Animal models of idiosyncratic drug reactions

Idiosyncratic drug reactions represent a major problem. In most cases the mechanisms of these reactions are unknown, but circumstantial evidence points to the involvement of reactive metabolites and the characteristics of the reactions suggest involvement of the immune system. If progress is to be made in dealing with these adverse reactions it is essential that we have a better understanding of their mechanisms, and it is hard to imagine testing mechanistic hypotheses without good animal models. Unfortunately, idiosyncratic reactions are also idiosyncratic in animals so few good models exist. The best models, in which a rodent develops a clinical syndrome similar to that which occurs in humans, appear to be penicillamine-induced autoimmunity in Brown Norway rats and nevirapine-induced skin rash in rats. Sulfamethoxazole-induced hypersensitivity in dogs and propylthiouracil-induced autoimmunity in cats are also similar to adverse reactions that occur in people, but they have practical limitations. Halothane-induced liver toxicity in guinea pigs and amodiaquine-induced bone marrow and liver toxicity in rats represent models in which there is an immune response and mild, reversible toxicity. It is possible that the development of immune tolerance is what limits the toxicity in these models, and if this is true, interventions that prevent tolerance might lead to good models. Although the history of developing animal models of idiosyncratic drug reactions is mostly one of failure, such models are essential. A better understanding of immune tolerance may greatly facilitate the development of better models; transgenic technology may also provide an important tool.     

Metabolic, idiosyncratic toxicity of drugs: overview of the hepatic toxicity induced by the anxiolytic, panadiplon

Preclinical drug safety evaluation studies, typically conducted in two or more animal species, reveal and define dose-dependent toxicities and undesirable effects related to pharmacological mechanism of action. Idiosyncratic toxic responses are often not detected during this phase in development due to their relative rarity in incidence and differences in species sensitivity. This paper reviews and discusses the metabolic idiosyncratic toxicity and species differences observed for the experimental non-benzodiazepine anxiolytic, panadiplon. This compound produced evidence of hepatic toxicity in Phase 1 clinical trial volunteers that was not predicted by rat, dog or monkey preclinical studies. However, subsequent studies in Dutch-belted rabbits revealed a hepatic toxic syndrome consistent with a Reye's Syndrome-like idiosyncratic response. Investigations into the mechanism of toxicity using rabbits and cultured hepatocytes from several species, including human, provided a sketch of the complex pathway required to produce hepatic injury. This pathway includes drug metabolism to a carboxylic acid metabolite (cyclopropane carboxylic acid), inhibition of mitochondrial fatty acid beta-oxidation, and effects on intermediary metabolism including depletion of glycogen and disruption of glucose homeostasis. We also provide evidence suggesting that the carboxylic acid metabolite decreases the availability of liver CoA and carnitine secondary to the formation of unusual acyl derivatives. Hepatic toxicity could be ameliorated by administration of carnitine, and to a lesser extent by pantothenate. These hepatocellular pathway defects, though not directly resulting in cell death, rendered hepatocytes sensitive to secondary stress, which subsequently produced apoptosis and hepatocellular necrosis. Not all rabbits showed evidence of hepatic toxicity, suggesting that individual or species differences in any step along this pathway may account for idiosyncratic responses. These differences may be roughly applied to other metabolic idiosyncratic hepatotoxic responses and include variations in drug metabolism, effects on mitochondrial function, nutritional status, and health or underlying disease.     

Adverse hepatic drug reactions: inflammatory episodes as consequence and contributor

Susceptibility to drug toxicity is influenced by a variety of factors, both genetic and environmental. The focus of this article is the evidence addressing the hypothesis that inflammation is both a result of and a susceptibility factor for drug toxicity, with an emphasis on liver as a target organ. Results of studies suggesting a role for inflammatory mediators in the hepatotoxicity caused by acetaminophen or ethanol are discussed. For several drugs, the evidence from animal models that concurrent inflammation increases injury is presented. In addition, the occurrence of adverse drug reactions in people with preexisting inflammatory diseases is considered. The special case of idiosyncratic drug reactions is discussed and the potential raised for development of animal models for this type of drug toxicity. The conclusion is that inflammatory factors should be considered as determinants of sensitivity to adverse drug reactions.     

In vitro assessment of pharmacogenetic susceptibility to toxic drug metabolites in humans

An experimental approach to the pharmacogenetics of human idiosyncratic drug reactions requires an assay for determining individual differences in susceptibility that does not expose patients to further drug-related risk. We have developed an in vitro drug toxicity assay designed to test the hypothesis that differences in susceptibility may be based on genetic abnormalities in the detoxification of electrophilic drug metabolites. Lymphocytes are challenged with metabolites generated by a murine hepatic microsomal system. By using cells from patients deficient in glutathione synthetase, we found that cells with decreased glutathione defenses are more sensitive to toxicity from metabolites of drugs such as acetaminophen, nitrofurantoin, and metronidazole. The assay was then applied to studying the pharmacogenetics of phenytoin hepatotoxicity. We found an inherited defect in the detoxification of phenytoin arene oxide metabolites in cells from patients and their relatives. The studies have led to an elucidation of a genetically heterogeneous group of detoxification defects for arene oxide metabolites of various aromatic drugs. Such experimental approaches may be useful in diagnosing idiosyncratic drug reactions, in establishing their pharmacogenetic basis, and perhaps in predicting toxicity potential of drugs for selected patients and families.     

DITOP: drug-induced toxicity related protein database

MOTIVATION: Drug-induced toxicity related proteins (DITRPs) are proteins that mediate adverse drug reactions (ADRs) or toxicities through their binding to drugs or reactive metabolites. Collection of these proteins facilitates better understanding of the molecular mechanisms of drug-induced toxicity and the rational drug discovery. Drug-induced toxicity related protein database (DITOP) is such a database that is intending to provide comprehensive information of DITRPs. Currently, DITOP contains 1501 records, covering 618 distinct literature-reported DITRPs, 529 drugs/ligands and 418 distinct toxicity terms. These proteins were confirmed experimentally to interact with drugs or their reactive metabolites, thus directly or indirectly cause adverse effects or toxicities. Five major types of drug-induced toxicities or ADRs are included in DITOP, which are the idiosyncratic adverse drug reactions, the dose-dependent toxicities, the drug-drug interactions, the immune-mediated adverse drug effects (IMADEs) and the toxicities caused by genetic susceptibility. Molecular mechanisms underlying the toxicity and cross-links to related resources are also provided while available. Moreover, a series of user-friendly interfaces were designed for flexible retrieval of DITRPs-related information. The DITOP can be accessed freely at http://bioinf.xmu.edu.cn/databases/ADR/index.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Mechanisms of idiosyncratic drug reactions: the case of felbamate

Idiosyncratic drug reactions (IDR) are a specific type of drug toxicity characterized by their delayed onset, low incidence and reactive metabolite formation with little, if any, correlation between pharmacokinetics or pharmacodynamics and the toxicological outcome. As the name implies, IDR are unpredictable and often result in the post marketing failure of otherwise useful therapies. Examples of drugs, which have failed as a result of IDR in recent years, include trovafloxacin, zileuton, troglitazone, tolcapone and felbamate. To date there exists no pre-clinical model to predict these adverse drug reactions and a mechanistic understanding of these toxicities remains limited. In an attempt to better understand this class of drug toxicities and gain mechanistic insight, we have studied the IDR associated with a model compound, felbamate. Our studies with felbamate are consistent with the theory that compounds which cause IDR undergo bioactivation to a highly reactive electrophilic metabolite that is capable of forming covalent protein adducts in vivo. In additon, our data suggest that under normal physiological conditions glutathione plays a protective role in preventing IDR during felbamate therapy, further emphasizing a correlation between reactive metabolite formation and a toxic outcome. Clinical studies with felbamate have been able to demonstrate an association between reactive metabolite formation and a clinically relevant toxicity; however, additional research is required to more fully understand the link between reactive metabolite formation and the events which elicit toxicity. Going forward, it seems reasonable that screening for reactive metabolite formation in early drug discovery may be an important tool in eliminating the post-marketing failure of otherwise useful therapies.     

Structural Alerts,Reactive Metabolites,and Protein Covalent Binding: How Reliable Are These Attributes as Predictors of Drug Toxicity?

In an increasing number of cases, a deeper understanding of the biochemical basis for idiosyncratic adverse drug reactions (IADRs) has aided to replace a vague perception of a chemical class effect with a sharper picture of individual molecular peculiarity. Considering that IADRs are too complex to duplicate in a test tube, and their idiosyncratic nature precludes prospective clinical studies, it is currently impossible to predict which new drugs will be associated with a significant incidence of toxicity. Because it is now widely appreciated that reactive metabolites, as opposed to the parent molecules from which they are derived, are responsible for the pathogenesis of some IADRs, the propensity of drug candidates to form reactive metabolites is generally considered a liability. Procedures have been implemented to monitor reactive‐metabolite formation in discovery with the ultimate goal of eliminating or minimizing the liability via rational structural modification of the problematic chemical series. While such mechanistic studies have provided retrospective insight into the metabolic pathways which lead to reactive metabolite formation with toxic compounds, their ability to accurately predict the IADR potential of new drug candidates has been challenged. There are several instances of drugs that form reactive metabolites, but only a fraction thereof cause toxicity. This review article will outline current approaches to evaluate bioactivation potential of new compounds with particular emphasis on the advantages and limitation of these assays. Plausible reason(s) for the excellent safety record of certain drugs susceptible to bioactivation will also be explored and should provide valuable guidance in the use of reactive‐metabolite assessments when nominating drug candidates for development.     

The Schistosoma mansoni Cytochrome P450 (CYP3050A1) Is Essential for Worm Survival and Egg Development

Schistosomiasis affects millions of people in developing countries and is responsible for more than 200,000 deaths annually. Because of toxicity and limited spectrum of activity of alternatives, there is effectively only one drug, praziquantel, available for its treatment. Recent data suggest that drug resistance could soon be a problem. There is therefore the need to identify new drug targets and develop drugs for the treatment of schistosomiasis. Analysis of the Schistosoma mansoni genome sequence for proteins involved in detoxification processes found that it encodes a single cytochrome P450 (CYP450) gene. Here we report that the 1452 bp open reading frame has a characteristic heme-binding region in its catalytic domain with a conserved heme ligating cysteine, a hydrophobic leader sequence present as the membrane interacting region, and overall structural conservation. The highest sequence identity to human CYP450s is 22%. Double stranded RNA (dsRNA) silencing of S. mansoni (Sm)CYP450 in schistosomula results in worm death. Treating larval or adult worms with antifungal azole CYP450 inhibitors results in worm death at low micromolar concentrations. In addition, combinations of SmCYP450-specific dsRNA and miconazole show additive schistosomicidal effects supporting the hypothesis that SmCYP450 is the target of miconazole. Treatment of developing S. mansoni eggs with miconazole results in a dose dependent arrest in embryonic development. Our results indicate that SmCYP450 is essential for worm survival and egg development and validates it as a novel drug target. Preliminary structure-activity relationship suggests that the 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol moiety of miconazole is necessary for activity and that miconazole activity and selectivity could be improved by rational drug design.     

Risk assessment and mitigation strategies for reactive metabolites in drug discovery and development

Drug toxicity is a leading cause of attrition of candidate drugs during drug development as well as of withdrawal of drugs post-licensing due to adverse drug reactions in man. These adverse drug reactions cause a broad range of clinically severe conditions including both highly reproducible and dose dependent toxicities as well as relatively infrequent and idiosyncratic adverse events. The underlying risk factors can be split into two groups: (1) drug-related and (2) patient-related. The drug-related risk factors include metabolic factors that determine the propensity of a molecule to form toxic reactive metabolites (RMs), and the RM and non-RM mediated mechanisms which cause cell and tissue injury. Patient related risk factors may vary markedly between individuals, and encompass genetic and non-genetic processes, e.g. environmental, that influence the disposition of drugs and their metabolites, the nature of the adverse responses elicited and the resulting biological consequences. We describe a new strategy, which builds upon the strategies used currently within numerous pharmaceutical companies to avoid and minimize RM formation during drug discovery, and that is intended to reduce the likelihood that candidate drugs will cause toxicity in the human population. The new strategy addresses drug-related safety hazards, but not patient-related risk factors. A common target organ of toxicity is the liver and to decrease the likelihood that candidate drugs will cause liver toxicity (both non-idiosyncratic and idiosyncratic), we propose use of an in vitro Hepatic Liability Panel alongside in vitro methods for the detection of RMs. This will enable design and selection of compounds in discovery that have reduced propensity to cause liver toxicity. In vitro Hepatic Liability is assessed using toxicity assays that quantify: CYP 450 dependent and CYP 450 independent cell toxicity; mitochondrial impairment; and inhibition of the Bile Salt Export Pump. Prior to progression into development, a Hepatotoxicity Hazard Matrix combines data from the Hepatic Liability Panel with the Estimated RM Body Burden. The latter is defined as the level of covalent binding of radiolabelled drug to human hepatocyte proteins in vitro adjusted for the predicted human dose. We exemplify the potential value of this approach by consideration of the thiazolidinedione class of drugs.     

The ecological characteristics of idiosyncratic and nested diatoms

Nestedness represents the degree to which species assemblages are proper subsets of larger assemblages. Highly nested assemblages are characterized by a high number of species conforming to nested subset pattern and a low number of species which depart from nested pattern, that is, idiosyncratic species. The main aims of this paper were to (i) examine whether local stream habitat factors and spatial variables govern the number of nested stream diatom species at sites, (ii) to examine whether nested and idiosyncratic species differ in ecological characteristics, and (iii) to study if these distinctions have implications for turnover in stream diatom communities. Stream diatom communities showed a highly significant nested pattern, with observed matrix temperature of 28.4 degrees C. However, number of idiosyncratic species was high, constituting 44% of total species number of the survey. The number of nested species at sites was not related to any of the measured habitat factors or geographical location of the sampling sites. Idiosyncratic species were significantly (ANOVA: P=0.002) more widely distributed than nested species. Partial mantel tests showed that idiosyncratic species exhibited faster turnover along geographical and environmental distance than nested species. These data showed that although the degree of nestedness was highly significant, stream diatom communities were nevertheless characterized by a number of idiosyncratic species departing from the nested subset pattern. It seems that the compositional turnover is faster in communities that are dominated by idiosyncratic species suggesting that turnover diversity in diatoms may be governed more by the distribution of idiosyncratic species.     

Interactions between prostaglandins and calcium: the importance of bell-shaped dose-response curves

Biological responses to PGs show two basic forms of dose/response relationship, plateau and bell-types. Although bell-shaped dose/response curves are well documented their possible occurrence is almost always ignored in the design and interpretation of experiments on PGs and related substances. This may lead to serious errors, several types of which are described. The ignoring of a well-documented phenomenon may take place because there is no accepted hypothesis which attempts to explain the bell-type curves. A hypothesis is proposed which accounts for both plateau and bell type responses. It is developed primarily with respect to PG-calcium interactions but may be applicable to some PG-cyclic nucleotide interactions as well. The model leads to precise predictions which can be experimentally tested in many systems.     

Acaricide,Fungicide and Drug Interactions in Honey Bees (Apis mellifera)

Background

Chemical analysis shows that honey bees (Apis mellifera) and hive products contain many pesticides derived from various sources. The most abundant pesticides are acaricides applied by beekeepers to control Varroa destructor. Beekeepers also apply antimicrobial drugs to control bacterial and microsporidial diseases. Fungicides may enter the hive when applied to nearby flowering crops. Acaricides, antimicrobial drugs and fungicides are not highly toxic to bees alone, but in combination there is potential for heightened toxicity due to interactive effects.

Methodology/Principal Findings

Laboratory bioassays based on mortality rates in adult worker bees demonstrated interactive effects among acaricides, as well as between acaricides and antimicrobial drugs and between acaricides and fungicides. Toxicity of the acaricide tau-fluvalinate increased in combination with other acaricides and most other compounds tested (15 of 17) while amitraz toxicity was mostly unchanged (1 of 15). The sterol biosynthesis inhibiting (SBI) fungicide prochloraz elevated the toxicity of the acaricides tau-fluvalinate, coumaphos and fenpyroximate, likely through inhibition of detoxicative cytochrome P450 monooxygenase activity. Four other SBI fungicides increased the toxicity of tau-fluvalinate in a dose-dependent manner, although possible evidence of P450 induction was observed at the lowest fungicide doses. Non-transitive interactions between some acaricides were observed. Sublethal amitraz pre-treatment increased the toxicity of the three P450-detoxified acaricides, but amitraz toxicity was not changed by sublethal treatment with the same three acaricides. A two-fold change in the toxicity of tau-fluvalinate was observed between years, suggesting a possible change in the genetic composition of the bees tested.

Conclusions/Significance

Interactions with acaricides in honey bees are similar to drug interactions in other animals in that P450-mediated detoxication appears to play an important role. Evidence of non-transivity, year-to-year variation and induction of detoxication enzymes indicates that pesticide interactions in bees may be as complex as drug interactions in mammals.     

On the relevance of mitochondrial fusions for the accumulation of mitochondrial deletion mutants: a modelling study

The molecular mechanisms underlying the aging process are still unclear, but the clonal accumulation of mitochondrial deletion mutants is one of the prime candidates. An important question for the mitochondrial theory of aging is to discover how defective organelles might be selected at the expense of wild-type mitochondria. We propose that mitochondrial fission and fusion events are of critical importance for resolving this apparent contradiction. We show that the occurrence of fusions removes the problems associated with the idea that smaller DNA molecules accumulate because they replicate in a shorter time--the survival of the tiny (SOT) hypothesis. Furthermore, stochastic simulations of mitochondrial replication, mutation and degradation show that two important experimental findings, namely the overall low mosaic pattern of oxidative phosphorylation (OXPHOS) impaired cells in old organisms and the distribution of deletion sizes, can be reproduced and explained by this hypothesis. Finally, we make predictions that can be tested experimentally to further verify our explanation for the age-related accumulation of mitochondrial deletion mutants.     

Tartrazine: a potentially hazardous dye in Canadian drugs.

The literature was reviewed to determine the incidence of idiosyncratic reactions to tartrazine. From 4% to 14% of individuals with asthma or allergies or both and from 7% to 20% of persons who are sensitive to acetylsalicylic acid may react to this dye. The mechanism of such reactions is unknown. Pharmaceutical manufacturers and distributors were surveyed and a list was prepared of approximately 450 Canadian pharmaceuticals that contain tartrazine. The 53 pharmaceutical and manufacturers and distributors whose drug products do not contain this dye were also listed. It is recommended that information concerning the tartrazine content of drugs be included on package labels.     

In vitro cytotoxicity assay to evaluate the toxicity of an electrophilic reactive metabolite using glutathione-depleted rat primary cultured hepatocytes

Glutathione plays an important role as not only a scavenger of reactive oxygen species but also in the conjugation or detoxification of electrophilic reactive metabolites, which has been thought to be one of the causes for idiosyncratic drug toxicity (IDT). Therefore, toxic responses to the reactive metabolites have been expected to be expressed more strongly in a glutathione-depleted condition. In the present study, we attempted to establish an in vitro cytotoxicity assay method to evaluate the toxicity of the reactive metabolite using rat primary cultured hepatocytes with cellular glutathione depletion by l-buthionine-S,R-sulfoximine. Also, we investigated whether the IDT risk is predictable by comparing the cytotoxic sensitivity between glutathione-depleted hepatocytes and untreated hepatocytes. Consequently, 10 drugs of 42 approved drugs, which were classified into 4 IDT categories (Withdrawn, Black box warning, Warning, and Safe), demonstrated higher cytotoxic sensitivity in the glutathione-depleted hepatocytes. Furthermore, a correlation was observed between the incidence of drugs with higher cytotoxic sensitivity in the glutathione-depleted hepatocytes and the IDT risk. The incidence was 50% in the Withdrawn category, 38% in the Black box warning category, 22% in the Warning category, and 8% in the Safe category. These results suggest that the IDT risk of some drugs may be predicted by comparing the cytotoxic sensitivity between them. Additionally, this method may be useful as a screening in the early stage of drug development where leads/candidates are optimized.     

The use of cultured hepatocytes to investigate the metabolism of drugs and mechanisms of drug hepatotoxicity

Hepatotoxins can be classified as intrinsic when they exert their effects on all individuals in a dose-dependent manner, and as idiosyncratic when their effects are the consequence of an abnormal metabolism of the drug by susceptible individuals (metabolic idiosyncrasy) or of an immune-mediated injury to hepatocytes (allergic hepatitis). Some xenobiotics are electrophilic, and others are biotransformed by the liver into highly reactive metabolites that are usually more toxic than the parent compound. This activation process is the key to many hepatotoxic phenomena. Mitochondria are a frequent target of hepatotoxic drugs, and the alteration of their function has immediate effects on the energy balance of cells (depletion of ATP). Lipid peroxidation, oxidative stress, alteration of Ca(2+) homeostasis, and covalent binding to cell macromolecules are the molecular mechanisms that are frequently involved in the toxicity of xenobiotics. Against these potential hazards, cells have their own defence mechanisms (for example, glutathione, DNA repair, suicide inactivation). Ultimately, toxicity is the balance between bioactivation and detoxification, which determines whether a reactive metabolite elicits a toxic effect. The ultimate goal of in vitro experiments is to generate the type of scientific information needed to identify compounds that are potentially toxic to man. For this purpose, both the design of the experiments and the interpretation of the results are critical.]     

Development of a whole-cell screening system for evaluation of the human CYP1A2-mediated metabolism

Cytochrome P450 1A2 (CYP1A2) is an important member of cytochrome P450 involved in drug metabolism. In this study, a cell line, Huh7-1A2-I-E, with high expression level of CYP1A2 is established based on Huh7 cells. To achieve this, we constructed a recombinant lentiviral vector, pLenti-1A2-I-E, containing a single promoter encoding CYP1A2 followed by an internal ribosome entry site (IRES) to permit the translation of enhanced green fluorescence protein (EGFP). Such a design has greatly facilitated the selection of stable cell lines because the translations of CYP1A2 and EGFP proteins would be based on a single bi-cistronic mRNA. The Huh7-1A2-I-E cells were evaluated as a cell-based model for identification of CYP1A2 inhibitors and for studies of cytotoxicity resulted from CYP-mediated drug metabolism. Treatment of Huh7-1A2-I-E cells and the Huh7-E control cells with aflatoxin B1 showed that cells with CYP1A2 expression are much more sensitive to aflatoxin B1 and the cellular toxicity of aflatoxin B1 in Huh7-1A2-I-E cells could be prevented by furafylline, a CYP1A2 inhibitor. A collection of approximately 200 drugs were screened using this system and results indicate that for most drugs the metabolism by CYP1A2 is unlikely to have made a major contribution to the in vitro cytotoxicity except for thimerosal and evoxine. Several previously unidentified CYP1A2 inhibitors such as evoxine and berberine were also identified in this study.     

Role of genetics and drug metabolism in human cancer risk

The research field concerning responses to drugs having a hereditary basis is called 'pharmacogenetics'. At least 5 dozen pharmacogenetic polymorphisms have been described in clinical medicine; many are responsible for marked differences in genetic predisposition toward toxicity or cancer. Three are detailed here: the acetylation, the debrisoquine, and the AH locus polymorphism. All 3 are very common among the United States' population: 1 in 2 is a 'slow acetylator', 1 in 12 is a 'poor metabolizer' for more than 2 dozen commonly prescribed drugs in the debrisoquine panel, and the CYP1A1 and CYP1A2 (cytochromes P(1)450 and P(3)450) genes are highly inducible by cigarette smoke in 1 of 10 patients. Differences in xenobiotic metabolism between individuals in the same family can be greater than 200-fold, suggesting that occupationally hazardous chemicals, as well as prescribed drugs having a narrow therapeutic window, might cause strikingly dissimilar effects between patients of differing genotypes. Our ultimate goal is 'preventive toxicology', i.e. the development of simple, inexpensive, unequivocal and sensitive assays to predict individual risk of toxicity or cancer. These tests could help the individual in choosing a safer life style or place of work and might aid the physician in deciding which drug to prescribe.     

Interaction between prostaglandins and calcium: The importance of bell-shaped dose-response curves

Biological response to PGs show two basic forms of dose/response relationship, plateau and bell-types. although bell-shaped dose/response curves are well documented their possible occurrence is almost always ignored on the design and interpretation of experiments on PGs and related substances. This may lead to serious errors, several types of which are described. The ignoring of a well-documented phenomenon may take place because there is no accepted hypothesis which attempts to explain the bell-type curves. A hypothesis is proposed which accounts for both plateau and bell type responses. It is developed primarily with respect to PG-calcium interactions but may be applicable to some PG-cyclic nucleotide interactions as well. The model leads to precise predictions which can be experimentally tested in many systems.