Discovery of an Extremely Potent Thiazine-Based ß-Secretase Inhibitor with Reduced Cardiovascular and Liver Toxicity at a Low Projected Human Dose.

Genetic evidence points to deposition of amyloid-ß (Aß) as a causal factor for Alzheimer's disease. Aß generation is initiated when ß-secretase (BACE1) cleaves the amyloid precursor protein. Starting with an oxazine lead 1, we describe the discovery of a thiazine-based BACE1 inhibitor 5 with robust Aß reduction in vivo at low concentrations, leading to a low projected human dose of 14 mg/day where 5 achieved sustained Aß reduction of 80% at trough level.

Impact of calcium-sensitive dyes on the beating properties and pharmacological responses of human iPS-derived cardiomyocytes using the calcium transient assay.

INTRODUCTION: Calcium-based screening of hiPS-CMs is a useful preclinical safety evaluation platform with the ability to generate robust signals that facilitates high-throughput screening and data analysis. However, due to the potential inherent toxicities, it is important to understand potential effects of different calcium-sensitive dyes on the hiPS-CMs model. METHODS: We compared three calcium-sensitive fluorescence dyes (Cal520, ACTOne and Calcium 5) for their impact on the variability, the beating properties and the pharmacological responses of hiPS-CMs using the Hamamatsu FDSS/µCell imaging platform. Direct effects of three dyes on the electrophysiological properties of hiPS-CMs were evaluated with the multi-electrode array (MEA) Axion Maestro platform. RESULTS: We propose a specific experimental protocol for each dye which gives the most optimal assay conditions to minimize variability and possible adverse effects. We showed that Cal520 had the smallest effect on hiPS-CMs together with the longest-lasting stable amplitude signal (up to 4 h). Although all dyes had a (minor) acute effect on hiPS-CMs, in the form of reduced beat rate and prolonged field potential duration, the selection of the dye did not influence the pharmacological response of four cardioactive drugs (dofetilide, moxifloxacin, nimodipine and isoprenaline). DISCUSSION: In conclusion, we have documented that different calcium sensitive dyes have only minor direct (acute) effects on hiPS-CMs with Cal520 showing the least effects and the longest lasting signal amplitude. Importantly, drug-induced pharmacological responses in hiPS-CMs were comparable between the three dyes. These findings should help further improve the robustness of the hiPS-CMs-based calcium transient assay as a predictive, preclinical cardiac safety evaluation tool.

Chronic drug-induced effects on contractile motion properties and cardiac biomarkers in human induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND AND PURPOSE: In the pharmaceutical industry risk assessments of chronic cardiac safety liabilities are mostly performed during late stages of preclinical drug development using in vivo animal models. Here, we explored the potential of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to detect chronic cardiac risks such as drug-induced cardiomyocyte toxicity. EXPERIMENTAL APPROACH: Video microscopy-based motion field imaging was applied to evaluate the chronic effect (over 72 h) of cardiotoxic drugs on the contractile motion of hiPS-CMs. In parallel, the release of cardiac troponin I (cTnI), heart fatty acid binding protein (FABP3) and N-terminal pro-brain natriuretic peptide (NT-proBNP) was analysed from cell medium, and transcriptional profiling of hiPS-CMs was done at the end of the experiment. KEY RESULTS: Different cardiotoxic drugs altered the contractile motion properties of hiPS-CMs together with increasing the release of cardiac biomarkers. FABP3 and cTnI were shown to be potential surrogates to predict cardiotoxicity in hiPS-CMs, whereas NT-proBNP seemed to be a less valuable biomarker. Furthermore, drug-induced cardiotoxicity produced by chronic exposure of hiPS-CMs to arsenic trioxide, doxorubicin or panobinostat was associated with different profiles of changes in contractile parameters, biomarker release and transcriptional expression. CONCLUSION AND IMPLICATIONS: We have shown that a parallel assessment of motion field imaging-derived contractile properties, release of biomarkers and transcriptional changes can detect diverse mechanisms of chronic drug-induced cardiac liabilities in hiPS-CMs. Hence, hiPS-CMs could potentially improve and accelerate cardiovascular de-risking of compounds at earlier stages of drug discovery. LINKED ARTICLES: This article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc.

The concordance between nonclinical and phase I clinical cardiovascular assessment from a cross-company data sharing initiative.

It is widely accepted that more needs to be done to bring new, safe, and efficacious drugs to the market. Cardiovascular toxicity detected both in early drug discovery as well as in the clinic, is a major contributor to the high failure rate of new molecules. The growth of translational safety offers a promising approach to improve the probability of success for new molecules. Here we describe a cross-company initiative to determine the concordance between the conscious telemetered dog and phase I outcome for 3 cardiovascular parameters. The data indicate that, in the context of the methods applied in this analysis, the ability to detect compounds that affect the corrected QT interval (QTc) was good within the 10-30x exposure range but the predictive or detective value for heart rate and diastolic blood pressure was poor. These findings may highlight opportunities to refine both the animal and the clinical study designs, as well as refocusing the assessment of value of dog cardiovascular assessments beyond phase 1. This investigation has also highlighted key considerations for cross-company data sharing and presents a unique learning opportunity to improve future translational projects.

The fentanyl/etomidate-anesthetized beagle (FEAB) model in safety pharmacology assessment.

This unit describes a procedure for performing safety studies in the anesthetized beagle dog. Detailed are the anesthetic regime, the surgical procedure, and all materials needed to perform cardiovascular, central nervous system, and respiratory safety studies in these animals. An overview of all parameters that can be measured and calculated is provided, as are experimental protocols. Endpoints discussed include hemodynamic, electrocardiological, respiratory, arterial blood, and electroencephalogical parameters. Also presented are a formula to correct QT interval for changes in core body temperature and an overview of changes in ECG, MAP, and EEG traces that may occur during safety studies. The information provided yields a multiparametric model for performing reliable safety studies in anesthetized dogs.

Blockade of the I(Ks) potassium channel: an overlooked cardiovascular liability in drug safety screening?

The problem of drug-induced hERG channel blockade, which can lead to acquired long QT syndrome and potentially fatal arrhythmias, has exercised drug developers and regulatory authorities for over 10 years, and exacting guidelines have been put into place to test for this liability both preclinically (ICH S7B) and clinically (ICH E14). However, the I(Ks) channel, which along with the transient outward current (I(to)) is the other main potassium channel affecting cardiac repolarisation and thus the length of the QT interval, has received little attention, and potent I(Ks) blocking drugs with serious side effects could potentially enter into human testing without being detected by the existing regulatory core battery and standard screening strategies. Here we review the pharmacology of cardiac I(Ks) channel blockade and describe the discovery of a potent I(Ks) blocker whose activity was not detected by standard hERG or invitro action potential screens, but subsequently evoked unprovoked torsades de pointes (TdP) invivo in our anaesthetised dog model. We have exploited this molecule to develop a ligand binding assay to detect I(Ks) blockade at an earlier stage in drug discovery, and note that several other laboratories developing new drugs have also developed higher throughput screens to detect I(Ks) blockade (e.g., [Trepakova, E. S., Malik, M. G., Imredy, J. P., Penniman, J. R., Dech, S. J., & Salata, J. J. (2007) Application of PatchXpress planar patch clamp technology to the screening of new drug candidates for cardiac KCNQ1/KCNE1 (I(Ks)) activity. Assay Drug Development Technology 5, 617-627]). Because of the presence of I(Ks) channels in other tissues, including blood vessels and in the epithelia of intestine, kidney, lung and the cochlea, I(Ks) blockade has the potential to cause extensive side effects in addition to QT prolongation and arrhythmias. We therefore suggest that compounds selected for development should also be examined for I(Ks) liability before testing in humans. The possibility of undetected I(Ks) blockade is therefore an additional gap to that identified earlier [Lu, H. R., Vlaminckx, E., Hermans, A. N., Rohrbacher, J., Van Ammel, K., Towart, R., et al. (2008) Predicting drug-induced changes in QT interval and arrhythmias: QT-shortening drugs point to gaps in the ICH S7B Guidelines. British Journal of Pharmacology, 154, 1427-1438] in the ICH S7B regulatory guidelines.

Application of semiparametric mixed models and simultaneous confidence bands in a cardiovascular safety experiment with longitudinal data.

Several pharmacological studies involve experiments aimed at testing for a difference between experimental groups wherein the data are longitudinal in nature, frequently with long sequences per subject. Oftentimes, treatment effect, if present, is not constant over time. In such situations, imposing a parametric mean structure can be too complicated and/or restrictive. A more flexible approach is to model the mean using a semiparametric smooth function, estimated using, for example, penalized smoothing splines. We formulate a series of models exhibiting how the group-specific mean profiles could possibly differ. Once an appropriate model is chosen, interest lies in identifying specific time points where the groups differ. For this purpose, we propose the use of simultaneous confidence bands around the fitted models wherein the bands take into account within and between-subject variability, as well as variability arising from smoothing.

Choice of cardiac tissue plays an important role in the evaluation of drug-induced prolongation of the QT interval in vitro in rabbit.

INTRODUCTION: Regulatory guidelines (CPMP/986/96, ICHS7B) recommend the use of isolated cardiac tissues, including Purkinje fibers, papillary muscles and ventricular trabeculae, for detecting potential drug-induced long QT. However, the differential sensitivity of these tissues in experimental drug-induced long QT is relatively unknown. We investigated the electrophysiological characteristics of these tissue types in vitro together with their different responses to drugs that are known to induce prolongation of the QT interval in man. METHODS: Electrophysiological parameters were measured in vitro using a micro-electrode technique. The isolated rabbit Purkinje fibers, papillary muscles or ventricular trabeculae were superperfused with Tyrode's solution and stimulated according to different stimulation protocols. The effects of dofetilide (1 x 10(-8) M), sertindole (1 x 10(-6) M), erythromycin (3 x 10(-4) M) and sparfloxacin (1 x 10(-4) M) were evaluated relative to solvent (n=8 to 12 for each group). RESULTS: In isolated Purkinje fibers, action potential duration at 90% repolarization (APD(90) at 1 Hz) was markedly prolonged by 55% (erythromycin), 103% (dofetilide), 118% (sertindole) and 88% (sparfloxacin) from baseline. The prolongation of APD(90) caused by these 4 compounds was associated with a 28% to 78% incidence of early afterdepolarizations (EADs) at 0.2 Hz only in the Purkinje fiber. In contrast, APD(90) was altered by erythromycin, dofetilide, sertindole and sparfloxacin only by +15%, +6%, -7% or +15%, respectively, in isolated papillary muscles, and by 33%, +28%, +4% or +16%, respectively, in ventricular trabeculae. EADs were not induced by these four compounds in papillary muscles or in trabeculae. Reducing the stimulation rate to 0.2 Hz resulted in a 33% prolongation of APD(90) in Purkinje fibers, while APD(90) was shortened by 10% in papillary muscles and by 20% in ventricular trabeculae. CONCLUSION: The present study demonstrates that the differential sensitivity of tissue types play an important role in the detection of drug-induced long APD and EADs. Indeed the Purkinje fiber was the only tissue type to display the well known phenomenon associated with I(kr) channel blockade (inverse-use dependence), when the stimulation rate was decreased below 1 Hz. Rabbit Purkinje fibers constitute the most sensitive tissue type for detecting drug-induced long action potential duration and EADs. As such the selection of tissue type needs to be taken into careful consideration in cardiac safety assessments when exploring drug induced long QT.