An Automated Approach To Solving Pharma's Cardiac Toxicity Conundrum
The pharmaceutical industry is facing ever-growing difficulties in developing new drugs and bringing them to market (1,2). Many factors stand in the way of R&D productivity, not least of which are shrinking budgets. Yet one of the most pressing challenges continues to be the issue of ensuring that new drug candidates have an acceptable safety profile.
In an effort to avoid surprises far along the drug-development road, pharmaceutical companies are making it a priority earlier in the process to test for toxicity – especially for highprevalence liabilities such as hERG cardiac toxicity (3).
But these tests are feasible only by using higher throughput methods that demand a small amount of compound (4). The recent introduction of automated patch clamp platforms that deliver high-quality data has enabled this much-needed hERG profiling earlier in drug discovery during lead optimisation, helping pharmaceutical companies spend their resources wisely (5).
This review discusses the application of automated patch clamp platforms during lead optimisation and thoroughly explores advantages and disadvantages of various platforms in use today. Several examples from the evaluation of the automated patch clamp platform CytoPatch (Figure 1) at WIL Research, a global contract research organisation, are also shown.
The business risk of innovative drug candidates
Pharmaceutical companies spend tremendously on drug research and development, pouring resources into every phase: target finding, lead finding, lead optimisation, preclinical development, and Phase IIII clinical development. And that is before marketing costs are factored in.
Yet, despite a marked increase in spending over the past decade, the industry is putting forth fewer new drug candidates; the number of applications filed to FDA’s Center of Drug Evaluation and Research declined to 23 in 2010 from 45 in 1996. Analysis of a database with 28,000 R&D projects shows that the attrition rates in all phases of drug development have increased significantly (2).
The fall in R&D productivity corresponds with the pharmaceutical industry’s focus on creating new therapeutic targets that will have less post-launch competition. The drawback of this approach, however, is the difficulty in developing such drug candidates with high efficacy and low safety risk. Due to this, the approach tends to come with lower success rates yet higher development costs.
Why aren’t these innovative drug candidates meeting safety requirements? A review of drug development data6 shows that cardiovascular toxicity is often to blame, accounting for approximately 27% of drug failures due to toxicity in the preclinical phase. Phase I clinical studies are relatively safer in terms of cardiovascular toxicity, with only 9% showing serious adverse drug reactions.
The overall attrition rate due to cardiovascular events in clinical development is 21%, indicating that several cardiovascular effects occur in Phase II and III clinical trials which are not detected in the preclinical studies or earlier clinical trials (6). There are several different types of cardiovascular toxicity; one major type is toxicity caused by drug effects on cardiac ion channels like hERG.
It’s all about hERG: the human Ether-à-go-go Related Gene (hERG) ion channel
The sum of the action potentials of the different parts of the heart is clinically monitored using surface electrodes and results in an electrocardiogram, or ECG. Drugs that affect ion channels in the heart can change ECG parameters such as the QT-interval, which represents the time from the depolarisation of the ventricles to the repolarisation of the ventricles. Many drugs showing cardiac toxicity prolong the QT-interval in the ECG (6,7).
Drug induced prolongation of the QT-interval can lead to torsade de pointes, a life-threatening ventricular arrhythmia that can cause sudden cardiac death. Drugs prolonging the QT interval appear to consistently inhibit the outward, rapid-delayed rectifier K+ current (IKr) conveyed by the hERG (human Ether-à-go-go Related Gene, or the KCNH2 gene in the modern nomenclature) channel. Therefore an in vitro study to assess a compound’s potential to inhibit this channel is an essential part of the non-clinical regulatory testing battery.
The hERG gene encodes for the pore-forming subunit of the voltage-gated potassium (K+) channel that controls the outward potassium current during a cardiac contraction. The hERG ion channel has long been known to be the target of class III anti-arrhythmic drugs such as amiodarone. Unfortunately, the hERG channel also interacts with a variety of noncardiovascular drugs.
Several drugs, in fact, have been withdrawn from the market due to adverse cardiac effects. Examples include astemizole, an antihistamine, and cisapride, a gastroprokinetic drug (7). As a result, a direct assay of hERG channel inhibition is now an expected part of the safety pharmacology package conducted to support initiation of First-in-Man clinical trials.
Regulatory studies for non-clinical cardiovascular safety testing are described in the ICH guidelines S7A and S7B. ICH S7A describes safety Pharmacology Studies for Human Pharmaceuticals. ICH S7B extends and complements this guideline and describes studies for the assessment of QT-interval prolongation. Studies described in the ICH S7B guideline identify the potential of a test substance and its metabolites to delay ventricular repolarisation (QT-prolongation), and assess the dose relationship between the compound concentration and the effect.
The manual patch clamp technique: the current standard
Today’s standard for measuring hERG inhibition is the manual patch clamp technique. Through this technique, a cell present in a bath is approached with a glass pipette containing an electrode by using a micromanipulator. The principle of the manual patch clamp technique is shown in the upper part of Figure 2.
During this technique, positive pressure is applied to the glass pipette to keep the tip of the pipette clean. Subsequently, mild suction is applied when the tip of the pipette touches the cell. As a result, the membrane of the cell enters the pipette and a tight seal forms between the cell membrane and the inner surface of the pipette. Finally, more suction is applied to disrupt the membrane and an electric circuit is established between the electrode in the micropipette and the cytoplasm.
In this way, the potential difference between a bath electrode and the electrode in the pipette directly reflects the membrane potential. By measuring cells that overexpress the hERG ion channel (such as HEK-293 cells stably transfected with hERG-1 cDNA), it is possible to accurately assess the effect that a drug candidate has on the current IKr that is conducted through the hERG ion channel.
An estimated 25-40% of all drug candidates show some level of hERG-related inhibition, which results in a high level of attrition due to QT-prolongation in the preclinical phase (8). The early detection of hERG cardiac toxicity and use of the data for compound development with better safety profiles has therefore been proposed by the pharmaceutical industry as a useful strategy (9).
Toxicity screening in the lead optimisation phase requires assays and techniques that have a relative high throughput and need a very small amount of test sample (4). Yet, the manual patch clamp technique is low throughput and requires a high amount of compound, which makes this standard technique less suitable during the lead optimisation phase of drug development.
A new option: automated patch clamping
There are several automated patch clamp platforms on the market (9-12) that are designed to address the drawbacks of manual methods. All have their own characteristics, throughput and data quality.
Automated systems suspend cells that are injected into a recording chamber containing a planar substrate with one or more small apertures. In this chamber, the cells are captured by negative pressure and directed on to the patch aperture. The negative pressure is then increased to help form a tight seal between the cell membrane and the chamber (10).
The formation of a tight seal is prerequisite for high-quality patch clamp recordings (good voltage control of the cell as well as a stable measurement). An electrical circuit is established after disrupting the membrane either by membrane rupture or the application of pore forming chemicals. By using a specific voltage protocol and cells with an overexpression of the hERG ion channel, researchers can determine a drug candidate’s potential to inhibit the IKr current that is conducted through the hERG ion channel.
To address the pharmaceutical industry’s shift to safety testing earlier in drug development, WIL Research has invested in automated patch clamping services. WIL Research acquired the automated patch clamp platform CytoPatch (Figure 1) due to the high quality of the platform and data it generates (10). This platform is unique in that it includes a chip made out of quartz glass containing a patch clamp pipette. This ensures tight binding between the pipette and cells, resulting in stable measurement and high-quality data. The quartz chip contains two microfluidic channels (Figure 2).
The first channel is present in the glass pipette and is filled with an intracellular solution. The second channel, called the cytocentring channel, captures cells by applying negative pressure. The quartz chips are embedded by plastic packaging. This packaging contains a third channel that applies extracellular buffer or a test compound, such as a drug candidate. The lower part of Figure 2 depicts the principle of a patch clamp measurement with a CytoPatch chip.
With exception of the process of cell catching, the principle of the CytoPatch resembles the manual patch clamping technique. Before cell catching, positive pressure is applied to the pipette to keep it clean. The cells are then captured by the cytocentring channel. Subsequently, negative pressure is applied to the pipette to form a tight seal. Finally, the negative pressure in the pipette is further increased to rupture the membrane. Electrical access to the cells is obtained and recordings can start. During a measurement there is continuous flow with extracellular solution, which stabilises the seal.
Evaluation of the automated patch clamp platform cytopatch
To demonstrate proper operation of the CytoPatch system, WIL Research and Cytocentrics Bioscience GmbH have performed an Installation Qualification (IQ), Operational Qualification (OQ) and Performance Qualification (PQ). The CytoPatch software includes an audit trail, user access administration and is 21 CFR part 11 and GLP compliant. The IQ, OQ and PQ passed all acceptance criteria.
CytoPatch was evaluated by testing eight reference hERG inhibitors (Table 1). The IC50 values were calculated and compared with manual patch clamp data obtained in-house or from Cytocentrics (Table 1).
The obtained IC50 values were similar for both methods. The only difference between IC50 values was with E4031, which exhibited an 8.5-fold difference. This difference could be explained by the use of different batches of E4031. The correlation between the IC50 values determined with the CytoPatch and those determined using manual patch clamp is depicted in Figure 3.
A high correlation of 0.97 between the two methods was observed representing the high data quality obtained with the CytoPatch instrument.
How automated patch clamps differ
Automated methods have a higher throughput than manual patch clamp methods, demand a smaller amount of test article (a few milligrams) and only require trained technicians rather than electrophysiologists to operate the devices. Based on these characteristics, the automated methods can be easily integrated in lead optimisation programmes for drug development. Table 2 shows a comparison of various automated patch clamp platforms and manual patch clamping.
The CytoPatch platform delivers, in comparison to the other automated patch clamp platforms, a lower throughput (approximately 20 data points per day versus up to about 100 to 1,000 data points per day for the other platforms when looking at hERG screening). But because the CytoPatch platform is designed with a modular concept, up to 20 devices can be connected to form one multichannel CytoPatch instrument.
Using this concept, the throughput can be increased to approximately 400 data points per day. Thus, when throughput alone is the most important criterion for selecting a patch clamp platform, the PatchXpress (Molecular Devices) or Qpatch (Sophion) platforms have the best characteristics.
Other characteristics such as the presence of a glass pipette, the ability to form gigaseals, fast and continuous perfusion, and 21 CFR part 11 and GLP compliancy are key criteria for measuring the quality of automated patch clamp platforms, as summarised in Table 2. All automated patch clamp devices in Table 2 have the characteristic that gigaseals are formed.
In contrast to other platforms the CytoPatch does not demand fluoride buffers to enhance seal formation, which results in a more physiological model. Of the automated systems, the CytoPatch is the only system that is 21 CFR part 11 and GLP compliant, that makes use of a real glass pipette and contains a fast and continuous perfusion system that allows for voltage gated and ligand gated ion channels tests. Therefore the CytoPatch ensures the highest data quality and similarity with the manual patch clamp method.
Future outlook: translating in vitro safety studies into clinical QTprolongation
The vast majority of the clinical cases of druginduced QT-prolongation have been observed to be due to blockade of the hERG ion channel. However, as more and more data becomes available about drug-induced QT-prolongation, it is apparent that there is not always a correlation between hERG inhibition and QT-prolongation. There are numerous examples of false positives and false negatives in the in vitro hERG assay (13-21) (Table 3).
Therefore, it can be concluded that additional (in vitro) assays are needed to better predict the cardiac safety profiles of compounds.
The main reason for erroneous conclusions of in vitro hERG assays is that several compounds affect ion channels other than hERG or affect multiple ion channels. Since the ventricular action potential is coordinated by interplay of ion channels – of which Nav1.5, Cav1.2, Kv4.2/4.3, Kv7.1, hERG and Kir2.1 are the main ones – drug interaction with one of these other channels can affect the QT-time.
To get a better cardiac safety profile of a drug candidate, screening of a compound against a panel of cell lines with overexpression of the main ion channels involved in the ventricular action potential has been proposed (22). Performing such a profiling by using manual patch clamping would be too laborious for screening purposes; however, the availability of automated patch clamp platforms has made it possible to perform such a screening (5).
A new development is the application of induced pluripotent stem (iPS) cell-derived human cardiomyocytes. These cells express several relevant cardiac ion channels at comparable levels to primary human cardiomyocytes and can be used for a total ion channel measurement. Future application of these iPS cell-derived human cardiomyocytes may give a better prediction for cardiac toxicity. Performing a total ion channel measurement is feasible with the automated patch clamp methods discussed here.
Drug-induced QT prolongation is recognised as a major hurdle in the successful development of drug candidates. The main cause of drug-induced QT FALSE prolongation is due to hERG channel blockade. A direct assay of hERG channel inhibition is therefore routinely part of the safety pharmacology package conducted to support initiation of clinical trials. Several medium or high-throughput automated patch clamp platforms have become available that make it possible to move the hERG assay earlier in the preclinical phase to early lead optimisation. This strategy can help to improve the success rate of drug candidates.
To support this new testing strategy, WIL Research now offers the CytoPatch automated patch clamp platform. Evaluation of this platform has shown that there is a high correlation with manual patch clamping. Of the automated patch clamp platforms tested, the CytoPatch platform most closely mimics manual patch clamping. Since the correlation between hERG inhibition and drug-induced long QT syndrome is not perfect, the testing of drug effects on other ion channels is recommended to be included in future in vitro testing strategies.
Beppy van de Waart is Head of the In Vitro & Environmental Toxicology Department at WIL Research, a global CRO whose focus is to custom design product safety toxicological research. Under her responsibility genetic toxicology, in vitro toxicology, in vitro ADME, in vitro safety pharmacology and ecotox studies (GLP and non- GLP) are designed, executed and reported and new studies are developed.
Dr Walter Westerink is Study Director at the In Vitro & Environmental Toxicology Department at WIL Research. He is involved in genotoxicity, in vitro safety pharmacology and in vitro toxicology studies and development of new studies. Before joining WIL Research in September 2011, he held various positions in the pharmaceutical industry where he was involved in mechanistic and investigative toxicology and early ADME and toxicity screening.
Dr Nuria Piñeiro Costas has been Study Director at the In Vitro & Environmental Toxicology Department at WIL Research since 2006. She is involved in all in vitro ADME studies and in vitro safety pharmacology studies. Before joining WIL Research, she held different research positions in university and industry.
1 Kola, I, Landis, J. Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov 2004; 3(8):711-715.
2 Pammolli, F, Magazzini, L, Riccaboni, M. The productivity crisis in pharmaceutical R&D. Nat Rev Drug Discov 2011; 10(6):428-438.
3 Thomas, CE, Will, Y. The impact of assay technology as applied to safety assessment in reducing compound attrition in drug discovery. Expert Opin Drug Discov 2012; 7(2): 109-122.
4 Schoonen, WG, Westerink, WM, Horbach, GJ. Highthroughput screening for analysis of in vitro toxicity. EXS 2009; 99:401-452.
5 Moller, C, Witchel, H. Automated electrophysiology makes the pace for cardiac ion channel safety screening. Front Pharmacol 2011; 2:73.
6 Laverty, H, Benson, C, Cartwright, E, Cross, M, Garland, C, Hammond, T et al. How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines? Br J Pharmacol 2011; 163(4): 675-693.
7 Chiang, C. Drug-induced long QT syndrome. J Med Biol Eng 2006; 26(3):107-113.
8 Stummann, TC, Beilmann, M, Duker, G, Dumotier, B, Fredriksson, JM, Jones, RL et al. Report and recommendations of the workshop of the European Centre for the Validation of Alternative Methods for Drug-Induced Cardiotoxicity. Cardiovasc Toxicol 2009; 9(3):107-125.
9 Polonchuk, L. Toward a New Gold Standard for Early Safety: Automated Temperature- Controlled hERG Test on the PatchLiner. Front Pharmacol 2012; 3:3.
10 Scheel, O, Himmel, H, Rascher-Eggstein, G, Knott, T. Introduction of a modular automated voltage-clamp platform and its correlation with manual human Ether-ago- go related gene voltageclamp data. Assay Drug Dev Technol 2011; 9(6):600-607.
11 Stoelzle, S, Obergrussberger, A, Bruggemann, A, Haarmann, C, George, M, Kettenhofen, R et al. State-of-the-Art Automated Patch Clamp Devices: Heat Activation, Action Potentials and High Throughput in Ion Channel Screening. Front Pharmacol 2011; 2:76.
12 Yang, N. Cardiac drug safety and hERG channel. T Bio/Phar Ind 2012; 1:30-35.
13 Zhang, S, Zhou, Z, Gong, Q, Makielski, JC, January, CT. Mechanism of block and identification of the verapamil binding domain to HERG potassium channels. Circ Res 1999; 84(9):989-998.
14 Yuill, KH, Borg, JJ, Ridley, JM, Milnes, JT, Witchel, HJ, Paul, AA et al. Potent inhibition of human cardiac potassium (HERG) channels by the antiestrogen agent clomiphenewithout QT interval prolongation. Biochem Biophys Res Commun 2004; 318(2):556-561.
15 Ridley, JM, Milnes, JT, Hancox, JC, Witchel, HJ. Clemastine, a conventional antihistamine, is a high potency inhibitor of the HERG K+ channel. J Mol Cell Cardiol 2006; 40(1):107-118.
16 Pacher, P, Magyar, J, Szigligeti, P, Banyasz, T, Pankucsi, C, Korom, Z et al. Electrophysiological effects of fluoxetine in mammalian cardiac tissues. Naunyn Schmiedebergs Arch Pharmacol 2000; 361(1):67-73.
17 Witchel, HJ, Pabbathi, VK, Hofmann, G, Paul, AA, Hancox, JC. Inhibitory actions of the selective serotonin re-uptake inhibitor citalopram on HERG and ventricular L-type calcium currents. FEBS Lett 2002; 512(1-3):59-66.
18 Lu, HR, Vlaminckx, E, Van de Water, A, Rohrbacher, J, Hermans, A, Gallacher, DJ. Invitro experimental models for the risk assessment of antibiotic-induced QT prolongation. Eur J Pharmacol 2007; 577(1-3):222-232.
19 Antzelevitch, C, Belardinelli, L, Zygmunt, AC, Burashnikov, A, Di Diego, JM, Fish, JM et al. Electrophysiological effects of ranolazine, a novel antianginal agent with antiarrhythmic properties. Circulation 2004; 110(8):904-910.
20 Lacerda, AE, Kuryshev, YA, Chen, Y, Renganathan, M, Eng, H, Danthi, SJ et al. Alfuzosin delays cardiac repolarization by a novel mechanism. J Pharmacol Exp Ther 2008; 324(2):427-433.
21 Rodriguez-Menchaca, AA, Navarro-Polanco, RA, Ferrer- Villada, T, Rupp, J, Sachse, FB, Tristani-Firouzi, M et al. The molecular basis of chloroquine block of the inward rectifier Kir2.1 channel. Proc Natl Acad Sci U S A 2008; 105(4): 1364-1368.
22 Gintant, G. An evaluation of hERG current assay performance: Translating preclinical safety studies to clinical QT prolongation. Pharmacol Ther 2011; 129(2):109-119.