Adverse effects due to off-target activity can be predicted by careful comparison of the relationship between expected plasma concentration and off-target activity of the test compound with that of reference drugs targeting that receptor for their therapeutic efficacy. The ratio between plasma concentration (unbound) and the K-i at the receptor is a surrogate measure reflecting receptor occupancy. Where data are available for reference drugs, we have curated and evaluated this at 100 receptors, 72 of which can involve both negative and positive modulations by drugs: a total of 172 'receptor modulations'. This provides a quantitative framework upon which to achieve consistent risk assessment of off-target interactions across receptors, across compounds and between assessors. It therefore represents a significant departure from an opinion-based to an evidence-based approach to secondary pharmacology. Demonstration of proof-of-principle was achieved for one of the receptor interactions (alpha(1A)-adrenoceptor antagonism leading to postural hypotension in clinical use) due to the availability of high-quality off-target K-i data for >30 drugs at this receptor.

WHAT IS KNOWN AND OBJECTIVE:Fenspiride, a drug that had been used for decades for the treatment of respiratory diseases, was recently withdrawn from the market due to the potential risk of QT prolongation and proarrhythmia. This is the first such withdrawal for many years and hence poses a question whether such risk could have been predicted and to what degree non-drug-specific parameters play a role in the reported QT prolongation and cases of TdP. The study aim was to test various 'what-if' scenarios to assess the influence of age, gender, heart rate, and plasma potassium concentration on QT interval prolongation due to various doses of fenspiride with the use of mechanistic mathematical modelling.METHODS:Concentration-time profiles were simulated with the use of a PBPK model developed based on published physico-chemical data, data from in vitro ADME experiments, and in vivo PK study results. Pharmacodynamic effect, that is, drug-triggered pseudoECG signal modification was simulated using a biophysically detailed model of human cardiac myocytes. Analysis of the qNet metric was also performed to classify proarrhythmic risk related to fenspiride.RESULTS:In the simulation study, arrhythmia was not observed even in the 'what-if' scenarios with extreme exposure, age, heart rate, and plasma potassium concentration. The qNet metric value positioned fenspiride in the intermediate risk class.WHAT IS NEW AND CONCLUSION:It can be hypothesized that the clinically observed arrhythmia cases were not directly caused by fenspiride alone but a combination of multiple factors, including comedications.

IntroductionThe anaesthetised guinea pig is a well characterised assay for early assessment of drug effects on ventricular repolarisation and risk of Torsade de Pointes (TdP). We assessed whether a selective hERG blocker with known TdP risk could be differentiated from lower risk, balanced ion channel blockers in the guinea pig, using corrected QT (QTc) interval alongside novel electrocardiogram (ECG) biomarkers J-Tpeakc and Tpeak-Tend. Effects were compared with previous clinical investigations at similar plasma concentrations and with another index of TdP risk, the electromechanical window (EMW).MethodsTwenty-two Dunkin Hartley guinea pigs anaesthetised with sodium pentobarbitone were instrumented for haemodynamic measurement and ECG recording. Three ascending doses of vehicle (n = 6), dofetilide (2, 6 or 20 μg/kg; n = 7), ranolazine (2, 6 or 20 mg/kg; n = 5) or verapamil (0.1, 0.3 or 1.0 mg/kg; n = 4) were administered intravenously.ResultsAs reported in previous clinical studies, dofetilide induced dose-dependent increases in QTc interval, with increases in both J-TpeakC or Tpeak-Tend, while verapamil caused no significant increase in QTc interval, J-TpeakC or Tpeak-Tend. Ranolazine caused dose-dependent increases in QTc interval and corrected J-Tpeakc, but had no effect on Tpeak-Tend, which is in contrast to the effects reported in humans at similar concentrations. Only dofetilide caused a clear, dose-related decrease in the EMW.DiscussionThese findings suggest that measurements of J-Tpeakc and Tpeak-Tend in addition to QT interval, may help differentiate pure hERG channel blockers with high risk of TdP from lower risk, multichannel blockers.

There has been significant focus on drug-induced QT interval prolongation caused by block of the human ether-a-go-go-related gene (hERG)-encoded potassium channel. Regulatory guidance has been implemented to assess QT interval prolongation risk: preclinical guidance requires a candidate drug's potency as a hERG channel blocker to be defined and also its effect on QT interval in a non-rodent species; clinical guidance requires a "Thorough QT Study" during development, although some QT prolonging compounds are identified earlier via a Phase I study. Clinical, heart rate-corrected QT interval (QTc) data on 24 compounds (13 positives; 11 negatives) were compared with their effect on dog QTc and the concentration of compound causing 50% inhibition (IC50) of hERG current. Concordance was assessed by calculating sensitivity and specificity across a range of decision thresholds, thus yielding receiver operating characteristic curves of sensitivity versus (1-specificity). The area under the curve of ROC curves (for which 0.5 and 1 indicate chance and perfect concordance, respectively) was used to summarize concordance. Three aspects of preclinical data were compared with the clinical outcome (receiver operating characteristic area under the curve values shown in brackets): absolute hERG IC50 (0.78); safety margin between hERG IC50 and clinical peak free plasma exposure (0.80); safety margin between QTc effects in dogs and clinical peak free plasma exposure (0.81). Positive and negative predictive values of absolute hERG IC50 indicated that from an early drug discovery perspective, low potency compounds can be progressed on the basis of a low risk of causing a QTc increase.

Drug toxicity is a major source of attrition in drug discovery and development. Pharmaceutical companies routinely use preclinical data to predict clinical outcomes and continue to invest in new assays to improve predictions. However, there are many open questions about how to make the best use of available data, combine diverse data, quantify risk, and communicate risk and uncertainty to enable good decisions. The costs of suboptimal decisions are clear: resources are wasted and patients may be put at risk. We argue that Bayesian methods provide answers to all of these problems and use hERG-mediated QT prolongation as a case study. Benefits of Bayesian machine learning models include intuitive probabilistic statements of risk that incorporate all sources of uncertainty, the option to include diverse data and external information, and visualizations that have a clear link between the output from a statistical model and what this means for risk. Furthermore, Bayesian methods are easy to use with modern software, making their adoption for safety screening straightforward. We include R and Python code to encourage the adoption of these methods.

Cardiotoxicity is a common cause of attrition in preclinical and clinical drug development. Current in vitro approaches have two main limitations, they either are limited to low throughput methods not amendable to drug discovery or lack the physiological responses to allow an integrated risk assessment. A human 3D cardiac microtissue containing humaninduced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), cardiac endothelial cells and cardiac fibroblast were used to assess their suitability to detect drug induced changes in cardiomyocyte contraction. These cardiac microtissues, have a uniform size, spontaneously beat, lack a hypoxic core, and contain key markers of each cell type. Application of field stimulation and measurement of cardiac contraction confirm cardiac microtissues to be a suitable model to investigate drug-induced changes in cardiomyocyte contractility. Using a bespoke image acquisition work flow and optical flow analysis method to test 29 inotroptic and 13 non-inotroptic compounds in vivo. We report that cardiac microtissues provide a high-throughput experimental model that is both able to detect changes in cardiac contraction with a sensitivity and specificity of 80 and 91%, respectively, and provide insight into the direction of the inotropic response. Allowing improved in vitro cardiac contractility risk assessment. Moreover, our data provide evidence of the detection of this liability at therapeutically relevant concentrations with a throughput amenable to drug discovery.

Cardiovascular toxicity is a prominent reason for failures in drug development, resulting in the demand for assays that can predict this liability in early drug discovery. We investigated whether iCell® cardiomyocytes have utility as an early QT/TdP screen. Thirty clinical drugs with known QT/TdP outcomes were evaluated blind using label-free microelectrode array (parameters measured were beating period (BP), field potential duration (FPD), fast Na+ amplitude and slope) and live cell, fast kinetic fluorescent Ca2+ transient FLIPR® Tetra (parameters measured were peak count, width, amplitude) systems. Many FPD-altering drugs also altered BP. Correction for BP, using a Log-Log (LL) model, was required to appropriately interpret direct drug effects on FPD. In comparison with human QT effects and when drug activity was to be predicted at top test concentration (TTC), LL-corrected FPD and peak count had poor assay sensitivity and specificity values: 13%/64% and 65%/11%, respectively. If effective free therapeutic plasma concentration (EFTPC) was used instead of TTC, the values were 0%/100% and 6%/100%, respectively. When compared to LL-corrected FPD and peak count, predictive values of uncorrected FPD, BP, width and amplitude were not much different. If pro-arrhythmic risk was to be predicted using Ca2+ transient data, the values were 67%/100% and 78%/53% at EFTPC and TTC, respectively. Thus, iCell® cardiomyocytes have limited value as an integrated QT/TdP assay, highlighting the urgent need for improved experimental alternatives that may offer an accurate integrated cardiomyocyte safety model for supporting the development of new drugs without QT/TdP effects.

Introduction: Changes in blood pressure (BP) are now proactively examined throughout the drug development process as an integral aspect of safety monitoring. This is because hypertension is a very strong risk factor for cardiovascular events and drug-induced increases in BP have attracted increased regulatory attention. However, there is currently no guidance from regulatory agencies on the minimum BP data required for submissions, and there are no specific criteria for what constitutes a safety signal for increased BP in non clinical studies.Areas covered: Evaluation of BP increases through the drug discovery and development process.Expert opinion: Research into the effects of drugs should begin before clinical development is initiated and continue throughout the clinical trial program. Non clinical studies should inform a benefit-risk analysis that will aid decision-making of whether to enter the drug into Phase I development. The degree of acceptable risk will vary according to the therapy area, treatment indication and intended population for the new drug, and the approach to BP assessment and risk mitigation should be tailored accordingly. However, BP monitoring should always be included in clinical trials, and data collected from multiple studies, to convincingly prove or refute a suspicion of BP effects.

Cardiotoxicity is a common cause of attrition in preclinical and clinical drug development. Current in vitro approaches have two main limitations, they either are limited to low throughput methods not amendable to drug discovery or lack the physiological responses to allow an integrated risk assessment. A human 3D cardiac microtissue containing human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), cardiac endothelial cells and cardiac fibroblast were used to assess their suitability to detect drug induced changes in cardiomyocyte contraction. These cardiac microtissues, have a uniform size, spontaneously beat, lack a hypoxic core, and contain key markers of each cell type. Application of field stimulation and measurement of cardiac contraction confirm cardiac microtissues to be a suitable model to investigate drug-induced changes in cardiomyocyte contractility. Using a bespoke image acquisition work flow and optical flow analysis method to test 29 inotroptic and 13 non-inotroptic compounds in vivo. We report that cardiac microtissues provide a high-throughput experimental model that is both able to detect changes in cardiac contraction with a sensitivity and specificity of 80 and 91%, respectively, and provide insight into the direction of the inotropic response. Allowing improved in vitro cardiac contractility risk assessment. Moreover, our data provide evidence of the detection of this liability at therapeutically relevant concentrations with a throughput amenable to drug discovery.

We sought to investigate whether drug-induced changes in contractility were affected by pacing rates that represent the range of heart rates encountered in vivo. Using the cell geometry measurement system (IonOptix), we paced dog cardiomyocytes at different cycle lengths (CLs) of 2000, 1000, 500, and 333.3 ms, before and after exposure to 13 inotropic drugs. Time course data using vehicle control (0.1% dimethyl sulfoxide (DMSO)) demonstrated stability of the system at all CLs tested. Seven positive inotropes (eg isoproterenol) exerted rate-dependent increases in sarcomere shortening (Sarc. short.; maximal effect at a CL of 333.3 ms [0.1 µM isoproterenol increased Sarc. short. by 41.1% and 145.9% at 2000 and 333.3 ms, respectively]). Omecamtiv mecarbil showed an atypical profile (increased Sarc. short. at 2000 ms [106.9%] and decreased at 333.3 ms [IC(50) = 0.64 µM]). Four negative inotropes (eg flecainide) showed rate-independent inhibition of Sarc. short. (IC(50)s: 3.3 µM [2000 ms] versus 2.3 µM [333.3 ms]). The remaining negative inotropes, verapamil, and BTS (N-benzyl-p-toluene sulphonamide) produced an increase (IC(50)s: 3.9 µM [2000 ms] versus 0.043 µM [333.3ms]) and decrease (IC(50)s: 18.3 µM [2000 ms] versus 34.0 µM [333.3 ms]) in potency, respectively. Negative inotropes (eg flecainide, BTS, and verapamil) decreased the area of the Ca(2+) transient versus Sarc. short. hysteresis loop, although rate dependency was seen with verapamil only. Positive inotropes (eg isoproterenol and levosimendan) induced a rate-dependent increase in the area, however Omecamtiv mecarbil increased and decreased the area at CLs of 2000 and 333.3 ms, respectively. Thus, the use of different pacing rates may improve the detection of inotropes in early drug discovery and illustrate the potential for finger-printing different mechanisms of action.

Background and PurposeWe aimed to characterize the pharmacology and electrophysiology of N‐[3‐(1H‐benzimidazol‐2‐yl)‐4‐chloro‐phenyl]pyridine‐3‐carboxamide (AZSMO‐23), an activator of the human ether‐a‐go‐go‐related gene (hERG)‐encoded K+ channel (Kv11.1).Experimental ApproachAutomated electrophysiology was used to study the pharmacology of AZSMO‐23 on wild‐type (WT), Y652A, F656T or G628C/S631C hERG, and on other cardiac ion channels. Its mechanism of action was characterized with conventional electrophysiology.Key ResultsAZSMO‐23 activated WT hERG pre‐pulse and tail current with EC50 values of 28.6 and 11.2 μM respectively. At 100 μM, pre‐pulse current at +40 mV was increased by 952 ± 41% and tail current at −30 mV by 238 ± 13% compared with vehicle values. The primary mechanism for this effect was a 74.5 mV depolarizing shift in the voltage dependence of inactivation, without any shift in the voltage dependence of activation. Structure–activity relationships for this effect were remarkably subtle, with close analogues of AZSMO‐23 acting as hERG inhibitors. AZSMO‐23 blocked the mutant channel, hERG Y652A, but against another mutant channel, hERG F656T, its activator activity was enhanced. It inhibited activity of the G628C/S631C non‐inactivating hERG mutant channel. AZSMO‐23 was not hERG selective, as it blocked hKv4.3‐hKChIP2.2, hCav3.2 and hKv1.5 and activated hCav1.2/β2/α2δ channels.Conclusion and ImplicationsThe activity of AZSMO‐23 and those of its close analogues suggest these compounds may be of value to elucidate the mechanism of type 2 hERG activators to better understand the pharmacology of this area from both a safety perspective and in relation to treatment of congenital long QT syndrome.

Entry into the crucial preclinical good laboratory practice (GLP) stage of toxicology testing triggers significant R&D investment yet >20% of AstraZeneca's potential new medicines have been stopped for safety reasons in this GLP phase alone. How could we avoid at least some of these costly failures? An analysis of historical toxicities that caused stopping ('stopping toxicities') showed that >50% were attributable to target organ toxicities emerging within 2 weeks of repeat dosing or to acute cardiovascular risks. By frontloading 2-week repeat-dose toxicity studies and a comprehensive assessment of cardiovascular safety, we anticipate a potential 50% reduction in attrition in the GLP phase. This will reduce animal use overall, save significant R&D costs and improve drug pipeline quality.

Functional changes to cardiomyocytes are a common cause of attrition in preclinical and clinical drug development. Current approaches to assess cardiomyocyte contractility in vitro are limited to low-throughput methods not amenable to early drug discovery. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) were used to assess their suitability to detect drug-induced changes in cardiomyocyte contraction. Application of field stimulation and measurement of cardiac contraction (IonOptix edge detection) and Ca(2+) transients confirmed hiPS-CMs to be a suitable model to investigate drug-induced changes in cardiomyocyte contractility. Using a live cell, fast kinetic fluorescent assay with a Ca(2+) sensitive dye to test 31 inotropic and 20 non-inotropic compounds in vivo, we report that hiPS-CMs provide a high-throughput experimental model to detect changes in cardiomyocyte contraction that is applicable to early drug discovery with a sensitivity and specificity of 87% and 70%, respectively. Moreover, our data provide evidence of the detection of this liability at therapeutically relevant concentrations with throughput amenable to influencing chemical design in drug discovery. Measurement of multiple parameters of the Ca(2+) transient in addition to the number of Ca(2+) transients offered no insight into the mechanism of cardiomyocyte contraction.