Microphysiological heart-liver body-on-a-chip system with a skin mimic for evaluating topical drug delivery.

Body-on-a-chip in vitro systems are a promising technology that aims to increase the predictive power of drug efficacy and toxicity in humans when compared to traditional animal models. Here, we developed a new heart-liver body-on-a-chip system with a skin surrogate to assess the toxicity of drugs that are topically administered. In order to test the utility of the system, diclofenac, ketoconazole, hydrocortisone and acetaminophen were applied topically through a synthetic skin surrogate (Strat-M membrane) and the toxicity results were compared to those of acute drug exposure from systemically applying the compounds. The heart-liver system was successful in predicting the effects for both cardiac and liver functions changes due to the compounds. The difference in the concentrations of drugs applied topically compared to systemically indicates that the barrier properties of the skin surrogate were efficient. One important advantage of this heart-liver system was the capability of showing differential effects of acute and chronic drug exposure which is necessary as part of the International Conference in Harmonisation (ICH) tri-partate guidelines. In conclusion, this work indicates a promising heart-liver body-on-a-chip system that can be used for the assessment of potential drug toxicity from dermal absorption as well as evaluate transport dynamics through the skin in the same system.

Long-Term Electrical and Mechanical Function Monitoring of a Human-on-a-Chip System.

The goal of human-on-a-chip systems is to capture multi-organ complexity and predict the human response to compounds within physiologically relevant platforms. The generation and characterization of such systems is currently a focal point of research given the long-standing inadequacies of conventional techniques for predicting human outcome. Functional systems can measure and quantify key cellular mechanisms that correlate with the physiological status of a tissue, and can be used to evaluate therapeutic challenges utilizing many of the same endpoints used in animal experiments or clinical trials. Culturing multiple organ compartments in a platform creates a more physiologic environment (organ-organ communication). Here is reported a human 4-organ system composed of heart, liver, skeletal muscle and nervous system modules that maintains cellular viability and function over 28 days in serum-free conditions using a pumpless system. The integration of non-invasive electrical evaluation of neurons and cardiac cells and mechanical determination of cardiac and skeletal muscle contraction allows the monitoring of cellular function especially for chronic toxicity studies in vitro. The 28 day period is the minimum timeframe for animal studies to evaluate repeat dose toxicity. This technology could be a relevant alternative to animal testing by monitoring multi-organ function upon long term chemical exposure.

Investigation of the effect of hepatic metabolism on off-target cardiotoxicity in a multi-organ human-on-a-chip system.

Regulation of cosmetic testing and poor predictivity of preclinical drug studies has spurred efforts to develop new methods for systemic toxicity. Current in vitro assays do not fully represent physiology, often lacking xenobiotic metabolism. Functional human multi-organ systems containing iPSC derived cardiomyocytes and primary hepatocytes were maintained under flow using a low-volume pumpless system in a serum-free medium. The functional readouts for contractile force and electrical conductivity enabled the non-invasive study of cardiac function. The presence of the hepatocytes in the system induced cardiotoxic effects from cyclophosphamide and reduced them for terfenadine due to drug metabolism, as expected from each compound's pharmacology. A computational fluid dynamics simulation enabled the prediction of terfenadine-fexofenadine pharmacokinetics, which was validated by HPLC-MS. This in vitro platform recapitulates primary aspects of the in vivo crosstalk between heart and liver and enables pharmacological studies, involving both organs in a single in vitro platform. The system enables non-invasive readouts of cardiotoxicity of drugs and their metabolites. Hepatotoxicity can also be evaluated by biomarker analysis and change in metabolic function. Integration of metabolic function in toxicology models can improve adverse effects prediction in preclinical studies and this system could also be used for chronic studies as well.

Microphysiological flux balance platform unravels the dynamics of drug induced steatosis.

Drug development is currently hampered by the inability of animal experiments to accurately predict human response. While emerging organ on chip technology offers to reduce risk using microfluidic models of human tissues, the technology still mostly relies on end-point assays and biomarker measurements to assess tissue damage resulting in limited mechanistic information and difficulties to detect adverse effects occurring below the threshold of cellular damage. Here we present a sensor-integrated liver on chip array in which oxygen is monitored using two-frequency phase modulation of tissue-embedded microprobes, while glucose, lactate and temperature are measured in real time using microfluidic electrochemical sensors. Our microphysiological platform permits the calculation of dynamic changes in metabolic fluxes around central carbon metabolism, producing a unique metabolic fingerprint of the liver's response to stimuli. Using our platform, we studied the dynamics of human liver response to the epilepsy drug Valproate (Depakine™) and the antiretroviral medication Stavudine (Zerit™). Using E6/E7LOW hepatocytes, we show TC50 of 2.5 and 0.8 mM, respectively, coupled with a significant induction of steatosis in 2D and 3D cultures. Time to onset analysis showed slow progressive damage starting only 15-20 hours post-exposure. However, flux analysis showed a rapid disruption of metabolic homeostasis occurring below the threshold of cellular damage. While Valproate exposure led to a sustained 15% increase in lipogenesis followed by mitochondrial stress, Stavudine exposure showed only a transient increase in lipogenesis suggesting disruption of ß-oxidation. Our data demonstrates the importance of tracking metabolic stress as a predictor of clinical outcome.

Development of a mechanistic SAR model for the detection of phototoxic chemicals and use in an integrated testing strategy.

Phototoxicity is of increasing concern in dermatology, since modern lifestyle is often associated with exposure to sunlight. The most commonly reported process is via oxidative reactions. Therefore characterizing the "photo-pro-oxidant" potential of a compound early in its industrial development is of utmost interest, especially for compounds likely to undergo sunlight exposure in skin. Today there is a need for filtering compounds to be tested in the 3T3 neutral red uptake in vitro test for phototoxicity since testing requires resources. A computational model aiming at predicting the mechanisms that imply the generation of reactive oxygen species was developed using a diverse set of 56 chemicals having 3T3 NRU data. An historical mechanistic (Q)SAR model developed for polycyclic aromatic hydrocarbons was used to derive the new mechanistic model: descriptors were selected upfront to describe the modeled phenomenon. The historical parabolic relationships between phototoxicity and the energy gap (E(GAP)) between energies of the highest occupied molecular orbital and the lowest unoccupied molecular orbital was confirmed. The model predicts chemicals to be "phototoxic or photodegradable", or "non-phototoxic and non-photodegradable". A four-step testing strategy is proposed to enable the reduction of experimental testing with the in silico model implemented as a first screen.

Developing structure-activity relationships for the prediction of hepatotoxicity.

Drug-induced liver injury is a major issue of concern and has led to the withdrawal of a significant number of marketed drugs. An understanding of structure-activity relationships (SARs) of chemicals can make a significant contribution to the identification of potential toxic effects early in the drug development process and aid in avoiding such problems. This process can be supported by the use of existing toxicity data and mechanistic understanding of the biological processes for related compounds. In the published literature, this information is often spread across diverse sources and can be varied and unstructured in quality and content. The current work has explored whether it is feasible to collect and use such data for the development of new SARs for the hepatotoxicity endpoint and expand upon the limited information currently available in this area. Reviews of hepatotoxicity data were used to build a structure-searchable database, which was analyzed to identify chemical classes associated with an adverse effect on the liver. Searches of the published literature were then undertaken to identify additional supporting evidence, and the resulting information was incorporated into the database. This collated information was evaluated and used to determine the scope of the SARs for each class identified. Data for over 1266 chemicals were collected, and SARs for 38 classes were developed. The SARs have been implemented as structural alerts using Derek for Windows (DfW), a knowledge-based expert system, to allow clearly supported and transparent predictions. An evaluation exercise performed using a customized DfW version 10 knowledge base demonstrated an overall concordance of 56% and specificity and sensitivity values of 73% and 46%, respectively. The approach taken demonstrates that SARs for complex endpoints can be derived from the published data for use in the in silico toxicity assessment of new compounds.

An expert system approach to the assessment of hepatotoxic potential.

Hepatotoxicity is a major cause of pharmaceutical drug attrition and is also a concern within other chemical industries. In silico approaches to the prediction of hepatotoxicity are an important tool in the early identification of adverse effects in the liver associated with exposure to a chemical. Here, we describe work in progress to develop an expert system approach to the prediction of hepatotoxicity, focussing particularly on the identification of structural alerts associated with its occurrence. The development of 74 such structural alerts based on public-domain literature and proprietary data sets is described. Evaluation results indicate that, whilst these structural alerts are effective in identifying the hepatotoxicity of many chemicals, further research is needed to develop additional structural alerts to account for the hepatotoxicity of a number of chemicals which is not currently predicted. Preliminary results also suggest that the specificity of the structural alerts may be improved by the combined use of applicability domains based on physicochemical properties such as log P and molecular weight. In the longer term, the performance of predictive models is likely to benefit from the further integration of diverse data and prediction model types.

Mitochondrial and metabolic effects of nucleoside reverse transcriptase inhibitors (NRTIs) in mice receiving one of five single- and three dual-NRTI treatments.

Although treatments with nucleoside reverse transcriptase inhibitors (NRTIs) can modify fat metabolism and fat distribution in humans, the mechanisms of these modifications and the roles of diverse NRTIs are unknown. We studied the mitochondrial and metabolic effects of stavudine (d4T), zidovudine (AZT), didanosine (ddI), lamivudine (3TC), zalcitabine (ddC), and three combinations (AZT-3TC, d4T-3TC, and d4T-ddI) in mice treated for 2 weeks with daily doses equivalent to the human dose per body area. Concentrations of AZT and d4T in plasma were lower when these drugs were administered with 3TC or ddI. Whatever the treatment, mitochondrial DNA was not significantly decreased in muscle, heart, brain, or white adipose tissue but was moderately decreased in liver tissue after the administration of AZT, 3TC, or d4T alone. Blood lactate was unchanged, even when NRTIs were administered at supratherapeutic doses. In contrast, the level of plasma ketone bodies increased with the administration of AZT or high doses of d4T but not with ddC, 3TC, or ddI, suggesting that the thymine moiety could be involved. Indeed, the levels of plasma ketone bodies increased in mice treated with beta-aminoisobutyric acid, a thymine catabolite. Treatment with AZT, d4T, or beta-aminoisobutyric acid increased hepatic carnitine palmitoyltransferase I (CPT-I) mRNA expression and the mitochondrial generation of ketone bodies from palmitate. In conclusion, therapeutic doses of NRTIs have no or moderate effects on mitochondrial DNA and no effects on plasma lactate in mice. However, AZT and high doses of d4T increase the levels of hepatic CPT-I, mitochondrial fatty acid beta-oxidation, and ketone bodies, and these catabolic effects are reproduced by beta-aminoisobutyric acid, a thymine metabolite.