Monica R. Metea

Evaluation of respiratory function in freely moving Beagle dogs using implanted impedance technology

INTRODUCTION The Safety Pharmacology ICH S7A guidelines mandate the preclinical evaluation of drug effects on respiratory function. Chronic measurements of potential drug effects are commonly performed in rodents due to lack of a viable alternative in large animals. Presently, although the value and validity of these standard methods cannot be refuted, each method presents inherent limitations; such as the introduction of restraint stress (e.g. head-out rodent plethysmography and the pneumotachograph-equipped dog face mask), or sensitivity issues (e.g. whole body plethysmography). Since these approaches may limit the number of time points tested or affect respiratory parameters, new and accurate methods are needed for assessing respiratory function in conscious, freely moving animals. METHODS We evaluated a new surgically implanted telemetry device, which adds an impedance sensor for the chronic measurement of respiratory parameters to the standard device used for safety pharmacology cardiovascular studies. The feasibility of the implantable device was assessed based on concordance of respiratory data with pneumotachograph-recorded parameters in conscious Beagle dogs following intravenous administration of a positive control (4 mg/kg doxapram). RESULTS Linear regression analysis of data collected under restrained conditions showed a high correlation (R(2) 0.95) between impedance-derived respiratory parameters (tidal volume and respiratory frequency) and direct measurements of respiration via pneumotachograph. The correlation was reproduced when animals were challenged under the same dosing regimen. Volume changes similar to those obtained during the restrained collection were observed during the ambulatory collection following doxapram administration. Calibration of impedance-based values was adequate using both individual and population-based baseline conversion factors, both approximating actual mean respiratory variables collected with the pneumotachograph. DISCUSSION The benefit of this model is the accurate, continuous measurement of respiratory endpoints in restrained, as well as ambulatory settings. Assessment of multiple physiological parameters collected concurrently and the use of population-based calibrations may enable the maximization of resources and shortened timelines in drug development.

In Vitro Screening for Seizure Liability Using Microelectrode Array Technology

Drug-induced seizure liabilities produce significant compound attrition during drug discovery. Currently available in vitro cytotoxicity assays cannot predict all toxicity mechanisms due to the failure of these assays to predict sublethal target-specific electrophysiological liabilities. Identification of seizurogenic and other electrophysiological effects at early stages of the drug development process is important to ensure that safe candidate compounds can be developed while chemical design is taking place, long before these liabilities are discovered in costly preclinical in vivo studies. The development of a high throughput and reliable in vitro assay to screen compounds for seizure liabilities would de-risk compounds significantly earlier in the drug discovery process and with greater dependability. Here we describe a method for screening compounds that utilizes rat cortical neurons plated onto multiwell microelectrode array plates to identify compounds that cause neurophysiological disruptions. Changes in 12 electrophysiological parameters (spike train descriptors) were measured after application of known seizurogenic compounds and the response pattern was mapped relative to negative controls, vehicle control and neurotoxic controls. Twenty chemicals with a variety of therapeutic indications and targets, including GABAA antagonists, glycine receptor antagonists, ion channel blockers, muscarinic agonist, δ-opioid receptor agonist, dopaminergic D2/adrenergic receptor blocker and nonsteroidal anti-inflammatory drugs, were tested to assess this system. Sixteen of the seventeen seizurogenic/neurotoxic compounds tested positive for seizure liability or neurotoxicity, moreover, different endpoint response patterns for firing rate, burst characteristics and synchrony that distinguished the chemicals into groups relating to target and seizurogenic response emerged from the data. The negative and vehicle control compounds had no effect on neural activity. In conclusion, the multiwell microelectrode array platform using cryopreserved rat cortical neurons is a highly effective high throughput method for reliably screening seizure liabilities within an early de-risking drug development paradigm.