The functional observational battery and modified Irwin test as global neurobehavioral assessments in the rat: Pharmacological validation data and a comparison of methods.

BACKGROUND: Evaluation of the effects of candidate drugs on the nervous system in preclinical safety pharmacology studies utilises a global neurobehavioral assessment, usually in the rat. This either takes the form of the functional observational battery (FOB) or modified Irwin Test, both of which evaluate effects across 4 functional domains: autonomic, neuromuscular, sensorimotor and behavioral. Although there is a great deal of overlap in the parameters they address, the two tests approach the assessments slightly differently. We undertook a broad pharmacological validation of both the FOB and the Irwin test, and compared the two outcomes. METHODS: Male rats (6 per treatment group) were used to assess each of 12 reference drugs alongside vehicle controls in separate FOB and Irwin studies. The drugs compared in the two study types were chlorpromazine, chlordiazepoxide, clonidine, baclofen, (+)-amphetamine, harmaline, 8-hydroxy-2-(di-n-propylamino)tetralin, buspirone, physostigmine, picrotoxin, yohimbine and atropine. There is a high degree of semantic equivalence in the parameters assessed in the autonomic domain between the two tests, with a lower degree of equivalence for neuromuscular and behavioral domains, whereas sensorimotor reflex testing in the FOB is far more extensive than in the Irwin test. RESULTS: Across the set of reference drugs, concordance between the two tests was generally good across the 4 functional domains at the 'domain' level (i.e., detecting 'an effect'), whereas there was generally a poor concordance at the individual parameter level. However, this was partially explained by variability between repeated studies on a single reference drug using the same test (FOB or Irwin). CONCLUSIONS: Both tests are 'fit-for-purpose' in detecting effects of candidate drugs on the nervous system. We would encourage the global safety pharmacology community to consider whether (a) the tests could be combined into one industry standard; (b) candidate drugs could be triaged according to CNS penetration, with the level of scrutiny in the CNS core battery assessment adjusted accordingly and (c) whether new home cage technology could be applied to semi-automate the preclinical neurobehavioral assessment.

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.

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.

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.

An evaluation of the non-invasive faecal pellet assessment method as an early drug discovery screen for gastrointestinal liability.

INTRODUCTION: Gastrointestinal adverse effects contribute significantly to drug attrition as well as reduced patient compliance. Determination of gastrointestinal liability early in a compound's preclinical development would be a valuable tool. We evaluated the non-invasive faecal pellet method in the rat, assessed the feasibility of adding the endpoint to other study types and investigated correlation with the charcoal meal method. METHODS: Han Wistar rats, pair housed in metabolism cages, received a single dose of vehicle, atropine, bethanechol, loperamide or metoclopramide. The number, weight and appearance of pellets produced were assessed over 10 h and at 24 h post-dose. The endpoint was also added to a modified Irwin screen (testing atropine, theophylline, clonidine, amphetamine, baclofen or quinine) and a whole body plethysmography study (testing theophylline or bethanechol). Pellets were collected from home cages out to 4 h post-dose (Irwin) or following a 45 minutes plethysmography session. To assess correlation with stomach emptying and intestinal transit charcoal meal data was generated where published data was not available. RESULTS: Atropine decreased, while bethanechol and metoclopramide increased the number and weight of faecal pellets produced. Atropine produced darker, harder pellets and bethanechol lighter, softer pellets. Loperamide reduced pellet production at later time points only. Theophylline increased (Irwin and plethysmography) and atropine (Irwin) decreased pellet number and weight. Effects were maximal at the T(max) and detected in all study environments. Primary data generation was not affected by pellet collection. Pellet findings were generally comparable to charcoal meal transit data, with compounds showing an inhibition (atropine, loperamide, amphetamine, baclofen, clonidine, quinine) or stimulation (bethanechol) in both models. DISCUSSION: We have demonstrated that the faecal pellet method can detect expected reference compound induced changes in pellet transit. The technique is a useful non-invasive 'add-on' to other study types allowing gastrointestinal effects to be flagged earlier in preclinical development.

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.

Psychiatric patient assault and staff victim gender: fifteen-year analysis of the Assaulted Staff Action Program (ASAP).

Extensive, largely cross-sectional research has documented the continued occurrence of patient assaults on male and female staff. Different studies report either male or female staff to be at highest risk. Studies of same/different gender assaults which might more fully answer this question have been few. In these latter studies both male and female staff were at high risk for same gender assaults. In community settings males were at risk from same gender assaults but females were at risk from assaults by both patient genders. The present 15-year longitudinal retrospective study examined same/different gender assaults over time. Since the health care system under study experienced several major policy changes during these years, data were also examined at 5-year intervals to assess the stability of findings across time. Male and female staff were at increased risk from same gender assaults over time in both inpatient and community settings. The findings and their implications are discussed. In addition, a cost-effective, comprehensive risk management strategy for containing assaults is outlined.