Investigating mitochondrial dysfunction to increase drug safety in the pharmaceutical industry

Drug-induced mitochondrial dysfunction is a contributor to both late-stage compound attrition and post-market drug withdrawals. This review outlines the mechanisms which lead to drug-induced mitochondrial dysfunction and discusses the tremendous advances that have been made in the development of in vitro methods to identify mitochondrial impairment. Potentially useful animal models and in vivo methods to detect drug-induced mitochondrial impairment are also discussed.     

Drug-induced cardiac mitochondrial toxicity and protection: from doxorubicin to carvedilol

Mitochondria have long been involved in several cellular processes beyond its role in energy production. The importance of this organelle for cardiac tissue homeostasis has been greatly investigated and its impairment can lead to cell death and consequent organ failure. Several compounds have been described in the literature as having direct effects on cardiac mitochondria which can provide a mechanistic explanation for their toxicological or pharmacological effects. The present review describes one classic example of drug-induced cardiac mitochondrial toxicity and another case of drug-induced mitochondrial protection. For the former, we present the case for doxorubicin, an anticancer agent whose treatment is associated with a cumulative and dose-dependent cardiomyopathy with a mitochondrial etiology. Following this, we present the case of carvedilol, a β-blocker with intrinsic antioxidant activity, which has been described to protect cardiac mitochondria from oxidative injury. The final part of the review integrates information from the previous chapters, demonstrating how carvedilol can contribute to reduce doxorubicin toxicity on cardiac mitochondria. The two referred examples result in important take-home messages: a) drug-induced cardiac mitochondrial dysfunction is an important contributor for drug-associated organ failure, b) protection of mitochondrial function is involved in the beneficial impact of some clinically-used drugs and c) a more accurate prediction of toxic vs. beneficial effects should be an important component of drug development by the pharmaceutical industry.     

A high-throughput dual parameter assay for assessing drug-induced mitochondrial dysfunction provides additional predictivity over two established mitochondrial toxicity assays

Mitochondrial toxicity is a major reason for safety-related compound attrition and post-market drug withdrawals, highlighting the necessity for higher-throughput screens that can identify this mechanism of toxicity during the early stages of drug discovery. Here, we present the validation of a 384-well dual parameter plate-based assay capable of measuring oxygen consumption and extracellular acidification in intact cells simultaneously. The assay showed good reproducibility and robustness and is suitable for use with both suspension cells and adherent cells. To determine if the assay provides additional value in detecting mitochondrial toxicity over existing platforms, 200 commercially available drugs were tested in the assay using HL60 suspension cells as well as in two conventional mitochondrial toxicity assays: an oxygen consumption assay that uses isolated mitochondria and a cell-based assay that uses HepG2 cells grown in glucose and galactose media. The combination of the dual parameter assay and the isolated mitochondrial oxygen consumption assay identified more compounds that caused mitochondrial impairment than any other combination of the three assays or each of the three assays on its own. Furthermore, novel information was obtained from the dual parameter assay on drugs not previously reported to cause mitochondrial impairment.     

Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro

As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction.     

Identification of early proteomic markers for hepatic steatosis

The identification of biomarkers for disease state, drug efficacy, and toxicity is becoming increasingly important for drug discovery and development. We have used two-dimensional differential in-gel electrophoresis and mass spectrometry to identify proteomic markers associated with hepatocellular steatosis in rats after dosing with a compound (CDA) in preclinical development. Rats were dosed daily for up to 5 days with CDA for measurement of blood biochemical parameters, histological, and proteomic analysis. Alterations in plasma glucose and liver transaminases were detected from dosing day 3 onward, and livers showed trace levels of hepatocellular vacuolation from 6 h which increased in extent and severity over the 5 day time course. The number of significantly altered protein spots increased over the 5 day time course, and Ingenuity Pathway Analysis showed that the predominant functions altered by CDA treatment were cell death and cellular assembly and organization. This included alterations in secreted proteins, endoplasmic reticulum and mitochondrial chaperones, antioxidant proteins, and enzymes involved in fatty acid biosynthesis. Comparative in vitro dosing studies showed similar alterations to the proteome, neutral lipid accumulation, and mitochondrial dehydrogenase activity in response to CDA treatment of cultured rat hepatocytes. The finding that several proteins showed significant changes in abundance before the onset of overt toxicity in vivo suggested that these could serve as predictive biomarkers of compounds with a propensity to induce liver steatosis. These markers underwent further direct analysis in the in vitro hepatocyte toxicity model to determine their utility in the development of high throughput assays for drug-induced steatosis.     

Screening for reactive intermediates and toxicity assessment in drug discovery

Moving forward in the 21st century, toxicity remains a significant cause for failures during drug development, and limited correlations exist between preclinical in vitro cellular toxicity and/or in vivo animal toxicity models, and human toxicity. This is due, in part, to the fact that drug candidates generally target multiple tissues rather than single organs, and result in a series of inter-related biochemical events. Drug-induced toxicities, such as idiosyncratic drug reactions, represent a serious complication of drug therapy that needs to be addressed at the drug discovery stage. Many robust toxicity screening methods, however, have been developed, and are described in the literature. Although biochemical mechanisms of drug-induced toxicities are complex, by combining these screening methods into a panel of assays, a risk assessment profile/decision-making guide can be obtained for each drug candidate. The panel of assays covered by this review broadly includes in silico prediction of reactive intermediates, detection and structural characterization of reactive intermediates, covalent binding of reactive intermediates to macromolecules, and the impact of reactive intermediates on cellular functions. Current and future strategies for the proper sequencing of these assays in a drug discovery environment are also discussed.     

Paradigm Shift in Toxicity Testing and Modeling

The limitations of traditional toxicity testing characterized by high-cost animal models with low-throughput readouts, inconsistent responses, ethical issues, and extrapolability to humans call for alternative strategies for chemical risk assessment. A new strategy using in vitro human cell-based assays has been designed to identify key toxicity pathways and molecular mechanisms leading to the prediction of an in vivo response. The emergence of quantitative high-throughput screening (qHTS) technology has proved to be an efficient way to decompose complex toxicological end points to specific pathways of targeted organs. In addition, qHTS has made a significant impact on computational toxicology in two aspects. First, the ease of mechanism of action identification brought about by in vitro assays has enhanced the simplicity and effectiveness of machine learning, and second, the high-throughput nature and high reproducibility of qHTS have greatly improved the data quality and increased the quantity of training datasets available for predictive model construction. In this review, the benefits of qHTS routinely used in the US Tox21 program will be highlighted. Quantitative structure-activity relationships models built on traditional in vivo data and new qHTS data will be compared and analyzed. In conjunction with the transition from the pilot phase to the production phase of the Tox21 program, more qHTS data will be made available that will enrich the data pool for predictive toxicology. It is perceivable that new in silico toxicity models based on high-quality qHTS data will achieve unprecedented reliability and robustness, thus becoming a valuable tool for risk assessment and drug discovery.     

Mitochondrial off targets of drug therapy

The bioenergetic features of mitochondria have long been exploited in the design of pharmacological agents suited to accomplish a desired physiological effect; uncoupling of oxidative phosphorylation to induce weight loss, for example. However, more recent experience demonstrates mitochondria to be unintended off targets of other drug therapies and responsible, at least in part, for the dose-limiting adverse events associated with a large array of pharmaceuticals. Review of the fundamentals of mitochondrial molecular biology and bioenergetics reveals a multiplicity of off targets that can be invoked to explain drug-induced mitochondrial failure. It is this redundancy of mitochondrial off targets that complicates identification of discrete mechanisms of toxicity and confounds QSAR-based design of new small molecules devoid of this potential for mitochondrial toxicity. The present review article briefly reviews the molecular biology and biophysics of mitochondrial bioenergetics, which then serves as a platform for identifying the various potential off targets for drug-induced mitochondrial toxicity.     

The role of pathology in the identification of drug-induced hepatic toxicity

Pathologists play a central role in the recognition and prevention of drug-induced toxicity. Pathologists engaged in clinical practice must identify a pattern of histological lesions that are interpreted in concert with a variety of clinical data to determine the probability of drug-induced toxicity versus background disease processes and the most likely drug, often of many, to have caused the specific injury. Toxicological pathologists, working in concert with other scientists, have the responsibility of preventing drug-induced toxicity in humans by identifying potentially toxic drugs and keeping them from the marketplace. In this process of drug development, a broad array of in vivo testing using a number of animal species and in vitro assays are used. Technological advances require pathologists to integrate molecular-based mechanistic data effectively with traditional morphological evaluation to develop a more detailed grasp of the pathogenesis of drug-induced injury. All pathologists have the responsibility to effectively and accurately communicate their findings and interpretations to the appropriate audiences.     

The significance of mitochondrial toxicity testing in drug development

Mitochondrial dysfunction is increasingly implicated in the etiology of drug-induced toxicities. Members of diverse drug classes undermine mitochondrial function, and among the most potent are drugs that have been withdrawn from the market, or have received Black Box warnings from the FDA. To avoid mitochondrial liabilities, routine screens need to be positioned within the drug-development process. Assays for mitochondrial function, cell models that better report mitochondrial impairment, and new animal models that more faithfully reflect clinical manifestations of mitochondrial dysfunction are discussed in the context of how such data can reduce late stage attrition of drug candidates and can yield safer drugs in the future.     

Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: State of the science

In vitro, high-throughput methods have been widely recommended as an approach to screen chemicals for the potential to cause developmental neurotoxicity and prioritize them for additional testing. The choice of cellular models for such an approach will have important ramifications for the accuracy, predictivity and sensitivity of the screening assays. In recent years neuroprogenitor cells from rodents and humans have become more widely available and may offer useful models having advantages over primary neuronal cultures and/or transformed cell lines. To date, these models have been utilized in only a limited number of toxicity studies. This review summarizes the state of the science regarding stem and neuroprogenitor models that could be used for screening assays, provides researchers in this field with examples of how these cells have been utilized to date, and discusses the advantages, limitations and knowledge gaps regarding these models. Data are available from both rodent and human stem and neuroprogenitor cell models that indicate that these models will be a valid and useful tool for developmental neurotoxicity testing. Full potential of these models will only be achieved following advances in neurobiology that elucidate differentiation pathways more clearly, and following further evaluation of larger sets of developmentally neurotoxic and non-toxic chemicals to define the sensitivity and predictivity of assays based on stem or progenitor cell models.     

Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants.

Many highly proliferative cells generate almost all ATP via glycolysis despite abundant O(2) and a normal complement of fully functional mitochondria, a circumstance known as the Crabtree effect. Such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, such as the inhibitors rotenone, antimycin, oligomycin, and compounds like carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that uncouple the respiratory electron transfer system from phosphorylation. These cells are also resistant to the toxicity of many drugs whose deleterious side effect profiles are either caused, or exacerbated, by impairment of mitochondrial function. Drug-induced mitochondrial toxicity is shown by members of important drug classes, including the thiazolidinediones, statins, fibrates, antivirals, antibiotics, and anticancer agents. To increase detection of drug-induced mitochondrial effects in a preclinical cell-based assay, HepG2 cells were forced to rely on mitochondrial oxidative phosphorylation rather than glycolysis by substituting galactose for glucose in the growth media. Oxygen consumption doubles in galactose-grown HepG2 cells and their susceptibility to canonical mitochondrial toxicants correspondingly increases. Similarly, toxicity of several drugs with known mitochondrial liabilities is more readily apparent in aerobically poised HepG2 cells compared to glucose-grown cells. Some drugs were equally toxic to both glucose- and galactose-grown cells, suggesting that mitochondrial impairment is likely secondary to other cytotoxic mechanisms.     

Zebrafish for drug toxicity screening: bridging the in vitro cell-based models and in vivo mammalian models

INTRODUCTION: Over the past decade, zebrafish have been tasked to play important roles from modeling human diseases to finding cures for them. Inadvertently, these fish now find themselves swimming along the drug development pipeline. A number of studies have been conducted to see if these small fish are up to the task of drug toxicity testing, an important rite of passage along the pharmaceutical pipeline. AREAS COVERED: This review covers the recent publications (2008 - 2010) on the state-of-the-art applications that couple advanced technologies with the unique advantages of zebrafish for drug toxicity screening. The paper looks at the several automated high-throughput platforms that have been developed for zebrafish teratogenicity, cardiotoxicity and neuro-sensory organ toxicity assays over the past 3 years as well as the important studies related to metabolism and biotransformation of selected drugs that have been initiated. This paper also reviews their mechanistic and predictive omics applications. EXPERT OPINION: While there have been a number of developments over the past 3 years and indeed over the last 10 years, challenges and limitations still exist, which, unless overcome, will prevent zebrafish from truly reaching their full potential as a drug toxicological model. That being said, recent developments have suggested that zebrafish could play a role in bridging the gap between in vitro cell-based models and in vivo mammalian models.     

New Perspectives for in Vitro Risk Assessment of Multiwalled Carbon Nanotubes: Application of Coculture and Bioinformatics

Nanotechnology is a rapidly expanding field with wide application for industrial and medical use; therefore, understanding the toxicity of engineered nanomaterials is critical for their commercialization. While short-term in vivo studies have been performed to understand the toxicity profile of various nanomaterials, there is a current effort to shift toxicological testing from in vivo observational models to predictive and high-throughput in vitro models. However, conventional monoculture results of nanoparticle exposure are often disparate and not predictive of in vivo toxic effects. A coculture system of multiple cell types allows for cross-talk between cells and better mimics the in vivo environment. This review proposes that advanced coculture models, combined with integrated analysis of genome-wide in vivo and in vitro toxicogenomic data, may lead to development of predictive multigene expression-based models to better determine toxicity profiles of nanomaterials and consequent potential human health risk due to exposure to these compounds.     

Human-based in vitro experimental systems for the evaluation of human drug safety

Accurate prediction of human drug safety remains a major challenge for drug development. Species-difference in drug toxicity represents a main reason for the difficulty in the prediction of human drug toxicity with nonhuman animal models. A combined in vitro-in vivo strategy (IVIVS), using human-based in vitro experimental systems to derive human-specific information, and animal systems for in vivo variables, may lead to a more accurate prediction of human in vivo drug toxicity. The success of IVIVS requires in vitro models with human-specific drug metabolism, appropriate target cell populations, and relevant endpoints. A novel theory, the Target Cell Initiation Theory for drug-induced organ failure (TACIT), is proposed to support the IVIVS. Based on TACIT, toxicity that requires chronic administration and multiple secondary changes may be defined by the evaluation of changes in target cells that initiate the cascade of secondary events. A novel in vitro experimental system, the Integrated Discrete Multiple Organ Co-culture (IdMOC) system, which allows the evaluation of multiple organ toxicity under conditions allowing multiple organ interactions, is described as a promising technology.     

Hypoxia/reoxygenation exacerbates drug-induced cytotoxicity by opening mitochondrial permeability transition pore: Possible application for toxicity screening

Recently, mitochondrial dysfunction is thought of as an important factor leading to a drug-induced liver injury. Our previous reports show that mitochondria-related toxicity, including respiratory chain inhibition (RCI) and reactive oxygen species (ROS) induction, can be detected by the modification of sugar resource substitution and high oxygen condition. However, this in vitro model does not detect mitochondrial permeability transition (MPT)-induced toxicity. Another study with a lipopolysaccharide-pre-administered rodent model showed that ischemia/reperfusion induced ROS, sensitized the susceptibility of MPT pore opening and, finally developed drug-induced liver toxicity. Based on this result, the present study investigated the effect of hypoxia/reoxygenation (H/R) treatment mimicking the ischemia/reperfusion on MPT-dependent toxicity, aiming to construct a system that can evaluate MPT by drugs in hepatocytes. Mitochondrial ROS were enhanced by H/R treatment only in the galactose culture condition. Amiodarone, benzbromarone, flutamide and troglitazone which induced MPT pore opening led to hepatocyte death only in combination with H/R and galactose. Moreover, this alteration was significantly suppressed in hepatocytes lacking cyclophilin D. In conclusion, MPT-induced cytotoxicity can be detected by activating mitochondrial function and H/R. This cell-based assay system could evaluate MPT induced-cytotoxicity by drugs, besides RCI and ROS induction.     

Research advances in drug-induced mitochondrial toxicity北大核心CSCD

Mitochondria perform important functions in energy production, balancing redox reactions, maintaining homeostasis, cell proliferation and apoptosis. Mitochondrial function is essential for human health. This is evidenced by a large number of diseases caused by mutations in the mitochondrial genome and the key role of mitochondrial dysfunction in many chronic diseases. A number of commonly used drugs can impair mitochondrial function, leading to adverse reactions or toxicity. Drug-induced mitochondrial dysfunction includes mitochondrial DNA damage, impaired mitochondrial respiration, increased production of reactive oxygen species and altered mitochondrial permeability. Based on data from experimental and clinical research, this review focuses on toxicity and mechanisms of common drugs such as analgesics, anticancer drugs and hypolipidemic drugs on mitochondria in order to provide guidance for clinical medication safety and new thoughts for analysis and screening of drug-induced mitochondrial toxicity. © 2017 Chinese Journal of Pharmacology and Toxicology. All rights reserved.     

A possible cellular mechanism of cisplatin-induced nephrotoxicity

Cisplatin, a relatively new antitumor agent, is associated with renal function impairment. The mechanism of cisplatin-induced nephrotoxicity is unknown. A mouse model was used to examine nephrotoxicity induced by cisplatin. This study demonstrates both morphologically and biochemically that mitochondrial damage may be associated with cisplatin-induced cellular toxicity. The morphological changes are evident after 72 h following a single 10 mg/kg i.p. dose of cisplatin. Biochemical changes also follow the morphological abbreviations. In vitro incubation of cisplatin with cells also shows a decline in Rhodamine 123 fluorescence with time, which is indicative of mitochondrial damage. The present findings suggest the possibility that the nephrotoxic effects of cisplatin may be related to a mitochondrial damage.