The Role of the Airway and Gut Microbiome in the Development of Chronic Lung Disease of Prematurity.

Chronic lung disease (CLD) of prematurity, a common cause of morbidity and mortality in preterm-born infants, has a multifactorial aetiology. This review summarizes the current evidence for the effect of the gut and airway microbiota on the development of CLD, highlighting the differences in the early colonisation patterns in preterm-born infants compared to term-born infants. Stool samples from preterm-born infants who develop CLD have less diversity than those who do not develop CLD. Pulmonary inflammation, which is a hallmark in the development of CLD, may potentially be influenced by gut bacteria. The respiratory microbiota is less abundant than the stool microbiota in preterm-born infants. There is a lack of clear evidence for the role of the respiratory microbiota in the development of CLD, with results from individual studies not replicated. A common finding is the presence of a single predominant bacterial genus in the lungs of preterm-born infants who develop CLD. Probiotic preparations have been proposed as a potential therapeutic strategy to modify the gut or lung microbiota with the aim of reducing rates of CLD but additional robust evidence is required before this treatment is introduced into routine clinical practice.

Acetabular fracture pattern is altered by pre-injury sacroiliac joint autofusion.

PURPOSE: Acetabular fracture shape is determined by the direction of force applied. We perceive an anecdotally observed connection between pre-existing autofused sacroiliac joints (aSIJ) and high anterior column (HAC) injuries. The purpose of this study was to compare variations in acetabular fracture patterns sustained in patients with and without pre-injury sacroiliac (SI) joint autofusion. METHODS: All adult patients receiving unilateral acetabular fixation (level 1 academic trauma; 2008-2018) were reviewed. Injury radiographs and CT scans were reviewed for fracture patterns and pre-existing aSIJ. Fracture types were subgrouped presence of HAC injury (includes anterior column (AC), anterior column posterior hemitransverse (ACPHT), or associated both column (ABC)). ANALYSIS: Logistic regression determined the association between aSIJ and HAC. RESULTS: A total of 371 patients received unilateral acetabular fixation (2008-2018); 61 (16%) demonstrated CT evidence of idiopathic aSIJ. These patients were older (64.1 vs. 47.4, p < 0.01), more likely to be male (95% vs. 71%, p < 0.01), less likely to be smokers (19.0% vs. 44.8%, p < 0.01), and were injured from lower energy mechanisms (21.3% vs. 8.4%, p = 0.01). The most common patterns with autofusion were ACPHT (n = 13, 21%) and ABC (n = 25, 41%). Autofusion was associated with greater odds of patterns involving a high anterior column injury (ABC, ACPHT, or isolated anterior column; OR = 4.97, p < 0.01). After adjusting for age, mechanism, and body mass index, the connection between autofusion and high anterior column injuries remained significant (OR = 2.60, p = 0.01). CONCLUSIONS: SI joint autofusion appears to change mode of failure in acetabular injuries; a more rigid posterior ring may precipitate a high anterior column injury. LEVEL OF EVIDENCE: Prognostic level III.

Radiographic bladder shift is a harbinger of intraoperative blood loss in acetabular surgical fixation.

PURPOSE: The purpose of this study was to characterize the relationship between a novel radiographic measurement on initial AP pelvis radiograph (termed "bladder shift," BS) to intraoperative blood loss (IBL) during acetabular surgical fixation. METHODS: All adult patients receiving unilateral acetabular fixation (Level 1 academic trauma; 2008-18) were reviewed. AP pelvis radiographs were reviewed for visible bladder outlines and then measured to determine the percentage deformation toward the midline. Hemoglobin & hematocrit data were then used to calculate quantitative blood loss between pre- and post- operative blood counts for data analysis. RESULTS: 371 patients with unilateral traumatic acetabular fractures requiring fixation were reviewed; 99 of these had visible bladder outlines, complete blood count and transfusion data (2008-2018; 66% associated patterns). Median bladder shift (BS) was 13.3%. Every 10% of bladder shift was associated with 123 mL greater IBL. Patients with full bladder shift to midline sustained a median 1.5L IBL (interquartile range [IQR] 0.8 to 1.6). Associated patterns had a threefold greater median BS (associated: 16.5% [15.4 to 45.9] vs. elementary: 5.6% [1.1 to 15.4], p < 0.05) and received intraoperative pRBC twice as frequently (57% vs. 24%, p < 0.01). CONCLUSIONS: Radiographic bladder shift is an easily available visual marker, in patients sustaining acetabular fractures, that may predict intraoperative hemorrhage and need for transfusions.

Methamphetamines and Acetabular Reoperation Rates: Poor Outcomes From the Front Lines.

OBJECTIVES: To compare acetabular fracture reoperation rates within 1 year of surgery in methamphetamine ("meth") abusers and abstainers. DESIGN: Retrospective database analysis. SETTING: Level 1 academic trauma facility, 2008-2018. PATIENTS/PARTICIPANTS: Three hundred seventy-one patients who underwent unilateral traumatic acetabular open reduction internal fixation during the study period, 36 of whom abused methamphetamines through self-report or toxicology. One hundred four were excluded for indeterminate abuse histories. INTERVENTION: Open reduction internal fixation. MAIN OUTCOME MEASUREMENTS: Reoperation resulting from major surgical complications, including hematoma, seroma, deep wound infection, failure of fixation, or arthrosis with conversion to arthroplasty. RESULTS: More than 10% of our cohort used meth, representing patients who were a mean 8 years younger and sustained a higher rate of high-energy mechanisms than sober peers. Meth abusers had a greater than 2-fold reoperation rate at 90 days and 1 year compared with abstainers (17% vs. 7% and 25% vs. 11%, respectively). The adjusted odds ratio of 1-year reoperation in meth users was 3.2 (confidence interval 1.2-8.5, P = 0.03). The adjusted 1-year survival of native hip after acetabular fractures in meth users approaches 55%. CONCLUSIONS: Methamphetamine use is a nonmodifiable factor associated with a 3-fold increase in adjusted odds for 1-year reoperation after surgical fixation of acetabular fractures. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Dissimilarity of the gut-lung axis and dysbiosis of the lower airways in ventilated preterm infants.

BACKGROUND: Chronic lung disease of prematurity (CLD), also called bronchopulmonary dysplasia, is a major consequence of preterm birth, but the role of the microbiome in its development remains unclear. Therefore, we assessed the progression of the bacterial community in ventilated preterm infants over time in the upper and lower airways, and assessed the gut-lung axis by comparing bacterial communities in the upper and lower airways with stool findings. Finally, we assessed whether the bacterial communities were associated with lung inflammation to suggest dysbiosis. METHODS: We serially sampled multiple anatomical sites including the upper airway (nasopharyngeal aspirates), lower airways (tracheal aspirate fluid and bronchoalveolar lavage fluid) and the gut (stool) of ventilated preterm-born infants. Bacterial DNA load was measured in all samples and sequenced using the V3-V4 region of the 16S rRNA gene. RESULTS: From 1102 (539 nasopharyngeal aspirates, 276 tracheal aspirate fluid, 89 bronchoalveolar lavage, 198 stool) samples from 55 preterm infants, 352 (32%) amplified suitably for 16S RNA gene sequencing. Bacterial load was low at birth and quickly increased with time, but was associated with predominant operational taxonomic units (OTUs) in all sample types. There was dissimilarity in bacterial communities between the upper and lower airways and the gut, with a separate dysbiotic inflammatory process occurring in the lower airways of infants. Individual OTUs were associated with increased inflammatory markers. CONCLUSIONS: Taken together, these findings suggest that targeted treatment of the predominant organisms, including those not routinely treated, such as Ureaplasma spp., may decrease the development of CLD in preterm-born infants.

Monitoring reperfused myocardial infarction with delayed left ventricular systolic dysfunction in rabbits by longitudinal imaging.

BACKGROUND: An experimental imaging platform for longitudinal monitoring and evaluation of cardiac morphology-function changes has been long desired. We sought to establish such a platform by using a rabbit model of reperfused myocardial infarction (MI) that develops chronic left ventricle systolic dysfunction (LVSD) within 7 weeks. METHODS: Fifty-five New Zeeland white (NZW) rabbits received sham-operated or 60-min left circumflex coronary artery (LCx) ligation followed by reperfusion. Cardiac magnetic resonance imaging (cMRI), transthoracic echocardiography (echo), and blood samples were collected at baseline, in acute (48 hours or 1 week) and chronic (7 weeks) stage subsequent to MI for in vivo assessment of infarct size, cardiac morphology, LV function, and myocardial enzymes. Seven weeks post MI, animals were sacrificed and heart tissues were processed for histopathological staining. RESULTS: The success rate of surgical operation was 87.27%. The animal mortality rates were 12.7% and 3.6% both in acute and chronic stage separately. Serum levels of the myocardial enzyme cardiac Troponin T (cTnT) were significantly increased in MI rabbits as compared with sham animals after 4 hours of operation (P<0.05). According to cardiac morphology and function changes, 4 groups could be distinguished: sham rabbits (n=12), and MI rabbits with no (MI_NO_LVSD; n=10), moderate (MI_M_LVSD; n=9) and severe (MI_S_LVSD; n=15) LVSD. No significant differences in cardiac function or wall thickening between sham and MI_NO_LVSD rabbits were observed at both stages using both cMRI and echo methods. cMRI data showed that MI_M_LVSD rabbits exhibited a reduction of ejection fraction (EF) and an increase in end-systolic volume (ESV) at the acute phase, while at the chronic stage these parameters did not change further. Moreover, in MI_S_LVSD animals, these observations were more striking at the acute stage followed by a further decline in EF and increase in ESV at the chronic stage. Lateral wall thickening determined by cMRI was significantly decreased in MI_M_LVSD versus MI_NO_LVSD animals at both stages (P<0.05). As for MI_S_LVSD versus MI_M_LVSD rabbits, the thickening of anterior, inferior and lateral walls was significantly more decreased at both stages (P<0.05). Echo confirmed the findings of cMRI. Furthermore, these in vivo outcomes including those from vivid cine cMRI could be supported by exactly matched ex vivo histomorphological evidences. CONCLUSIONS: Our findings indicate that chronic LVSD developed over time after surgery-induced MI in rabbits can be longitudinally evaluated using non-invasive imaging techniques and confirmed by the entire-heart-slice histomorphology. This experimental LVSD platform in rabbits may interest researchers in the field of experimental cardiology and help strengthen drug development and translational research for the management of cardiovascular diseases.

Evaluation of cardiac arrhythmic risks using a rabbit model of left ventricular systolic dysfunction.

Patients with heart disease have a higher risk to develop cardiac arrhythmias, either spontaneously or drug-induced. In this study, we have used a rabbit model of myocardial infarction (MI) with severe left ventricular systolic dysfunction (LVSD) to study potential drug-induced cardiac risks with N-(piperidin-2-ylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide (flecainide). Upon ligation of the left circumflex arteries, male New Zealand White rabbits developed a large MI and moderate or severe LVSD 7 weeks after surgery, in comparison to SHAM-operated animals. Subsequently, animals were exposed to escalating doses of flecainide (0.25-4 mg/kg) or solvent. Electrocardiograms (ECG) were recorded before surgery, 1 and 7 weeks after surgery and continuously during the drug protocol. The ECG biomarker iCEB (index of Cardio-Electrophysiological Balance = QT/QRS ratio) was calculated. During the ECG recording at week 1 and week 7 post MI, rabbits had no spontaneous cardiac arrhythmias. When rabbits were exposed to escalating doses of flecainide, 2 out of 5 rabbits with MI and moderate LVSD versus 0 out of 5 solvent-treated rabbits developed arrhythmias, such as ventricular tachycardia/ventricular fibrillation. These were preceded by a marked decrease of iCEB just before the onset (from 4.09 to 2.42 and from 5.56 to 2.25, respectively). Furthermore, 1 out of 5 MI rabbits with moderate LVSD and 1 out of 7 MI rabbits with severe LVSD developed total atrioventricular block after flecainide infusion and died. This rabbit model of MI and severe LVSD may be useful for preclinical evaluation of drug (similar mechanism as flecainide)-induced arrhythmic risks, which might be predicted by iCEB.

Population change of Trillium govanianum (Melanthiaceae) amid altered indigenous harvesting practices in the Indian Himalayas.

ETHNOBOTANICAL RELEVANCE: Trillium govanianum Wall. ex D. Don (Melanthiaceae) is valued as a traditional medicinal herb in the Himalayan region. Applications include treatment of cancer, hypertension, neurasthenia, giddiness, arthritis, dysentery, inflammation, sepsis and reproductive disorders. Its range is highly specific, and limited to cold, shaded and moist habitats at 2400-3500 m a.s.l. Rhizomes are gathered from wild populations for trade, and this has recently emerged as a significant source of income among indigenous people of the Indian Himalayan region. AIM: To assess the impact of changing rhizome prices on T. govanianum in the Indian Himalayan region by observing the status of existing populations and assessing the typical methods used for gathering and trade of rhizomes. MATERIAL AND METHODS: Ecological attributes were assessed in 17 sites from Tirthan Valley (Himachal Pradesh), Munsiyari and Tunghnath (Uttarakhand). Socio-economic attributes assessed through communal focus groups and interviews with 579 medicinal plants gatherers and 19 traders, using semi-structured open ended questionnaires. RESULTS: Population decline was highest in Munsiyari, followed by Tirthan Valley and Tunghnath, indicating that the species may become locally extinct in some areas. Methods used for gathering did not consider regeneration. CONCLUSION: Gathering of T. govanianum from wild populations is unselective and unmanaged, raising the potential threat of local extinctions. Sustainable utilization and effective conservation is needed to protect the species and maintain community incomes. A status of 'threatened' is justified for the species in the Indian Himalayan region, due to its slow life cycle, specific habitat requirement, low population density, and commercial value.

Chronic drug-induced effects on contractile motion properties and cardiac biomarkers in human induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND AND PURPOSE: In the pharmaceutical industry risk assessments of chronic cardiac safety liabilities are mostly performed during late stages of preclinical drug development using in vivo animal models. Here, we explored the potential of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to detect chronic cardiac risks such as drug-induced cardiomyocyte toxicity. EXPERIMENTAL APPROACH: Video microscopy-based motion field imaging was applied to evaluate the chronic effect (over 72 h) of cardiotoxic drugs on the contractile motion of hiPS-CMs. In parallel, the release of cardiac troponin I (cTnI), heart fatty acid binding protein (FABP3) and N-terminal pro-brain natriuretic peptide (NT-proBNP) was analysed from cell medium, and transcriptional profiling of hiPS-CMs was done at the end of the experiment. KEY RESULTS: Different cardiotoxic drugs altered the contractile motion properties of hiPS-CMs together with increasing the release of cardiac biomarkers. FABP3 and cTnI were shown to be potential surrogates to predict cardiotoxicity in hiPS-CMs, whereas NT-proBNP seemed to be a less valuable biomarker. Furthermore, drug-induced cardiotoxicity produced by chronic exposure of hiPS-CMs to arsenic trioxide, doxorubicin or panobinostat was associated with different profiles of changes in contractile parameters, biomarker release and transcriptional expression. CONCLUSION AND IMPLICATIONS: We have shown that a parallel assessment of motion field imaging-derived contractile properties, release of biomarkers and transcriptional changes can detect diverse mechanisms of chronic drug-induced cardiac liabilities in hiPS-CMs. Hence, hiPS-CMs could potentially improve and accelerate cardiovascular de-risking of compounds at earlier stages of drug discovery. LINKED ARTICLES: This article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc.

Assessment of drug-induced proarrhythmia: The importance of study design in the rabbit left ventricular wedge model.

In the present study, we investigated an impact of the stimulation rate on the detection of the proarrhythmic potential of 10 reference compounds with effects on different cardiac ion channels in the isolated arterially-perfused rabbit left ventricular wedge preparation. The compounds were tested in the wedge model using two distinct protocols; including baseline stimulation at 1-Hz followed by a brief period at 0.5-Hz, either without an additional brief period of 2-Hz stimulation (i.e. Protocol 1) or with 2-Hz stimulation (i.e. Protocol 2). As expected, QT-prolonging drugs (ibutilide and quinidine) prolonged the QT interval, similarly increased the Torsades de Pointes (TdP) score, and elicited early afterdepolarizations (EADs) in both protocols. HMR1556 and JNJ-303 (IKs blockers) also prolonged the QT interval up to 1µM similarly in both protocols. Nifedipine (Ca(2+) antagonist) shortened the QT interval, and reduced force of contraction similarly in both protocols. However, Na(+) channel blockers (Ia, Ib, Ic) widened the QRS duration more in Protocol 2 than in Protocol 1. Furthermore, it was only possible to detect non-TdP-like ventricular tachycardia/fibrillation (VT/VF) induced by Na(+) blockers and by QT-shortening drugs (levcromakalim and mallotoxin) using the 2-Hz stimulation (Protocol 2). Our data suggest that the inclusion of a brief period of fast stimulation at 2Hz is critical for detecting drug-induced slowing of conduction (QRS widening), QT shortening and associated (non-TdP-like) VT/VF, which are distinct from the QT prolongation/TdP proarrhythmia in isolated, arterially-perfused rabbit left ventricular wedges.

Functional and Transcriptional Characterization of Histone Deacetylase Inhibitor-Mediated Cardiac Adverse Effects in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

UNLABELLED: Histone deacetylase (HDAC) inhibitors possess therapeutic potential to reverse aberrant epigenetic changes associated with cancers, neurological diseases, and immune disorders. Unfortunately, clinical studies with some HDAC inhibitors displayed delayed cardiac adverse effects, such as atrial fibrillation and ventricular tachycardia. However, the underlying molecular mechanism(s) of HDAC inhibitor-mediated cardiotoxicity remains poorly understood and is difficult to detect in the early stages of preclinical drug development because of a delayed onset of effects. In the present study, we show for the first time in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) that HDAC inhibitors (dacinostat, panobinostat, vorinostat, entinostat, and tubastatin-a) induce delayed dose-related cardiac dysfunction at therapeutic concentrations associated with cardiac adverse effects in humans. HDAC inhibitor-mediated delayed effects on the beating properties of hiPS-CMs developed after 12 hours by decreasing the beat rate, shortening the field potential duration, and inducing arrhythmic behavior under form of sustained contractions and fibrillation-like patterns. Transcriptional changes that are common between the cardiotoxic HDAC inhibitors but different from noncardiotoxic treatments identified cardiac-specific genes and pathways related to structural and functional changes in cardiomyocytes. Combining the functional data with epigenetic changes in hiPS-CMs allowed us to identify molecular targets that might explain HDAC inhibitor-mediated cardiac adverse effects in humans. Therefore, hiPS-CMs represent a valuable translational model to assess HDAC inhibitor-mediated cardiotoxicity and support identification of better HDAC inhibitors with an improved benefit-risk profile. SIGNIFICANCE: Histone deacetylase (HDAC) inhibitors are a promising class of drugs to treat certain cancers, autoimmune, and neurodegenerative diseases. However, treated patients can experience various cardiac adverse events such as hearth rhythm disorders. This study found that human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) can predict cardiac adverse events in patients caused by HDAC inhibitors. Furthermore, transcriptional changes at the level of gene expression supported the effects on the beating properties of hiPS-CMs and highlight targets that might cause these cardiac adverse effects. hiPS-CMs represent a valuable translational model to assess HDAC inhibitor-mediated cardiotoxicity and to support development of safer HDAC inhibitors.

Respiratory Microbiome of New-Born Infants.

The respiratory tract, once believed to be sterile, harbors diverse bacterial communities. The role of microorganisms within health and disease is slowly being unraveled. Evidence points to the neonatal period as a critical time for establishing stable bacterial communities and influencing immune responses important for long-term respiratory health. This review summarizes the evidence of early airway and lung bacterial colonization and the role the microbiome has on respiratory health in the short and long term. The challenges of neonatal respiratory microbiome studies and future research directions are also discussed.

Modelling of drug-induced QT-interval prolongation: estimation approaches and translational opportunities.

Safety pharmacology studies are performed to assess whether compounds may provoke severe arrhythmias (e.g. Torsades de Pointes, TdP) and sudden death in man. Although there is strong evidence that drugs inducing TdP in man prolong the QT interval in vivo and block the human ether-a-go-go-related gene (hERG) ion channel in vitro, not all drugs affecting the QT interval or the hERG will induce TdP. Nevertheless, QT-interval prolongation and hERG blockade currently represent the most accepted early risk biomarkers to deselect drugs. An extensive pharmacokinetic/pharmacodynamic (PK/PD) analysis is developed to understand moxifloxacin's-induced effects on the QT interval by comparing the relationship between results of an in vitro patch-clamp model to in vivo models. The frequentist and the fully Bayesian estimation procedures were compared and provided similar performances when the best model selected in NONMEM is subsequently implemented in WinBUGS, which guarantees a straightforward calculation of the probability of QT-interval prolongation greater than 2.5 % (10 ms). The use of the percent threshold to account for the intrinsic differences between species and a new calculation of the probability curve are introduced. The concentration providing the 50 % probability indicates that dogs are more sensitive than humans to QT-interval prolongation. However, based on the drug effect, a clear distinction between species cannot be made. An operational PK/PD model of agonism was used to investigate the relationship between effects on the hERG and QT-interval prolongation in dogs. The proposed analysis contributes to establish a translational relationship that could potentially reduce the need for thorough QT studies.

Discovery of 1-butyl-3-chloro-4-(4-phenyl-1-piperidinyl)-(1H)-pyridone (JNJ-40411813): a novel positive allosteric modulator of the metabotropic glutamate 2 receptor.

We previously reported the discovery of 4-aryl-substituted pyridones with mGlu2 PAM activity starting from the HTS hit 5. In this article, we describe a different exploration from 5 that led to the discovery of a novel subseries of phenylpiperidine-substituted pyridones. The optimization strategy involved the introduction of different spacers between the pyridone core and the phenyl ring of 5. The fine tuning of metabolism and hERG followed by differentiation of advanced leads that were identified on the basis of PK profiles and in vivo potency converged on lead compound 36 (JNJ-40411813). Full in vitro and in vivo profiles indicate that 36 displayed an optimal interplay between potency, selectivity, favorable ADMET/PK and cardiovascular safety profile, and central EEG activity. Compound 36 has been investigated in the clinic for schizophrenia and anxious depression disorders.

Discovery of 3-cyclopropylmethyl-7-(4-phenylpiperidin-1-yl)-8-trifluoromethyl[1,2,4]triazolo[4,3-a]pyridine (JNJ-42153605): a positive allosteric modulator of the metabotropic glutamate 2 receptor.

Advanced leads from a series of 1,2,4-triazolo[4,3-a]pyridines with mGlu2 receptor PAM activity are reported. By modification of the analogous imidazo[1,2-a]pyridine series, the newly reported leads have improved potency, in vitro ADMET, and hERG as well as good in vivo PK profile. The optimization of the series focused on improving metabolic stability while controlling lipophilicity by introducing small modifications to the scaffold substituents. Analysis of this series combined with our previously reported mGlu2 receptor PAMs showed how lipophilic ligand efficiency was improved during the course of the program. Among the best compounds, example 20 (JNJ-42153605) showed a central in vivo efficacy by inhibition of REM sleep state at a dose of 3 mg/kg po in the rat sleep-wake EEG paradigm, a phenomenon shown earlier to be mGlu2 mediated. In mice, compound 20 reversed PCP-induced hyperlocomotion with an ED50 of 5.4 mg/kg sc, indicative of antipsychotic activity.

Direct effects of arsenic trioxide on action potentials in isolated cardiac tissues: importance of the choice of species, type of cardiac tissue and perfusion time.

INTRODUCTION: The aim of the present study was to evaluate direct/acute effects of arsenic trioxide on action potentials (APs) in isolated cardiac tissues, and to investigate if the choice of species and tissue and the duration of the perfusion play a role in arsenic-induced acute/direct prolongation of AP/QT. METHODS AND RESULTS: Direct electrophysiological effects of arsenic trioxide were measured in cardiac tissues isolated from four different species using micro-electrode recording. Arsenic (after 30 to 95 min perfusion at 10 µM) significantly prolonged APD(90), increased triangulation of the AP and elicited early afterdepolarizations (EADs) only in isolated guinea-pig and dog Purkinje fibers but not in rabbit and porcine (minipig) Purkinje fibers. Arsenic induced a prolongation of the APD(90) and increases in triangulation and the occurrence of EADs was not observed in papillary muscles of guinea-pigs and rabbits. Arsenic at 4 increasing concentrations from 0.1 µM to 10 µM at the standard perfusion-time of 15 min per concentration, and after a continuous 90-min perfusion at 1 µM and 1 Hz did not induce these direct effects on APD(90), triangulation and EADs in isolated guinea-pig Purkinje fibers, but it at 1 µM elicited EADs in 2 out of 7 preparations after 90 min at 0.2 Hz. DISCUSSION: The present study demonstrates that the choice of species and cardiac tissue as well as perfusion-time play important roles in arsenic-induced direct/acute effects on APD(90) and induction of EADs in vitro.

Does terfenadine-induced ventricular tachycardia/fibrillation directly relate to its QT prolongation and Torsades de Pointes?

BACKGROUND AND PURPOSE: Terfenadine has been reported to cause cardiac death. Hence, we investigated its pro-arrhythmic potential in various in vitro models. EXPERIMENTAL APPROACH: Pro-arrhythmic effects of terfenadine were investigated in rabbit isolated hearts and left ventricular wedge preparations. Also, using whole-cell patch-clamp recording, we examined its effect on the human ether-à-go-go-related gene (hERG) current in HEK293 cells transfected with hERG and on the I(Na) current in rabbit ventricular cells and human atrial myocytes. KEY RESULTS: Terfenadine concentration- and use-dependently inhibited I(Na) in rabbit myocytes and in human atrial myocytes and also inhibited the hERG. In both the rabbit left ventricular wedge and heart preparations, terfenadine at 1 µM only slightly prolonged the QT- and JT-intervals but at 10 µM, it caused a marked widening of the QRS complex, cardiac wavelength shortening, incidences of in-excitability and non-TdP-like ventricular tachycardia/fibrillation (VT/VF) without prolongation of the QT/JT-interval. At 10 µM terfenadine elicited a lower incidence of early afterdepolarizations versus non- Torsades de Pointes (TdP)-like VT/VF (100% incidence), and did not induce TdPs. Although the concentration of terfenadine in the tissue-bath was low, it accumulated within the heart tissue. CONCLUSION AND IMPLICATIONS: Our data suggest that: (i) the induction of non-TdP-like VT/VF, which is caused by slowing of conduction via blockade of I(Na) (like Class Ic flecainide), may constitute a more important risk for terfenadine-induced cardiac death; (ii) although terfenadine is a potent hERG blocker, the risk for non-TdP-like VT/VF exceeds the risk for TdPs; and (iii) cardiac wavelength (λ) could serve as a biomarker to predict terfenadine-induced VT/VF.

ECG arrhythmias in non-implanted vs. telemetry-implanted dogs: need for screening before and sufficient recovery time after implantation.

INTRODUCTION: The purpose of this study was: (1) to characterize and assess the incidence of spontaneous arrhythmias in totally naive Beagle dogs (n=51; 10 males+41 females): (2) to study the effects of permanent ECG- and LVP-probe telemetry implants both acutely and up to 233days after surgery in a subset of 11 female Beagle dogs. METHODS: Naive ECG assessments were conducted by means of 6 external telemetry leads in jacketed dogs. Telemetry ECG recordings were captured by means of implanted telemetry devices suitable for ECG, LVP and aortic blood pressure recording. Experienced laboratory personnel visually evaluated all 22h ECG recordings at different time points after implantation and evaluated the incidence and type of arrhythmia. RESULTS: The 51 healthy and totally naive Beagle dogs showed a prevalence of: 49.0% 2°AVB; 58.8% single atrial premature complexes; 17.6% junctional tachycardia; 27.5% ventricular complexes; 13.7% ventricular escape complexes; 21.6% ventricular premature complexes; 3.9% runs of ventricular complexes; 3.9% runs of ventricular escape complexes. As such, a high percentage of clinically normal Beagle dogs showed different types of arrhythmias when ECG's obtained by external telemetry leads were fully evaluated for a 22h period. The chronic implantation of a ventricular probe through the apex of the heart in 11 dogs only, resulted in higher incidences and frequencies of ventricular episodes, which (in some dogs) extended up to 8weeks. Eight months after surgery none of the implanted dogs showed ventricular tachycardia and only 10% had single ventricular or ventricular premature complexes at low frequencies. DISCUSSION: A thorough evaluation of the ECG's of Beagle dogs selected for telemetry implantation can help to avoid inherent arrhythmia-burdened dogs being implanted and used in studies where these arrhythmias will confound drug assessment by increasing the number of potential false positives.

Predicting drug-induced slowing of conduction and pro-arrhythmia: identifying the 'bad' sodium current blockers.

BACKGROUND AND PURPOSE: The regulatory guidelines (ICHS7B) for the identification of only drug-induced long QT and pro-arrhythmias have certain limitations. EXPERIMENTAL APPROACH: Conduction time (CT) was measured in isolated Purkinje fibres, left ventricular perfused wedges and perfused hearts from rabbits, and sodium current was measured in Chinese hamster ovary cells, transfected with Na(v)1.5 channels. KEY RESULTS: A total of 355 compounds were screened for their effects on CT: 32% of these compounds slowed conduction, 65% had no effect and 3% accelerated conduction. Lidocaine and flecainide, which slow conduction, were tested in more detail as reference compounds. In isolated Purkinje fibres, flecainide largely slowed conduction and markedly increased triangulation, while lidocaine slightly slowed conduction and did not produce significant triangulation. Also in isolated left ventricular wedge preparations, flecainide largely slowed conduction in a rate-dependent manner, and elicited ventricular tachycardia (VT). Lidocaine slightly slowed conduction, reduced Tp-Te and did not induce VT. Similarly in isolated hearts, flecainide markedly slowed conduction, increased Tp-Te and elicited VT or ventricular fibrillation (VF). The slowing of conduction and induction of VT/VF with flecainide was much more evident in a condition of ischaemia/reperfusion. Lidocaine abolished ischaemia/reperfusion-induced VT/VF. Flecainide blocked sodium current (I(Na)) preferentially in the activated state (i.e. open channel) with slow binding and dissociation rates in a use-dependent manner, and lidocaine weakly blocked I(Na). CONCLUSION AND IMPLICATIONS: Slowing conduction by blocking I(Na) could be potentially pro-arrhythmic. It is possible to differentiate between compounds with 'good' (lidocaine-like) and 'bad' (flecainide-like) I(Na) blocking activities in these models.

Blockade of the I(Ks) potassium channel: an overlooked cardiovascular liability in drug safety screening?

The problem of drug-induced hERG channel blockade, which can lead to acquired long QT syndrome and potentially fatal arrhythmias, has exercised drug developers and regulatory authorities for over 10 years, and exacting guidelines have been put into place to test for this liability both preclinically (ICH S7B) and clinically (ICH E14). However, the I(Ks) channel, which along with the transient outward current (I(to)) is the other main potassium channel affecting cardiac repolarisation and thus the length of the QT interval, has received little attention, and potent I(Ks) blocking drugs with serious side effects could potentially enter into human testing without being detected by the existing regulatory core battery and standard screening strategies. Here we review the pharmacology of cardiac I(Ks) channel blockade and describe the discovery of a potent I(Ks) blocker whose activity was not detected by standard hERG or invitro action potential screens, but subsequently evoked unprovoked torsades de pointes (TdP) invivo in our anaesthetised dog model. We have exploited this molecule to develop a ligand binding assay to detect I(Ks) blockade at an earlier stage in drug discovery, and note that several other laboratories developing new drugs have also developed higher throughput screens to detect I(Ks) blockade (e.g., [Trepakova, E. S., Malik, M. G., Imredy, J. P., Penniman, J. R., Dech, S. J., & Salata, J. J. (2007) Application of PatchXpress planar patch clamp technology to the screening of new drug candidates for cardiac KCNQ1/KCNE1 (I(Ks)) activity. Assay Drug Development Technology 5, 617-627]). Because of the presence of I(Ks) channels in other tissues, including blood vessels and in the epithelia of intestine, kidney, lung and the cochlea, I(Ks) blockade has the potential to cause extensive side effects in addition to QT prolongation and arrhythmias. We therefore suggest that compounds selected for development should also be examined for I(Ks) liability before testing in humans. The possibility of undetected I(Ks) blockade is therefore an additional gap to that identified earlier [Lu, H. R., Vlaminckx, E., Hermans, A. N., Rohrbacher, J., Van Ammel, K., Towart, R., et al. (2008) Predicting drug-induced changes in QT interval and arrhythmias: QT-shortening drugs point to gaps in the ICH S7B Guidelines. British Journal of Pharmacology, 154, 1427-1438] in the ICH S7B regulatory guidelines.