Preclinical safety and biodistribution assessment of Ad-KCNH2-G628S administered via atrial painting in New Zealand white rabbits.

Post-operative atrial fibrillation (POAF) is the most common complication after cardiac surgery. Despite implementation of several pharmacological strategies, incidence of POAF remains at approximately 30%. An adenovirus vector encoding KCNH2-G628S has proven efficacious in a porcine model of AF. In this preclinical study, 1.5 × 1010 or 1.5 × 1012 Ad-KCNH2-G628S vector particles (vp) were applied to the atrial epicardium or 1.5 × 1012 vp were applied to the whole epicardial surface of New Zealand White rabbits. Saline and vector vehicle served as procedure controls. Animals were followed for up to 42 days. Vector genomes persisted in the atria up to 42 days, with no distribution to extra-thoracic organs. There were no adverse effects attributable to test article on standard toxicological endpoints or on blood pressure, left atrial or ventricular ejection fractions, electrocardiographic parameters, or serum IL-6 or troponin concentrations. Mononuclear infiltration of the myocardium of the atrial free walls of low-dose, but not high-dose animals was observed at 7 and 21 days, but these changes did not persist or affect cardiac function. After scaling for heart size, results indicate the test article is safe at doses up to 25 times the maximum proposed for the human clinical trial.

Modeling long QT syndrome type 2 on-a-chip via in-depth assessment of isogenic gene-edited 3D cardiac tissues.

Long QT syndrome (LQTS) is a cardiovascular disease characterized by QT interval prolongation that can lead to sudden cardiac death. Many mutations with heterogeneous mechanisms have been identified in KCNH2, the gene that encodes for hERG (Kv11.1), which lead to onset of LQTS type 2 (LQTS2). In this work, we developed a LQTS2-diseased tissue-on-a-chip model, using 3D coculture of isogenic stem cell-derived cardiomyocytes (CMs) and cardiac fibroblasts (CFs) within an organotypic microfluidic chip technology. Primarily, we created a hiPSC line with R531W mutation in KCNH2 using CRISPR-Cas9 gene-editing technique and characterized the resultant differentiated CMs and CFs. A deficiency in hERG trafficking was identified in KCNH2-edited hiPSC-CMs, revealing a possible mechanism of R531W mutation in LQTS2 pathophysiology. Following creation of a 3D LQTS2 tissue-on-a-chip, the tissues were extensively characterized, through analysis of calcium handling and response to ß-agonist. Furthermore, attempted phenotypic rescue via pharmacological intervention of LQTS2 on a chip was investigated.

Electrocardiogram-based index for the assessment of drug-induced hERG potassium channel block.

INTRODUCTION: Drug-induced block of the hERG potassium channel could predispose to torsade de pointes, depending on occurrence of concomitant blocks of the calcium and/or sodium channels. Since the hERG potassium channel block affects cardiac repolarization, the aim of this study was to propose a new reliable index for non-invasive assessment of drug-induced hERG potassium channel block based on electrocardiographic T-wave features. METHODS: ERD30% (early repolarization duration) and TS/A (down-going T-wave slope to T-wave amplitude ratio) features were measured in 22 healthy subjects who received, in different days, doses of dofetilide, ranolazine, verapamil and quinidine (all being hERG potassium channel blockers and the latter three being also blockers of calcium and/or sodium channels) while undergoing continuous electrocardiographic acquisition from which ERD30% and TS/A were evaluated in fifteen time points during the 24 h following drug administration ("ECG Effects of Ranolazine, Dofetilide, Verapamil, and Quinidine in Healthy Subjects" database by Physionet). A total of 1320 pairs of ERD30% and TS/A measurements, divided in training (50%) and testing (50%) datasets, were obtained. Drug-induced hERG potassium channel block was modelled by the regression equation BECG(%) = a·ERD30% + b·TS/A+ c·ERD30%·TS/A + d; BECG(%) values were compared to plasma-based measurements, BREF(%). RESULTS: Regression coefficients values, obtained on the training dataset, were: a = -561.0 s-1, b = -9.7 s, c = 77.2 and d = 138.9. In the testing dataset, correlation coefficient between BECG(%) and BREF(%) was 0.67 (p < 10-81); estimation error was -11.5 ± 16.7%. CONCLUSION: BECG(%) is a reliable non-invasive index for the assessment of drug-induced hERG potassium channel block, independently from concomitant blocks of other ions.

Assessment of proarrhythmogenic risk for chloroquine and hydroxychloroquine using the CiPA concept.

Chloroquine and hydroxychloroquine have been proposed recently as therapy for SARS-CoV-2-infected patients, but during 3 months of extensive use concerns were raised related to their clinical effectiveness and arrhythmogenic risk. Therefore, we estimated for these compounds several proarrhythmogenic risk predictors according to the Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm. Experiments were performed with either CytoPatch™2 automated or manual patch-clamp setups on HEK293T cells stably or transiently transfected with hERG1, hNav1.5, hKir2.1, hKv7.1+hMinK, and on Pluricyte® cardiomyocytes (Ncardia), using physiological solutions. Dose-response plots of hERG1 inhibition fitted with Hill functions yielded IC50 values in the low micromolar range for both compounds. We found hyperpolarizing shifts of tens of mV, larger for chloroquine, in the voltage-dependent activation but not inactivation, as well as a voltage-dependent block of hERG current, larger at positive potentials. We also found inhibitory effects on peak and late INa and on IK1, with IC50 of tens of µM and larger for chloroquine. The two compounds, tested on Pluricyte® cardiomyocytes using the ß-escin-perforated method, inhibited IKr, ICaL, INa peak, but had no effect on If. In current-clamp they caused action potential prolongation. Our data and those from literature for Ito were used to compute proarrhythmogenic risk predictors Bnet (Mistry HB, 2018) and Qnet (Dutta S et al., 2017), with hERG1 blocking/unblocking rates estimated from time constants of fractional block. Although the two antimalarials are successfully used in autoimmune diseases, and chloroquine may be effective in atrial fibrillation, assays place these drugs in the intermediate proarrhythmogenic risk group.

Intracellular uptake of agents that block the hERG channel can confound the assessment of QT interval prolongation and arrhythmic risk.

BACKGROUND: Oliceridine is a biased ligand at the µ-opioid receptor recently approved for the treatment of acute pain. In a thorough QT study, corrected QT (QTc) prolongation displayed peaks at 2.5 and 60 minutes after a supratherapeutic dose. The mean plasma concentration peaked at 5 minutes, declining rapidly thereafter. OBJECTIVE: The purpose of this study was to examine the basis for the delayed effect of oliceridine to prolong the QTc interval. METHODS: Repolarization parameters and tissue accumulation of oliceridine were evaluated in rabbit left ventricular wedge preparations over a period of 5 hours. The effects of oliceridine on ion channel currents were evaluated in human embryonic kidney and Chinese hamster ovary cells. Quinidine was used as a control. RESULTS: Oliceridine and quinidine produced a progressive prolongation of the QTc interval and action potential duration over a period of 5 hours, paralleling slow progressive tissue uptake of the drugs. Oliceridine caused modest prolongation of these parameters, whereas quinidine produced a prominent prolongation of action potential duration and QTc interval as well as development of early afterdepolarization (after 2 hours), resulting in a high torsades de pointes score. The 50% inhibitory concentration values for the oliceridine inhibition of the rapidly activating delayed rectifier current (human ether a-go-go current) and late sodium channel current were 2.2 and 3.45 µM when assessed after traditional acute exposure but much lower after 3 hours of drug exposure. CONCLUSION: Our findings suggest that a gradual increase of intracellular access of drugs to the hERG channels as a result of their intracellular uptake and accumulation can significantly delay effects on repolarization, thus confounding the assessment of QT interval prolongation and arrhythmic risk when studied acutely. The multi-ion channel effects of oliceridine, late sodium channel current inhibition in particular, point to a low risk of devloping torsades de pointes.

Toward in vivo-relevant hERG safety assessment and mitigation strategies based on relationships between non-equilibrium blocker binding, three-dimensional channel-blocker interactions, dynamic occupancy, dynamic exposure, and cellular arrhythmia.

The human ether-a-go-go-related voltage-gated cardiac ion channel (commonly known as hERG) conducts the rapid outward repolarizing potassium current in cardiomyocytes (IKr). Inadvertent blockade of this channel by drug-like molecules represents a key challenge in pharmaceutical R&D due to frequent overlap between the structure-activity relationships of hERG and many primary targets. Building on our previous work, together with recent cryo-EM structures of hERG, we set about to better understand the energetic and structural basis of promiscuous blocker-hERG binding in the context of Biodynamics theory. We propose a two-step blocker binding process consisting of: The initial capture step: diffusion of a single fully solvated blocker copy into a large cavity lined by the intra-cellular cyclic nucleotide binding homology domain (CNBHD). Occupation of this cavity is a necessary but insufficient condition for ion current disruption.The IKr disruption step: translocation of the captured blocker along the channel axis, such that: The head group, consisting of a quasi-rod-shaped moiety, projects into the open pore, accompanied by partial de-solvation of the binding interface.One tail moiety packs along a kink between the S6 helix and proximal C-linker helix adjacent to the intra-cellular entrance of the pore, likewise accompanied by mutual de-solvation of the binding interface (noting that the association barrier is comprised largely of the total head + tail group de-solvation cost).Blockers containing a highly planar moiety that projects into a putative constriction zone within the closed channel become trapped upon closing, as do blockers terminating prior to this region.A single captured blocker copy may conceivably associate and dissociate to/from the pore many times before exiting the CNBHD cavity. Lastly, we highlight possible flaws in the current hERG safety index (SI), and propose an alternate in vivo-relevant strategy factoring in: Benefit/risk.The predicted arrhythmogenic fractional hERG occupancy (based on action potential (AP) simulations of the undiseased human ventricular cardiomyocyte).Alteration of the safety threshold due to underlying disease.Risk of exposure escalation toward the predicted arrhythmic limit due to patient-to-patient pharmacokinetic (PK) variability, drug-drug interactions, overdose, and use for off-label indications in which the hERG safety parameters may differ from their on-label counterparts.

Safety assessment of metarrestin in dogs: A clinical candidate targeting a subnuclear structure unique to metastatic cancer cells.

Pancreatic cancer is a leading cause of cancer-related deaths in the U.S. Ninety percent of patients with stage IV pancreatic cancer die within one year of diagnosis due to complications of metastasis. A metastatic potential of cancer cells has been shown to be closely associated with formation of perinucleolar compartment (PNC). Metarrestin, a first-in-class PNC inhibitor, was evaluated for its toxicity, toxicokinetics, and safety pharmacology in beagle dogs following every other day oral (capsule) administration for 28 days to support its introduction into clinical trials. The study consisted of four dose groups: vehicle; 0.25, 0.75 and 1.50 mg/kg/dose. Metarrestin reached its maximum concentration in blood at 3 h (overall median Tmax) across all doses with a mean t1/2 over 168 h of 55.5 h. Dose dependent increase in systemic exposure (Cmax and AUClast) with no sex difference was observed on days 1 and 27. Metarrestin accumulated from Day 1 to Day 27 at all dose levels and in both sexes by an overall factor of about 2.34. No mortality occurred during the dosing period; however, treatment-related clinical signs of toxicity consisting of hypoactivity, shaking/shivering, thinness, irritability, salivation, abnormal gait, tremors, ataxia and intermittent seizure-like activity were seen in both sexes at mid and high dose groups. Treatment-related effects on body weight and food consumption were seen at the mid and high dose levels. Safety pharmacology study showed no treatment-related effects on blood pressure, heart rate, corrected QT, PR, RR, or QRS intervals, or respiratory function parameters (respiratory rate, tidal volume, minute volume). There were no histopathological changes observed, with the exception of transient thymic atrophy which was considered to be non-adverse. Based primarily on clinical signs of toxicity, the No Observed Adverse Effect Level (NOAEL) in dogs was considered to be 0.25 mg/kg metarrestin after every other day dosing for 28 days with a mean of male and female Cmax = 82.5 ng/mL and AUClast = 2521 h*ng/mL, on Day 27.

A rapid solubility assay of protein domain misfolding for pathogenicity assessment of rare DNA sequence variants.

PURPOSE: DNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care. METHODS: We report an E. coli-based solubility assay for assessing the effects of variants on protein domain stability for three disease-associated proteins. RESULTS: First, we examined variants in the Kv11.1 channel PAS domain (PASD) associated with inherited long QT syndrome type 2 and found that protein solubility correlated well with reported in vitro protein stabilities. A comprehensive solubility analysis of 56 Kv11.1 PASD variants revealed that disruption of membrane trafficking, the dominant loss-of-function disease mechanism, is largely determined by domain stability. We further validated this assay by using it to identify second-site suppressor PASD variants that improve domain stability and Kv11.1 protein trafficking. Finally, we applied this assay to several cancer-linked P53 tumor suppressor DNA-binding domain and myopathy-linked Lamin A/C Ig-like domain variants, which also correlated well with reported protein stabilities and functional analyses. CONCLUSION: This simple solubility assay can aid in determining the likelihood of pathogenicity for sequence variants due to protein misfolding in structured domains of disease-associated genes as well as provide insights into the structural basis of disease.

Reliable identification of cardiac liability in drug discovery using automated patch clamp: Benchmarking best practices and calibration standards for improved proarrhythmic assessment.

INTRODUCTION: Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an alternative to manual patch clamp in order to increase throughput whilst maintaining data quality. In order to standardize APC experiments, we have investigated the effects on IC50 values under different conditions using several devices across multiple sites. METHODS: APC instruments SyncroPatch 384i, SyncroPatch 384PE and Patchliner, were used to record hERG expressed in HEK or CHO cells. Up to 27 CiPA compounds were used to investigate effects of voltage protocol, incubation time, labware and time between compound preparation and experiment on IC50 values. RESULTS: All IC50 values of 21 compounds recorded on the SyncroPatch 384PE correlated well with IC50 values from the literature (Kramer et al., 2013) regardless of voltage protocol or labware, when compounds were used immediately after preparation, but potency of astemizole decreased if prepared in Teflon or polypropylene (PP) compound plates 2-3 h prior to experiments. Slow acting compounds such as dofetilide, astemizole, and terfenadine required extended incubation times of at least 6 min to reach steady state and therefore, stable IC50 values. DISCUSSION: Assessing the influence of different experimental conditions on hERG assay reliability, we conclude that either the step-ramp protocol recommended by CiPA or a standard 2-s step-pulse protocol can be used to record hERG; a minimum incubation time of 5 min should be used and although glass, Teflon, PP or polystyrene (PS) compound plates can be used for experiments, caution should be taken if using Teflon, PS or PP vessels as some adsorption can occur if experiments are not performed immediately after preparation. Our recommendations are not limited to the APC devices described in this report, but could also be extended to other APC devices.

Quantitative T-wave morphology assessment from surface ECG is linked with cardiac events risk in genotype-positive KCNH2 mutation carriers with normal QTc values.

INTRODUCTION: Long QT syndrome (LQTS) mutation carriers have elevated the risk of cardiac events even in the absence of QTc prolongation; however, mutation penetrance in patients with normal QTc may be reflected in abnormal T-wave shape, particularly in KCNH2 mutation carriers. We aimed to assess whether the magnitude of a three-dimensional T-wave vector (TwVM) will identify KCNH2-mutation carriers with normal QTc at risk for cardiac events. METHODS: Adult LQT2 patients with QTc < 460 ms in men and <470 ms in women (n = 113, age 42 ± 16 years, 43% male) were compared with genotype-negative family members (n = 1007). The TwVM was calculated using T-wave amplitudes in leads V6, II, and V2 as the square root of (TV62 + TII2 + (0.5*TV2)2 ). Cox regression analysis adjusted for gender and time-dependent beta-blocker use was performed to assess cardiac event (CE) risk, defined as syncope, aborted cardiac arrest, implantable cardioverter-defibrillator therapy, or sudden death. RESULTS: Dichotomized by median of 0.30 mV, lower TwVM was associated with elevated CE risk compared to those with high TwVM (HR = 2.95, 95% CI, 1.25-6.98, P = .014) and also remained significant after including sex and time-dependent beta-blocker usage in the Cox regression analysis (HR = 2.64, 95% CI, 1.64-4.24, P < .001). However, these associations were found only in women but not in men who had low event rates. CONCLUSION: T-wave morphology quantified as repolarization vector magnitude using T-wave amplitudes retrieved from standard 12-lead electrocardiogram predicts cardiac events risk in LQT2 women and appears useful for risk stratification of KCNH2-mutation carriers without QTc prolongation.

Assessment of an In Silico Mechanistic Model for Proarrhythmia Risk Prediction Under the CiPA Initiative.

The International Council on Harmonization (ICH) S7B and E14 regulatory guidelines are sensitive but not specific for predicting which drugs are pro-arrhythmic. In response, the Comprehensive In Vitro Proarrhythmia Assay (CiPA) was proposed that integrates multi-ion channel pharmacology data in vitro into a human cardiomyocyte model in silico for proarrhythmia risk assessment. Previously, we reported the model optimization and proarrhythmia metric selection based on CiPA training drugs. In this study, we report the application of the prespecified model and metric to independent CiPA validation drugs. Over two validation datasets, the CiPA model performance meets all pre-specified measures for ranking and classifying validation drugs, and outperforms alternatives, despite some in vitro data differences between the two datasets due to different experimental conditions and quality control procedures. This suggests that the current CiPA model/metric may be fit for regulatory use, and standardization of experimental protocols and quality control criteria could increase the model prediction accuracy even further.

Electrophysiological evaluation of pentamidine and 17-AAG in human stem cell-derived cardiomyocytes for safety assessment.

Human ether-a-go-go-related gene (hERG) trafficking inhibition is known to be one of the mechanisms of indirect hERG inhibition, resulting in QT prolongation and lethal arrhythmia. Pentamidine, an antiprotozoal drug, causes QT prolongation/Torsades de Pointes (TdP) via hERG trafficking inhibition, but 17-AAG, a geldanamycin derivative heat shock protein 90 (Hsp90) inhibitor, has not shown torsadogenic potential clinically, despite Hsp90 inhibitors generally being hypothesized to cause TdP by hERG trafficking inhibition. In the present study, we investigated the underlying mechanisms of both drugs' actions on hERG channels using hERG-overexpressing CHO cells (hERG-CHOs) and human embryonic stem cell-derived cardiomyocytes (hES-CMs). The effects on hERG tail current and protein levels were evaluated using population patch clamp and Western blotting in hERG-CHOs. The effects on field potential duration (FPD) were recorded by a multi-electrode array (MEA) in hES-CMs. Neither drug affected hERG tail current acutely. Chronic treatment with each drug inhibited hERG tail current and decreased the mature form of hERG protein in hERG-CHOs, whereas the immature form of hERG protein was increased by pentamidine but decreased by 17-AAG. In MEA assays using hES-CMs, pentamidine time-dependently prolonged FPD, but 17-AAG shortened it. The FPD prolongation in hES-CMs upon chronic pentamidine exposure is relevant to its clinically reported arrhythmic risk. Cav1.2 or Nav1.5 current were not reduced by chronic application of either drug at a relevant concentration to hERG trafficking inhibition in human embryonic kidney (HEK293) cells. Therefore, the reason why chronic 17-AAG shortened the FPD despite the hERG trafficking inhibition occur is still unknown.

Towards Bridging Translational Gap in Cardiotoxicity Prediction: an Application of Progressive Cardiac Risk Assessment Strategy in TdP Risk Assessment of Moxifloxacin.

Drug-induced cardiac arrhythmia, especially occurrence of torsade de pointes (TdP), has been a leading cause of attrition and post-approval re-labeling and withdrawal of many drugs. TdP is a multifactorial event, reflecting more than just drug-induced cardiac ion channel inhibition and QT interval prolongation. This presents a translational gap in extrapolating pre-clinical and clinical cardiac safety assessment to estimate TdP risk reliably, especially when the drug of interest is used in combination with other QT-prolonging drugs for treatment of diseases such as tuberculosis. A multi-scale mechanistic modeling framework consisting of physiologically based pharmacokinetics (PBPK) simulations of clinically relevant drug exposures combined with Quantitative Systems Toxicology (QST) models of cardiac electro-physiology could bridge this gap. We illustrate this PBPK-QST approach in cardiac risk assessment as exemplified by moxifloxacin, an anti-tuberculosis drug with abundant clinical cardiac safety data. PBPK simulations of moxifloxacin concentrations (systemic circulation and estimated in heart tissue) were linked with in vitro measurements of cardiac ion channel inhibition to predict the magnitude of QT prolongation in healthy individuals. Predictions closely reproduced the clinically observed QT interval prolongation, but no arrhythmia was observed, even at ×10 exposure. However, the same exposure levels in presence of physiological risk factors, e.g., hypokalemia and tachycardia, led to arrhythmic event in simulations, consistent with reported moxifloxacin-related TdP events. Application of a progressive PBPK-QST cardiac risk assessment paradigm starting in early development could guide drug development decisions and later define a clinical "safe space" for post-approval risk management to identify high-risk clinical scenarios.

Drug interaction at hERG channel: In vitro assessment of the electrophysiological consequences of drug combinations and comparison against theoretical models.

Drugs carry a proarrhythmic risk, which gets even greater when they are used in combination. In vitro assessment of the proarrhythmic potential of drugs is limited to one compound and thus neglects the potential of drug-drug interactions, including those involving active metabolites. Here we present the results of an in vitro study of potential drug-drug interactions at the level of the hERG channel for the combination of up to three compounds: loratadine, desloratadine and ketoconazole. Experiments were performed at room temperature on an automated patch-clamp device CytoPatch 2, with the use of heterogeneously, stably transfected HEK cells. Single drugs, pairs and triplets were used. The results provided as the inhibition of the IKr current for pairs were compared against the calculated theoretical interaction. Models applied to calculate the combined effect of inhibitory actions of simultaneously given drugs include: (1) simple additive model with a maximal inhibition limit of 1 (all channels blocked in 100%); (2) Bliss independence; and (3) Loewe additivity. The observed IC50 values for loratadine, desloratadine and ketoconazole were 5.15, 1.95 and 0.74 µm respectively. For the combination of drugs tested in pairs, the effect was concentration dependent. In lower concentrations, the synergistic effect was observed, while for the highest tested concentrations it was subadditive. To triple the effect, it was subadditive regardless of concentrations. The square root of sum of squares of differences between the observed and predicted total inhibition was calculated to assess the theoretical interaction models. For most of the drugs, the allotopic model offered the best fit.

Real Patient and its Virtual Twin: Application of Quantitative Systems Toxicology Modelling in the Cardiac Safety Assessment of Citalopram.

A quantitative systems toxicology (QST) model for citalopram was established to simulate, in silico, a 'virtual twin' of a real patient to predict the occurrence of cardiotoxic events previously reported in patients under various clinical conditions. The QST model considers the effects of citalopram and its most notable electrophysiologically active primary (desmethylcitalopram) and secondary (didesmethylcitalopram) metabolites, on cardiac electrophysiology. The in vitro cardiac ion channel current inhibition data was coupled with the biophysically detailed model of human cardiac electrophysiology to investigate the impact of (i) the inhibition of multiple ion currents (IKr, IKs, ICaL); (ii) the inclusion of metabolites in the QST model; and (iii) unbound or total plasma as the operating drug concentration, in predicting clinically observed QT prolongation. The inclusion of multiple ion channel current inhibition and metabolites in the simulation with unbound plasma citalopram concentration provided the lowest prediction error. The predictive performance of the model was verified with three additional therapeutic and supra-therapeutic drug exposure clinical cases. The results indicate that considering only the hERG ion channel inhibition of only the parent drug is potentially misleading, and the inclusion of active metabolite data and the influence of other ion channel currents should be considered to improve the prediction of potential cardiac toxicity. Mechanistic modelling can help bridge the gaps existing in the quantitative translation from preclinical cardiac safety assessment to clinical toxicology. Moreover, this study shows that the QST models, in combination with appropriate drug and systems parameters, can pave the way towards personalised safety assessment.

Nonclinical safety assessment of SPX-101, a novel peptide promoter of epithelial sodium channel internalization for the treatment of cystic fibrosis.

BACKGROUND: ENaC inhibition has long been an attractive therapeutic target for the treatment of cystic fibrosis. However, previous attempts at developing ENaC inhibitors have been unsuccessful due to complications arising from systemic circulation of the compounds. Here, we describe the preclinical toxicology assessment of a new inhaled peptide promoter of ENaC internalization delivered as a nebulized aerosol. METHODS: Preclinical assessment of SPX-101 safety was determined using an in vitro hERG assay, bolus injection of SPX-101 in a canine cardiovascular and respiratory safety pharmacology model and 28-day inhalation toxicology studies of nebulized drug in rats and dogs. RESULTS: SPX101 had no effects on the respiratory, cardiac or central nervous systems. The 28-day inhalation toxicology studies of nebulized SPX-101 in rats and dogs revealed no drug-related adverse events. Plasma levels of SPX-101 peaked less than 1 h after the end of treatment in rats and were below the limit of detection in canine models. CONCLUSIONS: SPX-101, a novel peptide promoter of ENaC internalization, elicited no adverse effects at doses up to the MFD and in excess of the highest preclinical efficacious and expected clinical doses. In contrast to channel blockers like amiloride and derivative small molecules, SPX-101 does not achieve significant systemic circulation, thus doses are not limited due to toxic side effects like hyperkalemia and weight loss.

In silico assessment of the effects of quinidine, disopyramide and E-4031 on short QT syndrome variant 1 in the human ventricles.

AIMS: Short QT syndrome (SQTS) is an inherited disorder associated with abnormally abbreviated QT intervals and an increased incidence of atrial and ventricular arrhythmias. SQT1 variant (linked to the rapid delayed rectifier potassium channel current, IKr) of SQTS, results from an inactivation-attenuated, gain-of-function mutation (N588K) in the KCNH2-encoded potassium channels. Pro-arrhythmogenic effects of SQT1 have been well characterized, but less is known about the possible pharmacological antiarrhythmic treatment of SQT1. Therefore, this study aimed to assess the potential effects of E-4031, disopyramide and quinidine on SQT1 using a mathematical model of human ventricular electrophysiology. METHODS: The ten Tusscher et al. biophysically detailed model of the human ventricular action potential (AP) was modified to incorporate IKr Markov chain (MC) formulations based on experimental data of the kinetics of the N588K mutation of the KCNH2-encoded subunit of the IKr channels. The modified ventricular cell model was then integrated into one-dimensional (1D) strand, 2D regular and realistic tissues with transmural heterogeneities. The channel-blocking effect of the drugs on ion currents in healthy and SQT1 cells was modeled using half-maximal inhibitory concentration (IC50) and Hill coefficient (nH) values from literatures. Effects of drugs on cell AP duration (APD), effective refractory period (ERP) and pseudo-ECG traces were calculated. Effects of drugs on the ventricular temporal and spatial vulnerability to re-entrant excitation waves were measured. Re-entry was simulated in both 2D regular and realistic ventricular tissue. RESULTS: At the single cell level, the drugs E-4031 and disopyramide had hardly noticeable effects on the ventricular cell APD at 90% repolarization (APD90), whereas quinidine caused a significant prolongation of APD90. Quinidine prolonged and decreased the maximal transmural AP heterogeneity (δV); this led to the decreased transmural heterogeneity of APD across the 1D strand. Quinidine caused QT prolongation and a decrease in the T-wave amplitude, and increased ERP and decreased temporal susceptibility of the tissue to the initiation of re-entry and increased the minimum substrate size necessary to prevent re-entry in the 2D regular model, and further terminated re-entrant waves in the 2D realistic model. Quinidine exhibited significantly better therapeutic effects on SQT1 than E-4031 and disopyramide. CONCLUSIONS: The simulated pharmacological actions of quinidine exhibited antiarrhythmic effects on SQT1. This study substantiates a causal link between quinidine and QT interval prolongation in SQT1 and suggests that quinidine may be a potential pharmacological agent for treating SQT1 patients.

Recapitulation of Clinical Individual Susceptibility to Drug-Induced QT Prolongation in Healthy Subjects Using iPSC-Derived Cardiomyocytes.

To predict drug-induced serious adverse events (SAE) in clinical trials, a model using a panel of cells derived from human induced pluripotent stem cells (hiPSCs) of individuals with different susceptibilities could facilitate major advancements in translational research in terms of safety and pharmaco-economics. However, it is unclear whether hiPSC-derived cells can recapitulate interindividual differences in drug-induced SAE susceptibility in populations not having genetic disorders such as healthy subjects. Here, we evaluated individual differences in SAE susceptibility based on an in vitro model using hiPSC-derived cardiomyocytes (hiPSC-CMs) as a pilot study. hiPSCs were generated from blood samples of ten healthy volunteers with different susceptibilities to moxifloxacin (Mox)-induced QT prolongation. Different Mox-induced field potential duration (FPD) prolongation values were observed in the hiPSC-CMs from each individual. Interestingly, the QT interval was significantly positively correlated with FPD at clinically relevant concentrations (r > 0.66) in multiple analyses including concentration-QT analysis. Genomic analysis showed no interindividual significant differences in known target-binding sites for Mox and other drugs such as the hERG channel subunit, and baseline QT ranges were normal. The results suggest that hiPSC-CMs from healthy subjects recapitulate susceptibility to Mox-induced QT prolongation and provide proof of concept for in vitro preclinical trials.

Monte Carlo method for predicting of cardiac toxicity: hERG blocker compounds.

The estimation of the cardiotoxicity of compounds is an important task for the drug discovery as well as for the risk assessment in ecological aspect. The experimental estimation of the above endpoint is complex and expensive. Hence, the theoretical computational methods are very attractive alternative of the direct experiment. A model for cardiac toxicity of 400 hERG blocker compounds (pIC50) is built up using the Monte Carlo method. Three different splits into the visible training set (in fact, the training set plus the calibration set) and invisible validation sets examined. The predictive potential is very good for all examined splits. The statistical characteristics for the external validation set are (i) the coefficient of determination r(2)=(0.90-0.93); and (ii) root-mean squared error s=(0.30-0.40).

CSAHi study: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes -assessment of inter-facility and cells lot-to-lot-variability.

In vitro screening of hERG channels are recommended under ICH S7B guidelines to predict drug-induced QT prolongation and Torsade de Pointes (TdP), whereas proarrhythmia is known to be evoked by blockage of other ion channels involved in cardiac contraction and compensation mechanisms. A consortium for drug safety assessment using human iPS cells-derived cardiomyocytes (hiPS-CMs), CSAHi, has been organized to establish a novel in vitro test system that would enable better prediction of drug-induced proarrhythmia and QT prolongation. Here we report the inter-facility and cells lot-to-lot variability evaluated with FPDc (corrected field potential duration), FPDc10 (10% FPDc change concentration), beat rate and incidence of arrhythmia-like waveform or arrest on hiPS-CMs in a multi-electrode array system. Arrhythmia-like waveforms were evident for all test compounds, other than chromanol 293B, that evoked FPDc prolongation in this system and are reported to induce TdP in clinical practice. There was no apparent cells lot-to-lot variability, while inter-facility variabilities were limited within ranges from 3.9- to 20-folds for FPDc10 and about 10-folds for the minimum concentration inducing arrhythmia-like waveform or arrests. In conclusion, the new assay model reported here would enable accurate prediction of a drug potential for proarrhythmia.