生物标志物在药物性肝损伤中应用的研究进展

摘 要药物性肝损伤是伴随着药物使用而出现的最常见不良反应之一,也是药物研发失败及药物撤市或限制使用的主要原因。生物标志物检测是临床前药物肝毒性评价和临床患者潜在肝损伤诊断的重要技术手段,而传统的肝损伤指标因缺乏特异性和灵敏性等因素,限制了其在早期评价中的应用。因此,寻求及验证新的生物标志物势在必行。本文主要对结合现代组学等技术探索出具有科研及临床价值的新型生物标志物研究现状予以概述。以期为科研人员、临床医师、新药研发机构等预测药物的肝毒性及可能的发展趋势,判断药物引起的肝损伤程度等提供参考。     

Metabolic activation and drug‐induced liver injury: in vitro approaches for the safety risk assessment of new drugs

Drug‐induced liver injury (DILI) is a significant leading cause of hepatic dysfunction, drug failure during clinical trials and post‐market withdrawal of approved drugs. Many cases of DILI are unexpected reactions of an idiosyncratic nature that occur in a small group of susceptible individuals. Intensive research efforts have been made to understand better the idiosyncratic DILI and to identify potential risk factors. Metabolic bioactivation of drugs to form reactive metabolites is considered an initiation mechanism for idiosyncratic DILI. Reactive species may interact irreversibly with cell macromolecules (covalent binding, oxidative damage), and alter their structure and activity. This review focuses on proposed in vitro screening strategies to predict and reduce idiosyncratic hepatotoxicity associated with drug bioactivation. Compound incubation with metabolically competent biological systems (liver‐derived cells, subcellular fractions), in combination with methods to reveal the formation of reactive intermediates (e.g., formation of adducts with liver proteins, metabolite trapping or enzyme inhibition assays), are approaches commonly used to screen the reactivity of new molecules in early drug development. Several cell‐based assays have also been proposed for the safety risk assessment of bioactivable compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Ariadne's ChemEffect and Pathway Studio knowledge base

Importance of the field: Drug discovery and development is a very complex and costly process. Understanding the detailed molecular mechanisms of a disease and drug actions can make it more efficient not only for new target discovery but also for lead prioritization, drug repositioning and development of biomarkers for drug efficacy and safety. Access to formalized knowledge about functions of proteins and small molecules is crucial for rationalization of the drug development process, and scientific publications are the main source of this knowledge. Protein knowledge networks capturing protein functions, protein–protein relations and organization of proteins in complex cellular sub-systems are making their way into modern drug discovery. Chemical networks representing multiple aspects of chemical functional information integrated into a protein systems biology network is even more advanced and promising paradigm.

Areas covered in this review: This review describes utilization of literature-derived protein and chemical functional knowledge bases in drug development.

What the reader will gain: Readers will gain an understanding of how integrated protein and chemical knowledge networks can be used for understanding and building the models of cellular events, disease mechanisms, and drug actions, finding biomarkers of drug efficacy and safety, as well as interpretation of high-throughput gene expression, proteomic and metabolomic experiments.

Take home message: Integrated literature-derived protein and chemical knowledge bases can rationalize many aspects of drug development process including drug repositioning and biomarker design.     

The discovery and development of proteomic safety biomarkers for the detection of drug-induced liver toxicity

Biomarkers are biometric measurements that provide critical quantitative information about the biological condition of the animal or individual being tested. In drug safety studies, established toxicity biomarkers are used along with other conventional study data to determine dose-limiting organ toxicity, and to define species sensitivity for new chemical entities intended for possible use as human medicines. A continuing goal of drug safety scientists in the pharmaceutical industry is to discover and develop better trans-species biomarkers that can be used to determine target organ toxicities for preclinical species in short-term studies at dose levels that are some multiple of the intended human dose and again later in full development for monitoring clinical trials at lower therapeutic doses. Of particular value are early, predictive, noninvasive biomarkers that have in vitro, in vivo, and clinical transferability. Such translational biomarkers bridge animal testing used in preclinical science and human studies that are part of subsequent clinical testing. Although suitable for in vivo preclinical regulatory studies, conventional hepatic safety biomarkers are basically confirmatory markers because they signal organ toxicity after some pathological damage has occurred, and are therefore not well-suited for short-term, predictive screening assays early in the discovery-to-development progression of new chemical entities (NCEs) available in limited quantities. Efforts between regulatory agencies and the pharmaceutical industry are underway for the coordinated discovery, qualification, verification and validation of early predictive toxicity biomarkers. Early predictive safety biomarkers are those that are detectable and quantifiable prior to the onset of irreversible tissue injury and which are associated with a mechanism of action relevant to a specific type of potential hepatic injury. Potential drug toxicity biomarkers are typically endogenous macromolecules in biological fluids with varying immunoreactivity which can present bioanalytical challenges when first discovered. The potential success of these efforts is greatly enhanced by recent advances in two closely linked technologies, toxicoproteomics and targeted, quantitative mass spectrometry. This review focuses on the examination of the current status of these technologies as they relate to the discovery and development of novel preclinical biomarkers of hepatotoxicity. A critical assessment of the current literature reveals two distinct lines of safety biomarker investigation, (1) peripheral fluid biomarkers of organ toxicity and (2) tissue or cell-based toxicity signatures. Improved peripheral fluid biomarkers should allow the sensitive detection of potential organ toxicity prior to the onset of overt organ pathology. Advancements in tissue or cell-based toxicity biomarkers will provide sensitive in vitro or ex vivo screening systems based on toxicity pathway markers. An examination of the current practices in clinical pathology and the critical evaluation of some recently proposed biomarker candidates in comparison to the desired characteristics of an ideal toxicity biomarker lead this author to conclude that a combination of selected biomarkers will be more informative if not predictive of potential animal organ toxicity than any single biomarker, new or old. For the practical assessment of combinations of conventional and/or novel toxicity biomarkers in rodent and large animal preclinical species, mass spectrometry has emerged as the premier analytical tool compared to specific immunoassays or functional assays. Selected and multiple reaction monitoring mass spectrometry applications make it possible for this same basic technology to be used in the progressive stages of biomarker discovery, development, and more importantly, routine study applications without the use of specific antibody reagents. This technology combined with other “omics” technologies can provide added selectivity and sensitivity in preclinical drug safety testing.     

Cellular responses associated with dibucaine-induced phospholipidosis

A wide range of cationic amphiphilic drugs (CADs) from different therapeutic areas are known to cause phospholipidosis both in vivo and in vitro. Although the relevance of this storage disorder for human health remains uncertain, CADs have been repeatedly associated with clinical side effects, and as a result, phospholipidosis is of major concern for drug development in the pharmaceutical industry. An important unresolved question in this field is whether phospholipidosis is really linked to cellular toxicity. This work was focused on studying cellular responses associated with CAD-induced phospholipidosis in cultured mammalian kidney cells. Dibucaine (2-butoxy-N-[2-diethylaminoethyl]quinoline-4-carboxamide), an amide-type anesthetic with poorly defined cytotoxic effects, was used to induce phospholipidosis in Vero cells. The results from several assays that measure cell viability, proliferation, and morphological changes indicated that dibucaine-induced lysosomal phospholipidosis was accompanied by cellular defense responses such as transient growth arrest and autophagy, under mild stress conditions. Conversely, when tolerance limits were exceeded treated Vero cells underwent extensive and irreparable injury, leading ultimately to cell death. Our data provide additional information that may be of considerable interest for drug safety assessment.     

Histologically defined biomarkers in toxicology

Histopathology is the gold standard when defining toxicological effects, but it is invasive, time consuming and expensive. Using biomarkers linked to distinct, defined cell types and tissues may provide a direct link to histopathology without its drawbacks and it also provides increased sensitivity and specificity. Furthermore, as histological testing is often impractical in human subjects, using biomarkers with a known histological distribution may fill the need of localising toxic injury to distinct organs or tissues. This paper discusses how, by using biomarkers with a known cellular origin (histologically defined biomarkers), toxic effects may be found earlier and at lower doses of compound, leading to potential savings in drug development.     

Nanomaterial and toxicity: what can proteomics tell us about the nanotoxicology?

1.?In the last few years, a substantial scientific work is focused to identify the potential toxicity of nanomaterials by studying the cellular pathways under in vitro and in vivo conditions. Owing to high surface area to volume ratio nanoparticles (NPs) can pass through cell membranes which might be responsible for creating adverse interactions in biological systems. Simultaneously, researchers are also interested to assess the fate of NP inside the living system, which may lead to altered protein expression as well as protein corona formation.

2.?According to published reports, NP-mediated toxicity involves altered cellular system including cell morphology, cell differentiation, cell metabolism, cell mobility, cellular immunity, which is derived from the side effects of nanoformulation and leading to apoptosis and necrosis. These results indicate the existence of potential toxic effect of these particles to human health.

3.?The advent of proteomics with sophisticated technical improvement coupled with advanced bioinformatics has led to identify altered proteins due to nanomaterial exposure that could provide a new avenue to biomarker discovery.

4.?This review aims to provide the current status of safe production and use of nanomaterials.     

The Endoplasmic Reticulum in Xenobiotic Toxicity

The endoplasmic reticulum (ER) is involved in an array of cellular functions that play important roles in xenobiotic toxicity. The ER contains the majority of cytochrome P450 enzymes involved in xenobiotic metabolism, as well as a number of conjugating enzymes. In addition to its role in drug bioactivation and detoxification, the ER can be a target for damage by reactive intermediates leading to cell death or immune-mediated toxicity. The ER contains a set of luminal proteins referred to as ER stress proteins (including GRP78, GRP94, protein disulfide isomerase, and calreticulin). These proteins help regulate protein processing and folding of membrane and secretory proteins in the ER, calcium homeostasis, and ER-associated apoptotic pathways. They are induced in response to ER stress. This review discusses the importance of the ER in molecular events leading to cell death following xenobiotic exposure. Data showing that the ER is important in both renal and hepatic toxicity will be discussed.     

The endoplasmic reticulum in xenobiotic toxicity

The endoplasmic reticulum (ER) is involved in an array of cellular functions that play important roles in xenobiotic toxicity. The ER contains the majority of cytochrome P450 enzymes involved in xenobiotic metabolism, as well as a number of conjugating enzymes. In addition to its role in drug bioactivation and detoxification, the ER can be a target for damage by reactive intermediates leading to cell death or immune-mediated toxicity. The ER contains a set of luminal proteins referred to as ER stress proteins (including GRP78, GRP94, protein disulfide isomerase, and calreticulin). These proteins help regulate protein processing and folding of membrane and secretory proteins in the ER, calcium homeostasis, and ER-associated apoptotic pathways. They are induced in response to ER stress. This review discusses the importance of the ER in molecular events leading to cell death following xenobiotic exposure. Data showing that the ER is important in both renal and hepatic toxicity will be discussed.     

Aluminum in liver cells – the element species matters

Abstract

Aluminum (Al) can be ingested from food and released from packaging and can reach key organs involved in human metabolism, including the liver via systemic distribution. Recent studies discuss the occurrence of chemically distinct Al-species and their interconversion by contact with biological fluids. These Al species can vary with regard to their intestinal uptake, systemic transport, and therefore could have species-specific effects on different organs and tissues. This work aims to assess the in vitro hepatotoxic hazard potential of three different relevant Al species: soluble AlCl₃ and two nanoparticulate Al species were applied, representing for the first time an investigation of metallic nanoparticles besides to mineral bound γ-Al₂O₃ on hepatic cell lines. To investigate the uptake and toxicological properties of the Al species, we used two different human hepatic cell lines: HepG2 and differentiated HepaRG cells. Cellular uptake was determined by different methods including light microscopy, transmission electron microscopy, side-scatter analysis, and elemental analysis. Oxidative stress, mitochondrial dysfunction, cell death mechanisms, and DNA damage were monitored as cellular parameters. While cellular uptake into hepatic cell lines occurred predominantly in the particle form, only ionic AlCl₃ caused cellular effects. Since it is known, that Al species can convert one into another, and mechanisms including ‘trojan-horse’-like uptake can lead to an Al accumulation in the cells. This could result in the slow release of Al ions, for which reason further hazard cannot be excluded. Therefore, individual investigation of the different Al species is necessary to assess the toxicological potential of Al particles.     

Organotypic liver culture models: meeting current challenges in toxicity testing

Prediction of chemical-induced hepatotoxicity in humans from in vitro data continues to be a significant challenge for the pharmaceutical and chemical industries. Generally, conventional in vitro hepatic model systems (i.e. 2-D static monocultures of primary or immortalized hepatocytes) are limited by their inability to maintain histotypic and phenotypic characteristics over time in culture, including stable expression of clearance and bioactivation pathways, as well as complex adaptive responses to chemical exposure. These systems are less than ideal for longer-term toxicity evaluations and elucidation of key cellular and molecular events involved in primary and secondary adaptation to chemical exposure, or for identification of important mediators of inflammation, proliferation and apoptosis. Progress in implementing a more effective strategy for in vitro-in vivo extrapolation and human risk assessment depends on significant advances in tissue culture technology and increasing their level of biological complexity. This article describes the current and ongoing need for more relevant, organotypic in vitro surrogate systems of human liver and recent efforts to recreate the multicellular architecture and hemodynamic properties of the liver using novel culture platforms. As these systems become more widely used for chemical and drug toxicity testing, there will be a corresponding need to establish standardized testing conditions, endpoint analyses and acceptance criteria. In the future, a balanced approach between sample throughput and biological relevance should provide better in vitro tools that are complementary with animal testing and assist in conducting more predictive human risk assessment.     

Effect of Aegle marmelos on DEN initiated and 2-AAF promoted hepatocarcinogenesis: a chemopreventive study

In this study, we examined the inhibitory effects of Aegle marmelos methanolic extract on diethylnitrosamine (DEN) initiated and 2-acetyl aminofluorene (2-AAF) promoted liver carcinogenesis in male Wistar rats. Interestingly, it was found that A. marmelos (25 and 50?mg/kg body weight) resulted in a marked reduction of the incidence of liver tumors, which was further confirmed with histopathology. Furthermore to understand the underlying mechanisms of chemoprevention potential of A. marmelos, we evaluated the levels of hepatic antioxidant defence enzymes, ornithine decarboxylase (ODC) activity and hepatic DNA synthesis as a marker for tumor promotion since a direct correlation between these marker parameters and carcinogenicity have been well documented. Treatment of male Wistar rats for five consecutive days with 2-AAF induced significant hepatic toxicity, oxidative stress and hyper-proliferation. Pretreatment of A. marmelos extract (25 and 50?mg/kg body weight) prevented oxidative stress and toxicity by restoring the levels of antioxidant enzymes at both the doses. The promotion parameters (ODC activity and DNA synthesis) induced by 2-AAF administration in diet with partial hepatectomy (PH) were also significantly suppressed dose-dependently by A. marmelos. Therefore, we can conclude that ultimately the protection against liver carcinogenesis by A. marmelos methanolic extract might be mediated by multiple actions, which include restoration of cellular antioxidant enzymes, detoxifying enzymes, ODC activity and DNA synthesis.     

Primary liver tumors: origin and target therapy

Background: The liver is the largest gland and chief metabolic organ of the human body possessing a unique ability to regenerate. The general interest of primary liver tumors is noteworthy because of their increasing worldwide incidence and mortality. Recent studies have focused on the ancestors of mature hepatocytes, which are capable of proliferating, differentiating and self-renewing. Objective: To provide a brief and up-to-date review on the cellular origin of primary liver tumors and to examine the use of stem cells in potential future therapeutic attempts. Methods: A review of relevant literature. Results: It is clear that hepatic progenitor cells (HPCs) could be the basis of some hepatocellular carcinomas (HCC), cholangiocarcinomas (CHC), hepatocellular adenomas and hepatoblastomas. Cancer stem cell (CSC) theory emphasizes the role of hepatic stem cells in the development and progression of liver tumors. Conclusion: The expression of HPCs markers may be used as new independent prognostic factors in HCC. Conventional treatments for HCC do not seem to be beneficial for the majority of patients and new therapeutic approaches such as gene therapy and targeted drug therapy are of great clinical interest.     

二至丸及其组成药味保肝药理作用研究进展

二至丸由女贞子和墨旱莲两味中药组成,这两味药及其组方均有不同程度的保肝作用。二至丸中含有多种保肝有效成分,主要有三萜类、环烯醚萜类、苯乙醇-苷类、香豆素醚类等,它们能对抗多种化学性肝损伤、抗缺血再灌注性肝损伤以及抗肝纤维化等,其保肝作用机制包括抗氧化应激、抗炎、抑制肝星状细胞增殖和活化、促进肝星状细胞凋亡以及影响肝细胞代谢过程等。总结二至丸及其组成药味女贞子和墨旱莲的保肝作用,探讨其保肝作用机制,以期为进一步研究二至丸药效物质基础提供参考。     

药物导致肝损伤、药物处置及其代谢产物分析(英文)

药物导致肝损伤是药物研发失败和上市药退市的一个主要原因。药物导致肝损伤的频发跟肝生理功能密切相关,因为大部分药物分子在体内的消除依赖于药物代谢或胆汁排泄的肝清除。虽然不少发病机制已有广泛的研究,但是大部分与药物导致肝损伤相关的机制仍然未明确。从这个意义上讲,代谢组学将成为研究药物导致肝损伤强有力的手段以有助于更好地了解其机制并进行相关生物标志物的鉴定。     

A toxicologist's guide to biomarkers of hepatic response

Biological markers (biomarkers) are used to recognize, characterize and monitor treatment-related responses following exposure to xenobiotics. Biomarkers serve three primary applications in toxicology: 1) to confirm exposure to a deleterious agent, 2) to provide a system for monitoring individual susceptibility to a toxicant, and 3) to quantitatively assess deleterious effects of a toxicant to an organism or individual. Because the liver is a general target for adverse effects of drugs and other chemicals, biomarkers of untoward hepatic response to xenobiotics are of particular interest to the pharmaceutical toxicologist. General requirements for the latter category of biomarkers are sample availability, target organ specificity, sensitivity for the toxicity of interest, accessibility, a relatively short half-life, and available detection systems. Biomarkers that can be assayed in biological fluids from both human and animal subjects are particularly desirable. Histologically, acute and subacute hepatic toxicity commonly involves necrosis, steatosis, cholestasis, vascular disorders, or multiple lesions. The purpose of this review is to summarize reported applications using clinical analytes and biochemical indicators of hepatic dysfunction with emphasis on those that show promise of supplementing or improving upon standard laboratory procedures. Liver function markers refer to peripheral indicators of hepatic synthetic and secretory activities, enterohepatic function, or perturbations of the hepatic uptake and clearance of circulating biomolecules. Liver injury biomarkers include various peripheral proteins released in response to a cellular damage or locally, proteins that are significantly altered within the liver. These include both circulating cytosolic, mitochondrial, or canalicular membrane markers, and the up-regulation or depletion of radical scavengers, modulators, and stabilizers of intracellular damage. Subsequent recovery from a toxic insult involves repair, regenerative, and proliferative responses that constitute the third class of biomarkers. Of these, protein markers found either in sera, plasma, or urine either during or just prior to the early manifestation of histological hepatic lesions are of greatest interest. Examples of a number of these markers, their documented applications in humans or animals, and potential advantages as well as limitations are presented.     

Cellular glutathione in fatty liver in vitro models

The range of non-alcoholic fatty liver disease (NAFLD) includes simple hepatic steatosis, the inflammatory non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. The accumulation of specific lipids in hepatocytes has been associated with oxidative stress and progression of the disease. Elevated serum free fatty acids and hepatocyte lipotoxicity can be studied in an in vitro cellular model. For this purpose, we cultured the human liver cell line, HepG2/C3A, in medium supplemented with increasing amounts of oleic acid (C18:1) and evaluated oxidative stress by measuring the content of the cellular antioxidant, glutathione (GSH). We observed a dose-dependent steatosis, as determined by Nile Red staining, with concurrent increases of GSH; similar findings were also observed in cultured human hepatocytes. Cells cultured with palmitic acid (C16:0) or the combination oleic/palmitic acids (2:1 ratio) also exhibited a dose-dependent increase of GSH; however palmitic-supplemented cultures did not sustain the GSH increase after 24 h. We also detected an increase in the formation of lipid peroxides (LPO) indicating that the increase of GSH was a cellular mechanism that may be related to the high exposure of fatty acids. The results of this in vitro study suggest an antioxidant response against fat overloading and indicate potential differences in response to specific fatty acid-induced hepatic steatosis and associated lipotoxicity.     

1 例肾病综合征合并药物性肝损伤患儿的药学监护

目的:探讨临床药师在肾病综合征合并药物性肝损伤患儿药学监护中的作用。 方法:1 例肾病综合征患儿在治疗中出现 肝功能异常,临床药师分析患儿肝功能异常的原因,排除其他因素后考虑药物引起。 参考美国 LiverTox 数据库、中国 Hepatox 数 据库以及相关指南和文献,临床药师建议医师停用怀疑药物,并提供替代治疗方案,开展药物不良反应监测和电话随访。 结果: 临床药师判断卡托普利致患儿肝功能异常可能性大,医师采纳建议并停用卡托普利,选用福辛普利作为替代治疗;此外,临床药 师建议医师将泼尼松调整为甲泼尼龙长期给药、选用复方甘草酸苷进行保肝治疗,调整为上述治疗方案后患儿肝功能逐渐恢复 正常。 结论:临床药师可协助医师查找患儿肝损伤的药物因素、提供治疗方案调整策略,降低患儿发生严重肝损伤的风险。