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

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 (C max ). However, when drug effects were extrapolated above 10× C max , 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 IC 50 -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 IC 50 -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.

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., (Published by Elsevier Inc.)

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.

1. Topical anesthesia with benzocaine or lidocaine occasionally causes methemoglobinemia, an uncommon but potentially fatal disorder where the blood has a reduced ability to transport oxygen. Previous in vitro studies using human whole blood have shown that benzocaine causes more methemoglobin (MetHb) formation than lidocaine, and that both compounds require metabolic transformation to form the MetHb producing species. In the current investigation, the active species of benzocaine forming the MetHb was investigated. 2. HPLC analysis of benzocaine samples incubated with human hepatic S9 showed the formation of a peak with the same UV spectrum and retention time as benzocaine hydroxylamine (BenzNOH). To confirm the activity of BenzNOH, MetHb production following exposure to the compound was determined in whole human blood using an Avoximeter 4000 CO-oximeter. 3. BenzNOH produced MetHb in a concentration dependent manner without the need for metabolic activation. Benzocaine in the presence of metabolic activation required a concentration of 500 μM to produce a similar degree of MetHb formation as 20 μM BenzNOH without activation. Previous work suggested that two metabolites of lidocaine may also form MetHb; N-hydroxyxylidine and 4-hydroxyxylidine. Of these two metabolites 4-hydroxyxylidine produced the most MetHb in whole blood in vitro in the absence of metabolic activation, however BenzNOH produced up to 14.2 times more MetHb than 4-hydroxyxylidine at a similar concentration. 4. These results suggest that the ability of benzocaine to form MetHb is likely to be mediated through its hydroxylamine metabolite and that this metabolite is inherently more active than the potentially MetHb-forming metabolites of lidocaine.

Background: The current proarrhythmia safety testing paradigm, although highly efficient in preventing new torsadogenic drugs from entering the market, has important limitations that can restrict the development and use of valuable new therapeutics. The CiPA (Comprehensive in vitro Proarrhythmia Assay) proposes to overcome these limitations by evaluating drug effects on multiple cardiac ion channels in vitro and using these data in a predictive in silico model of the adult human ventricular myocyte. A set of drugs with known clinical torsade de pointes risk was selected to develop and calibrate the in silico model., Methods and Results: Manual patch-clamp data assessing drug effects on expressed cardiac ion channels were integrated into the O'Hara-Rudy myocyte model modified to include dynamic drug-hERG channel (human Ether-à-go-go-Related Gene) interactions. Together with multichannel pharmacology data, this model predicts that compounds with high torsadogenic risk are more likely to be trapped within the hERG channel and show stronger reverse use dependency of action potential prolongation. Furthermore, drug-induced changes in the amount of electronic charge carried by the late sodium and L-type calcium currents was evaluated as a potential metric for assigning torsadogenic risk., Conclusions: Modeling dynamic drug-hERG channel interactions and multi-ion channel pharmacology improves the prediction of torsadogenic risk. With further development, these methods have the potential to improve the regulatory assessment of drug safety models under the CiPA paradigm., (© 2017 American Heart Association, Inc.)

The implementation of the ICH S7B and E14 guidelines has been successful in preventing the introduction of potentially torsadogenic drugs to the market, but it has also unduly constrained drug development by focusing on hERG block and QT prolongation as essential determinants of proarrhythmia risk. The Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative was established to develop a new paradigm for assessing proarrhythmic risk, building on the emergence of new technologies and an expanded understanding of torsadogenic mechanisms beyond hERG block. An international multi-disciplinary team of regulatory, industry and academic scientists are working together to develop and validate a set of predominantly nonclinical assays and methods that eliminate the need for the thorough-QT study and enable a more precise prediction of clinical proarrhythmia risk. The CiPA effort is led by a Steering Team that provides guidance, expertise and oversight to the various working groups and includes partners from US FDA, HESI, CSRC, SPS, EMA, Health Canada, Japan NIHS, and PMDA. The working groups address the three pillars of CiPA that evaluate drug effects on: 1) human ventricular ionic channel currents in heterologous expression systems, 2) in silico integration of cellular electrophysiologic effects based on ionic current effects, the ion channel effects, and 3) fully integrated biological systems (stem-cell-derived cardiac myocytes and the human ECG). This article provides an update on the progress of the initiative towards its target date of December 2017 for completing validation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Current regulatory guidelines for assessing the risk of QT prolongation include in vitro assays assessing drug effects on the human ether-à-go-go-related (hERG; also known as Kv11.1) channel expressed in cell lines. These assays are typically conducted at room temperature to promote the ease and stability of recording hERG currents. However, the new Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm proposes to use an in silico model of the human ventricular myocyte to assess risk, requiring as input hERG channel pharmacology data obtained at physiological temperatures. To accommodate current industry safety pharmacology practices for measuring hERG channel activity, an in silico model of hERG channel that allows for the extrapolation of hERG assay data across different temperatures is desired. Because temperature may have an effect on both channel gating and drug binding rate, such models may need to have two components: a base model dealing with temperature-dependent gating changes without drug, and a pharmacodynamic component simulating temperature-dependent drug binding kinetics. As a first step, a base mode that can capture temperature effects on hERG channel gating without drug is needed., Methods and Results: To meet this need for a temperature-dependent base model, a Markov model of the hERG channel with state transition rates explicitly dependent on temperature was developed and calibrated using data from a variety of published experiments conducted over a range of temperatures. The model was able to reproduce observed temperature-dependent changes in key channel gating properties and also to predict the results obtained in independent sets of new experiments., Discussion: This new temperature-sensitive model of hERG gating represents an attempt to improve the predictivity of safety pharmacology testing by enabling the translation of room temperature hERG assay data to more physiological conditions. With further development, this model can be incorporated into the CiPA paradigm and also be used as a tool for developing insights into the thermodynamics of hERG channel gating mechanisms and the temperature-dependence of hERG channel block by drugs., Competing Interests: Statement The authors declared no conflict of interest., (Published by Elsevier Inc.)

For the past decade, cardiac safety screening to evaluate the propensity of drugs to produce QT interval prolongation and Torsades de Pointes (TdP) arrhythmia has been conducted according to ICH S7B and ICH E14 guidelines. Central to the existing approach are hERG channel assays and in vivo QT measurements. Although effective, the present paradigm carries a risk of unnecessary compound attrition and high cost, especially when considering costly thorough QT (TQT) studies conducted later in drug development. The C: omprehensive I: n Vitro P: roarrhythmia A: ssay (CiPA) initiative is a public-private collaboration with the aim of updating the existing cardiac safety testing paradigm to better evaluate arrhythmia risk and remove the need for TQT studies. It is hoped that CiPA will produce a standardized ion channel assay approach, incorporating defined tests against major cardiac ion channels, the results of which then inform evaluation of proarrhythmic actions in silico, using human ventricular action potential reconstructions. Results are then to be confirmed using human (stem cell-derived) cardiomyocytes. This perspective article reviews the rationale, progress of, and challenges for the CiPA initiative, if this new paradigm is to replace existing practice and, in time, lead to improved and widely accepted cardiac safety testing guidelines., (© 2015 Society for Laboratory Automation and Screening.)

Attempts to characterize and formally qualify biomarkers for regulatory purposes have raised questions about how histological and histopathological methods impact the evaluation of biomarker performance. A group of pathologists was asked to analyze digitized images prepared from rodent kidney injury experiments in studies designed to investigate sources of variability in histopathology evaluations. Study A maximized variability by using samples from diverse studies and providing minimal guidance, contextual information, or opportunities for pathologist interaction. Study B was designed to limit interpathologist variability by using more uniform image sets from different locations within the same kidneys and allowing pathologist selected interactions to discuss and identify the location and injury to be evaluated but without providing a lexicon or peer review. Results from this study suggest that differences between pathologists and across models of disease are the largest sources of variability in evaluations and that blind evaluations do not generally make a significant difference. Results of this study generally align with recommendations from both industry and the U.S. Food and Drug Administration and should inform future studies examining the effects of common lexicons and scoring criteria, peer review, and blind evaluations in the context of biomarker performance assessment., (© 2014 by The Author(s).)

Medical-product development has become increasingly challenging and resource-intensive. In 2004, the Food and Drug Administration (FDA) described critical challenges facing medical-product development by establishing the critical path initiative [1]. Priorities identified included the need for improved modeling and simulation tools, further emphasized in FDA's 2011 Strategic Plan for Regulatory Science [Appendix]. In an effort to support and advance model-informed medical-product development (MIMPD), the Critical Path Institute (C-Path) [www.c-path.org], FDA, and International Society of Pharmacometrics [www.go-isop.org] co-sponsored a workshop in Washington, D.C. on September 26, 2013, to examine integrated approaches to developing and applying model- MIMPD. The workshop brought together an international group of scientists from industry, academia, FDA, and the European Medicines Agency to discuss MIMPD strategies and their applications. A commentary on the proceedings of that workshop is presented here.

The clinical use of local anesthetic products to anesthetize mucous membranes has been associated with methemoglobinemia (MetHba), a serious condition in which the blood has reduced capacity to carry oxygen. An evaluation of spontaneous adverse event reporting of MetHba submitted to FDA through 2013 identified 375 reports associated with benzocaine and 16 reports associated with lidocaine. The current study was performed to determine the relative ability of benzocaine and lidocaine to produce methemoglobin (MetHb) in vitro. Incubation of 500μM benzocaine with whole human blood and pooled human liver S9 over 5h resulted in MetHb levels equaling 39.8±1.2% of the total hemoglobin. No MetHb formation was detected for 500μM lidocaine under the same conditions. Because liver S9 does not readily form lidocaine hydrolytic metabolites based on xylidine, a primary metabolic pathway, 500μM xylidine was directly incubated with whole blood and S9. Under these conditions MetHb levels of 4.4±0.4% were reached by 5h. Studies with recombinant cytochrome P450 revealed benzocaine to be extensively metabolized by CYP 1A2, with 2B6, 2C19, 2D6, and 2E1 also having activity. We conclude that benzocaine produces much more MetHb in in vitro systems than lidocaine or xylidine and that benzocaine should be more likely to cause MetHba in vivo as well., (Published by Elsevier Inc.)

Influenza virus infections represent a serious public health problem worldwide, due to the rapid emergence of drug resistance. One strategy to improve treatment efficacy is to combine drugs that act synergistically. Potentially useful drug combinations are typically identified through empirical testing using in vitro and animal models, but the complexity of the clinical situation warrants the use of more careful analysis and sophisticated approaches. To explore new approaches, we constructed a mechanistic model representing the interaction of antiviral drugs with the viral replication pathway and human immune responses. Simulation of combination therapy using oseltamivir and amantadine predicted significant therapeutic synergy only when immune response was included, in agreement with previous in vitro and in vivo studies using amantadine-resistant strains. Our model can be used to predict the optimal doses for combination therapy, and also raises questions about current drug evaluation methods that do not account for immune system interactions.

Phospholipidosis (PLD), an abnormal accumulation of phospholipids within tissues, is observed during the preclinical testing of many pharmaceutical drugs. Diagnosis of PLD is currently based on morphologic criteria. An understanding of the clinical incidence of PLD and its possible relationship to adverse drug reactions has been hampered by the absence of noninvasive biomarkers for PLD. The uncommon phospholipid di-docosahexaenoyl bis(monoacylglycerol) phosphate (di-22:6-BMP) has been proposed as a potential urinary biomarker for PLD, but data on the utility of serum di-22:6-BMP measurements in diagnosing PLD is more limited. In this report, we compared the performance of serum and urinary di-22:6-BMP as biomarkers for PLD in rats treated with the PLD-inducing drugs amiodarone and 4,4'-diethylaminoethoxyhexestrol dihydrochloride or the hepatotoxicant acetaminophen (APAP). Serum levels of di-22:6-BMP showed a higher correlation to a generalized PLD incidence score than did levels in urine, but were unexpectedly elevated in rats with marked levels of APAP-induced liver necrosis. When samples were filtered based on serum ALT or liver histopathology thresholds, the diagnostic accuracy of serum di-22:6-BMP for PLD improved to the high level observed for urinary di-22:6-BMP without sample exclusion. These data help define the potential context-of-use of serum di-22:6-BMP as a non-clinical biomarker of PLD., (Published by Elsevier Ireland Ltd.)

Cationic amphiphilic drugs and aminoglycoside antibiotics can induce phospholipidosis (PLD), an abnormal accumulation of phospholipids in lysosome-derived vesicles, in preclinical studies. The incidence of PLD in patients and its clinical relevance are difficult to assess without noninvasive biomarkers. Di-docosahexaenoyl bis(monoacylglycerol)phosphate (di-22:6-BMP) is a phospholipid that is enriched in lysosomal membranes and a proposed urinary biomarker of drug-induced PLD. The specificity of di-22:6-BMP for PLD was compared to other phospholipid species that can increase in urine with nephrotoxicity. Using liquid chromatography coupled to mass spectrometry, 12 phospholipids were assayed in the urine of rats treated with drugs that induced PLD or caused renal or skeletal muscle injury. In receiver operating curve analyses, urinary di-22:6-BMP was a significantly better predictor of PLD and the least predictive of tissue injury of the phospholipids assayed. The data provide evidence supporting the use of di-22:6-BMP as a urinary biomarker of PLD in rats.

The safe disposal of unused opioid drugs is an area of regulatory concern. While toilet flushing is recommended for some drugs to prevent accidental exposure, there is a need for data that can support a more consistent disposal policy based on an assessment of relative risk. For drugs acting at the Mu-opioid receptor (MOR), published measurements of binding affinity (K(i)) are incomplete and inconsistent due to differences in methodology and assay system, leading to a wide range of values for the same drug thus precluding a simple and meaningful relative ranking of drug potency. Experiments were conducted to obtain K(i)'s for 19 approved opioid drugs using a single binding assay in a cell membrane preparation expressing recombinant human MOR. The K(i) values obtained ranged from 0.1380 nM (sufentanil) to 12.486 μM (tramadol). The drugs were separated into three categories based upon their K(i) values: K(i) > 100 nM (tramadol, codeine, meperidine, propoxyphene and pentazocine), K(i)=1-100 nM (hydrocodone, oxycodone, diphenoxylate, alfentanil, methadone, nalbuphine, fentanyl and morphine) and K(i) < 1 nM (butorphanol, levorphanol, oxymorphone, hydromorphone, buprenorphine and sufentanil). These data add to the understanding of the pharmacology of opioid drugs and support the development of a more consistent labeling policies regarding safe disposal., (Published by Elsevier Inc.)

Drug-induced vascular injury (DIVI) is a nonclinical finding that often confounds the toxicological evaluation of investigational drugs, but there is an absence of qualified biomarkers that can be used to detect and monitor its appearance in animals and patients during drug development and clinical use. It is well known that endothelial cell (EC) activation plays a key role in the expression and evolution of DIVI, and the various immunological and inflammatory factors involved in its expression may serve as potential biomarker candidates. Activated ECs change their morphology and gene expression, generating endothelial adhesion molecules, pro-coagulant molecules, cytokines, chemokines, vasodilators, nitric oxide, and acute-phase reactants. This review provides a brief historical background of EC activation and the search for biomarkers of early EC activation for monitoring DIVI. At present, no biomarkers of EC activation have been qualified to predict DIVI in the nonclinical or clinical context, and a robust pathologic foundation for their use is still lacking. We propose three categories of EC activation biomarkers: recommended surrogate markers, potentially useful markers, and emerging candidate markers. This review alerts pharmaceutical companies, research institutions, and regulatory agencies to the continuing need for reliable biomarkers of EC activation in drug development.

Objective: The fate of an impulse arising from stimulation is determined by the ability of the wave front to recruit sufficient Na channels from adjacent cells. Previous numerical studies of mutant Na channels revealed both the velocity of a conditioning wave and the recruiting capacity of the front as determinants of the vulnerable period (VP), an interval within which excitation results in unidirectional conduction. Drugs that block excitatory Na channels in a voltage dependent manner, such as antiarrhythmics, abused substances and antidepressants, slow the restoration of Na conductance trailing an action potential and are associated with proarrhythmia and sudden cardiac death. We hypothesized that drug-induced slowing of Na conductance recovery would flatten the Na conductance restoration gradient thereby reducing the recruiting capacity of a front, extending the VP and increasing the probability of unidirectional propagation., Methods: In a cable of ventricular cells, we explored the sensitivity of the VP to voltage-dependent blockade. While varying the unbinding time constant from 100 ms to 5 s, we measured the Na conductance restoration gradient, the liminal length, the refractory period (RP) and the VP., Results: Reducing the rate of drug unbinding flattened the restoration gradient, diminished the recruiting capacity of a premature impulse and extended the liminal length, RP and the VP. The VP was linearly dependent on the drug unbinding time constant. Rapidly unbinding drugs (time constant <1 s) reduced the liminal length below that of a quiescent cable., Conclusions: Slowing the unbinding rate of voltage-dependent drug block of Na channels extended the RP and the VP. Drugs with unbinding time constants greater than 1 s dramatically increased the probability of unidirectional propagation, reflecting increases in both the RP and the VP. This study provides a new mechanism linking Na channel function, compromised by voltage-dependent Na channel drug block, with proarrhythmic conditions that can lead to sudden cardiac death following premature stimulation.

The effects of the ATP-dependent potassium channel agonists ZD6169, celikalim, and WAY-133537 on bladder contractile function were examined in vitro on isolated bladder strips and in vivo on spontaneous bladder contractions. All three compounds produced a concentration-dependent relaxation of isolated rat detrusor strips (IC50 values = 0.93, 0.03, and 0.09 microM, respectively for ZD6169, celikalim, and WAY-133537. Contractile inhibition by all three compounds was fully reversed by 6 microM glyburide. These compounds also effectively inhibited spontaneous bladder contractions in the rat hypertrophied bladder model of detrusor instability. We also examined the electrophysiological properties of WAY-133537 on isolated rat bladder detrusor myocytes. Myocytes had an average resting membrane potential of -40 mV. Under patch current-clamp conditions, WAY-133537 (0.3 and 1.0 microM, n = 4-5) produced a significant hyperpolarization of 21 and 26 mV, respectively. Hyperpolarization was reversed by the addition of 5 microM glyburide. In patch voltage-clamp studies, WAY-133537 (0.3 microM, n = 3) significantly increased outward current in response to both voltage step and ramp protocols consistent with activation of the ATP-dependent potassium channel. In the detrusor instability model, WAY-133537 and celikalim had similar oral potencies (ED50 = 0.13 and 0.3 mg/kg, respectively), whereas ZD6169 was less potent (ED50 = 2.4 mg/kg). The antihypertensive agent celikalim exerted effects on the bladder at doses that significantly reduced systemic blood pressure. In contrast, both WAY-133537 and ZD6169 inhibited bladder hyperactivity at doses that produced minimal changes in both mean arterial blood pressure and heart rate. These data suggest that both WAY-133537 and ZD6169 may be useful in the treatment of bladder instability at doses associated with minimal hemodynamic side effects.

Mutations in the HERG gene are linked to the LQT2 form of the inherited long-QT syndrome. Transgenic mice were generated expressing high myocardial levels of a particularly severe form of LQT2-associated HERG mutation (G628S). Hearts from G628S mice appeared normal except for a modest enlargement seen only in females. Ventricular myocytes isolated from adult wild-type hearts consistently exhibited an inwardly rectifying E-4031-sensitive K+ current resembling the rapidly activating cardiac delayed rectifier K+ current (Ikr) in its time and voltage dependence; this current was not found in cells isolated from G628S mice. Action potential duration was significantly prolonged in single myocytes from G628S ventricle (cycle length=1 second, 26 degrees C) but not in recordings from intact ventricular strips studied at more physiological rates and temperature (200 to 400 bpm, 37 degrees C). ECG intervals, including QT duration, were unchanged, although minor aberrancies were noted in 20% (16/80) of the G628S mice studied, primarily involving the QRS complex and, more rarely, T-wave morphology. The aberrations were more commonly observed in females than males but could not be correlated with sex-based differences in action potential duration. These results establish the presence of IKr in the adult mouse ventricle and demonstrate the ability of the G628S mutation to exert a dominant negative effect on endogenous IKr in vivo, leading to the expected LQT2 phenotype of prolonged repolarization at the single cell level but not QT prolongation in the intact animal. The model may be useful in dissecting repolarization currents in the mouse heart and as a means of examining the mechanism(s) by which the G628S mutation exerts its dominant negative effect on native cardiac cells in vivo.

Background: The genes for the long QT syndrome (LQTS) linked to chromosomes 3 (LQT3) and 7 (LQT2) were identified as SCN5A, the cardiac Na+ channel gene, and as HERG, a K+ channel gene. These findings opened the possibility of attempting gene-specific control of ventricular repolarization. We tested the hypothesis that the QT interval would shorten more in LQT3 than in LQT2 patients in response to mexiletine and also in response to increases in heart rate., Methods and Results: Fifteen LQTS patients were studied. Six LQT3 and 7 LQT2 patients were treated with mexiletine, and its effects on QT and QTc were measured. Mexiletine significantly shortened the QT interval among LQT3 patients (QTc from 535 +/- 32 to 445 +/- 31 ms, P < .005) but not among LQT2 patients (QTc from 530 +/- 79 to 503 +/- 60 ms, P = NS). LQT3 patients (n = 7) shortened their QT interval in response to increases in heart rate much more than LQT2 patients (n = 4) and also more than 18 healthy control subjects (9.45 +/- 3.3 versus 3.95 +/- 1.97 and 2.83 +/- 1.33, P < .05; data expressed as percent reduction in QT per 100-ms shortening in RR). Among these patients, there is also a trend for LQT2 patients to have syncope or cardiac arrest under emotional or physical stress and for LQT3 patients to have cardiac events either at rest or during sleep., Conclusions: This is the first study to demonstrate differential responses of LQTS patients to interventions targeted to their specific genetic defect. These findings also suggest that LQT3 patients may be more likely to benefit from Na+ channel blockers and from cardiac pacing because they would be at higher risk of arrhythmia at slow heart rates. Conversely, LQT2 patients may be at higher risk to develop syncope under stressful conditions because of the combined arrhythmogenic effect of catecholamines with the insufficient adaptation of their QT interval when heart rate increases.

The antihypertensive agents pinacidil, cromakalim and celikalim lower blood pressure by opening potassium channels in vascular smooth muscle. The role of these compounds in inhibiting human platelet aggregation and preventing white thrombus formation in a rabbit arteriovenous shunt model was examined. None of these agents (100 microM), substantially inhibited platelet aggregation induced by epinephrine or arachidonic acid. Only celikalim (100 microM) inhibited collagen (45%), ADP (56%), or serotonin (61%) induced platelet aggregation and ADP- (41%) or epinephrine-potentiated (61%) serotonin-induced platelet aggregation. Celikalim inhibited white thrombus formation at i.v. doses of 0.25 mg/kg (46% inhibition) but not 0.1 mg/kg; (14.6%); equihypotensive doses of pinacidil (0.5 mg/kg; 21.7%) and cromakalim (0.2 mg/kg, 7.5%; 0.4 mg/kg, 33%) were less effective. Glyburide (i.v. dose of 0.5 mg/kg) inhibited the antithrombotic activity of celikalim and to a lesser extent cromakalim. The greater antithrombotic activity of celikalim in vivo may be related to beneficial effects on blood rheology and reduced red blood cell deformability.

Objective: The objectives were (a) to evaluate the effects of WAY-123,398, a new class III antiarrhythmic agent, on the action potential of canine Purkinje fibres in comparison with dofetilide, E-4031, and dl-sotalol, and (b) to characterise the mechanism of the class III action by studying its effects on several ionic currents in isolated cat myocytes., Methods: Transmembrane potentials in Purkinje fibres were studied with standard microelectrodes filled with 3M KCl. Myocytes were isolated by enzymatic disaggregation with collagenase and current recordings were obtained by voltage clamp with either the nystatin perforated patch technique or the usual whole cell configuration., Results: WAY-123,398 prolonged action potential duration (APD) in Purkinje fibres and in cat ventricular myocytes without altering other variables of the action potential; in Purkinje fibres the concentration producing a 20% prolongation of APD-60 mV at a basic cycle length of 1000 ms was 0.2 microM. After depolarising voltage steps, the delayed rectifier (IK) peak tail currents in cat myocytes were blocked with IC50 = 0.1 microM. The block was unaffected by varying the duration (200 to 500 ms) or the frequency (0.4 to 2.5 Hz) of the depolarising steps. A much higher concentration of WAY-123,398 (10 microM) did not have effects on the L type Ca current (ICa-L), and on the inward rectifier (IK1) and transient outward (I(to)) K currents., Conclusions: The results indicate that WAY-123,398 is an effective and specific class III agent devoid of class I activity, and suggest that WAY-123,398 prolongs cardiac repolarisation by specifically blocking the delayed rectifier current (IK). The block was unchanged over a range of frequencies and duration of depolarisation, showing no evidence of "reverse use dependence" of block.

Previous studies in isolated ventricular myocytes showed that WAY-123,398 is a selective blocker of the delayed rectifier K+ current (IK). In this report, we studied the electrophysiological and hemodynamic effects of WAY-123,398 in open-chest anesthetized dogs. WAY-123,398 prolonged atrial and ventricular refractoriness without affecting conduction; WAY-123,398 was as effective as UK-68798, E-4031, and dl-sotalol, but less potent than UK-68798 and E-4031. The increase in atrial refractoriness was approximately twice as large as the ventricular increase with all compounds. The hemodynamic effects of WAY-123,398 were similar to those of UK-68798; at the ED20 for increasing ventricular refractoriness, WAY-123,398 did not affect the mean arterial pressure and decreased the heart rate by 20%. In a different series of experiments, all four compounds produced large and comparable increases in the ventricular fibrillation threshold in anesthetized dogs; WAY-123,398 and UK-68798 induced defibrillation and restoration of sinus rhythm in two of six dogs each and E-4031 in one of six dogs. No episodes of drug-induced restoration to sinus rhythm were observed in dogs treated with sotalol or vehicle. In conclusion, WAY-123,398 is an effective Class III agent without Class I actions and with a favorable hemodynamic profile.

Objective: The aim was to compare the effects of the class Ic antiarrhythmic drugs flecainide, encainide, and recainam on the delayed rectifier current, IK., Methods: Membrane currents were studied using the single suction pipette voltage clamp technique in freshly dissociated cat ventricular myocytes bathed in HEPES buffered physiological saline at 32 degrees C., Results: Flecainide and encainide decreased IK with IC50 values of 2.1 microM and 6 microM, respectively. Recainam (100 microM) reduced IK by only 7 (SEM 3)% after 20-30 min exposure and by 19% after an 80 min exposure (IC50 > 400 microM). None of the compounds blocked the inward rectifier, IK1. Block of IK by flecainide and encainide increased with depolarisation following a voltage dependence similar to that describing channel activation. Flecainide and encainide also slowed the time course of the IK tail currents, consistent with drug dissociating from open channels., Conclusions: The observed voltage dependence for IK block by flecainide and encainide resembles the interaction reported between these agents and the excitatory sodium channel, ie, depolarisation enhances block while repolarisation leads to removal of block. The results further suggest that the electrophysiological profile of class Ic agents can have a markedly different ionic basis, ie, K+ channel block by flecainide and encainide is balanced by a potent block of sodium channels, while recainam appears to be a weak but relatively specific blocker of sodium channels only. These differences are not readily accommodated by the current Harrison-Vaughan-Williams classification scheme, and suggest the possibility that potentially important drug specific differences can exist within the same antiarrhythmic drug class.

Celikalim (WAY-120,491) is a putative potassium channel activator that has been shown to lower blood pressure in animal models and humans. In the present study, we have examined the effects of celikalim on contractility and ionic currents in feline cardiac muscle. Celikalim was found to decrease contractility in electrically stimulated (2 Hz frequency) left atrial and right ventricular papillary muscle preparations with IC50 values of 0.95 +/- 0.12 microM (n = 6) and 0.29 +/- 0.07 microM (n = 5), respectively. Glyburide (1 microM) reversed the celikalim-induced negative inotropy (left atrial halves). Celikalim was also shown to activate a glyburide-sensitive current in voltage-clamped isolated ventricular myocytes that reversed close to the calculated value of the potassium equilibrium potential (n = 4 cells). In addition, celikalim was found to inhibit voltage-activated calcium current (L-type) in isolated ventricular myocytes (51 +/- 2% inhibition at 1 microM; n = 4 cells). We conclude that celikalim is a potassium channel activator and hypothesize that both the negative inotropy and the glyburide-sensitive current evoked by this drug are mediated by ATP-regulated potassium channels. Inhibition of voltage-activated calcium channels by celikalim may also contribute to the negative inotropy induced by this drug.

The cardioprotective effects of the K channel activator drugs celikalim (WAY-120,491) and cromakalim were studied in a canine model of myocardial infarction consisting of 90 min of ischemia and 5 h of reperfusion. Intracoronary infusion of cromakalim and celikalim at 0.2 microgram/kg/min beginning 10 min before occlusion of the left circumflex coronary artery and continuing throughout the duration of the reperfusion period appeared to exacerbate ischemic injury. Infarct size (percent of risk area) was 27.7 +/- 5.6% in vehicle control animals (n = 5), 40.3 +/- 6.2% for cromakalim (n = 5) and 55.7 +/- 6.4% (p less than 0.05 vs. vehicle) for celikalim-treated animals (n = 5). When these compounds were administered intravenously, using doses shown to increase total coronary flow in nonoccluded control animals, no exacerbation of ischemic injury was observed. Anatomic infarct size was 32.8 +/- 7.1% for vehicle animals (n = 5) and 32.6 +/- 13.3 and 30.9 +/- 9.8% for cromakalim- (n = 6) and celikalim-treated (n = 5) animals, respectively. Intravenous diltiazem decreased myocardial infarct size to 16.3 +/- 7.3% (n = 5) of area at risk (p = NS vs. vehicle). The anatomic area at risk was similar in all three treatment groups, and no significant differences in rate-pressure product were observed. Results of this study suggest that K-channel-activating drugs such as cromakalim and celikalim may not be effective agents in the acute therapeutic management of myocardial ischemic injury.

The effects of cadmium on the delayed outward potassium current (IK) were investigated in isolated cat ventricular myocytes using the single suction pipette voltage-clamp technique. IK activation was examined using peak tail currents elicited after 750-ms voltage-clamp steps to selected membrane potentials from a holding potential of -40 mV. In the presence of Cd2+ (0.2 mM), peak tail currents increased from a control value of 85 +/- 12 to 125 +/- 18 pA (n = 4). Activation curves constructed from the average peak tail-current measurements in all experiments showed that Cd2+ shifted the voltage dependence of activation to more positive potentials by 16.4 +/- 2.0 mV and increased the slope factor of the activation curve from 6.1 +/- 0.2 to 6.9 +/- 0.2 mV. In the absence of Cd2+, increases in holding potential from -30 to -70 mV had no effect on the magnitude of the peak tail currents, suggesting that the Cd(2+)-induced increase was not the result of a voltage-dependent increase in the number of available K+ channels at the holding potential. Slow voltage ramps from -70 to +70 mV revealed that Cd2+ increased the outward current at membrane potentials positive to +20 mV and shifted the voltage range in which IK inwardly rectified to more positive potentials. The fully activated current-voltage relationship was also shifted to more positive potentials by Cd2+. Cd2+ did not alter channel selectivity for K+.(ABSTRACT TRUNCATED AT 250 WORDS)

The phosphodiesterase (PDE) inhibitors AY-31,390, milrinone and pelrinone (AY-28,768) were analyzed in human platelet aggregatory systems and in a rabbit arteriovenous shunt model to delineate their activity. AY-31,390 showed a remarkably potent capacity to inhibit human antithrombotic platelet aggregation. AY-31,390 inhibited arachidonic acid, U46619, collagen, epinephrine (second phase) and adenosine diphosphate (second phase) induced platelet aggregation (PA) with IC50 values of 0.18, 0.21, 0.54, 0.43 and 0.20 microM, respectively. Milrinone, although less potent than AY-31,390, inhibited PA with IC50 values of 2.1, 2.0, 5.4, 3.7 and 4.1 microM and pelrinone's IC50 values were 2.8, 6.6, 13.3, 18.6 and 11.8 microM, respectively. Platelets which were incubated with AY-31,390, milrinone or pelrinone, washed with Hanks' balanced salt solution and then resuspended in platelet poor plasma, lost their inhibitory activity in collagen and arachidonic acid PA systems. These results suggested that AY-31,390, milrinone and pelrinone did not bind tightly to cAMP PDE. If human platelet-rich plasma was pretreated with adenosine deaminase, an enzyme that degrades adenosine, the inhibitory effect of milrinone and to a lesser extent pelrinone was reversed. AY-31,390 did not produce a loss of activity with adenosine deaminase in the arachidonic acid system and only a small loss in the collagen system. Adenosine did not appear to be a meaningful factor in AY-31,390's inhibitory activity. Pelrinone, milrinone to a greater extent, and AY-31,390 to the greatest extent were effective inhibitors of white thrombus formation in the in vivo rabbit arteriovenous shunt model. These PDE III inhibitors were potent deterrants of platelet aggregation and white thrombus formation; these agents would be expected to be efficacious therapeutic antithrombotics.

WAY-120,491 [(-)-(3S-trans)-2-[3,4-dihydro-3-hydroxy-2,2-dimethyl-6-(trifluoromet hox y)- 2H-1-benzopyran-4-yl]-2,3-dihydro-1H-isoindol-1-one] is a novel antihypertensive agent. We have investigated the effects of this compound on contractile force and 86Rb efflux, using the rabbit aorta, in order to assess its K channel activator properties. K channel blockers and ionic conditions thought to modulate specific K channel types have been used to provide insight into the K channel(s) affected by this compound. WAY-120,491 evoked relaxation of precontracted rabbit aortic rings and increased the rate of 86Rb efflux from strips of rabbit aorta; both effects occurring in a concentration-dependent manner. The WAY-120,491 (1 microM)-induced 86Rb efflux was inhibited by tetraethylammonium (IC50 = 0.38 mM), indicating that the increased efflux was mediated by K channels. Glyburide completely blocked the WAY-120,491 (1 microM)-evoked 86Rb efflux with 50% block occurring at a concentration of 0.48 microM. Glyburide also antagonized the WAY-120,491-induced relaxation of aortic rings. Omission of Ca from the solution bathing the aorta did not inhibit the WAY-120,491 induced 86Rb efflux but rather caused an augmentation of the response. It is concluded that WAY-120,491 may be classified as a K channel opener. Furthermore, the K channel upon which WAY-120,491 acts exhibits some characteristics normally associated with the ATP regulated K channel although the involvement of other K channel types has not been ruled out.

The antithrombotic activity of pelrinone, a phosphodiesterase III inhibitor was examined in a canine model of coronary thrombosis that uses electrical current to injure the coronary endothelium. Ninety percent of vehicle treated animals developed complete coronary occlusion and thrombus mass was 32.0 +/- 5.8 mg. In a group of animals treated with zomepirac, 10 mg/kg i.v., included as a positive control, thrombus mass was decreased to 10.3 +/- 3.3 mg and incidence of occlusion was reduced to 37.5%. Pelrinone, 5.0 mg/kg i.v. decreased the incidence of occlusion to 50%, thrombus mass to 21.3 +/- 8.3 mg and inhibited platelet aggregation to collagen, ADP and arachidonic acid by 80%, 54% and 87% of baseline, respectively. When yohimbine, an alpha 2-adrenergic antagonist, was co-administered (2.0 mg/kg at the beginning of the experiment +0.5 mg/kg halfway through the experiment) with the same dose of pelrinone, thrombus mass was decreased to 1.0 +/- 0.5 mg and none of the animals developed coronary occlusion. Yohimbine administration by itself at 2.0-3.0 mg/kg showed no evidence of antithrombotic activity (thrombus mass = 32.8 +/- 8.0 mg, incidence of occlusion = 100%). This dose of yohimbine inhibited significantly ADP-induced aggregation in the presence of epinephrine. These results demonstrate that, even though this dose of pelrinone elicited near maximal inhibition of platelet aggregation, the concurrent administration of an alpha 2-adrenergic antagonist was able to potentiate markedly the phosphodiesterase inhibitor antithrombotic activity.

Although work on class III antiarrhythmics remains at an early stage, these agents still appear to possess greater efficacy and less proarrhythmia than conventional class I agents in those experimental arrhythmia models considered to be most representative of the clinical situation. Although prolongation of repolarization carries with its own tendency for pause-dependent arrhythmogenesis (i.e., torsade de pointes), available data suggest that this may be a function of nonspecificity in potassium channel block rather than a general characteristic of class III activity. The availability of new and more selective blockers of specific cardiac potassium channels under development as class III agents have already helped to clarify basic questions about the ionic mechanism of repolarization in the heart, and one hopes that a growing clinical data base will eventually determine the relative safety and efficacy of these agents in preventing symptomatic and life-threatening arrhythmias.

Block of the delayed rectifier potassium current, IK, by the class IC antiarrhythmic agent, flecainide, and by the novel selective class III antiarrhythmic agent, E-4031, were compared in isolated cat ventricular myocytes using the single suction-pipette, voltage-clamp technique. Flecainide (10 microM) markedly reduced IK elicited on depolarization steps to plateau voltages (+10 mV) and nearly completely blocked the "tail currents" elicited on repolarization to -40 mV (93 +/- 4% block at +40 mV, n = 3). E-4031 (1 microM) produced similar effects (96 +/- 3% block at +40 mV, n = 3). Slow voltage ramps from -100 to +40 mV confirmed inward rectifying properties of IK and showed that flecainide and E-4031 have no effects on the background potassium current, IK1. Thus, the results demonstrate that block of IK is a common feature of flecainide and E-4031. IK block by E-4031 most likely underlies the drug's potent class III antiarrhythmic properties. On the other hand, flecainide block of IK during an action potential would tend to prolong repolarization, but this effect may be obscured by concomitant block of plateau Na+ channels to produce little or no change in action potential duration, consistent with its class IC classification.

The cardiac electrophysiologic effects of the potassium channel activators cromakalim, pinacidil and nicorandil were determined in anesthetized open-chest normotensive dogs using conventional surface electrogram recording techniques. Intravenous administration of cromakalim (0.025-0.5 mg/kg), pinacidil (0.1-2.0 mg/kg) and nicorandil (0.1-2.5 mg/kg) produced large dose-dependent decreases in arterial blood pressure accompanied by smaller reductions of atrial and ventricular effective refractory periods. The shortening of refractoriness was more pronounced in the atrium than in the ventricle and was similar for all three compounds at a given level of hypotension. Effects on other electrophysiological parameters were minimal. Atrial arrhythmias could be induced during electrical pacing at doses of cromakalim and pinacidil producing excessive (greater than or equal to 40%) decreases in mean arterial pressure. No arrhythmias were observed with nicorandil. Induction of the arrhythmias appeared to be closely coupled to the extrastimuli (S2) used to determine refractory periods and was associated with a significant reduction in atrial refractory period (greater than or equal to 30%). No ventricular arrhythmias were observed in this study with any of the compounds tested. Although the plasma levels reached in this study are likely to be higher than those seen clinically, the results nevertheless suggest the potential for cardiac electrophysiologic effects by these agents.

The passive electrical properties of subendocardial Purkinje fibers surviving in infarcted regions of canine ventricle 24 hours after coronary ligation were studied by using microelectrode techniques and cable theory. In normal hearts, cells within the subendocardial Purkinje fiber strands were found to be well coupled to each other but electrically isolated from neighboring myocardium. Voltage response to intracellular current injection was consistent with one-dimensional cable behavior and yielded estimates of passive electrical properties in general agreement with previous work on free-running Purkinje strands (membrane length constant, 1.2 +/- 0.1 mm; membrane time constant, 7.3 +/- 0.8 msec; input resistance, 67.4 +/- 7.4 K omega; membrane resistance, 8.2 +/- 0.7 K omega.cm; axial resistance, 0.52 +/- 0.06 M omega/cm; membrane capacitance, 960 +/- 102 nF/cm) (n = 21). On the day after coronary ligation, subendocardial Purkinje fiber action potentials were prolonged and slightly depolarized. Significant increases were measured in input resistance (+40.5%), membrane resistance (+43.9%), and axial resistance (+47.5%), whereas membrane capacitance was found to be significantly decreased (-24.3%) (n = 19). Conduction velocity, membrane length constant, membrane time constant, and the time constant and capacitance for the foot of the action potential remained unchanged. These results are consistent with electrical uncoupling between adjacent cells, which will increase internal resistivity, accompanied by changes in cellular phospholipid content, which can increase membrane resistance and alter membrane capacitance. Alternatively, the results can be explained by a simple model in which the apparent electrical structure is altered by changes in electrical coupling alone, with specific electrical properties remaining constant. Although the mechanisms underlying the observed changes remain uncertain, the present study indicates that myocardial infarction is associated with alterations in the passive electrical structure of surviving subendocardial Purkinje fibers, which, together with changes in action potential configuration, may provide a substrate for the generation of ventricular arrhythmias 24 hours after coronary ligation.

1. Voltage clamp studies of the excitatory sodium current, INa, were carried out in rabbit cardiac Purkinje fibres using th two-micro-electrode technique. Previous work has shown the rabbit Purkinje fibre to have relatively simple morphology (Sommer & Johnson, 1968) and electrical structure (Colatsky & Tsien, 1979a) compared to other cardiac preparations. 2. Non-uniformities in membrane potential were kept small by reducing the size of INa to less than 50 microA/cm2 of total membrane surface area through prepulse inactivation or removal of external sodium, Nao. Temporal resolution was improved by cooling to 10-26 degrees C. These adjustments did not greatly alter the measured properties of the sodium channel. 3. Under these conditions, sodium currents were recorded satisfying a number of criteria for adequate voltage control. Direct measurement of longitudinal non-uniformity using a second voltage electrode showed only small deviations at the time of peak current. 4. The properties of the sodium channel were examined using conventional protocols. Both peak sodium permeability, PNa, and steady-state sodium inactivation, h infinity, showed a sigmoidal dependence on membrane potential. PNa rose steeply with small depolarizations, increasing roughly e-fold per 3.2 mV, and reaching half-maximal activation at -30 +/- 2 mV. The h infinity -V curve had a midpoint of -74.9 +/- 2 mV and a reciprocal slope of 4.56 +/- 0.13 mV at temperatures of 10-19.5 degrees C, and showed a dependence on temperature, shifting to more negative potentials with cooling (approximately 3 mV/10 degrees C). Recovery of INa from inactivation in double pulse experiments followed a single exponential time course with time constants of 108-200 msec at 19 degrees C for holding potentials near -80 mV. No attempt was made to describe the activation kinetics because of uncertainties about the early time course of the current. 5. These data predict a maximum duration for INa of less than 1-2 msec and a maximum peak current density of about 500 microA/cm2 under physiological conditions, i.e. 37 degrees C and 150 mM-Nao. This current magnitude is sufficient to discharge the membrane capacitance at rates comparable to those measured experimentally (311 +/- 27 V/sec, Colatsky & Tsien, 1979a). 6. The limitations of the method are discussed. The major problem is the longitudinal cable delay which limits the speed of voltage control. This makes it difficult to separate the activation of INa from the decay of the capacity transient for potentials positive to -15 mV. 7. It is concluded that the approach described is valid for measurements of sodium currents in the potential range where action potentials are initiated, making it possible to study cardiac sodium channels in an adult mammalian preparation which is free of enzymatic treatment.

Tetrodotoxin (TTX) block of cardiac sodium channels was studied in rabbit Purkinje fibers using a two-microelectrode voltage clamp to measure sodium current. INa decreases with TTX as if one toxin molecule blocks one channel with a dissociation constant KD approximately equal to 1 microM. KD remains unchanged when INa is partially inactivated by steady depolarization. Thus, TTX binding and channel inactivation are independent at equilibrium. Interactions between toxin binding and gating were revealed, however, by kinetic behavior that depends on rates of equilibration. For example, frequent suprathreshold pulses produce extra use-dependent block beyond the tonic block seen with widely spaced stimuli. Such lingering aftereffects of depolarization were characterized by double-pulse experiments. The extra block decays slowly enough (tau approximately equal to 5 s) to be easily separated from normal recovery from inactivation (tau less than 0.2 s at 18 degrees C). The amount of extra block increases to a saturating level with conditioning depolarizations that produce inactivation without detectable activation. Stronger depolarizations that clearly open channels give the same final level of extra block, but its development includes a fast phase whose voltage- and time-dependence resemble channel activation. Thus, TTX block and channel gating are not independent, as believed for nerve. Kinetically, TTX resembles local anesthetics, but its affinity remains unchanged during maintained depolarization. On this last point, comparison of our INa results and earlier upstroke velocity (Vmax) measurements illustrates how much these approaches can differ.