Technological Advances in Cardiovascular Safety Assessment Decrease Preclinical Animal Use and Improve Clinical Relevance.

Cardiovascular (CV) safety liabilities are significant concerns for drug developers and preclinical animal studies are predominately where those liabilities are characterized before patient exposures. Steady progress in technology and laboratory capabilities is enabling a more refined and informative use of animals in those studies. The application of surgically implantable and telemetered instrumentation in the acute assessment of drug effects on CV function has significantly improved historical approaches that involved anesthetized or restrained animals. More chronically instrumented animals and application of common clinical imaging assessments like echocardiography and MRI extend functional and in-life structural assessments into the repeat-dose setting. A growing portfolio of circulating CV biomarkers is allowing longitudinal and repeated measures of cardiac and vascular injury and dysfunction better informing an understanding of temporal pathogenesis and allowing earlier detection of undesirable effects. In vitro modeling systems of the past were limited by their lack of biological relevance to the in vivo human condition. Advances in stem cell technology and more complex in vitro modeling platforms are quickly creating more opportunity to supplant animals in our earliest assessments for liabilities. Continuing improvement in our capabilities in both animal and nonanimal modeling should support a steady decrease in animal use for primary liability identification and optimize the translational relevance of the animal studies we continue to do.

The evaluation of drug-induced changes in cardiac inotropy in dogs: Results from a HESI-sponsored consortium.

INTRODUCTION: Drug-induced effects on the cardiovascular system remain a major cause of drug attrition. While hemodynamic (blood pressure (BP) and heart rate (HR)) and electrophysiological methods have been used in testing drug safety for years, animal models for assessing myocardial contractility are used less frequently and their translation to humans has not been established. The goal of these studies was to determine whether assessment of contractility and hemodynamics, when measured across different laboratories using the same protocol, could consistently detect drug-induced changes in the inotropic state of the heart using drugs known to have clinically relevant positive and negative effects on myocardial contractility. METHODS: A 4×4 double Latin square design (n=8) design using Beagle dogs was developed. Drugs were administrated orally. Arterial blood pressure, left ventricular pressure (LVP) and the electrocardiogram were assessed. Each of the six laboratories studied at least 2 drugs (one positive inotrope (pimobendan or amrinone) and one negative inotrope) (itraconazole or atenolol) at 3 doses selected to match clinical exposure data and a vehicle control. Animals were instrumented with an ITS telemetry system, DSI's D70-PCTP system or DSI's Physiotel Digital system. Data acquisition and analysis systems were Ponemah, Notocord or EMKA. RESULTS: Derived parameters included: diastolic, systolic and mean arterial BP, peak systolic LVP, HR, end-diastolic LVP, and LVdP/dtmax as the primary contractility index. Blood samples were drawn to confirm drug exposures predicted from independent pharmacokinetic studies. Across the laboratories, a consistent change in LVdP/dtmax was captured despite some differences in the absolute values of some of the hemodynamic parameters prior to treatment. DISCUSSION: These findings indicate that this experimental model, using the chronically instrumented conscious dog, can accurately and consistently detect changes in cardiac contractility, across multiple sites and instrumentation systems, and that data obtained in this model may also translate to clinical outcomes.

Cardiovascular pressure measurement in safety assessment studies: technology requirements and potential errors.

In the early days of in vivo nonclinical pressure measurement, most laboratories were required to have considerable technical/engineering expertise to configure and maintain pressure transducers, amplifiers, tape recorders, chart recorders, etc. Graduate students and postdoctoral fellows typically had some training in the requirements and limitations of the technology they used and were closely engaged in the collection and evaluation of data from their own experiments. More recently, pressure sensing telemetry and data acquisition/analysis systems are provided by vendors as turnkey systems, often resulting in a situation where users are less familiar with the technicalities of their operation. Also, investigators are now more likely to be absent and rely on technical staff for the collection of raw in vivo pressure data from their experiments than in the past. Depending on the goals of an experiment, an investigator may require the measurement of a variety of different pressure parameters, over varying periods of time. A basic understanding of the requirements and limitations that can affect the accuracy and precision of these parameters is important to ensure that the results and conclusions from an experiment are reliable. Factors to consider include the possibility of hydrostatic pressure effects from blood inside the vasculature of the animal, depending on the location of the sensor, as well as from fluid inside a fluid-filled catheter system; long-term stability (lack of drift) of a sensor over time, which can affect the interpretation of absolute pressure changes over a prolonged experiment; frequency response of the sensor and associated electronics; and the phase shift that occurs depending on location of the sensor in the vasculature or because of a fluid-filled catheter system. Each of these factors is discussed, and the particular requirements of frequency response as applied to the measurement of cardiac left ventricular pressure are emphasized. When these factors are understood, a pressure sensing and measurement system can be selected that is optimized for the experimental model being studied, thus eliminating errors or inaccurate results.

A public-private consortium advances cardiac safety evaluation: achievements of the HESI Cardiac Safety Technical Committee.

INTRODUCTION: The evaluation of cardiovascular side-effects is a critical element in the development of all new drugs and chemicals. Cardiac safety issues are a major cause of attrition and withdrawal due to adverse drug reactions (ADRs) in pharmaceutical drug development. METHODS: The evolution of the HESI Technical Committee on Cardiac Safety from 2000-2013 is presented as an example of an effective international consortium of academic, government, and industry scientists working to improve cardiac safety. RESULTS AND DISCUSSION: The HESI Technical Committee Working Groups facilitated the development of a variety of platforms for resource sharing and communication among experts that led to innovative strategies for improved drug safety. The positive impacts arising from these Working Groups are described in this article.

Left ventricular pressure, contractility and dP/dt(max) in nonclinical drug safety assessment studies.

Increasing or decreasing cardiac contractility is an undesirable property of drugs being developed for noncardiovascular indications. The International Conference on Harmonization (ICH) Topic S7A and S7B guidelines only require the assessment of heart rate, blood pressure and the electrocardiogram in nonclinical in vivo safety pharmacology studies. Assessment of drug effects on contractility is only suggested as an optional follow-up study. However, these nonclinical safety assessment studies can detect these effects if properly designed and conducted using appropriate instrumentation. Left ventricular dP/dt is the first derivative of left ventricular pressure, which is computed by software algorithms by using calculus. Its peak value, dP/dt(max), is a common, robust and sensitive indicator of changes in cardiac contractility if experimental parameters such as preload, afterload and heart rate are well controlled. In order to ensure accuracy and avoid errors in the measurement of contractility in experimental animals, the frequency response of the pressure sensing system and the sample rate of the data acquisition system must be optimized for the signal. For dogs, nonhuman primates, and normotensive rats, all important information in a left ventricular pressure signal can be captured with a system with a frequency response of 100 Hz. Although systems with much higher frequency response can be used to measure left ventricular pressure, the output of these devices must be filtered to allow no frequencies to be acquired that are higher than one-half the sample rate of the acquisition system. Stated conversely, the sample rate of the acquisition system must be at least 2× the highest frequency contained in the signal. Failure to follow these principals can lead to incorrect results due to measurement artifacts from high frequency noise, which could be present but not detectable by the investigator. This manuscript has been written for biologists who do not have advanced knowledge of physics and/or engineering and is therefore less technical and more simplified than what would be found in the engineering literature.

Cardiovascular function in nonclinical drug safety assessment: current issues and opportunities.

There are several recent examples where clinically significant, safety-related, drug effects on hemodynamics or cardiac function were not apparent until large clinical trials were completed or the drugs entered the consumer market. Such late-stage safety issues can have significant impact on patient health and consumer confidence, as well as ramifications for the regulatory, pharmaceutical, and financial communities. This manuscript provides recommendations that evolved from a 2009 HESI workshop on the need for improved translation of nonclinical cardiovascular effects to the clinical arena. The authors conclude that expanded and improved efforts to perform sensitive yet specific evaluations of functional cardiovascular parameters in nonclinical studies will allow pharmaceutical companies to identify suspect drugs early in the discovery and development process while allowing promising drugs to proceed into clinical development.

Evaluation of blood pressure measurement using a miniature blood pressure transmitter with jacketed external telemetry in cynomolgus monkeys.

INTRODUCTION: Current techniques used to accurately determine arterial blood pressure (BP) in conscious, unrestrained monkeys require invasive telemetry. This study evaluated the functionality of an implanted miniature telemetry blood pressure transmitter for the collection of BP measurements in conjunction with electrocardiographic measurements using a jacketed external telemetry (JET) system in conscious, unrestrained cynomolgus monkeys. METHODS: Twenty-four animals were surgically implanted with the transmitter in the right femoral artery. Local tolerability to the implant, signal quality, and variability in hemodynamic values were evaluated. On alternate weeks, animals were given single doses of positive control agents to produce hypotensive (clonidine hydrochloride) or hypertensive (L-NAME) effects. Undisturbed telemetry BP data were continuously collected for at least 24h following dosing and analyzed. RESULTS: While exhibiting remarkably high signal quality ( approximately 95% data points retained over 24h of data collection) and moderate variability across study weeks in baseline pulse height measurements (changes as small as < 0 mmHg), nine of 18 transmitters were nonfunctional by 19 weeks post-surgery, most likely due to migration of the catheter out of the artery. In animals given positive control agents, L-NAME induced a statistically significant increase (> or = + 8 mmHg) and clonidine hydrochloride induced a statistically significant decrease (-11 mmHg) in mean arterial pressures. Histological analysis revealed femoral arterial thickening near the sites of implantation. DISCUSSION: These results demonstrate the ability of the miniature BP transmitter, in conjunction with the JET system, to detect small changes in hemodynamic data continuously collected in conscious unrestrained monkeys. Future optimization of the transmitter includes the addition of a suture rib to the transmitter body and increased catheter size to prevent catheter migration out of the artery, the root cause of failed transmitters. The miniature blood pressure transmitter evaluated provides a minimally invasive technique for continuous collection of hemodynamic data in a toxicology study environment.

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.

Sensitivity of two noninvasive blood pressure measurement techniques compared to telemetry in cynomolgus monkeys and beagle dogs.

INTRODUCTION: Animals are commonly used in toxicological research for the evaluation of drug effects on the cardiovascular system. Accurate and reproducible determination of blood pressure (BP) in conscious, manually restrained monkeys and dogs is a challenge with current non-invasive cuff techniques. The High Definition Oscillometry (HDO) technique enables real time measurements with immediate visual feedback via PC screen on data validity. HDO measurements are considerably faster with a duration of approximately 8 to 15s than conventional cuff methods that can take several minutes. METHODS: HDO Memo Diagnostic Model Science and Cardell BP Monitor Model 9401 measurements were compared for accuracy and reliability with simultaneously recorded direct blood pressure data captured via radiotelemetry. Six monkeys and six dogs implanted with DSI PCT telemetry transmitters were used; BP data were collected by all methods under manual constraint and compared. Measurements were performed with HDO and Cardell in the presence of a BP lowering drug (hexamethonium bromide). Systolic, diastolic, mean arterial pressure, and pulse rate were determined before, during and following up to 10mg/kg hexamethonium administration via intravenous slow bolus injection. RESULTS: Drug induced hemodynamic changes could be detected in monkeys and dogs with the HDO method but only in dogs with the Cardell method. Correlation coefficients were generally higher for HDO versus Telemetry than Cardell versus Telemetry comparisons, indicating that this novel, non-invasive technique produces reliable blood pressure data and is able to detect drug-induced hemodynamic changes. DISCUSSION: HDO provides an alternative approach for invasive telemetry surgeries to obtain reliable hemodynamic data in animal models for cardiovascular research when invasive techniques are not warranted.

Standing on the shoulders of giants: Dean Franklin and his remarkable contributions to physiological measurements in animals.

The use of electronic instrumentation to monitor physiological function in conscious research animals and humans has become routine. Beyond basic research, animal studies using these methods are required by government regulatory agencies worldwide before human testing of potential new drugs. Living, as we do, in an age of miniaturized high-tech electronic devices, we are accustomed to believing this technology is easy; however, this has not always been the case. While a broad supporting cast of engineers, physiologists, fellows, and technicians was involved, the true innovators were Dr. Robert Rushmer, Dr. Robert Van Citters, and Mr. Dean Franklin. Before Dean Franklin's death in 2007, the primary author recorded approximately 5 h of interviews with him at his home in Columbia, MO. An additional approximate 1.5-h interview was recorded with Dr. Van Citters via telephone. The information contained herein is based on the recollections of these men as recorded in their interviews.

Effects of antipsychotic drugs on I(to), I (Na), I (sus), I (K1), and hERG: QT prolongation, structure activity relationship, and network analysis.

PURPOSE: To evaluate in vitro and computationally model the effects of selected antipsychotic drugs on several ionic currents that contribute to changes in the action potential in cardiac tissue. METHODS: Fourteen antipsychotic drugs or metabolites were examined to determine whether QT interval prolongation could be accounted for by an effect on one or more myocardial ion channels [I(to), I(Na), I(sus), I(K1), and human ether-a-go-go related gene (hERG)]. Using the patch clamp technique, drug effects on these human cardiac currents were tested. RESULTS: All molecules had little inhibitory effect on ion channels (blocking at concentrations >5 microM) other than hERG. A significant correlation was observed between the estimated hERG blockade and the increase in corrected QT for five of the antipsychotics. Molecular modeling identified hydrophobic features related to the interaction with hERG and correctly rank-ordered the test set molecules olanzapine and its metabolites. A network analysis of ligand and protein interactions around hERG using MetaCore (GeneGo Inc., St. Joseph, MI, USA) was used to visualize antipsychotics with affinity for this channel and their interactions with other proteins in this database. CONCLUSION: The antipsychotics do not inhibit the ion channels I(to), I(Na), I(sus), I(K1) to any appreciable extent; however, blockade of hERG is a likely mechanism for the prolongation of the QT interval.

Statistical power analysis for hemodynamic cardiovascular safety pharmacology studies in beagle dogs.

INTRODUCTION: We studied the statistical power of a replicated Latin square design where eight animals each receive a vehicle control and three dose levels of a drug on four separate dosing days. Cardiovascular parameters evaluated in the study were systolic arterial pressure, diastolic arterial pressure, left ventricular heart rate, and dP/dt(max). METHODS: Observations were simulated based on historical data and drug response profiles from cardiovascular safety pharmacology studies conducted at Lilly Research Laboratories. Statistical analysis for treatment effects was performed using a linear mixed model. Monotonicity of dose response was examined using sequential linear trend tests based on ordinal spacing of dose levels. RESULTS: The replicated Latin square design for cardiovascular safety pharmacology studies is shown to have at least an 80% power of detecting changes from control of at least a 10% increment in systolic and diastolic pressure and a 15% increment in heart rate and dP/dt(max). The power is not sensitive to the shape of dose response profile over time. DISCUSSION: Several unique features of our statistical power evaluation include the comparison of different covariance structures and drug response profiles. The procedure can also be applied to future power evaluations of other cardiovascular parameters, such as the QT interval, and the loss of statistical power due to missing observations.

Three-dimensional quantitative structure-activity relationship for inhibition of human ether-a-go-go-related gene potassium channel.

The protein product of the human ether-a-go-go gene (hERG) is a potassium channel that when inhibited by some drugs may lead to cardiac arrhythmia. Previously, a three-dimensional quantitative structure-activity relationship (3D-QSAR) pharmacophore model was constructed using Catalyst with in vitro inhibition data for antipsychotic agents. The rationale of the current study was to use a combination of in vitro and in silico technologies to further test the pharmacophore model and qualitatively predict whether molecules are likely to inhibit this potassium channel. These predictions were assessed with the experimental data using the Spearman's rho rank correlation. The antipsychotic-based hERG inhibitor model produced a statistically significant Spearman's rho of 0.71 for 11 molecules. In addition, 15 molecules from the literature were used as a further test set and were also well ranked by the same model with a statistically significant Spearman's rho value of 0.76. A Catalyst General hERG pharmacophore model was generated with these literature molecules, which contained four hydrophobic features and one positive ionizable feature. Linear regression of log-transformed observed versus predicted IC(50) values for this training set resulted in an r(2) value of 0.90. The model based on literature data was evaluated with the in vitro data generated for the original 22 molecules (including the antipsychotics) and illustrated a significant Spearman's rho of 0.77. Thus, the Catalyst 3D-QSAR approach provides useful qualitative predictions for test set molecules. The model based on literature data therefore provides a potentially valuable tool for discovery chemistry as future molecules may be synthesized that are less likely to inhibit hERG based on information provided by a pharmacophore for the inhibition of this potassium channel.