In vitro pharmacological profiling is increasingly being used earlier in the drug discovery process to identify undesirable off-target activity profiles that could hinder or halt the development of candidate drugs or even lead to market withdrawal if discovered after a drug is approved. Here, for the first time, the rationale, strategies and methodologies for in vitro pharmacological profiling at four major pharmaceutical companies (AstraZeneca, GlaxoSmithKline, Novartis and Pfizer) are presented and illustrated with examples of their impact on the drug discovery process. We hope that this will enable other companies and academic institutions to benefit from this knowledge and consider joining us in our collaborative knowledge sharing.

Reperfusion of the ischemic myocardium results in the generation of oxygen-derived free radicals, NO, and presumably peroxynitrite. These, in turn, may cause strand breaks in DNA, which activate the nuclear enzyme poly(ADP ribose) synthetase (PARS). This results in a rapid depletion of intracellular NAD and ATP. When this reaction is excessive, there is ultimately cell death. Here we demonstrate that 3-aminobenzamide (and several other, chemically distinct, inhibitors of PARS activity) reduces the infarct size caused by ischemia and reperfusion of the heart or skeletal muscle of the rabbit. Inhibition of PARS activity also attenuates the myocardial dysfunction caused by global ischemia and reperfusion in the isolated, perfused heart of the rabbit. In skeletal muscle, inhibition of the activity of neuronal NO synthase reduces infarct size, indicating that the formation of NO contributes to the activation of PARS there. There is no significant neuronal NO synthase activity in the heart, and hence NO synthase inhibitors did not reduce myocardial infarct size. Thus, activation of PARS contributes to the cell death caused by ischemia-reperfusion, and PARS inhibitors may constitute a novel therapy for ischemia-reperfusion injury.Poly(ADP ribose) synthetase (PARS; EC 2.4.2.30) is a chromatin-bound enzyme, which plays a physiological role in the repair of strand breaks in DNA (1). PARS is located in the nuclei of cells of various tissues, including the heart and skeletal muscle (2). When activated by strand breaks in DNA, PARS catalyzes the transfer of ADP ribose moieties from NAD with the concomitant formation of nicotinamide. This results in a substantial depletion of NAD. Nicotinamide, which inhibits PARS activity by negative feedback, can be recycled to NAD in a reaction that consumes ATP. As NAD is essential to mitochondrial electron transport, depletion of NAD rapidly leads to a fall in ATP, and ultimately cell death (3-5). Radicals including superoxide anions, hydrogen peroxide or hydroxyl radicals (3-5), and NO or peroxynitrite (6-8) cause the breakage of DNA strands and activation of PARS. Inhibitors of PARS activity attenuate the fall in NAD and ATP and improve survival of cultured cells (e.g., fibroblasts, endothelial cells, and smooth muscle cells) exposed to oxygen-derived free radicals (3-5, 9), NO (6-8), or peroxynitrite (8). The formation of radicals contributes to the ''reperfusion injury'' of previously ischemic organs, including the heart (10) and the skeletal muscle (11). Here we demonstrate that several inhibitors of PARS activity reduce the infarct size caused by ischemia-reperfusion of the heart and skeletal muscle. MATERIALS AND METHODS Determination of Myocardial Infarct Size (in Vivo).Male New Zealand white rabbits were premedicated [Hypnorm (Janssen, Saunderton, U.K.), 0.1 ml⅐kg Ϫ1 i.m.; containing 0.315 mg⅐ml Ϫ1 fentanyl citrate and 10 mg⅐mlϪ1 fluanisone] and anesthetized (pentobarbitone, 20 mg⅐kg Ϫ1 i.v.). Following tracheotomy and ventilation (36-40 strokes⅐min Ϫ1 , tidal volume: 18...

Drug-induced liver injury (DILI) is a major cause of failed drug development, withdrawal and restricted usage. Therefore screening assays which aid selection of candidate drugs with reduced propensity to cause DILI are required. We have investigated the toxicity of 144 drugs, 108 of which caused DILI, using assays identified in the literature as having some predictivity for hepatotoxicity. The validated assays utilised either HepG2 cells, HepG2 cells in the presence of rat S9 fraction or isolated human hepatocytes. All parameters were quantified by multiplexed and automated high content fluorescence microscopy, at appropriate time points after compound administration (4, 24 or 48h). The individual endpoint which identified drugs that caused DILI with greatest precision was maximal fold induction in CM-H2DFFDA staining in hepatocytes after 24h (41% sensitivity, 86% specificity). However, hierarchical clustering analysis of all endpoints provided the most sensitive identification of drugs which caused DILI (58% sensitivity, 75% specificity). We conclude that multi-parametric high content cell toxicity assays can enable in vitro detection of drugs that have high propensity to cause DILI in vivo but that many DILI compounds exhibit few in vitro signals when evaluated using these assays.

No abstract

Functional changes to cardiomyocytes are a common cause of attrition in preclinical and clinical drug development. Current approaches to assess cardiomyocyte contractility in vitro are limited to low-throughput methods not amenable to early drug discovery. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) were used to assess their suitability to detect drug-induced changes in cardiomyocyte contraction. Application of field stimulation and measurement of cardiac contraction (IonOptix edge detection) and Ca(2+) transients confirmed hiPS-CMs to be a suitable model to investigate drug-induced changes in cardiomyocyte contractility. Using a live cell, fast kinetic fluorescent assay with a Ca(2+) sensitive dye to test 31 inotropic and 20 non-inotropic compounds in vivo, we report that hiPS-CMs provide a high-throughput experimental model to detect changes in cardiomyocyte contraction that is applicable to early drug discovery with a sensitivity and specificity of 87% and 70%, respectively. Moreover, our data provide evidence of the detection of this liability at therapeutically relevant concentrations with throughput amenable to influencing chemical design in drug discovery. Measurement of multiple parameters of the Ca(2+) transient in addition to the number of Ca(2+) transients offered no insight into the mechanism of cardiomyocyte contraction.

1 Poly (ADP-ribose) synthetase (PARS) is a nuclear enzyme activated by strand breaks in DNA which are caused by reactive oxygen species (ROS). Inhibitors of PARS activity reduce the degree of reperfusion injury of the heart in vivo and in vitro. Here we investigate the role of PARS in the cell death of human cardiac myoblasts caused by hydrogen peroxide. 2 Exposure of human cardiac myoblasts to hydrogen peroxide caused a time-and concentrationdependent reduction in mitochondrial respiration (cell injury), an increase in cell death (LDH release), as well as an increase in PARS activity. 3 The PARS inhibitors 3-aminobenzamide (3 mM), 1,5-dehydroxyisoquinoline (300 mM) or nicotinamide (3 mM) attenuated the cell injury and death as well as the increase in PARS activity caused by hydrogen peroxide (3 mM; 4 h for cell injury/death, 60 min for PARS activity) in human cardiac myoblasts. In contrast, the inactive analogues 3-aminobenzoic acid (3 mM) or nicotinic acid (3 mM) were without e ect. 4 The iron chelator deferoxamine (1 ± 10 mM) caused a concentration-dependent reduction in the cell injury and death caused by hydrogen peroxide in these human cardiac myoblasts. 5 Thus, the cell injury/death caused by hydrogen peroxide in human cardiac myoblasts is secondary to the formation of hydroxyl radicals and due to an increase in PARS activity. We therefore propose that activation of PARS contributes to the cell injury/cell death associated with oxidant stress in the heart.

Inhibition of the activity of PARS attenuates the cell death associated with oxidant stress in rat cardiac myoblasts and heart.

1 The cardioprotective properties of inhibition of poly (ADP-ribose) synthetase (PARS) were investigated in the isolated perfused heart of the rat. Hearts were perfused in the Langendor mode and subjected to 23 min total global ischaemia and reperfused for 60 min. 2 Left ventricular function was assessed by means of an intra-ventricular balloon. High energy phosphates were measured by 31 P-NMR spectroscopy. Intracellular levels of NAD were measured by capillary electrophoresis of perchloric acid extracts of hearts at the end of reperfusion. 3 Reperfusion in the presence of the PARS inhibitor 1,5 didroxyisoquinoline (ISO, 100 mM) attenuated the mechanical dysfunction observed following 1 h of reperfusion; 27+13 and 65+8% recovery of preischaemic rate pressure product for control and 100 mM ISO, respectively. 4 This cardioprotection was accompanied by a preservation of intracellular high-energy phosphates during reperfusion; 38+2 vs 58+4% (P50.05) of preischaemic levels of phosphocreatine (PCr) for control and 100 mM ISO respectively and 23+1 vs 31+3% (P50.05) of preischaemic levels of ATP for control and 100 mM ISO respectively. 5 Cellular levels of NAD were higher in ISO treated hearts at the end of reperfusion; 2.56+0.45 vs 4.76+1.12 mmoles g 71 dry weight (P50.05) for control and ISO treated. 6 These results demonstrate that the cardioprotection a orded by inhibition of PARS activity with ISO is accompanied by a preservation of high-energy phosphates and cellular NAD levels and suggest that the mechanism responsible for this cardioprotection may involve prevention of intracellular ATP depletion.