Russell Bialecki

A framework to assess the translation of safety pharmacology data to humans

This article outlines a strategy for collecting accurate data for the determination of the sensitivity, specificity and predictive value of safety pharmacology models. This entails performing a retrospective analysis on commonly used safety pharmacology endpoints and an objective assessment of new non-clinical models. Such assessments require a systematic quantitative analysis of safety pharmacology parameters as well as clinical Phase I adverse events. Once the sensitivity, specificity and predictive capacity of models have been determined, they can be aligned within specific phases of the drug discovery and development pipeline for maximal impact, or removed from the screening cascade altogether. Furthermore, data will contribute to evidence-based decision-making based on the knowledge of the model sensitivity and specificity. This strategy should therefore contribute to the reduction of candidate drug attrition and a more appropriate use of animals. More data are needed to increase the power of analysis and enable more accurate comparisons of models e.g. pharmacokinetic/phamacodynamic (PK/PD) relationships as well as non-clinical and clinical outcomes for determining concordance. This task requires the collaboration and agreement of pharmaceutical companies to share data anonymously on proprietary and candidate drugs.

Approaches to seizure risk assessment in preclinical drug discovery

Assessment of seizure risk traditionally occurs late in the drug discovery process using low-throughput, resource intensive in vivo assays. Such approaches do not allow sufficient time to mitigate risk by influencing chemical design. Early identification using cheaper, higher throughput assays with lower animal and compound requirements would be preferable. Here we review the current techniques available to assess this issue and describe how they may be combined in a rational step-wise cascade allowing more effective assessment of seizure risk.

Discovery of novel, orally active dual NK1/NK2 antagonists.

Exploration of the SAR around selective NK2 antagonists, SR48968 and ZD7944, led to the discovery that naphth-1-amide analogues provide potent dual NK1 and NK2 antagonists. ZD6021 inhibited binding of [3H]-NKA or [3H]-SP to human NK1 and NK2 receptors, with high-affinity (K(i)=0.12 and 0.62nM, respectively). In functional assays ZD6021 had, at 10(-7)M, in human pulmonary artery pK(B)=8.9 and in human bronchus pK(B)=7.3, for NK1 and NK2, respectively. Oral administration of ZD6021 to guinea pigs dose-dependently attenuated ASMSP induced extravasation of plasma proteins, ED(50)=0.5mg/kg, and NK2 mediated bronchoconstriction, ED(50)=13mg/kg.

Hypoxic exposure time dependently modulates endothelin- induced contraction of pulmonary artery smooth muscle

Endothelins (ETs) have been implicated in the pathogenesis of hypoxia-induced pulmonary hypertension. We determined whether hypoxic exposure of rats (10% O2-90% N2, 1 atm, 1-48 days) altered contraction to ET in isolated segments of endothelium-denuded extralobar branch pulmonary artery (PA) and aorta. Hypoxic exposure increased hematocrit, right ventricular hypertrophy, and ET-1 plasma concentration. Hypoxia also caused a sustained decrease in PA but not in aorta sensitivity to ET-1. In comparison, hypoxic exposure throughout 12 days decreased time dependently the maximum contraction of PA to ET-1, BaCl2, and KCl. The hypoxia-induced decrease in maximum contraction of PA to ET-1 returned toward normal levels by 21 days and approximated control levels by 48 days. After 14 days of hypoxia, right ventricular hypertrophy correlated with decreased sensitivity of PA to ET-1. After 21 days of hypoxia, PA sensitivity to ET-2 and ET-3 was decreased, and sarafotoxin S6c-induced contraction was abolished. In conclusion, hypoxic exposure time dependently modulates the responsiveness of PA smooth muscle to ETs, BaCl2, and KCl. The hypoxia-induced changes in tissue responsiveness to ET-1 may be associated with increased plasma concentrations of this peptide.Endothelins (ETs) have been implicated in the pathogenesis of hypoxia-induced pulmonary hypertension. We determined whether hypoxic exposure of rats (10% O2-90% N2, 1 atm, 1-48 days) altered contraction to ET in isolated segments of endothelium-denuded extralobar branch pulmonary artery (PA) and aorta. Hypoxic exposure increased hematocrit, right ventricular hypertrophy, and ET-1 plasma concentration. Hypoxia also caused a sustained decrease in PA but not in aorta sensitivity to ET-1. In comparison, hypoxic exposure throughout 12 days decreased time dependently the maximum contraction of PA to ET-1, BaCl2, and KCl. The hypoxia-induced decrease in maximum contraction of PA to ET-1 returned toward normal levels by 21 days and approximated control levels by 48 days. After 14 days of hypoxia, right ventricular hypertrophy correlated with decreased sensitivity of PA to ET-1. After 21 days of hypoxia, PA sensitivity to ET-2 and ET-3 was decreased, and sarafotoxin S6c-induced contraction was abolished. In conclusion, hypoxic exposure time dependently modulates the responsiveness of PA smooth muscle to ETs, BaCl2, and KCl. The hypoxia-induced changes in tissue responsiveness to ET-1 may be associated with increased plasma concentrations of this peptide.

The discovery of non-basic atrial natriuretic peptide clearance receptor antagonists. Part 1

The cyclic peptide ANP 4-23 and the linear peptide analogue AP-811 have been shown to be selective ANP-CR antagonists. Via alanine scanning and truncation studies we sought to determine which residues in these molecules were important in their binding to the clearance receptor and the relationship between these two molecules. These studies show that several modifications to these compounds are possible which improve physical properties of these molecules while retaining high affinity for the ANP-CR.

Assessment of cisplatin-induced kidney injury using an integrated rodent platform

Current diagnosis of drug-induced kidney injury (DIKI) primarily relies on detection of elevated plasma creatinine (Cr) or blood urea nitrogen (BUN) levels; however, both are indices of overall kidney function and changes are delayed with respect to onset of nephron injury. Our aim was to investigate whether early changes in new urinary DIKI biomarkers predict plasma Cr, BUN, renal hemodynamic and kidney morphological changes associated with kidney injury following a single dose of cisplatin (CDDP) using an integrated platform in rodent. Conscious surgically prepared male Han Wistar rats were given a single intraperitoneal dose of CDDP (15mg/kg). Glomerular filtration rate (GFR), effective renal plasma flow (ERPF), urinalysis, DIKI biomarkers, CDDP pharmacokinetics, blood pressures, heart rate, body temperature and electroencephalogram (EEG) were measured in the same vehicle- or CDDP-treated animals over 72h. Plasma chemistry (including Cr and BUN) and renal tissues were examined at study termination. Cisplatin caused progressive reductions of GFR, ERPF, heart rate and body temperature from day 1 (0-24h). DIKI biomarkers including alpha-glutathione S-transferase (α-GST) significantly increased as early as 6h post-dose, which preceded significant declines of GFR and ERPF (24h), increased plasma Cr and BUN (72h), and associated with renal acute tubular necrosis at 72h post-dose. The present study adds to the current understanding of CDDP action by demonstrating that early increases in urinary excretion of α-GST predict DIKI risk following acute exposure to CDDP in rats, before changes in traditional DIKI markers are evident.

Combining radio telemetry and automated blood sampling: a novel approach for integrative pharmacology and toxicology studies.

INTRODUCTION A novel automated blood sampling and telemetry (ABST) system was developed to integrate pharmacokinetic (PK), pharmacodynamic (PD) and toxicology studies. The goals of this investigation were to determine: 1) optimal feeding conditions and minimal acclimation times for recording PD parameters (blood pressure, heart rate, and temperature) after animals arrived on-site; 2) stress hormone levels in ABST-housed rats; 3) the feasibility of simultaneously recording cardiovascular parameters with electroencephalogram (EEG); 4) the equivalence of renal endpoints from ABST-housed rats with those in the metabolic cage, and 5) the cardiovascular responses to baclofen. METHODS Body weight, blood pressure, temperature, stress biomarkers, urine chemistries, renal biomarkers and responses to vehicle or baclofen (10mg/kg) were compared in awake and freely moving rats housed in the ABST system, home cage (HC) or metabolic cage. RESULTS Fasted rats lost 5+/-1% and 7+/-1% body weight when housed in ABST and metabolic cages, respectively. Weight loss was reversed by supplementing regular diet with hydration and nutritional supplements. Based on PD parameters, the minimum acclimation time required for both ABST and HC rats was 3days. The feasibility of simultaneously measuring multiple parameters, such as EEG with cardiovascular parameters in ABST was demonstrated. Renal function and biomarkers in rats continuously housed in the ABST and metabolic cages were equivalent (p>0.05) on days 1, 3, and 7. Baclofen-induced quantitatively and qualitatively similar (p>0.05) PK, mean arterial pressure, heart rate and temperature in ABST- and HC-housed rats. DISCUSSION These studies demonstrate for the first time that drug-induced PD responses can be recorded simultaneously with time-matched pharmacokinetic, biochemical and metabolic parameters in the same animal. The ABST system has the added advantage of blood sampling without the need for satellite PK animals. ABST is a useful and novel tool for establishing efficacy and safety margins using an in vivo integrative pharmacology approach.

Low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine causes inflammatory activation of astrocytes in nuclear factor-κB reporter mice prior to loss of dopaminergic neurons.

Neuroinflammation is implicated in the progression of numerous disease states of the CNS, but early inflammatory signaling events in glial cells that may predispose neurons to injury are not easily characterized in vivo. To address this question, we exposed transgenic mice expressing a nuclear factor‐κB (NF‐κB)‐driven enhanced green fluorescent protein (EGFP) reporter construct to low doses of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and examined inflammatory activation of astrocytes in relation to neurobehavioral and neuropathological outcomes. The highest dose of MPTP (60 mg/kg total dose) caused a decrease in locomotor activity and a reduction in stride length. No significant loss of dopaminergic neurons in the substantia nigra was apparent at any dose. In contrast, expression of tyrosine hydroxylase in striatal fibers was reduced at 60 mg/kg MPTP, as were levels of dopamine and DOPAC. Colocalized expression of EGFP and inducible nitric oxide synthase (NOS2) occurred in astrocytes at 30 and 60 mg/kg MPTP and was associated with increased protein nitration in nigral dopaminergic neurons. Inhibition of NF‐κB in primary astrocytes by expression of mutant IκBα suppressed expression of NOS2 and protected cocultured neurons from astrocyte‐mediated apoptosis. These data indicate that inflammatory activation of astrocytes and enhanced nitrosative stress occurs at low doses of MPTP prior to loss of dopaminergic neurons. NF‐κB‐mediated expression of NOS2 appears to be a sensitive indicator of neuroinflammation that correlates with MPTP‐induced neurochemical and neurobehavioral deficits prior to loss of dopaminergic neurons in the subtantia nigra.

An integrative pharmacological approach to radio telemetry and blood sampling in pharmaceutical drug discovery and safety assessment

BackgroundA successful integration of the automated blood sampling (ABS) and telemetry (ABST) system is described. The new ABST system facilitates concomitant collection of physiological variables with blood and urine samples for determination of drug concentrations and other biochemical measures in the same rat without handling artifact.MethodIntegration was achieved by designing a 13 inch circular receiving antenna that operates as a plug-in replacement for the existing pair of DSIs orthogonal antennas which is compatible with the rotating cage and open floor design of the BASi Culex® ABS system. The circular receiving antennas electrical configuration consists of a pair of electrically orthogonal half-toroids that reinforce reception of a dipole transmitter operating within the coils interior while reducing both external noise pickup and interference from other adjacent dipole transmitters.ResultsFor validation, measured baclofen concentration (ABST vs. satellite (μM): 69.6 ± 23.8 vs. 76.6 ± 19.5, p = NS) and mean arterial pressure (ABST vs. traditional DSI telemetry (mm Hg): 150 ± 5 vs.147 ± 4, p = NS) variables were quantitatively and qualitatively similar between rats housed in the ABST system and traditional home cage approaches.ConclusionThe ABST system offers unique advantages over traditional between-group study paradigms that include improved data quality and significantly reduced animal use. The superior within-group model facilitates assessment of multiple physiological and biochemical responses to test compounds in the same animal. The ABST also provides opportunities to evaluate temporal relations between parameters and to investigate anomalous outlier events because drug concentrations, physiological and biochemical measures for each animal are available for comparisons.

CNS Adverse Effects: From Functional Observation Battery/Irwin Tests to Electrophysiology.

This chapter describes various approaches for the preclinical assessment of drug-induced central nervous system (CNS) adverse effects. Traditionally, methods to evaluate CNS effects have consisted of observing and scoring behavioral responses of animals after drug is administered. Among several behavioral testing paradigms, the Irwin and the functional observational battery (FOB) are the most commonly used assays for the assessment of CNS effects. The Irwin and FOB are considered good first-tier assays to satisfy the ICH S7A guidance for the preclinical evaluation of new chemical entities (NCE) intended for humans. However, experts have expressed concern about the subjectivity and lack of quantitation that is derived from behavioral testing. More importantly, it is difficult to gain insight into potential mechanisms of toxicity by assessing behavioral outcomes. As a complement to behavioral testing, we propose using electrophysiology-based assays, both in vivo and in vitro, such as electroencephalograms and brain slice field-potential recordings. To better illustrate these approaches, we discuss the implementation of electrophysiology-based techniques in drug-induced assessment of seizure risk, sleep disruption, and cognitive impairment.