ABSTRACT
INTRODUCTION: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. METHODS: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. RESULTS: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. DISCUSSION: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.
Subject(s)
ERG1 Potassium Channel/antagonists & inhibitors , Risk Assessment/methods , Torsades de Pointes/chemically induced , Bayes Theorem , Computer Simulation , Humans , Models, Biological , Patch-Clamp Techniques , Potassium Channel Blockers/pharmacology , Safety , Torsades de Pointes/physiopathologyABSTRACT
Corrected QT interval (QTc) prolongation in humans is usually predictable based on results from preclinical findings. This study confirms the signal from preclinical cardiac repolarization models (human ether-a-go-go-related gene, guinea pig monophasic action potential, and dog telemetry) on the clinical effects on the QTc interval. A thorough QT/QTc study is generally required for bioavailable pharmaceutical compounds to determine whether or not a drug shows a QTc effect above a threshold of regulatory interest. However, as demonstrated in this AZD3839 [(S)-1-(2-(difluoromethyl)pyridin-4-yl)-4-fluoro-1-(3-(pyrimidin-5-yl)phenyl)-1H-isoindol-3-amine hemifumarate] single-ascending-dose (SAD) study, high-resolution digital electrocardiogram data, in combination with adequate efficacy biomarker and pharmacokinetic data and nonlinear mixed effects modeling, can provide the basis to safely explore the margins to allow for robust modeling of clinical effect versus the electrophysiological risk marker. We also conclude that a carefully conducted SAD study may provide reliable data for effective early strategic decision making ahead of the thorough QT/QTc study.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Electrocardiography/drug effects , Indoles/pharmacology , Pyrimidines/pharmacology , Animals , Arterial Pressure/drug effects , Dogs , Dose-Response Relationship, Drug , Double-Blind Method , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/genetics , Guinea Pigs , Humans , Indoles/adverse effects , Male , Models, Biological , Pyrimidines/adverse effectsABSTRACT
The role of the outermost helix (M4) in the pentameric ligand-gated ion channel (pLGIC) family is currently not fully understood. It is known that M4 is important for receptor assembly, possibly via interactions with neighboring M1 and M3 helices. M4 can also transmit information on the lipid content of the membrane to the gating mechanism, and it may form a link to the extracellular domain via the Cys-loop. Our previous study examining the α4ß2 nACh receptor M4 helix using HEK cells indicated M4 here is more sensitive to change than those of other pLGIC. Many of these other studies, however, were performed in Xenopus oocytes. Here we examine the nine previously identified nonfunctional α4ß2 nACh receptor M4 mutant receptors using this system. The data reveal that seven of these mutant receptors do function when expressed in oocytes, with only 2, the conserved Asp at the intracellular end of M4 and a Phe in the center, having a similar phenotype (nonfunctional) in both HEK cells and oocytes. The oocyte data are more consistent with studies in other pLGIC and demonstrate the importance of the expression system used. Of the many differences between these two expression systems, we suggest that the different lipid content of the plasma membrane is a possible candidate for explaining these discrepancies.
ABSTRACT
Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC50 values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug's cardiac safety margin. We determined variability of APC data from multiple sites that measured blocking potency of 12 blinded drugs (with different levels of proarrhythmic risk) against four human cardiac currents (hERG [IKr], hCav1.2 [L-Type ICa], peak hNav1.5, [Peak INa], late hNav1.5 [Late INa]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC50 variability (25/75 percentiles) differed for drugs and currents (e.g., 10.4-fold for dofetilide block of hERG current and 4-fold for mexiletine block of hNav1.5 current). Within-platform variance predominated for 4 of 12 hERG blocking drugs and 4 of 6 hNav1.5 blocking drugs. hERG and hNav1.5 block. Bland-Altman plots depicted varying agreement across APC platforms. A follow-up survey suggested multiple sources of experimental variability that could be further minimized by stricter adherence to standard protocols. Adoption of best practices would ensure less variable APC datasets and improved safety margins and proarrhythmic risk assessments.
ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
For the past decade, cardiac safety screening to evaluate the propensity of drugs to produce QT interval prolongation and Torsades de Pointes (TdP) arrhythmia has been conducted according to ICH S7B and ICH E14 guidelines. Central to the existing approach are hERG channel assays and in vivo QT measurements. Although effective, the present paradigm carries a risk of unnecessary compound attrition and high cost, especially when considering costly thorough QT (TQT) studies conducted later in drug development. The C: omprehensive I: n Vitro P: roarrhythmia A: ssay (CiPA) initiative is a public-private collaboration with the aim of updating the existing cardiac safety testing paradigm to better evaluate arrhythmia risk and remove the need for TQT studies. It is hoped that CiPA will produce a standardized ion channel assay approach, incorporating defined tests against major cardiac ion channels, the results of which then inform evaluation of proarrhythmic actions in silico, using human ventricular action potential reconstructions. Results are then to be confirmed using human (stem cell-derived) cardiomyocytes. This perspective article reviews the rationale, progress of, and challenges for the CiPA initiative, if this new paradigm is to replace existing practice and, in time, lead to improved and widely accepted cardiac safety testing guidelines.
Subject(s)
Arrhythmias, Cardiac/chemically induced , Arrhythmias, Cardiac/diagnosis , Drug-Related Side Effects and Adverse Reactions/diagnosis , Drug-Related Side Effects and Adverse Reactions/etiology , Heart/drug effects , Animals , Humans , Long QT Syndrome/chemically induced , Long QT Syndrome/diagnosis , Torsades de Pointes/chemically induced , Torsades de Pointes/diagnosisABSTRACT
Evaluating whether a new medication prolongs QT intervals is a critical safety activity that is conducted in a sensitive animal model during non-clinical drug development. The importance of QT liability detection has been reinforced by non-clinical [International Conference on Harmonization (ICH) S7B] and clinical (ICH E14) regulatory guidance from the International Conference on Harmonization. A key challenge for the cardiovascular safety community is to understand how the finding from a non-clinical in vivoâ QT assay in animals predicts the outcomes of a clinical QT evaluation in humans. The Health and Environmental Sciences Institute Pro-Arrhythmia Working Group performed a literature search (1960-2011) to identify both human and non-rodent animal studies that assessed QT signal concordance between species and identified drugs that prolonged or did not prolong the QT interval. The main finding was the excellent agreement between QT results in humans and non-rodent animals. Ninety-one percent (21 of 23) of drugs that prolonged the QT interval in humans also did so in animals, and 88% (15 of 17) of drugs that did not prolong the QT interval in humans had no effect on animals. This suggests that QT interval data derived from relevant non-rodent models has a 90% chance of predicting QT findings in humans. Disagreement can occur, but in the limited cases of QT discordance we identified, there appeared to be plausible explanations for the underlying disconnect between the human and non-rodent animal QT outcomes.
Subject(s)
Long QT Syndrome/chemically induced , Animals , Drug Evaluation, Preclinical , Humans , Sensitivity and SpecificityABSTRACT
Drug toxicity to T-antigen-immortalized human liver epithelial (THLE) cells stably transfected with plasmid vectors that encoded human cytochrome P450s 1A2, 2C9, 2C19, 2D6, or 3A4, or an empty plasmid vector (THLE-Null), was investigated. An automated screening platform, which included 1% dimethyl sulfoxide (DMSO) vehicle, 2.7% bovine serum in the culture medium, and assessed 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reduction, was used to evaluate the cytotoxicity of 103 drugs after 24h. Twenty-two drugs caused cytotoxicity to THLE-Null cells, with EC50 ≤ 200 µM; 21 of these drugs (95%) have been reported to cause human liver injury. Eleven drugs exhibited lower EC50 values in cells transfected with CYP3A4 (THLE-3A4 cells) than in THLE-Null cells; 10 of these drugs (91%) caused human liver injury. An additional 8 drugs, all of which caused human liver injury, exhibited potentiated THLE-3A4 cell toxicity when evaluated using a manual protocol that included 0.2% or 1% DMSO, but not bovine serum. Fourteen of the drugs that exhibited potentiated THLE-3A4 cell toxicity are known to be metabolized by P450s to reactive intermediates. These drugs included troglitazone, which was shown to undergo metabolic bioactivation and covalent binding to proteins in THLE-3A4 cells. A single drug (rimonabant) exhibited marked THLE cell toxicity but did not cause human liver injury; this drug had very low reported plasma exposure. These results indicate that evaluation of toxicity to THLE-Null and THLE-3A4 cell lines during drug discovery may aid selection of drugs with reduced propensity to cause drug-induced liver injury and that consideration of human exposure is required to enhance data interpretation.
Subject(s)
Chemical and Drug Induced Liver Injury/etiology , Cytochrome P-450 Enzyme System/metabolism , Epithelial Cells/drug effects , Liver/drug effects , Biological Assay , Biotransformation , Cell Line , Cell Survival/drug effects , Chemical and Drug Induced Liver Injury/enzymology , Chemical and Drug Induced Liver Injury/pathology , Cytochrome P-450 Enzyme System/genetics , Dose-Response Relationship, Drug , Epithelial Cells/enzymology , Humans , Inhibitory Concentration 50 , Liver/enzymology , Liver/pathology , Risk Assessment , Risk Factors , Toxicity Tests , TransfectionABSTRACT
INTRODUCTION: Detection of drug-induced pro-arrhythmic risk is a primary concern for pharmaceutical companies and regulators. Increased risk is linked to prolongation of the QT interval on the body surface ECG. Recent studies have shown that multiple ion channel interactions can be required to predict changes in ventricular repolarisation and therefore QT intervals. In this study we attempt to predict the result of the human clinical Thorough QT (TQT) study, using multiple ion channel screening which is available early in drug development. METHODS: Ion current reduction was measured, in the presence of marketed drugs which have had a TQT study, for channels encoded by hERG, CaV1.2, NaV1.5, KCNQ1/MinK, and Kv4.3/KChIP2.2. The screen was performed on two platforms - IonWorks Quattro (all 5 channels, 34 compounds), and IonWorks Barracuda (hERG & CaV1.2, 26 compounds). Concentration-effect curves were fitted to the resulting data, and used to calculate a percentage reduction in each current at a given concentration. Action potential simulations were then performed using the ten Tusscher and Panfilov (2006), Grandi et al. (2010) and O'Hara et al. (2011) human ventricular action potential models, pacing at 1Hz and running to steady state, for a range of concentrations. RESULTS: We compared simulated action potential duration predictions with the QT prolongation observed in the TQT studies. At the estimated concentrations, simulations tended to underestimate any observed QT prolongation. When considering a wider range of concentrations, and conventional patch clamp rather than screening data for hERG, prolongation of ≥5ms was predicted with up to 79% sensitivity and 100% specificity. DISCUSSION: This study provides a proof-of-principle for the prediction of human TQT study results using data available early in drug development. We highlight a number of areas that need refinement to improve the method's predictive power, but the results suggest that such approaches will provide a useful tool in cardiac safety assessment.
Subject(s)
Action Potentials/drug effects , Drug Evaluation, Preclinical/adverse effects , Electrocardiography/drug effects , Ion Channels/metabolism , Long QT Syndrome/chemically induced , Dose-Response Relationship, Drug , HumansABSTRACT
Matched molecular pairs analysis (MMPA)1,2 is an inverse quantitative structure activity relationship (QSAR) technique that is rapidly gaining popularity in the retrospective analysis of large experimental datasets.3,4 While much of the recent focus has been on the differences in properties between structurally related groups of existing compounds, attempts to extend this methodology to the de-novo design of novel structures have been limited. To our knowledge the aggregate effect of multiple transformations, all suggesting the same molecular structure, has only ever being considered within a very limited dataset.5 We therefore sought to test this exciting new approach to the design (and absolute property prediction - effectively QSAR-by-MMPA) of novel chemical entities based on a larger, more diverse dataset, and couple these designs to MMPA-based predictions of antitarget activity.
ABSTRACT
INTRODUCTION: Evaluation of drug candidates in in-vitro assays of action potential duration (APD) is one component of preclinical safety assessment. Current assays are limited by technically-demanding, time-consuming electrophysiological methods. This study aimed to assess whether a voltage-sensitive dye-based assay could be used instead. METHODS: Optical APs were recorded using di-4-ANEPPS in electrically field stimulated beagle left ventricular midmyocardial myocytes (LVMMs). Pharmacological properties of di-4-ANEPPS on the main cardiac ion channels that shape the ventricular AP were investigated using IonWorks and conventional electrophysiology. Effects of 9 reference drugs (dofetilide, E4031, D-sotalol, ATXII, cisapride, terfenadine, alfuzosin, diltiazem and pinacidil) with known APD-modulating effects were assessed on optically measured APD at 1 Hz. RESULTS: Under optimum conditions, 0.1 microM di-4-ANEPPS could be used to monitor APs paced at 1 Hz during nine, 5 s exposures without altering APD. di-4-ANEPPS had no effect on either hI(ERG), hI(Na), hI(Ks) and hI(to) currents in transfected CHO cells (up to 10 microM) or I(Ca,L) current in LVMMs (at 16 microM). di-4-ANEPPS had no effect on APs recorded with microelectrodes at 1 or 0.5 Hz over a period of 30 min di-4-ANEPPS displayed the sensitivity to record changes in optically measured APD in response to altered pacing frequencies and sequential vehicle additions did not affect the optically measured APD. APD data obtained with 9 reference drugs were as expected except (i) D-sotalol-induced increases in duration were smaller than those caused by other I(Kr) blockers and (ii) increases in APD were not detected using low concentrations of terfenadine. DISCUSSION: Early in drug discovery, the di-4-ANEPPS-based method can reliably be used to assess drug effects on APD as part of a cardiac risk assessment strategy.