Optimized J to T peak and T peak to T end measurements in nonclinical species administered moxifloxacin and amiodarone.

INTRODUCTION: Cardiovascular safety and the risk of developing the potentially fatal ventricular tachyarrhythmia, Torsades de Pointes (TdP), have long been major concerns of drug development. TdP is associated with a delayed ventricular repolarization represented by QT interval prolongation in the electrocardiogram (ECG), typically due to block of the potassium channel encoded by the human ether-a-go-go related gene (hERG). Importantly however, not all drugs that prolong the QT interval are torsadagenic and not all hERG blockers prolong the QT interval. Recent clinical reports suggest that partitioning the QT interval into early (J to T peak; JTp) and late repolarization (T peak to T end; TpTe) components may be valuable for distinguishing low-risk mixed ion channel blockers (hERG plus calcium and/or late sodium currents) from high-risk pure hERG channel blockers. This strategy, if true for nonclinical animal models, could be used to de-risk QT prolonging compounds earlier in the drug development process. METHODS: To explore this, we investigated JTp and TpTe in ECG data collected from telemetered dogs and/or monkeys administered moxifloxacin or amiodarone at doses targeting relevant clinical exposures. An optimized placement of the Tpeak fiducial mark was utilized, and all intervals were corrected for heart rate (QTc, JTpc, TpTec). RESULTS: Increases in QTc and JTpc intervals with administration of the pure hERG blocker moxifloxacin and an initial QTc and JTpc shortening followed by prolongation with the mixed ion channel blocker amiodarone were detected as expected, aligning with clinical data. However, anticipated increases in TpTec by both standard agents were not detected. DISCUSSION: The inability to detect changes in TpTec reduces the utility of these subintervals for prediction of arrhythmias using continuous single­lead ECGs collected from freely moving dogs and monkeys.

Evaluation of cardiovascular changes in dogs administered three positive controls using jacketed external telemetry-blood pressure (JET-BP).

INTRODUCTION: Nonclinical safety studies are increasingly incorporating cardiac safety endpoints to discover potential cardiovascular liabilities. This trend for more thorough cardiovascular nonclinical safety evaluation is prudent given the high attrition rate of potential therapeutics due to unexpected cardiovascular liabilities discovered in late-stage clinical trials or post-market approval. In particular, the causal relationship of blood pressure changes that lead to risk of major adverse cardiac events suggests hemodynamic changes should be critically evaluated in preclinical studies of novel therapeutics. METHODS: Jacketed external telemetry with an implanted miniature blood pressure transmitter (JET-BP) was used to characterize the tolerability, functionality, and sensitivity of this study design in dogs. Thirty-six male or female beagles (n=6 dogs/sex/group) were administered vehicle control (reverse osmosis water) or etilefrine (1, 10mg/kg), sotalol (3, 30mg/kg), and hydralazine (1, 10mg/kg) on separate days. Telemetry data were evaluated for positive control article-related changes and retrospective power analysis was also completed. Animals were evaluated for instrumentation-related changes in clinical and anatomic pathology endpoints. RESULTS: All three positive controls elicited the expected pharmacologic responses that were statistically different at high and low doses. Retrospective power analysis confirmed this study design was able to statistically differentiate minor (approximately 5 to 15%) changes in electrocardiography and blood pressure values. This study also demonstrated the potential advantages of combining cardiovascular data across sex when the test article exposure and pharmacodynamics were consistent. Data collection using miniature telemetry blood pressure transmitters did not result in anatomic or clinical pathology findings that would prevent their use in general toxicology studies. DISCUSSION: This characterization study indicates that JET-BP in dogs offers a scientifically-robust method to evaluate novel therapeutics for potential cardiovascular liabilities.

Intravenous solid tip ECG lead placement in telemetry implanted dogs: Part 2: High quality telemetry signals yield high sensitivity to drug-induced changes.

INTRODUCTION: Dogs are commonly used in cardiovascular drug safety assessment, and implanted telemetry models include subcutaneous or epicardial electrocardiogram (ECG) electrode placements. The purpose of this study was to determine the sensitivity of a canine telemetry model with intravenous ECG lead placement: the negative ECG lead (solid tip) inserted into the jugular vein and the positive lead sutured to the diaphragm. Reference drugs were administered to test the sensitivity to drug-induced changes. METHODS: Twenty-four dogs were implanted with PCT or PCTP transmitters [Data Sciences International (DSI)]. Three reference drugs were administered: sotalol to eight PCT and milrinone to eight PCTP transmitter-implanted dogs. Twenty-four dogs received moxifloxacin (12 dogs/transmitter type). Telemetry data were collected for 25h and analyzed using double Latin squares for sotalol and milrinone data or a 4×4 or 3×6 parallel design for moxifloxacin data. Evaluated parameters were PR, QT, corrected QT (QTc), QRS, heart rate, left ventricular function, and hemodynamic data. Various correction factors for QTc interval were tested. Retrospective power analysis was performed to detect minimal absolute changes comparing a single to a double Latin square or the two parallel designs. RESULTS: Expected changes on ECG and hemodynamic parameters were observed after administration of all reference drugs. The individual animal corrected QT (QTci) interval provided the optimal correction factor. Retrospective power analysis confirmed detection of smaller differences in double versus single Latin squares. Minimal detectable differences were smaller in both Latin squares compared to parallel designs, with smaller detectable differences in a 3×6 compared to a 4×4 parallel design. DISCUSSION: The solid tip intravenous ECG lead configuration in dogs is a viable radiotelemetry model to detect drug-induced changes with high sensitivity. This model yields comparable signal quality and represents a refinement over epicardial ECG leads and allows for possible reduction in the number of animals if study design and size are selected based on needed assay sensitivity.

Left thoracotomy surgical approach for chronic instrumentation in dogs and monkeys providing high-quality electrocardiogram signals.

INTRODUCTION: Assessment of cardiovascular parameters, including the electrocardiogram (ECG) is required by the regulatory guidelines. In safety pharmacology studies, this is typically done using chronically implanted radiotelemetry devices in non-rodent species. METHODS: We compared ECG signal quality from ten male beagle dogs and 10 male cynomolgus monkeys with telemetry transmitters implanted using two surgical approaches: i) epicardial ECG lead placement via single incision, left side thoracotomy or ii) subcutaneous ECG lead placement via laparotomy. In addition, epicardial leads and semi-automated scoring were used in combination to detect changes in ECG values caused by moxifloxacin. Telemetry-instrumented male beagle dogs (n=8) and male cynomolgus monkeys (n=8) were given moxifloxacin at 10, 30, or 100 mg/kg (dogs) and 10, 50, or 175 mg/kg (monkeys) as a single dose by oral gavage. RESULTS: ECG signals were of excellent quality with epicardial lead placement, and human activity in the room did not significantly alter signal quality. Administration of moxifloxacin was associated with prolongation of QTc interval, in both dogs and monkeys in a dose-dependent pattern. Dogs given 30 mg/kg and 100 mg/kg, the maximum QTcf interval prolongations were 22 ms (+9%, 8 h postdose) and 60 ms (+24%, 15 h postdose). In monkeys given 50 and 175 mg/kg, the QTcb interval was significantly prolonged from 1 to 6h postdose, and QTcb interval prolongation persisted in monkeys given 175 mg/kg through 19 h postdose. In monkeys given 175 mg/kg, the maximum QTcb interval prolongation was 43 ms (+12.9%, 16 h postdose). DISCUSSION: The present study demonstrated that placing leads directly on the epicardium drastically diminishes signal disruption due to room disturbances and subsequent animal excitement. This novel surgical model demonstrated adequate sensitivity to detect changes in ECG parameters, specifically QTc interval prolongation in both the dog and monkey.

Kinetics of selected di-n-butyl phthalate metabolites and fetal testosterone following repeated and single administration in pregnant rats.

Human exposure to phthalic acid diesters occurs through a variety of pathways as a result of their widespread use in consumer products and plastics. Repeated doses of di-n-butyl phthalate (DBP) from gestation day (GD) 12 to 19 disrupt testosterone synthesis and male sexual development in the fetal rat. Currently little is known about the disposition of DBP metabolites, such as monobutyl phthalate (MBP) and its glucuronide conjugate (MBP-G), during gestation after repeated exposure to DBP. In order to gain a better understanding of the effect of repeated dosing on maternal and fetal metabolism and distribution, pregnant Sprague-Dawley rats were given a single dose of 500 mg/kg DBP on GD 19 or daily doses of 50, 100, and 500 mg/(kg day) from GD 12 to 19 via corn oil gavage. Dose-response evaluation revealed a non-linear increase in maternal and fetal plasma concentrations of MBP. Maternal and fetal MBP levels were slightly lower in animals after 8 days of dosing at 500 mg/(kg day). Fetal plasma MBP levels closely followed maternal plasma, while the appearance and elimination of MBP-G in fetal plasma were significantly delayed. MBP-G accumulated over time in the amniotic fluid. Inhibition of testosterone was rapid in fetal testes when exposed to DBP (500 mg/(kg day)) on GD 19. Within 24h, the level of inhibition in the fetus was similar between animals exposed to a single or multiple daily doses of 500 mg/(kg day). Examination of testosterone time-course data indicates a rapid recovery to normal levels within 24h post-dosing at DBP doses of 50 and 100 mg/(kg day), with a rebound to higher than normal concentrations at later time-points. MBP kinetics in fetal testes allows direct comparison of active metabolite concentrations and testosterone response in the fetal testes.

Tissue exposures to free and glucuronidated monobutylyphthalate in the pregnant and fetal rat following exposure to di-n-butylphthalate: evaluation with a PBPK model.

Human exposure to phthalic acid diesters occurs through a variety of pathways as a result of their widespread use in plastics. Repeated doses of di-n-butylphthalate (DBP) from gestation day (GD) 12 to 19 disrupt testosterone synthesis and male sexual development in the fetal rat. To gain a better understanding of the relationship of the target tissue (testes) dose to observed developmental effects, the pharmacokinetics of monobutyl phthalate (MBP) and its glucuronide (MBP-G) were examined in pregnant and fetal rats following single and repeated administration of DBP from GD 12-19. These data, together with results from previously published studies, were used to develop a physiologically based pharmacokinetic model for DBP and its metabolites in the male, pregnant and fetal rat. The model structure accounts for the major metabolic (hydrolysis, glucuronidation, oxidative metabolism) and transport processes (enterohepatic recirculation, urinary and fecal excretion, placental transfer). Extrapolation of the validated adult male rat model to gestation successfully predicts MBP and MBP-G levels in maternal plasma, placenta and urine, as well as the fetal plasma and testes. Sensitivity analysis indicates that plasma MBP kinetics are particularly sensitive to glucuronidation and enterohepatic recirculation: a decrease in the uridine 5'-diphospho-glucuronosyltransferase (UDPGT) capacity during gestation results in an increased MBP residence time, and saturation of UDPGT at the highest doses (> 100 mg/kg/day) causes a flattening out of the plasma time course data. Oxidative metabolism plays a significant role in elimination only at low doses (< 50 mg/kg DBP). Insights gained from modeling of the rat data will be used to support development of a human PBPK model for DBP.

Pharmacokinetics of monobutylphthalate, the active metabolite of di-n-butylphthalate, in pregnant rats.

Di-n-butylphthalate (DBP) is a phthalic acid ester used as a plasticizer and solvent. DBP is a developmental toxicant in rats and mice, with adverse effects arising from the monoester metabolite monobutyl phthalate (MBP). The objective of this study was to evaluate the pharmacokinetics of MBP and monobutyl phthalate glucuronide (MBP-G) in pregnant rats following intravenous (i.v.) dosing with MBP. Pregnant dams were i.v. dosed with aqueous MBP (10, 30, or 50mg MBP/kg body weight) on gestation day (GD) 19. The pharmacokinetics of MBP and MBP-G were rapid: MBP was metabolized to MBP-G within 5 min, and MBP and MBP-G disappeared from maternal and fetal plasma within 24h of dosing. Results were consistent with two previous studies that utilized oral doses of DBP, suggesting that chemical (DBP versus MBP), vehicle (oil versus aqueous), dose level, and route (oral versus i.v.) have minimal effects on the maternal pharmacokinetics of MBP and MBP-G. This study provides direct pharmacokinetic analysis for MBP and MBP-G in pregnant rats during fetal male reproductive development, and indicates that future pharmacokinetic or toxicology studies can reliably utilize oral dosing with DBP.

Kinetics of adsorption of beta-amyloid peptide Abeta(1-40) to lipid bilayers.

The Alzheimer's disease-related peptide beta-amyloid (Abeta) is toxic to neurons. The toxicity of the peptide appears to require conversion of the monomeric form to an aggregated fibrillar species. The interaction of Abeta with cell membranes has attracted interest as one plausible mechanism by which the peptide exerts its toxic activity. We developed two methods to measure the adsorption of fresh (monomeric) and aged (aggregated) Abeta to lipid bilayers. In one method, the kinetics of Abeta adsorption and desorption to liposomes deposited onto a dextran-coated surface was measured using surface plasmon resonance. In the other method, Abeta was contacted with liposome-coated magnetic beads; adsorbed Abeta was separated from solution-phase peptide by use of a magnetic field. Monomeric Abeta adsorbed quickly but reversibly to lipid bilayers with low affinity, while aggregated Abeta adsorbed slowly but irreversibly. These two methods provide complementary means of quantifying the adsorption of aggregating proteins to membranes. The results correlate strongly with previous observations that fibrillar, but not monomeric, Abeta restricts the motion of acyl tails in phospholipid bilayers. The methods should be useful for further elucidation of the role of membrane adsorption in mediating Abeta toxicity, and in the search for inhibitors of toxicity.