as a demonstration of acceptable model sensitivity and robust pharmacological validation. During the process of outsourcing safety pharmacology studies to Charles River Laboratories, AstraZeneca set out to ensure that models were performed identically in each facility and saw this as an opportunity to review the inter-laboratory variability of these essential models. METHODS: The five in vivo studies outsourced were the conscious dog telemetry model for cardiovascular assessment, the rat whole body plethysmography model for respiratory assessment, the rat modified Irwin screen for central nervous system assessment, the rat charcoal meal study for gastrointestinal assessment and the rat metabolic cage study for assessment of renal function. Each study was validated with known reference compounds and data were compared across facilities. Statistical power was also calculated for each model. RESULTS: The results obtained indicated that each of the studies could be performed with comparable statistical power and could achieve a similar outcome, independent of facility. DISCUSSION: The consistency of results obtained from these models across multiple facilities was high thus providing confidence that the models can be run in different facilities and maintain compliance with ICH S7A and B.

Discovery of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides as selective antagonists of the kappa opioid receptor. Part 1.

Initial high throughput screening efforts identified highly potent and selective kappa opioid receptor antagonist 3 (κ IC(50)=77 nM; µ:κ and δ:κ IC(50) ratios>400) which lacked CNS exposure in vivo. Modification of this scaffold resulted in development of a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides showing potent and selectivity κ antagonism as well as good brain exposure. Analog 6c (κ IC(50)=20 nM; µ:κ=36, δ:κ=415) was also shown to reverse κ-agonist induced rat diuresis in vivo.