Derek J. Leishman

Mechanism of hERG K+ channel blockade by the fluoroquinolone antibiotic moxifloxacin

The fluoroquinolone antibiotic moxifloxacin has been associated with the acquired long QT syndrome and is used as a positive control in the evaluation of the QT‐interval prolonging potential of new drugs. In common with other QT‐prolonging agents, moxifloxacin is known to inhibit the hERG potassium K+ channel, but at present there is little mechanistic information available on this action. This study was conducted in order to characterise the inhibition of hERG current (IhERG) by moxifloxacin, and to determine the role in drug binding of the S6 aromatic amino‐acid residues Tyr652 and Phe656. hERG currents were studied using whole‐cell patch clamp (at room temperature and at 35–37°C) in an HEK293 cell line stably expressing hERG channels. Moxifloxacin reversibly inhibited currents in a dose‐dependent manner. We investigated the effects of different voltage commands to elicit hERG currents on moxifloxacin potency. Using a ‘step‐ramp’ protocol, the IC50 was 65 μM at room temperature and 29 μM at 35°C. When a ventricular action potential waveform was used to elicit currents, the IC50 was 114 μM. Block of hERG by moxifloxacin was found to be voltage‐dependent, occurred rapidly and was independent of stimulation frequency. Mutagenesis of the S6 helix residue Phe656 to Ala failed to eliminate or reduce the moxifloxacin‐mediated block whereas mutation of Tyr652 to Ala reduced moxifloxacin block by ∼66%. Our data demonstrate that moxifloxacin blocks the hERG channel with a preference for the activated channel state. The Tyr652 but not Phe656 S6 residue is involved in moxifloxacin block of hERG, concordant with an interaction in the channel inner cavity.

Best practice in the conduct of key nonclinical cardiovascular assessments in drug development: Current recommendations from the Safety Pharmacology Society☆

A cardiovascular safety pharmacology assessment is routinely conducted prior to first administration of a new chemical entity or biopharmaceutical to man. These assessments are used to inform clinicians of potential effects in those initial clinical studies. They may also indicate more subtle effects having more relevance for longer term patient treatment studies such as a potential effect in a Thorough QT (TQT) study or a small persistent increase in blood pressure. Many pharmaceutical companies use the nonclinical studies for early decision making to avoid the clinical development of any compound likely to have a positive signal in a TQT study. These latter purposes generally require more sensitive assay systems and a confidence in their translation to man. At present it is often unclear whether any given study meets the standard required to convincingly detect these subtle effects. The Safety Pharmacology Society (SPS) brought together a group of over 50 experts to discuss best practices for dog and monkey cardiovascular assessments in safety pharmacology and toxicology studies in order to build overall confidence in the ability of a study to test a given hypothesis. It is clearly impossible to dictate a very specific standard practice for assays which are conducted globally in very different facilities using different equipment. However it was clear that a framework could be described to improve comparison and interpretation. Recommendations can be summarized on the basis of three key criteria: 1) know your study population quantitatively and qualitatively, 2) know how well your current study matches the historical data and 3) support your conclusions on the basis of the specific studys determined ability to detect change.

Investigating dynamic protocol-dependence of hERG potassium channel inhibition at 37 °C: Cisapride versus dofetilide

INTRODUCTION Pharmacological inhibition of cardiac potassium channels encoded by hERG (human ether-à-go-go-related gene) is associated with QT interval prolongation and torsades de pointes arrhythmia. Electrophysiological assays of hERG channel inhibition are integral to the safety testing of novel drug candidates. This study was conducted to compare, for the high affinity hERG inhibitors dofetilide and cisapride, hERG blockade between action potential (AP) and conventional (step and step-ramp) screening waveforms. Furthermore, it evaluated dynamic (pulse-by-pulse) protocol-dependence of hERG channel inhibition by these drugs. METHODS Whole-cell patch-clamp recordings were made at 37 degrees C from hERG-expressing HEK 293 cells. Half-maximal inhibitory concentrations (IC(50) values) for I(hERG) blockade were obtained using conventional voltage clamp and action potential clamp, using previously digitised ventricular and Purkinje fibre (PF) AP waveforms. RESULTS A more marked variation in IC(50) values with different command waveforms was observed for cisapride (ranging from 7 to 72 nM) than for dofetilide (ranging from 4 to 15 nM), with higher IC(50)s obtained with AP than step or step-ramp commands. The two drugs differed little from one another in effects on voltage-dependent activation; however, I(hERG) blockade by each drug was initially voltage-dependent, but at steady-state was only voltage-dependent for cisapride. There was comparatively little difference between the two drugs in effects on I(hERG) availability or time constants of development of inactivation. Features of time-dependence of blockade and the use of protocols employing varying rest periods in drug or commands of alternating duration highlighted a pronounced ability of cisapride, but not dofetilide, to dissociate and reassociate from hERG on a pulse-by-pulse basis. DISCUSSION Protocols described here that demonstrated dynamic variation (drug dissociation/reassociation) in hERG channel current blockade at 37 degrees C for cisapride may have future value for investigating drug interactions with the hERG channel. Downloadable digitised ventricular and PF AP waveforms that can be used in AP clamp experiments also accompany this article.

The Concordance between Nonclinical and Phase I Clinical Cardiovascular Assessment from a Cross-Company Data Sharing Initiative

It is widely accepted that more needs to be done to bring new, safe, and efficacious drugs to the market. Cardiovascular toxicity detected both in early drug discovery as well as in the clinic, is a major contributor to the high failure rate of new molecules. The growth of translational safety offers a promising approach to improve the probability of success for new molecules. Here we describe a cross-company initiative to determine the concordance between the conscious telemetered dog and phase I outcome for 3 cardiovascular parameters. The data indicate that, in the context of the methods applied in this analysis, the ability to detect compounds that affect the corrected QT interval (QTc) was good within the 10-30x exposure range but the predictive or detective value for heart rate and diastolic blood pressure was poor. These findings may highlight opportunities to refine both the animal and the clinical study designs, as well as refocusing the assessment of value of dog cardiovascular assessments beyond phase 1. This investigation has also highlighted key considerations for cross-company data sharing and presents a unique learning opportunity to improve future translational projects.

Overcoming hERG Affinity in the Discovery of Maraviroc; A CCR5 Antagonist for the Treatment of HIV

Avoiding cardiac liability associated with blockade of hERG (human ether a go-go) is key for successful drug discovery and development. This paper describes the work undertaken in the discovery of a potent CCR5 antagonist, maraviroc 34, for the treatment of HIV. In particular the use of a pharmacophore model of the hERG channel and a high throughput binding assay for the hERG channel are described that were critical to elucidate SAR to overcome hERG liabilities. The key SAR involves the introduction of polar substituents into regions of the molecule where it is postulated to undergo hydrophobic interactions with the ion channel. Within the CCR5 project there appeared to be no strong correlation between hERG affinity and physiochemical parameters such as pKa or lipophilicity. It is believed that chemists could apply these same strategies early in drug discovery to remove hERG interactions associated with lead compounds while retaining potency at the primary target.

Effects of the Class III antiarrhythmic agent dofetilide (UK-68,798) on L-type calcium current from rabbit ventricular myocytes

The methanesulphonanilide agent dofetilide (UK‐68,798) exerts Class III antiarrhythmic effects by inhibiting the cardiac rapid delayed rectifier potassium current (IKr) encoded by HERG. The aim of the present study was to determine whether dofetilide also exhibits Class IV (L‐type calcium‐channel blocking) effects. L‐type calcium current (ICa,L) was measured from rabbit isolated ventricular myocytes, using the whole‐cell patch‐clamp technique under selective recording conditions. Positive control experiments demonstrated inhibition of ICa,L elicited by pulses to + 10 mV by both nifedipine and externally applied Ni2+ ions. Three concentrations of dofetilide were tested: 100 nm, 1 μm and 10 μm. ICa,L magnitude was not significantly reduced by any of the concentrations tested (P>0.05; n = minimum of seven cells per drug concentration). The inactivation time‐course of ICa,L was also unaffected by 10 μm dofetilide. Heterologously expressed HERG current (IHERG) recorded from Chinese Hamster Ovary cells was extensively inhibited by 100 nm and 1 μm dofetilide, with inhibition at 1 μm not significantly different from 100% (P> 0.1). It is concluded that dofetilide produced no ICa,L blocking effects at concentrations up to and exceeding that required for maximal IHERG inhibition. The findings support the notion that dofetilide is a highly selective Class III antiarrhythmic agent, devoid of Class IV antiarrhythmic activity.

Proarrhythmia liability assessment and the comprehensive in vitro Proarrhythmia Assay (CiPA): An industry survey on current practice

INTRODUCTION The Safety Pharmacology Society (SPS) has conducted a survey of its membership to identify industry practices related to testing considered in the Comprehensive In vitro Proarrhythmia Assay (CiPA). METHODS Survey topics included nonclinical approaches to address proarrhythmia issues, conduct of in silico studies, in vitro ion channel testing methods used, drugs used as positive controls during the conduct of cardiac ion channel studies, types of arrhythmias observed in non-clinical studies and use of the anticipated CiPA ion channel assay. RESULTS In silico studies were used to evaluate effects on ventricular action potentials by only 15% of responders. In vitro assays were used mostly to assess QT prolongation (95%), cardiac Ca2+ and Na+ channel blockade (82%) and QT shortening or QRS prolongation (53%). For de-risking of candidate drugs for proarrhythmia, those assays most relevant to CiPA including cell lines stably expressing ion channels used to determine potency of drug block were most frequently used (89%) and human stem cell-derived or induced pluripotent stem cell cardiomyocytes (46%). Those in vivo assays related to general proarrhythmia derisking include ECG recording using implanted telemetry technology (88%), jacketed external telemetry (62%) and anesthetized animal models (53%). While the CiPA initiative was supported by 92% of responders, there may be some disconnect between current practice and future expectations, as explained. DISCUSSION Proarrhythmia liability assessment in drug development presently includes study types consistent with CiPA. It is anticipated that CiPA will develop into a workable solution to the concern that proarrhythmia liability testing remains suboptimal.

Drug Discovery vs hERG

A number of marketed drugs have been withdrawn and numerous clinical and preclinical compounds discontinued due to prolongation of the time interval between Q and T waves of the electrocardiogram (long QT syndrome). Drug-induced QT interval prolongation, which may lead to life-threatening cardiac arrhythmia torsades de pointes (TdP), has been linked to blockade of a cardiac potassium channel, a product of the human ether-a-go-go-related gene (hERG). Consequently, drug discovery efforts across the pharmaceutical industry have been utilizing hERG in vitro assays to predict and minimize the risk of QT prolongation. This chapter discusses in silico, in vitro, ex vivo, and in vivo methods employed to measure blockade of hERG and QT prolongation, as well as regulatory recommendations, and medicinal chemistry strategies utilized to circumvent interactions with hERG.

Heartbeat dynamics in adrenergic blocker treated conscious beagle dogs

INTRODUCTION Adrenergic blockade as a treatment for chronic heart failure (CHF) has proved effective, but its pharmacological mechanism on CHF remains unclear. In the past two decades, studies on heart rate variability (HRV) have reported that CHF patients generally have a reduced temporal complexity in heart rate variability. On the other hand, adrenergic blockers have been shown to restore such complexity. Fractal analysis is a novel and efficient tool to explore the adrenergic blockade effect on HRV. This paper applies the detrended fluctuation analysis (DFA) and multifractal DFA (MF-DFA) methods in an attempt to understand the effect of adrenergic blockade on cardiac dynamics in conscious beagle dogs. METHODS DFA and MF-DFA analysis are conducted on RR interval data generated from telemetry instrumented dogs receiving a combination of 15 mg/kg nadolol and 5 mg/kg phenoxybenzamine orally administered at the 22nd and 34th hour in a parallel design (n=12). All dogs had approximately 48 h of beat-to-beat heart rate measurements recorded in the left ventricle. Complexity measures for heartbeat series are compared between the blocker and vehicle group. We also compute traditional statistics for HRV and spectral parameters and examine their correlation with fractal analysis. RESULTS When compared to the vehicle group, the adrenergic blocker group had: 1) longer RR intervals (p=0.02) and lower beat-to-beat variability (p=0.04); 2) decreased low frequency (LF) and high frequency (HF) power (p=0.03), and higher LF-to-HF ratio; 3) larger middle-range scaling exponents (p<0.01); 4) broader multifractal spectra (p=0.03) with higher dominant singularity indices (p=0.02). DISCUSSION Our results show that 1) adrenergic blockade alters the sympathovagal balance; 2) adrenergic blockers enhance the complexity of the cardiac dynamics; 3) the adrenergic blockade effect on cardiac dynamics is primarily the attenuation of small fluctuations in RR intervals. Fractal analysis also has the potential to be applied to early QT diagnosis.

The Diplomate in Safety Pharmacology (DSP) certification scheme.

As with other professional disciplines there is a growing need from within industry as well as global regulatory authorities for implementation of a certification process in order to assure that appropriate expertise is developed and quality standards are identified for professionals involved in the practice of Safety Pharmacology (SP). In order to meet this need, the Safety Pharmacology Society (SPS) has developed the Diplomate in Safety Pharmacology (DSP) certification process. There are many benefits to certification including authentication of the discipline within the overall pharmaceutical community and with regulatory authorities. It also encourages participation in SPS activities by other professionals (toxicologists, clinicians, academics) who wish to broaden their professional expertise. It provides an opportunity for candidates to strengthen their fundamental scientific knowledge, and stimulates the sharing of data, methods and model development in the form of publications and presentations on relevant topics in SP. Accreditation in SP occurs after candidates successfully complete a written certification examination conducted at the annual SPS meeting. The DSP exam consists primarily of material pertinent to the conduct of SP vital function core battery studies (i.e., cardiovascular, respiratory and central nervous systems), supplemental SP studies (i.e., renal/urinary, gastrointestinal, immunology, and hematology), Regulatory Guidelines (ICH Guidelines) as well as relevant cross-functional knowledge (e.g., physiology, pharmacology, toxicology, biochemistry, pathology, pharmacokinetics, dosing formulation, analytical methods, and statistics). Maintenance of the DSP certification results from the accrual of credits which are gained from a range of educational and scientific contributions. Eligibility requirements include a combination of at least a bachelor degree in science and two years of relevant professional SP experience and one poster presentation on a SP topic as first author at a recognized major scientific meeting.