Detecting drug-induced changes in ECG parameters using jacketed telemetry: Effect of different data reduction techniques.

INTRODUCTION: Continuous cardiovascular data is routinely collected during preclinical safety assessment of new medicines. This generates large datasets, which must be summarised to analyse and interpret drug effects. We assessed four methods of data reduction of continuous electrocardiogram (ECG) data and examined the impact on the statistical power of the assay. METHODS: Continuous ECG data were collected from a validation study in 6 cynomolgus monkeys using jacketed telemetry. Animals received either vehicle or vehicle followed by ascending doses of moxifloxacin each on a different dosing day. Recordings made for 25h on each dosing day were reduced to discrete time-points using: 1-min average snapshots, 15-min average snapshots, large duration averages (0.5-4h) or super-intervals (3.5-9h averages). RESULTS: There was no difference in the ability to detect moxifloxacin-induced QTc prolongation between the 1- and 15-min snapshots and the large duration averages data reduction methods (minimum detectable change in QTca of 20, 17 and 18ms, respectively at 80% power). The super-intervals method detected slightly smaller changes in QTc (15ms), but did not detect a statistically significant increase in QTc after the lowest dose of moxifloxacin, in contrast to the other methods. There were fewer statistically significant differences between dosing days in animals given vehicle when the large duration averages and super-interval reduction techniques were used. DISCUSSION: There is no marked difference in the power of detection of drug-induced ECG changes in cynomolgus monkeys when using either small duration average or large duration average data reduction techniques. Use of larger duration averages or super-intervals may facilitate data interpretation by reducing the incidence of spurious significant differences that occur by chance between dosing days.

Assessment of extracellular field potential and Ca2+ transient signals for early QT/pro-arrhythmia detection using human induced pluripotent stem cell-derived cardiomyocytes.

Cardiovascular toxicity is a prominent reason for failures in drug development, resulting in the demand for assays that can predict this liability in early drug discovery. We investigated whether iCell® cardiomyocytes have utility as an early QT/TdP screen. Thirty clinical drugs with known QT/TdP outcomes were evaluated blind using label-free microelectrode array (parameters measured were beating period (BP), field potential duration (FPD), fast Na+ amplitude and slope) and live cell, fast kinetic fluorescent Ca2+ transient FLIPR® Tetra (parameters measured were peak count, width, amplitude) systems. Many FPD-altering drugs also altered BP. Correction for BP, using a Log-Log (LL) model, was required to appropriately interpret direct drug effects on FPD. In comparison with human QT effects and when drug activity was to be predicted at top test concentration (TTC), LL-corrected FPD and peak count had poor assay sensitivity and specificity values: 13%/64% and 65%/11%, respectively. If effective free therapeutic plasma concentration (EFTPC) was used instead of TTC, the values were 0%/100% and 6%/100%, respectively. When compared to LL-corrected FPD and peak count, predictive values of uncorrected FPD, BP, width and amplitude were not much different. If pro-arrhythmic risk was to be predicted using Ca2+ transient data, the values were 67%/100% and 78%/53% at EFTPC and TTC, respectively. Thus, iCell® cardiomyocytes have limited value as an integrated QT/TdP assay, highlighting the urgent need for improved experimental alternatives that may offer an accurate integrated cardiomyocyte safety model for supporting the development of new drugs without QT/TdP effects.

Prevention of fostamatinib-induced blood pressure elevation by antihypertensive agents.

Fostamatinib is a tyrosine kinase inhibitor with activity against spleen tyrosine kinase which has completed clinical trials for patients with rheumatoid arthritis. In clinical studies fostamatinib treatment was associated with a small elevation of systemic arterial blood pressure (BP), a similar finding to that seen with other kinase inhibitors, especially those that inhibit VEGFR2 signaling. We have investigated the link between fostamatinib-induced blood pressure elevation and plasma levels of the fostamatinib-active metabolite R940406 in conscious rats and found the time course of the BP effect correlated closely with changes in R940406 plasma concentration, indicating a direct pharmacological relationship. Free plasma levels of R940406 produced in these studies (up to 346 nmol/L) span the clinically observed mean peak free plasma concentration of 49 nmol/L. We have demonstrated that the blood pressure elevation induced by fostamatinib dosing can be successfully controlled by a variety of methods, notably simple drug withdrawal or codosing with a range of standard antihypertensive agents such as atenolol, captopril, and nifedipine. These findings support potential methods of maintaining patient safety while on fostamatinib therapy. Furthermore, we have demonstrated, using nifedipine as an example agent, that this blood pressure control was not achieved by reduction in plasma exposure of R940406, suggesting that potential benefits from the pharmacology of the investigational drug can be maintained while blood pressure control is managed by use of standard comedications.

Effects of group housing on ECG assessment in conscious cynomolgus monkeys.

INTRODUCTION: Assessing the cardiovascular safety of new chemical or biological entities is important during pre-clinical development. Electrocardiogram (ECG) assessments in non-human primate (NHP) toxicology studies are often made using non-invasive telemetry systems. We investigated whether ECG recording was feasible during group housing of NHPs, rather than the usual single housed arrangement, and whether it would impact the data collected or affect the ability to detect drug-induced changes in QTc interval. METHODS: Following a period of acclimatisation to jackets, cynomolgus monkeys (3 males and 3 females) were housed in same sex groups of 3. Female monkeys were administered 4 doses of vehicle while male monkeys were administered vehicle, 15, 45, and 135mg/kg moxifloxacin. Each dose was administered on a separate dosing day. The same dosing protocol was repeated with the animals singly housed and the results from the two phases were compared including assessment of statistical power. RESULTS: Heart rate (HR) was significantly lower, and PR and QT intervals were significantly higher, at multiple time points when the animals were group housed compared with the singly housed phase. QRS duration and QTc interval were less affected. Moxifloxacin increased QT and QTc intervals but had no consistent effect on HR, QRS duration or PR interval under group housed or singly housed conditions. Power analysis suggested that group housing did not adversely affect the magnitude of detectable changes of ECG parameters. In general, detection of slightly smaller changes was achieved under conditions of group housing. DISCUSSION: The current study shows group housing to be technically possible during non-invasive ECG recording, resulting in lower resting heart rates and small improvements in sensitivity of detection of drug-induced effects. Given the psychological benefits of group housing for NHPs, it is a refinement that should be considered when conducting ECG assessments in NHP toxicology studies.

Effects of group housing on ECG assessment in conscious cynomolgus monkeys.

INTRODUCTION: Assessing the cardiovascular safety of new chemical or biological entities is important during pre-clinical development. Electrocardiogram (ECG) assessments in non-human primate (NHP) toxicology studies are often made using non-invasive telemetry systems. We investigated whether ECG recording was feasible during group housing of NHPs, rather than the usual single housed arrangement, and whether it would impact the data collected or affect the ability to detect drug-induced changes in QTc interval. METHODS: Following a period of acclimatisation to jackets, cynomolgus monkeys (3 males and 3 females) were housed in same sex groups of 3. Female monkeys were administered 4 doses of vehicle whilst male monkeys were administered vehicle, 15, 45 and 135mg/kg moxifloxacin. Each dose was administered on a separate dosing day. The same dosing protocol was repeated with the animals singly housed and the results from the two phases were compared including assessment of statistical power. RESULTS: Heart rate (HR) was significantly lower, and PR and QT interval significantly higher, at multiple time points when the animals were group housed compared with the singly housed phase. QRS duration and QTc interval were less affected. Moxifloxacin increased QT and QTc intervals but had no consistent effect on HR, QRS duration or PR interval under group housed or singly housed conditions. Power analysis suggested that group housing did not adversely affect the magnitude of detectable changes of ECG parameters. In general, detection of slightly smaller changes was achieved under conditions of group housing. DISCUSSION: The current study shows group housing to be technically possible during non-invasive ECG recording, resulting in lower resting heart rates and small improvements in sensitivity of detection of drug-induced effects. Given the psychological benefits of group housing for NHPs, it is a refinement that should be considered when conducting ECG assessments in NHP toxicology studies.

Enhanced characterization of contractility in cardiomyocytes during early drug safety assessment.

We sought to investigate whether drug-induced changes in contractility were affected by pacing rates that represent the range of heart rates encountered in vivo. Using the cell geometry measurement system (IonOptix), we paced dog cardiomyocytes at different cycle lengths (CLs) of 2000, 1000, 500, and 333.3 ms, before and after exposure to 13 inotropic drugs. Time course data using vehicle control (0.1% dimethyl sulfoxide (DMSO)) demonstrated stability of the system at all CLs tested. Seven positive inotropes (eg isoproterenol) exerted rate-dependent increases in sarcomere shortening (Sarc. short.; maximal effect at a CL of 333.3 ms [0.1 µM isoproterenol increased Sarc. short. by 41.1% and 145.9% at 2000 and 333.3 ms, respectively]). Omecamtiv mecarbil showed an atypical profile (increased Sarc. short. at 2000 ms [106.9%] and decreased at 333.3 ms [IC(50) = 0.64 µM]). Four negative inotropes (eg flecainide) showed rate-independent inhibition of Sarc. short. (IC(50)s: 3.3 µM [2000 ms] versus 2.3 µM [333.3 ms]). The remaining negative inotropes, verapamil, and BTS (N-benzyl-p-toluene sulphonamide) produced an increase (IC(50)s: 3.9 µM [2000 ms] versus 0.043 µM [333.3ms]) and decrease (IC(50)s: 18.3 µM [2000 ms] versus 34.0 µM [333.3 ms]) in potency, respectively. Negative inotropes (eg flecainide, BTS, and verapamil) decreased the area of the Ca(2+) transient versus Sarc. short. hysteresis loop, although rate dependency was seen with verapamil only. Positive inotropes (eg isoproterenol and levosimendan) induced a rate-dependent increase in the area, however Omecamtiv mecarbil increased and decreased the area at CLs of 2000 and 333.3 ms, respectively. Thus, the use of different pacing rates may improve the detection of inotropes in early drug discovery and illustrate the potential for finger-printing different mechanisms of action.

Preservation of cardiomyocytes from the adult heart.

Cardiomyocytes represent one of the most useful models to conduct cardiac research. A single adult heart yields millions of cardiomyocytes, but these cells do not survive for long after isolation. We aimed to determine whether inhibition of myosin II ATPase that is essential for muscle contraction may preserve fully differentiated adult cardiomyocytes. Using inhibitors of the myosin II ATPase, blebbistatin and N-benzyl-p-toluene sulphonamide (BTS), we preserved freshly isolated fully differentiated adult primary cardiomyocytes that were stored at a refrigerated temperature. Specifically, preserved cardiomyocytes stayed viable for a 2-week period with a stable expression of cardiac genes and retained the expression of key markers characteristic of cardiomyocytes. Furthermore, voltage-clamp, action potential, calcium transient and contractility studies confirmed that the preserved cardiomyocytes are comparable to freshly isolated cells. Long-term exposure of preserved cardiomyocytes to four tyrosine kinase inhibitors, sunitinib malate, dasatinib, sorafenib tosylate and imatinib mesylate, revealed their potential to induce cardiac toxicity that was manifested with a decrease in contractility and induction of cell death, but this toxicity was not observed in acute experiments conducted over the time course amenable to freshly prepared cardiomyocytes. This study introduces the concept that the inhibition of myosin II ATPase safeguards the structure and function of fully differentiated adult cardiomyocytes. The fact that these preserved cardiomyocytes can be used for numerous days after preparation makes them a robust and versatile tool in cardiac research and allows the investigation of long-term exposure to novel drugs on cardiomyocyte function.

A multi-site comparison of in vivo safety pharmacology studies conducted to support ICH S7A & B regulatory submissions.

INTRODUCTION: Parts A and B of the ICH S7 guidelines on safety pharmacology describe the in vivo studies that must be conducted prior to first time in man administration of any new pharmaceutical. ICH S7A requires a consideration of the sensitivity and reproducibility of the test systems used. This could encompass maintaining a dataset of historical pre-dose values, power analyses, as well as a demonstration of acceptable model sensitivity and robust pharmacological validation. During the process of outsourcing safety pharmacology studies to Charles River Laboratories, AstraZeneca set out to ensure that models were performed identically in each facility and saw this as an opportunity to review the inter-laboratory variability of these essential models. METHODS: The five in vivo studies outsourced were the conscious dog telemetry model for cardiovascular assessment, the rat whole body plethysmography model for respiratory assessment, the rat modified Irwin screen for central nervous system assessment, the rat charcoal meal study for gastrointestinal assessment and the rat metabolic cage study for assessment of renal function. Each study was validated with known reference compounds and data were compared across facilities. Statistical power was also calculated for each model. RESULTS: The results obtained indicated that each of the studies could be performed with comparable statistical power and could achieve a similar outcome, independent of facility. DISCUSSION: The consistency of results obtained from these models across multiple facilities was high thus providing confidence that the models can be run in different facilities and maintain compliance with ICH S7A and B.

The rat bone marrow micronucleus test--study design and statistical power.

Although the rodent bone marrow micronucleus test has been in routine use for over 20 years, little work has been published to support its experimental design and all this has used the mouse rather than the rat. When it was decided to change the strain of rat routinely used in this laboratory to the Han Wistar, a preliminary study was performed to investigate the possible factors influencing experimental variability and to use statistical tools to examine possible study designs. Subsequently, a historical database comprising of vehicle controls accumulated from 65 studies was used to establish test acceptance criteria and a strategy for analysing equivocal results. The following conclusions were made: (i) no statistically significant differences were observed in experimental variability within or between control animals; although not statistically significant, the majority of experimental variability seen was found to be between separate counts on the same slide, with minimal differences found between duplicate slides from the same rat or between individual rats; (ii) power analyses showed that, if an equivocal result is obtained after scoring 2000 immature erythrocytes (IE), it is appropriate to re-code the slides and score an additional 4000 IE, i.e. analysing a total of 6000 IE; no meaningful increase in statistical power is gained by scoring >6000 IE; this is consistent with the variability observed between separate counts on the same slide; (iii) there was no significant difference between the control micronucleated immature erythrocyte (MIE) values at 24 and 48 h after dosing or between males and females; therefore, if an unusually low control value at either time point results in apparent small increases in MIE in a treated group, it is valid to pool control values from both time points for clarification and (iv) similar statistical power can be achieved by scoring 2000 IE from seven rats or 4000 IE from five rats, respectively. However, this is based only on control animals and does not consider possible differences in responses between animals to treatment with a potential genotoxin. In order to minimize the possible influence of responders and non-responders, the preferred study design in this laboratory is to score 2000 IE from groups of seven rats. Study data obtained over time confirmed observations made in the control study. Also from an ethical viewpoint, clarifying equivocal responses by combining control data from the 24- and 48-h time points and/or increasing the number of IE scored per animal has minimized the numbers of repeat studies necessary to determine the genotoxic status of a novel compound. However, before any laboratory can use these procedures, experimental data must be generated to demonstrate their validity.