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Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC50 values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug’s cardiac safety margin. We determined variability of APC data from multiple sites that measured blocking potency of 12 blinded drugs (with different levels of proarrhythmic risk) against four human cardiac currents (hERG [IKr], hCav1.2 [L-Type ICa], peak hNav1.5, [Peak INa], late hNav1.5 [Late INa]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC50 variability (25/75 percentiles) differed for drugs and currents (e.g., 10.4-fold for dofetilide block of hERG current and 4-fold for mexiletine block of hNav1.5 current). Within-platform variance predominated for 4 of 12 hERG blocking drugs and 4 of 6 hNav1.5 blocking drugs. hERG and hNav1.5 block. Bland-Altman plots depicted varying agreement across APC platforms. A follow-up survey suggested multiple sources of experimental variability that could be further minimized by stricter adherence to standard protocols. Adoption of best practices would ensure less variable APC datasets and improved safety margins and proarrhythmic risk assessments.

We have developed an automated patch clamp module for high-throughput ion channel screening, recording from 384 cells simultaneously. The module is incorporated into a laboratory pipetting robot and uses a 384-channel pipettor head for application of cells and compounds. The module contains 384 amplifier channels for fully parallel recordings using a digital amplifier. Success rates for completed experiments (1- to 4-point concentration-response curves for cells satisfying defined quality control parameters) of greater than 85% have been routinely achieved with, for example, HEK, CHO, and RBL cell lines expressing hNaV1.7, hERG, Kir2.1, GABA, or glutamate receptors. Pharmacology experiments are recorded and analyzed using specialized software, and the pharmacology of hNaV1.7 and hERG is described. Fast external solution exchange rates of 200 MΩ, and recordings of voltage-gated Na+ and Ca2+ are shown.

Introduction Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an alternative to manual patch clamp in order to increase throughput whilst maintaining data quality. In order to standardize APC experiments, we have investigated the effects on IC50 values under different conditions using several devices across multiple sites. Methods APC instruments SyncroPatch 384i, SyncroPatch 384PE and Patchliner, were used to record hERG expressed in HEK or CHO cells. Up to 27 CiPA compounds were used to investigate effects of voltage protocol, incubation time, labware and time between compound preparation and experiment on IC50 values. Results All IC50 values of 21 compounds recorded on the SyncroPatch 384PE correlated well with IC50 values from the literature ( Kramer et al., 2013 ) regardless of voltage protocol or labware, when compounds were used immediately after preparation, but potency of astemizole decreased if prepared in Teflon or polypropylene (PP) compound plates 2–3 h prior to experiments. Slow acting compounds such as dofetilide, astemizole, and terfenadine required extended incubation times of at least 6 min to reach steady state and therefore, stable IC50 values. Discussion Assessing the influence of different experimental conditions on hERG assay reliability, we conclude that either the step-ramp protocol recommended by CiPA or a standard 2-s step-pulse protocol can be used to record hERG; a minimum incubation time of 5 min should be used and although glass, Teflon, PP or polystyrene (PS) compound plates can be used for experiments, caution should be taken if using Teflon, PS or PP vessels as some adsorption can occur if experiments are not performed immediately after preparation. Our recommendations are not limited to the APC devices described in this report, but could also be extended to other APC devices.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The NaV1.8 gene (originally named PN3 or SNS; gene symbol SCN10A) encodes a voltage-gated sodium (NaV) channel, selectively expressed in dorsal root ganglion (DRG) neurons. In contrast to the fast and rapidly inactivating TTX-sensitive channels, Nav1.8 is TTX-resisitant and exhibits slower kinetics with a depolarized voltage-dependence of activation and inactivation (1). NaV1.8 is an interesting drug target for inflammatory and neuropathic pain, because modulation of NaV1.8 by inflammatory mediators seems to be a key mechanism of DRG nociceptor sensitization and activation (2). Interestingly, the development of potent and selective Nav1.8 inhibitors demonstrating efficacy in reducing neurophathic pain in animal models fueled the interest in the search for selective Nav1.8 inhibitors (1).The obstacle for drug discovery on ion channels are usually electrophysiological assays.Nanion's SyncroPatch 384PE offers a high throughput gigaeal platform which records up to 384/768 experiments at the same time, and therefore helps filling the gap. It does not only allow to record high quality data with reliable pharmacology, but also more biophyiscal characterization of the protein. A temperature control feature supports measurements at physiological temperatures. Our results show consistent current-voltage relationships and very stable recordings using multihole-chips. Furthermore, we show activation of NaV1.8 from different states that show clearly separated compound affinity. Our assay development demonstrates the ability of Nanion's SyncroPatch 384PE to record the difficult and low expressing target NaV1.8 at a high throughput.

Background 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. Methods In this study, high-throughput electrophysiological recordings of human embryonic kidney (HEK 293) cells and Chinese hamster ovary (CHO) cells stably expressing human ether-a-go-go related gene (hERG) were performed utilizing an automated 384-well-patch-clamp system, which records up to 384 cells simultaneously. hERG channel inhibition, which is closely related to a drug-induced QT prolongation and is increasing the risk of sudden cardiac death, was investigated in the high-throughput screening patch-clamp system. Results In the automated patch-clamp measurements performed here, Kv currents were investigated with high efficiency. Various hERG channel blockers showed concentration-dependent inhibition, the 50 % inhibitory concentrations (IC50) of those blockers were in good agreement with previous reports. Conclusions The high-throughput patch-clamp system has a high potential in the field of pharmacology, toxicology, and cardiac physiology, and will contribute to the acceleration of pharmaceutical drug development and drug safety testing.

Cycle-resolved residual gas fraction measurements were made inside a heavy-duty diesel engine using an infrared absorption method. An in situ laser infrared absorption method was developed using an optical fiber sensor and a 4.301-μm quantum cascade laser (QCL) as the light source. We discuss the feasibility of obtaining in situ CO2 concentration measurements inside the engine combustion chamber using the newly developed optical fiber sensor system. Lambert–Beer’s law can be applied for the case of a single absorption line of CO2, and the dependence of the CO2 molar absorption coefficient on the ambient pressure and temperature of was determined using a constant volume vessel. This coefficient decreased with increasing pressure, indicating almost constant at pressures over 1.0 MPa. CO2 concentration measurements were made in a compression–expansion engine in order to calibrate the measurement system. The feasibility of the optical fiber sensor system was then investigated in a heavy-duty diesel engine. We were able to measure the CO2 concentration inside the combustion chamber under various engine load conditions and were able to determine the internal exhaust gas recirculation (EGR) ratio. This measurement technique proved to be valuable in obtaining the cycle-to-cycle CO2 concentration of the residual gas in a heavy-duty diesel engine.

The effects of terfenadine and pentamidine on the human ether-a-go-go related gene (hERG) channel current and its intracellular trafficking were evaluated. Green fluorescent protein (GFP)-linked hERG channels were expressed in HEK293 cells, and the membrane current was measured by an automated whole cell voltage clamp system. To evaluate drug effects on channel trafficking to the cell membrane, the fraction of channel present on the cell membrane was quantified by current measurement after drug washout and confocal microscopy. Terfenadine directly blocked the hERG channel current but had no effect on trafficking of hERG channels to the cell membrane after application in culture medium for 2 d. In contrast, pentamidine had no direct effect on the hERG channel current but reduced trafficking of hERG channels. The two drugs inhibited hERG channel function through different mechanisms: terfenadine through direct channel blockade and pentamidine through inhibition of channel trafficking to the cell membrane. Combined use of automated voltage clamp and confocal microscopic analyses would provide insights into the mechanisms of drug-induced QT-prolongation and arrhythmogenesis.

Cystic fibrosis is caused by malfunction of the chloride channel Cystic Fibrosis Transmembrane Regulator (CFTR). CFTR is expressed in the apical membrane of epithelial cells where it is involved in the regulation of fluid transport across the epithelium. A large number of mutations in the protein are known to cause CFTR to become dysfunctional and only a few pharmaceutical compounds have been developed to treat the disease by restoring the chloride conductance of the channel (1). CFTR is activated by cAMP dependent phosphorylation and is gated by ATP. Activation is typically achieved using forskolin that activates adenylate cyclase which then leads to phosphorylation of the channel via protein kinase A (PKA). Screening for CFTR activators is usually done in the absence of forskolin, but in the presence of low concentrations of cAMP. Studies in animal models support the development of CFTR inhibitors for antisecretory therapy of enterotoxin-mediated diarrheas and polycystic kidney disease. For this purpose an alternative approach can be used. In the presence of internal fluoride CFTR is activated as to the same degree as after forskolin activation. We here present data from Nanion's SyncroPatch 384PE where we activate the channel with either forskolin under fluoride free conditions or by internal solution exchange where the fluoride is washed into the intracellular solution. Our results show that the activation of CFTR by either forskolin or intracellular F- is sensitive to glibenclamide and CFTRInh-172 in a dose - and/or voltage dependent manner. Taken together, these experiments show a stable and cost optimized approach to study the pharmacology of CFTR at high throughput that might empowers new ways in the drug discovery on CFTR.

Cloperastine is an antitussive drug, which can be received as an over-the-counter cold medicine. The chemical structure of cloperastine is quite similar to that of the antihistamine drug diphenhydramine, which is reported to inhibit hERG K+ channels and clinically induce long QT syndrome after overdose. To analyze its proarrhythmic potential, we compared effects of cloperastine and diphenhydramine on the hERG K+ channels expressed in HEK293 cells. We further assessed their effects on the halothane-anesthetized guinea-pig heart under the monitoring of monophasic action potential (MAP) of the ventricle. Cloperastine inhibited the hERG K+ currents in a concentration-dependent manner with an IC50 value of 0.027 μM, whose potency was 100 times greater than that of diphenhydramine (IC50; 2.7 μM). In the anesthetized guinea pigs, cloperastine at a therapeutic dose of 1 mg/kg prolonged the QT interval and MAP duration without affecting PR interval or QRS width. Diphenhydramine at a therapeutic dose of 10 mg/kg prolonged the QT interval and MAP duration together with increase in PR interval and QRS width. The present results suggest that cloperastine may be categorized as a QT-prolonging drug that possibly induces arrhythmia at overdoses like diphenhydramine does. Keywords:: cloperastine, antitussive drug, hERG K+ channel, QT interval, monophasic action potential

In order to establish a technique which will properly account for the LMFBR core deformation under various reactor operating conditions, two separate computer programs have been developed, and coupled together. The first of the two, “FEMCRP”, is a finite element creep analysis code for a long, straight, hexagonal tube. The second program owes its origin to BOX-V developed by ANL, but was later modified considerably by the authors to include the effect of stainless steel swelling. The computer program, HICODEM, is the cross between the two. It can analyze the equilibrium configuration of the core consisting of fuel subassemblies of various burnup histories, with corresponding degree of swelling and creep. The code can assume a mechanical clamping on the periphery of the core during the power operation, and after a desired length of time, it can bring the entire core to the refueling temperature and unclamp the constraint, the resultant equilibrium core configuration being successively obtained. The results can be obtained by the ordinary digital print out and/or by the X−Y plotter as the vertical cross section of the core. HICODEM code has been succesfully checked out against available literature and against itself by taking different computational paths. The code consists of about 6,000 source cards, and takes about 20 minutes on a HITAC 5020F (or IBM 360) for a typical computation simulating one entire refueling cycle (about 146 days for Monju).