An overview of the safety pharmacology society strategic plan.

Safety Pharmacology studies are conducted to characterize the confidence by which biologically active new chemical entities (NCE) may be anticipated as safe. Non-clinical safety pharmacology studies aim to detect and characterize potentially undesirable pharmacodynamic activities using an array of in silico, in vitro and in vivo animal models. While a broad spectrum of methodological innovation and advancement of the science occurs within the Safety Pharmacology Society, the society also focuses on partnerships with health authorities and technology providers and facilitates interaction with organizations of common interest such as pharmacology, physiology, neuroscience, cardiology and toxicology. Education remains a primary emphasis for the society through content derived from regional and annual meetings, webinars and publication of its works it seeks to inform the general scientific and regulatory community. In considering the future of safety pharmacology the society has developed a strategy to successfully navigate forward and not be mired in stagnation of the discipline. Strategy can be defined in numerous ways but generally involves establishing and setting goals, determining what actions are needed to achieve those goals, and mobilizing resources within the society to accomplish the actions. The discipline remains in rapid evolution and its coverage is certain to expand to provide better guidance for more systems in the next few years. This overview from the Safety Pharmacology Society will outline the strategic plan from 2016 to 2018 and beyond and provide insight into the future of the discipline which builds upon a previous strategic plan established in 2009.

Gastrointestinal Safety Pharmacology in Drug Discovery and Development.

Although the basic structure of the gastrointestinal tract (GIT) is similar across species, there are significant differences in the anatomy, physiology, and biochemistry between humans and laboratory animals, which should be taken into account when conducting a gastrointestinal (GI) assessment. Historically, the percentage of cases of drug attrition associated with GI-related adverse effects is small; however, this incidence has increased over the last few years. Drug-related GI effects are very diverse, usually functional in nature, and not limited to a single pharmacological class. The most common GI signs are nausea and vomiting, diarrhea, constipation, and gastric ulceration. Despite being generally not life-threatening, they can greatly affect patient compliance and quality of life. There is therefore a real need for improved and/or more extensive GI screening of candidate drugs in preclinical development, which may help to better predict clinical effects. Models to identify drug effects on GI function cover GI motility, nausea and emesis liability, secretory function (mainly gastric secretion), and absorption aspects. Both in vitro and in vivo assessments are described in this chapter. Drug-induced effects on GI function can be assessed in stand-alone safety pharmacology studies or as endpoints integrated into toxicology studies. In silico approaches are also being developed, such as the gut-on-a-chip model, but await further optimization and validation before routine use in drug development. GI injuries are still in their infancy with regard to biomarkers, probably due to their greater diversity. Nevertheless, several potential blood, stool, and breath biomarkers have been investigated. However, additional validation studies are necessary to assess the relevance of these biomarkers and their predictive value for GI injuries.

Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays.

Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies - radioligand-binding or automated electrophysiology - was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost.

Translational potential of a mouse in vitro bioassay in predicting gastrointestinal adverse drug reactions in Phase I clinical trials.

BACKGROUND: Motility-related gastrointestinal (GI) adverse drug reactions (GADRs) such as diarrhea and constipation are a common and deleterious feature associated with drug development. Novel biomarkers of GI function are therefore required to aid decision making on the GI liability of compounds in development. METHODS: Fifteen compounds associated with or without clinical GADRs were used to assess the ability of an in vitro colonic motility bioassay to predict motility-related GADRs. Compounds were examined in a blinded fashion for their effects on mouse colonic peristaltic motor complexes in vitro. For each compound concentration-response relationships were determined and the results compared to clinical data. Compounds were also assessed using GI transit measurements obtained using an in vivo rat charcoal meal model. KEY RESULTS: Within a clinically relevant dosing range, the in vitro assay identified five true and three false positives, four true and three false negatives, which gave a predictive capacity of 60%. The in vivo assay detected four true and four false positives, four false and three true negatives, giving rise to a predictive capacity for this model of 47%. CONCLUSIONS & INFERENCES: Overall these results imply that both assays are poor predictors of GADRs. Further analysis would benefit from a larger compound set, but the data show a clear need for improved models for use in safety pharmacology assessment of GI motility.

Detecting drug-induced prolongation of the QRS complex: new insights for cardiac safety assessment.

BACKGROUND: Drugs slowing the conduction of the cardiac action potential and prolonging QRS complex duration by blocking the sodium current (I(Na)) may carry pro-arrhythmic risks. Due to the frequency-dependent block of I(Na), this study assesses whether activity-related spontaneous increases in heart rate (HR) occurring during standard dog telemetry studies can be used to optimise the detection of class I antiarrhythmic-induced QRS prolongation. METHODS: Telemetered dogs were orally dosed with quinidine (class Ia), mexiletine (class Ib) or flecainide (class Ic). QRS duration was determined standardly (5 beats averaged at rest) but also prior to and at the plateau of each acute increase in HR (3 beats averaged at steady state), and averaged over 1h period from 1h pre-dose to 5h post-dose. RESULTS: Compared to time-matched vehicle, at rest, only quinidine and flecainide induced increases in QRS duration (E(max) 13% and 20% respectively, P<0.01-0.001) whereas mexiletine had no effect. Importantly, the increase in QRS duration was enhanced at peak HR with an additional effect of +0.7 ± 0.5 ms (quinidine, NS), +1.8 ± 0.8 ms (mexiletine, P<0.05) and +2.8 ± 0.8 ms (flecainide, P<0.01) (calculated as QRS at basal HR-QRS at high HR). CONCLUSION: Electrocardiogram recordings during elevated HR, not considered during routine analysis optimised for detecting QT prolongation, can be used to sensitise the detection of QRS prolongation. This could prove useful when borderline QRS effects are detected. Analysing during acute increases in HR could also be useful for detecting drug-induced effects on other aspects of cardiac function.

Validation of an in vitro contractility assay using canine ventricular myocytes.

Measurement of cardiac contractility is a logical part of pre-clinical safety assessment in a drug discovery project, particularly if a risk has been identified or is suspected based on the primary- or non-target pharmacology. However, there are limited validated assays available that can be used to screen several compounds in order to identify and eliminate inotropic liability from a chemical series. We have therefore sought to develop an in vitro model with sufficient throughput for this purpose. Dog ventricular myocytes were isolated using a collagenase perfusion technique and placed in a perfused recording chamber on the stage of a microscope at ~36 °C. Myocytes were stimulated to contract at a pacing frequency of 1 Hz and a digital, cell geometry measurement system (IonOptix™) was used to measure sarcomere shortening in single myocytes. After perfusion with vehicle (0.1% DMSO), concentration-effect curves were constructed for each compound in 4-30 myocytes taken from 1 or 2 dog hearts. The validation test-set was 22 negative and 8 positive inotropes, and 21 inactive compounds, as defined by their effect in dog, cynolomolgous monkey or humans. By comparing the outcome of the assay to the known in vivo contractility effects, the assay sensitivity was 81%, specificity was 75%, and accuracy was 78%. With a throughput of 6-8 compounds/week from 1 cell isolation, this assay may be of value to drug discovery projects to screen for direct contractility effects and, if a hazard is identified, help identify inactive compounds.

An in silico canine cardiac midmyocardial action potential duration model as a tool for early drug safety assessment.

Cell lines expressing ion channels (IC) and the advent of plate-based electrophysiology device have enabled a molecular understanding of the action potential (AP) as a means of early QT assessment. We sought to develop an in silico AP (isAP) model that provides an assessment of the effect of a compound on the myocyte AP duration (APD) using concentration-effect curve data from a panel of five ICs (hNav1.5, hCav1.2, hKv4.3/hKChIP2.2, hKv7.1/hminK, hKv11.1). A test set of 53 compounds was selected to cover a range of selective and mixed IC modulators that were tested for their effects on optically measured APD. A threshold of >10% change in APD at 90% repolarization (APD(90)) was used to signify an effect at the top test concentration. To capture the variations observed in left ventricular midmyocardial myocyte APD data from 19 different dogs, the isAP model was calibrated to produce an ensemble of 19 model variants that could capture the shape and form of the APs and also quantitatively replicate dofetilide- and diltiazem-induced APD(90) changes. Provided with IC panel data only, the isAP model was then used, blinded, to predict APD(90) changes greater than 10%. At a simulated concentration of 30 µM and based on a criterion that six of the variants had to agree, isAP prediction was scored as showing greater than 80% predictivity of compound activity. Thus, early in drug discovery, the isAP model allows integrating separate IC data and is amenable to the throughput required for use as a virtual screen.

hERG subunit composition determines differential drug sensitivity.

BACKGROUND AND PURPOSE: The majority of human ether-a-go-go-related gene (hERG) screens aiming to minimize the risk of drug-induced long QT syndrome have been conducted using heterologous systems expressing the hERG 1a subunit, although both hERG 1a and 1b subunits contribute to the K+ channels producing the repolarizing current I(Kr) . We tested a range of compounds selected for their diversity to determine whether hERG 1a and 1a/1b channels exhibit different sensitivities that may influence safety margins or contribute to a stratified risk analysis. EXPERIMENTAL APPROACH: We used the IonWorks™ plate-based electrophysiology device to compare sensitivity of hERG 1a and 1a/1b channels stably expressed in HEK293 cells to 50 compounds previously shown to target hERG channels. Potency was determined as IC50 values (µM) obtained from non-cumulative, eight-point concentration-effect curves of normalized data, fitted to the Hill equation. To minimize possible sources of variability, compound potency was assessed using test plates arranged in alternating columns of cells expressing hERG 1a and 1a/1b. KEY RESULTS: Although the potency of most compounds was similar for the two targets, some surprising differences were observed. Fluoxetine (Prozac) was more potent at blocking hERG 1a/1b than 1a channels, yielding a corresponding reduction in the safety margin. In contrast, E-4031 was a more potent blocker of hERG 1a compared with 1a/1b channels, as previously reported, as was dofetilide, another high-affinity blocker. CONCLUSIONS AND IMPLICATIONS: The current assays may underestimate the risk of some drugs to cause torsades de pointes arrhythmia, and overestimate the risk of others.

On the relationship between block of the cardiac Na⁺ channel and drug-induced prolongation of the QRS complex.

BACKGROUND AND PURPOSE: Inhibition of the human cardiac Na(+) channel (hNa(v) 1.5) can prolong the QRS complex and has been associated with increased mortality in patients with underlying cardiovascular disease. The safety implications of blocking hNa(v) 1.5 channels suggest the need to test for this activity early in drug discovery in order to design out any potential liability. However, interpretation of hNa(v) 1.5 blocking potency requires knowledge of how hNa(v) 1.5 block translates into prolongation of the QRS complex. EXPERIMENTAL APPROACH: We tested Class I anti-arrhythmics, other known QRS prolonging drugs and drugs not reported to prolong the QRS complex. Their block of hNa(v) 1.5 channels (as IC(50) values) was measured in an automated electrophysiology-based assay. These IC(50) values were compared with published reports of the corresponding unbound (free) plasma concentrations attained during clinical use (fC(max)) to provide an IC(50) : fC(max) ratio. KEY RESULTS For 42 Class I anti-arrhythmics and other QRS prolonging drugs, 67% had IC(50) : fC(max) ratios <30. For 55 non-QRS prolonging drugs tested, 72% had ratios >100. Finally, we determined the relationship between the IC(50) value and the free drug concentration associated with prolongation of the QRS complex in humans. For 37 drugs, QRS complex prolongation was observed at free plasma concentrations that were about 15-fold lower than the corresponding IC(50) at hNa(v) 1.5 channels. CONCLUSIONS AND IMPLICATIONS: A margin of 30- to 100-fold between hNa(v) 1.5 IC(50) and fC(max) appears to confer an acceptable degree of safety from QRS prolongation. QRS prolongation occurs on average at free plasma levels 15-fold below the IC(50) at hNa(v) 1.5 channels. LINKED ARTICLE: This article is commented on by Gintant et al., pp. 254-259 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01433.x.

An introduction to QT interval prolongation and non-clinical approaches to assessing and reducing risk.

Owing to its association with Torsades de Pointes, drug-induced QT interval prolongation has been and remains a significant hurdle to the development of safe, effective medicines. Genetic and pharmacological evidence highlighting the pivotal role the human ether-a-go-go-related gene (hERG) channel was a critical step in understanding how to start addressing this issue. It led to the development of hERG assays with the rapid throughput needed for the short timescales required in early drug discovery. The resulting volume of hERG data has fostered in silico models to help chemists design compounds with reduced hERG potency. In early drug discovery, a pragmatic approach based on exceeding a given potency value has been required to decide when a compound is likely to carry a low QT risk, to support its progression to late-stage discovery. At this point, the in vivo efficacy and metabolism characteristics of the potential drug are generally defined, as well its safety profile, which includes usually a dog study to assess QT interval prolongation risk. The hERG and in vivo QT data, combined with the likely indication and the estimated free drug level for efficacy, are put together to assess the risk that the potential drug will prolong QT in man. Further data may be required to refine the risk assessment before making the major investment decisions for full development. The non-clinical data are essential to inform decisions about compound progression and to optimize the design of clinical QT studies.

Pharmacological and electrophysiological characterization of nine, single nucleotide polymorphisms of the hERG-encoded potassium channel.

BACKGROUND AND PURPOSE: Potencies of compounds blocking K(V)11.1 [human ether-ago-go-related gene (hERG)] are commonly assessed using cell lines expressing the Caucasian wild-type (WT) variant. Here we tested whether such potencies would be different for hERG single nucleotide polymorphisms (SNPs). EXPERIMENTAL APPROACH: SNPs (R176W, R181Q, Del187-189, P347S, K897T, A915V, P917L, R1047L, A1116V) and a binding-site mutant (Y652A) were expressed in Tet-On CHO-K1 cells. Potencies [mean IC(50); lower/upper 95% confidence limit (CL)] of 48 hERG blockers was estimated by automated electrophysiology [IonWorks HT (IW)]. In phase one, rapid potency comparison of each WT-SNP combination was made for each compound. In phase two, any compound-SNP combinations from phase one where the WT upper/lower CL did not overlap with those of the SNPs were re-examined. Electrophysiological WT and SNP parameters were determined using conventional electrophysiology. KEY RESULTS: IW detected the expected sixfold potency decrease for propafenone in Y652A. In phase one, the WT lower/upper CL did not overlap with those of the SNPs for 77 compound-SNP combinations. In phase two, 62/77 cases no longer yielded IC(50) values with non-overlapping CLs. For seven of the remaining 15 cases, there were non-overlapping CLs but in the opposite direction. For the eight compound-SNP combinations with non-overlapping CLs in the same direction as for phase 1, potencies were never more than twofold apart. The only statistically significant electrophysiological difference was the voltage dependence of activation of R1047L. CONCLUSION AND IMPLICATIONS: Potencies of hERG channel blockers defined using the Caucasian WT sequence, in this in vitro assay, were representative of potencies for common SNPs.

Comparison of electrocardiographic analysis for risk of QT interval prolongation using safety pharmacology and toxicological studies.

Testing for possible cardiovascular side effects of new drugs has been an essential part of drug development for years. A more detailed analysis of the electrocardiogram (ECG) to detect effects on ventricular repolarization (effects on the QT interval), as a marker for possible proarrhythmic potential has been added to that evaluation in recent years. State-of-the art evaluation of drug-induced effects on the QT interval have evolved, but due to the complexity of the assessment, the trend in safety pharmacology studies has been to collect large numbers of high quality ECGs to allow for a robust assessment including the influence of heart rate on the QT interval apart from possible drug-induced effects. Since an assessment of the ECG is often included in toxicological studies, one can consider making such an assessment using ECG data from routine toxicological studies. This review summarizes various aspects of both safety pharmacology and toxicology studies with regards to their impact on the quality and quantity of ECG data that one can reasonably derive. We conclude that ECG data from toxicological studies can offer complementary ECG data that can strengthen a risk assessment. However, for the great majority of standard toxicity studies conducted, the ECG data collected do not permit an adequate assessment of drug-induced effects on the QT interval with the sensitivity expected from the ICH S7B guidelines. Furthermore, sponsors should be discouraged from performing any analyses on low quality ECGs to avoid generating misleading data. Substantial improvements in ECG quality and quantity are available, thereby making a QT interval assessment within the context of a standard toxicological study feasible, but these methods may require a larger commitment of resources from the sponsor. From the viewpoint of risk mitigation and limiting the attrition of promising new therapies, a commitment of resources to insure ECG data quality in either toxicology or safety pharmacology studies may be well justified.

International Life Sciences Institute (Health and Environmental Sciences Institute, HESI) initiative on moving towards better predictors of drug-induced torsades de pointes.

Knowledge of the cardiac safety of emerging new drugs is an important aspect of assuring the expeditious advancement of the best candidates targeted at unmet medical needs while also assuring the safety of clinical trial subjects or patients. Present methodologies for assessing drug-induced torsades de pointes (TdP) are woefully inadequate in terms of their specificity to select pharmaceutical agents, which are human arrhythmia toxicants. Thus, the critical challenge in the pharmaceutical industry today is to identify experimental models, composite strategies, or biomarkers of cardiac risk that can distinguish a drug, which prolongs cardiac ventricular repolarization, but is not proarrhythmic, from one that prolongs the QT interval and leads to TdP. To that end, the HESI Proarrhythmia Models Project Committee recognized that there was little practical understanding of the relationship between drug effects on cardiac ventricular repolarization and the rare clinical event of TdP. It was on that basis that a workshop was convened in Virginia, USA at which four topics were introduced by invited subject matter experts in the following fields: Molecular and Cellular Biology Underlying TdP, Dynamics of Periodicity, Models of TdP Proarrhythmia, and Key Considerations for Demonstrating Utility of Pre-Clinical Models. Contained in this special issue of the British Journal of Pharmacology are reports from each of the presenters that set out the background and key areas of discussion in each of these topic areas. Based on this information, the scientific community is encouraged to consider the ideas advanced in this workshop and to contribute to these important areas of investigations over the next several years.

Validation of the use of zebrafish larvae in visual safety assessment.

INTRODUCTION: The use of zebrafish (Danio rerio) larvae was investigated to predict adverse visual effects and to establish the potential application of this organism in early drug safety assessment. METHODS: Following a comparison of the effects of 4 compounds in TL and WIK strains of zebrafish larvae, a blinded validation set of 27 compounds was tested on WIK strain of larval zebrafish in the optomotor response (OMR) assay. Selected compounds were also tested in the optokinetic response (OKR) and locomotor assays. Larvae were exposed from 3-8 days post-fertilisation (d.p.f.) by immersion in embryo culture media (E3) containing the compound in 1% DMSO (v/v). At 8 d.p.f. toxicity was assessed and the OMR or OKR assays were undertaken at non-toxic treatment levels. Compounds were then rated as 'red', 'amber' or 'green' according to their effects on visual function prior to unblinding of the identities of the test compounds. RESULTS: Overall, the OMR assay revealed a good concordance between the effects of compounds in WIK strain zebrafish with the data available from other in vivo and in vitro models or the clinic: thirteen out of nineteen positive compounds produced the expected effect while six of the eight negative compounds were correctly predicted. This gave an overall predictivity of 70% with a sensitivity of 68% and a specificity of 75%. The two false positive compounds were further tested in locomotor and optokinetic response assays and it was shown that a motility defect, rather than an effect on vision, had given rise to the positive result in the OMR assays. Therefore, the OMR assay would best be employed with other techniques to identify false positives. Further studies on two of the false negatives at higher concentrations suggested that the initial concentrations tested were too low. Therefore, it should be ensured that the maximum tolerated concentration is tested in the OMR assay. A comparison of four standard compounds in the OMR assay in WIK and TL zebrafish wild type strains revealed no difference in sensitivity between the strains. DISCUSSION: Overall, these results suggest that the OMR assay in zebrafish could be useful in predicting the adverse effects of drugs on visual function in man and would support its potential as a screen for 'frontloading' safety pharmacology assessment of this endpoint in vivo.

In vitro models of proarrhythmia.

Proarrhythmia models use electrophysiological markers to predict the risk of torsade de pointes (TdP) in patients. The set of variables used by each model to predict the torsadogenic propensity of a drug has been reported to correlate with clinical outcome; however, these reports should be interpreted cautiously as no model has been independently assessed. Each model is discussed along with its merits and shortcomings; none, as yet, having shown a predictive value that makes it clearly superior to the others. As predictive as these models may become, extrapolation of results directly to the clinic must be exercised with caution. The use of in silico models, from subcellular to whole system, is rapidly beginning to form the first line of screening activity in many drug discovery programmes, indicating that biological experimentation may become secondary to analysis by simulation. In vitro proarrhythmia models challenge current perceptions of appropriate surrogates for TdP in man and question existing non-clinical strategies for assessing proarrhythmic risk. The rapid emergence of such models, compounded by the lack of a clear understanding of the key proarrhythmic mechanisms has resulted in a regulatory reluctance to embrace such models. The wider acceptance of proarrhythmia models is likely to occur when there is a clear understanding and agreement on the key proarrhythmia mechanisms. With greater acceptance and ongoing improvements, these models have the potential to unravel the complex mechanisms underlying TdP.

Moving towards better predictors of drug-induced torsades de pointes.

Drug-induced torsades de pointes (TdP) remains a significant public health concern that has challenged scientists who have the responsibility of advancing new medicines through development to the patient, while assuring public safety. As a result, from the point of discovering a new molecule to the time of its registration, significant efforts are made to recognize potential liabilities, including the potential for TdP. With this background, the ILSI (HESI) Proarrhythmia Models Project Committee recognized that there was little practical understanding of the relationship between drug effects on cardiac ventricular repolarization and the rare clinical event of TdP. A workshop was therefore convened at which four topics were considered including: Molecular and Cellular Biology Underlying TdP, Dynamics of Periodicity, Models of TdP Proarrhythmia and Key Considerations for Demonstrating Utility of Pre-Clinical Models. The series of publications in this special edition has established the background, areas of debate and those that deserve scientific pursuit. This is intented to encourage the research community to contribute to these important areas of investigation in advancing the science and our understanding of drug-induced proarrhythmia.

Strategies to reduce the risk of drug-induced QT interval prolongation: a pharmaceutical company perspective.

Drug-induced prolongation of the QT interval is having a significant impact on the ability of the pharmaceutical industry to develop new drugs. The development implications for a compound causing a significant effect in the 'Thorough QT/QTc Study' -- as defined in the clinical regulatory guidance (ICH E14) -- are substantial. In view of this, and the fact that QT interval prolongation is linked to direct inhibition of the hERG channel, in the early stages of drug discovery the focus is on testing for and screening out hERG activity. This has led to understanding of how to produce low potency hERG blockers whilst retaining desirable properties. Despite this, a number of factors mean that when an integrated risk assessment is generated towards the end of the discovery phase (by conducting at least an in vivo QT assessment) a QT interval prolongation risk is still often apparent; inhibition of hERG channel trafficking and partitioning into cardiac tissue are just two confounding factors. However, emerging information suggests that hERG safety margins have high predictive value and that when hERG and in vivo non-clinical data are combined, their predictive value to man, whilst not perfect, is >80%. Although understanding the anomalies is important and is being addressed, of greater importance is developing a better understanding of TdP, with the aim of being able to predict TdP rather than using an imperfect surrogate marker (QT interval prolongation). Without an understanding of how to predict TdP risk, high-benefit drugs for serious indications may never be marketed.

Optimisation and validation of a medium-throughput electrophysiology-based hNav1.5 assay using IonWorks.

INTRODUCTION: The safety implications of blocking the human cardiac Na(+) channel (hNav1.5) make it prudent to test for this activity early in the drug discovery process and design-out any potential liability. This needs a method with adequate throughput and a demonstrable predictive value to effects in native cardiac tissues. Here we describe the validation of a method that combines the ability to screen tens of compounds a day, with direct assessment of channel function. METHODS: The electrophysiological and pharmacological properties of hNav1.5 were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks HT). A pharmacological comparison was also made between IonWorks HT and canine cardiac Purkinje Fibre action potential upstroke data. RESULTS: Activation curve parameters for hNav1.5 in IonWorks HT were not statistically different (p>0.05) from those generated using conventional electrophysiology. IonWorks HT V(1/2)=-22+/-0.8 mV, slope=6.9+/-0.2 (n=11); conventional electrophysiology V(1/2)=-20+/-1.6 mV, slope=6.4+/-0.3 (n=11). Potency values for a range of hNav1.5 blockers determined using IonWorks HT correlated closely with those obtained using conventional electrophysiology (R=0.967, p<0.001). The assay was able to distinguish between highly use-dependent blockers (e.g. tetracaine) and blockers that do not display strong use-dependence (e.g. quinidine). Comparison of the degree of hNav1.5 inhibition and decrease in canine Purkinje fibre action potential upstroke velocity (V(max)) showed that the IonWorks HT assay would have predicted the outcome in Purkinje fibres in the majority of cases, with false negative and positive rates estimated at 8 and 7%, respectively. Finally, hNav1.5 pharmacology was similar when determined using either IonWorks HT or IonWorks Quattro, although the latter yielded more consistent data. DISCUSSION: The assay described combines a functional assessment of hNav1.5 with medium-throughput. Furthermore the assay was able to reveal information on the use-dependency of compound block, as well as predicting Na(+) channel effects in more integrated systems such as the cardiac Purkinje fibre action potential. This makes it possible to determine quantitative potency data, and mechanistic information about use-dependence, in a timeframe short enough to influence medicinal chemistry.

Pharmacological validation of a semi-automated in vitro hippocampal brain slice assay for assessment of seizure liability.

INTRODUCTION: Drug-induced seizures are a serious, life-threatening adverse drug reaction (ADR) that can result in the failure of drugs to be licensed for clinical use or withdrawn from the market. Seizure liability of potential drugs is traditionally assessed using animal models run during the later phases of the drug discovery process. Given the low throughput, high animal usage and high compound requirement associated with these assays, it would be advantageous to identify higher throughput, in vitro models that could be used to give an earlier assessment of seizure liability. The hippocampal brain slice is one possibility but conventionally allows recording from only one slice at a time. The aim of this study was to validate a semi-automated system (Slicemaster, Scientifica UK Ltd) which allows concurrent electrophysiological recording from multiple brain slices. METHODS: Conventional electrophysiological recording techniques were used to record electrically evoked synaptic activity from rat hippocampal brain slices. Population spikes (PS) were evoked at 30 s intervals by electrical stimulation of the Schaffer collateral pathway and were recorded using extracellular electrodes positioned in the CA1 cell body layer. Responses were quantified as PS areas (the area above and below the 0 mV line). The effects of eight validation compounds known to cause seizures in vivo and/or in the clinic were assessed. RESULTS: Seven out of eight compounds evoked a concentration-dependent increase in population spike (PS) area that was statistically significant at higher concentrations (P<0.05; ANOVA). At the highest test concentration the percentage effects (mean+/-s.e.m.), relative to vehicle, were: picrotoxin 212.9+/-28.8, pentylenetrazole (PTZ) 181.4+/-24.7, 4-AP 328.9+/-48.6, aminophylline 124.5+/-5.9, chlorpromazine 122.1+/-9.8, SNC-80 132.1+/-12.6 and penicillin 174.7+/-14.1. Physostigmine had no significant effect on PS area although a concentration-dependent change in the morphology of the response was evident. DISCUSSION: All validation compounds evoked a statistically significant effect on synaptic activity in the rat hippocampal slice. Although similar effects have been described previously, this is the first time that the effects of a pharmacologically diverse set of compounds have been assessed using a standardised brain slice assay. Given the low compound usage and relatively high throughput associated with this assay, the hippocampal brain slice assay may facilitate earlier testing of convulsant liability than is currently possible using in vivo models.