Use of toxicogenomics to understand mechanisms of drug-induced hepatotoxicity during drug discovery and development

Hepatotoxicity is a common cause of failure in drug discovery and development and is also frequently the source of adverse drug reactions. Therefore, a better prediction, characterization and understanding of drug-induced hepatotoxicity could result in safer drugs and a more efficient drug discovery and development process. Among the 'omics technologies, toxicogenomics (or the use of gene expression profiling in toxicology) represents an attractive approach to predict toxicity and to gain a mechanistic understanding of toxic changes. In this review, we illustrate, using selected examples, how toxicogenomics can be applied to investigate drug-induced hepatotoxicity in animal models and in vitro systems. In general, this technology can not only improve the discipline of toxicology and risk assessment but also represent an extremely effective, hypothesis-generating alternative to rapidly understand mechanisms of hepatotoxicity.     

cDNA phage display as a novel tool to screen for cellular targets of chemical compounds

cDNA phage display is frequently used in drug development to screen for cellular target of drugs. However, in toxicology, cDNA phage display remains unexplored, although it has large potential in this field. In this study, cDNA phage display is demonstrated as a novel tool to screen for interactions between chemical compounds and cellular targets. The knowledge of these target interactions is valuable to have a more complete understanding of the mechanisms of action of chemical compounds.Bisphenol A (BPA) was selected as a model compound for this study. By selection of the cellular proteins that bind BPA with cDNA phage display, it was possible to identify a known cellular target of BPA, tubulin α and a possible novel cellular target of BPA, transforming acidic coiled-coil containing protein 3. Both these cellular proteins are involved in the mechanism of cell division. The disruption of cell division is a known non-genomic effect of BPA. Non-genomic effects are not mediated by differences in gene expression and therefore important mechanistic information might be missed with the widely used differential gene expression techniques for mode of action research. This cDNA phage display technique can provide important additional information about the interaction of chemical compounds with cellular targets that mediates these non-genomic actions and therefore gives complementary information to toxicogenomic studies to obtain a more complete understanding of the mechanism of action of chemical compounds.     

高内涵分析在新药发现毒理学中的应用进展

在新药发现早期开展发现毒理学研究是提高新药研发效率的重要策略之一。高内涵分析(HCA)是基于高效新药筛选需求发展起来的一项新技术,其主要特点是基于活细胞、多参数、实时、高通量,能够实现化合物多种生物活性、毒性的早期、快速地检测,为发现毒理学研究提供了高效的技术手段。目前,HCA已用于多种靶器官细胞毒性、遗传毒性、神经毒性、血管毒性、生殖毒性等检测以及毒理学分子机制的研究,本文就HCA在新药发现毒理学方面的应用进展进行综述。     

Genomics: Applications in mechanism elucidation

The inability to predict the pharmacology and toxicology of drug candidates in preclinical studies has led to the decline in the number of new drugs which make it to market and the rise in cost associated with drug development. Identifying molecular interactions associated with therapeutic and toxic drug effects early in development is a top priority. Traditional mechanism elucidation strategies are narrow, often focusing on the identification of solely the molecular target. Methods which can offer additional insight into wide-ranging molecular interactions required for drug effect and the biochemical consequences of these interactions are in demand. Genomic strategies have made impressive advances in defining a more global view of drug action and are expected to increasingly be used as a complimentary tool in drug discovery and development.     

蛋白质组学技术在药物肝毒性研究中的应用

蛋白质组学技术的发展促进了许多学科的发展,毒理蛋白质组学(toxicoproteomics)整合了经典毒理学、病理学和蛋白质差异表达分析技术,使毒理学研究上升到了一个新的高度。药物性肝损伤是肝脏疾病的一个重要原因,也是药物开发所面临的一项重大挑战。现就蛋白质组学的主要技术,包括双向凝胶电泳-质谱(2-DE-MS)、液相色谱-串联质谱(LC-MS-MS)、保留色谱-质谱(RC-MS)和蛋白质阵列,以及其在药物肝毒性研究中的应用,包括毒性蛋白标志物的筛选、毒性机制研究、毒性预测与毒性蛋白质数据库的建立方面进行综述。     

Gene expression profiling for pharmaceutical safety assessment

Toxicogenomics is the application of gene expression profiling technology to toxicology. This results in the generation of very large and complex gene expression data sets associated with the development of toxicities. It is widely assumed that this data can be deconvoluted to reveal novel insights into toxicological processes that are of value to the task of risk assessment. More specifically, it is hoped that toxicogenomics will aid in the prediction of the toxic potential and mechanisms of toxicity of novel chemical entities. On the basis of such promise, the pharmaceutical industry has invested heavily in this area, as the perceived rewards in terms of improved pipeline efficiency and safer drugs are immense. Consequently, a great deal of groundwork has been done over the past several years to establish working methods in toxicogenomics, both within industry and academia, demonstrating utility in proof-of-concept studies, generating the databases on which some approaches depend, and developing new data analysis tools. Despite such activity, there is little reported evidence to suggest that toxicogenomics is making a significant impact on the discovery and development of drugs. This may partly reflect the understandable reluctance of pharmaceutical industries to share information in a competitive environment. It may also partly reflect difficulties in bridging the gap between theory and practice, as is required to deliver real value to the industry. This review will assess the successes and shortcomings of toxicogenomics, and consider how it can be usefully applied to a drug discovery pipeline.     

Gene expression microarray analysis in cancer biology, pharmacology, and drug development: progress and potential.

With the imminent completion of the Human Genome Project, biomedical research is being revolutionised by the ability to carry out investigations on a genome wide scale. This is particularly important in cancer, a disease that is caused by accumulating abnormalities in the sequence and expression of a number of critical genes. Gene expression microarray technology is gaining increasingly widespread use as a means to determine the expression of potentially all human genes at the level of messenger RNA. In this commentary, we review developments in gene expression microarray technology and illustrate the progress and potential of the methodology in cancer biology, pharmacology, and drug development. Important applications include: (a) development of a more global understanding of the gene expression abnormalities that contribute to malignant progression; (b) discovery of new diagnostic and prognostic indicators and biomarkers of therapeutic response; (c) identification and validation of new molecular targets for drug development; (d) provision of an improved understanding of the molecular mode of action during lead identification and optimisation, including structure-activity relationships for on-target versus off-target effects; (e) prediction of potential side-effects during preclinical development and toxicology studies; (f) confirmation of a molecular mode of action during hypothesis-testing clinical trials; (g) identification of genes involved in conferring drug sensitivity and resistance; and (h) prediction of patients most likely to benefit from the drug and use in general pharmacogenomic studies. As a result of further technological improvements and decreasing costs, the use of microarrays will become an essential and potentially routine tool for cancer and biomedical research.     

Application of genomics in preclinical drug safety evaluation

Understanding the response of biological systems to xenobiotics is fundamental to the evaluation of drug safety. Toxicologists have traditionally gathered pathological, morphological, chemical and biochemical information from in vivo studies of preclinical species in order to assess drug safety and to determine how new drugs can be safely administered to the human patient population. In recent years the emerging "-omics" technologies have been developed and integrated into preclinical studies in order to better assess drug safety by gaining information on the cellular and molecular events underlying adverse drug reactions. Genomics approaches in particular have become readily available and are being applied in several stages of drug development. The burgeoning literature on what has become known as "toxicogenomics" has for the most part highlighted successful applications of gene expression profiling in predictive toxicology, enabling decisions to be made on the developability of a compound early in the drug development process. It is also becoming apparent that toxicogenomic approaches are good starting points to develop experiments designed to gain a mechanistic insight into drug toxicities within and across species. Gene expression arrays permit the measurement of responses of essentially all the genes in the entire genome to be monitored, and knowledge of the function of the genes affected can identify the potential mechanisms to then be confirmed using conventional biochemical, toxicological and pathological approaches. As toxicologists put these technologies into practice they build up a knowledge base to better characterize toxicities at the molecular level and to make the search for much needed, novel biomarkers of toxicity more achievable.     

Relationship between hepatic gene expression profiles and hepatotoxicity in five typical hepatotoxicant-administered rats.

In the field of gene expression analysis, DNA microarray technology has a major impact on many different areas including toxicogenomics, such as in predicting the adverse effects of new drug candidates and improving the process of risk assessment and safety evaluation. In this study, we investigated whether there is relationship between the hepatotoxic phenotypes and gene expression profiles of hepatotoxic chemicals measured by DNA microarray analyses. Sprague-Dawley rats (6 weeks old) were administered five hepatotoxicants: acetaminophen (APAP), bromobenzene, carbon tetrachloride, dimethylnitrosamine, and thioacetamide. Serum biochemical markers for liver toxicity were measured to estimate the maximal toxic time of each chemical. Hepatic mRNA was isolated, and the gene expression profiles were analyzed by DNA microarray containing 1,097 drug response genes, such as cytochrome P450s, other phase I and phase II enzymes, nuclear receptors, signal transducers, and transporters. All the chemicals tested generated specific gene expression patterns. APAP was sorted to a different cluster from the other four chemicals. From the gene expression profiles and maximal toxic time estimated by serum biochemical markers, we identified 10 up-regulated genes and 10 down-regulated genes as potential markers of hepatotoxicity. By Quality-Threshold (QT) clustering analysis, we identified major up- and down-regulated expression patterns in each group. Interestingly, the average gene expression patterns from the QT clustering were correlated with the mean value profiles from the biochemical markers. Furthermore, this correlation was observed at any extent of hepatotoxicity. In this study, we identified 17 potential toxicity markers, and those expression profiles could estimate the maximal toxic time independently of the hepatotoxicity levels. This expression profile analysis could be one of the useful tools for evaluating a potential hepatotoxicant in the drug development process.     

Development of an in vitro liver toxicity prediction model based on longer term primary rat hepatocyte culture

The assumption that compounds with similar toxic endpoints generate unique gene expression signatures has led to attempts to classify unknown compounds according to their genomic profile. However, studies reported so far have mostly been conducted in vivo. In this proof of concept study, we assessed the use of rat hepatocyte sandwich cultures in combination with a toxicogenomic classification method to generate a predictive in vitro toxicity classification model.After pre-incubation for 3 days, primary rat hepatocytes were treated for up to 9 days with 13 well known model compounds, changes in the global gene expression profile were measured and subsequently used for the establishment of a predictive classification model. A subset of 724 genes was capable of discriminating compounds with a misclassification rate of 7.5%. As a preliminary verification, the resulting classifier was applied to two blinded control compounds. The classification of compounds according to transient changes in global gene expression allowed the correct prediction independently from the knowledge of their underlying toxic mechanisms.The results of this first pilot study demonstrate the possibility of in vitro gene expression models to contribute to candidate selection early in drug discovery by improving the predictivity of toxicological studies and thereby reducing animal usage in toxicology.     

药物毒理学研究新进展

药物毒理学是现代毒理学中研究药物的毒副作用机制、评价新药安全性的分支学科,主要目的在于指导药物合成和临床合理用药,降低药物的毒副作用及减少因毒性导致的新药研发失败。现就药物毒理学研究的新思路、发现毒理学的发展和全程式新药安全性研究评价新模式的特征以及药物毒理学研究的新方法作简要阐述。     

Application of toxicogenomics in drug development

The recognition of toxicological potential in new chemical entities early on in development would be highly desirable in streamlining and reducing the cost of drug development. Analysis of gene expression profiles using microarrays is one of the most fashionable methods for achieving this objective. The procedure relies on developing a gene expression profile related to compound exposure and then matching this against a database of profiles associated with known mechanisms of toxicity. Studies profiling tumors and associating these with pathology and phenotype have shown the potential of the technique. However, application of the technique in toxicology is only at the preliminary stage. This article examines current state of development in toxicology and its future potential.     

药物毒理学技术方法研究进展

药物是否安全和有效是药物研发成功与否的决定性因素。就药物研发的整个流程而言,毒性是终止药物研发的重要原因之一。药物毒理学研究贯穿于新药发现阶段、临床前安全性评价和上市后跟踪监督的全过程,因此药物的毒理学研究至关重要。简要综述了药物毒理学研究过程中所涉及的技术方法以及研究新动向,并主要介绍了组学技术的发展。     

Identification of end points relevant to detection of potentially adverse drug reactions

Expectations are high that the use of proteomics, gene arrays and metabonomics will improve risk assessment and enable prediction of toxicity early in drug development. These molecular profiling techniques may be used to classify compounds and to identify predictive markers that can be used to screen large numbers of chemicals. One of the challenges for the scientific community is to discriminate between changes in gene/protein expression and metabolic profiles reflecting physiological/adaptive responses, and changes related to pathology and toxicology. In these proceedings we provide a brief overview of the technologies with focus on proteomics and the possible applications to mechanistic and predictive toxicology. The discussion also includes strengths and limitations of molecular profiling technologies.     

毒理芯片技术在中药毒理学研究中的应用及前景

通过介绍毒理芯片技术的概念、工作原理及其在中药毒理学研究中的应用状况，分析毒理芯片技术在中药毒理学研究中的应用前景及优缺点,提出借助毒理芯片技术进行中药毒理学研究，可在基因转录表达水平对中药进行准确、快速、高效地筛选与安全性评价，从而使在分子水平。掌握中药毒理作用的靶点、方式及代谢途径等成为可能。     

蛋白质组学在药物研究中的应用

近年来,蛋白质组学技术飞速发展,尤其在药物的靶点确认、药物作用机制等研究中,发挥出了其极大的技术优势,明显地提高了药物发现的效率。该文对蛋白质组学的基本方法、新技术以及它在药物靶点的发现和确认、阐明药物作用机制、药物毒理学、耐药相关机制研究、临床医药研究等方面的应用进行综述。     

新药毒性机制研究进展

新药毒性机制研究是准确预测新药临床毒性的重要手段之一，必须及早且完整深入地进行，并贯穿新药研发的各个时期，结合药理学、化学、药动学、毒代动力学、毒理学及危险因素评估等诸多学科共同进行。现代科研技术为毒性机制研究提供了强有力的保证。     

Use of Transgenic Animals in Understanding Molecular Mechanisms of Toxicity*

Understanding molecular mechanisms of chemical toxicity and the potential risks of drugs to man is a pivotal part of the drug development process. With the dramatic increase in the number of new chemical entities arising from high throughput screening, there is an urgent need to develop systems for the rapid evaluation of potential drugs so that those agents which are most likely to be free of adverse effects can be identified at the earliest possible stage in drug development. The complex mechanisms of action of chemical toxins has made it extremely difficult to evaluate the precise toxic mechanism and also the relative role of specific genes in either potentiating or ameliorating the toxic effect. This problem can be addressed by the application of genetic strategies. Such strategies can exploit strain differences in susceptibility to specific toxic agents or, with the rapidly developing technologies, can exploit the use of transgenic animals where specific genes can be manipulated and subsequent effects on chemical toxicity evaluated. Transgenic animals can be exploited in a variety of ways to understand mechanisms of chemical toxicity. For example, a human gene encoding a drug metabolizing enzyme can be directly introduced and the effects on toxic response evaluated. Alternatively, specific genes can be deleted from the mouse genome and the consequences on toxicological response determined. Many toxic chemical agents modulate patterns of gene expression within target cells. This can be used to screen for responses to different types of toxic insult. In such experiments the promotor of a stress-regulated gene can be ligated to a suitable reporter gene, such as lacZ, or green fluorescent protein, and inserted into the genome of an appropriate test species. On administration of a chemical agent, cells which are sensitive to the toxic effects of that chemical will express the reporter, which can then be identified using an appropriate assay system. This latter strategy provides the potential for screening a large number of compounds rapidly for their potential toxic effects and also provides information on tissue and cellular specificity. Experiments using transgenic animals can be complex, and care must be taken to ensure that the results are not affected by background activities within the species being used. For example, the introduction of a specific human cytochrome P450 gene may have no effect on the metabolic disposition of a drug or toxin because of the background activity within the mouse. As the toxicity of a chemical agent is determined by a wide range of different factors including drug uptake, metabolism, detoxification and repair, differences between man and the species being used could potentially generate a toxic response in the animal model whereas no toxicity may be observed in man. In spite of these confounding factors, the application of transgenic animals to toxicological issues has enormous potential for speeding up the drug discovery process and will undoubtedly become part of this process in the future.