Frontiers early identification of herg liability in drug. The milnes clamp protocol 14 provides detailed descriptions of. The recent years have witnessed automatization and parallelization of the manual patchclamp technique, allowing higher throughput screening on recombinant. We introduce a novel screening system for druginduced arrhythmogenic risk that combines in vitro pharmacological assays and a multiscale heart simulator. That technique required a huge effort, training, luckiness and patience, but good results acceptable for publication were not easily obtained.
The protocol described in this unit is designed to assess the effects of new chemical entities after acute or long. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patchclamp electrophysiology studies. He has an experience of herg assay from scratch at a cro, where he was responsible for herg assay in terms of creating and supervising patch clamp platform, sops, protocol. Verification of cipa recommended voltage protocols in. We decided that the traditional approach of specific fixed voltage steps which neatly decouples time and voltagedependence was a bit slow and tricky to assemble into a coherent model. A comment on the milnes dynamic block voltage clamp protocol for assessing kinetics of herg current block. This ion channel sometimes simply denoted as herg is best known for its contribution to the electrical activity of the heart. Patch clamp technique is the gold standard for carrying out the herg assay. Ionic current measurements request a study outline from ipst wholecell current amplitude and kinetics measurements verify the results of the interaction of a test article with a selected ionic current. The intercellline reproducibility of herg assay using.
In this assay, cho herg cells or hek293 herg cells are used to specifically assess the effect of test compounds on the herg channel. Borosilicate glass chips are used as the patch clamp substrate, ensuring excellent voltage control of the cell membrane and high quality seals. The current fluctuations are so high that i cant even test a single conc. In silico this is a nice robust validation of modifications to a mature model, i worry about. There are two main modes in patch clamp recordings. Voltage gated channels such as herg expressed in hek293 have been used to validate the system. Patch clamp technique is the gold standard for carrying. Pdf automated patchclamp methods for the herg cardiac. Crosssite and crossplatform variability of automated. Measuring kinetics and potency of herg block for cipa. Automated patch clamp electrophysiology is an emerging technique for identifying cardiac ion channel liabilities. Role of herg potassium channel assays in drug development. However, a number of potential sources of errors in setting up herg channel assays by automated patch clamp can lead to misinterpretation of data or false effects being reported.
In hek293herg cells, conventional patch clamp measurements revealed that venlafaxine, a serotonin and norepinephrine reuptake inhibitor antidepressant, inhibits herg channels with a potency of 28m. This article describes protocols for automated electrophysiology screening of compound effects on the herg channel current. Screening system for druginduced arrhythmogenic risk. View verification of cipa recommended voltage protocols in patch clamp assay for herg, cav1. Protocols are available for single dose testing and doseresponse curves. Manual patch clamp evaluation of herg channel pharmacology. So we made up some new sinusoidbased protocols for the patch clamp amplifier to rapidly probe the voltage and timedynamics of the currents. Cipa herg protocol this protocol was used for herg studies on the patchliner and syncropatch 384pe. Sucrose gap method with 2 electrodes was the standard voltage clamp technique when used the cardiac tissue.
This chapter describes how to record herg currents in a recombinant cell line using the wholecell patch clamp technique. The human etheragogo related gene herg encodes the inward rectifying. Since activation and inactivation of herg is dependent on membrane potential, voltage clamp recordings can differentiate between compounds that preferentially interact with different states of the channel. I am doing herg voltage clamp protocol using port a patch but i am unable to get a steady current. The predictor herg fluoresence polarization assay uses a proprietary fluorescent tracer and membrane preparation to create a highly reproducible assay. The current fluctuations are so high that i cant even test a. Early identification of herg liability in drug discovery. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or longterm exposure on the amplitude of ikr in human embryonic kidney 293 hek293 cells stably transfected with the herg channel wholecell configuration of the patch clamp technique. We present a new highinformation 15 s protocol to characterise herg kv11. Patch clamp recording is an extremely useful technique for investigating the biophysical properties of the ion channels that control neuronal activation. The intercellline reproducibility of herg assay using the wholecell patch clamping 1. Wholecell configuration of the patchclamp technique in the herg. Predicting how pharmaceuticals may affect heart rhythm is a crucial step in drug development and requires a deep understanding of a compounds action on ion channels.
Ion channel targets remain a top priority for many pharma and biotechs with most looking to increase their screening efforts in 2006. High throughput herg assay using the automated patch. Fluicell dynaflow resolve system ion channel screening. The syncropatch 384768pe is the highest throughput patch clamp. The herg safety service is performed at our parent company, evotec, and is a cellbased assay which employs the qpatch htx system sophion bioscience as or the syncropatch 384pe nanion technologies as automated patch clamp electrophysiology measurements. We demonstrate its use on the nanion syncropatch 384pe, a 384 well automated patch clamp platform, by applying it to cho cells stably expressing herg1a. The expertise of our technicians allows us to provide customized assays. The impact of automated patch clamping apc on ion channel screening is now. Blockade of the human ether agogorelated gene herg. In this study, an automated patch clamp system, patchxpress 7000a, was used to evaluate effects of drug on the herg channels transfected in hek 293 cells. These traces show the raw current responses of a single cell to a herg iv pulse protocol. Wholecell configuration of the patchclamp technique in the herg channel assay to predict the ability of a compound to prolong qt interval. B a manual patchclamp protocol composed of an 8 s voltageclamp protocol designed for rapid characterization of ion channel kinetics by. Characterization of herg channel blockers using the flipr.
Various voltage protocols to induce herg currents were determined and the. The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. Wholecell configuration of the patchclamp technique in. The impact of automated patch clamping apc on ion channel screening is now evident, particularly in early noncompliant herg liability testing, where apc is rapidly becoming the new gold standard technology. It also provides typical voltage protocols used for assessing the basic electrophysiological properties of these currents and for assessing the effects of compounds on herg. The human etheragogorelated gene herg encodes the poreforming subunit of the rapidly activating delayed rectifier potassium channel ikr. Verification of cipa recommended voltage protocols in patch clamp assay for herg, cav1. The membrane potential was clamped to a holding potential of 60 mv, then depolarized to 20 mv for 2000 ms, before stepping to 60 mv. Protocol details and the translation of criteria known from manual patch clamp experiments to automated patch clamp experiments to achieve good quality data are emphasized.
In vitro electrophysiological safety studies have become an integral part of the drug development process since, in many instances, compound. Herg channel dysfunction revealed by dynamic action potential. The syncropatch 384pe upgradeable to 768pe is a high throughput patch clamp instrument recording from up to 384 or 768 cells simultaneously. The comprehensive in vitro proarrhythmia assay cipa. The procedure involves pressing a glass micropipette against a cell in order to isolate a small patch of membrane that contains one or more ion channels. The assay is based on the principle of fluorescence polarization where a redshifted fluorescent tracer is displaced from the herg channel by compounds that bind to the channel. Short and rich voltageclamp protocols mathematical. While standardized patch clamp protocols and new multichannel pharmacology data sets were being generated by the icwg, initial in silico efforts used ic 50 s for the various cardiac ion channels of interest from the patch clamp data set generated by crumb et al. Our manual and automated patch clamp assays reveal interactions of compounds with cardiac herg channels herg currents are recorded from stably transfected cho cells. Both drugs were previously shown by manual patch clamp to not exhibit drug trapping milnes et. The popular herg assay is an example of ionic current assay, saimed specifically at the wellknown herg channel. Automated electrophysiological patch clamp allows assessment of herg channel effects early in drug development to aid medicinal chemistry programs and has become routine in pharmaceutical companies. Why are there current fluctuations in herg patch clamp assay using. Automated patchclamp methods for the herg cardiac potassium channel.
Introduction the wholecell patch clamp technique using cell lines transfected with human etheragogorelated gene herg has become more important recently for prediction of a. For 12 drugs reported to have varying cardiotoxicity risks, doseinhibition curves were determined for six ion channels using automated patch clamp systems. Why are there current fluctuations in herg patch clamp. The performance of the dynaflow system in this study demonstrates its suitability for conducting safety studies. Automated patchclamp methods for the herg cardiac potassium. In vitro herg channel current recordings are an important step in evaluating the proarrhythmic potential of small molecules and are now routinely performed using automated highthroughput patch clamp platforms. What is the benefit of tail current in patch clamp recordings. Precompound current and postcompound current are measured by patch clamp, and applied to the calculation of herg inhibition. To achieve this, the protocol should ideally be able to measure both potency and kinetics of drug binding to herg and be amenable to data acquisition in the high throughput automated patch clamp systems that are currently employed within industry. Protocol details and the translation of criteria known from manual patch clamp experiments to automated patch clamp experiments to. Examples of results obtained with terfenadine, arsenic, pentamidine, erythromycin, and sotalol are provided for illustrative purposes. The results confirm the importance of herg as the most studied ion channel with. The results were obtained by eliciting herg current responses with a voltage pulse protocol. Technical validation through characterization of voltagegated potassium channels and ligandgated ion channels.
95 530 300 626 887 704 356 279 982 502 1341 415 235 726 47 31 109 1015 147 365 504 837 499 1410 341 1222 1189 1334 1022 24 788 758