An impedance-based approach using human iPSC-derived cardiomyocytes significantly improves in vitro prediction of in vivo cardiotox liabilities.

Current in vitro approaches to cardiac safety testing typically focus on mechanistic ion channel testing to predict in vivo proarrhythmic potential. Outside of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, structural and functional cardiotoxicity related to chronic dosing effects are of great concern as these effects can impact compound attrition. Development and implementation of an in vitro cardiotoxicity screening platform that effectively identifies these liabilities early in the discovery process should reduce costly attrition and decrease preclinical development time. Impedence platforms have the potential to accurately identify structural and functional cardiotoxicity and have sufficient throughput to be included in a multi-parametric optimization approach. Human induced pluripotent stem cell cardiomyocytes (hIPSC-CMs) have demonstrated utility in cardiac safety and toxicity screening. The work described here leverages these advantages to assess the predictive value of data generated by two impedance platforms. The response of hIPSC-CMs to compounds with known or predicted cardiac functional or structural toxicity was determined. The compounds elicited cardiac activities and/or effects on "macro" impedance often associated with overt structural or cellular toxicity, detachment, or hypertrophy. These assays correctly predicted in vivo cardiotox findings for 81% of the compounds tested and did not identify false positives. In addition, internal or literature Cmax values from in vivo studies correlated within 4 fold of the in vitro observations. The work presented here demonstrates the predictive power of impedance platforms with hIPSC-CMs and provides a means toward accelerating lead candidate selection by assessing preclinical cardiac safety earlier in the drug discovery process.

Electrical alternans and hemodynamics in the anesthetized guinea pig can discriminate the cardiac safety of antidepressants.

INTRODUCTION: The arrhythmogenic risk of fluoxetine, citalopram, and venlafaxine were evaluated through preclinical assays measuring hERG, blood pressure and electrical alternans over their respective clinical unbound concentration ranges. METHODS: Anesthetized guinea pigs were instrumented with jugular and carotid cannulae for drug infusion and blood pressure monitoring respectively; a thoracotomy was performed for placement of a monophasic action potential probe on the left ventricle and for placement of pacing wires on the left ventricular apex. Drugs were infused as a 5-min loading dose immediately followed by a 10-min maintenance dose to achieve clinically relevant plasma concentrations; blood samples were taken at the end of each maintenance dose. Ventricular pacing was performed twice at baseline and at each dose level as follows: 50 preconditioning-beats at S1=220 (or 240) ms immediately followed by 30 test-beats at S2=200 ms. This S1-S2 protocol was repeated for S2=190 to 140 ms. HERG and calcium current measurements were recorded in HEK-293 cells stably expressing hERG potassium currents and freshly isolated guinea pig cardiac myocytes using the whole-cell configuration of the patch clamp technique. RESULTS: Physiologically relevant inhibition (IC(20)) of hERG occurred at concentrations 22-fold (fluoxetine), 9-fold (citalopram), and 11-fold (venlafaxine) beyond their respective clinically effective concentration (C(eff)). At the highest achievable levels, fluoxetine (20-fold C(eff)) and citalopram (28-fold C(eff)) significantly decreased heart rate and/or blood pressure as well as increasing electrical alternans by 5 and 18 ms respectively. Venlafaxine increased blood pressure at only 1.3-fold C(eff), but did not increase electrical alternans at the highest achievable dose (3.1-fold C(eff)). DISCUSSION: These data suggest that evaluating other dose limiting side effects in relation to a drug's therapeutic range may be crucial for accurate assessment of arrhythmia liability.

Structure-activity relationships of triazolopyridine oxazole p38 inhibitors: identification of candidates for clinical development.

The synthesis, structure-activity relationship, in vivo activity, and metabolic profile for a series of triazolopyridine-oxazole based p38 inhibitors are described. The deficiencies of the lead structure in the series, CP-808844, were overcome by changes to the C4 aryl group and the triazole side-chain culminating in the identification of several potential clinical candidates.

Novel potent human ether-a-go-go-related gene (hERG) potassium channel enhancers and their in vitro antiarrhythmic activity.

A variety of drugs has been reported to cause acquired long QT syndrome through inhibition of the IKr channel. Screening compounds in early discovery and development stages against their ability to inhibit IKr or the hERG channel has therefore become an indispensable procedure in the pharmaceutical industry. In contrast to numerous hERG channel blockers discovered during screening, only (3R,4R)-4-[3-(6-methoxyquinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid (RPR260243) has been reported so far to enhance the hERG current. In this article, we describe several potent mechanistically distinct hERG channel enhancers. One example is PD-118057 (2-{4-[2-(3,4-dichloro-phenyl)-ethyl]-phenylamino}-benzoic acid) which produced average increases of 5.5 +/- 1.1, 44.8 +/- 3.1, and 111.1 +/- 21.7% in the peak tail hERG current at 1, 3, and 10 muM, respectively, in human embryonic kidney 293 cells. PD-118057 did not affect the voltage dependence and kinetics of gating parameters, nor did it require open conformation of the channel. In isolated guinea pig cardiomyocytes, PD-118057 showed no major effect on I(Na), I(Ca,L), I(K1), and I(Ks). PD-118057 shortened the action potential duration and QT interval in arterially perfused rabbit ventricular wedge preparation in a concentration-dependent manner. The presence of 3 muM PD-118057 prevented action potential duration and QT prolongation caused by dofetilide. "Early after-depolarizations" induced by dofetilide were also completely eliminated by 3 microM PD-118057. Although further investigation is warranted to evaluate the therapeutic value and safety profile of these compounds, our data support the notion that hERG activation by pharmaceuticals may offer a new approach in the treatment of delayed repolarization conditions, which may occur in patients with inherited or acquired long QT syndrome, congestive heart failure, and diabetes.

Blockade of human cardiac potassium channel human ether-a-go-go-related gene (HERG) by macrolide antibiotics.

Several macrolides have been reported to cause QT prolongation and ventricular arrhythmias such as torsades de pointes. To clarify the underlying ionic mechanisms, we examined the effects of six macrolides on the human ether-a-go-go-related gene (HERG)-encoded potassium current stably expressed in human embryonic kidney-293 cells. All six drugs showed a concentration-dependent inhibition of the current with the following IC(50) values: clarithromycin, 32.9 microM; roxithromycin, 36.5 microM; erythromycin, 72.2 microM; josamycin, 102.4 microM; erythromycylamine, 273.9 microM; and oleandomycin, 339.6 microM. A metabolite of erythromycin, des-methyl erythromycin, was also found to inhibit HERG current with an IC(50) of 147.1 microM. These findings imply that the blockade of HERG may be a common feature of macrolides and may contribute to the QT prolongation observed clinically with some of these compounds. Mechanistic studies showed that inhibition of HERG current by clarithromycin did not require activation of the channel and was both voltage- and time-dependent. The blocking time course could be described by a first-order reaction between the drug and the channel. Both binding and unbinding processes appeared to speed up as the membrane was more depolarized, indicating that the drug-channel interaction may be affected by electrostatic responses.