Use of an Esophageal Heat Exchanger to Maintain Core Temperature during Burn Excisions and to Attenuate Pyrexia on the Burns Intensive Care Unit.

Introduction. Burns patients are vulnerable to hyperthermia due to sepsis and SIRS and to hypothermia due to heat loss during excision surgery. Both states are associated with increased morbidity and mortality. We describe the first use of a novel esophageal heat exchange device in combination with a heater/cooler unit to manage perioperative hypothermia and postoperative pyrexia. Material and Methods. The device was used in three patients with full thickness burns of 51%, 49%, and 45% body surface area to reduce perioperative hypothermia during surgeries of >6 h duration and subsequently to control hyperthermia in one of the patients who developed pyrexia of 40°C on the 22nd postoperative day due to E. coli/Candida septicaemia which was unresponsive to conventional cooling strategies. Results. Perioperative core temperature was maintained at 37°C for all three patients, and it was possible to reduce ambient temperature to 26°C to increase comfort levels for the operating team. The core temperature of the pyrexial patient was reduced to 38.5°C within 2.5 h of instituting the device and maintained around this value thereafter. Conclusion. The device was easy to use with no adverse incidents and helped maintain normothermia in all cases.

MicroRNA-122: a novel hepatocyte-enriched in vitro marker of drug-induced cellular toxicity.

Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular coculture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury.

Assessment of cardiomyocyte contraction in human-induced pluripotent stem cell-derived cardiomyocytes.

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.

Evaluation of the use of imaging parameters for the detection of compound-induced hepatotoxicity in 384-well cultures of HepG2 cells and cryopreserved primary human hepatocytes.

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.

Reducing safety-related drug attrition: the use of in vitro pharmacological profiling.

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.