An Industry Survey With Focus on Cardiovascular Safety Pharmacology Study Design and Data Interpretation.

INTRODUCTION: The Safety Pharmacology Society (SPS) conducted a membership survey to examine industry practices related mainly to cardiovascular (CV) safety pharmacology (SP). METHODS: Questions addressed nonclinical study design, data analysis methods, drug-induced effects, and conventional and novel CV assays. RESULTS: The most frequent therapeutic area targeted by drugs developed by the companies/institutions that employ survey responders was oncology. The most frequently observed drug-mediated effects included an increased heart rate, increased arterial blood pressure, hERG (IKr) block, decreased arterial blood pressure, decreased heart rate, QTc prolongation, and changes in body temperature. Broadly implemented study practices included Latin square crossover study design with n = 4 for nonrodent CV studies, statistical analysis of data (eg, analysis of variance), use of arrhythmia detection software, and the inclusion of data from all study animals when integrating SP studies into toxicology studies. Most responders frequently used individual animal housing conditions. Responders commonly evaluated drug effects on multiple ion channels, but in silico modeling methods were used much less frequently. Most responders rarely measured the J-Tpeak interval in CV studies. Uncertainties relative to Standard for Exchange of Nonclinical Data applications for data derived from CV SP studies were common. Although available, the use of human induced pluripotent stem cell cardiomyocytes remains rare. The respiratory SP study was rarely involved with identifying drug-induced functional issues. Responders indicated that the study-derived no observed effect level was more frequently determined than the no observed adverse effect level in CV SP studies; however, a large proportion of survey responders used neither.

A Proof-of-Concept Evaluation of JTPc and Tp-Tec as Proarrhythmia Biomarkers in Preclinical Species: A Retrospective Analysis by an HESI-Sponsored Consortium.

INTRODUCTION: Based on the ICH S7B and E14 guidance documents, QT interval (QTc) is used as the primary in vivo biomarker to assess the risk of drug-induced torsades de pointes (TdP). Clinical and nonclinical data suggest that drugs that prolong the corrected QTc with balanced multiple ion channel inhibition (most importantly the l-type calcium, Cav1.2, and persistent or late inward sodium current, Nav1.5, in addition to human Ether-à-go-go-Related Gene [hERG] IKr or Kv11.1) may have limited proarrhythmic liability. The heart rate-corrected J to T-peak (JTpc) measurement in particular may be considered to discriminate selective hERG blockers from multi-ion channel blockers. METHODS: Telemetry data from Beagle dogs given dofetilide (0.3 mg/kg), sotalol (32 mg/kg), and verapamil (30 mg/kg) orally and Cynomolgus monkeys given medetomidine (0.4 mg/kg) orally were retrospectively analyzed for effects on QTca, JTpca, and T-peak to T-end covariate adjusted (Tpeca) interval using individual rate correction and super intervals (calculated from 0-6, 6-12, 12-18, and 18-24 hours postdose). RESULTS: Dofetilide and cisapride (IKr or Kv11.1 blockers) were associated with significant increases in QTca and JTpca, while sotalol was associated with significant increases in QTca, JTpca, and Tpeca. Verapamil (a Kv11.1 and Cav1.2 blocker) resulted in a reduction in QTca and JTpca, however, and increased Tpeca. Medetomidine was associated with a reduction in Tpeca and increase in JTpca. DISCUSSION: Results from this limited retrospective electrocardiogram analysis suggest that JTpca and Tpeca may discriminate selective IKr blockers and multichannel blockers and could be considered in the context of an integrated comprehensive proarrhythmic risk assessment.

The evaluation of endpoint variability and implications for study statistical power and sample size in conscious instrumented dogs.

INTRODUCTION: The sensitivity of a given test to detect a treatment-induced effect in a variable of interest is intrinsically related to the variability of that variable observed without treatment and the number of observations made in the study (i.e. number of animals). To evaluate test sensitivity to detect drug-induced changes in myocardial contractility using the variable LVdP/dtmax, a HESI-supported consortium designed and conducted studies in chronically instrumented, conscious dogs using telemetry. This paper evaluated the inherent variability of the primary endpoint, LVdP/dtmax, over time in individual animals as well as the variability between animals for a given laboratory. An approach is described to evaluate test system variability and thereby test sensitivity which may be used to support the selection of the number of animals for a given study, based on the desired test sensitivity. METHODS: A double 4 × 4 Latin square study design where eight animals each received a vehicle control and three dose levels of a test compound was conducted at six independent laboratories. LVdP/dtmax was assessed via implanted telemetry systems in Beagle dogs (N = 8) using the same protocol and each of the six laboratories conducted between two and four studies. Vehicle data from each study was used to evaluate the between-animal and within-animal variability in different time averaging windows. Simulations were conducted to evaluate statistical power and type I error for LVdP/dtmax based on the estimated variability and assumed treatment effects in hourly-interval, bi-hourly interval, or drug-specific super interval. RESULTS: We observe that the within-animal variability can be reduced by as much as 30% through the use of a larger time averaging window. Laboratory is a significant source of animal-to-animal variability as between-animal variability is laboratory-dependent and is less impacted by the use of different time averaging windows. The statistical power analysis shows that with N = 8, the double Latin square design has over 90% power to detect a minimal time profile with a maximum change of up to 15% or approximately 450 mm Hg/s in LVdP/dtmax. With N = 4, the single Latin square design has over 80% power to detect a minimal time profile with a maximum change of up to 20% or approximately 600 mm Hg/s in LVdP/dtmax. DISCUSSION: We describe a statistical procedure to quantitatively evaluate the acute cardiac effects from studies conducted across six sites and objectively examine the variability and sensitivity that were difficult or impossible to calculate consistently based on previous works. Although this report focuses on the evaluation on LVdP/dtmax, this approach is appropriate for other variables such as heart rate, arterial blood pressure, or variables derived from the ECG.

Proarrhythmia liability assessment and the comprehensive in vitro Proarrhythmia Assay (CiPA): An industry survey on current practice.

INTRODUCTION: The Safety Pharmacology Society (SPS) has conducted a survey of its membership to identify industry practices related to testing considered in the Comprehensive In vitro Proarrhythmia Assay (CiPA). METHODS: Survey topics included nonclinical approaches to address proarrhythmia issues, conduct of in silico studies, in vitro ion channel testing methods used, drugs used as positive controls during the conduct of cardiac ion channel studies, types of arrhythmias observed in non-clinical studies and use of the anticipated CiPA ion channel assay. RESULTS: In silico studies were used to evaluate effects on ventricular action potentials by only 15% of responders. In vitro assays were used mostly to assess QT prolongation (95%), cardiac Ca2+ and Na+ channel blockade (82%) and QT shortening or QRS prolongation (53%). For de-risking of candidate drugs for proarrhythmia, those assays most relevant to CiPA including cell lines stably expressing ion channels used to determine potency of drug block were most frequently used (89%) and human stem cell-derived or induced pluripotent stem cell cardiomyocytes (46%). Those in vivo assays related to general proarrhythmia derisking include ECG recording using implanted telemetry technology (88%), jacketed external telemetry (62%) and anesthetized animal models (53%). While the CiPA initiative was supported by 92% of responders, there may be some disconnect between current practice and future expectations, as explained. DISCUSSION: Proarrhythmia liability assessment in drug development presently includes study types consistent with CiPA. It is anticipated that CiPA will develop into a workable solution to the concern that proarrhythmia liability testing remains suboptimal.

An evaluation of the utility of LVdP/dt40, QA interval, LVdP/dtmin and Tau as indicators of drug-induced changes in contractility and lusitropy in dogs.

INTRODUCTION: The importance of drug-induced effects on the inotropic state of the heart is well known. Unlike hemodynamic and cardiac electrophysiological methods, which have been routinely used in drug safety testing for years, the non-clinical assessment of drug effects on myocardial contractility is used less frequently with no established translation to humans. The goal of these studies was to determine whether assessment of alternate measures of cardiac inotropy could detect drug-induced changes in the contractile state of the heart using drugs known to have clinically relevant positive and negative effects on myocardial contractility. This study also evaluated drug-induced effects on lusitropy (relaxation) parameters of the heart. METHODS: A double 4×4 Latin square study design using Beagle dogs (n=8) was conducted. Drugs were administrated orally. Arterial blood pressure (BP), left ventricular pressure (LVP) and the electrocardiogram (ECG) were assessed across different laboratories using the same protocol. 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 or DSI's D70-PCTP or PhysioTel™ Digital system. The data acquisition and analysis systems used were Ponemah, Notocord or EMKA. RESULTS: The derived inotropic and lusitropic parameters evaluated included peak systolic and end diastolic LVP, LVdP/dtmax, LVdP/dt40, QA interval, LVdP/dtmin and Tau. This study showed that LVdP/dt40 provided essentially identical results to LVdP/dtmax qualifying it as an index to assess drug effects on cardiac contractility. LVdP/dt40 provided an essentially identical assessment to that of LVdP/dtmax. The QA interval did not react sensitively to the drugs tested in this study; however, it did detect large effects and could be useful in early cardiovascular safety studies. The lusitropic parameter, LVdP/dtmin, was modestly decreased, and Tau was increased, by atenolol and itraconazole. At the doses tested, amrinone and pimobendan produced no changes in LVdP/dtmin while Tau was modestly increased. The drugs did not produce effects on BP, HR or the ECG at the doses tested. Blood samples were drawn to confirm drug exposures predicted from independent pharmacokinetic studies. DISCUSSION: These findings indicate that this experimental model can accurately and consistently detect changes in cardiac contractility, across multiple sites and instrumentation systems. While LVdP/dt40 produced responses similar to LVdP/dtmax, the QA interval and lusitropic parameters LVdP/dtmin and Tau were not markedly changed at the dose of drugs tested. Further studies with drugs that affect early diastolic relaxation through calcium handling are needed to better evaluate drug-induced changes on lusitropic properties of the heart.

The Diplomate in Safety Pharmacology (DSP) certification scheme.

As with other professional disciplines there is a growing need from within industry as well as global regulatory authorities for implementation of a certification process in order to assure that appropriate expertise is developed and quality standards are identified for professionals involved in the practice of Safety Pharmacology (SP). In order to meet this need, the Safety Pharmacology Society (SPS) has developed the Diplomate in Safety Pharmacology (DSP) certification process. There are many benefits to certification including authentication of the discipline within the overall pharmaceutical community and with regulatory authorities. It also encourages participation in SPS activities by other professionals (toxicologists, clinicians, academics) who wish to broaden their professional expertise. It provides an opportunity for candidates to strengthen their fundamental scientific knowledge, and stimulates the sharing of data, methods and model development in the form of publications and presentations on relevant topics in SP. Accreditation in SP occurs after candidates successfully complete a written certification examination conducted at the annual SPS meeting. The DSP exam consists primarily of material pertinent to the conduct of SP vital function core battery studies (i.e., cardiovascular, respiratory and central nervous systems), supplemental SP studies (i.e., renal/urinary, gastrointestinal, immunology, and hematology), Regulatory Guidelines (ICH Guidelines) as well as relevant cross-functional knowledge (e.g., physiology, pharmacology, toxicology, biochemistry, pathology, pharmacokinetics, dosing formulation, analytical methods, and statistics). Maintenance of the DSP certification results from the accrual of credits which are gained from a range of educational and scientific contributions. Eligibility requirements include a combination of at least a bachelor degree in science and two years of relevant professional SP experience and one poster presentation on a SP topic as first author at a recognized major scientific meeting.

SK&F 95654-induced acute cardiovascular toxicity in Sprague-Dawley rats--histopathologic, electron microscopic, and immunohistochemical studies.

The characteristics and pathogenesis of the cardiovascular toxicity induced by the type III selective phosphodiesterase inhibitor SK&F 95654 were examined in 2 studies. Sprague-Dawley rats received either a single sc injection of 50, 100, or 200 mg/kg SK&F 95654 and were euthanized at 24 hours after administration of the drug (Study 1), or were given a single subcutaneous (sc) injection of 100 mg/kg SK&F 95654 and euthanized at 1, 2, 4, 6, 8,12, 24 hours, or 2 weeks after treatment (Study 2). Control rats received either DMSO or saline. Myocardial lesions and vascular lesions of the mesentery, spleen, and pancreas were seen 24 hours after dosing with either 50,100, or 200 mg/kg SK&F 95654. The frequency and severity of these lesions (evaluated after the 100 mg/kg dose) increased with time over a period of 1 to 24 hours. By 2 weeks, the lesions subsided. Cardiac lesions consisted of myocyte necrosis with hypercontraction bands, inflammatory cell infiltration, interstitial hemorrhage, and interstitial edema. Vascular lesions of the mesentery were most prominent and consisted of vasodilatation and inflammation in the small-sized vessels, arterial medial necrosis and hemorrhage, and venous thrombosis. The vascular lesions included: leukocyte adhesion to endothelial cells, transendothelial migration of leukocytes, and inflammatory cell infiltration into vessel walls. Affected vessels included arteries, terminal arterioles, capillaries, postcapillary venules, and veins. Apoptosis of endothelial and smooth muscle cells was detected in the mesenteric vasculature by both TUNEL assay and electron microscopy. Evidence of endothelial cell activation in the mesenteric arteries and veins was also observed by electron microscopy. Immunohistochemical staining detected enhanced endothelial cell expression of intercellular adhesion molecule- 1 (ICAM- 1) and von Willebrand factor (vWF) in the mesenteric arteries and veins. Mast cells were noted to be more prevalent in affected mesenteric tissue from drug-treated animals. The present findings suggest that apoptosis of endothelial and smooth muscle cells, activation of endothelial cells, recruitment of mast cells, and increased expression of adhesion molecules are important factors to the overall pathogenesis of SK&F 95654-induced vasculitis.