Internodal conduction pathways: revisiting a century-long debate on their existence, morphology, and location in the context of 2023 best science.

Internodal conduction pathways are the communication apparatus of the cardiac conduction system conveying sinus node action potentials (APs) to the atrioventricular node and atrial working myocytes. In 1910, at the Deutschen Pathologischen Gesellschaft held in Erlangen, Charles Thörel related his discovery of an internodal bundle structured with Purkinje-like cells, which was rejected by the participants, who endorsed the then-leading doctrine that atrial contractile cardiomyocytes operate as internodal routes. Starting in 1963 and for five subsequent decades, two groups have revisited this issue. The first group, led by Thomas James, defended the histological existence of uninsulated, high-speed cordlike internodal conduction tracts. Although not supported by robust experimental data, this hypothesis achieved the status of a physiological creed in the scientific community. The second group, led by Robert Anderson, systematically refuted this stance and adopted anisotropy as an innovative internodal conduction mechanism operating via spindle-shaped atrial contractile myocytes structured in anisotropic muscular bundles relaying sinoatrial node (SAN) APs to their atrial destinations at physiologically required (fit for purpose) velocities to enable electromechanical synchrony of atrial systoles. Modern imaging and electrophysiological techniques can now clearly visualize muscular internodal and interatrial tracts and SAN depolarization electrical waves, confirming the existence of atrial conduction paths consisting of anisotropically arranged contractile cardiomyocytes. Mastery of the current best-science anatomy and physiology of the human atria is required for electrophysiologists to safely perform atrial radiofrequency ablation interventions to restore sinus rhythm in patients distressed by supraventricular arrhythmias.NEW & NOTEWORTHY This report reexamines disparate historical views on internodal pathway existence and nature. The conflicting research, ongoing since 1963, regarding these issues, is appraised. The discovery of myocytes anisotropically structured in atrial bundles relaying sinoatrial node (SAN) action potentials (APs) to atrioventricular node (AVN) and atrial wall destinations is emphasized, since it is still a relatively unfamiliar subject in physiology teaching. Modern imaging technologies can now visualize internodal pathways as muscular bundles displaying SAN electrical waves traversing the atrial walls.

Remembering the canonical discoverers of the core components of the mammalian cardiac conduction system: Keith and Flack, Aschoff and Tawara, His, and Purkinje.

The mammalian cardiac conduction system (CCS) is a multifaceted continuum of electrically distinct, interconnected constructs within the myocardial mass. The key components are the sinus node (SAN), the atrioventricular node (AVN), the His bundle (HB), and the Purkinje fiber network (PF), the latter serendipitously discovered by Jan Evangelista Purkinje in 1839 in the sheep ventricle. In 1893, Wilhelm His, Jr. described a ventricular muscular tract conveying SAN-generated action potentials from the AVN (discovered by Sunao Tawara and Karl Albert Aschoff in 1906) to the PF. In 1906, Keith and Flack completed these explorations by localizing the SAN, the primum movens of CCS, which functions as a biologic oscillator emitting cadenced impulses that travel via the Bachmann bundle to the atrial myocytes and, via internodal pathways, to the AVN. Here these impulses are briefly delayed, enabling atrial systole before continuing via the AVN and the high-speed His-Purkinje conduction axis to signal ventricular contraction. The CCS canonical discoverers (Keith and Flack, Aschoff and Tawara, His, and Purkinje), historical controversies, fundamental notions of anatomy, physiology, and pathology, and therapeutic interventions pertaining to the CCS are the main themes of this review. Any scientist mentioned or unmentioned in this report who contributed directly or indirectly, with correct or inaccurate hypotheses, to the characterization of the CCS deserves our deepest gratitude for the long and painstaking hours spent microscopically scrutinizing heart specimens from multiple mammalian species, including humans.NEW & NOTEWORTHY This report presents the first comprehensive summary of the factors enabling the discovery of the cardiac conduction system (CCS). Biographical highlights and achievements of the CCS canonical discoverers, hypotheses concerning mechanisms underlying sinus node (SAN) automaticity, use of eponyms to denominate a discovery, famous controversies, possible reproachable behavior (e.g., intellectual support for eugenics in post-World War I Germany) by two of the discoverers, and examples of historical mentor-pupil relationships are discussed.

Nonclinical cardiovascular safety assessment of thioridazine: Impact of autonomic tone, body temperature, and choice of species.

Pending updates to ICH S7B/E14 guidelines will enable the substitution of human TQT studies with support from concomitant negative hERG and non-rodent CV studies. This retrospective analysis compared the effects of thioridazine (THD) (5-20 mg/kg) on heart rate (HR), blood pressure (BP), body temperature (Tc), and QT in the dog (n = 6), cynomolgus monkey (n = 4), and Goettingen minipig (n = 4) with data from previously completed studies employing crossover designs. As QT measurements are confounded by HR and Tc changes, QT effects were individually corrected for changes in HR (QTca) and Tc (QTcaT). THD-induced hemodynamic changes seen in humans were most accurately reflected in the monkey and, to a lesser extent, the dog, but not in the minipig. The minipig was most sensitive to THD QTc effects. When QTca was adjusted for THD-associated Tc decreases in minipigs and monkeys, the minipig revealed a lessened but pronounced QTcaT increase (48 ms). In the monkey, a persistent QTca increase was reduced to only a transient (0.5-3 h) QTcaT increase (20 ms). The dog's lack of THD QTca effects triggered co-administration of atenolol (AT) to attenuate THD-induced HR increases in the dog and monkey. THD + AT revealed peak QTcaT increases of 32 ms in the dog and 40 ms in the monkey, suggesting potential autonomic nervous system (ANS) interference in detecting repolarization changes. These results highlight critical species-specific differences in the outcome of parallel safety investigations. Species selection for nonclinical safety studies should consider the potential impact of Tc and ANS effects to avoid false-negative or overly positive outcomes.

19th Annual Meeting of the Safety Pharmacology Society: regulatory and safety perspectives for advanced therapy medicinal products (cellular and gene therapy products).

This report summarizes and discusses talks delivered at an educational course offered during the 2019 Annual Meeting of the Safety Pharmacology Society on advanced therapy medicinal products (ATMPs) and cell gene therapeutic products (CGTPs). ATMPs and CGTPs comprise gene and cell therapy medicinal products, tissue-engineered products, or the incorporation of one of these products into a medical device. Cited examples of ATMPs are autologous CD34+ cells encoding for the ßA-T87Q-globin gene, CAR (chimeric antigen receptor)-T cell immunotherapy medicines, genome editing products, and engineered heart muscle patches constructed from induced human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for remuscularization of the failing human heart. The nonclinical assessment of efficacy and safety of ATMPs for undertaking human clinical trials requires innovative, product-specific strategies. In order to succeed in gaining marketing approval for these novel medicines, sponsors should establish well-defined collaborative relationships with the appropriate regulatory authorities.

18th Annual Meeting of the Safety Pharmacology Society: drug safety assessment on gastrointestinal system functions.

Introduction: The 18th Annual Meeting of the Safety Pharmacology Society included a session dedicated to the assessment of drug safety on the gastrointestinal (GI) system.Areas covered: GI anatomy, physiology, adverse effects (AEs) of chemical and biological therapies, and approaches to mitigate them.Expert opinion: GI AEs, albeit common and generally of minor intensity, may prolong clinical development time and reduce patient compliance.

18th world congress of basic and clinical pharmacology: thought-provoking lectures on drug safety issues.

Introduction: Pharmacology of the Future for Science, Drug Development and Therapeutics was the leitmotif which guided the presentations at the 18th World Congress of Basic and Clinical Pharmacology held in Kyoto in July 2018 (WPC2018).Areas covered: Of the 380 invited lectures, this report reviews the opening presentation on immune checkpoint inhibitors and three talks dealing with drug safety issues (irreproducibility of nonclinical data, clinical Phase 1 catastrophes by TGN1214 and BIA-102,474-101 in healthy volunteers, and Phase I sentinel dosing to reduce risk to clinical study participants).Expert opinion: The nonclinical safety assessment of drug candidates preceding clinical investigations requires the adoption of more human predictable biological assays and a careful and critical analysis of all available knowledge on a candidate to ensure the safety of clinical trial participants.

Human organotypic bioconstructs from organ-on-chip devices for human-predictive biological insights on drug candidates.

INTRODUCTION: Historically, drug development and marketing failures have been experienced by pharma organizations largely from insufficient human-predictability of biological data. AREAS COVERED: Organs-on-chips (OOCs) are emerging, cutting edge microphysiology systems for in vitro production of microengineered three-dimensional, miniature organotypic constructs obtained by cultivating small amounts of human primary, or induced pluripotent stem, cells in native-like microhabitats. These preparations circumvent experimental limitations inherent to animal assays and two-dimensional monolayers, the mainstay core biological assays of traditional drug research. This report reviews the fundamental tenets, key components (chip plate, biomaterials, cell differentiation approaches, and monitoring sensors) and issues concerning OOC systems (engineered top-down and bottom-up strategies for tissue/organ assembly, public aids to OOC development, regulatory validation, advantages, limitations, prospective and perspective of OOCs, ethics). Examples of OOC platforms (cancer-, lung-, blood-brain barrier-, heart-, intestine-, kidney-, liver-, pharmacokinetics-, placenta and vessel-on-chip) and their importance for drug research and development are presented. EXPERT OPINION: OOC device-generated bioconstructs hold great promise as experimental human tissue and organ platforms for generating human-pertinent knowledge on drug candidates for clinical assessment and reducing reliance on animal models. MPS technologies currently enable ready-to-assemble tissue patches and, hopefully, in coming decades, full-size, patient-personalized organs for regenerative medical interventions.

Reminiscing about Jan Evangelista Purkinje: a pioneer of modern experimental physiology.

This article reminisces about the life and key scientific achievements of Jan Evangelista Purkinje (1787-1869), a versatile 19th century Czech pioneer of modern experimental physiology. In 1804, after completing senior high school, Purkinje joined the Piarist monk order, but, after a 3-yr novitiate, he gave up the religious calling "to deal more freely with science." In 1818, he earned a Medical Doctor degree from Prague University by defending a dissertation on intraocular phenomena observed in oneself. In 1823, Purkinje became a Physiology and Pathology professor at the Prussian Medical University in Breslau, where he innovated the traditional teaching methods of physiology. Purkinje's contributions to physiology were manifold: accurate descriptions of various visual phenomena (e.g., Purkinje-Sanson images, Purkinje phenomenon), discovery of the terminal network of the cardiac conduction system (Purkinje fibers), identification of cerebellar neuronal bodies (Purkinje cells), formulation of the vertigo law (Purkinje's law), discovery of criteria to classify human fingerprints, etc. In 1850, Purkinje accepted and held until his death the Physiology chair at Prague Medical Faculty. During this period, he succeeded in introducing the Czech idiom (in addition to long-established German and Latin) as a Medical Faculty teaching language. Additionally, as a zealous Czech patriot, he actively contributed to the naissance and consolidation of a national Czech identity conscience. Purkinje was a trend-setting scientist who, throughout his career, worked to pave the way for the renovation of physiology from a speculative discipline, ancilla of anatomy, into a factual, autonomous science committed to the discovery of mechanisms governing in-life functions.

15th Annual Meeting of the Safety Pharmacology Society: Focus on traditional sensory systems.

INTRODUCTION: This report summarizes and comments key talks on the five traditional senses (ear, vestibular system, vision, taste, olfaction, and touch) which were delivered during the 2015 Annual Meeting of the Safety Pharmacology (SP) Society. AREAS COVERED: The functional observational battery (FOB) can detect major candidate drug liabilities only on ear, touch and vision. Anatomy, physiology, pharmacology, and pathology notions on each sensory system introduce speaker talks. Techniques for evaluating drug effects on hearing functions are reviewed. Nonclinical approaches to assess vestibular toxicity leading to balance deficits are presented. Retinal explants studied with multielectrode arrays allow the identification of drug liability sites on the retina. Routinely performed Safety Pharmacology assays are not powered to address candidate drug-induced disturbances on taste and smell. This weakness needs correction since unintended pharmacological impairment of these sensorial functions may have serious health consequences. Neuropathy produced by chemotherapeutic agents may cause multiple sensorial perception distortions. CONCLUSIONS: Safety Pharmacology studies should ensure the safety of any candidate drug on the five sensorial systems.

Comprehensive in vitro Proarrhythmia Assay (CiPA): Pending issues for successful validation and implementation.

INTRODUCTION: The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a nonclinical Safety Pharmacology paradigm for discovering electrophysiological mechanisms that are likely to confer proarrhythmic liability to drug candidates intended for human use. TOPICS COVERED: Key talks delivered at the 'CiPA on my mind' session, held during the 2015 Annual Meeting of the Safety Pharmacology Society (SPS), are summarized. Issues and potential solutions relating to crucial constituents [e.g., biological materials (ion channels and pluripotent stem cell-derived cardiomyocytes), study platforms, drug solutions, and data analysis] of CiPA core assays are critically examined. DISCUSSION: In order to advance the CiPA paradigm from the current testing and validation stages to a research and regulatory drug development strategy, systematic guidance by CiPA stakeholders is necessary to expedite solutions to pending and newly arising issues. Once a study protocol is proved to yield robust and reproducible results within and across laboratories, it can be implemented as qualified regulatory procedure.

The prospective IQ-CSRC trial: A prototype early clinical proarrhythmia assessment investigation for replacing the ICH E14 thorough QTc (TQT) study.

INTRODUCTION: Early clinical Phase I ECG investigations designed to replace the currently applied thorough QT (TQT) study are reviewed to examine how they could complement and verify the conclusions of nonclinical investigations and, in particular, the Comprehensive in vitro Proarrhythmia Assay (CiPA). TOPICS: The IQ-CSRC trial is a prospective ascending multiple-dose first in human (FIH) type investigation performed as a possible replacement for the thorough QT study (TQT). Designed in accordance with the results of a simulation study by the FDA QT Interdisciplinary Review Team (IRT), it succeeded in correctly categorizing 5/5 established QTc-prolonging agents free of notable heart rate effects (dofetilide, dolasetron, moxifloxacin, ondansetron, and quinine) and the QTc-negative drug, levocetirizine. DISCUSSION: The positive results obtained with the IQ-CSRC study require additional confirmation with threshold QTc-positive and negative drugs and established QTc prolongers producing both increases and decreases in heart rate. In the future, similar studies should also adopt and validate innovative proarrhythmic metrics, in addition to, or instead of, the traditional proarrhythmic surrogate of QTc, to assess the proarrhythmic safety of candidate drugs.

14th Annual Meeting of the Safety Pharmacology Society: Threading through peripheral and central nervous system presentations.

The 2014 Annual Meeting of the Safety Pharmacology Society discussed pathophysiological mechanisms and novel investigational approaches to assess drug safety. The plenary keynote reviewed past, present, and future research on Alzheimer's disease. Polysomnography tools can uncover drug-induced sleep disturbances. FDA examiners currently assess proconvulsive liabilities on a case-by-case basis due to the lack of official guidance. In contrast, abuse liability potential is determined according to established paradigms. The FDA guideline on opioid deterrent formulations was discussed. The mechanisms and treatments of obstructive sleep apnea (OSA) and diabetes-induced neuropathic pain were reviewed. There were salient points arising from the CNS presentations but from a pharmacological point of view we note in particular that safety pharmacology should move to routinely apply polysomnographic technologies to determine whether candidate drugs exert deleterious effects on sleep quality and architecture that may markedly decrease quality of life and impair cognitive functions, including alertness and reaction time.

CiPA: Ongoing testing, future qualification procedures, and pending issues.

INTRODUCTION: The comprehensive in vitro proarrhythmia assay (CiPA) is a nonclinical, mechanism-based paradigm for assessing drug proarrhythmic liability. TOPICS COVERED: The first CiPA assay determines effects on cloned human cardiac ion channels. The second investigates whether the latter study-generated metrics engender proarrhythmic markers on a computationally reconstructed human ventricular action potential. The third evaluates conclusions from, and searches possibly missed effects by in silico analysis, in human stem cell-derived cardiomyocytes (hSC-CMs). CiPA ad hoc Expert-Working Groups have proposed patch clamp protocols for seven cardiac ion channels, a modified O'Hara-Rudy model for in silico analysis, detailed procedures for field (MEA) and action potential (VSD) measurements in hSC-CMs, and 29 reference drugs for CiPA assay testing and validation. DISCUSSION: CiPA adoption as drug development tool for identifying electrophysiological mechanisms conferring proarrhythmic liability to candidate drugs is a complex, multi-functional task requiring significant time, reflection, and efforts to be fully achieved.

Telemetric assessment of social and single housing: Evaluation of electrocardiographic intervals in jacketed cynomolgus monkeys.

INTRODUCTION: Proactive efforts to socially house laboratory animals are a contemporary, important focus for enhancing animal welfare. Jacketing cynomolgus monkeys has been traditionally considered an exclusionary criterion for social housing based on unsubstantiated concerns that study conduct or telemetry equipment might be compromised. Our objective was to evaluate the effects of jacketing naïve, adolescent cynomolgus monkeys in different single and social housing types based on parallel comparisons of heart rate. METHODS: Eight naive cynomolgus monkeys were randomized into pairs and ECG data were collected for 24h from each animal in each housing condition using a crossover design. Caging paradigms consisted of standard individual, standard pair, quaternary pair (4 linked cages), and European-style pair housing in non-sequential order varied by pair to control for possible time bias. Dosing and blood collection procedures were performed to characterize any effects of housing on ECG data during study conduct. RESULTS: There was no increase in the incidence of equipment damage in pair vs. individually housed animals. Further, animals in all 4 housing paradigms showed similar acclimation assessed as heart rate (mean 139-154 beats per minute), and maintained similar diurnal rhythms, with an expected slowing of the heart rate at night (aggregate lights out HR 110±4bpm compared to daytime 146±7bpm). DISCUSSION: This study demonstrates the effects of different social access and housing types on the study-naïve cynomolgus monkeys during jacketed cardiovascular telemetry data collection in a repeat-dose toxicology study design. There were no discernible effects of social housing on baseline ECG parameters collected via jacketed telemetry, and all animals maintained expected diurnal rhythms in all housing settings tested. These data demonstrate that cynomolgus monkeys can be socially housed during data collection as a standard practice, consistent with global efforts to improve study animal welfare.

Comprehensive in vitro Proarrhythmia Assay, a novel in vitro/in silico paradigm to detect ventricular proarrhythmic liability: a visionary 21st century initiative.

INTRODUCTION: The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a novel safety screening proposal intended to replace the 2005 regulatory strategy recommended by the International Conference of Harmonization S7B guideline. AREAS COVERED: CiPA consists of three components. The first assay evaluates candidate drug effects on key cardiac ion channels. Then, simulations test whether the channel dataset yields proarrhythmic markers on a computationally reconstructed human ventricular cardiomyocyte action potential. Finally, the relevance of in silico conclusions is verified by determining the electrical activity of human stem cell-derived ventricular cardiomyocytes. EXPERT OPINION: The CiPA initiative is intended to move safety pharmacology from a predominantly traditional pharmacodynamics approach to in silico and in vitro drug toxicity assessment. In practice, CiPA assays will have to be compliant with regulatory safety pharmacology tenets. The latter will necessitate international consensus on assay protocols, method standardization and validation and, thus, is likely to involve protracted discussions to achieve agreement. As such, full CiPA implementation by July 2015, as currently envisaged, to supplant E14 guidance for a thorough QT/QTc interval study as prerequisite for noncardiac drug marketing approval, appears to be difficult. Nevertheless, safety stakeholders should do their best to validate and implement the CiPA initiative in the shortest possible time.

Preclinical QT safety assessment: cross-species comparisons and human translation from an industry consortium.

INTRODUCTION: In vivo models have been required to demonstrate relative cardiac safety, but model sensitivity has not been systematically investigated. Cross-species and human translation of repolarization delay, assessed as QT/QTc prolongation, has not been compared employing common methodologies across multiple species and sites. Therefore, the accurate translation of repolarization results within and between preclinical species, and to man, remains problematic. METHODS: Six pharmaceutical companies entered into an informal consortium designed to collect high-resolution telemetered data in multiple species (dog; n=34, cynomolgus; n=37, minipig; n=12, marmoset; n=14, guinea pig; n=5, and man; n=57). All animals received vehicle and varying doses of moxifloxacin (3-100 mg/kg, p.o.) with telemetered ECGs (≥500 Hz) obtained for 20-24h post-dose. Individual probabilistic QT-RR relationships were derived for each subject. The rate-correction efficacies of the individual (QTca) and generic correction formulae (Bazett, Fridericia, and Van de Water) were objectively assessed as the mean squared slopes of the QTc-RR relationships. Normalized moxifloxacin QTca responses (Veh Δ%/µM) were derived for 1h centered on the moxifloxacin Tmax. RESULTS: All QT-RR ranges demonstrated probabilistic uncertainty; slopes varied distinctly by species where dog and human exhibited the lowest QT rate-dependence, which was much steeper in the cynomolgus and guinea pig. Incorporating probabilistic uncertainty, the normalized QTca-moxifloxacin responses were similarly conserved across all species, including man. DISCUSSION: The current results provide the first unambiguous evidence that all preclinical in vivo repolarization assays, when accurately modeled and evaluated, yield results that are consistent with the conservation of moxifloxacin-induced QT prolongation across all common preclinical species. Furthermore, these outcomes are directly transferable across all species including man. The consortium results indicate that the implementation of standardized QTc data presentation, QTc reference cycle lengths, and rate-correction coefficients can markedly improve the concordance of preclinical and clinical outcomes in most preclinical species.

Comparative TQT analysis with three fully-automated platforms: comparison to core laboratory semi-automated results.

BACKGROUND AND PURPOSE: Technological advances in machine-read QT measurement now enable detailed and precise cardiac repolarization assessments. This study assessed the applicability of three state-of-art ECG measurement applications to provide reliable continuous analyses from data obtained in a positive thorough QT study previously characterized with sparse semi-automated measurements performed by an ECG core laboratory. METHODS: Continuous RR, QT, QTc measurements, and individual QT/RR relationships and their associated intra- and inter-subject variability were derived in parallel with BioQT, Ponemah PRO, and WinAtrec analysis software. RESULTS: Despite slight vendor-specific differences in measurement variability and QTc, all machine-read methods demonstrated requisite assay sensitivity and yielded similar conclusions in accordance with SA analysis. CONCLUSIONS: Three commercially available ECG analytical software applications reliably detected the drug-induced QT prolonging effects and replicated the SA core-laboratory conclusions, with greatly improved temporal resolution and reduced analytical costs. With broader experience, these data suggest that current SA methodologies could be effectively replaced by fully automated ECG analysis.

Pharmaceutical toxicology: designing studies to reduce animal use, while maximizing human translation.

Evaluation of the safety of new chemicals and pharmaceuticals requires the combination of information from various sources (e.g. in vitro, in silico and in vivo) to provide an assessment of risk to human health and the environment. The authors have identified opportunities to maximize the predictivity of this information to humans while reducing animal use in four key areas; (i) accelerating the uptake of in vitro methods; (ii) incorporating the latest science into safety pharmacology assessments; (iii) optimizing rodent study design in biological development and (iv) consolidating approaches in developmental and reproductive toxicology. Through providing a forum for open discussion of novel proposals, reviewing current research and obtaining expert opinion in each of the four areas, the authors have developed recommendations on good practice and future strategy.

Pattern recognition analysis of digital ECGs: decreased QT measurement error and improved precision compared to semi-automated methods.

BACKGROUND AND PURPOSE: Machine-read QT measurements employing T-wave detection algorithms (ALG) are not accepted by regulatory agencies for the primary analysis of thorough QT (TQT) studies. Newly developed pattern recognition software (PRO) which matches ECG waveforms to user-defined templates may improve this situation. METHODS: We compared RR, QT, QTc, QT variability, T-end measurement errors, and individual QT rate correction factors and their associated coefficients of determination (R(2)) following ALG and PRO analysis. Machine-read QTc values were compared with core laboratory semi-automated (SA) values for verification. RESULTS: Compared to ALG, PRO reduced the frequency of T-end measurement errors (5.6% vs. 0.1%), reduced the intra-individual QT variability (12.6±5.9 vs. 4.9±1.1ms) and allowed the recovery of 3/58 subjects that exhibited an unacceptable (<0.9) R(2). CONCLUSIONS: PRO adjusted for ALG-based T-end measurement errors and provided an accurate and precise automated method for continuous QT analysis, thus offering an alternative to resource-intensive semi-automated analyses currently performed by ECG core laboratories.

Characterization of the human QT interval: novel distribution-based assessment of the repolarization effects of moxifloxacin.

The authors have previously demonstrated rate-independent QT variability in the dog and cynomolgus monkey, where the QT associated with any RR was a normally distributed value that was accurately evaluated as the distribution mean. The present study investigated the rate-independent characteristics of the human QT. Digital electrocardiographs (1000 Hz) were collected for 24 hours in 51 patients (thorough QT study) and analyzed by computer. Distribution-based analysis was applied to the placebo and moxifloxacin (400 mg) arms to characterize the nature of the QT interval and to assess the efficacy of distribution-based analysis for QTc determination. Novel statistics using continuous means and bootstrapped 95% confidence intervals were developed to facilitate QT analysis. Machine-read QT values were compared with core laboratory semiautomated values for verification. RR intervals demonstrated repetitive protocol-dependent variations (50-250 milliseconds); QT intervals were normally distributed, spanning 60 to 100 milliseconds for each RR interval. Distribution-based analysis detected a moxifloxacin response identical to semiautomated analysis, but with reduced variability and improved statistical power, where n = 12 satisfied the ICH E14 criteria for a positive control. Distribution-based analysis has the potential to provide a universal method for clinical QT heart rate correction, enabling accurate detection of QT changes when limited numbers of volunteers are exposed to drug.