Louise Marks

The role of the anaesthetised guinea-pig in the preclinical cardiac safety evaluation of drug candidate compounds

Despite rigorous preclinical and clinical safety evaluation, adverse cardiac effects remain a leading cause of drug attrition and post-approval drug withdrawal. A number of cardiovascular screens exist within preclinical development. These screens do not, however, provide a thorough cardiac liability profile and, in many cases, are not preventing the progression of high risk compounds. We evaluated the suitability of the anaesthetised guinea-pig for the assessment of drug-induced changes in cardiovascular parameters. Sodium pentobarbitone anaesthetised male guinea-pigs received three 15 minute intravenous infusions of ascending doses of amoxicillin, atenolol, clonidine, dobutamine, dofetilide, flecainide, isoprenaline, levosimendan, milrinone, moxifloxacin, nifedipine, paracetamol, verapamil or vehicle, followed by a 30 minute washout. Dose levels were targeted to cover clinical exposure and above, with plasma samples obtained to evaluate effect/exposure relationships. Arterial blood pressure, heart rate, contractility function (left ventricular dP/dt(max) and QA interval) and lead II electrocardiogram were recorded throughout. In general, the expected reference compound induced effects on haemodynamic, contractility and electrocardiographic parameters were detected confirming that all three endpoints can be measured accurately and simultaneously in one small animal. Plasma exposures obtained were within, or close to the expected clinical range of therapeutic plasma levels. Concentration-effect curves were produced which allowed a more complete understanding of the margins for effects at different plasma exposures. This single in vivo screen provides a significant amount of information pertaining to the cardiovascular risk of drug candidates, ultimately strengthening strategies addressing cardiovascular-mediated compound attrition and drug withdrawal.

Optimising conditions for studying the acute effects of drugs on indices of cardiac contractility and on haemodynamics in anaesthetized guinea pigs.

INTRODUCTION Detecting adverse effects of drugs on cardiac contractility is becoming a priority in pre-clinical safety pharmacology. The aim of this work was to optimise conditions and explore the potential of using the anaesthetized guinea pig as an in vivo model. METHODS Guinea pigs were anaesthetized with Hypnorm/Hypnovel, isoflurane, pentobarbital or fentanyl/pentobarbital. The electrocardiogram (ECG), heart rate, arterial blood pressure and indices of cardiac contractility were recorded. In further experiments in fentanyl/pentobarbital anaesthetized guinea pigs the influence of bilateral versus unilateral carotid artery occlusion on haemodynamic responses was investigated and the effects of inotropic drugs on left ventricular (LV) dP/dt(max) and the QA interval were determined. RESULTS Pentobarbital, given alone or after fentanyl, provided suitable anaesthesia for these experiments. Bilateral carotid artery occlusion did not alter heart rate or arterial blood pressure responses to isoprenaline or angiotensin II. Isoprenaline and ouabain increased LVdP/dt(max) and decreased the QA interval whereas verapamil had opposite effects and strong inverse correlations between LVdP/dt(max) and the QA interval were found. DISCUSSION Conditions can be optimised to allow the pentobarbital-anaesthetized guinea pig to be used for simultaneous measurement of the effects of drugs on the ECG, haemodynamics and indices of cardiac contractility. The use of this small animal model in early pre-clinical safety pharmacology should contribute to improvements in detecting unwanted actions on the heart during the drug development process.

Incorporation of capillary microsampling into whole body plethysmography and modified Irwin safety pharmacology studies in rats

UNLABELLED Limited toxicokinetic data is generated during modified Irwin screen and whole body plethysmography safety pharmacology studies, due to disturbance of primary endpoints by standard blood sampling methods. We evaluated if incorporation of microsampling would impact on data quality, as providing toxicokinetic data from main test animals could reduce the need for satellite animals. METHODS A modified Irwin screen was performed, testing oral clonidine (0, 0.03, 0.1, or 0.3 mg/kg). One cohort of rats per dose level underwent standard blood sampling (post-4 h Irwin assessment), and another cohort underwent microsampling after the 0.5, 1, 2, 4 and 24 h Irwin assessments. The respiratory effects of oral theophylline (0 or 10 mg/kg) and oral baclofen (0 or 5 mg/kg), were tested using whole body plethysmography. Groups of animals underwent standard blood sampling (at end of recording at 4 h post-dose) or microsampling at either 0.5 h or 1 h intervals (4 or 8 microsamples, respectively). RESULTS Microsampling did not impact on the quality of the data generated. The expected effects of clonidine on behavioural parameters, and of theophylline and baclofen on changes in ventilatory parameters were observed at a similar magnitude and duration independent of sampling method. DISCUSSION The incorporation of multiple capillary microsamples into the modified Irwin or respiratory study did not adversely affect the primary endpoints. The capillary microsampling method is a refinement in blood sampling technique which can easily be adapted into safety pharmacology studies to generate pharmacokinetic profiles within the same animal as the primary data, thus enhancing scientific robustness and reducing the satellite animals required.

Gastrointestinal Safety Pharmacology in Drug Discovery and Development

Although the basic structure of the gastrointestinal tract (GIT) is similar across species, there are significant differences in the anatomy, physiology, and biochemistry between humans and laboratory animals, which should be taken into account when conducting a gastrointestinal (GI) assessment. Historically, the percentage of cases of drug attrition associated with GI-related adverse effects is small; however, this incidence has increased over the last few years. Drug-related GI effects are very diverse, usually functional in nature, and not limited to a single pharmacological class. The most common GI signs are nausea and vomiting, diarrhea, constipation, and gastric ulceration. Despite being generally not life-threatening, they can greatly affect patient compliance and quality of life. There is therefore a real need for improved and/or more extensive GI screening of candidate drugs in preclinical development, which may help to better predict clinical effects. Models to identify drug effects on GI function cover GI motility, nausea and emesis liability, secretory function (mainly gastric secretion), and absorption aspects. Both in vitro and in vivo assessments are described in this chapter. Drug-induced effects on GI function can be assessed in stand-alone safety pharmacology studies or as endpoints integrated into toxicology studies. In silico approaches are also being developed, such as the gut-on-a-chip model, but await further optimization and validation before routine use in drug development. GI injuries are still in their infancy with regard to biomarkers, probably due to their greater diversity. Nevertheless, several potential blood, stool, and breath biomarkers have been investigated. However, additional validation studies are necessary to assess the relevance of these biomarkers and their predictive value for GI injuries.

Abstract 3676: Influence of early toxicology assessment on the selection of azd9291

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Discovery toxicology is the concept of evaluating the safety of molecules early in the drug development process when there is chemical scope to refine candidate drug quality. The aim is to identify and mitigate safety liabilities to maximize the chance of clinical success. This poster describes the impact of discovery toxicology on the selection of AZD9291, an irreversible inhibitor of both EGFR-TKI sensitising and resistance mutations. Although, maintaining a margin of selectivity to wild type EGFR was a key medicinal chemistry goal, early in vitro selectivity evaluation of the lead series compounds revealed significant off-target inhibition against the insulin and insulin-like growth factor receptors (IR/IGFR). Further exploration of this liability in a rat in vivo study revealed that our early lead compound (Cmpd 1) was associated with severe hyperglycemia, rapid body weight loss and evidence of reduced gastric emptying. The reduction in bodyweight and the inhibition of gastric emptying was dose limiting and we hypothesized this was caused by the hyperglycaemia. A broader selection of compounds based on the same scaffold was tested both in vitro and in vivo in the hope of identifying one or more compounds without this liability. Compounds were tested in a modified single dose rat study, exploring insulin and glucose homeostasis and effects on gastric emptying. Compounds within the shortlist caused increases in insulin of up to 80-fold, 3-fold increases in glucose and up to a 60% reduction in gastric emptying. There was a clear link between in vitro IR/IGF inhibition, hyperinsulinemia and inhibition of gastric emptying. One of the shortlisted candidates (now selected as AZD9291), had up to a 30 fold improvement in in vitro IGFR cellular potency compared to previous candidates and had no effect on insulin/glucose homeostasis in vivo. AZD9291 has demonstrated tumor shrinkage in patients at doses that have been very well tolerated and to date, no effect on glucose homeostasis has been reported. View this table: Citation Format: Mark J. Anderton, Richard A. Ward, Paula Daunt, Anne Galer, Darren A. E. Cross, Louise Marks, M. Raymond V. Finlay. Influence of early toxicology assessment on the selection of azd9291. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3676. doi:10.1158/1538-7445.AM2014-3676

Provision of food and water in rodent whole body plethysmography safety pharmacology respiratory studies – Impact on animal welfare and data quality

INTRODUCTION We evaluated the feasibility of providing food and water to rodents during whole body plethysmography (WBP) studies as a welfare improvement to standard conditions. METHODS Male Han Wistar rats or CD1 mice (n=8) were placed in WBP chambers and respiratory parameters recorded for approximately 6h on four separate occasions. On each occasion the animals were exposed to a different plethysmography chamber environment using a randomised design: no food/water (the standard conditions), water bottle, hydrating gel and wet food. In a further session, rats (n=8) were administered theophylline, or vehicle and respiratory parameters measured in the plethysmography chamber containing wet food. RESULTS Respiratory parameters of rats were not significantly altered by the provision of water or food. Providing wet food resulted in reduced body weight loss. Administration of theophylline caused the expected increase in respiratory rate. When mice were given access to hydrating gel or wet food the respiratory parameters were significantly affected; respiratory rate and tidal volume were increased. Providing wet food resulted in reduced bodyweight loss. DISCUSSION The provision of food and water did not impact on respiratory parameters in rats placed in WBP chambers. When provided with wet food, rats lost less bodyweight. Therefore, to improve welfare conditions for rats during WBP respiratory studies wet food should be provided when appropriate to the study design. In mice, provision of food and water led to changes in respiratory parameters, therefore these improvements in welfare conditions are not suitable for mice.