The evolving role of investigative toxicology in the pharmaceutical industry.

For decades, preclinical toxicology was essentially a descriptive discipline in which treatment-related effects were carefully reported and used as a basis to calculate safety margins for drug candidates. In recent years, however, technological advances have increasingly enabled researchers to gain insights into toxicity mechanisms, supporting greater understanding of species relevance and translatability to humans, prediction of safety events, mitigation of side effects and development of safety biomarkers. Consequently, investigative (or mechanistic) toxicology has been gaining momentum and is now a key capability in the pharmaceutical industry. Here, we provide an overview of the current status of the field using case studies and discuss the potential impact of ongoing technological developments, based on a survey of investigative toxicologists from 14 European-based medium-sized to large pharmaceutical companies.

Is there a role for the no observed adverse effect level in safety pharmacology?

In nonclinical toxicology the highest dose or exposure without test article-related adverse effects, known as the No Observed Adverse Effect Level (NOAEL), is a variable that may be determined. In safety pharmacology the vast majority of the endpoints measured are quantitative numeric functional endpoints such as changes in heart rate, blood pressure or respiratory frequency, endpoints that are usually not assessed using a defined framework of adversity. Therefore, we asked the question: is there a role for the NOAEL in safety pharmacology? To help answer this question, we conducted a survey via the Safety Pharmacology Society. We found that within safety pharmacology there is no formal definition of adversity and no guidance on defining NOAEL. We also found, perhaps unsurprisingly, there is no agreed rubric for using a NOAEL in safety pharmacology and we learned that the NOAEL is not a requirement in order to progress a new investigational drug through the regulatory process. Thus, a summary label such as NOAEL lacks nuance and disregards context in relation to the nature and the severity of the safety pharmacology findings. Consequently, defining 'adversity' and determining a NOAEL in safety pharmacology studies are not recommended since the range of functional endpoints investigated do not conform to a binary 'toxic/non-toxic' rubric. Focusing on describing test article-related effects on safety pharmacology endpoints, using reasoned arguments as part of an integrated risk assessment, will ensure that the clinical pharmacologists and regulatory bodies see a clear description of relevant findings at each dose or exposure level.

Optimizing drug discovery by Investigative Toxicology: Current and future trends.

Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an "Investigative Toxicology Think-Tank", which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology's position by identifying key challenges and perspectives.

Novel selective PDE type 1 inhibitors cause vasodilatation and lower blood pressure in rats.

BACKGROUND AND PURPOSE: The PDE enzymes (PDE1-11) hydrolyse and thus inactivate cyclic nucleotides and are important in the regulation of the cardiovascular system. Here,we have investigated the effects on the cardiovascular system, of two novel selective PDE1 inhibitors, Lu AF41228 and Lu AF58027. EXPERIMENTAL APPROACH: We used rat mesenteric small arteries (internal diameters of 200-300 µm), RT-PCR and measured isometric wall tension. Effects of Lu AF41228 and Lu AF58027 on heart rate and BP were assessed in both anaesthetized and conscious male rats. KEY RESULTS: Nanomolar concentrations of Lu AF41228 and Lu AF58027 inhibited PDE1A, PDE1B and PDE1C enzyme activity, while micromolar concentrations were required to observe inhibitory effects at other PDEs. RT-PCR revealed expression of PDE1A, PDE1B and PDE1C in rat brain, heart and aorta, but only PDE1A and PDE1B in mesenteric arteries. In rat isolated mesenteric arteries contracted with phenylephrine or U46619, Lu AF41228 and Lu AF58027 induced concentration-dependent relaxations which were markedly reduced by inhibitors of guanylate cyclase, ODQ, and adenylate cyclase, SQ22536, and in preparations without endothelium. In anaesthetized rats, Lu AF41228 and Lu AF58027 dose-dependently lowered mean BP and increased heart rate. In conscious rats with telemetric pressure transducers, repeated dosing with Lu AF41228 lowered mean arterial BP 10-15 mmHg and increased heart rate. CONCLUSIONS AND IMPLICATIONS: These novel PDE1 inhibitors induce vasodilation and lower BP, suggesting a potential use of these vasodilators in the treatment of hypertension and vasospasm.

Ion currents of cardiomyocytes in different regions of the Göttingen minipig heart.

INTRODUCTION: The Göttingen minipig is a promising model for pharmacological safety assessment and for translational research in cardiology. We have examined the main ion currents in cardiomyocytes of the minipig heart. METHODS: Cardiac cells were isolated from different cardiac regions (endo-, mid- and epicardial left ventricle and right ventricle) from Göttingen minipigs and examined using the whole cell patch clamp technique combined with pharmacological interventions. RESULTS: The inward rectifier (IK1), the delayed rectifier (IK), with the rapid and slow components, (IKr, IKs) and the L-type Ca2+ channel (ICa,L) were identified in the different regions of the heart, whereas the Ca2+-independent transient outward current (Ito1) was observed in only a few cells. IK1 was similar in the cardiac regions with a slightly lower value in the epicardial cells. IKs was smaller in epi- and endo-cardial regions. DISCUSSION: The equivalents of the main human cardiac ion currents are present in the minipig cardiomyocytes with the exception of the Ca2+-independent Ito1. The study provides further evidence that the minipig is a valid model for investigating cardiovascular pharmacology.

Assessment of anti-arrhythmic activity of antipsychotic drugs in an animal model: influence of non-cardiac α1-adrenergic receptors.

Torsades de Pointes (TdP) is a potentially lethal cardiac arrhythmia and a known adverse effect of many drugs secondary to block of the rapidly activating delayed rectifier potassium current (IKr). In animal models antipsychotic drugs have shown reduced pro-arrhythmic potential compared to drugs with comparable IKr-blocking characteristics. The reduced pro-arrhythmic properties of antipsychotic drugs has been attributed to a variety of different causes e.g., effects on α1-adrenergic receptors, ß-adrenergic receptors, muscarinic receptors or cardiac ion channels like Ca(2+)- and Na(+)-channels. Since only limited experimental information exists about the effects of α1-adrenergic receptor activity of antipsychotic drugs in pro-arrhythmic models, we have decided to investigate this. In this study we show that four antipsychotic drugs all have high affinity for α1-adrenergic receptor (sertindole>risperidone>haloperidol>olanzapine) and all block IKr (sertindole>haloperidol>risperidone>olanzapine). In canine Purkinje fibres, α1-adrenergic stimulation prolonged action potential duration; however, the stimulation does not cause afterdepolarizations, even in the presence of dofetilide-induced delayed repolarization. We showed for the first time in an in vivo pro-arrhythmic rabbit model that several antipsychotic drugs in accordance with their known α1-adrenergic receptor blocking properties reduced the incidence of drug-induced TdP and that the overall ability of the antipsychotic drugs to prevent TdP was associated with prevention of methoxamine induced increase in blood pressure. Further investigations are required to clarify the relative importance of α1-adrenergic receptor antagonism in conjunction with the additional effects of antipsychotic drugs on various receptors and ion channels.

Validation of a high throughput flow cytometric in vitro micronucleus assay including assessment of metabolic activation in TK6 cells.

Genotoxicity is an unacceptable property for new drug candidates and we employ three screening assays during the drug discovery process to identify genotoxicity early and optimize chemical series. One of these methods is the flow cytometric in vitro micronucleus assay for which protocol optimizations have been described recently. Here, we report further validation of the assay in TK6 cells including assessment of metabolic activation. We first optimized assay conditions to allow for testing with and without metabolic activation in parallel in a 96-well plate format. Then, we tested a set of 48 compounds carefully selected to contain known in vivo genotoxins, nongenotoxins and drugs. Avoidance of irrelevant positives, a known issue with mammalian cell-based genotoxicity assays, is important to prevent early deselection of potentially promising compounds. Therefore, we enriched the validation set with compounds that were previously reported to produce irrelevant positive results in mammalian cell-based genotoxicity assays. The resulting dataset was used to set the relevant cut-off values for scoring a compound positive or negative, such that we obtained an optimal balance of high sensitivity (88%) and high specificity (87%). Finally, we tested an additional set of 16 drugs to further probe assay performance and 14 of them were classified correctly. To our knowledge, the present study is the most comprehensive validation of the in vitro flow cytometric micronucleus assay and the first to report parallel assessment with metabolic activation in reasonable throughput. The assay allows for rapidly screening novel compounds for genotoxicity and is therefore well-suited for use in early drug discovery projects. Environ.

High-content analysis/screening for predictive toxicology: application to hepatotoxicity and genotoxicity.

High-content imaging/analysis has emerged as a powerful tool for predictive toxicology as it can be used for identifying and mitigating potential safety risks during drug discovery. By careful selection of end-points, some cellular assays can show better predictivity than routine animal toxicity testing for certain adverse events. Here, we present the perhaps most utilized high-content screening assays for predictive toxicology in the pharmaceutical industry. Multi-parametric imaging of cell health in simple and cost-effective model systems can be used to predict human hepatotoxicity and elucidate mechanisms of toxicity, and imaging of bile salt transport inhibition in sandwich-cultured hepatocytes can be used to predict cholestasis-inducing compounds. Imaging of micronuclei formation in simple cell models can be used to detect genotoxic potential and elucidate anuegenic or clastogenic mode of actions. The hope is that application of these relatively predictive assays during drug discovery will reduce toxicity and safety-related attrition of drug development programmes at later stages.

Exploratory toxicology as an integrated part of drug discovery. Part I: Why and how.

Toxicity and clinical safety have major impact on drug development success. Moving toxicological studies into earlier phases of the R&D chain prevents drug candidates with a safety risk from entering clinical development. However, to identify candidates without such risk, safety has to be designed actively. Therefore, we argue that toxicology should be fully integrated into the discovery process. We describe our strategy, including safety assessment of novel targets, selection of chemical series without inherent liabilities, designing out risk factors and profiling of candidates, and we discuss considerations regarding what to screen for. We aim to provide timely go/no-go decisions (fail early) and direction to the discovery teams, by steering away from safety risk (showing what will not fail).

Exploratory toxicology as an integrated part of drug discovery. Part II: Screening strategies.

In an effort to reduce toxicity-related attrition, different strategies have been implemented throughout the pharmaceutical industry. Previously (in Part I), we have outlined our 'integrated toxicology' strategy, which aims to provide timely go/no-go decisions (fail early) but also to show a direction to the drug discovery teams (showing what will not fail). In this review (Part II of the series) we describe our compound testing strategies with respect to cardiovascular safety, hepatotoxicity, genotoxicity, immunotoxicity and exploratory in vivo toxicity. We discuss the in vitro, ex vivo and in vivo assays and models we employ to assess safety risks and optimize compound series during the drug discovery process, including their predictivity and the decisions they generate.

A high content screening assay to predict human drug-induced liver injury during drug discovery.

INTRODUCTION: Adverse drug reactions are a major cause for failures of drug development programs, drug withdrawals and use restrictions. Early hazard identification and diligent risk avoidance strategies are therefore essential. For drug-induced liver injury (DILI), this is difficult using conventional safety testing. To reduce the risk for DILI, drug candidates with a high risk need to be identified and deselected. And, to produce drug candidates without that risk associated, risk factors need to be assessed early during drug discovery, such that lead series can be optimized on safety parameters. This requires methods that allow for medium-to-high throughput compound profiling and that generate quantitative results suitable to establish structure-activity-relationships during lead optimization programs. METHODS: We present the validation of such a method, a novel high content screening assay based on six parameters (nuclei counts, nuclear area, plasma membrane integrity, lysosomal activity, mitochondrial membrane potential (MMP), and mitochondrial area) using ~100 drugs of which the clinical hepatotoxicity profile is known. RESULTS DISCUSSION: We find that a 100-fold TI between the lowest toxic concentration and the therapeutic Cmax is optimal to classify compounds as hepatotoxic or non-hepatotoxic, based on the individual parameters. Most parameters have ~50% sensitivity and ~90% specificity. Drugs hitting ≥2 parameters at a concentration below 100-fold their Cmax are typically hepatotoxic, whereas non-hepatotoxic drugs typically hit <2 parameters within that 100-fold TI. In a zone classification model, based on nuclei count, MMP and human Cmax, we identified an area without a single false positive, while maintaining 45% sensitivity. Hierarchical clustering using the multi-parametric dataset roughly separates toxic from non-toxic compounds. We employ the assay in discovery projects to prioritize novel compound series during hit-to-lead, to steer away from a DILI risk during lead optimization, for risk assessment towards candidate selection and to provide guidance of safe human exposure levels.

Keeping the rhythm -- pro-arrhythmic investigations in isolated Göttingen minipig hearts.

INTRODUCTION: Cardiac arrhythmia is a potentially lethal condition. A better prediction and mechanistic understanding of human cardiac arrhythmia is dependent on the development of good animal models. Minipigs are increasingly being used in cardiovascular research and we hypothesize that Langendorff-perfused minipig hearts could serve as a novel model of pro-arrhythmia. METHODS: We studied arrhythmic biomarkers and overt arrhythmia using sharp electrode recordings and Langendorff-perfusion of minipig and dog hearts. Hearts were subjected to pharmacological blockade of repolarizing currents, chemical and surgical AV-block, and programmed electrical stimulation. RESULTS: Minipigs have significantly longer cardiac action potentials and QT intervals than dogs at comparable cycle lengths. In addition, minipigs were relatively resistant to arrhythmias in a variety of settings and arrhythmias were only observed in response to programmed electrical S1-S2 stimulation after surgical AV-ablation and combined I(Kr) and I(Ks) blockade. Accordingly, the short term variability of the action potential remained very low in minipigs. In addition, the electro-mechanical window (EMw) remained positive in both dog and minipig experiments, although the EMw approached negative values in dogs during I(Ks) blockade and ß-adrenergic stimulation. Isoprenaline- or pacing-induced changes in frequency resulted in paradoxical transient effects on repolarization. DISCUSSION: The evidence presented indicates that Langendorff-perfused Göttingen minipig hearts are resistant to the development of arrhythmias in experimental settings known to be pro-arrhythmic in other species including dogs. A possible mechanistic explanation could be the long electrical systole compared to dogs, combined with a short vulnerable window, recorded in minipig hearts. We also find that EMw recordings from Langendorff perfused hearts are less sensitive to I(Ks) blockade than what has been reported from in vivo experiments, most likely due to the mechanical properties of the recording apparatus.

Characterization of cardiac repolarization in the Göttingen minipig.

INTRODUCTION: The minipig represents an attractive experimental animal within cardiovascular research due to its extensive similarities to the human heart in terms of anatomy and physiology. Although minipigs have been used for cardiovascular research for decades no thorough characterization of the minipig cardiac electrophysiology has been performed. Therefore, we have for the first time characterized the minipig cardiac repolarization in a series of experiments ranging from mRNA quantification to in vivo studies. METHODS: Göttingen minipigs were used throughout the study. Cardiac mRNA quantification was performed using quantitative PCR methods. For ex vivo experiments, hearts were excised using cardioplegic procedures and Langendorff and microelectrode action potential recordings were performed. Effects of temperature in vivo were recorded in anesthetized animals. RESULTS: On the mRNA level the expression profile of major cardiac ion channel proteins in both atria and ventricle was very similar to what has been reported for humans. In both intact isolated heart and isolated endocardial strips the I(Kr) blocker dofetilide increased action potential duration (APD). The I(Ks) blocker HMR1556 increased APD and triangulation only when I(Kr) was blocked with dofetilide. In the presence of I(Kr) and I(Ks) blockade a reduction of [K+](e) resulted in a marked increase in APD(90) in isolated hearts. I(K1) blockade with Ba²+ increased APD in whole heart and isolated endocardium. In isolated endocardium, ß-adrenergic stimulation with isoprenaline resulted in an increase in APD and potential amplitude but a decrease in triangulation. There was a rate-dependent decrease in APD in both whole heart and isolated endocardium. In vivo and ex vivo investigations revealed a negative correlation between temperature and duration of cardiac repolarization. DISCUSSION: Our results point toward the minipig being a promising species for cardiac safety research.