Longitudinal trends and subgroup analysis in publication patterns for preclinical data of newly approved drugs.

Having observed a large variation in the number and type of original preclinical publications for newly registered drugs, we have explored whether longitudinal trends and/or factors specific for certain drugs or their manufacturers may explain such variation. Our analysis is based on 1954 articles related to 170 newly approved drugs. The number of preclinical publications per compound declined from a median of 10.5 in 1991 to 3 in 2011. A similar trend was observed for the number of in vivo studies in general, but not in the subset of in vivo studies in animal models of disease. The percentage of compounds with studies using isolated human cells or cell lines almost doubled over time from 37 to 72%. Number of publications did not exhibit major differences between compounds intended for human versus veterinary use, therapeutic areas, small molecules versus biologicals, or innovator versus follow-up compounds; however, some companies may publish fewer studies per compound than others. However, there were qualitative differences in the types of models being used depending on the therapeutic area; specifically, compounds for use in oncology very often used isolated cells and cell lines, often from human origin. We conclude that the large variation in number and type of reported preclinical data is not easily explained. We propose that pharmaceutical companies should consistently provide a comprehensive documentation of the preclinical data they generate as part of their development programs in the public domain to enable a better understanding of the drugs they intend to market.

CiPA: Ongoing testing, future qualification procedures, and pending issues.

INTRODUCTION: The comprehensive in vitro proarrhythmia assay (CiPA) is a nonclinical, mechanism-based paradigm for assessing drug proarrhythmic liability. TOPICS COVERED: The first CiPA assay determines effects on cloned human cardiac ion channels. The second investigates whether the latter study-generated metrics engender proarrhythmic markers on a computationally reconstructed human ventricular action potential. The third evaluates conclusions from, and searches possibly missed effects by in silico analysis, in human stem cell-derived cardiomyocytes (hSC-CMs). CiPA ad hoc Expert-Working Groups have proposed patch clamp protocols for seven cardiac ion channels, a modified O'Hara-Rudy model for in silico analysis, detailed procedures for field (MEA) and action potential (VSD) measurements in hSC-CMs, and 29 reference drugs for CiPA assay testing and validation. DISCUSSION: CiPA adoption as drug development tool for identifying electrophysiological mechanisms conferring proarrhythmic liability to candidate drugs is a complex, multi-functional task requiring significant time, reflection, and efforts to be fully achieved.

Advances in laser scanning imaging cytometry for high-content screening.

The advent of high-content screening more than a decade ago remodeled drug discovery workflows by recasting the role of cell-based approaches in target identification, primary screening, lead optimization, and toxicity. The ability to identify and quantify compound effects on multiple cellular functions allows for rapid characterization of chemical libraries. Laser scanning imaging cytometry (LSIC) is one of the technologies that is being applied to a broad range of assays utilizing fluorescent labeling, at throughputs compatible with primary screening campaigns. Cellular resolution is achieved using laser scanning excitation through a specialized F-theta scan lens. This configuration results in rapid whole well scanning and large depth of field. The recent availability of systems equipped with multiple sources of laser excitation and arrays of detectors for spectral analysis has significantly increased its applicability through enabling more fluorescent reagents and higher levels of multiplexing. LSIC is being used most extensively for phenotypic screening especially in areas such as cell health, RNA interference (RNAi) screening, and three-dimensional cell models. This review communicates advances in LSIC and how it is being applied by presenting an overview of the technology and a range of real-world case studies.

High-content pSTAT3/1 imaging assays to screen for selective inhibitors of STAT3 pathway activation in head and neck cancer cell lines.

The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is hyperactivated in most cancers and represents a plausible therapeutic target. In the absence of STAT3-selective small-molecule inhibitors, we sought to develop pSTAT3/1 high-content imaging (HCS) assays to screen for selective inhibitors of STAT3 pathway activation in head and neck squamous cell carcinomas (HNSCC) tumor cell lines. Based on the expression of the interleukin-6 (IL-6)Rα and gp130 subunits of the IL-6 receptor complex and STAT3, we selected the Cal33 HNSCC cell line as our model. After developing image acquisition and analysis procedures, we rigorously investigated the cytokine activation responses to optimize the dynamic ranges of both assays and demonstrated that the pan-Janus kinase inhibitor pyridone 6 nonselectively inhibited pSTAT3 and pSTAT1 activation with 50% inhibition concentrations of 7.19 ± 4.08 and 16.38 ± 8.45 nM, respectively. The optimized pSTAT3 HCS assay performed very well in a pilot screen of 1,726 compounds from the Library of Pharmacologically Active Compounds and the National Institutes of Health clinical collection sets, and we identified 51 inhibitors of IL-6-induced pSTAT3 activation. However, only three of the primary HCS actives selectively inhibited STAT3 compared with STAT1. Our follow-up studies indicated that the nonselective inhibition of cytokine induced pSTAT3 and pSTAT1 activation by G-alpha stimulatory subunit-coupled G-protein-coupled receptor agonists, and forskolin was likely due to cyclic adenosine monophosphate-mediated up-regulation of suppressors of cytokine signaling 3. Azelastine, an H1 receptor antagonist approved for the treatment of seasonal allergic rhinitis, nonallergic vasomotor rhinitis, and ocular conjunctivitis, was subsequently confirmed as a selective inhibitor of IL-6-induced pSTAT3 activation that also reduced the growth of HNSCC cell lines. These data illustrate the power of a chemical biology approach to lead generation that utilizes fully developed and optimized HCS assays as phenotypic screens to interrogate specific signaling pathways.

Ligand screening system using fusion proteins of G protein-coupled receptors with G protein alpha subunits.

G protein-coupled receptors (GPCRs) constitute one of the largest families of genes in the human genome, and are the largest targets for drug development. Although a large number of GPCR genes have recently been identified, ligands have not yet been identified for many of them. Various assay systems have been employed to identify ligands for orphan GPCRs, but there is still no simple and general method to screen for ligands of such GPCRs, particularly of G(i)-coupled receptors. We have examined whether fusion proteins of GPCRs with G protein alpha subunit (Galpha) could be utilized for ligand screening and showed that the fusion proteins provide an effective method for the purpose. This article focuses on the followings: (1) characterization of GPCR genes and GPCRs, (2) identification of ligands for orphan GPCRs, (3) characterization of GPCR-Galpha fusion proteins, and (4) identification of ligands for orphan GPCRs using GPCR-Galpha fusion proteins.

Improving success rates for lead generation using affinity binding technologies.

Affinity technologies have been applied at several stages of the drug discovery process, ranging from target identification and purification to the identification of preclinical candidates. The detection of ligand-macromolecule interactions in lead discovery is the best studied and most powerful of these techniques. Although affinity methods have been in widespread use for about a decade, only recently have many reports emerged on their utility. Primary affinity screens of large libraries of small molecules or fragments have begun to produce results for challenging targets. Furthermore, in secondary assays affinity methods are opening new avenues to tackle important medicinal chemistry tasks.