Drug Development
Table 1: Reference hERG inhibitors used for the evaluation of the automated patch clamp platform CytoPatch COMPOUND DESCRIPTION E4031 Cisapride Astemizole Caffeine Dofetilide Terfenadine Quinidine Verapamil
Class III antiarrhythmic drug
Gastroprokinetic agent
Antihistamine drug
Central nervous system stimulant
Class III antiarrhythmic drug
Antihistamine
Class I antiarrhythmic drug
L-type calcium channel blocker
CYTOPATCH IC50 70.3nM 32.8nM 3.50nM 7.42mM 19.2nM 36.7nM 0.97µM 0.56µM
IC50 MANUAL PATCH
CLAMP 8.3nM 13.6nM 2.1nM 10.5mM 25.5nM 47.5nM 2µM 0.37µM 8.5 2.4 1.7 0.71 0.75 0.77 0.49 1.5 RATIO
potential to inhibit the IKr current that is conduct- ed 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 clamp- ing services. WIL Research acquired the automated patch clamp platform CytoPatch (Figure 1) due to the high quality of the platform and data it gener- ates10. 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 measure- ment and high-quality data. The quartz chip con- tains 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, pos-
Drug Discovery World Spring 2013
itive pressure is applied to the pipette to keep it clean. The cells are then captured by the cytocen- tring 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 refer-
ence 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
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References 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. High- throughput 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.
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