In Silico Classifiers for the Assessment of Drug Proarrhythmicity.

Drug-induced torsade de pointes (TdP) is a life-threatening ventricular arrhythmia responsible for the withdrawal of many drugs from the market. Although currently used TdP risk-assessment methods are effective, they are expensive and prone to produce false positives. In recent years, in silico cardiac simulations have proven to be a valuable tool for the prediction of drug effects. The objective of this work is to evaluate different biomarkers of drug-induced proarrhythmic risk and to develop an in silico risk classifier. Cellular simulations were performed using a modified version of the O'Hara et al. ventricular action potential model and existing pharmacological data (IC50 and effective free therapeutic plasma concentration, EFTPC) for 109 drugs of known torsadogenic risk (51 positive). For each compound, four biomarkers were tested: Tx (drug concentration leading to a 10% prolongation of the action potential over the EFTPC), TqNet (net charge carried by ionic currents when exposed to 10 times the EFTPC with respect to the net charge in control), Ttriang (triangulation for a drug concentration of 10 times the EFTPC over triangulation in control), and TEAD (drug concentration originating early afterdepolarizations over EFTPC). Receiver operating characteristic (ROC) curves were built for each biomarker to evaluate their individual predictive quality. At the optimal cutoff point, accuracies for Tx, TqNet, Ttriang, and TEAD were 89.9, 91.7, 90.8, and 78.9% respectively. The resulting accuracy of the hERG IC50 test (current biomarker) was 78.9%. When combining Tx, TqNet and Ttriang into a classifier based on decision trees, the prediction improves, achieving an accuracy of 94.5%. The sensitivity analysis revealed that most of the effects on the action potential are mainly due to changes in IKr, ICaL, INaL and IKs. In fact, considering that drugs affect only these four currents, TdP risk classification can be as accurate as when considering effects on the seven main currents proposed by the CiPA initiative. Finally, we built a ready-to-use tool (based on more than 450 000 simulations), which can be used to quickly assess the proarrhythmic risk of a compound. In conclusion, our in silico tool can be useful for the preclinical assessment of TdP-risk and to reduce costs related with new drug development. The TdP risk-assessment tool and the software used in this work are available at https://riunet.upv.es/handle/10251/136919.

When Does the IC50 Accurately Assess the Blocking Potency of a Drug?

Preclinical assessment of drug-induced proarrhythmicity is typically evaluated by the potency of the drug to block the potassium human ether-à-go-go-related gene (hERG) channels, which is currently quantified by the IC50. However, channel block depends on the experimental conditions. Our aim is to improve the evaluation of the blocking potency of drugs by designing experimental stimulation protocols to measure the IC50 that will help to decide whether the IC50 is representative enough. We used the state-of-the-art mathematical models of the cardiac electrophysiological activity to design three stimulation protocols that enhance the differences in the probabilities to occupy a certain conformational state of the channel and, therefore, the potential differences in the blocking effects of a compound. We simulated an extensive set of 144 in silico IKr blockers with different kinetics and affinities to conformational states of the channel and we also experimentally validated our key predictions. Our results show that the IC50 protocol dependency relied on the tested compounds. Some of them showed no differences or small differences on the IC50 value, which suggests that the IC50 could be a good indicator of the blocking potency in these cases. However, others provided highly protocol dependent IC50 values, which could differ by even 2 orders of magnitude. Moreover, the protocols yielding the maximum IC50 and minimum IC50 depended on the drug, which complicates the definition of a "standard" protocol to minimize the influence of the stimulation protocol on the IC50 measurement in safety pharmacology. As a conclusion, we propose the adoption of our three-protocol IC50 assay to estimate the potency to block hERG in vitro. If the IC50 values obtained for a compound are similar, then the IC50 could be used as an indicator of its blocking potency, otherwise kinetics and state-dependent binding properties should be accounted.

In Silico QT and APD Prolongation Assay for Early Screening of Drug-Induced Proarrhythmic Risk.

Drug-induced proarrhythmicity is a major concern for regulators and pharmaceutical companies. For novel drug candidates, the standard assessment involves the evaluation of the potassium hERG channels block and the in vivo prolongation of the QT interval. However, this method is known to be too restrictive and to stop the development of potentially valuable therapeutic drugs. The aim of this work is to create an in silico tool for early detection of drug-induced proarrhythmic risk. The system is based on simulations of how different compounds affect the action potential duration (APD) of isolated endocardial, midmyocardial, and epicardial cells as well as the QT prolongation in a virtual tissue. Multiple channel-drug interactions and state-of-the-art human ventricular action potential models ( O'Hara , T. , PLos Comput. Biol. 2011 , 7 , e1002061 ) were used in our simulations. Specifically, 206.766 cellular and 7072 tissue simulations were performed by blocking the slow and the fast components of the delayed rectifier current ( IKs and IKr, respectively) and the L-type calcium current ( ICaL) at different levels. The performance of our system was validated by classifying the proarrhythmic risk of 84 compounds, 40 of which present torsadogenic properties. On the basis of these results, we propose the use of a new index (Tx) for discriminating torsadogenic compounds, defined as the ratio of the drug concentrations producing 10% prolongation of the cellular endocardial, midmyocardial, and epicardial APDs and the QT interval, over the maximum effective free therapeutic plasma concentration (EFTPC). Our results show that the Tx index outperforms standard methods for early identification of torsadogenic compounds. Indeed, for the analyzed compounds, the Tx tests accuracy was in the range of 87-88% compared with a 73% accuracy of the hERG IC50 based test.

Experimentally validated modeling of dynamic drug-hERG channel interactions reproducing the binding mechanisms and its importance in action potential duration.

BACKGROUND AND OBJECTIVE: Assessment of drug cardiotoxicity is critical in the development of new compounds and modeling of drug-binding dynamics to hERG can improve early cardiotoxicity assessment. We previously developed a methodology to generate Markovian models reproducing preferential state-dependent binding properties, trapping dynamics and the onset of IKr block using simple voltage clamp protocols. Here, we test this methodology with real IKr blockers and investigate the impact of drug dynamics on action potential prolongation. METHODS: Experiments were performed on HEK cells stably transfected with hERG and using the Nanion SyncroPatch 384i. Three protocols, P-80, P0 and P 40, were applied to obtain the experimental data from the drugs and the Markovian models were generated using our pipeline. The corresponding static models were also generated and a modified version of the O´Hara-Rudy action potential model was used to simulate the action potential duration. RESULTS: The experimental Hill plots and the onset of IKr block of ten compounds were obtained using our voltage clamp protocols and the models generated successfully mimicked these experimental data, unlike the CiPA dynamic models. Marked differences in APD prolongation were observed when drug effects were simulated using the dynamic models and the static models. CONCLUSIONS: These new dynamic models of ten well-known IKr blockers constitute a validation of our methodology to model dynamic drug-hERG channel interactions and highlight the importance of state-dependent binding, trapping dynamics and the time-course of IKr block to assess drug effects even at the steady-state.

Automatic modeling of dynamic drug-hERG channel interactions using three voltage protocols and machine learning techniques: A simulation study.

BACKGROUND: Assessment of drug cardiac safety is critical in the development of new compounds and is commonly addressed by evaluating the half-maximal blocking concentration of the potassium human ether-à-go-go related gene (hERG) channels. However, recent works have evidenced that the modelling of drug-binding dynamics to hERG can help to improve early cardiac safety assessment. Our goal is to develop a methodology to automatically generate Markovian models of the drug-hERG channel interactions. METHODS: The training and the test sets consisted of 20800 and 5200 virtual drugs, respectively, distributed into 104 groups with different affinities and kinetics to the conformational states of the channel. In our system, drugs may bind to any state (individually or simultaneously), with different degrees of preference for a conformational state and the change of the conformational state of the drug bound channels may be restricted or allowed. To model such a wide range of possibilities, 12 Markovian chains are considered. Our approach uses the response of the drugs to our three previously developed voltage clamp protocols, which enhance the differences in the probabilities of occupying a certain conformational state of the channel (open, closed and inactivated). The computing tool is comprised of a classifier and a parameter optimizer and uses linear interpolation, support vector machines and a simplex method for function minimization. RESULTS: We propose a novel methodology that automatically generates dynamic drug models using Markov model formulations and that elucidates the states where the drug binds and unbinds and the preferential binding state using data obtained from simple voltage clamp protocols that captures the preferential state-dependent binding properties, the relative affinities, trapping and non-trapping dynamics and the onset of IKr block. Overall, the tool correctly predicted the class of 92.04% of the drugs and the model provided by the tool accurately fitted the response of the target compound, the mean accuracy being 97.53%. Moreover, generation of the dynamic model of an IKr blocker from its response to our voltage clamp protocols usually takes less than an hour on a common desktop computer. CONCLUSION: Our methodology could be very useful to model and simulate dynamic drug-hERG channel interactions. It would contribute to the improvement of the preclinical assessment of the proarrhythmic risk of drugs that inhibit IKr and the efficacy of antiarrhythmic IKr blockers.

A multiscale simulation system for the prediction of drug-induced cardiotoxicity.

The preclinical assessment of drug-induced ventricular arrhythmia, a major concern for regulators, is typically based on experimental or computational models focused on the potassium channel hERG (human ether-a-go-go-related gene, K(v)11.1). Even if the role of this ion channel in the ventricular repolarization is of critical importance, the complexity of the events involved make the cardiac safety assessment based only on hERG has a high risk of producing either false positive or negative results. We introduce a multiscale simulation system aiming to produce a better cardiotoxicity assessment. At the molecular scale, the proposed system uses a combination of docking simulations on two potassium channels, hERG and KCNQ1, plus three-dimensional quantitative structure-activity relationship modeling for predicting how the tested compound will block the potassium currents IK(r) and IK(s). The obtained results have been introduced in electrophysiological models of the cardiomyocytes and the ventricular tissue, allowing the direct prediction of the drug effects on electrocardiogram simulations. The usefulness of the whole method is illustrated by predicting the cardiotoxic effect of several compounds, including some examples in which classic hERG-based models produce false positive or negative results, yielding correct predictions for all of them. These results can be considered a proof of concept, suggesting that multiscale prediction systems can be suitable for being used for preliminary screening in lead discovery, before the compound is physically available, or in early preclinical development when they can be fed with experimentally obtained data.

Changes in nutrient balance, methane emissions, physiologic biomarkers, and production performance in goats fed different forage-to-concentrate ratios during lactation.

The objective was to determine the effect forage-to-concentrate (F:C) ratio and stage of lactation on methane emissions, digestibility, nutrient balance, lactation performance, and metabolic responses in lactating goats. Twenty Murciano-Granadina dairy goats were used in an experiment divided into 3 periods: early (30 d), mid (100 d), and late (170 d) lactation. All goats were fed a diet with 35:65 F:C (FCL) during early-lactation. Then, 1 group (n = 10 goats) remained on FCL through mid- and late-lactation while the other group (n = 10 goats) was fed a diet with 50:50 F:C at mid-lactation (FCM) and 65:35 (FCH) at late lactation. A greater proportion of concentrate in the diet was associated with greater overall intake and digestibility (P < 0.05). Energy balance was negative in early-lactation (-77 kJ/kg of BW0.75, on average) and positive for FCL at mid- and late-lactation (13 and 35 kJ/kg of BW0.75, respectively). Goats fed FCM and FCH maintained negative energy balance throughout lactation. Plasma concentrations of non-esterified fatty acids at mid-lactation were greater for FCM than FCL (680 mEq/L), and at late-lactation concentrations were greater for FCH and FCL (856 mEq/L). A similar response was detected for plasma ß-hydroxybutyrate. Methane emission was greater (P < 0.05) for FCM than FCH (1.7 g CH4/d). This study demonstrated that differences in F:C across stages of lactation lead to distinct metabolic responses at the level of the rumen and tissues.

A new protocol test for physical activity research in obese children (etiobe project).

A new protocol is presented to validate TIPS (portable physiological monitoring device designed by I3BH that can get respiration, ecg and activity of the patient) for physical habits detection. Physiological and activity parameters and data from questionnaires have been acquired from a group of obese & non-obese children (n=20). Children completed activities from sedentary level to vigorous level. Preliminary results show variability on the response of children's effort and feasibility of TIPS platform as an ambulatory tool.

An Open-Circuit Indirect Calorimetry Head Hood System for Measuring Methane Emission and Energy Metabolism in Small Ruminants.

Methane (CH4) is a natural by-product of microbial fermentation in the rumen and is a powerful greenhouse gas. An open-circuit indirect calorimetry system for continuous determination of CH4 and CO2 production and O2 consumption and, thereafter, heat production (HP) calculation for small ruminants was described and validated. The system consisted of a computerized control, data acquisition and recording system for gases and air flux. The average value ± standard deviation for the calibration factors in the system were 1.005 ± 0.0007 (n = 6), 1.013 ± 0.0012 (n = 6) and 0.988 ± 0.0035 (n = 6) for O2, CO2 and CH4, respectively. Calibration factors close to 1 confirmed the absence of leaks in the indirect calorimetry system. In addition, an experimental test with 8 goats at mid lactation was conducted to validate the system. The repeatability for CH4 and heat production measured with the open-circuit indirect calorimetry system was 79% and 61%, respectively. Daily average HP measured by indirect calorimetry (Respiration Quotient method) was close to the average HP determined from Carbon-Nitrogen balance (CN method), accounting for 685 and 667 kJ per kg metabolic body weight, respectively. Therefore, discrepancies averaged 1.92%, a rather satisfactory value considering the substantial amount of technical and analytical work involved. The close agreement found between both methods can be considered as being indicative of the absence of systematic error. Two diets with different forage were tested: 40% was either alfalfa hay (HAY) or alfalfa silage (SIL), and the proportion of concentrate was the same in both groups (60%). The experimental trial shown that HP and CH4 were higher in HAY than SIL diet (differences between treatments of 28 kJ of HP per kg of metabolic body weight and 7.1 L CH4/day were found). The data acquisition and recording device developed improved the accuracy of the indirect calorimetry system by reducing the work involved in managing output data and refining the functionality for measuring gas exchange and energy metabolism in small ruminants.

In silico assessment of drug safety in human heart applied to late sodium current blockers.

Drug-induced action potential (AP) prolongation leading to Torsade de Pointes is a major concern for the development of anti-arrhythmic drugs. Nevertheless the development of improved anti-arrhythmic agents, some of which may block different channels, remains an important opportunity. Partial block of the late sodium current (I(NaL)) has emerged as a novel anti-arrhythmic mechanism. It can be effective in the settings of free radical challenge or hypoxia. In addition, this approach can attenuate pro-arrhythmic effects of blocking the rapid delayed rectifying K(+) current (I(Kr)). The main goal of our computational work was to develop an in-silico tool for preclinical anti-arrhythmic drug safety assessment, by illustrating the impact of I(Kr)/I(NaL) ratio of steady-state block of drug candidates on "torsadogenic" biomarkers. The O'Hara et al. AP model for human ventricular myocytes was used. Biomarkers for arrhythmic risk, i.e., AP duration, triangulation, reverse rate-dependence, transmural dispersion of repolarization and electrocardiogram QT intervals, were calculated using single myocyte and one-dimensional strand simulations. Predetermined amounts of block of I(NaL) and I(Kr) were evaluated. "Safety plots" were developed to illustrate the value of the specific biomarker for selected combinations of IC(50)s for I(Kr) and I(NaL) of potential drugs. The reference biomarkers at baseline changed depending on the "drug" specificity for these two ion channel targets. Ranolazine and GS967 (a novel potent inhibitor of I(NaL)) yielded a biomarker data set that is considered safe by standard regulatory criteria. This novel in-silico approach is useful for evaluating pro-arrhythmic potential of drugs and drug candidates in the human ventricle.

Effects of the antiarrhythmic drug dofetilide on transmural dispersion of repolarization in ventriculum. A computer modeling study.

Dofetilide is a class-III drug that inhibits the rapid component of the delayed potassium current ( I(Kr)). Experimental studies have shown that the different layers of ventricular muscle present differences in action potential duration (APD) and different responses to class III agents. It has been suggested that it contributes to APD heterogeneity in the ventricles. However, in vivo studies suggest that the strong cellular coupling reduces APD dispersion in intact heart. The aim of this paper is to study the effect of dofetilide on the action potentials (APs) in isolated ventricular cells and on APD dispersion in a strand of ventricular tissue. A mathematical model of dofetilide effects on I(Kr) has been developed and incorporated into the Luo--Rudy dynamic model of ventricular AP. Our results show that dofetilide induces in midmyocardium cells a faster time-course inhibition of I(Kr) than in endocardial or epicardial cells, and periods of instability with beat-to-beat APs variability. This behavior could favor temporal dispersion of repolarization between the different cells. The results also indicate that although dofetilide increases, the transmural gradient of APD in the ventricular wall, early afterdepolarizations (EADs) did not appear even under strong uncoupling conditions. However, reduced repolarization reserve favors the induction of EADs, even under normal coupling conditions.

Sex and age related differences in drug induced QT prolongation by dofetilide under reduced repolarization reserve in simulated ventricular cells.

The aim of this study was to investigate sex and age related differences in drug induced QT prolongation by dofetilide under reduced repolarization reserve in simulated ventricular cells. Left ventricular endocardial action potentials were simulated using a modified Luo Rudy model. Sex, age and regional differences in currents I(CaL), IK(r), IK(s), and I(to) were incorporated into the model by modifying the equations representing them. A model of dofetilide, a class III antiarrhythmic drug, was developed and included into a ventricular cell models. The reduced repolarization reserve was reproduced decreasing the IKs current. Our results shown that the adult female cells had longer action potentials, a steeper APD-BCL relationship and a higher susceptibility to EADs than adult male cells, under control, drug induced and reduced repolarization reserve conditions. On the other hand, young female and young male cells had similar action potentials under control conditions. However, young male cells had longer action potentials and higher susceptibility to EADs than young female cells under drug induced and reduced repolarization reserve conditions. Sex and age dependent differences in I(CaL), IKr, IKs, and Ito may explain the age and sex disparities in prolongation of APD by the action of dofetilide.

Role of Ca2+-dependent Cl- current on delayed afterdepolarizations. A simulation study.

Calcium-dependent chloride current (I (Cl,Ca)) is the second component (I (to2)) of the transient outward current (I (to)) that provokes the action potential (AP) phase 1 repolarization. This current contributes to the transient inward current (I (ti)) that generates delayed afterdepolarizations (DAD) in several pathological conditions. The present work uses a computer AP model of rabbit atrial myocyte and a one-dimensional (1D) tissue model of 400 cells to study the role of I (Cl,Ca) on the generation of DAD and triggered activity under calcium-overload conditions. A mathematical model describing the dependence of I (Cl,Ca) on intracellular Ca(2+) is proposed. This model takes into account the experimentally recorded characteristics of I (Cl,Ca): (1) calcium dependence, (2) voltage-dependent inactivation, and (3) I-V field-diffusion relation. Our results support the hypothesis that I (Cl,Ca) plays an important role in action potential repolarization, mainly at high frequencies. In the calcium-overload conditions tested in this work, I (Cl,Ca) represents between 28% and 44% of the total I (ti) that provokes DADs. Our simulations also show that the blockage of I (Cl,Ca) reduces the calcium overload range in which DADs provoke triggered activity.