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.

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.

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.

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.

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 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.

Analysis of the nonclinical telemetered ECG: impact of logging rate and RR bin width in the dog and cynomolgus monkey.

INTRODUCTION: Species-dependent ECG differences may affect QT interval analysis. Among these are the range of QT and RR values, heart rate variability, and respiratory sinus arrhythmia (SA). Importantly, the effects of data logging rates and RR bin width (BW) on QT analysis have not been characterized in dogs and nonhuman primates. METHODS: Digital ECGs were collected for 18-21 h in telemetered dogs (n=7) and cynomolgus monkeys (n=7) employing epicardial ECG leads and analyzed by computerized algorithms. ECG intervals were determined employing beat-to-beat (1 ms BW) and 10 s logging rates (for 1, 10, 20, and 50 ms BW). Diurnal heart rate variability was defined as the beat-to-beat RR ratio (VR) where SA was defined as >+/-10% change in VR. RESULTS: Canine beat-to-beat QT-RR data were curvilinear and plateaued, with multiple RR values associated to any given QT for RR>or=900 ms. Cynomolgus QT-RR data collected as beat-to-beat or at a 10 s logging rate were linear for all RR intervals. During the day, SA comprised 62.9+/-2.4% and 11.4+/-6.1% of total beats in the dog and cynomolgus, respectively, with no diurnal/nocturnal differences in either species. QT interval changes varied with BW such that 5 ms resolution was maintained for BW

Application of a probabilistic method for the determination of drug-induced QT prolongation in telemetered cynomolgus monkeys: effects of moxifloxacin.

INTRODUCTION: Moxifloxacin is the most widely used positive reference agent in clinical cardiac repolarization safety studies, but it has not been characterized in the cynomolgus monkey. This important experimental animal species exhibits pronounced heart rate variability, complicating the temporal evaluation of QT interval data. METHODS: Digitized epicardial ECGs and aortic blood pressures were collected for 20 h in telemetered cynomolgus monkeys (n=6) following the administration of either vehicle or moxifloxacin (10 or 50 mg/kg, p.o.). Moxifloxacin plasma concentrations were determined 4 h postdose. ECG intervals were analyzed by computerized algorithms. Individual probabilistic QT rate-corrections (QTc) were derived from the slopes of predose log-transformed QT-RR data where each QT value was the mean of >250 beats/RR increment. The resulting QTc was used to determine the repolarization effects of moxifloxacin, expressed as the placebo-adjusted change in QTc (DeltaQTc), and as the integrated response from 0 to 12 h (AUC(0-->12)) postdose. RESULTS: No DeltaQTc effect was produced by 10 mg/kg moxifloxacin. However, moxifloxacin (50 mg/kg; 5.86+/-0.5 microg/mL C(max)) significantly prolonged the RR interval by 50 to 112 ms from 3.5 to 7.5 h postdose and DeltaQTc by >or=7.2 ms from 1.83 to 9.17 h, with a maximal DeltaQTc effect of +26.4 ms. No notable effects on either systemic blood pressure or body temperature occurred with either dose. DISCUSSION: Probabilistic QT rate-corrections appear to have eliminated the confounding effects of heart rate, provided for a stable QTc baseline, and enabled the demonstration of an exposure-dependent QTc prolongation by moxifloxacin. The duration and magnitude of the QTc effect paralleled moxifloxacin pharmacokinetics, and C(max) values were similar to those achieved clinically in thorough QT/QTc studies. Thus, novel probabilistic QT rate-corrections may offer highly robust assessments of repolarization risk in both nonclinical and clinical investigations.

Novel probabilistic method for precisely correcting the QT interval for heart rate in telemetered dogs and cynomolgus monkeys.

INTRODUCTION: QT intervals are not regulated on a beat-to-beat cadence, but are strongly influenced by the preceding heart rate history (hysteresis). ECG sampling, when performed over sufficiently long periods, results in the detection of ranges of different QT values for each discrete RR interval. Given the potential impact of QT hysteresis in QT interval rate-correction procedures, we hypothesized that, physiologically, the QT interval exists as a probabilistic variable where the exact value corresponding to any RR interval is precisely estimated from the associated QT population. METHODS: Digital ECGs were collected for 18-21 h in telemetered dogs (n=7) and cynomolgus monkeys (n=7) employing epicardial ECG leads for accurate T(end) detection, and analyzed by computerized algorithms. Descriptive statistics were calculated for raw QT values in 10 ms RR increments. Individual rate-corrected QT (QTc) formulae were derived from the slopes of log-transformed QT-RR data where each QT point was the mean of >250 beats/RR increment. The aptness of this QTc model was assessed by residual analysis. RESULTS: Beat-to-beat ECG analysis demonstrated that for all discrete cycle lengths, the associated raw QT intervals were normally distributed populations, spanning approximately 30-40 and 45-100 ms in the dog and cynomolgus monkey, respectively. In both species, QTc was stable (< or =5 ms variation) over all physiological RR intervals. DISCUSSION: The probabilistic treatment of raw QT interval populations natively associated to any RR interval provides hysteresis-free raw QT estimates which can be accurately modeled, allowing the derivation of a precise QTc value. Previous unawareness of the probabilistic nature of the QT interval explains the historical failure of numerous QT rate-correction formulae to correctly solve this scientific issue. Importantly, QT distribution analysis has the potential to provide, for the first time, a universal and sensitive method for QT heart rate-correction, providing a robust method for nonclinical and clinical cardiac safety investigations of repolarization delay.

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.

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.

Comparison of telemetry and high-definition oscillometry for blood pressure measurements in conscious dogs: effects of torcetrapib.

This study compared torcetrapib-induced blood pressure (BP) changes simultaneously obtained by high-definition oscillometry (HDO) and telemetry. Male beagles (n = 6) received single oral doses of vehicle or torcetrapib at 10 or 30 mg/kg; BP were acquired simultaneously by HDO and telemetry from 2 h before dosage until 7 h afterward. Systolic, diastolic, and mean arterial pressures (MAP) and heart rate were compared by using Altman-Bland agreement analysis. Dogs were allocated into subgroups according to temperament and baseline MAP (less than 110 mm Hg and 110 mm Hg or greater). Both methods demonstrated high precision. HDO recordings exhibited higher variability for all parameters (inclusive MAP SDs were 7.0 +/- 2.7 mm Hg for HDO compared with 3.4 +/- 1.9 mm Hg for telemetry), accompanied by a positive bias for all pressures (systolic, 10.4 mm Hg; diastolic, 5.7 mm Hg; MAP, 1.9 mm Hg). Both methods detected similar maximal increases in MAP with 30 mg/kg torcetrapib (HDO, 15.8 +/- 10.4 mm Hg; telemetry, 15.8 +/- 5.3 mm Hg). No significant effects were noted for heart rate. Torcetrapib elicited a dose-dependent increase in BP in dogs with baseline MAP of less than 110 mm Hg, whereas increases were maximal with 10 mg/kg in the other group, and dose-dependence was no longer observed. BP changes were influenced by animal temperament, demonstrating that HDO results must be interpreted with caution. HDO may provide a useful and accurate method for noninvasive BP measurements in canine studies.

Application of probabilistic analysis for precisely correcting the QT interval for heart rate in telemetered common marmosets.

INTRODUCTION: QT intervals are strongly influenced by preceeding heart rate history and are also characterized by rate-independent variability, leading to difficulty in precise rate-correction of the raw QT interval. The present study elucidates a novel analytical method that effectively addresses this problematic phenomenon in telemetered common marmosets. METHODS: ECGs were collected from telemetered common marmosets (male and female) and analyzed by computerized algorithms. Descriptive statistics were calculated from the mean of QT intervals for 5-ms increments of RR. The QT interval was corrected for the RR interval by applying Bazett's, Fridericia's, and individual probabilistic QT rate-correction formulae. RESULTS: The linear regression of log-transformed QT and RR intervals derived from a probabilistic approach yielded a well-correlated QT-RR fit. Assessed as the slope of the QTc-RR interval, application of individual probabilistic QT rate-corrections resulted in the most effective dissociation of the effects of rate from the raw QT interval, compared to generic rate-correction formulae. Using individual corrections, the QTc was stable while the interquartile range (IQR) of the QTc distribution was stable, spanning 5-10 ms for each subject over all physiological RR intervals. Heart rate variability distributions were centered about unity during both photoperiods and sinus arrhythmia was far less pronounced compared with measurements in dogs. DISCUSSION: Probabilistic QT rate-correction eliminated the confounding effects of heart rate and provided a stable QTc baseline. These results indicate that application of this method of analysis in telemetered common marmosets results in a high degree of sensitivity for the consistent detection of small (5-10 ms) changes in the QTc interval.

Sensitivity of common marmosets to detect drug-induced QT interval prolongation: moxifloxacin case study.

INTRODUCTION: Moxifloxacin is the most widely used positive reference agent in clinical cardiac repolarization studies, but it has not been characterized in common marmosets which are uniquely suited to studies in early-stage development due to their small size and minimal test article requirements. The purpose of this study was to evaluate the sensitivity of the common marmoset to detect moxifloxacin-associated QT interval prolongation. METHODS: Eight telemetered common marmosets were monitored for 24 h following oral administration of moxifloxacin by gavage at 0, 10, 30, and 100 mg/kg using a Latin square design. Concurrently, a pharmacokinetic evaluation in 8 non-telemetered animals was conducted. A rate-corrected QT (QTc) interval was derived using an individual probabilistic QT rate-correction. QTc (placebo-adjusted QTc change from the individual baseline) was calculated and the relationship between pharmacokinetics (PK) and pharmacodynamics (PD) was analyzed. RESULTS: A slight, but not significant, increase in QTc was detected with 10 mg/kg of moxifloxacin. Moxifloxacin at 30 and 100 mg/kg elicited dose-dependent increases in QTc of 14.0+/-3.6 and 35.0+/-6.2 ms, respectively, with associated total moxifloxacin C(max) values of 6.5+/-0.5 and 16.5+/-1.6 microg/mL, respectively. From the PK/PD relationship, the plasma concentration which would attain QTc of 5 to 10 ms was estimated to be 1.67-3.73 microg/mL. The results were consistent with typical clinical trial results (QTc of 6.6-14.8 ms at 2.5-3.5 microg/mL). CONCLUSIONS: The present study demonstrates that the common marmoset is highly sensitive to moxifloxacin-associated changes in cardiac repolarization, assessed as QTc. As such, this species is suitable for precise and reliable detection of small, but significant, drug-associated increases in QTc interval. Thus, the common marmoset should be regarded as a validated animal model for the detection of QT risk in early-stage drug development and represents an important addition to the current in vivo armamentarium.

Vasopeptidase inhibition in a canine model of exercise-induced left ventricular dysfunction.

1. The present study compared the acute efficacies of vasopeptidase inhibition with omapatrilat, nitroglycerin and angiotensin-converting enzyme (ACE) inhibition in exercise-induced myocardial dysfunction. Omapatrilat, a vasopeptidase inhibitor, inhibits both neutral endopeptidase and ACE. Whereas vasopeptidase inhibitors have demonstrated clinical efficacy in hypertension and heart failure, their effects in myocardial ischaemia remain unclear. 2. Omapatrilat (0.3 mg/kg) was compared with vehicle (saline), an ACE inhibitor (fosinoprilat; 0.44 mg/kg) and nitroglycerin (8.0 microg/kg per min), in an established canine model of exercise-induced myocardial dysfunction induced by progressive closure of an ameroid constrictor placed about the proximal circumflex coronary artery. Maximal treadmill exercise tests, terminated when heart rate failed to increase with increasing workload or failure to continue exercise, were performed in chronically instrumented dogs. 3. During exercise, omapatrilat and nitroglycerin similarly increased ischaemic wall thickening (P< or = 0.0001, ANOVA, 12 d.f.), whereas fosinoprilat and vehicle were without effect. Ischaemic zone ST changes were decreased with nitroglycerin (P = 0.0006, ANOVA, 12 d.f.) and tended to decrease with omapatrilat (P = 0.07, ANOVA, 12 d.f.). Peak exercise capacity was increased with nitroglycerin (9.7 +/- 1.1 vs 11.2 +/- 1.0 kcal, control vs 4 h, respectively; n = 6) and omapatrilat (9.7 +/- 0.8 vs 11.4 +/- 0.6 kcal, control vs 4 h, respectively; n = 6) and was unchanged with ACE inhibition (9.0 +/- 1.2 vs 9.5 +/- 1.1 kcal, control vs 4 h, respectively; n = 7). Omapatrilat differentially increased double product during exercise (P = 0.001, ANOVA, 12 d.f.) compared with other treatments. 4. During exercise-induced myocardial dysfunction, acute ACE inhibition did not attenuate ischaemic changes and failed to improve exercise capacity. Increased exercise capacity with omapatrilat was accompanied by a differential increase in double product, consistent with increased oxygen supply and demand. Improvements in ischaemic function were comparable between omapatrilat and nitroglycerin, suggesting that omapatrilat may represent a novel therapy in demand-induced ischaemia.