The Safety Pharmacology Society (SPS) held a West Coast Regional Meeting in Foster City, CA on November 14, 2018 at the Gilead Sciences Inc. site. The meeting was attended by scientists from the pharmaceutical and biotechnology industry, contract research organizations (CROs) and academia. A variety of scientific topics were presented by speakers, covering a broad variety of topics in the fields of safety risk assessment; from pro-arrhythmia and contractility risk evaluation, to models of heart failure and seizure in-a-dish; and discovery sciences; from stem cells and precision medicine, to models of inherited cardiomyopathy and precision cut tissue slices. The present review summarizes the highlights of the presentations and provides an overview of the high level of innovation currently underlying many frontiers in safety pharmacology.

The Nav1.9 voltage dependent sodium channel has long been a desirable target for potential pain therapeutics owing to the highly restricted expression in peripheral sensory neurons. More recently human genetic evidence has been produced which directly links Nav1.9 to human pain disorders, increasing the motivation to develop pharmacological screening methodologies. The primary barrier to assay development has been removed with the generation of robust recombinant Nav1.9 cell lines which recapitulate the unique biophysical properties of the channel. We have previously reported the generation and characterization of human, mouse and rat Nav1.9 channels stably expressed in human HEK-293 cells which exhibit the slowly activating and inactivating inward sodium channel currents that are characteristic of native Nav1.9 (Lin et al, 2016). Nanion's SyncroPatch 384PE high throughput electrophysiology (HTEP) system allows for high fidelity assessment and detailed biophysical characterization of NaV channels with up to 384 simultaneous recordings. To accelerate the pharmacological characterization of NaV1.9 we have sought to develop HTEP assays on the Syncropatch platform. Here we report that SyncroPatch recordings of recombinant Nav1.9 channels can recapitulate the pharmacological properties of Nav1.9. Our results show stable recordings of human and rat orthologs of Nav1.9. Recordings in the absence and presence of known Nav1.9 inhibitors (lidocaine, tetracaine, TC-N 1752) were robust and stable, allowing for accurate measurements of compound IC50s. Additionally, biophysical properties of voltage-dependent activation and inactivation properties were characterized. Collectively, these results show that Nav1.9 channels can studied on high throughput electrophysiological systems, facilitating the search for novel pain therapeutics.

One of the significant challenges for automated patch clamp has been to incorporate amplifiers with the ability of adjusting to changes in membrane capacitance, series resistance (Rs) and voltage-clamp time constants without user intervention. If these adjustments are not made correctly there is a risk of generating un-precise data or even losing data due to oscillations in the recorded trace. Rs compensation capability of the patch clamp amplifier of QPatch is presented. A new "clip detector feature" allowing Rs to be temporarily turned off avoiding loss of cells due to fatal oscillations, and thereby increasing the data throughput while maintaining high quality recordings, is presented. Clamping whole-cells can be a challenge depending on the magnitude of Rs. The series resistance produces two undesired effect on whole cell voltage clamp recordings: i) The cells not being clamped to the desired potential due to the voltage drop over the Rs (Vdrop= Rs x I), ii) The resolution of the voltage clamp in fast physiological processes being affected by the Rs. It is essential to reach adequate series compensation in a timely manner to obtain accurate recordings. As such, the quality of data depends on the quality of the patch clamp amplifier and its features. In order to make fast 100% series resistance compensation a fast patch clamp amplifier is needed. The embedded amplifier in the QPatch utilizes a patented technique invented by Adam Sherman (Alembic Instruments). QPatch data from human SK potassium and fast sodium channels are shown. The data demonstrates that i) QPatch measures the fastest sodium channels with accurate clamp, and ii) the patch clamp amplifier in QPatch has the required speed to record the correct current amplitude when the current through an open SK channel is reversed.

The conventional patch clamp has long been considered the best approach for studying ion channel function and pharmacology. However, its low throughput has been a major hurdle to overcome for ion channel drug discovery. The recent emergence of higher throughput, automated patch clamp technology begins to break this bottleneck by providing medicinal chemists with high-quality, information-rich data in a more timely fashion. As such, these technologies have the potential to bridge a critical missing link between high-throughput primary screening and meaningful ion channel drug discovery programs. One of these technologies, the QPatch automated patch clamp system developed by Sophion Bioscience, records whole-cell ion channel currents from 16 or 48 individual cells in a parallel fashion. Here, we review the general applicability of the QPatch to studying a wide variety of ion channel types (voltage-/ligand-gated cationic/anionic channels) in various expression systems. The success rate of gigaseals, formation of the whole-cell configuration and usable cells ranged from 40-80%, depending on a number of factors including the cell line used, ion channel expressed, assay development or optimization time and expression level in these studies. We present detailed analyses of the QPatch features and results in case studies in which secondary screening assays were successfully developed for a voltage-gated calcium channel and a ligand-gated TRP channel. The increase in throughput compared to conventional patch clamp with the same cells was approximately 10-fold. We conclude that the QPatch, combining high data quality and speed with user friendliness and suitability for a wide array of ion channels, resides on the cutting edge of automated patch clamp technology and plays a pivotal role in expediting ion channel drug discovery.

Alpha7 nicotinic acetylcholine receptor channels are important ligand-gated ion channels that are fast desensitizing, cation selective and have been implicated in the pathophysiology of schizophrenia and Alzheimer's disease. We report here high quality alpha7 parallel patch clamp recordings using the QPatch automated patch clamp system. The QPatch patch clamps up to 48 cells in parallel with the same high fidelity as conventional patch clamp. EC(50) and IC(50) values were comparable to values obtained with conventional patch clamp. The EC(50) value for acetylcholine (ACh) on the QPatch with area under the curve (AUC) analysis was 26microM compared to a value of 29microM determined from conventional patch clamp experiments. Sequential additions of ACh can be made with minimal decay of the peak amplitude. The competitive alpha7 antagonist methyllycaconitine (MLA) blocked currents with an IC(50) value of 0.25nM which is similar to published IC(50) values for MLA. Finally, two different classes of positive allosteric modulators represented by PNU-120596 and NS-1738 elicited characteristic responses, thus allowing accurate characterization of modulation and measurements of potency. These results demonstrate that alpha7 nicotinic acetylcholine receptor channels can be studied reliably in a higher throughput, parallel manner with the QPatch automated patch clamp system.

The suitability of an automated patch clamp for the characterization and pharmacological screening of calcium release-activated calcium (CRAC) channels endogenously expressed in RBL-2H3 cells was explored with the QPatch system. CRAC currents (I( CRAC)) are small, and thus precise recordings require high signal-to-noise ratios obtained by high seal resistances. Automated whole-cell establishment resulted in membrane resistances of 1728 +/- 226 MOmega (n = 44). CRAC channels were activated by a number of methods that raise intracellular calcium concentration, including EGTA, ionomycin, Ins(1,4,5)P3, and thapsigargin. I(CRAC) whole-cell currents ranged from 30 to 120 pA with rise times of 40 to 150 s. An initial delay in current activation was observed in particular when I(CRAC) was activated by passive store depletion using EGTA. Apparent rundown of I(CRAC) was commonly observed, and the current could be reactivated by subsequent addition of thapsigargin. I(CRAC) was blocked by SKF-96365 and 2-APB with IC50 values of 4.7 +/- 1.1 microM (n = 9) and 7.5 +/- 0.7 (n = 9) microM, respectively. The potencies of these blockers were similar to values reported for I(CRAC) in similar conventional patch-clamp experiments. The study demonstrates that CRAC channels can be rapidly and efficiently targeted with automated patch-clamp techniques for characterization of physiological and pharmacological properties.

The QPatch 16 significantly increases throughput for gigaseal patch clamp experiments, making direct measurements in ion channel drug discovery and safety testing feasible. Released to the market in the Autumn of 2004 by Sophion Bioscience, the QPatch originated from work done at NeuroSearch (Denmark) in the early days of automated patch clamp. Today, the QPatch provides many unique features. For example, only the QPatch includes an automated cell preparation station making several hours of unattended operation possible. The 16-channel electrode array, called the QPlate, includes glass-coated microfluidic channels for less compound absorption and, hence, more accurate IC50 values. The microfluidic pathways also allow for very small amounts of compound used for each experiment (∼ 5 µl per addition). Only the QPatch has four independent pipetting heads for more efficient liquid handling (especially for ligand-gated ion channel experiments). Patch clamp recordings with the QPatch match the high quality of conventional patch clamp and in some cases the results are even better. For example, only the QPatch includes 100% series resistance compensation for the elimination of false positives due to voltage errors. Finally, the modular QPatch 16 was designed with more channels in mind. The upgrade pathway to 48-channels (the QPatch HT) will be discussed.

The 2nd Annual Ion Channel Targets (ICT) Conference (by Select Bioscience LLC) was held in Boston on 12-13 September 2006. A healthy mixture of scientists from pharma, biotech and academic sectors attended the meeting. The speaker list reflected this mixture. In general, the conference focused on new ion channel targets and the methods for studying them in detail. Keynote lectures from Professors David Clapham (Harvard Medical School, USA) and Reinhold Penner (University of Hawaii, USA) set the tone by highlighting recent findings with a voltage-gated proton channel (Clapham), cation channel in sperm (Clapham) and the calcium-release-activated calcium channel (Penner). Also described at ICT were voltage-gated sodium, potassium, transmembrane-receptor-potential channels, as well as ligand-gated nicotinic acetylcholine (nAChR) and GABA type A receptors.

Effector functions and proliferation of T helper (Th) cells are influenced by cytokines in the environment. Th1 cells respond to a synergistic effect of interleukin-12 (IL-12) and interleukin-18 (IL-18) to secrete interferon-gamma (IFN-gamma). In contrast, Th2 cells respond to interleukin-4 (IL-4) to secrete IL-4, interleukin-13 (IL-13), interleukin-5 (IL-5), and interleukin-10 (IL-10). The authors were interested in identifying nonpeptide inhibitors of the Th1 response selective for the IL-12/IL-18-mediated secretion of IFN-gamma while leaving the IL-4-mediated Th2 cytokine secretion relatively intact. The authors established a screening protocol using human peripheral blood mononuclear cells (PBMCs) and identified the hydrazino anthranilate compound 1 as a potent inhibitor of IL-12/IL-18-mediated IFN-gamma secretion from CD3(+) cells with an IC(50) around 200 nM. The inhibitor was specific because it had virtually no effect on IL-4-mediated IL-13 release from the same population of cells. Further work established that compound 1 was a potent intracellular iron chelator that inhibited both IL-12/IL-18- and IL-4-mediated T cell proliferation. Iron chelation affects multiple cellular pathways in T cells. Thus, the IL-12/IL-18-mediated proliferation and IFN-gamma secretion are very sensitive to intracellular iron concentration. However, the IL-4-mediated IL-13 secretion does not correlate with proliferation and is partially resistant to potent iron chelation.

Highlights from the second annual Ion Chanels conference, held 14-15 July 2003 in Philadelphia, USA

A B S TRACT The relationship between the deplet ion of IP3-releasable intracellular Ca z+ stores and the activation of Ca2+-selective membrane current was determined dur ing the st imulation of M1 muscarinic receptors in N1E-115 neuroblastoma cells. External Ca 2+ is required for refilling Ca 2+ stores and the voltage-independent , receptor-regulated Ca 2+ current represents a significant Ca 2+ source for refilling. The time course of Ca z+ store deplet ion was measured with fura-2 fluorescence imaging, and it was compared with the time course of Ca 2+ current activation measured with nystatin patch voltage clamp. At the time of max imum current density (0.18 + .03 pA/pF; n = 48), the Ca ~+ conten t of the IP3-releasable Ca z+ pool is reduced to 39 + 3 % (n = 10) of its resting value. Calcium stores deplete rapidly, reaching a m i n i m u m Ca 2+ conten t in 15-30 s. The activation of Ca 2+ current is delayed by 10-15 s after the beg inn ing of Ca z+ release and continues to gradually increase for nearly 60 s, long after Ca 2+ release has peaked and subsided. The delay in the appearance of the current is consistent with the idea that the product ion and accumulat ion of a second messenger is the rate-limiting step in current activation. The time course of Ca 2+ store deplet ion was also measured after adding thapsigargin to block intracellular Ca 2+ ATPase. After 15 min in thapsigargin, IP3-releasable Ca z+ stores are depleted by >90% and the Ca z+ current is maximal (0.19 + 0.05 pA/pF; n = 6). Intracellular loading with the Ca 2+ buffer EGTA/AM (10 p~M; 30 min) depletes 1P3-releasable Ca 2+ stores by between 25 and 50%, and it activates a vol tage-independent inward current with properties similar to the cur ren t activated by agonist or thapsigargin. The current density after EGTA/AM loading (0.61 + 0.32 pA/pF; n = 4) is three times greater than the current density in response to agonist or thapsigargin. This could result from partial removal of Ca2+-dependent inactivation.

A B S TRACT The relationship between the deplet ion of IP3-releasable intracellular Ca z+ stores and the activation of Ca2+-selective membrane current was determined dur ing the st imulation of M1 muscarinic receptors in N1E-115 neuroblastoma cells. External Ca 2+ is required for refilling Ca 2+ stores and the voltage-independent , receptor-regulated Ca 2+ current represents a significant Ca 2+ source for refilling. The time course of Ca z+ store deplet ion was measured with fura-2 fluorescence imaging, and it was compared with the time course of Ca 2+ current activation measured with nystatin patch voltage clamp. At the time of max imum current density (0.18 + .03 pA/pF; n = 48), the Ca ~+ conten t of the IP3-releasable Ca z+ pool is reduced to 39 + 3 % (n = 10) of its resting value. Calcium stores deplete rapidly, reaching a m i n i m u m Ca 2+ conten t in 15-30 s. The activation of Ca 2+ current is delayed by 10-15 s after the beg inn ing of Ca z+ release and continues to gradually increase for nearly 60 s, long after Ca 2+ release has peaked and subsided. The delay in the appearance of the current is consistent with the idea that the product ion and accumulat ion of a second messenger is the rate-limiting step in current activation. The time course of Ca 2+ store deplet ion was also measured after adding thapsigargin to block intracellular Ca 2+ ATPase. After 15 min in thapsigargin, IP3-releasable Ca z+ stores are depleted by >90% and the Ca z+ current is maximal (0.19 + 0.05 pA/pF; n = 6). Intracellular loading with the Ca 2+ buffer EGTA/AM (10 p~M; 30 min) depletes 1P3-releasable Ca 2+ stores by between 25 and 50%, and it activates a vol tage-independent inward current with properties similar to the cur ren t activated by agonist or thapsigargin. The current density after EGTA/AM loading (0.61 + 0.32 pA/pF; n = 4) is three times greater than the current density in response to agonist or thapsigargin. This could result from partial removal of Ca2+-dependent inactivation.