Improved throughput of patchxpress herg assay using. An in vitro electrophysiological assay system, which can assess compound effects and thus show cardiotoxicity including arrhythmia risks of test drugs, is an essential method in the field of drug development and toxicology. The comprehensive in vitro proarrhythmia assay cipa initiative was established to develop a new paradigm for assessing proarrhythmic risk, building on the emergence of new technologies and an expanded understanding of torsadogenic mechanisms beyond herg block. Automated patchclamp methods for the herg cardiac potassium. This time we have investigated the applicability of those protocols. In preclinical drug development, seldane gave no cardiac risk signals no effects on cardiac action potential or electrocardiographic qt interval.
Manual patch clamp assay manual patch clamping is the goldstandard for the investigation of ion channel activity. Complementary to these technologies has been the development of lower capacity systems that fully automate conventional manual patchclamp. Since manual patch clamping has long been regarded as the gold standard for evaluating herg safety by fda and other regulatory institutions, creative biolabs provides firstclass manual patch clamp electrophysiology measurement of herg inhibition with high accuracy. Crosssite and crossplatform variability of automated patch. Pdf improved throughput of patchxpress herg assay using. Our manual and automated patch clamp assays reveal interactions of compounds with cardiac herg channels herg currents are recorded from stably transfected cho cells protocols are available for single dose testing and doseresponse curves throughput according to our customers needs herg patch clamp. Cipa comprehensive in vitro proarrhythmia assay sophion. In addition to confirming the activity of potential hits from high or medium throughput screens, manual patchclamping can be used to assess mechanism of action of compounds and to determine the effects of compounds on the.
Early identification of herg liability in drug discovery. This new paradigm will consist of four interrelated components. In addition to confirming the activity of potential hits from high or medium throughput screens, manual patch clamping can be used to assess mechanism of action of compounds and to determine the effects of compounds on the. Role of herg potassium channel assays in drug development. The assay should be conducted by manual patch clamp rather than automated patch clamp and variants, and be conducted at physiological temperature to reproduce the in vivo kinetics of activation. Precompound current and postcompound current are measured by patch clamp, and applied to the calculation of herg inhibition. Measurement of k v currents in an automated 384wellpatchclamp system. I am doing herg voltage clamp protocol using port a patch but i am unable to get a steady current. A broad range of in vitro services for screening, efficacy evaluation and preliminary assessement of safety of new drugs and formulations a key area of our expertise is represented by in vitro electrophysiology studies cardiac safety and high throughput automated patch clamp. Although manual patch clamp testing is the gold standard for this purpose, biochemical herg assay allows checking multiple compounds at the earliest stages of drug discovery. However, prediction of in vivo effects from in vitro measurements of herg block may be complicated in.
Manual wholecell patchclamping of the herg cardiac k channel. In the automated patchclamp measurements performed here, kv. Although manual patchclamp testing is the gold standard for this purpose, biochemical herg assay allows checking multiple compounds at the earliest stages of drug discovery. Automated electrophysiological patch clamp allows assessment of herg.
Of the seven ion currents studied, herg was the most often blocked current followed by nav1. The majority of survey respondents are making greatest use of automated herg patch clamp and manual herg patch clamp systems to undertake in vitro cardiotoxicity assessment. An invitro patchclamp assay using hek293 cells transfected with the human erg gene, which encodes the channel allowing the ikr potassium current. Ion currents were evaluated in expression systems using the manual whole cell patch clamp technique. Manual patch clamping is the goldstandard for the investigation of ion channel activity. The closest to this ideal would be voltage clamp recordings in cardiac myocytes using a. Protocol details and the translation of criteria known from manual patch clamp. Cipa comprehensive in vitro proarrhythmia assay the comprehensive in vitro proarrhythmia assay cipa initiative aims to replace the preclinical herg current assay required under the ich s7b safety pharmacology guidelines and clinical tqt study, which provides a surrogate marker of proarrhythmia, with more translationally relevant assessments of proarrhythmic risk sager et al. Mice increased accuracy to 93% and significantly reduced types 1 and 2 errors. Growing interest in outsourcing ion channel services. In this study, highthroughput electrophysiological recordings of human embryonic kidney hek 293 cells and chinese hamster ovary cho cells stably expressing. As, copenhagen, denmark technology, many of the assay and protocol. In vitro assessment of drug effects on multiple ionic currents. During a patch clamp recording, a hollow glass tube known as a micropipette or patch pipette filled with an electrolyte solution and a recording electrode connected to an amplifier is brought into contact with the membrane of an isolated cell.
In vitro herg patch clamp assays have become standard components in. We constructed logistic regression models to compare the herg and mice assays. Mice vs herg assays the in vitro herg assay has 70% accuracy for predicting tdp type 1 errors are most frequent but type 2 errors occur we proposed a multiple ion channel effects mice assay as a better predictor we measured concentrationresponses of herg, hnav1. A key area of our expertise is represented by in vitro electrophysiology studies cardiac safety and high throughput automated patch clamp.
The comprehensive in vitro proarrhythmia assay cipa guide. In the aftermath of recent drug withdrawals, regulatory agencies now require in vitro herg screening of all pharmaceutical compounds that are targeted for human use. A patchclamp investigation of hek 293 cells or cho cells expressing herg was performed in an automated way by using a 384wellpatchclamp system. Guidelines for nonclinical studies of drugs manual in 1995. Electrophysiological analysis of mammalian cells expressing herg. Wholecell recordings or singlechannel analysis in various expression systems hek 293, cho, neuroblastoma etc. The qpatch performs up to 48 independent patch clamp experiments in parallel in a 48channel chip array with microfluidic chambers qplate.
Verification of cipa recommended voltage protocols in. Electrophysiological analysis of mammalian cells expressing. We offer manual patch clamp as well as automated patch clamp services. While this article focuses on herg channel recordings using the qpatch sophion as, copenhagen, denmark technology, many of the assay and protocol details given in this article can be transferred for setting up different ion channel assays by automated patch clamp and are similar on other planar patch clamp platforms.
The qpatch htx is a highthroughput and automated electrophysiology platform. In this assay, cho herg cells or hek293 herg cells are used to specifically assess the effect of test compounds on the herg channel. An evaluation of 30 clinical drugs against the comprehensive. However, prediction of in vivo effects from in vitro measurements of herg block may be complicated in the case of drugs that are strongly protein bound. Technologies as automated patch clamp electrophysiology measurements. Manual patch clamp herg inhibition assay performed in 100% fetal bovine serum.
From singlecell manual patch clamp, the gold standard in ion channel work, to high throughput 384well automated electrophysiology instruments, we use our cells on a variety of platforms to provide the throughput or depth of analysis that matches your needs. The comprehensive in vitro proarrhythmia assay cipa. To minimize the potential for failure in laterstage drug development, many pharmaceutical and biotechnology companies have begun to use automated patch clamp systems with higher. Manual patch clamp evaluation of herg channel pharmacology. The predictor herg fluorescence polarization assay kit provides a set of validated components to perform herg channel biochemical binding studies in the absence of radioligand. In this assay, choherg cells or hek293herg cells are used to specifically assess the effect of test compounds on the herg channel. Improved throughput of patchxpress herg assay using intracellular potassium fluoride. Dec 16, 2015 measurement of k v currents in an automated 384wellpatchclamp system.
Acroscell experienced technicians allow us to provide customized assays. These data were used for model optimization and metric selection, as detailed in several manuscripts. With ongoing investments in stateoftheart equipment and scientific expertise to support your specific needs, we are focused on providing costeffective, highquality, reproducible data, and flexible solutions with fast turnaround times. Data has a very good correlation with fdas manual patch clamp.
Verification of cipa recommended voltage protocols in patch. Ion channel targets remain a top priority for many pharma and biotechs with most looking to increase their screening efforts in 2006. Sep 02, 2014 while this article focuses on herg channel recordings using the qpatch sophion as, copenhagen, denmark technology, many of the assay and protocol details given in this article can be transferred for setting up different ion channel assays by automated patch clamp and are similar on other planar patch clamp platforms. Please email us to discuss which platform is most appropriate according to your needs, or get in touch with the contact person on the relevant subpage. Complementary to these technologies has been the development of lower capacity systems that fully automate conventional manual patch clamp. Screening compounds for hazardous side effects on herg channel early in the drug development process is likely to cut the drug development costs. In vitro herg patch clamp assays have become standard components in cardiac safety evaluation during nonclinical drug development. Our cardiac panel of ion channels include all relevant cardiac ion channels defined by the cipa consortium, including herg, na v 1. The comprehensive in vitro proarrhythmia assay cipa guide sedating antihistamine for symptomatic treatment of allergic rhinitis sneezing, itchy or runny nose, watery eyes. Metrion offers nonglp herg screening services using the qpatch automated gigaseal patch clamp platform. An in vitro electrophysiological assay system, which can assess compound. 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. We offer fourpoint and eightpoint concentration response herg screening assays that are performed at room temperature.
In contrast, the measured value for cisapride using this assay is threefold. Other systems, eg herg receptor binding methods and purkinje fibre analysis were less widely used to undertake in vitro cardiotoxicity assessment. Early identification of herg liability in drug discovery programs by. Currents were elicited using either a ventricular action potential waveform or stepramp voltage protocols. However, the multiwell plate format of automatized patchclamp does not allow visual detection of potential microprecipitation of poorly soluble compounds. Many factors can impact data quality hence reproducibility. Applied strict data quality criteria and compared results generated by 3 electrophysiologists using different styles of manual patch clamp rigs.
A typical test pulse protocol for a manual patch clamp study of hek293 cells. Ntp42, a novel antagonist of the thromboxane receptor, attenuates experimentally induced pulmonary arterial hypertension. A broad range of in vitro services for screening, efficacy evaluation and preliminary assessement of safety of new drugs and formulations. Medium and high throughput herg assays the ideal herg assay provides a linear measure of channel activity under physiologically relevant conditions.
An in vitro patch clamp assay using hek293 cells transfected with the human erg gene, which encodes the channel allowing the ikr potassium current. Completed herg channel pharmacology using 12 cipatraining drugs and manual patch clamp rigs. This assay is recommended by the cipa committee since it can detect changes in herg amplitude and decay kinetics due to drug binding and trapping. This version of model and metric were frozen and their performance are to be evaluated using independent. Review preclinical cardiovascular risk assessment in modern.
Why are there current fluctuations in herg patch clamp assay. We use the industry leading platform qpatch for ion channel screening. Patch clamp herg safety testing services herg human etheragogorelated gene, in italics, or kcnh2 in the new nomenclature is a gene that encodes the poreforming alpha subunit of a voltagegated potassium channel expressed in the heart and in nervous tissue. In this chapter we describe bench procedures for the culture and preparation of hergexpressing cho cells for recording on an automated patchclamp workstation. The assay was designed to identify potential herg channel blockers by producing data that accurately correlates with patchclamp electrophysiology studies. Depending on the application scheme and the type of ion channel the manual setups at bsys allow the measurement of 2050 compounds per day. The milnes protocol can easily be run on qpatch and the assay meets the fdas cipa requirements for improved arrhythmia prediction. The current fluctuations are so high that i cant even test a single conc. Pdf early identification of herg liability in drug discovery. Conventional manual patch clamp measurements of the concentrationresponse relationship in ion channels stably expressed in mammalian cells provide. The protocol described in this unit is designed to assess the effects of new chemical entities after acute or long. A typical test pulse protocol for a manual patch clamp study of. Whether you want to explore an ion channel as a potential new target or understand cardiovascular liability, or other liabilities, apconix can help you with our electrophysiology expertise, customerfocused flexibility and rapid delivery of high data quality. The assay should be conducted by manual patchclamp rather than automated patchclamp and variants, and be conducted at physiological temperature to reproduce the invivo kinetics of activation.