Clinical and nonclinical safety liabilities remain a major cause of adverse drug reactions, candidate drug attrition, delays during development, labelling restrictions, non-approval, and product withdrawal. Many of the toxicities are functional in nature and/or in origin. Whereas pharmacological responses tend to be fairly rapid in onset, and are therefore detectable after a single dose, some diminish on repeated dosing, whereas others increase in magnitude and therefore can be missed or underestimated in single-dose safety pharmacology studies. Functional measurements can be incorporated into repeat-dose toxicity studies, either routinely or on an ad hoc basis. Drivers for this are both scientific (see above), and regulatory (e.g., ICH S6, S7, S9). There are inherent challenges in achieving this: the availability of suitable technical and scientific expertise in the test facility; unsuitable laboratory conditions; use of simultaneous (as opposed to staggered) dosing; requirement for toxicokinetic sampling; unsuitability of certain techniques (e.g., use of anaesthesia; surgical implantation; food restriction); equipment availability at close proximity; sensitivity of the methods to detect small, clinically relevant, changes. Nonetheless, 'fit-for-purpose' data can still be acquired without requiring additional animals. Examples include assessment of behaviour, sensorimotor, visual, and autonomic functions, ambulatory ECG and blood pressure, echocardiography, respiratory, gastrointestinal, renal and hepatic functions. This is entirely achievable if functional measurements are relatively unobtrusive, both with respect to the animals and to the toxicology study itself.Careful pharmacological validation of any methods used, and establishing their detection sensitivity, is vital to ensure the credibility of generated data.

Non-clinical QT-related assays aligned to the pharmaceutical drug discovery and development phases are used in several ways. During the early discovery phases, assays are used for hazard identification and wherever possible for hazard elimination. The data generated enable us to: (i) establish structure-activity relationships and thereby; (ii) influence the medicinal chemistry design and provide tools for effective decision making; and provide structure-activity data for in silico predictive databases; (iii) solve problems earlier; (iv) provide reassurance for compound or project to progress; and (v) refine strategies as scientific and technical knowledge grows. For compounds progressing into pre-clinical development, the 'core battery' QT-related data enable an integrated risk assessment to: (i) fulfil regulatory requirements; (ii) assess the safety and risk-benefit for compound progression to man; (iii) contribute to defining the starting dose during the phase I clinical trials; (iv) influence the design of the phase I clinical trials; (v) identify clinically relevant safety biomarkers; and (vi) contribute to the patient risk management plan. Once a compound progresses into clinical development, QT-related data can be applied in the context of risk management and risk mitigation. The data from 'follow-up' studies can be used to: (i) support regulatory approval; (ii) investigate discrepancies that may have emerged within and/or between non-clinical and clinical data; (iii) understand the mechanism of an undesirable pharmacodynamic effect; (iv) provide reassurance for progression into multiple dosing in humans and/or large-scale clinical trials; and (v) assess drug-drug interactions. Based on emerging data, the integrated risk assessment is then reviewed in this article, and the benefit-risk for compound progression was re-assessed. Project examples are provided to illustrate the impact of non-clinical data to support compound progression throughout the drug discovery and development phases, and regulatory approval.

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Sulfur mustard (SM) is known to induce cutaneous injury and to cause acute damage to the respiratory tract. Although skin vesication has been demonstrated on human epidermal keratinocytes in culture, no study has been carried out to analyze the effects of SM on the ultrastructural and functional activity of surface respiratory epithelial cells. To evaluate this SM toxicity, we developed an in vitro model of respiratory epithelial cells in primary culture. The study was performed on surface epithelial cells from rabbit trachea cultured according to the explant-outgrowth technique. The functional activity of the cultures was evaluated by measuring the ciliary beating frequency (CBF) of the ciliated cells with a videomicroscopic method. The morphological aspects of the cells were analyzed by light and electron microscopy. Addition of 0.1 mM SM directly into the culture medium produced a sudden and irreversible CBF inhibition, first observed after 2 hr on the ciliated cells of the outgrowth periphery. The arrest of the ciliary beating progressively reached the whole surface of the outgrowth and was simultaneously observed with a detachment of the outgrowth cells. It began at the outgrowth border, leading to the exfoliation of cell sheets, and then to the whole culture after 48 hr. Morphological damage was expressed by intense vacuolisation and disorganization of cytoplasmic and nuclear structures. These findings suggest that the detachment of the respiratory epithelial cells from the matrix represents a major toxic effect of 0.1 mM SM. SM dramatically affects the viability of respiratory epithelial cells in culture. Moreover, the sudden CBF inhibition is more likely due to the death of the ciliated cells than to a specific ciliotoxic effect of SM.

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