Pharmacological Characterization of a Novel 5-Hydroxybenzothiazolone-Derived ß 2-Adrenoceptor Agonist with Functional Selectivity for Anabolic Effects on Skeletal Muscle Resulting in a Wider Cardiovascular Safety Window in Preclinical Studies.

The anabolic effects of ß 2-adrenoceptor (ß 2-AR) agonists on skeletal muscle have been demonstrated in various species. However, the clinical use of ß 2-AR agonists for skeletal muscle wasting conditions has been limited by their undesired cardiovascular effects. Here, we describe the preclinical pharmacological profile of a novel 5-hydroxybenzothiazolone (5-HOB) derived ß 2-AR agonist in comparison with formoterol as a representative ß 2-AR agonist that have been well characterized. In vitro, 5-HOB has nanomolar affinity for the human ß 2-AR and selectivity over the ß 1-AR and ß 3-AR. 5-HOB also shows potent agonistic activity at the ß 2-AR in primary skeletal muscle myotubes and induces hypertrophy of skeletal muscle myotubes. Compared with formoterol, 5-HOB demonstrates comparable full-agonist activity on cAMP production in skeletal muscle cells and skeletal muscle tissue-derived membranes. In contrast, a greatly reduced intrinsic activity was determined in cardiomyocytes and cell membranes prepared from the rat heart. In addition, 5-HOB shows weak effects on chronotropy, inotropy, and vascular relaxation compared with formoterol. In vivo, 5-HOB significantly increases hind limb muscle weight in rats with attenuated effects on heart weight and ejection fraction, unlike formoterol. Furthermore, changes in cardiovascular parameters after bolus subcutaneous treatment in rats and rhesus monkeys are significantly lower with 5-HOB compared with formoterol. In conclusion, the pharmacological profile of 5-HOB indicates superior tissue selectivity compared with the conventional ß 2-AR agonist formoterol in preclinical studies and supports the notion that such tissue-selective agonists should be investigated for the safe treatment of muscle-wasting conditions without cardiovascular limiting effects.

Republished: A straightforward guide to the basic science behind arrhythmogenesis.

The underlying mechanisms behind cardiac arrhythmias are described in this manuscript. In clinical practice, significant arrhythmias are unpredictable, and under some conditions, potentially life-threatening. How can basic science help improve our understanding of molecular entities and factors behind the arrhythmia to advance, develop, adapt or deliver available medications? Structural heart disease and remodelling (e.g., heart failure, cardiomyopathy), the presence of modulating factors (i.e., diabetes mellitus, autonomic nervous system), genetic predispositions (i.e., channelopathies) are considerable preconditions, and influence the development of an arrhythmia. Cardiac arrhythmias may indeed share common basic mechanisms, while elements and substrates perpetuating these may be different and ultimately manifest as various ECG abnormalities. This article lists cellular and subcellular iatrogenic disorders responsible for abnormal impulse generation, or conduction disturbances, including the latest development in theories and biological research, for a better understanding of cellular disorders behind arrhythmogenesis.

Preclinical mitigation of 5-HT2B agonism-related cardiac valvulopathy revisited.

Cardiac valvulopathy (Cardiac Valve Disease; CVD) associated with off-target activation of the 5-hydroxytryptamine (5-HT) 2B receptor has been well recognized, but is still poorly predicted during drug development. The regulatory guidance proposes the use of 5-HT2B binding data (i.e., Ki values) and free maximum therapeutic exposure (Cmax) to calculate safety margins as a threshold of detection (>10) for eliminating the risk of drug-induced cardiac valvulopathy. In this paper, we provide additional recommendations for preclinical prediction of CVD risk based on clinical pharmacodynamic and pharmacokinetic data obtained from drugs with or without 5-HT2B receptor activation. Our investigations showed that 5-HT2B agonist affinity of molecules tested in an in vitro 5-HT2B cell-based functional assay, placed in perspective to their sustained plasma exposure (AUCs) and not to their peak plasma exposure, Cmax (i.e., maximum therapeutic exposure) provide a solid basis for interpreting 5-HT2B data, for calculating safety margins and then, accurately differentiate drugs associated with a clinical risk of CVD from those which are not (despite having some agonist 5-HT2B activity). In addition, we discuss the risk of multi-organ fibrosis linked to 5-HT2B receptor activation, often underestimated, however well reported in FAERS for 5-HT2B agonists. We believe that our recommendations have the potential to mitigate the risk for the clinical development of CVD and fibrosis.

The state of the art in secondary pharmacology and its impact on the safety of new medicines.

Secondary pharmacology screening of investigational small-molecule drugs for potentially adverse off-target activities has become standard practice in pharmaceutical research and development, and regulatory agencies are increasingly requesting data on activity against targets with recognized adverse effect relationships. However, the screening strategies and target panels used by pharmaceutical companies may vary substantially. To help identify commonalities and differences, as well as to highlight opportunities for further optimization of secondary pharmacology assessment, we conducted a broad-ranging survey across 18 companies under the auspices of the DruSafe leadership group of the International Consortium for Innovation and Quality in Pharmaceutical Development. Based on our analysis of this survey and discussions and additional research within the group, we present here an overview of the current state of the art in secondary pharmacology screening. We discuss best practices, including additional safety-associated targets not covered by most current screening panels, and present approaches for interpreting and reporting off-target activities. We also provide an assessment of the safety impact of secondary pharmacology screening, and a perspective on opportunities and challenges in this rapidly developing field.

Optimization of a Class of Dihydrobenzofurane Analogs toward Orally Efficacious YAP-TEAD Protein-Protein Interaction Inhibitors.

The inhibition of the YAP-TEAD protein-protein interaction constitutes a promising therapeutic approach for the treatment of cancers linked to the dysregulation of the Hippo signaling pathway. The identification of a class of small molecules which potently inhibit the YAP-TEAD interaction by binding tightly to the Ω-loop pocket of TEAD has previously been communicated. This report details the further multi-parameter optimization of this class of compounds resulting in advanced analogs combining nanomolar cellular potency with a balanced ADME and off-target profile, and efficacy of these compounds in tumor bearing mice is demonstrated for the first time.

Drug-induced QTC prolongation dangerously underestimates proarrhythmic potential: lessons from terfenadine.

BACKGROUND: Terfenadine's proarrhythmia prompted market withdrawal; therapeutic antihistaminic concentration is less than 1 nM, whereas IC50 of IKr and INa exceed 200 nM. METHODS AND RESULTS: Rabbit hearts were perfused with terfenadine (1-10,000 nM; 10-450 minutes). A dosage of 1 nM tended to shorten action potential duration (APD60) (-30 ± 30.5 ms; n = 6); 10 nM (450 minutes) significantly prolonged APD60 (46 ± 11 ms; n = 6), but after 1 hour washout, APD60 further prolonged. Above 30 nM, APD60 shortening was followed by prolongation; net effect depended on exposure time (n = 33). In the µM range, cardiac wavelength (λ) shortened (APD60 shortened, conduction slowed; P < 0.05). Terfenadine induced triangulation, reverse use dependence, instability and dispersion of repolarization (TRIaD) at 1 to 1000 nM, increasing with concentration (450 minutes: 1 nM yielded 50% of hearts, 10 nM 100%) and exposure (100 nM: 10 minutes yielded 16%, 30 minutes 33%, 150 minutes 66%, 450 minutes 100%). TRIaD with APD prolongation preceded two Torsade de Pointes, with shortening seven ventricular tachycardia and five ventricular fibrillation. Terfenadine causes normally little QTc prolongation in patients and Food and Drug Administration records suggest that incidence of ventricular tachycardia/ventricular fibrillation exceeds Torsade de Pointes. CONCLUSION: For terfenadine, TRIaD predicts drug-induced proarrhythmia: with λ prolongation, Torsade de Pointes is preferred, otherwise ventricular tachycardia/ventricular fibrillation. APD/QTc alone is clearly inadequate for proarrhythmia evaluation.

Cardiotoxic Potential of Hydroxychloroquine, Chloroquine and Azithromycin in Adult Human Primary Cardiomyocytes.

Substantial efforts have been recently committed to develop coronavirus disease-2019 (COVID-19) medications, and Hydroxychloroquine alone or in combination with Azithromycin has been promoted as a repurposed treatment. Although these drugs may increase cardiac toxicity risk, cardiomyocyte mechanisms underlying this risk remain poorly understood in humans. Therefore, we evaluated the proarrhythmia risk and inotropic effects of these drugs in the cardiomyocyte contractility-based model of the human heart. We found Hydroxychloroquine to have a low proarrhythmia risk, whereas Chloroquine and Azithromycin were associated with high risk. Hydroxychloroquine proarrhythmia risk changed to high with low level of K+, whereas high level of Mg2+ protected against proarrhythmic effect of high Hydroxychloroquine concentrations. Moreover, therapeutic concentration of Hydroxychloroquine caused no enhancement of elevated temperature-induced proarrhythmia. Polytherapy of Hydroxychloroquine plus Azithromycin and sequential application of these drugs were also found to influence proarrhythmia risk categorization. Hydroxychloroquine proarrhythmia risk changed to high when combined with Azithromycin at therapeutic concentration. However, Hydroxychloroquine at therapeutic concentration impacted the cardiac safety profile of Azithromycin and its proarrhythmia risk only at concentrations above therapeutic level. We also report that Hydroxychloroquine and Chloroquine, but not Azithromycin, decreased contractility while exhibiting multi-ion channel block features, and Hydroxychloroquine's contractility effect was abolished by Azithromycin. Thus, this study has the potential to inform clinical studies evaluating repurposed therapies, including those in the COVID-19 context. Additionally, it demonstrates the translational value of the human cardiomyocyte contractility-based model as a key early discovery path to inform decisions on novel therapies for COVID-19, malaria, and inflammatory diseases.

Preclinical cardiac safety assessment of pharmaceutical compounds using an integrated systems-based computer model of the heart.

Blockade of the delayed rectifier potassium channel current, I(Kr), has been associated with drug-induced QT prolongation in the electrocardiogram and life-threatening cardiac arrhythmias. However, it is increasingly clear that compound-induced interactions with multiple cardiac ion channels may significantly affect QT prolongation that would result from inhibition of only I(Kr) [Redfern, W.S., Carlsson, L., et al., 2003. Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development. Cardiovasc. Res. 58(1), 32-45]. Such an assessment may not be feasible in vitro, due to multi-factorial processes that are also time-dependent and highly non-linear. Limited preclinical data, I(Kr) hERG assay and canine Purkinje fiber (PF) action potentials (APs) [Gintant, G.A., Limberis, J.T., McDermott, J.S., Wegner, C.D., Cox, B.F., 2001. The canine Purkinje fiber: an in vitro model system for acquired long QT syndrome and drug-induced arrhythmogenesis. J. Cardiovasc. Pharmacol. 37(5), 607-618], were used for two test compounds in a systems-based modeling platform of cardiac electrophysiology [Muzikant, A.L., Penland, R.C., 2002. Models for profiling the potential QT prolongation risk of drugs. Curr. Opin. Drug. Discov. Dev. 5(1), 127-35] to: (i) convert a canine myocyte model to a PF model by training functional current parameters to the AP data; (ii) reverse engineer the compounds' effects on five channel currents other than I(Kr), predicting significant IC(50) values for I(Na+), sustained and I(Ca2+), L-type , which were subsequently experimentally validated; (iii) use the predicted (I(Na+), sustained and I(Ca2+), L-type) and measured (I(Kr)) IC(50) values to simulate dose-dependent effects of the compounds on APs in endocardial, mid-myocardial, and epicardiac ventricular cells; and (iv) integrate the three types of cellular responses into a tissue-level spatial model, which quantifiably predicted no potential for the test compounds to induce either QT prolongation or increased transmural dispersion of repolarization in a dose-dependent and reverse rate-dependent fashion, despite their inhibition of I(Kr) in vitro.

Blinded Contractility Analysis in hiPSC-Cardiomyocytes in Engineered Heart Tissue Format: Comparison With Human Atrial Trabeculae.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) may serve as a new assay for drug testing in a human context, but their validity particularly for the evaluation of inotropic drug effects remains unclear. In this blinded analysis, we compared the effects of 10 indicator compounds with known inotropic effects in electrically stimulated (1.5 Hz) hiPSC-CM-derived 3-dimensional engineered heart tissue (EHT) and human atrial trabeculae (hAT). Human EHTs were prepared from iCell hiPSC-CM, hAT obtained at routine heart surgery. Mean intra-batch variation coefficient in baseline force measurement was 17% for EHT and 49% for hAT. The PDE-inhibitor milrinone did not affect EHT contraction force, but increased force in hAT. Citalopram (selective serotonin reuptake inhibitor), nifedipine (LTCC-blocker) and lidocaine (Na+ channel-blocker) had negative inotropic effects on EHT and hAT. Formoterol (beta-2 agonist) had positive lusitropic but no inotropic effect in EHT, and positive clinotropic, lusitropic, and inotropic effects in hAT. Tacrolimus (calcineurin-inhibitor) had a negative inotropic effect in EHTs, but no effect in hAT. Digoxin (Na+-K+-ATPase-inhibitor) showed a positive inotropic effect only in EHTs, but no effect in hAT probably due to short incubation time. Ryanodine (ryanodine receptor-inhibitor) reduced contraction force in both models. Rolipram and acetylsalicylic acid showed noninterpretable results in hAT. Contraction amplitude and kinetics were more stable over time and less variable in hiPSC-EHTs than hAT. HiPSC-EHT faithfully detected cAMP-dependent and -independent positive and negative inotropic effects, but limited beta-2 adrenergic or PDE3 effects, compatible with an immature CM phenotype.

Xenobiotic CAR Activators Induce Dlk1-Dio3 Locus Noncoding RNA Expression in Mouse Liver.

Derisking xenobiotic-induced nongenotoxic carcinogenesis (NGC) represents a significant challenge during the safety assessment of chemicals and therapeutic drugs. The identification of robust mechanism-based NGC biomarkers has the potential to enhance cancer hazard identification. We previously demonstrated Constitutive Androstane Receptor (CAR) and WNT signaling-dependent up-regulation of the pluripotency associated Dlk1-Dio3 imprinted gene cluster noncoding RNAs (ncRNAs) in the liver of mice treated with tumor-promoting doses of phenobarbital (PB). Here, we have compared phenotypic, transcriptional ,and proteomic data from wild-type, CAR/PXR double knock-out and CAR/PXR double humanized mice treated with either PB or chlordane, and show that hepatic Dlk1-Dio3 locus long ncRNAs are upregulated in a CAR/PXR-dependent manner by two structurally distinct CAR activators. We further explored the specificity of Dlk1-Dio3 locus ncRNAs as hepatic NGC biomarkers in mice treated with additional compounds working through distinct NGC modes of action. We propose that up-regulation of Dlk1-Dio3 cluster ncRNAs can serve as an early biomarker for CAR activator-induced nongenotoxic hepatocarcinogenesis and thus may contribute to mechanism-based assessments of carcinogenicity risk for chemicals and novel therapeutics.

Secondary pharmacology: screening and interpretation of off-target activities - focus on translation.

Secondary pharmacology is an essential component of drug discovery and is used extensively in the pharmaceutical industry for achieving optimal specificity of new drugs via early hazard identification and off-target mitigation. The importance of this discipline has been achieved by increasing its translational value, based on the recognition of biological target-drug molecule-adverse drug reaction (ADR) associations and integration of secondary pharmacology data with pharmacokinetic parameters. Information obtained from clinical ADRs, from recognition of specific phenotypes of animal models and from hereditary diseases provides increasing regulatory confidence in the target-based approach to ADR prediction and mitigation. Here, we review the progress of secondary pharmacology during the past decade and highlight and demonstrate its applications and impact in drug discovery.

Inhibition of hERG K+ currents by antimalarial drugs in stably transfected HEK293 cells.

Several antimalarial drugs are known to produce a QT interval prolongation via a blockade of the rapidly activating delayed rectifier K+ current (IKr), encoded by the human-ether-a-go-go-related gene (hERG). We investigated the influence of lumefantrine and its major metabolite desbutyl-lumefantrine, as well as halofantrine, chloroquine, and mefloquine, on wild type hERG K+ channels in stably transfected human embryonic kidney cells (HEK293) using the whole cell patch-clamp technique. All of the tested antimalarial drugs inhibited the hERG K+ channels in a concentration- and time-dependent manner. Only halofantrine blocked hERG tail currents voltage-dependently. The ranking of the half-maximal inhibitory concentrations (IC50) of the antimalarials was: halofantrine (0.04 microM)

A straightforward guide to the basic science behind arrhythmogenesis.

The underlying mechanisms behind cardiac arrhythmias are described in this manuscript. In clinical practice, significant arrhythmias are unpredictable, and under some conditions, potentially life-threatening. How can basic science help improve our understanding of molecular entities and factors behind the arrhythmia to advance, develop, adapt or deliver available medications? Structural heart disease and remodelling (eg, heart failure, cardiomyopathy), the presence of modulating factors (ie, diabetes mellitus, autonomic nervous system), genetic predispositions (ie, channelopathies) are considerable preconditions, and influence the development of an arrhythmia. Cardiac arrhythmias may indeed share common basic mechanisms, while elements and substrates perpetuating these may be different and ultimately manifest as various ECG abnormalities. This article lists cellular and subcellular iatrogenic disorders responsible for abnormal impulse generation, or conduction disturbances, including the latest development in theories and biological research, for a better understanding of cellular disorders behind arrhythmogenesis.

Analysis of unipolar electrograms in rabbit heart demonstrated the key role of ventricular apicobasal dispersion in arrhythmogenicity.

Reduced repolarization reserve and increased transmural dispersion of repolarization (TDR) are known risk factors for Torsade de Pointes development, but less is known about the role of apex-to-base (apicobasal) repolarization in arrhythmogenesis. Three needles were inserted in rabbit left ventricle to record unipolar electrograms from endocardium to epicardium and base to apex. Total repolarization interval (TRI) and peak-to-end repolarization interval (Tp) were assessed after quinidine (n = 6) and D,L-sotalol (n = 6) perfusion in combination with the IKs inhibitor chromanol 293B. About 30 µM D,L-sotalol increased TRI and Tp more at the base (TRI + 40 ± 4 %; Tp +89 ± 11 %) relative to the apex (TRI + 28 ± 3 %, Tp + 30 ± 8 %). Similar results were obtained with quinidine: TRI and Tp increased more at the base compared to the apex. No significant differences were recorded from the endocardium to the epicardium. Our results show that combined IKr + IKs block prolonged TRI and Tp significantly more at the ventricular base than at the apex, in the absence of transmural dispersion of refractoriness. Regional changes in TRI and Tp indicate the contribution of apicobasal dispersion to arrhythmogenicity compared to TDR in a rabbit heart model.

Conformational refinement of hydroxamate-based histone deacetylase inhibitors and exploration of 3-piperidin-3-ylindole analogues of dacinostat (LAQ824).

Inspired by natural product HDAC inhibitors, we prepared a series of conformationally restrained HDAC inhibitors based on the hydroxamic acid dacinostat (LAQ824, 7). Several scaffolds with improved biochemical and cellular potency, as well as attenuated hERG inhibition, were identified, suggesting that the introduction of molecular rigidity is a viable strategy to enhance HDAC binding and mitigate hERG liability. Further SAR studies around a 3-piperidin-3-ylindole moiety resulted in the discovery of compound 30, for which a unique conformation was speculated to contribute to overcoming increased lipophilicity and attenuating hERG binding. Separation of racemate 30 afforded 32, the R enantiomer, which demonstrated improved potency in both enzyme and cellular assays compared to dacinostat.

Modeling and simulation of preclinical cardiac safety: towards an integrative framework.

Despite an impressive battery of preclinical cardiac electrophysiology experimental models and the assessment of QT during clinical trials, the risk of Torsades de Pointes (TdP), a potentially lethal ventricular arrhythmia, remains among the common reasons for drug market withdrawal or lack of approval. Due to the low prevalence of TdP, development of statistical evidence that other clinical markers could be better predictors of TdP has proven challenging. Preclinical studies have provided a deeper understanding of torsadogenic mechanisms and potential pro-arrhythmic markers to assess. Translating these preclinical insights into a quantitative clinical risk assessment remains challenging because of (i) species differences in cardiac electrophysiology and drug pharmacokinetics; and (ii) the inability to measure clinically specific cardiac electrophysiology metrics, and therefore ascertain the full predictive value of earlier preclinical components of the risk assessment process. The integrative capacity of cardiac electrophysiology modeling to span time and length scales may provide a quantitative and predictive framework, to complement expert-based preclinical-to-clinical cardiac risk assessment process. In this review, we present salient elements of this risk assessment process and describe essential components of cardiac electrophysiology modeling, to propose that a progressive integration of mechanistic components into a common quantitative framework may help improve the predictability of drug-induced TdP risk.

Report and recommendations of the workshop of the European Centre for the Validation of Alternative Methods for Drug-Induced Cardiotoxicity.

Cardiotoxicity is among the leading reasons for drug attrition and is therefore a core subject in non-clinical and clinical safety testing of new drugs. European Centre for the Validation of Alternative Methods held in March 2008 a workshop on "Alternative Methods for Drug-Induced Cardiotoxicity" in order to promote acceptance of alternative methods reducing, refining or replacing the use of laboratory animals in this field. This review reports the outcome of the workshop. The participants identified the major clinical manifestations, which are sensitive to conventional drugs, to be arrhythmias, contractility toxicity, ischaemia toxicity, secondary cardiotoxicity and valve toxicity. They gave an overview of the current use of alternative tests in cardiac safety assessments. Moreover, they elaborated on new cardiotoxicological endpoints for which alternative tests can have an impact and provided recommendations on how to cover them.

Antimalarial drugs: QT prolongation and cardiac arrhythmias.

Most available antimalarial drugs induce cardiac side effects. These side effects include various mild heart rate changes (amodiaquine) to excessive prolongation of the QT interval (halofantrine) which may lead to lethal arrhythmias such as Torsade de Pointes (TdP). The cellular mechanism of such events during antimalarial therapy is principally related to ion channel inhibition (e.g., human ether-a-go-go related gene channel) which may slow the repolarisation process and create a good substrate for arrhythmia (when dispersion of repolarisation is present). However, other antimalarial drugs do not show as potent cardiac side effects, like co-arthemeter and sulfadoxine-pyrimethamine. Considering that TdP are favoured by a complex combination of electrophysiological changes, a predictive cardiosafety strategy for new antimalarial drugs should comprise assays with an increasing level of information from ion channel level, cellular and organ level, to the whole organism. In this review, the actual knowledge on underlying mechanisms of QT prolongation and TdP is described, followed by the cardiac safety profiles of present antimalarial drugs.