Manual Patch Clamp Research Articles

Electrophysiology-Based Assays to Detect Subtype-Selective Modulation of Human Nicotinic Acetylcholine Receptors.

The Family Smoking Prevention and Tobacco Control Act of 2009 (Public Law 111-31) gave the US Food and Drug Administration (FDA) the responsibility for regulating tobacco products. Nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. Nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nAChRs), which function as ion channels in cholinergic modulation of neurotransmission. Subtypes within the family of neuronal nAChRs are defined by their α- and β-subunit composition. The subtype-selective profiles of tobacco constituents are largely unknown, but could be essential for understanding the physiological effects of tobacco products. In this report, we report the development and validation of electrophysiology-based high-throughput screens (e-HTS) for human nicotinic subtypes, α3β4, α3β4α5, α4β2, and α7 stably expressed in Chinese Hamster Ovary cells. Assessment of agonist sensitivity and acute desensitization gave results comparable to those obtained by conventional manual patch clamp electrophysiology assays. The potency of reference antagonists for inhibition of the receptor channels and selectivity of positive allosteric modulators also were very similar between e-HTS and conventional manual patch voltage clamp data. Further validation was obtained in pilot screening of a library of FDA-approved drugs that identified α7 subtype-selective positive allosteric modulation by novel compounds. These assays provide new tools for profiling of nicotinic receptor selectivity.

Assessment of drug-induced arrhythmic risk using limit cycle and autocorrelation analysis of human iPSC-cardiomyocyte contractility

Cardiac safety assays incorporating label-free detection of human stem-cell derived cardiomyocyte contractility provide human relevance and medium throughput screening to assess compound-induced cardiotoxicity. In an effort to provide quantitative analysis of the large kinetic datasets resulting from these real-time studies, we applied bioinformatic approaches based on nonlinear dynamical system analysis, including limit cycle analysis and autocorrelation function, to systematically assess beat irregularity. The algorithms were integrated into a software program to seamlessly generate results for 96-well impedance-based data. Our approach was validated by analyzing dose- and time-dependent changes in beat patterns induced by known proarrhythmic compounds and screening a cardiotoxicity library to rank order compounds based on their proarrhythmic potential. We demonstrate a strong correlation for dose-dependent beat irregularity monitored by electrical impedance and quantified by autocorrelation analysis to traditional manual patch clamp potency values for hERG blockers. In addition, our platform identifies non-hERG blockers known to cause clinical arrhythmia. Our method provides a novel suite of medium-throughput quantitative tools for assessing compound effects on cardiac contractility and predicting compounds with potential proarrhythmia and may be applied to in vitro paradigms for pre-clinical cardiac safety evaluation.

An evaluation of 30 clinical drugs against the comprehensive in vitro proarrhythmia assay (CiPA) proposed ion channel panel

IntroductionThe Comprehensive in vitro Proarrhythmia Assay (CiPA) is intended to address the misidentification of drug-associated torsade de pointes risk based solely on hERG and QT data. This new paradigm will consist of four interrelated components, one of which is a panel consisting of six ion channels whose currents are important in both depolarization and repolarization of the cardiac action potential. This study examined the effects of 30 clinical drugs on these ion channels. MethodsIon currents were evaluated in expression systems using the manual whole cell patch clamp technique. Currents were elicited using either a ventricular action potential waveform or step-ramp voltage protocols. ResultsOf the seven ion currents studied, hERG was the most often blocked current followed by Nav1.5-late, and Cav1.2. Using a 20% reduction in current amplitude as an arbitrary maker, at a free plasma Cmax concentration, no drug tested blocked Nav1.5-peak, KvLQT1/mink, Kir2.1 and Kv4.3 by that amount. At a 3x free plasma Cmax, every current except Kir2.1 had at least one drug reduce current amplitude by at least 20%. DiscussionThis is the first study of its kind to examine the effects of 30 clinical drugs against the seven ion currents currently proposed to makeup the CiPA ion channel panel. The results indicate the importance of drug-induced block of hERG, Nav1.5-late and Cav1.2 at clinically relevant concentrations, with low risk torsade drugs having equal or greater Nav1.5-late or Cav1.2 block compared to hERG block. In addition, the results of this study provide data which can be used to test the ability of various in silico models to predict drug-induced arrhythmias.

Catch and Patch: A Pipette-Based Approach for Automating Patch Clamp That Enables Cell Selection and Fast Compound Application.

Manual patch clamp, the gold standard of electrophysiology, represents a powerful and versatile toolbox to stimulate, modulate, and record ion channel activity from membrane fragments and whole cells. The electrophysiological readout can be combined with fluorescent or optogenetic methods and allows for ultrafast solution exchanges using specialized microfluidic tools. A hallmark of manual patch clamp is the intentional selection of individual cells for recording, often an essential prerequisite to generate meaningful data. So far, available automation solutions rely on random cell usage in the closed environment of a chip and thus sacrifice much of this versatility by design. To parallelize and automate the traditional patch clamp technique while perpetuating the full versatility of the method, we developed an approach to automation, which is based on active cell handling and targeted electrode placement rather than on random processes. This is achieved through an automated pipette positioning system, which guides the tips of recording pipettes with micrometer precision to a microfluidic cell handling device. Using a patch pipette array mounted on a conventional micromanipulator, our automated patch clamp process mimics the original manual patch clamp as closely as possible, yet achieving a configuration where recordings are obtained from many patch electrodes in parallel. In addition, our implementation is extensible by design to allow the easy integration of specialized equipment such as ultrafast compound application tools. The resulting system offers fully automated patch clamp on purposely selected cells and combines high-quality gigaseal recordings with solution switching in the millisecond timescale.

Manual Patch-Clamp Recording for Medium-Throughput Ion Channel Drug Screening: Assay Validation by Searching for a Blocker of Nuclear Monovalent Cationic Channel

The use of manual patch-clamp recording in drug discovery is limited by ultra-low throughput. The high cost of automated patch-clamp drug screening makes it unavailable for the majority of electrophysiological laboratories. Here we propose a drug screening strategy that combines manual patch-clamp technique with a specially designed drug cocktail application algorithm. This strategy allows the testing of up to 80 compounds per experiment with the effective average rate close to 40 drugs/experiment. The drug application algorithm was validated by screening a small library of 446 compounds in just 11 manual patch-clamp experiments while searching for a blocker of nuclear monovalent cationic channel (NMCC) expressed in the inner membrane of skeletal muscle nuclei. We found that one of the drugs, D11P6H2, blocked the NMCC activity completely and irreversibly at concentrations as low as 100 nM. We concluded that this method can be useful in drug screening of medium size libraries. If combined with an automated patch-clamp system, it can further increase the productivity and lower the cost of screening significantly.

Patient-specific hiPSC bioprocessing for drug screening: Bioprocess economics and optimisation

This paper describes a decisional tool that is designed to identify cost-effective process designs for drug screening products derived from human induced pluripotent stem cells (hiPSC). The decisional tool comprises a bioprocess economics model linked to a search algorithm to assess the financial impact of manual and automated bioprocessing strategies that use 2D-planar tissue culture technologies. The tool was applied to a case study that examines the production of patient-specific iPSC-derived neurons for drug screening. The production strategies were compared across three analytical drug screening methods, each requiring cell production at a distinct scale (manual patch-clamp analysis, high throughput screening and plate-based pharmacology), as well as different annual cell line utilization requirements (‘throughputs’) (between 10 and 100 lines) so as to represent different industry scenarios. The tool determined the critical cell line throughput where the most cost-effective production strategy switched from the manual to automated workflow. The key process economics driver was the number of iPSC expansion stages required. Stochastic modelling of the bioprocess illustrated that the automated was more robust than the manual workflow in the scenarios investigated. The tool predicted the level of performance improvements required in iPSC expansion and differentiation as well as reductions in indirect costs and media costs so as to achieve an acceptable cost of goods (COG).

Stem cell-derived cardiomyocyte toxicity testing using a novel label-free and contact-free imaging platform

• Pulse was able to detect dose-dependent changes in beating parameters including beat rate, duration, magnitude and arrhythmias using different classes of compounds.. • Our results provide the first clinically-relevant demonstration of a fully-automated and non-invasive imaging-based beating assay for characterization of cardiomyocytes and drug toxicity screening. We compared the output of Pulse’s image analysis algorithm to data collected by manual patch clamp (ChanTest), where imaging was performed simultaneously with patch recordings. Quantifying the Effects of Drugs

Abstract 245: Assessing Proarrhythmic Potential of Drugs Using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes and Optical Recordings With Voltage-sensitive Dyes

Background: Drug-induced arrhythmias have been a leading reason for withdrawing drugs from the market and abandoning the development of new drugs. For the past decade FDA has required new drugs to be tested for block of the hERG potassium channel and QT interval prolongation. While these requirements have prevented arrhythmia-related drug recalls, some new drugs affecting multiple ion channels are being dropped from development inappropriately. Human induced pluripotent stem cell derived cardiomyocytes (iPS-CMs) are a new technology for preclinical risk assessment; however they have not been fully characterized or validated. Methods: We studied 26 drugs and 3 drug combinations that block multiple cardiac ion channels with two commercially available iPS-CM lines from Axiogenesis and Cellular Dynamics using optical recordings of action potentials with voltage-sensitive dyes. Drug-induced action potential duration (APD) prolongation was compared to clinical QT prolongation from two FDA-sponsored clinical trials. The effects of the drugs on multiple individual cardiac ion channels were assessed using manual patch clamp of overexpressed cell lines. Cardiac ion channel gene expression in the iPS-CMs was quantified and compared to primary human heart cell controls. Results: Of 19 drugs with an FDA label of clinical QT prolongation, 15 exhibited iPS-CM APD prolongation after acute drug exposure. None of the 6 drugs without clinical QT prolongation caused iPS-CM APD prolongation. Of 14 drugs with arrhythmia risk on the FDA label, nine caused arrhythmias in iPS-CMs. iPS-CM response had good correlation with clinical QT data for drugs that block hERG and calcium channels, while drugs blocking the late sodium current had variable response. This was in line with gene expression data, which showed most robust expression of hERG and calcium channels. Conclusion: Optical recordings from iPS-CMs with voltage sensitive dyes is a promising technology for high-throughput toxicity assessment for drug-induced arrhythmias. This study provides a comprehensive characterization of the cardiac ion channel properties of multiple commercially available iPS-CMs to support a potential new paradigm for assessing the arrhythmia risk of all new drugs.

The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system

Kv2.1, the voltage-gated ion channel, is ubiquitously expressed in variety of tissues and dysfunction of this ion channel is responsible for multiple diseases. Electrophysiological properties of ion channels are so far characterized with eukaryotic cells using the manual patch clamp which requires skilful operators and expensive equipments. In this research, we created a simple and sensitive drug screen method using bacterial giant spheroplasts and the automated patch clamp which does not require special skills. We expressed a eukaryotic voltage-gated ion channel Kv2.1 in Escherichia coli using prokaryotic codon, and prepared giant spheroplasts large enough for the patch clamp. Human Kv2.1 currents were successfully recorded from giant spheroplasts with the automated system, and Kv2.1-expressed E. coli spheroplasts could steadily reacted to the dose–response assay with TEA and 4-AP. Collectively, our results indicate for the first time that the bacterial giant spheroplast can be applied for practical pharmaceutical assay using the automated patch clamp.

Hypaconitine-induced QT prolongation mediated through inhibition of KCNH2 (hERG) potassium channels in conscious dogs

Ethnopharmacological relevanceHypaconitine is one of the main aconitum alkaloids in traditional Chinese medicines prepared with herbs from the genus Acotinum. These herbs are widely used for the treatment of cardiac insufficiency and arrhythmias. However, Acotinum alkaloids are known for their toxicity as well as their pharmacological activity, especially cardiotoxicity including QT prolongation, and the mechanism of this toxicity is not clear. Material and methodsIn this study, hypaconitine was administered orally to conscious Beagle dogs, and electrocardiograms were recorded by telemetry. Pharmacokinetic studies (6h) were conducted to evaluate the relationship between QT prolongation and exposure level. HEK293 cells stably transfected with KCNH2 (hERG) cDNA were used to examine the effects of hypaconitine on the KCNH2 channel by using the manual patch clamp technique. ResultsIn the conscious dogs, all doses of hypaconitine induced QTcV (QT interval corrected according to the Van de Water formula) prolongation by more than 23% (67ms) of control in a dose-dependent manner. The maximum QTcV prolongation was observed at 2h after dosing. Maximum prolongation percentages were plotted against plasma concentrations of hypaconitine and showed a strong correlation (R2=0.789). In the in vitro study in HEK293 cells, hypaconitine inhibited the KCNH2 currents in a concentration-dependent manner with an IC50 of 8.1nM. ConclusionThese data suggest that hypaconitine inhibits KCNH2 potassium channels and this effect might be the molecular mechanism underlying QT prolongation in conscious dogs.

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy.

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

Pharmacological Characterization of NR1/NR2A and NR1/NR2B Glutamate Receptors using an Electrophysiology Based HTS Assay

Glutamate NMDA receptors are increasingly employed in drug discovery for numerous clinical indications. Multiple receptor isoforms with distinct brain distributions and properties arise by selective splicing of the NR1 transcripts and differential expression of NR2 (A-D) subunit subtypes of NMDA receptors. NR1/NR2A and NR1/NR2B represent the most abundant subtypes. Recent advances in electrophysiology-based HTS assays however, demand renewed characterization of these receptors on automated patch clamp platforms. In this study we used IonWorks Barracuda, an HTS platform that can operate in 384-well format, population patch clamp (PPC) mode. Side-by-side comparisons of pharmacological properties of NR1/NR2A and NR1/NR2B glutamate receptor responses to L-glutamate and NMDA, pharmacological profiling and comparison of eight competitive and uncompetitive inhibitors and comparison of kinetic of responses were obtained in “single hole” (SH) and PPC modes. Experiment protocols were developed to routinely produce signals ∼2 nA and ∼1 nA for NR1/NR2A and NR1/NR2B, respectively, in PPC mode. Under our experimental procedures both NR1/N2A and NR1/NR2B showed good uniformity with Z’ factor > 0.6. We found that EC50 concentrations for L-glutamate were 3.8 µM and 2.0 µM for NR1/NR2A and NR1/NR2B, respectively. NMDA appeared to be a less potent partial agonist at NR1/NR2B (>75% and EC50=18-20 µM), whereas NMDA appeared to be a full agonist at NR1/NR2A with EC50 = 100 µM. These EC50 values were consistent with literature data obtained with conventional manual patch clamp methods. Additionally, we confirmed selectivity of the uncompetitive inhibitor, ifenprodil, towards NR1/NR2B receptors with IC50Ifen= 19.3 µM, whereas NR1/NR2A receptors were unaffected. Potential variability in IC50 values of uncompetitive inhibitors due to open-pore dependence will be discussed. Our data demonstrate the potential of a robust HTS patch clamp assay for profiling new chemical entities against NMDA receptor subtypes.

Novel Voltage Protocols for Determining hERG Channel Kinetics

Drug interactions with cardiac ion channels can cause disturbances to normal electrical activity in the heart. In particular, hERG channel block has been linked with QT prolongation and increased pro-arrhythmic risk. Consequently, hERG channel screening, quantifying drug block at different drug concentrations, forms a routine part of cardiovascular safety assessment. Recent initiatives led by the US Food and Drug Administration (FDA) aim to reform the current safety assessment guidelines and in silico approaches are envisaged to form an integral part of the new regime (Sager et al., Am Heart J., 2014).Mathematical ion channel representations, embedded within action potential models, are typically constructed through fitting to patch clamp data that are acquired by applying a series of voltage-step protocols. But different models exhibit disparate behaviour under non-standard protocols; including physiologically relevant action potential clamps, or clamps mimicking arrhythmogenic behaviour. We demonstrate the influence the choice of hERG channel representation can have on action potentials simulated from a mathematical ventricular myocyte model when predicting drug-induced effects; indicating the necessity of including accurate descriptions of hERG channel kinetics.We present novel rapidly fluctuating voltage-clamp protocols, designed mathematically to enable a robust model of hERG channel kinetics to be determined, on a cell-specific basis. The protocols have been tested in manual patch clamp experiments, using hERG-1a transfected HEK 293 cells. The approach will be extended to determine compound-specific models describing hERG channel kinetics in the presence of different drugs, which may enhance the predictive ability of in silico approaches used for cardiovascular safety assessment.

Early identification of hERG liability in drug discovery programs by automated patch clamp.

Blockade of the cardiac ion channel coded by human ether-à-gogo-related gene (hERG) can lead to cardiac arrhythmia, which has become a major concern in drug discovery and development. 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. 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. This article describes protocols for automated electrophysiology screening of compound effects on the hERG channel current. 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. Typical pitfalls and artifacts that may lead to misinterpretation of data are discussed. While this article focuses on hERG channel recordings using the QPatch (Sophion A/S, 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.

Patch clamp recordings of fast‐acting ion channels in rat dorsal root ganglion cells (654.3)

Manual patch clamp recordings offer direct insight into ion channel properties through the characterization of ion channel activity. Due to the high quality data it is considered the gold standard for ion channel research. Still, the limited throughput means that maximizing data from each cell, especially sensitive primary cells, is important.We have developed a patch clamp‐based assay for characterization fast‐acting ion channels, in this case exemplified by P2X, in primary dorsal root ganglion (DRG) neurons. This assay is based on the use of primary DRG neurons in culture as a cell model for chronic pain. These neurons retain their sensory functionality and remain responsive to thermal, mechanical and functional stimuli, and when supplemented with nerve growth factor (NGF) they can be used to mimic peripheral sensitization. The assay utilizes a microfluidic perfusion system, Dynaflow Resolve, to facilitate a stable recording situation and fast and programmable solution exchange. At the start of the experiment, compounds and buffer are loaded in the 16 wells of the Dynaflow Resolve chip. Micro‐channels connect each well to a recording chamber where the cells are added. To be able to provide fast solution exchange the DRG neuron is lifted using the patch pipette and positioned in front of the micro‐channel outlets. Then, the cell is scanned through the discrete flow zones formed outside the channels.A comparison between conventional bath perfusion and the automated Dynaflow Resolve system showed that both systems have similar capacity when it comes to obtaining successful high resistance seals. However, for collecting full 5‐8 point dose‐responses from single cells, the success rate for the Dynaflow Resolve superseded the conventional system. This enables a dramatically increased productivity when performing more complex pharmacological characterizations.

Transforming TRP Channel Drug Discovery Using Medium-Throughput Electrophysiological Assays

Since the cloning of its first member in 1998, transient receptor potential (TRP) cation channels have become one of the most studied ion channel families in drug discovery. These channels, almost all calcium permeant, have been studied in many different (patho)-physiological and therapeutic areas as diverse as pain; neurodegenerative, cardiovascular, and inflammatory diseases; and cancer. At the same time, implementation of automated electrophysiology screening platforms has significantly increased the tractability of ion channels, mainly voltage gated, as drug targets. The work presented in this article shows the design and validation of TRP screening assays using the IonWorks Quattro platform (Molecular Devices, Sunnyvale, CA), allowing a significant increase in throughput to support drug discovery programs. This new player has a direct impact on resources and timelines by prioritizing potential candidates and reducing the number of molecules requiring final testing by manual patch-clamp, which is still today the gold standard technology for this challenging drug target class.

Automated Action Potential Recording in Stem Cell-Derived Cardiomyocytes for Proarrhythmia Assessment

Development of in vitro toxicity assays based on the human stem cell-derived cardiomyocytes (SC-CMs) provides an opportunity to assess the specific cardiac safety end-points early in drug development. The cardiac action potential (AP) is an established biomarker for cardiac safety and efficacy in humans and a good detector of proarrhythmic events. However, its utility has been mainly limited by a low throughput of manual patch clamp and a restricted access to healthy human cardiomyocytes. The recent progress in automated electrophysiology combined with availability of human SC-CMs provides an exciting possibility to measure AP in human cells expressing endogenous cardiac ion channels.Electrophysiological properties of human SC-CMs were investigated with special focus on establishment of automated cell-based cardiac safety screens.For the first time assay protocols and procedures were established to measure and characterize both cardiac AP and the main underlying currents in human SC-CMs using automated electrophysiology platforms Patchliner(r) and Synchropatch(r)96 (Nanion Technologies GmbH). Employment of the higher throughput systems allowed capturing and evaluating AP and cardiac current profiles in cardiomyocyte cultures revealing specific heterogeneity. Outward potassium current mainly consisting of slow and rapid delayed rectifier and Ca2+-activated components as well as inward cardiac sodium current were recorded and characterized. Density of L-type Ca2+ current varied with the type and differentiation status of cardiomyocytes. The inward rectifier potassium current and a pacemaker current could also be measured. These results demonstrate that functional cardiac currents and appropriate AP present in human SC-CMs can be successfully recorded by automated electrophysiology systems.The automated cardiac AP measurements in human SC-CMs exemplify a novel screening method for early and sensitive detection of multiple ion channel effects in vitro completing the current core battery hERG assay and animal ECG measurements in pre-clinical safety assessment.

Current Clamp of Stem Cell Derived Cardiomyocytes on Qpatch

The U.S. Food and Drug Administration (FDA) guidelines S7B mandate the measurement of inhibition of the human ether-a-go-go-related (hERG) ion channel by new drug candidates as it has been found that drug inhibition of the hERG channel can be associated with cardiac action potential (AP) prolongation and lethal cardiac arrhythmias. However, hERG is only one of multiple ion channels involved in forming the cardiac AP and therefore direct measurement of the cardiac AP has the potential to detect the effects of the candidate drug on multiple ion channels potentially affected. Stem cell-derived cardiomyocytes (SC-CM) or induced pluripotent stem cells (iPS-CM) have many of the phenotypic properties of authentic cardiomyocytes e.g. realistic ventricular action potentials. In this work we show how the QPatch can trigger action potentials using rapid switching from voltage to current clamp during recordings on the iPS-CM. We demonstrate that the current clamp recordings made on this automated patch clamp device are comparable to results obtained on standard manual patch clamp rigs. The integrated low volume glass flow channels of the QPatch minimize the consumption of iPS-CM cells - a valuable and often limiting consumable. Parallel recordings from up to 48 iPS-CM were made on the QPatch HT with full analysis of action potential parameters including action potential duration and upstroke velocity. We measured assay success rates, the stability of action potentials and pharmacological profiles of reference compounds. Current clamp measurements of these cardiomyocytes offer important information to the understanding of the complex pharmacological effect of compounds on ion channels involved in the AP. Our data presented here clearly demonstrate how automated patch clamp on QPatch can augment the throughput of current clamp to meet new demands in drug discovery.

Development of a Qpatch Automated Electrophysiology Assay for Identifying Kca3.1 Inhibitors and Activators

The intermediate-conductance Ca2+-activated K+ channel KCa3.1 (also known as KCNN4, IK1 or the Gardos channel) plays an important role in the activation of T- and B-cells, mast cells, macrophages and microglia by regulating membrane potential, cellular volume and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization (EDH) responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorder, while KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca2+ sensitivity of KCa3.1 was studied using varying intracellular Ca2+ concentrations. A free Ca2+ concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca2+ concentration (∼200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay.Supported by U54NS079202, R21NS072585 and T32-GM008799 from NIH.

Mitigating prolonged QT interval in cancer nanodrug development for accelerated clinical translation

BackgroundCardiac toxicity is the foremost reason for drug discontinuation from development to clinical evaluation and post market surveillance [Fung 35:293-317, 2001; Piccini 158:317-326 2009]. The Food and Drug Administration (FDA) has rejected many potential pharmaceutical agents due to QT prolongation effects. Since drug development and FDA approval takes an enormous amount of time, money and effort with high failure rates, there is an increased focus on rescuing drugs that cause QT prolongation. If these otherwise safe and potent drugs were formulated in a unique way so as to mitigate the QT prolongation associated with them, these potent drugs may get FDA approval for clinical use. Rescuing these compounds not only benefit the patients who need them but also require much less time and money thus leading to faster clinical translation. In this study, we chose curcumin as our drug of choice since it has been shown to posses anti-tumor properties against various cancers with limited toxicity. The major limitations with this pharmacologically active drug are (a) its ability to prolong QT by inhibiting the hERG channel and (b) its low bioavailability. In our previous studies, we found that lipids have protective actions against hERG channel inhibition and therefore QT prolongation.ResultsResults of the manual patch clamp assay of HEK 293 cells clearly illustrated that our hybrid nanocurcumin formulation prevented the curcumin induced inhibition of hERG K+ channel at concentrations higher than the therapeutic concentrations of curcumin. Comparing the percent inhibition, the hybrid nanocurcumin limited inhibition to 24.8% at a high curcumin equivalent concentration of 18 μM. Liposomal curcumin could only decrease this inhibition upto 30% only at lower curcumin concentration of 6 μM but not at 18 μM concentration.ConclusionsHere we show a curcumin encapsulated lipopolymeric hybrid nanoparticle formulation which could protect against QT prolongation and also render increased bioavailability and stability thereby overcoming the limitations associated with curcumin.