ICH S7B In Vitro Assays Do Not Address Mechanisms of QTC Prolongation for Peptides and Proteins - Data in Support of Not Needing Dedicated QTC Studies.

Current cardiac safety testing focuses on detecting drug-induced QTC prolongation as a surrogate for risk of Torsade de Pointes. The nonclinical strategy, described in International Conference on Harmonization (ICH) S7B, includes in vitro assessment of hERG block or ventricular repolarization delay and in vivo QT prolongation. Several studies have reported predictive values of ICH S7B results for clinical QTC outcomes for small molecules; none has examined peptides and proteins other than monoclonal antibodies. To address this knowledge gap, information for peptides and proteins submitted to the US Food and Drug Administration (FDA) was collected. Results of hERG assays, ventricular repolarization assays, and in vivo QT assessment were compared with clinical QTC study outcomes. The results show that 14% clinical QTC studies for approved and investigational products failed to exclude 10-ms QTC prolongation. Clinical QTC prolongation for these molecules lacked concentration-dependence which is expected for hERG block-mediated mechanism or QTC prolongation could not be excluded due to characterization in the clinical study. The hERG and ventricular repolarization assays do not identify clinical QTC prolongation potential for peptides and proteins. Lack of alignment between hERG and ventricular repolarization assay results and clinical QTC outcomes suggests that the mechanisms of QTC prolongation by some peptides and proteins are unrelated to direct cardiac ion channel block. Similar to large targeted proteins and monoclonal antibodies, peptides and proteins regardless of size have a low likelihood of direct cardiac ion channel interactions. This characteristic supports waiving the requirement for thorough QT assessment for products comprised of naturally occurring amino acids unless proarrhythmia potential is suggested by nonclinical or clinical data.

Experimental factors that impact CaV1.2 channel pharmacology-Effects of recording temperature, charge carrier, and quantification of drug effects on the step and ramp currents elicited by the "step-step-ramp" voltage protocol.

BACKGROUND AND PURPOSE: CaV1.2 channels contribute to action potential upstroke in pacemaker cells, plateau potential in working myocytes, and initiate excitation-contraction coupling. Understanding drug action on CaV1.2 channels may inform potential impact on cardiac function. However, literature shows large degrees of variability between CaV1.2 pharmacology generated by different laboratories, casting doubt regarding the utility of these data to predict or interpret clinical outcomes. This study examined experimental factors that may impact CaV1.2 pharmacology. EXPERIMENTAL APPROACH: Whole cell recordings were made on CaV1.2 overexpression cells. Current was evoked using a "step-step-ramp" waveform that elicited a step and a ramp current. Experimental factors examined were: 1) near physiological vs. room temperature for recording, 2) drug inhibition of the step vs. the ramp current, and 3) Ca2+ vs. Ba2+ as the charge carrier. Eight drugs were studied. KEY RESULTS: CaV1.2 current exhibited prominent rundown, exquisite temperature sensitivity, and required a high degree of series resistance compensation to optimize voltage control. Temperature-dependent effects were examined for verapamil and methadone. Verapamil's block potency shifted by up to 4X between room to near physiological temperature. Methadone exhibited facilitatory and inhibitory effects at near physiological temperature, and only inhibitory effect at room temperature. Most drugs inhibited the ramp current more potently than the step current-a preference enhanced when Ba2+ was the charge carrier. The slopes of the concentration-inhibition relationships for many drugs were shallow, temperature-dependent, and differed between the step and the ramp current. CONCLUSIONS AND IMPLICATIONS: All experimental factors examined affected CaV1.2 pharmacology. In addition, whole cell CaV1.2 current characteristics-rundown, temperature sensitivity, and impact of series resistance-are also factors that can impact pharmacology. Drug effects on CaV1.2 channels appear more complex than simple pore block mechanism. Normalizing laboratory-specific approaches is key to improve inter-laboratory data reproducibility. Releasing original electrophysiology records is essential to promote transparency and enable the independent evaluation of data quality.

hERG block potencies for 5 positive control drugs obtained per ICH E14/S7B Q&As best practices: Impact of recording temperature and drug loss.

According to the ICH S7B guideline, drug candidates are screened for hERG block prior to first-in-human testing to predict the likelihood of delayed repolarization associated with a rare, but life-threatening, ventricular tachyarrhythmia. The new ICH E14 Q&As guideline allows hERG results to be used in later clinical development for decision-making (Q&As 5.1 and 6.1). To pursue this path, the hERG assay should be conducted following the new ICH S7B Q&A 2.1 guideline, which calls for best practice considerations of the recording temperature, voltage protocol, stimulation frequency, recording/data quality, and concentration verification. This study investigated hERG block by cisapride, dofetilide, terfenadine, sotalol, and E-4031 - positive controls commonly used to demonstrate assay sensitivity - using the manual whole cell patch clamp method and an action potential-like voltage protocol presented at 0.2 Hz. Recordings were conducted at room and near physiological temperature. Drug concentrations were measured using samples collected during real patch clamp experiments and satellite experiments. Results showed temperature effects for E-4031, terfenadine, and sotalol, but not cisapride and dofetilide. Cisapride and terfenadine showed substantial concentration losses, largely due to nonspecific binding to the perfusion apparatus. Using concentrations measured from the real and satellite experiments to assess block potencies yielded comparable results, indicating that satellite sample collection may be viable for drugs with nonspecific binding concerns only. In summary, this study provides block potencies for 5 hERG positive controls, and serves as a case study for hERG assays conducted, and results illustrated in accordance with the new ICH E14/S7B Q&As.

New science, drug regulation, and emergent public health issues: The work of FDA's division of applied regulatory science.

The U.S. Food and Drug Administration (FDA) Division of Applied Regulatory Science (DARS) moves new science into the drug review process and addresses emergent regulatory and public health questions for the Agency. By forming interdisciplinary teams, DARS conducts mission-critical research to provide answers to scientific questions and solutions to regulatory challenges. Staffed by experts across the translational research spectrum, DARS forms synergies by pulling together scientists and experts from diverse backgrounds to collaborate in tackling some of the most complex challenges facing FDA. This includes (but is not limited to) assessing the systemic absorption of sunscreens, evaluating whether certain drugs can convert to carcinogens in people, studying drug interactions with opioids, optimizing opioid antagonist dosing in community settings, removing barriers to biosimilar and generic drug development, and advancing therapeutic development for rare diseases. FDA tasks DARS with wide ranging issues that encompass regulatory science; DARS, in turn, helps the Agency solve these challenges. The impact of DARS research is felt by patients, the pharmaceutical industry, and fellow regulators. This article reviews applied research projects and initiatives led by DARS and conducts a deeper dive into select examples illustrating the impactful work of the Division.

Translational Models and Tools to Reduce Clinical Trials and Improve Regulatory Decision Making for QTc and Proarrhythmia Risk (ICH E14/S7B Updates).

After multiple drugs were removed from the market secondary to drug-induced torsade de pointes (TdP) risk, the International Council for Harmonisation (ICH) released guidelines in 2005 that focused on the nonclinical (S7B) and clinical (E14) assessment of surrogate biomarkers for TdP. Recently, Vargas et al. published a pharmaceutical-industry perspective making the case that "double-negative" nonclinical data (negative in vitro hERG and in vivo heart-rate corrected QT (QTc) assays) are associated with such low probability of clinical QTc prolongation and TdP that potentially all double-negative drugs would not need detailed clinical QTc evaluation. Subsequently, the ICH released a new E14/S7B Draft Guideline containing Questions and Answers (Q&As) that defined ways that double-negative nonclinical data could be used to reduce the number of "Thorough QT" (TQT) studies and reach a low-risk determination when a TQT or equivalent could not be performed. We review the Vargas et al. proposal in the context of what was contained in the ICH E14/S7B Draft Guideline and what was proposed by the ICH E14/S7B working group for a "stage 2" of updates (potential expanded roles for nonclinical data and details for assessing TdP risk of QTc-prolonging drugs). Although we do not agree with the exact probability statistics in the Vargas et al. paper because of limitations in the underlying datasets, we show how more modest predictive value of individual assays could still result in low probability for TdP with double-negative findings. Furthermore, we expect that the predictive value of the nonclinical assays will improve with implementation of the new ICH E14/S7B Draft Guideline.

Mechanisms of QT prolongation by buprenorphine cannot be explained by direct hERG channel block.

Buprenorphine is a µ-opioid receptor (MOR) partial agonist used to manage pain and addiction. QTC prolongation that crosses the 10 msec threshold of regulatory concern was observed at a supratherapeutic dose in two thorough QT studies for the transdermal buprenorphine product BUTRANS®. Because QTC prolongation can be associated with Torsades de Pointes (TdP), a rare but potentially fatal ventricular arrhythmia, these results have led to further investigation of the electrophysiological effects of buprenorphine. Drug-induced QTC prolongation and TdP are most commonly caused by acute inhibition of hERG current (IhERG) that contribute to the repolarizing phase of the ventricular action potentials (APs). Concomitant inhibition of inward late Na+ (INaL) and/or L-type Ca2+ (ICaL) current can offer some protection against proarrhythmia. Therefore, we characterized the effects of buprenorphine and its major metabolite norbuprenorphine on cardiac hERG, Ca2+, and Na+ ion channels, as well as cardiac APs. For comparison, methadone, a MOR agonist associated with QTC prolongation and high TdP risk, and naltrexone and naloxone, two opioid receptor antagonists, were also studied. Whole cell recordings were performed at 37°C on cells stably expressing hERG, CaV1.2, and NaV1.5 proteins. Microelectrode array (MEA) recordings were made on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The results showed that buprenorphine, norbuprenorphine, naltrexone, and naloxone had no effect on IhERG, ICaL, INaL, and peak Na+ current (INaP) at clinically relevant concentrations. In contrast, methadone inhibited IhERG, ICaL, and INaL. Experiments on iPSC-CMs showed a lack of effect for buprenorphine, norbuprenorphine, naltrexone, and naloxone, and delayed repolarization for methadone at clinically relevant concentrations. The mechanism of QTC prolongation is opioid moiety-specific. This remains undefined for buprenorphine, while for methadone it involves direct hERG channel block. There is no evidence that buprenorphine use is associated with TdP. Whether this lack of TdP risk can be generalized to other drugs with QTC prolongation not mediated by acute hERG channel block warrants further study.

A general procedure to select calibration drugs for lab-specific validation and calibration of proarrhythmia risk prediction models: An illustrative example using the CiPA model.

INTRODUCTION: In response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development strategies to validate models, and implementation strategies to ensure a model developed by one lab can be used by other labs in a consistent manner in the presence of lab-to-lab experimental variability. While the development strategies were illustrated through the validation of the model under the Comprehensive In vitro Proarrhythmia Assay (CiPA), the implementation strategies have not been adopted yet. METHODS: The proposed implementation strategies were applied to the CiPA model by performing a sensitivity analysis to identify a subset of calibration drugs that were most critical in determining the classification thresholds for proarrhythmia risk prediction. RESULTS: The selected calibration drugs were able to recapitulate classification thresholds close to those calculated from the full list of CiPA drugs. Using an illustrative dataset it was shown that a new lab could use these calibration drugs to establish its own classification thresholds (lab-specific calibration), and verify that the model prediction accuracy in the new lab is comparable to that in the original lab where the model was developed (lab-specific validation). DISCUSSION: This work used the CiPA model as an example to illustrate how to adopt the proposed model implementation strategies to select calibration drugs and perform lab-specific calibration and lab-specific validation. Generic in nature, these strategies could be generally applied to different proarrhythmia risk prediction models using various experimental systems under the new paradigm.

A systematic strategy for estimating hERG block potency and its implications in a new cardiac safety paradigm.

INTRODUCTION: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. METHODS: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. RESULTS: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. DISCUSSION: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.

Transient Hypoxemia Disrupts Anatomical and Functional Maturation of Preterm Fetal Ovine CA1 Pyramidal Neurons.

Children who survive premature birth often exhibit reductions in hippocampal volumes and deficits in working memory. However, it is unclear whether synaptic plasticity and cellular mechanisms of learning and memory can be elicited or disrupted in the preterm fetal hippocampus. CA1 hippocampal neurons were exposed to two common insults to preterm brain: transient hypoxia-ischemia (HI) and hypoxia (Hx). We used a preterm fetal sheep model using both sexes in twin 0.65 gestation fetuses that reproduces the spectrum of injury and abnormal growth in preterm infants. Using Cavalieri measurements, hippocampal volumes were reduced in both Hx and HI fetuses compared with controls. This volume loss was not the result of neuronal cell death. Instead, morphometrics revealed alterations in both basal and apical dendritic arborization that were significantly associated with the level of systemic hypoxemia and metabolic stress regardless of etiology. Anatomical alterations of CA1 neurons were accompanied by reductions in probability of presynaptic glutamate release, long-term synaptic plasticity and intrinsic excitability. The reduction in intrinsic excitability was in part due to increased activity of the channels underlying the fast and slow component of the afterhyperpolarization in Hx and HI. Our studies suggest that even a single brief episode of hypoxemia can markedly disrupt hippocampal maturation. Hypoxemia may contribute to long-term working memory disturbances in preterm survivors by disrupting neuronal maturation with resultant functional disturbances in hippocampal action potential throughput. Strategies directed at limiting the duration or severity of hypoxemia during brain development may mitigate disturbances in hippocampal maturation.SIGNIFICANCE STATEMENT Premature infants commonly sustain hypoxia-ischemia, which results in reduced hippocampal growth and life-long disturbances in learning and memory. We demonstrate that the circuitry related to synaptic plasticity and cellular mechanisms of learning and memory (LTP) are already functional in the fetal hippocampus. Unlike adults, the fetal hippocampus is surprisingly resistant to cell death from hypoxia-ischemia. However, the hippocampus sustains robust structural and functional disturbances in the dendritic maturation of CA1 neurons that are significantly associated with the magnitude of a brief hypoxic stress. Since transient hypoxic episodes occur commonly in preterm survivors, our findings suggest that the learning problems that ensue may be related to the unique susceptibility of the hippocampus to brief episodes of hypoxemia.

Drug potency on inhibiting late Na+ current is sensitive to gating modifier and current region where drug effects were measured.

INTRODUCTION: Cardiac late Na+ current (INaL) contributes to ventricular action potential duration. Pathological increase in INaL is arrhythmogenic, and inhibition of INaL offers protection against ventricular repolarization disturbance. Recently, two INaL datasets generated by different laboratories that assessed current inhibition by a panel of clinical drugs as a part of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative were published. The results revealed a surprising degree of data variability despite of the use of a standardized voltage protocol. This study investigated whether remaining procedural differences related to experimental methods and data analysis associated with these datasets can produce differences in INaL pharmacology. METHODS: Whole cell voltage clamp recordings were performed on cells expressing NaV1.5 α- and ß1-subunits to study: 1) the impact of gating modifiers used to augment INaL (ATX-II vs. veratridine), internal solution composition (with vs. without ATP and GTP), and recording temperature (23 °C vs 37 °C) on stability of INaL measured across the duration of a patch clamp experiment; 2) mechanisms of each gating modifier on Na+ channels; and 3) effects of six drugs (lidocaine, mexiletine, chloroquine, ranolazine, ritonavir, and verapamil) on INaL induced by either gating modifier. RESULTS: Stability of INaL is affected by the choice of gating modifier, presence of nucleotides in the internal solution, and recording temperature. ATX-II and veratridine produced different changes in Na+ channel gating, inducing mechanistically distinct INaL. Drug potencies on inhibiting INaL were dependent on the choice of gating modifier and current region where drug effects were measured. DISCUSSION: INaL pharmacology can be impacted by all experimental factors examined in this study. The effect of gating modifier and current region used to quantify drug inhibition alone led to 30× difference in half inhibitory concentration (IC50) for ritonavir, demonstrating that substantial difference in drug inhibition can be produced. Drug potencies on inhibiting INaL derived from different patch clamp studies may thus not be generalizable. For INaL pharmacology to be useful for in silico modeling or interpreting drug-induced changes in cardiac action potentials or ECG, standardizing INaL experimental procedures including data analysis methods is necessary to minimize data variability.

Assessment of an In Silico Mechanistic Model for Proarrhythmia Risk Prediction Under the CiPA Initiative.

The International Council on Harmonization (ICH) S7B and E14 regulatory guidelines are sensitive but not specific for predicting which drugs are pro-arrhythmic. In response, the Comprehensive In Vitro Proarrhythmia Assay (CiPA) was proposed that integrates multi-ion channel pharmacology data in vitro into a human cardiomyocyte model in silico for proarrhythmia risk assessment. Previously, we reported the model optimization and proarrhythmia metric selection based on CiPA training drugs. In this study, we report the application of the prespecified model and metric to independent CiPA validation drugs. Over two validation datasets, the CiPA model performance meets all pre-specified measures for ranking and classifying validation drugs, and outperforms alternatives, despite some in vitro data differences between the two datasets due to different experimental conditions and quality control procedures. This suggests that the current CiPA model/metric may be fit for regulatory use, and standardization of experimental protocols and quality control criteria could increase the model prediction accuracy even further.

Corrigendum: Optimization of an In silico Cardiac Cell Model for Proarrhythmia Risk Assessment.

[This corrects the article on p. 616 in vol. 8, PMID: 28878692.].

Uncertainty Quantification Reveals the Importance of Data Variability and Experimental Design Considerations for in Silico Proarrhythmia Risk Assessment.

The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a global initiative intended to improve drug proarrhythmia risk assessment using a new paradigm of mechanistic assays. Under the CiPA paradigm, the relative risk of drug-induced Torsade de Pointes (TdP) is assessed using an in silico model of the human ventricular action potential (AP) that integrates in vitro pharmacology data from multiple ion channels. Thus, modeling predictions of cardiac risk liability will depend critically on the variability in pharmacology data, and uncertainty quantification (UQ) must comprise an essential component of the in silico assay. This study explores UQ methods that may be incorporated into the CiPA framework. Recently, we proposed a promising in silico TdP risk metric (qNet), which is derived from AP simulations and allows separation of a set of CiPA training compounds into Low, Intermediate, and High TdP risk categories. The purpose of this study was to use UQ to evaluate the robustness of TdP risk separation by qNet. Uncertainty in the model parameters used to describe drug binding and ionic current block was estimated using the non-parametric bootstrap method and a Bayesian inference approach. Uncertainty was then propagated through AP simulations to quantify uncertainty in qNet for each drug. UQ revealed lower uncertainty and more accurate TdP risk stratification by qNet when simulations were run at concentrations below 5× the maximum therapeutic exposure (Cmax). However, when drug effects were extrapolated above 10× Cmax, UQ showed that qNet could no longer clearly separate drugs by TdP risk. This was because for most of the pharmacology data, the amount of current block measured was <60%, preventing reliable estimation of IC50-values. The results of this study demonstrate that the accuracy of TdP risk prediction depends both on the intrinsic variability in ion channel pharmacology data as well as on experimental design considerations that preclude an accurate determination of drug IC50-values in vitro. Thus, we demonstrate that UQ provides valuable information about in silico modeling predictions that can inform future proarrhythmic risk evaluation of drugs under the CiPA paradigm.

Optimization of an In silico Cardiac Cell Model for Proarrhythmia Risk Assessment.

Drug-induced Torsade-de-Pointes (TdP) has been responsible for the withdrawal of many drugs from the market and is therefore of major concern to global regulatory agencies and the pharmaceutical industry. The Comprehensive in vitro Proarrhythmia Assay (CiPA) was proposed to improve prediction of TdP risk, using in silico models and in vitro multi-channel pharmacology data as integral parts of this initiative. Previously, we reported that combining dynamic interactions between drugs and the rapid delayed rectifier potassium current (IKr) with multi-channel pharmacology is important for TdP risk classification, and we modified the original O'Hara Rudy ventricular cell mathematical model to include a Markov model of IKr to represent dynamic drug-IKr interactions (IKr-dynamic ORd model). We also developed a novel metric that could separate drugs with different TdP liabilities at high concentrations based on total electronic charge carried by the major inward ionic currents during the action potential. In this study, we further optimized the IKr-dynamic ORd model by refining model parameters using published human cardiomyocyte experimental data under control and drug block conditions. Using this optimized model and manual patch clamp data, we developed an updated version of the metric that quantifies the net electronic charge carried by major inward and outward ionic currents during the steady state action potential, which could classify the level of drug-induced TdP risk across a wide range of concentrations and pacing rates. We also established a framework to quantitatively evaluate a system's robustness against the induction of early afterdepolarizations (EADs), and demonstrated that the new metric is correlated with the cell's robustness to the pro-EAD perturbation of IKr conductance reduction. In summary, in this work we present an optimized model that is more consistent with experimental data, an improved metric that can classify drugs at concentrations both near and higher than clinical exposure, and a physiological framework to check the relationship between a metric and EAD. These findings provide a solid foundation for using in silico models for the regulatory assessment of TdP risk under the CiPA paradigm.

Functional changes in hippocampal synaptic signaling in offspring survivors of a mouse model of intrauterine inflammation.

BACKGROUND: Recent evidence suggests that exposure to intrauterine inflammation causes acute fetal brain injury and is linked to a spectrum of neurobehavioral disorders. In a rodent model of intrauterine inflammation induced by lipopolysaccharide (LPS) exposure in utero, activated microglia can be detected in the hippocampus of offspring survivors, as late as 60 days postnatal (DPN). Given that the hippocampus is important for learning and memory, these results suggest that in utero inflammation underlies long-term cognitive deficits observed in children/survivors. METHODS: An established mouse model of LPS-induced intrauterine inflammation was used to study hippocampal function from offspring at 44-59 DPN. Microgliosis was examined at 45 DPN. Extracellular field recordings of synaptic transmission were performed on acute hippocampal slices. RESULTS: LPS offspring mice displayed persistent microglial activation and increased CA3-CA1 excitatory synaptic strength, which can be explained in part by an increase in the probability of glutamate release, and reduced long-term synaptic potentiation compared to control mice. CONCLUSIONS: These results offer a mechanistic explanation for the cognitive and behavioral deficits observed in survivors of preterm birth caused by intrauterine inflammation.

Characterization of loperamide-mediated block of hERG channels at physiological temperature and its proarrhythmia propensity.

BACKGROUND: Loperamide (Immodium®) is indicated for symptomatic control of diarrhea. It is a µ-opioid receptor agonist, and recently has been associated with misuse and abuse. At therapeutic doses loperamide has not been associated with cardiotoxicity. However, loperamide overdose is associated with proarrhythmia and death - two effects that are likely attributable to its block of cardiac ion channels that are critical for generating action potentials. In this study, we defined loperamide-hERG channel interaction characteristics, and used a ventricular myocyte action potential model to compare loperamide's proarrhythmia propensity to twelve drugs with defined levels of clinical risk. METHODS AND RESULTS: Whole-cell voltage-clamp recordings were performed at 37°C on a HEK293 cell line stably expressing the hERG channel proteins, and loperamide was bath-applied to assess its effects on hERG current. Loperamide suppressed hERG current in a use- and voltage-dependent but frequency-independent manner, with a half-maximal inhibitory concentration <90nM. The onset of current suppression was concentration-dependent and appeared to follow a first-order reaction. Loperamide also altered the voltage-dependence of steady state hERG current properties. Electrophysiological data were integrated into a myocyte model that simulated dynamic drug-hERG channel interaction to estimate Torsade de Pointes risk through comparisons with reference drugs with defined clinical risk. In the context of overdose that would result in loperamide levels far exceeding those produced by therapeutic doses, loperamide is placed in the high risk category, alongside quinidine, bepridil, dofetilide, and sotalol. CONCLUSIONS: The combined in vitro and in silico approach provides mechanistic insight regarding the potential for loperamide to generate cardiotoxicity in overdose situations. This strategy holds promise for improving cardiac safety assessment.

IK1 channels do not contribute to the slow afterhyperpolarization in pyramidal neurons.

In pyramidal neurons such as hippocampal area CA1 and basolateral amygdala, a slow afterhyperpolarization (sAHP) follows a burst of action potentials, which is a powerful regulator of neuronal excitability. The sAHP amplitude increases with aging and may underlie age related memory decline. The sAHP is due to a Ca(2+)-dependent, voltage-independent K(+) conductance, the molecular identity of which has remained elusive until a recent report suggested the Ca(2+)-activated K(+) channel, IK1 (KCNN4) as the sAHP channel in CA1 pyramidal neurons. The signature pharmacology of IK1, blockade by TRAM-34, was reported for the sAHP and underlying current. We have examined the sAHP and find no evidence that TRAM-34 affects either the current underling the sAHP or excitability of CA1 or basolateral amygdala pyramidal neurons. In addition, CA1 pyramidal neurons from IK1 null mice exhibit a characteristic sAHP current. Our results indicate that IK1 channels do not mediate the sAHP in pyramidal neurons.

Apamin Boosting of Synaptic Potentials in CaV2.3 R-Type Ca2+ Channel Null Mice.

SK2- and KV4.2-containing K+ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca2+ source that provides Ca2+-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca2+ entry through NMDARs, whereas KV4.2-containing channel availability is increased by Ca2+ entry through SNX-482 (SNX) sensitive CaV2.3 R-type Ca2+ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca2+ entry through R-type Ca2+ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca2+ channels and KV4.2-containing K+ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca2+ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from CaV2.3 null mice. The results show that in the absence of CaV2.3 channels, apamin application still boosted EPSPs. The boosting effect of CaV2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from CaV2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and KV4.2-containing channels to CaV2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.

Ovarian hormone loss impairs excitatory synaptic transmission at hippocampal CA3-CA1 synapses.

Premature and long-term ovarian hormone loss following ovariectomy (OVX) is associated with cognitive impairment. This condition is prevented by estradiol (E2) therapy when initiated shortly following OVX but not after substantial delay. To determine whether these clinical findings are correlated with changes in synaptic functions, we used adult OVX rats to evaluate the consequences of short-term (7-10 d, OVXControl) and long-term (∼5 months, OVXLT) ovarian hormone loss, as well as subsequent in vivo E2 treatment, on excitatory synaptic transmission at the hippocampal CA3-CA1 synapses important for learning and memory. The results show that ovarian hormone loss was associated with a marked decrease in synaptic strength. E2 treatment increased synaptic strength in OVXControl but not OVXLT rats, demonstrating a change in the efficacy for E2 5 months following OVX. E2 also had a more rapid effect: within minutes of bath application, E2 acutely increased synaptic strength in all groups except OVXLT rats that did not receive in vivo E2 treatment. E2's acute effect was mediated postsynaptically, and required Ca(2+) influx through the voltage-gated Ca(2+) channels. Despite E2's acute effect, synaptic strength of OVXLT rats remained significantly lower than that of OVXControl rats. Thus, changes in CA3-CA1 synaptic transmission associated with ovarian hormone loss cannot be fully reversed with delayed E2 treatment. Given that synaptic strength at CA3-CA1 synapses is related to the ability to learn hippocampus-dependent tasks, these findings provide additional insights for understanding cognitive impairment-associated long-term ovarian hormone loss and ineffectiveness for delayed E2 treatment to maintain cognitive functions.