INTRODUCTION:Pharmacokinetic (PK) and pharmacodynamic (PD) modeling can elucidate the temporal interplay between drug concentration and physiological effects. Its use in safety pharmacology studies has been hindered, since collecting an adequate number of blood samples from conscious animals poses challenges. This study introduces an automatic blood sampling system into a cardiovascular safety pharmacology study to enable the collection of frequent blood samples from conscious animals to allow for a PK/PD assessment. METHOD:For this investigation, we employed a telemetry-based approach to obtain cardiovascular data from 4 Beagle dogs. To assess the PK profile of the test compound, an automated blood sampling device was used to facilitate the collection of up to 20 blood samples at predefined time points over the 7-h study period. The cross-over study was conducted using conscious, freely-moving animals, with Moxifloxacin administered as the test compound. RESULTS:Moxifloxacin administered orally in doses of 10 mg/kg, 30 mg/kg and 100 mg/kg reached Cmax of 6340 ± 1087, 15,840 ± 5471 and 29,033 ± 6540 nM, respectively, resulting in a QTc prolongation of 7 ms for 10 mg/kg, 17 ms for 30 mg/kg and 35 ms for 100 mg/kg. Based on the PK/PD analysis, a 5 ms QTc prolongation occurs with a Moxifloxacin concentration of 6.2 μM and a 10 ms QTc prolongation occurs with 12 μM. The feasibility of integrating automated blood samples in a cardiovascular safety pharmacology study using conscious animals has been demonstrated, by collecting enough PK data to support a PK/PD evaluation. CONCLUSION:The implementation of the automatic blood sampling system provides a significant advancement in safety pharmacology studies, offering a potent instrument for supporting PK/PD modeling.

Conducting safety evaluations of new drugs using conscious animals has been a specialty of our working group for thirty years. In this article, we review the various technical challenges and solutions dealt with over the years to improve both the data quality and the well being of our animal subjects. Of particular interest for us has been the use of telemetry-based data acquisition for conducting studies on cardiovascular (CV) function. This includes the evolving technical aspects of the studies, as well as the development of new applications that take advantage of this technical approach.

INTRODUCTION:A safety pharmacology study detects and evaluates potential side effects of a new drug on physiological function at therapeutic levels and above and, in most cases, prior to the initiation of clinical trials. The aim of this study was to investigate the effects of environmental and biological factors on resting heart rate (HR), a representative cardiac parameter in cardiovascular safety pharmacology.METHODS:Over twenty years, 143 dogs (Beagles, Labradors and mongrels) received implanted telemetry transmitters to measure aortic pressure (AP), left ventricular pressure (LVP), Electrocardiogram (ECG) and body temperature. Throughout the 7-h period of data collection, data were continuously recorded without drug treatment and included the range of HRs resulting from spontaneous physiological changes. Statistics and visualizations were calculated using R and Spotfire.RESULTS:Beagles had a higher HR than the mongrels, while Labradors had a lower HR than mongrels. Labradors were found to have a sex-based difference in HR, with females having a higher HR. A higher HR was observed in young animals of all breeds when they were in contact with humans. The cage system affected the HR of Labradors and mongrels more than Beagles. Larger dogs (e.g. Labrador) have a lower HR than smaller dogs (Beagles). Animals that are younger were found to have more HR variability and have a higher HR than older animals. In addition, older animals reacted less to the application period and human interaction than younger animals. The HR response of animals inside a cage system may depend on the cage system in which they were bred. A familiar cage system typically has less impact on HR.DISCUSSION:This retrospective data base evaluation has demonstrated the impact of environmental and biological factors on cardiovascular parameters in the context of performing safety pharmacology studies. Breed, sex, age and the type of cage system used affected, at least in some cases, the HR and its variability. They should therefore be carefully considered when designing safety pharmacology studies to have the highest possible test sensitivity.

Introduction: A newly developed total implant telemetry system for cardiovascular (CV), electrophysiological and body temperature measurement was evaluated. A cloud-based transmission of the physiological signals allowed an assessment of the quality of the physiological signals despite the physical separation between the instrumented animals and the evaluating home laboratory. The new system is intended to be used for safety pharmacological evaluations of drug candidates in various species. Methods: Two female minipigs, 6 Labrador-mixed breed dogs and 4 female Cynomolgus monkeys were instrumented with a newly developed total implant system (TSE SYSTEMS). The implants feature a microprocessor, internal memory (1 GB), 2 solid state pressure-tipped catheters, amplifiers and a radio transmitter. Sampling rates for each measurement can be selected within a range between 0.1 and 1 kHz. Biological signals are selected in a programmable fashion on a session-by-session basis according to a user-defined protocol. The pressure sensors are at the tip of an electrical lead having a length customized to each species. Core temperature measurement and activity monitoring (3D accelerometer) are included in the system. Digital transmission range using a single antenna is 5 m with up to 16 animals held together and monitored simultaneously. The range can be expanded with more antennas in an array coupled to a single receiver. The antenna/receiver station consists of a single USB powered mobile unit connected to a PC or laptop. The battery life provides 110 days of continuous recording. The dogs and minipigs were instrumented and monitored in Germany. A novel cloud-based data transmission system was developed to monitor the physiological signals in real-time from the Cynomolgus monkeys, still kept in Mauritius, from the data evaluation laboratory in Germany. After recovery from the surgical implantation, aortic pressure (AP), left ventricular pressure (LVP), ECG and body temperature were recorded for 24 hr monitoring sessions in all animals. Additionally, moxifloxacin (10, 30 and 100 mg/kg) was tested in the dog model using a modified Latin square cross-over study design. Results: The implant was well tolerated and the animals recovered rapidly from the implantation procedure. Excellent signal quality was obtained and stable hemodynamic and electrophysiological parameters could be measured, with little signal artefact or drop-out, over 24 h in each species. After oral dosing of moxifloxacin to the dogs, a substantial, dose-dependent increase in the QT-interval duration could be shown, as anticipated for this agent. Cloud-based data acquisition from the animals in Mauritius and the data evaluation lab in Germany worked well. Conclusion: This new CV telemetry system provides a novel alternative to fluid-filled catheter telemetry systems and the coupling to a cloud-based data transmission allows for flexibility in the location of the instrumented animals and data acquisition and the location of the site for data analysis. For the first time it is technically feasible to conduct a CV safety pharmacology study in Cynomolgus monkeys without having to ship them long distances to the home laboratory.

Introduction: In order to differentiate heart rate (HR)-induced changes from drug-induced positive or negative inotropic effects, HR-dependent effects need to be taken into account. Left ventricular (LV) dP/dt(max), the maximal value of the first derivative of a left ventricular pressure signal, is a convenient index for LV contractile state. The objective of this study was to define the normal relationship between left ventricular LVdP/dt(max) and HR in chronically instrumented, conscious dogs, primates and minipigs in our laboratory and then to use these data as the basis for developing a LVdP/dt(max)-HR-correction formula for each species. Methods: Trained Labrador-mix dogs, cynomolgus monkeys and minipigs (Goettinger) were equipped with a fully implantable radiotelemetry-based system (ITS, Maryland, USA) for the measurement of aortic pressure (AP), left ventricular pressure (LVP), ECG (lead II) and body temperature. The contractility index LVdP/dt(max) was derived from the LV pressure signal. Notocord HEM 4.2 software was used for data acquisition. For each species the relationship between LVdP/dt(max) and HR was evaluated using spontaneous HRs throughout the observation period (8-24 h) without pharmacological intervention. The formulae for the LVdP/dt(max)-HR relationships were generated using the R-script software for statistical evaluations and then used as the basis for an automated software for data analysis. Additionally, two different validation compounds (1 negative inotrope and 1 positive inotrope) were then used to investigate the impact of these compounds on the LVdP/dt(max)-HR relationship. Results and Discussion: There was a direct and reproducible LVdP/dt(max)-HR relationship in all animals tested and formulae were derived to describe this relationship in each species. Inotropic agents (both positive and negative) demonstrated the expected shifts of this relationship. Using the formulae found for each species describing the LVdP/dt(max)-HR dependency, one can assess the inotropic effects of drugs independently from simultaneous changes in HR. (C) 2012 Elsevier Inc. All rights reserved.

Introduction: The QT interval of the electrocardiogram (ECG) reflects the duration of ventricular depolarization and repolarization. A drug-induced prolongation of ventricular repolarization, and thereby QT prolongation, is recognized to be a marker for an enhanced risk for ventricular arrhythmia. The assessment of a drug's effect on the QT interval has therefore become routine within pharmaceutical research and development. However, the heart rate has a major influence on the QT interval; the QT interval shortens as heart rate increases such that one needs to account for such heart rate-dependent changes when evaluating possible drug-induced effects on the QT interval. The relationship between the QT interval and heart rate can be modeled mathematically and using this function a so-called "corrected" QT interval (QTc) can be generated to assess drug-induced effects independent from heart rate-dependent effects. In the past few years, a large number of mathematical relationship have been described that supposedly best describe the heart rate-QT relationship. In this paper we describe a novel approach for selecting the optimal mathematical function for this purpose for a given individual. Methods: Mongrel, purpose-bred dogs (16, males and females) were instrumented with radiotelemetry transmitters (ITS) for measurement of aortic pressure (AP), left ventricular pressure (LVP), the lead II ECG and body temperature. ECGs were recorded continuously without drug treatment and include a range of HRs due to spontaneous, physiological changes over the 24 h of data acquisition. Various mathematical models (>20) were then used to evaluate the HR-QT relationship and these were compared statistically to objectively select the model best fitting the data set of each individual animal. Results: In this study a dynamic analysis algorithm was developed to find the optimal descriptor of the HR-QT relationship for a given individual animal under control conditions. The use of this optimal relationship provides the best possible approach for detecting drug-induced effects on the QT interval for compounds that also affect the heart rate. Discussion: Several numerical methods to optimize the correction functions and statistical procedures to perform significance tests were discussed and implemented in a QT/RR relationship analysis system, named QTana. Given a sample data set, QTana searches the best correction model(s) from the integrated 11 QT/RR relationship modeling functions. (C) 2011 Elsevier Inc. All rights reserved.

INTRODUCTION:The objective of this study was to use a newly established cardiovascular model using freely moving minipigs to document the hemodynamic and electrocardiographic effects of known pharmacological agents. The data generated are to serve as the basis of pharmacological drug safety evaluations using this new model.METHODS:6 Göttingen minipigs were equipped with a radiotelemetry system (ITS). Following a recovery period, aortic pressure (AP), left ventricular pressure (LVP), lead II of the ECG and body temperature were continuously recorded throughout an 8 h monitoring period following oral administration of one of the test agents or vehicle. Notocord HEM 4.2 software was used for data acquisition. One known hERG blocker (moxifloxacin (30, 100 or 300 mg/kg)) and one non-selective beta-adrenoreceptor antagonist (propranolol (3, 10 or 20 mg/kg)) were tested in the model using a cross-over study design in 6 pigs.RESULTS:We obtained high signal quality and found stable hemodynamic parameters with low intrinsic heart rates in the Göttingen minipig under resting, pre-treatment conditions. After oral dosing of moxifloxacin, a substantial, dose-dependent increase in the QT-interval duration could be shown, as anticipated for this agent. After propranolol administration, a decrease in HR and left ventricular dP/dt was detected as expected for a beta-adrenoceptor blocking agent.DISCUSSION:The present data demonstrate that using this model in conscious, chronically instrumented Göttingen minipigs, a cross-over study with six animals was sensitive enough to detect a dose-dependent QT prolongation when moxifloxacin was administered in oral doses leading to clinically relevant plasma drug concentrations. Additionally, we could demonstrate the expected propranolol-induced effects on heart rate and myocardial contractility, despite the low intrinsic resting heart rates in these minipigs. These data support the use of the Göttingen minipig as a sensitive cardiovascular and electrocardiographic model for the testing of new pharmaceutical agents.

INTRODUCTION:The objective of this study was to evaluate the normal cardiovascular and ECG parameters in freely moving minipigs and to use these data as the basis of pharmacological drug safety evaluation.METHODS:7 Göttingen Minipigs were equipped with radiotelemetry transmitters (ITS). Aortic pressure (AP), left ventricular pressure (LVP), lead II of the ECG and body temperature were continuously monitored. Notocord HEM 4.2 software was used for data acquisition. Power calculations for the various parameters were done to assess appropriate sample sizes.RESULTS:We obtained excellent signal quality and found stable hemodynamic parameters with a low intrinsic heart rate in the Göttingen Minipig. After oral dosing of vehicle, the hemodynamic parameters returned quickly to baseline values indicating that the procedure was well tolerated. The heart rate dependency of the QT interval had to be corrected individually. A sufficient power could be achieved with a sample size of 4 due to the low variability of the parameters measured.DISCUSSION:These are, to our knowledge, the first data documenting the course of systemic arterial and ventricular hemodynamic parameters in the freely moving Göttingen Minipig over 24 h. As such, they may serve as a basis for future studies in which drug effects are studied in these animals.

Introduction: The objective of this study was to define the normal LVdP/dt (an index of myocardial contractility)–heart rate relationship in telemetered conscious dogs, primates and mini-pigs in our laboratory and to use these data as the basis for an additional parameter useful in drug safety evaluation. Methods: Trained dogs, Rhesus monkeys, Cynomolgus monkeys and mini-pigs (Goettinger) were equipped with radiotelemetry transmitters (ITS). Aortic pressure (AP), left ventricular pressure (LVP), a lead II ECG and body temperature could be continuously monitored. The contractility index LVdP/dtmax was derived from the LVP signal. Notocord HEM 4.1 software was used for data acquisition. For each species an LVdP/dt–heart rate relationship was evaluated using spontaneous heart rates (HR) throughout the observation period. A validation compound with positive inotropic effects (pimobendan) was then used to investigate the LVdP/dt–heart rate relationship. Results: There was a clear LVdP/dt–HR relationship in the animals tested. The inotropic agent pimobendan demonstrated the expected shift in this relationship. Discussion: Contractility of the myocardium is regulated by autonomic input activating primarily myocardial β1-adrenoceptors, but it is also affected by the “force–frequency” relationship. Compounds can therefore either directly or indirectly affect the contractility of the heart. The chronotropic effects are routinely measured in preclinical studies; however, the inotropic effects are not routinely analysed in cardiovascular safety studies. Our experience strongly recommends including this evaluation for drug candidate selection. The evaluation of LVdP/dtmax, as an index of myocardial contractile state must, however, take into account its HR-dependency.

INTRODUCTION:The objective of this study was to test the influence of housing conditions on hemodynamics during cardiovascular general pharmacological studies. Our goal was to optimize both the quality of the data through an optimization of the physiological conditions, as well as to ensure the dog's well-being in general pharmacological studies. METHODS:Two groups of four dogs were equipped with radiotelemetry transmitters and continuously monitored in two different housing models. Model I consisted of 4 cages, two on each site of a corridor. Model II consisted of 4 cages positioned in a row, where the bordering cages were not separated with a metal plate. The physiological status of the dogs in the different housing models was based on the frequency of vocalizations and the average resting heart rate, as well as video monitoring. RESULTS:The housing arrangement during the study had a remarkable effect on the hemodynamics measured. The hemodynamic parameters were best when the dogs were housed with their usual run mate. In this setting, they have impressively low average heart rates of about 60 bpm during the entire study, was associated with fewer vocalizations. DISCUSSION:This study demonstrated that the quality of the acquired cardiovascular data for conscious dogs is dependent on the pen configuration and group make-up during a study.

Introduction: Estimation of possible cardiovascular side effects belongs to the safety assessment of every drug candidate. This paper describes a new strategy for treating conscious labrador dogs with drugs by inhalation using a specially designed mask and a novel inhaler device. Methods: Labrador dogs (male or female) were used that had transducers implanted for the measurement of left ventricular and descending aortic blood pressures and an ECG for use together with a telemetry system. Administration by inhalation was achieved using a novel delivery device. The Respimat device is a propellant-free inhaler to deliver aerosols from solutions. The new system was evaluated using Formoterol with four dogs using a 4×4 Latin square design. Three doses of Formoterol (0.6, 1.2, and 2.4 μg/kg, dissolved in 60% ethanol) were administered by inhalation together with a vehicle (60% ethanol) treatment by applying three inhalations, each consisting of 10 μl solution. Results: Formoterol increased HR, QRS-interval, QT-interval, and LVPdP/dtmax and dose-dependently decreased systolic and diastolic BP. This effect lasted up to 14 h. Discussion: Drug administration by inhalation in the conscious labrador dog using the Respimat is a useful new model for safety pharmacology studies of new drug candidates that are intended to be given by inhalation in the clinic.