ArticleLiterature Review

Hepatocytes-the choice to investigate drug metabolism and toxicity in man: In vitro variability as a reflection of in vivo

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Abstract

The pharmaceutical industry is committed to marketing safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. Drug metabolism is a major determinant of drug clearance and interindividual pharmacokinetic differences, and an indirect determinant of the clinical efficacy and toxicity of drugs. Progressive advances in the knowledge of metabolic routes and enzymes responsible for drug biotransformation have contributed to understanding the great metabolic variations existing in human beings. Phenotypic as well genotypic differences in the expression of the enzymes involved in drug metabolism are the main causes of this variability. However, only a minor part of phenotypic variability in man is attributable to gene polymorphisms, thus making the definition of a normal liver complex. At present, the use of human in vitro hepatic models at early preclinical stages means that the process of selecting drug candidates is becoming much more rational. Cultured human hepatocytes are considered to be the closest model to human liver. However, the fact that hepatocytes are located in a microenvironment that differs from that of the cell in the liver raises the question: to what extent does drug metabolism variability observed in vitro actually reflect that of the liver in vivo? By comparing the metabolism of a model compound both in vitro and in vivo in the same individual, a good correlation between the in vitro and in vivo relative abundance of oxidized metabolites and the hydrolysis of the compound was observed. Thus, it is reasonable to consider that the variability observed in human hepatocytes reflects the existing phenotypic heterogeneity of the P450 expression in human liver.

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... Thus far, several good reviews in the field have been published (2, [10][11][12][13][14][15][16]. The above mentioned papers, however, were mainly focused on single issues such as in vitro metabolic stability testing development, comparison of methods for the assessment of drug metabolic stability, and prediction of in vivo metabolic parameters using data from in vitro assays. ...
... Some of the most important advantages of using microsomes are accessibility, various species models, procedure simplicity, limited amount of test agents used in the study and the fact that microsmes can be stored for a long period of time. On the other hand, hepatocytes, which contain intact cell membranes and physiological concentrations of enzymes, provide the most physiologically relevant model for predicting hepatic clearance (2, 15,18,37,39,48,[55][56][57][58]. Some researchers use microsomes in conjunction with hepatocytes to obtain more comprehensive results (7,55,(59)(60)(61). ...
... -In the metabolic assay, hepatocytes coming from different species can be used (e.g., human, mouse, rat and dog). Hepatocyte cultures represent a complex metabolic system, equipped with natural orientation for linked enzymes, intact cell membranes and all the necessary physiological co-factors (14,15,37,(56)(57)(58)(59). On the other hand, one of the main concerns about using freshly isolated human hepatocytes for predicting metabolic stability is their longer-term availability and their de-differentiation in culture (16,31,39,48,77,78). ...
Article
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Determination of metabolic profiles of new chemical entities is a key step in the process of drug discovery, since it influences pharmacokinetic characteristics of therapeutic compounds. One of the main challenges of medicinal chemistry is not only to design compounds demonstrating beneficial activity, but also molecules exhibiting favourable pharmacokinetic parameters. Chemical compounds can be divided into those which are metabolized relatively fast and those which undergo slow biotransformation. Rapid biotransformation reduces exposure to the maternal compound and may lead to the generation of active, non-active or toxic metabolites. In contrast, high metabolic stability may promote interactions between drugs and lead to parent compound toxicity. In the present paper, issues of compound metabolic stability will be discussed, with special emphasis on its significance, in vitro metabolic stability testing, dilemmas regarding in vitro-in vivo extrapolation of the results and some aspects relating to different preclinical species used in in vitro metabolic stability assessment of compounds.
... Depending on the model, they can either detect/ identify the generated metabolites, evaluate the toxicity of the compound and/or the metabolites generated, demonstrate the inhibition or induction of enzymes, or attempt to predict in vivo pharmacokinetics. 34 For the aforementioned scientific methodologies, miscellaneous in vitro models, or instrumental tools, are used, such as (i) microsomes, 35 (ii) genetically engineered eukaryote and prokaryote cells, 36,37 (iii) primary hepatocytes, 34,38 (iv) hepatocyte cell lines, such as HepG2 and HepaRG, 39,40 (v) liver slices, (vi) isolated and perfused liver, 41 and (vii) monitoring redox processes (such as metabolism mimicking) in electrochemical cells (EC) in cyclic voltamperometry or combination between an EC cell and mass spectrometry (MS). 42,43 Nowadays, research trends promote innovative bioreactor-based approaches for high-throughput drug screening and discovery in the pharmaceutical field. ...
... Depending on the model, they can either detect/ identify the generated metabolites, evaluate the toxicity of the compound and/or the metabolites generated, demonstrate the inhibition or induction of enzymes, or attempt to predict in vivo pharmacokinetics. 34 For the aforementioned scientific methodologies, miscellaneous in vitro models, or instrumental tools, are used, such as (i) microsomes, 35 (ii) genetically engineered eukaryote and prokaryote cells, 36,37 (iii) primary hepatocytes, 34,38 (iv) hepatocyte cell lines, such as HepG2 and HepaRG, 39,40 (v) liver slices, (vi) isolated and perfused liver, 41 and (vii) monitoring redox processes (such as metabolism mimicking) in electrochemical cells (EC) in cyclic voltamperometry or combination between an EC cell and mass spectrometry (MS). 42,43 Nowadays, research trends promote innovative bioreactor-based approaches for high-throughput drug screening and discovery in the pharmaceutical field. ...
... Indeed, this molecule is described in the literature as a reference for targeting CYP2C9 and gives 4 0 -hydroxydiclofenac as a main metabolite. Moreover, its metabolism in HepaRG cells has already been reported in the literature 34,[58][59][60][61][62][63][64][65] Sunitinib was retained as a second reference substance because it is metabolized into a single primary metabolite (N-desethyl sunitinib). ...
Article
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The research for new in vitro screening tools for predictive metabolic profiling of drug candidates is of major interest in the pharmaceutical field. The main motivation is to avoid late rejection in drug development and to deliver safer drugs to the market. Thanks to the superparamagnetic properties of iron oxide nanoparticles, a flow bioreactor has been developed which is able to perform xenobiotic metabolism studies. The selected cell line (HepaRG) maintained its metabolic competencies once iron oxide nanoparticles were internalized. Based on magnetically trapped cells in a homemade immobilization chamber, through which a flow of circulating phase was injected to transport nutrients and/or the studied xenobiotic, off-line and online (when coupled to a high-performance liquid chromatography chain) metabolic assays were developed using diclofenac as a reference compound. The diclofenac demonstrated a similar metabolization profile chromatogram, both with the newly developed setup and with the control situation. Highly versatile, this pioneering and innovative instrumental design paves the way for a new approach in predictive metabolism studies.
... In the same way, also engineered cells or systems (e.g. microsomes) based on reporter gene transfer have been shown to be not sufficiently reliable and informative (for more exhaustive information on the principal in vitro hepatic models traditionally in use, see also Brandon et al., 2003, Zucco et al., 2004, Gómez-Lechón et al., 2007, Guillouzo & Guguen-Guillouzo, 2008. ...
... It is now well recognised that any experimental model that has to be used for reproducing in vitro the function of human liver, must be developed from human hepatocytes in primary culture (Gómez-Lechón et al., 2007). The scarce availability of tissue, its variable quality, and the difficulty to succeed, with the traditional techniques, in maintaining isolated hepatocytes in vitro, preserving their viability and functions for long-term studies, have strongly hindered the refinement of these models, limiting, in such a way, the significance of their use (Gómez-Lechón et al., 2007;Guillouzo & Guguen-Guillouzo, 2008). ...
... It is now well recognised that any experimental model that has to be used for reproducing in vitro the function of human liver, must be developed from human hepatocytes in primary culture (Gómez-Lechón et al., 2007). The scarce availability of tissue, its variable quality, and the difficulty to succeed, with the traditional techniques, in maintaining isolated hepatocytes in vitro, preserving their viability and functions for long-term studies, have strongly hindered the refinement of these models, limiting, in such a way, the significance of their use (Gómez-Lechón et al., 2007;Guillouzo & Guguen-Guillouzo, 2008). No result worthy of further consideration has been derived from the very large number of attempts performed to get well-differentiated hepatocytes from stem precursors, either originating from the liver itself (resident hepatic progenitors), or from extra-hepatic sites (e.g. ...
Book
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Preface: There are a myriad number of different types of risks that, as individuals, we face every day that impact human health. Exposure to hazards can result from the natural and build environments with modulation of this exposure due to other factors such as our biology (eg. genetics, age, gender and comorbid conditions) and social interactions (eg. lifestyle choices and our level of risk acceptance). A broad population health approach includes determinants of health, this is the collective label given to factors and conditions that are thought to have an influence on health. Using such an extended understanding of the risk issue and its determinants of health context are very important to the scope of risk assessment efforts. Many risks that we face on a daily basis may be unavoidable, so there is an expectation by individuals that the level of risk is being managed and reduced to safe levels through evidence-based risk assessments and public health interventions. There has been a growing recognition that risks need to be viewed in their public health context to ensure that the most important risks are prioritized and addressed. Under a broader public health imperative, risk assessments are used as an important process to quantify the probability of harmful effects to individuals, sub-populations (eg. vulnerable patient groups) or entire populations. Thus, both quantitative and qualitative risk assessments help to evaluate the risks associated with hazards, help to prioritize the risks, and allow for cost-effective option generation to eliminate or control the hazards. The completion of risk assessments, appropriate in scope, can help decision-makers to select the most efficient and effective evidence based strategies. With limited government health budgets challenged by an aging population demographic such an understanding can improve resource allocation. Risk assessment must be sufficiently broad to ensure adequate understanding of the risk and to identify effective risk management options. This book presents an interesting and diverse collection of health risk assessments and health risk management research for known, and emerging risks that span a continuum towards future developments that aim to improve risk assessment analyses. Two case studies for existing health risks are presented in the first section and utilize surveys and look-back modeling methods. The second section deals with emerging health risk and provides three case studies and demonstrates the difficulties of assessing new risks when the scientific evidence base is limited. The third section provides case studies that challenge traditional assessments to improve future risk assessment methods. Case studies for existing risks in the first section include drug use in Italian professional delivery truck drivers and using look-back risk assessment for syringe reuse in Canada. The first chapter by Oraby et al. describes the occurrence of syringe reuse reported in a Canadian health care setting on approximately 1,400 patients in the province of Alberta. Multiple syringe re-use events may act as a vector to transmit both RNA and DNA viruses. This look-back study analyzed the risks for Hepatitis B (HBV), Hepatitis C (HCV) and Human Immunodeficiency Virus (HIV) using a probabilistic model with sensitivity analyses. The second chapter case study by Rosso et al. presents a medical survey that tests professional drivers for the presence of various psychoactive substances. Positives were identified by using a commercially available immunoassay rapid kit test which indicates consumption of psychoactive drugs. This is of interest and important since the dependence on drugs may pose a risk to drivers in their profession affecting their reaction time and driving judgment. This is one of the first and important contributions to the literature in this area. The study is of high importance as drivers under the influence of psychoactive drugs may endanger themselves or others if impaired at the jobsite. Case studies for emerging risks in the second section include evidence-based precautionary interventions to safeguard blood supplies, the evaluation of nanoparticle deposition in the lung and nasal airways and the discourse surrounding emerging potential health risks of genetically modified animals consumed as food. The third chapter by Tyshenko et al. reviews a case study concerning xenotropic murine leukemia virus-related virus (XMRV) and its emergence as a potential new blood pathogen that occurred in 2009; a lack of information for decision-making confounded risk assessment and early management decision-making. The chapter provides insight into the early assessment process and the application of precaution, an often poorly described management action rarely captured in peer review literature. The situation surrounding this potential new threat to blood safety was largely resolved in mid-2011 when it was determined that the virus was an artifact from contaminated patient samples and from contamination stemming from a diagnostic test kit widely used by researchers. The case study provides good assessment and management insight into the application of early precautionary action and the use of expert opinion for proactive risk management of emerging blood-borne pathogens. The fourth chapter by Jinxiang et al. assesses the airflow and aerosol dynamic characteristics within the nasal cavity for three different individuals. The study assesses and models the physical dimensions of the nasal airway and lungs for an infant, child, and an adult to characterize breathing resistance, airflow dynamics, and particle transport/deposition during inhalation. Such a comprehensive model allows for the modeled deposition of submicrometer aerosols (nanosized particles and particulate matter sizes larger that nano to determine the total deposition as well as localized deposition of particles). The results are important, prospectively, since they may lead to a better understanding of the developmental respiratory physiology and the associated effects on children’s health response to environmental pollutants, or the medical outcome from inhalation therapy for infants and children from nanoparticlecontaining medicines. The model also has applications for adults who may experience high nanoparticle exposure through the workplace as an occupational hazard and may be exposed to synthetic nanoparticles of this concentration. The fifth chapter by Benessia and Barbiero discusses the epistemic and normative issues surrounding the uncertainty, risks and knowledge gaps of genetically modified organisms (GMOs). Genetically modified salmon is the first animal seeking regulatory approval but it is still unapproved after more than a decade of risk assessments. The authors use this as a case study to explore the context and "ways of knowing" surrounding risk assessments to show how regulatory oversight and policy that are framed for GMOs may be inadequate for providing assurances of long term environmental and health safety. The interplay between science, society and governance is important in the area of GMOs, which once approved and released into the environment, may have unexpected and uncontrollable impacts. The salmon contains genetic modifications for fast growth which could have unknown effects on wild populations should they be released into the wild. The issue focuses the concerns over global environmental safety and security stemming from the risk assessments of not only GM salmon but all follow-on genetic modifications to animals. The authors conclude that the way in which risk assessments are completed for GMOs presents a paradox to environmental and public safety. The solution to this problem suggested by the authors is to invoke a more transparent, wider public democratization of the issues surrounding GMOs to incorporate local, social, cultural and ecological public values. The chapter reinforces the theme of incorporating broader determinants of health similar to the other case studies dealing with emerging health risks. Looking towards the future of risk assessments, the final section deals with improving health risk assessments through the use of personalized genomics, new approaches using genomics data in risk assessments, and new in silico modeling for toxicogenomics analyses. The sixth chapter by Zhao et al. presents microarray expression profiling in breast cancer risk assessment. The chapter reviews and describes breast cancer microarrays, the algorithms used, the established gene signature, and the limitations with combining gene signatures for improved prediction of cancer therapy. The authors provide a potential improvement for breast cancer gene-expression signature analyses that will be of great interest to those involved in breast cancer therapy and gene expression profiling. The seventh chapter by Ageev et al. uses formaldehyde exposure as a case study. The formaldehyde exposure data is re-analyzed to reveal exposure effects on gene expression levels not previously observed with the datasets. This type of analysis can provide better estimates of gene expression activity at low doses for well characterized chemical hazards. The final chapter by Mazzoleni and Steimberg provides an excellent overview of current and new models for the study of liver toxicity with a focus on cultured cells and culture methods. The use of new 3D culture and emerging bioreactor models for toxicity testing fits well with the future paradigm of toxicity risk assessments that seek to move towards in vitro and in silico methods. Overall, the book is a collection of interesting case studies that provides a continuum of risk assessment methods and epistemology for known, emerging and future risks. The book will be of interest to risk assessors, epidemiologists, toxicologists, and anyone involved in health policy or health studies.
... Phase II reactions involve the conjugation of metabolites via glucuronidation, sulfation, methylation, or acetylation to create compounds that are much more soluble and, therefore, more easily eliminated [5][6][7]. Generally, metabolism leads to the detoxification of xenobiotics by creating inactive metabolites; however, intermediate products created during metabolism can be toxic and reactive [6,8,9]. Therefore, it is critical to understand and characterize the metabolism of xenobiotics to better predict potential toxicity. ...
... In vitro methods to study xenobiotic metabolism in liver-derived cells can address the limitations associated with in vivo testing by enabling high-throughput screening at much lower costs. Traditional assays such as hepatocyte suspensions and 2D cultures have been widely used for rapid screening and characterization of xenobiotic metabolism [8,17,18]; however, these approaches have limitations. Due to the time-dependent loss of cell function and viability in suspension assays, the metabolism of low-turnover compounds tends to be underestimated [17]. ...
Article
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The evaluation of exposure to multiple contaminants in a mixture presents a number of challenges. For example, the characterization of chemical metabolism in a mixture setting remains a research area with critical knowledge gaps. Studies of chemical metabolism typically utilize suspension cultures of primary human hepatocytes; however, this model is not suitable for studies of more extended exposures and donor-to-donor variability in a metabolic capacity is unavoidable. To address this issue, we utilized several in vitro models based on human-induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep) to characterize the metabolism of an equimolar (1 or 5 µM) mixture of 20 pesticides. We used iHep suspensions and 2D sandwich cultures, and a microphysiological system OrganoPlate® 2-lane 96 (MimetasTM) that also included endothelial cells and THP-1 cell-derived macrophages. When cell culture media were evaluated using gas and liquid chromatography coupled to tandem mass spectrometry methods, we found that the parent molecule concentrations diminished, consistent with metabolic activity. This effect was most pronounced in iHep suspensions with a 1 µM mixture, and was lowest in OrganoPlate® 2-lane 96 for both mixtures. Additionally, we used ion mobility spectrometry–mass spectrometry (IMS-MS) to screen for metabolite formation in these cultures. These analyses revealed the presence of five primary metabolites that allowed for a more comprehensive evaluation of chemical metabolism in vitro. These findings suggest that iHep-based suspension assays maintain higher metabolic activity compared to 2D sandwich and OrganoPlate® 2-lane 96 model. Moreover, this study illustrates that IMS-MS can characterize in vitro metabolite formation following exposure to mixtures of environmental contaminants.
... Diesbezüglich erbrachte eine Metabolisierungsstudie an PHH mit dem nicht-opioiden Analgetikum Aceclofenac von Ponsoda et al. den Nachweis, daß die Variabilität der Messwerte die unterschiedlichen Phänotypen der Leberzellspender wiederspiegelt[160]. Zu einem ähnlichen Ergebnis kam eine Studie von Gómez-Lechón et al. nach Messung der Heterogenität der P450-Expression[161]. Eine bedeutende Limitation für eine Ausweitung der Nutzung des humanen Fettlebermodells stellt neben ethischen Aspekten die eingeschränkte Verfügbarkeit von humanem Lebergewebe dar. ...
... Gesundes Leberparenchym steht nur selten für die Forschung zur Verfügung und ist in ständiger Konkurrenz zu klinischen Anwendungen, wie Hepatozytentransplantation oder artifiziellen Leberunterstützungssystemen. In diesem Zusammenhang zeigen sich humane Kadaverspenden, welche für eine Lebertransplantation ungeeignet sind, als eine potentielle Quelle für die Isolierung hochwertiger Hepatozyten[162,161]. Als weiterer Nachteil ist der sehr kosten-und zeitintensive Prozess der Hepatozytenisolierung anzusehen, der ein gut organisiertes Netzwerk zwischen Klinik und Forschung voraussetzt.6.6 Schlussfolgerung und AusblickDas in dieser Arbeit an primären humanen Hepatozyten etablierte Modell der Steatosis hepatis ist eine valide in-vitro-Abbildung der in-vivo-Gegebenheiten einer nicht-alkoholischen Fettlebererkrankung hinsichtlich Lipidgehalt, Lipotoxizität und metabolischer Kompetenz. ...
Thesis
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Introduction: Non-alcoholic fatty liver disease (NAFLD) is already the most common chronic liver disease in western countries. The complex disorder is regarded as the hepatic manifestation of metabolic syndrome and has been strongly associated with insulin resistance. Genistein (GEN), a natural soy isoflavone, has shown some potential benefits on NAFLD as well as insulin resistance in numerous epidemiological and experimental studies. Aim of the present study was the establishment and characterization of a human in-vitro fatty liver model and the evaluation of GEN effects on hepatic insulin signalling. Materials and Methods: Primary human hepatocytes (PHH) were isolated from resected liver tissues. Steatosis was induced by adding 1 mM oleic and palmitic acid (2:1) for 24h. Lipid content was quantified by Oilred O staining and cytotoxicity and metabolic activity was evaluated by enzymatic assays of AST, LDH and urea as well as XTT cell viability assay. Alterations in mRNA expression of insulin receptor (INSR) and glucose transporter type 2 (GLUT2) were measured by realtime-PCR. Furthermore, changes in protein phosphorylation of AKT, ERK1/2, FoxO1 and GSK3α/β besides stimulation with and without 100 nM insulin for 15 minutes were analysed by Western Blot. Steatotic and non-steatotic PHH were stimulated with GEN at different concentrations ranging from 1 to 100 µM for 24h. Results: PHH treated with free fatty acids showed a significant increase in cytoplasmic lipid vesicles. A moderate lipotoxicity was observed whereas metabolic activity was not changed. We observed decreased insulin-induced phosphorylation levels of AKT, ERK1/2, GSK3α/β and FoxO1 in steatotic PHH. Furthermore, lipid-overloaded hepatocytes showed a slight elevation of GLUT2 and INSR expression levels. Additional treatment with GEN showed cytotoxicity at 50 and 100 µM. In steatotic PHH, we observed increased phosphorylation of ERK1/2 and GSK3α/β as well as increased expression of GLUT2 and INSR in a dose-dependent manner of GEN. In non-steatotic PHH, we observed an increased phosphorylation of Akt, ERK1/2 and GSK3α/β as well as an increased expression of INSR in a concentration-dependent manner of GEN, respectively. Conclusion: Our data show that the human in-vitro model of NAFLD is a reliable reproduction of in-vivo conditions regarding fat accumulation, lipotoxicity and insulin resistance. Additional treatment with GEN showed insulinotropic effects in steatotic as well as non-steatotic PHH by an increased phosphorylation of insulin signalling related key proteins in a dose-dependent manner. Moreover, an increased expression of transmembrane transport proteins has been observed, suggesting an improvement of hepatic energy metabolism.
... The product of the ECF distribution of a chemical with an observed blood concentration of the chemical provides a distribution of external exposure concentrations corresponding to that blood level (Lyons et al. 2008). Unlike the study by Tan, Liao, and Clewell (2007) who performed Monte Carlo simulations, the reverse dosimetry approach developed in Lyons et al. (2008) consisted of a Bayesian inference using Markov chain Monte Carlo (MCMC), the standard method for Bayesian analysis (Gelman and Rubin 1996), and leading to an external exposure expressed as a probability distribution rather than a single value. The PBPK model used in all these reverse dosimetry studies was the standard one of an adult of 70 kg, with physiological input parameters of the model that were constants (and not expressed as equations as in the current study) even if a variability in these physiological parameters was simulated through Monte Carlo simulations or the Bayesian method. ...
... Additional information regarding exposure and lifestyle factors such as alcohol consumption, smoking, nutrition, and health status were not addressed in the model simulations that might help to better interpret biomonitoring data. These factors may modulate the relationship between the exposure dose of a chemical in human body and its response (body burden) (Dorne 2007;Renwick 2004, 2005;Gomez-Lechon, Castell, and Donato 2007;Kato et al. 2010;Parkinson et al. 2004;Pierce et al. 1998). ...
Article
Biomonitoring might provide useful estimates of population exposure to environmental chemicals. However, data uncertainties stemming from interindividual variability are common in large population biomonitoring surveys. Physiologically based pharmacokinetic (PBPK) models might be used to account for age- and gender-related variability in internal dose. The objective of this study was to reconstruct air concentrations consistent with blood toluene measures reported in the third Canadian Health Measures Survey using reverse dosimetry PBPK modeling techniques. Population distributions of model’s physiological parameters were described based upon age, weight, and size for four subpopulations (12–19, 20–39, 40–59, and 60–79 years old). Monte Carlo simulations applied to PBPK modeling allowed converting the distributions of venous blood measures of toluene obtained from CHMS into related air levels. Based upon blood levels observed at the 50th, 90th and 95th percentiles, corresponding air toluene concentrations were estimated for teenagers aged 12–19 years as being, respectively, 0.009, 0.04 and 0.06 ppm. Similarly, values were computed for adults aged 20–39 years (0.007, 0.036, and 0.06 ppm), 40–59 years (0.007, 0.036 and 0.06 ppm) and 60–79 years (0.006, 0.022 and 0.04 ppm). These estimations are well below Health Canada’s maximum recommended chronic air guidelines for toluene. In conclusion, PBPK modeling and reverse dosimetry may be combined to help interpret biomonitoring data for chemical exposure in large population surveys and estimate the associated toxicological health risk.
... 1,2 Because primary human hepatocytes (PHHs) retain the activities of drug metabolizing enzymes and transporters, PHHs are the main cell source used as a human liver model for preclinical in vitro studies of drug metabolism and toxicities. [3][4][5][6] However, PHHs have at least two problems: first, they cannot be cultured with sufficient hepatic functions for long periods of time, and second, they have supply limitations due to a lack of proliferating capacity in vitro. Therefore, many studies have been conducted to overcome these issues. ...
Article
Full-text available
Human liver organoids derived from primary human hepatocytes (PHHs) are expected to be a hepatocyte source for preclinical in vitro studies of drug metabolism and disposition. Because hepatic functions of these organoids remain low, it is necessary to enhance the hepatic functions. Here, we develop a novel method for two dimensional (2D)-cultured hepatic differentiation from PHH-derived organoids by screening several compounds, cytokines, and growth factors. Hepatic gene expressions in the hepatocyte-like cells differentiated from PHH-derived organoids (Org-HEPs) were greatly increased, compared to those in PHH-derived organoids. The metabolic activities of cytochrome P450 (CYP) 1A2, CYP2C8, CYP2C19, CYP2E1, and CYP3A4 were at levels comparable to those in PHHs. The cell viability of Org-HEPs treated with hepatotoxic drugs was almost the same as that of PHHs. Thus, PHH-derived organoids could be differentiated into highly functional hepatocytes in 2D culture. Thus, Org-HEPs will be useful for pharmaceutical research, including hepatotoxicity tests.
... Performing drug metabolism in vitro has been a keen interest of scientists for various reasons, including deciphering the metabolic routes, identification of metabolites, studying differences in metabolism between species, studying toxicity, assessing for potential in-vivo study, etc. In-vitro drug metabolism can be performed using cellular organelles such as liver slices or hepatocytes, subcellular fractions such as S9, cytosolic or microsomal fractions, and metabolizing enzymes such as CYP P450s [10]. Liver slices and hepatocytes mimic the in-vivo condition the most among different approaches available for drug metabolism, but contain both phase I and phase II drug metabolizing enzymes (DMEs) [11]. Three different fractions can be obtained from liver slices or hepatocytes through differential centrifugation: S9, cytosolic, and microsomal fractions. ...
Article
Full-text available
Microsomes are commonly used to perform in vitro drug metabolism, predominantly to form phase I drug metabolites. Pooled microsomes from multiple donors can contain microorganisms from underlying microbial diseases. Exposure to microbes can also occur during extraction if aseptic processing is compromised. Although microbial presence does not affect the metabolic activity of microsomes, presence of unwanted microorganisms can cause interference if the downstream application of microsomal drug metabolites is screening for antibacterial activity. In this work, traditional biochemical tests and advanced proteomics-based identification techniques were used to identify two gram-negative bacteria in pooled human liver microsomes. Several decontamination procedures were assessed to eradicate these two bacteria from the microsomes without affecting its metabolic capacity, and organic extraction was found to be the most convenient and efficient approach to decontaminate microsomes and screen drug metabolites for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).
... But the major limitations are the availability of liver donors, the disease status of the liver and person-to-person diversity. Moreover, individual variations in CYP expression and activity were already reported because of lifestyle differences, age group and hormonal status that affect the hepatocytes' activity [64]. ...
Article
Full-text available
Drug discovery and development have become a very time-consuming and expensive process. Preclinical animal models have become the gold standard for studying drug pharmacokinetic and toxicity parameters. However, the involvement of a huge number of animal subjects and inter-species pathophysiological variations between animals and humans has provoked a lot of debate, particularly because of ethical concerns. Although many efforts are being established by biotech and pharmaceutical companies for screening new chemical entities in vitro before preclinical trials, failures during clinical trials are still involved. Currently, a large number of two-dimensional (2D) in vitro assays have been developed and are being developed by researchers for the screening of compounds. Although these assays are helpful in screening a huge library of compounds and have shown perception, there is a significant lack in predicting human Absorption, Distribution, Metabolism, Excretion and Toxicology (ADME-Tox). As a result, these assays cannot completely replace animal models. The recent inventions in three-dimensional (3D) cell culture-based assays like organoids and micro-physiological systems have shown great potential alternative tools for predicting the compound pharmacokinetic and pharmacodynamic fate in humans. In this comprehensive review, we have summarized some of the most commonly used 2D in vitro assays and emphasized the achievements in next-generation 3D cell culture-based systems for predicting the compound ADME-Tox.
... Nowadays, the drug disease interaction is the center of contemplation among the drug regulatory agencies and drug development units. The expression of DMEs is anomalously modulated during inflammatory disorders and associated with toxic effects (Gomez-Lechon, Castell, & Donato, 2007). ...
Chapter
Pharmacokinetics properties of drugs are determined by drug-metabolizing enzymes (DMEs), participating in absorption, distribution, and biotransformation. Inflammation is a key feature of many disorders such as metabolic disorders, aging, age-related neurological disorders, and inflammatory bowel diseases. The inflammation is arbitrated by molecular mechanistic synchronization of DMEs and their anomalous expression in different diseases. In autoimmune diseases, the aberrant inflammatory signaling alters the contents and metabolizing capacity of CYP. In rheumatoid arthritis (RA), the level of proinflammatory cytokines is increased in systemic circulation and in synovial joints resulting in the downregulation of mRNA expression of hepatic enzymes such as CYP2B1, CYP2B2, CYP2C6/7, and CYP3A1, proceeding to decrease the effectiveness of statins, calcium channel, and β-blockers drugs. Antibodies against specific cytokines and NSAIDS are suitable therapeutic choice to modulate these aberrant changes in RA. The PXR activation suggests a novel drug target to treat inflammatory bowel diseases along with antiinflammatory agents and antibiotics. In aged population, most of drug detoxification enzymes are downregulated; however, the CYP content regulation is controversial as compared to normal age population. In type 2 diabetes and obesity models, transgenic diabetic models, and obesity-induced diabetes models, the expression of CAR, RXRα, PXR, HNF-4α, and NF-κB is significantly altered resulting in the alteration of drug kinetics. Neuroinflammation-induced neurotoxicity alters the expression of CYP2D6 resulting in altered metabolism of antipsychotic drugs. In this chapter, the underlying molecular mechanism behind these regulations has been discussed to unravel the clear picture and to anticipate the clear drug targets.
... Recombinant enzymes can also be used in the confirmation of reaction phenotyping studies. Conversely, the absence of the remaining phase I and phase II enzymes can be considered as a disadvantage of this type of system [2,66]. ...
Chapter
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Metabolic stability of a compound is an important factor to be considered during the early stages of drug discovery. If the compound has poor metabolic stability, it never becomes a drug even though it has promising pharmacological characteristics. For example, a drug is quickly metabolized in the body; it does not have sufficient in vivo exposure levels and leads to the production of toxic, non-active or active metabolites. A drug is slowly metabolized in the body it could remain longer periods in the body and lead to unwanted adverse reactions, toxicity or may cause drug interactions. Metabolic stability assay is performed to understand the susceptibility of the compound to undergo biotransformation in the body. Intrinsic clearance of the compound is measured by metabolic stability assays. Different in vitro test systems including liver microsomes, hepatocytes, S9 fractions, cytosol, recombinant expressed enzymes, and cell lines are used to investigate the metabolic stability of drugs. Metabolite profiling is a vital part of the drug discovery process and LC–MS plays a vital role. The development of high-resolution (HR) MS technologies with improved mass accuracy, in conjunction with novel data processing techniques, has significantly improved the metabolite detection and identification process. HR-MS based data acquisition (ion intensity-dependent acquisition, accurate-mass inclusion list-dependent acquisition, isotope pattern-dependent acquisition, pseudo neutral loss-dependent acquisition, and mass defect-dependent acquisition) and data mining techniques (extracted ion chromatogram, product ion filter, mass defect filter, isotope pattern filter, neutral loss filter, background subtraction, and control sample comparison) facilitate the drug metabolite identification process.
... However, primary human hepatocytes are scarce and expensive, and this is a major limiting factor for their use in drug screening. 9 As an alternative cell source, human pluripotent stem cells (hPSC) can be differentiated into many cell types consistently and indefinitely. 10,11 Vascular 12,13 and hepatic 14 cells generated from hPSC have exhibited their respective tissue-specific phenotypes and functions. ...
Article
Human pluripotent stem cell (hPSC) is a great resource for generating cell derivatives for drug efficiency testing. Metabolites of nutraceuticals can exert anti-inflammatory effects on blood vessels. However, the concentration of nutraceutical metabolites produced in hPSC-derived hepatocytes (hPSC-HEPs) is usually low. To enable the detection of these metabolites under the in vitro environment, we have developed a co-culture model consisting of parallel co-culture chambers and a recirculating microfluidic system with minimum fluid volume, optimal cell culture environment. The model allows cells to be exposed continuously to nutraceutical metabolites. In this perfused culturing model, hPSC-derived endothelial cells and hPSC-HEPs are co-cultured without physical contact. When an anti-inflammatory nutraceutical, quercetin, was administrated to the co-culture, higher levels of quercetin metabolites were detected on-chip compared with static control. We further induced inflammation with Interleukin-1β in the co-culture model and measured interleukin 8 (IL-8) generation. The IL-8 level was suppressed more significantly by quercetin metabolites in the perfusion co-culture, as compared to static culture. This is due to enhanced metabolites production on-chip. This microfluidic co-culture model enables in vitro screening of nutraceuticals using hPSC-derived cells.
... Human cell-based models and subcellular fractions (microsomes, cytosol, and S9 fractions) are considered superior to the preclinical animal models for the prediction of human drug metabolism, transport, and drug-drug interactions (DDIs) due to the large inter-species differences in these processes. [1][2][3][4] During the early drug development stage, the in vitro data are utilized for IVIVE and physiologically-based pharmacokinetic (PBPK) modeling is used to assist in clinical trial design, e.g., the first-in-human dose prediction. In the later stages of drug development, PBPK modeling is used for the prediction of inter-individual variability in pharmacokinetics, e.g., in special populations such as patients with liver and kidney dysfunctions. ...
Article
Quantitative proteomics is a powerful technique that provides important data for in vitro to in vivo extrapolation (IVIVE) and predicts interindividual variability in drug disposition.However, the conventional quantitative proteomics methods often require high initial total protein and enriched samples (e.g., microsomes and pure membrane fractions). The goal of this study was i) to develop a microflow based LC‐MS/MS method for quantification of clinically relevant drug metabolizing enzymes (DMEs) and transporters using small total protein (400 ng), and ii) to quantify these proteins in human liver and intestine S 9 samples.Targeted quantitation of DMEs and transporters was performed on trypsin digested pooled liver (N=200) and pooled intestinal (N=15) S 9 fractions (Xenotech, Kansas city). The digestion was performed using an optimized in house protocol. A total of 400 ng protein (1 μl injection volume) was injected through a trap‐column followed by peptide separation in a microflow column (BEH C18; 130 A°,1.7μm, 150 μm*50 mm) using M‐class UPLC system and detection by Xevo TQ‐XS MS instrument supported by ionkey interface. An optimized gradient elution was used with 3 μl/min flow rate. Data were acquired in scheduled MRM mode and the data analysis was performed using Skyline 19.1 software.72 DMEs and transporters were quantifiable using 400 ng of total protein injection. A total of 12 CYPs, 12 UGTs, 33 nonCYPs/nonUGTs (e.g., SULTs, GSTs, ADHs, ALDHs, etc.) and 16 transporters were detected. Out of the total detected proteins, 53 were in both the liver and intestine, 17 were only detected in the liver, and 2 were only quantifiable in the intestine. CYP2C8, CYP2B6, CYP2D6, CYP2A6, CYP2E1, UGT1A9, UGT1A4, UGT2B4, UGT2B15, SULT1E1, PON3, FMO3, AOX1, ADH1A, ADH1B, OATP1B1 and OATP1B3 were exclusively detected in the liver whereas UGT1A10 and UGT2A1 were only seen in the intestine. ALDH1A1, GSTT1, SULT4A1, SLC22A3, CYP3A4, ALDH1A3, SULT2A1, CYP1A1, SLC22A2, MATE1, ADH1C, SULT1C, POR, ABCC2 were within 0.75–1.5‐fold in the liver and intestine S 9 fractions. CES1, PON1, UGT1A6, CYP2C9 and UGT2B7 were significantly higher in the liver as compared to intestine, and SULT1B1, CES2, UGT2B17, FMO1 and UGT1A8 were amongst the top differentially expressed proteins in the intestine. We were able to quantify low abundant proteins such as BCRP, CYP1A1 and UGT1A8, which are generally difficult to detect by conventional LC‐MS/MS methods. In summary, the microflow‐LC‐MS/MS is an ultrasensitive approach for quantifying DMEs and transporters in crude tissue preparations such as homogenate and S 9 fractions. Since microsomes and pure membrane preparations require recovery factors that add batch‐to‐batch variability, quantification of proteins in homogenate and S 9 fractions using the ultrasensitive method would provide accurate scaling factors for IVIVE. This microflow‐LC/MS/MS method can be applied to small biopsy samples (e.g., tissue weight ~2.5 mg) thus providing broad applications in predicting interindividual variability (e.g., effect of diseases) in drug disposition using patient samples. Support or Funding Information Department of Pharmaceutical Sciences, Washington State University Number of proteins quantified in intestine and liver S9 fractions. Figure 1
... They offer a potential territory for checking of toxicants impacts and have pulled in a lot of consideration of researchers from different fields. The binding of the toxicant to specific enzymes leads to acceleration or inhibition of enzymatic activities and progression of various metabolic issues [16]. There are numerous toxicants that can bind to active site or near the active site of enzyme. ...
Article
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Chlorantraniliprole is a novel insecticide introduced by DuPont Crop Protection (Tokyo, Japan) in 2007 and now it is widely used to control agricultural pests from the Order Lepidopteron, Coleopteran, Dipterans and Hemiptera species. Due to run off water and soil erosion these insecticides get introduced at nearest water bodies and thereby adversely affects non-target organism like fishes. Alkaline phosphatase is a membrane bound glycoprotein present in tissues of fish and is involved in active membrane transport and carbohydrate metabolism. The aim of present study was to determine the toxic effect at lethal concentration of Chlorantraniliprole on alkaline phosphatase activity on freshwater fish Cirrhinus mrigala. Prior to experimental protocol, fingerlings were acclimatized in glass aquaria for seven days. After acclimatization, fingerlings were exposed to LC0 and LC50 concentration of Chlorantraniliprole (0.0025ppm and 0.01 ppm respectively) in twenty-liter test container for 96 hrs (acute toxicity). It was observed that the alkaline phosphatase activity in muscle, intestine and liver; significantly decreased at LC0 and LC50 concentration as compared to the control group. From the observed results, it can be concluded that, 'selected insecticide Chlorantraniliprole interferes with alkaline phosphatase activity in selected test fish Cirrhinus mrigala and retards the normal activity.
... Western blot assay Guzmán et al. (2003) Ostreopsis ovata from microalgae Cytotoxicity assay Exposure in human hepatocellular liver carcinoma (HepG-2, ATCC HB-8065) Gómez-Lechón et al. (2007), Nakamura et al. (2011) Haematococcus pluvialis Antioxidant assay 1. Cellular lipid peroxidation antioxidant activity assay 2. ...
... The class of salicylic acid derivatives has a widely applied usage in the pharmaceutical field for their antipyretic and anti-inflammatory properties (El-Ezaby 1981;Tavadyan 2002 andGómez-Lechón 2007) antioxidant (Şentürk 2009), in addition it can be act as antibacterial (Estevinho 2008 andChoi 2009) and antifungal (Han 2008 ). The most widely used in the pharmaceutical field is the acetylsalicylic acid (aspirin) which its already used as antipyretic (Subramanian 2008), antiinflammatory (Šoltés 2007), analgesic (El-Gazzar 2008) and also it is used extensively for the relief of headache (Lewis 2007), and recently employed in the treatment of heart attacks and strokes in the elderly (Rodondi 2008 andBernstein 2009), among others. ...
Article
The reaction of 5-Chlorosulfonyl-2-hydroxy-benzoic acid 1a with each sulfamethoxazole and sulfadimidine to produce compounds 1b and 1c respectively, these compounds can be undergo some type of reactions with amino-acid methyl esters followed by reaction with hydrazine hydrate to produce the corresponding hydrazide derivatives 2-9, which is easily directed toward the reaction with either ethyl isothiocyanate or phenylisothiocyanate, the thiosemicarbazides were produced 10-17, the latter synthesized compounds were cyclized in acidic medium to produce triazole derivatives compounds 18-25, some of the newly synthesized compounds were found to possess antimicrobial activities against two different series of microorganisms including gram-positive, gram-negative microorganisms Bacillius subtilis, salmonella typhi, Staphylococcus aureus, Escherichia coli and the fungi Candida utilis .
... Similarly, primary cultures of hepatic stellate cells have been instrumental in understanding the causes of liver fibrosis and identification of key fibrogenic mediators (25,26). In drug testing, primary human hepatocyte cell cultures are considered the "gold standard" because they display many phenotypic functions of the liver when compared to other in vitro models (27,28). However, this approach has been heavily criticized as suboptimal. ...
... As a model derived from individuals with different genetic backgrounds, the variability observed in PHH in vitro could better reflect that of the liver in vivo (Gomez-Lechon et al. 2007). Jetten et al. investigated the variability in response to APAP exposures (24 h) in PHH derived from five donors by incorporating multiple omics techniques (transcriptomics and metabolomics). ...
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Drug-induced liver injury (DILI) complicates safety assessment for new drugs and poses major threats to both patient health and drug development in the pharmaceutical industry. A number of human liver cell-based in vitro models combined with toxicogenomics methods have been developed as an alternative to animal testing for studying human DILI mechanisms. In this review, we discuss the in vitro human liver systems and their applications in omics-based drug-induced hepatotoxicity studies. We furthermore present bioinformatic approaches that are useful for analyzing toxicogenomic data generated from these models and discuss their current and potential contributions to the understanding of mechanisms of DILI. Human pluripotent stem cells, carrying donor-specific genetic information, hold great potential for advancing the study of individual-specific toxicological responses. When co-cultured with other liver-derived non-parenchymal cells in a microfluidic device, the resulting dynamic platform enables us to study immune-mediated drug hypersensitivity and accelerates personalized drug toxicology studies. A flexible microfluidic platform would also support the assembly of a more advanced organs-on-a-chip device, further bridging gap between in vitro and in vivo conditions. The standard transcriptomic analysis of these cell systems can be complemented with causality-inferring approaches to improve the understanding of DILI mechanisms. These approaches involve statistical techniques capable of elucidating regulatory interactions in parts of these mechanisms. The use of more elaborated human liver models, in harmony with causality-inferring bioinformatic approaches will pave the way for establishing a powerful methodology to systematically assess DILI mechanisms across a wide range of conditions.
... There is evidence, based both on experimental and modeling data, that exposure to chemical mixtures can result in metabolic interactions between different chemicals in the human or animal body, depending on the levels of exposure to the individual chemicals present in the mixtures (Badger, Huang, Ronis, & Lumpkin, 1993;Crespi, Miller, & Penman, 1997;Dennison et al., 2005;Dobrev, Andersen, & Yang, 2001Gomez-Lechon, Castell, & Donato, 2007;Haddad, Béliveau, Tardif, & Krishnan, 2001;Haddad, Tardif, et al., 2010;Haddad, Tardif, Charest-Tardif, & Krishnan, 1999;Krishnan & Anderson, 2010;Lin & Lu, 1998;Mumtaz et al., 2007;Mumtaz, De Rosa, Cibulas, & Falk, 2004;Purcell, Cason, Gargas, Andersen, & Travis, 1990;Sager et al., 2014;Tang, Donnelly, Tiffany-Castiglioni, & Mumtaz, 2003;Tardif, Charest-Tardif, Brodeur, & Krishnan, 1997;Tully, Cox, Mumtaz, Davis, & Chapin, 2000;Valcke & Haddad, 2015;Yang & Andersen, 1994). ...
Article
This study aimed to assess the impact of multi‐route co‐exposures to chemicals on interindividual variability in toxicokinetics. Probabilistic physiologically based pharmacokinetic multi‐route interaction models were developed for adults and four younger subpopulations. Drinking water‐mediated multi‐route exposures were simulated for benzene alone or in co‐exposure with toluene, ethylbenzene and m‐xylene, for trichloroethylene or vinyl chloride (VC), alone and in mixture. These simulations were performed for “low” and “high” exposure scenarios, involving respectively the US EPA's short‐term drinking water health advisories, and 10 times these advisory values. Distributions of relevant internal dose metrics for benzene, trichloroethylene and VC were obtained using Monte Carlo simulations. Intergroup variability indexes (VI) were computed for the “low” (VIL) and “high” (VIH) exposure scenarios, as the ratio between the 95th percentile in each subpopulation over the median in adults. Thus, for benzene, parent compound's area under the curve‐based VIL for single exposures vs. co‐exposures correspondingly varied between 1.7 (teenagers) and 2.8 (infants) vs. 1.9 and 3.1 respectively. VIH varied between 2.5 and 3.5 vs. 2.9 and 4.1. Inversely, VIL and VIH for the amount of benzene metabolized via CYP2E1 pathway decreased in co‐exposure compared to single exposure. For VC and trichloroethylene, similar results were obtained for the “high” exposure, but “low” co‐exposures did not impact the toxicokinetics of individual substances. In conclusion, multi‐route co‐exposures can have an impact on the toxicokinetics of individual substances, but to an extent, that does not seem to challenge the default values attributed to the factors deemed at reflecting interindividual or child/adult differences in toxicokinetics.
... Ensayos de citotoxicidad. Con el fin de evaluar la actividad citotòxica de los extractos, se utilizaron las células HepG2 las cuales son una de las líneas celulares más usadas para la evaluación de la citotoxicidad [27,28]. Estas células se cultivaron en medio DMEN (Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12), suplementado con 10% de suero fetal bovino inactivado, mantenidas a 37°C y CO 2 al 5% [29,30]. ...
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Malaria is a parasite disease with negative impact for the worldwide human health.This motivates research in new anti-malaric natural medicine, which have proved high potential in in vitro and in vivo experiments.The objective of this research in the in vitro evaluation is: taking into account the antimalaric activity, its cytotoxicity with 9 raw extracts are prepared after some of this plant leaves : Piper tricuspe, Plantago major, Solanum nudum, Gliricidia sepium found in the Colombian south Pacific in Tumaco, Nariño. The antiplasmodic activity was evaluated for the 3D7 Y FCR3 strains prepared with P. tricuspe y S. nudum. Ethanol had a CI50 24,41 y 16,59μg/mL for the strain FCR3 and 27,1 y 23,26 μg /mL for the strain 3D7, this, The cytotoxic activity valued in the cell line HepG2, bot extracts being low toxic. It should be noted also, the importance in the intention to find new antimalaric from the plants found in the communities in south Colombia.
... LO2 is a human normal liver cell. In the present study, LO2 cells were selected as the cultured cell representing tissue from the biggest digestive gland, the liver, where 90% of drugs are metabolized by cytochrome P450 enzymes (34) with the majority of them producing efficacy or toxicity afterwards (35). In addition, the liver is very sensitive to xenobiotics and is subject to damage by drug doses several times lower than that which damages other organ tissues (36). ...
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Ursodeoxycholic acid (UDCA) is a type of hydrophilic bile acid extracted from animal bile with a wide range of biological functions. The present results demonstrated that UDCA could effectively inhibit the proliferation of two human melanoma cell line (M14 and A375) with time- and concentration-dependence. Following exposure to various concentrations of UDCA, M14 cells exhibited typical morphological changes and weaker ability of colony forming. Flow cytometry analysis demonstrated that UDCA could induce a decrease of mitochondrial membrane potential and an increase in reactive oxygen species (ROS) levels in M14 cells. The cell cycle was arrested in the G2/M phase, which was confirmed by the decrease of cyclin-dependent kinase 1 and cyclinB1 at the protein level. However, when M14 cells were treated with UDCA and Z-VAD-FMK (caspase inhibitor) synchronously, the apoptosis rate of the cells was reduced significantly. In addition, it was demonstrated that UDCA induced apoptosis of human melanoma M14 cells through the ROS-triggered mitochondrial-associated pathway, which was indicated by the increased expression of cleaved-caspase-3, cleaved-caspase-9, apoptotic protease activating factor-1, cleaved-poly (ADP-ribose) polymerase 1 and the elevation of B cell lymphoma-2 (Bcl-2) associated X protein/Bcl-2 ratio associated with apoptosis. Therefore, UDCA may be a potential drug for the treatment of human melanoma.
... Ensayos de citotoxicidad. Con el fin de evaluar la actividad citotòxica de los extractos, se utilizaron las células HepG2 las cuales son una de las líneas celulares más usadas para la evaluación de la citotoxicidad [27,28]. Estas células se cultivaron en medio DMEN (Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12), suplementado con 10% de suero fetal bovino inactivado, mantenidas a 37°C y CO 2 al 5% [29,30]. ...
Article
Full-text available
La malaria es una enfermedad parasitaria con impacto negativo para la salud humanamundial, lo que motiva la búsqueda de nuevos antimalaricos a partir de plantasmedicinales que han mostrado gran potencial en experimentos in vivo e in vitro. Esteestudio tuvo por objetivo la evaluación in vitro de la actividad antimalárica y la citotoxicidadde nueve extractos crudos preparados a partir de las hojas de las plantas:Piper tricuspe, Plantago major, Solanum nudum, Gliricidia sepium del sur pacificocolombiano Tumaco Nariño. La actividad antiplasmódica se evaluó en las cepas 3D7Y FCR3 de Plasmodium falciparum y se encontró que los extractos preparados con etanol de P.tricuspe y S. nudum tuvieron una CI50 24,41 y 16,59 mg/mL para la cepa FCR3 y 27,1 y 23,26 mg/mL para la cepa 3D7 respectivamente; la actividad citotóxica se valoró en la línea celular HepG2, siendo ambos extractos bajamente toxico. Cabe resaltar la importancia de la intención por encontrar nuevos medicamentos antipalúdicos a partir de las plantas informadas por las comunidades del sur de Colombia.
... Human hepatocytes were isolated from small samples of the human liver tissue using a two-step perfusion technique as described in detail elsewhere. [20] Human liver samples were prepared from livers rejected for transplantation (i.e. prolonged cold ischaemic time, fibrosis, arteriosclerosis) under the supervision of the Hospital's Transplantation Coordination and in agreement with the rules of the hospital's Ethics Committee. ...
... Similarly, as depicted in Figure 15B, the expression level of CYP2E1 differs significantly in 18 human liver samples (391). This similarity has also been shown for other hepatic CYPs (171,392,393). ...
Thesis
The main part of the thesis is devoted to pharmacogenetic approach to the anti-inflammatory effects of thienopyridine therapy. Taking into the account that activated platelets play a central role in the inflammatory responses and that CYP2C19 gain- and loss-of-function polymorphisms (*2 and *17) are sources of inter-individual difference in response to the anti-platelet effects of thienopyridines, we hypothesized that *2 and/or *17 alleles are also associated with inter-individual variability in the potential inflammation-reducing effects of thienopyridines. The following markers were used to test the hypothesis: CRP, haptoglobin and orosomucoid acid. To be reliably interpretable in daily medical practice, genetic status should be considered for partitioning the reference values of haptoglobin. In a small healthy population, no significant association was observed between *2 allele and changes in levels of inflammatory markers from baseline to 7 days after administration of clopidogrel and our findings did not support the notion that the genetic variations of CYP epoxygenases are associated with the level of inflammatory markers. Also, in post-PCI population consisting of 1128 on-clopidogrel or on-prasugrel patients, CRP levels were observed to be regulated with a significant interaction between smoking and CYP2C19 polymorphisms; this effect was independent to the level of platelet aggregation. Additionally, in a large population of 1000 on-clopidogrel patients, whether there is a potential interaction between clopidogrel and calcium channel blockers. Collectively, we demonstrated in this thesis that inflammatory markers might be alternative tools for the prediction of response to thienopyridines
... In this study, LO2 cells are selected as the cultured cell representing tissue from the biggest digestive gland (Kmieć, 2001), the liver, where 90% of medicines are metabolized by cytochrome P450 enzymes (CYP450) (Dambach et al., 2005) with most of them producing efficacy or toxicity afterwards (Gómez-Lechón et al., 2007). Besides, the liver is super sensitive to xenobiotics and is subject to damage by medicine doses several times lower than that which damages other organ tissues (Burcham, 2014). ...
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To address the controversial issue of the toxicity of dental alloys and silver nanoparticles in medical applications, an in vivo-like LO2 3-D model was constructed within polyvinylidene fluoride hollow fiber materials to mimic the microenvironment of liver tissue. The use of microscopy methods and the measurement of liver-specific functions optimized the model for best cell performances and also proved the superiority of the 3-D LO2 model when compared with the traditional monolayer model. Toxicity tests were conducted using the newly constructed model, finding that four dental castings coated with silver nanoparticles were toxic to human hepatocytes after cell viability assays. In general, the toxicity of both the castings and the coated silver nanoparticles aggravated as time increased, yet the nanoparticles attenuated the general toxicity by preventing metal ion release, especially at high concentrations.
... Cytotoxicity was analysed on human hepatocellular liver carcinoma (HepG2) cells to evaluate the potential toxic effect of the extracts. Hepatocytes were chosen because they are good models for studying toxicity since the liver is the primary site for drug metabolism and biotransformation 41,42 . None of the active extracts altered hepatocyte HepG2 cell survival (Student's t-test, p > 0.05). ...
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Marine microalgae are considered a potentially new and valuable source of biologically active compounds for applications in several biotechnology sectors. They can be easily cultured, have short generation times and enable an environmentally-friendly approach to drug discovery by overcoming problems associated with the over-utilization of marine resources and the use of destructive collection practices. Considering the increasing rate of antibiotic-resistance bacteria and infections by fungi, 46 microalgae have been screened in this study for possible antibacterial and antifungal activities. Two different extraction methods have been used in order to increase the probability of finding positive hits. In particular, we screened microalgae in both control and nutrient stress conditions. We also tested different strains for 7 species in order to study potentially different bioactivities due to strain diversity. Results showed that extracts of two diatoms, Skeletonema tropicum and Chaetoceros pseudocurvise
... Cytotoxicity was analysed on human hepatocellular liver carcinoma (HepG2) cells to evaluate the potential toxic effect of the extracts. Hepatocytes are good models for studying toxicity since the liver is the primary site for drug metabolism and biotransformation 105,106 ...
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Dinoflagellates are phytoplanktonic organisms found in both freshwater and marine habitats. They are often studied because related to harmful algal blooms but they are also known to produce bioactive compounds for the treatment of human pathologies. The aim of this study was to sequence the full transcriptome of the dinoflagellate Amphidinium carterae in both nitrogen-starved and -replete culturing conditions (1) to evaluate the response to nitrogen starvation at the transcriptional level, (2) to look for possible polyketide synthases (PKSs) in the studied clone (genes that may be involved in the synthesis of bioactive compounds), (3) if present, to evaluate if nutrient starvation can influence PKS expression, (4) to look for other possible enzymes of biotechnological interest and (5) to test strain cytotoxicity on human cell lines. Results showed an increase in nitrogen metabolism and stress response in nitrogen-starved cells and confirmed the presence of a type I β-ketosynthase. In addition, L-asparaginase (used for the treatment of Leukemia and for acrylamide reduction in food industries) and cellulase (useful for biofuel production and other industrial applications) have been identified for the first time in this species, giving new insights into possible biotechnological applications of dinoflagellates.
... Considerable variations in the expression levels of both cytochrome P450 (CYP) and conjugating enzymes have been found in human livers (Gomez-Lechon et al. 2007). Genetic polymorphisms, gender, age, hormonal status and drug intake are the key factors responsible for such interindividual differences. ...
Article
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Drug-induced liver injury (DILI) has a considerable impact on human health and is a major challenge in drug safety assessments. DILI is a frequent cause of liver injury and a leading reason for post-approval drug regulatory actions. Considerable variations in the expression levels of both cytochrome P450 (CYP) and conjugating enzymes have been described in humans, which could be responsible for increased susceptibility to DILI in some individuals. We herein explored the feasibility of the combined use of HepG2 cells co-transduced with multiple adenoviruses that encode drug-metabolising enzymes, and a high-content screening assay to evaluate metabolism-dependent drug toxicity and to identify metabolic phenotypes with increased susceptibility to DILI. To this end, HepG2 cells with different expression levels of specific drug-metabolism enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, GSTM1 and UGT2B7) were exposed to nine drugs with reported hepatotoxicity. A panel of pre-lethal mechanistic parameters (mitochondrial superoxide production, mitochondrial membrane potential, ROS production, intracellular calcium concentration, apoptotic nuclei) was used. Significant differences were observed according to the level of expression and/or the combination of several drug-metabolism enzymes in the cells created ad hoc according to the enzymes implicated in drug toxicity. Additionally, the main mechanisms implicated in the toxicity of the compounds were also determined showing also differences between the different types of cells employed. This screening tool allowed to mimic the variability in drug metabolism in the population and showed a highly efficient system for predicting human DILI, identifying the metabolic phenotypes associated with increased DILI risk, and indicating the mechanisms implicated in their toxicity.
... Nevertheless, they fail in the mimesis of cell-cell and cell-ECM interactions, essential for hepatocyte viability and functions, resulting in cell dedifferentiation and loss of the majority of their phenotypic properties, including their drug-metabolizing capacity, thus hampering their application in drug testing (Mazzoleni and Steimberg, 2012;Mueller et al., 2013). Primary human hepatocytes are the gold standard in the design of in vitro models reproducing human liver functions for the investigation of drug metabolism and toxicity, as in the study of disease onset, progression and response to new therapies (Gómez-Lechón et al., 2007;Mazzoleni and Steimberg, 2012;Mueller et al., 2013;Willebrords et al., 2015). However, the capability to maintain liver native functions for only few days (about three days) under conventional cell culture conditions, together with tissue shortage and its high variability, have limited the establishment of 2D primary hepatocyte cell culture and consequently the use of these cells in the development of liver models (Guillouzo and Guguen-Guillouzo, 2008;Mazzoleni and Steimberg, 2012;Mueller et al., 2013;Soldatow et al., 2013). ...
Article
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In the tissue engineering (TE) paradigm, engineering and life sciences tools are combined to develop bioartificial substitutes for organs and tissues, which can in turn be applied in regenerative medicine, pharmaceutical, diagnostic, and basic research to elucidate fundamental aspects of cell functions in vivo or to identify mechanisms involved in aging processes and disease onset and progression. The complex three-dimensional (3D) microenvironment in which cells are organized in vivo allows the interaction between different cell types and between cells and the extracellular matrix, the composition of which varies as a function of the tissue, the degree of maturation, and health conditions. In this context, 3D in vitro models can more realistically reproduce a tissue or organ than two-dimensional (2D) models. Moreover, they can overcome the limitations of animal models and reduce the need for in vivo tests, according to the “3Rs” guiding principles for a more ethical research. The design of 3D engineered tissue models is currently in its development stage, showing high potential in overcoming the limitations of already available models. However, many issues are still opened, concerning the identification of the optimal scaffold-forming materials, cell source and biofabrication technology, and the best cell culture conditions (biochemical and physical cues) to finely replicate the native tissue and the surrounding environment. In the near future, 3D tissue-engineered models are expected to become useful tools in the preliminary testing and screening of drugs and therapies and in the investigation of the molecular mechanisms underpinning disease onset and progression. In this review, the application of TE principles to the design of in vitro 3D models will be surveyed, with a focus on the strengths and weaknesses of this emerging approach. In addition, a brief overview on the development of in vitro models of healthy and pathological bone, heart, pancreas, and liver will be presented.
... In response to inflammatory or biomechanical stimuli, hPSC-derived endothelial cells are able to model athero-susceptible phenotypes and display a breach of barrier integrity to allow leukocyte transmigration. Although incorporation of primary hepatocytes is most likely to replicate human liver function for drug testing in a metabolism-enabled cellular model, their scarcity and the high cost of freshly isolated or cryopreserved human primary hepatocytes limit their extensive use [13]. We and others have created hPSC-derived hepatocytes that express cytochrome P450 (CYP) drug-metabolizing enzymes and show sensitivity to drugs with known hepatotoxicity [14,15]. ...
Article
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Atherosclerosis underlies many cardiovascular and cerebrovascular diseases. Nutraceuticals are emerging as a therapeutic moiety for restoring vascular health. Unlike small-molecule drugs, the complexity of ingredients in nutraceuticals often confounds evaluation of their efficacy in preclinical evaluation. It is recognized that the liver is a vital organ in processing complex compounds into bioactive metabolites. In this work, we developed a coculture system of human pluripotent stem cell-derived endothelial cells (hPSC-ECs) and human pluripotent stem cell-derived hepatocytes (hPSC-HEPs) for predicting vascular-protective effects of nutraceuticals. To validate our model, two compounds (quercetin and genistein), known to have anti-inflammatory effects on vasculatures, were selected. We found that both quercetin and genistein were ineffective at suppressing inflammatory activation by interleukin-1b owing to limited metabolic activity of hPSC-ECs. Conversely, hPSC-HEPs demonstrated metabolic capacity to break down both nutraceuticals into primary and secondary metabolites. When hPSC-HEPs were cocultured with hPSC-ECs to permit paracrine interactions, the continuous turnover ofmetabolitesmitigated interleukin-1b stimulation on hPSC-ECs.We observed significant reductions in inflammatory gene expressions, nuclear translocation of nuclear factor kB, and interleukin-8 production. Thus, integration of hPSC-HEPs could accurately reproduce systemic effects involved in drug metabolism in vivo to unravel beneficial constituents in nutraceuticals. This physiologically relevant endothelial-hepatic platform would be a great resource in predicting the efficacy of complex nutraceuticals and mechanistic interrogation of vascular-targeting candidate compounds.
... 16,17 PHH mono-cultures are considered to be the gold standard for the investigation of hepatic metabolism and toxicity of xenobiotics. 18 However, detailed morphological and functional studies have demonstrated that these models are limited due to hepatocyte dedifferentiation and loss of functions within few days. 2 Additionally, mono-hepatocyte cultures have only limited abilities for the in vitro reproduction of hepatotoxic effects observed in vivo. ...
... A previous study has reported a faster degradation rate of (+)-Smetalaxyl in rat hepatic microsomes (Zhang et al. 2012). While, compared with hepatic microsomes, primary hepatocyte represented a more complete system with intact membranes and physiological level of cofactors and enzymes (Gomez-Lechon et al. 2007). Thus, the two in vitro models might exhibit a certain difference in degradation behavior of an exogenous chemical. ...
Article
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Enantiomers of chiral compounds often exhibit enantioselective adverse effects and biochemical processes in non-target organisms. In this study, enantioselective metabolism and toxic effects of metalaxyl enantiomers on primary rat hepatocytes were investigated. Stereoselectivity was observed on both degradation of metalaxyl and formation of metabolites. (−)-R-metalaxyl eliminated faster than (+)-S-metalaxyl, while the hydroxylmetalaxyl, demethylmetalaxyl, and didemethylmetalaxyl metabolites derived from 50-μM (+)-S-metalaxyl after 24 h of incubation were approximately 1.57, 1.43, and 1.86 times more than that of (−)-R-metalaxyl, respectively. According to the methyl tetrazolium (MTT) assay, the EC50 values (24 h) for rac-, (+)-S-, and (−)-R-metalaxyl were 1788.22, 2066.73, and 2263.71 μM, respectively. An accordant enantioselective effect on oxidative stress suggested that the enantioselective cytotoxicity induced by metalaxyl enantiomers may partly contribute to enantioselective oxidative damage and mitochondrial dysfunction. Such results could be of great importance for credible environmental and toxicological risk assessment of metalaxyl.
... 5-10% of adverse drug reactions are the result of liver toxicity and a third of all post-market drug withdrawals are because of liver toxicity (3). The central role of the liver has led to the use of liver cells (hepatocytes) as a major choice for in vitro testing systems (1,4). The FDA has already found drug testing with hepatocyte cell culture to be an acceptable preclinical tool (5). ...
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Even the finest state-of-the art preclinical drug testing, usually in primary hepatocytes, remains an imperfect science. Drugs continue to be withdrawn from the market due to unforeseen toxicity, side effects, and drug interactions. The space program may be able to provide a lifeline. Best known for rockets, space shuttles, astronauts and engineering, the space program has also delivered some serious medical science. Optimized suspension culture in NASA's specialized suspension culture devices, known as rotating wall vessels, uniquely maintains Phase I and Phase II drug metabolizing pathways in hepatocytes for weeks in cell culture. Previously prohibitively expensive, new materials and 3D printing techniques have the potential to make the NASA rotating wall vessel available inexpensively on an industrial scale. Here we address the tradeoffs inherent in the rotating wall vessel, limitations of alternative approaches for drug metabolism studies, and the market to be addressed. Better pre-clinical drug testing has the potential to significantly reduce the morbidity and mortality of one of the most common problems in modern medicine: adverse events related to pharmaceuticals.
Article
Background Tangeretin, present in citrus fruits, is a polymethoxy flavone with extensive pharmacological effects. It has been widely used in the clinic, but there were no detailed studies on the in vivo metabolism of tangeretin. Objective This study aimed to establish a rapid and effective strategy to identify the metabolites of tangeretin and evaluate the biotransformation pathways of tangeretin in rats. Methods The ultra-high performance liquid chromatography (UHPLC) equipped with a Q-Exactive Orbitrap mass spectrometer was used to identify the metabolites of tangeretin in plasma, urine and faeces of rats after intragastric administration. Based on high-resolution extracted ion chromatograms (HREICs) and parallel reaction monitoring mode (PRM), metabolites of tangeretin were identified by comparing the accurate mass, chromatographic retention times, diagnostic product ions (DPIs) and neutral loss fragments (NLFs) with those of tangeretin reference standard. Isomers were distinguished by ClogP values. Results An efficient and integrated strategy was established for the comprehensive screening and characterizing of tangeretin metabolites through Rapid Profiling. Based on this strategy, a total of 52 metabolites were detected and identified, among which 25 metabolites were found in rat plasma, while 48 and 16 metabolites were characterized from rat urine and faeces, respectively. These metabolites were produced by demethylation, demethoxylation, hydroxylation, methoxylation, glucuronidation, glycosylation, sulfation, and their composite reactions. Interestingly, tangeretin is easy to lose methyl in vivo and becomes an intermediate product, and then other phase I and phase II reactions occur. Moreover, the characteristic fragmentation pathways of tangeretin were summarized for the subsequent metabolite identification. Conclusion The analytical method based on UHPLC-Q-Exactive mass spectrometer has the ability to quickly clarify unknown metabolism. And the the comprehensive metabolism study of tangeretin provided an overall metabolic profile, which will be of great scientific basis for further studies on tangeretin in determining its pharmacokinetics, the bioactivity of the metabolites, and clinical applications. Conclusion An efficient and integrated strategy was established for the comprehensive screening and characterizing of tangeretin metabolites Rapid Profiling. The results showed that after oral administration of tangeretin, it was easy to become demethylated products in vivo, and then other phase I and phase II reactions occurred on the basis of its demethylated products. The findings from this study will provide a scientific basis for further studies on tangeretin in determining its pharmacokinetics, the bioactivity of the metabolites, and clinical applications. Other No
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Liver cultures may be used for disease modeling, testing therapies, and predicting drug‐induced injury. The complexity of the liver cultures has evolved from hepatocyte monocultures to co‐cultures with non‐parenchymal cells and finally to precision‐cut liver slices. The latter culture format retains liver's native biomolecular and cellular complexity and therefore holds considerable promise for in vitro testing. However, liver slices remain functional for ≈72 h in vitro and display limited utility for some disease modeling and therapy testing applications that require longer culture times. This paper describes a microfluidic device for longer‐term maintenance of functional organotypic liver cultures. This microfluidic culture system is designed to enable direct injection of liver tissue into a culture chamber through a valve‐enabled side port. Liver tissue is embedded in collagen and remained functional for up to 31 days, highlighted by continued production of albumin and urea. These organotypic cultures also express several enzymes involved in xenobiotic metabolism. Conversely, matched liver tissue embedded in collagen in a 96‐well plate loses its phenotype and function within 3–5 days. The microfluidic organotypic liver cultures described here represent a significant advance in liver cultivation and may be used for future modeling of liver diseases or for individualized liver‐directed therapies.
Chapter
This chapter discusses the concepts related to the use of drugs in vivo, specifically from the point of drug absorption, distribution throughout the body, metabolism by the hepatic (and other) systems, and excretion (primarily through the renal system). The combination of these processes in vivo is considered as well as the various in vitro assays are used to predict pharmacokinetics effects.
Article
Numerous attempts have been made to organize isolated primary hepatocytes into functional three-dimensional (3D) constructs, but technologies to introduce extracellular matrix (ECM) components into such assemblies have not been fully developed. Here we report a new approach to forming hepatocyte-based 3D tissues using fibrillized collagen microparticles (F-CMPs) as intercellular binders. We created thick tissues with a thickness of ∼200 μm simply by mixing F-CMPs with isolated primary rat hepatocytes and culturing them in cell culture inserts. Owing to the incorporated F-CMPs, the circular morphology of the formed tissues was stabilized, which was strong enough to be manually manipulated and retrieved from the chamber of the insert. We confirmed that the F-CMPs dramatically improved the cell viability and hepatocyte-specific functions such as albumin production and urea synthesis in the formed tissues. The presented approach provides a versatile strategy for hepatocyte-based tissue engineering, and will have a significant impact on biomedical applications and pharmaceutical research.
Research
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TOXICOLOGICAL EFFECT OF INSECTICIDE CHLORANTRANILIPROLE ON THE ACID PHOSPHATASE ACTIVITY OF FRESHWATER FISH CIRRHINUS MRIGALA
Chapter
There are many factors which are known to cause variability in human in vitro enzyme kinetic data. Factors such as the source of enzyme and how it was prepared, the genetics and background of the donor, how the in vitro studies are designed, and how the data are analyzed contribute to variability in the resulting kinetic parameters. It is important to consider not only the factors which cause variability within an experiment, such as selection of a probe substrate, but also those that cause variability when comparing kinetic data across studies and laboratories. For example, the artificial nature of the microsomal lipid membrane and microenvironment in some recombinantly expressed enzymes, relative to those found in native tissue microsomes, has been shown to influence enzyme activity and thus can be a source of variability when comparing across the two different systems. All of these factors, and several others, are discussed in detail in the chapter below. In addition, approaches which can be used to visualize the uncertainty arising from the use of enzyme kinetic data within the context of predicting human pharmacokinetics are discussed.
Article
Protein abundance data of drug-metabolizing enzymes and transporters (DMETs) are broadly applicable to the characterization of in vitro and in vivo models, in vitro to in vivo extrapolation (IVIVE), and interindividual variability prediction. However, the emerging need of DMET quantification in small sample volumes such as organ-on a chip effluent, organoids, and biopsies requires ultrasensitive protein quantification methods. We present an ultrasensitive method that relies on an optimized sample preparation approach involving acetone precipitation coupled with a microflow-based liquid chromatography-tandem mass spectrometry (µLC-MS/MS) for the DMET quantification using limited sample volume or protein concentration, i.e., liver tissues (1-100 mg), hepatocyte counts (∼4000 to 1 million cells), and microsomal protein concentration (0.01-1 mg/ml). The method was applied to quantify DMETs in differential tissue S9 fractions (liver, intestine, kidney, lung, and heart) and cryopreserved human intestinal mucosa (i.e., CHIM). The method successfully quantified >75% of the target DMETs in the trypsin digests of 1 mg tissue homogenate, 15,000 hepatocytes, and 0.06 mg/ml microsomal protein concentration. The precision of DMET quantification measured as the coefficient of variation across different tissue weights, cell counts, or microsomal protein concentration was within 30%. The method confirmed significant extrahepatic abundance of non-cytochrome P450 enzymes such as dihydropyridine dehydrogenase (DPYD), epoxide hydrolases (EPXs), arylacetamide deacetylase (AADAC), paraoxonases (PONs), and glutathione S-transferases (GSTs). The ultrasensitive method developed here is applicable to characterize emerging miniaturized in vitro models and small volume biopsies. In addition, the differential tissue abundance data of the understudied DMETs will be important for physiologically-based pharmacokinetic (PBPK) modeling of drugs.
Article
This study aimed to use a reverse dosimetry PBPK modeling approach to estimate toluene atmospheric exposure from urinary measurements of S-benzylmercapturic acid (BMA) in a small group of individuals and to evaluate the uncertainty associated to urinary spot-sampling compared to 24-hour collected urine samples. Each exposure assessment technique was developed namely to estimate toluene air exposure from BMA measurements in 24-hour urine samples (24-h-BMA) and from distributions of daily urinary BMA spot measurements (DUBSM). Model physiological parameters were described based upon age, weight, size and sex. Monte Carlo simulations with the PBPK model allowed converting DUBSM distribution (and 24-h-BMA) into toluene air levels. For the approach relying on DUBSM distribution, the ratio between the 95% probability of predicted toluene concentration and its 50% probability in each individual varied between 1.2 and 1.4, while that based on 24-h-BMA varied between 1.0 and 1.1. This suggests more variability in estimated exposure from spot measurements. Thus, estimating toluene exposure based on DUBSM distribution generated about 20% more uncertainty. Toluene levels estimated (0.0078-0.0138 ppm) are well below Health Canada's maximum chronic air guidelines. PBPK modeling and reverse dosimetry may be combined to interpret urinary metabolites data of VOCs and assess related uncertainties.
Article
• Individual differences in cytochrome P450 (CYP) enzymes contribute to responses to drugs and environmental chemicals. The expression of CYPs are influenced by sex, age and ethnicity. Human CYP studies are often conducted with human liver microsomes and liver cells to evaluate chemical induction and drug interactions. However, the basal or constitutive expression of CYP transcripts and enzyme activities in the intact liver are also important in our understanding of individual variation in CYPs. • This study utilized 100 human liver samples to profile the constitutive expression of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 4A11 enzyme activity and transcript levels. The mRNA expression of the CYPs and xenobiotic receptors AhR, CAR and PXR was examined via qPCR. • Results showed that there was greater inter-individual variation in mRNA expression than in enzyme activities, except for CYP2C19. Females had higher CYP3A4 activity than males. Children had lower CYP4A14 activity, while elderly had lower P450 oxidoreductase activity. Compared to Caucasians, Hispanics had higher CYP2C8 activity and higher CYP2B6, CYP2C9 and CYP2C19 mRNA expression, whereas African Americans had lower CYP2D6 mRNA expression. • These results add to our understanding of individual variations in xenobiotic metabolism and toxicology.
Article
Metabolic stability plays a crucial role in assessing the safety of potential drug candidates. Assays to designate in vitro half-life are well established and are focused on phase I metabolism using human liver microsomes. However, such an assay can easily be modified to include phase II metabolism. Yet, to authors best knowledge, no attempts at designating in vitro half-life values that includes phase II enzymes were performed. That means, that all current metabolic stability estimations reflect only partial information – as only phase I of metabolism takes place in currently used assays. The following study uses originally modified metabolic stability assay, coupled with LC–MS analysis to provide in vitro half-life values for four pirydo[1,2-c]pirymidine derivatives. Testosterone was used as a positive control. The assay was modified using alamethicin (a pore-forming agent which enhances membrane transport, allowing for phase II reactions) and UDPGA (Uridine 5′-diphosphoglucuronic acid) – co-factor for most common phase II biotransformation reaction – glucuronidation. In vitro half-life values estimated using two assays (a standard one comprising sole phase I enzymes and modified one – providing phase II reactions) have been compared using U Mann-Whitney’s test for statistical significance. Values for phases I + II were slightly higher, however, the difference was not statistically significant. Such results lead to the conclusion, that in vitro half-life values are not strongly influenced by phase II biotransformation in the case of the considered compounds.
Article
Sarcomas are a rare and heterogeneous cancer variant of mesenchymal origin. Their genetic heterogeneity coupled with uncertain histogenesis make them difficult to treat and is associated with poor prognosis. In this work, we show that structure-based drug discovery involving computational modeling can be used to identify new retinoid X receptor (RXR) agonist ligand with a bis(indolyl)methane scaffold. This agent co-self-assembles with an amphiphilic diblock-co-polymer resulting into nanoparticles (Nano-RXR) with excellent kinetic stability which was evaluated for efficacy and safety in transformed sarcoma cells, 63-3 Cre and 141-10 Cre of pig origin and in rodent xenograft models. Responses at gene and protein levels established the treatment approach as a highly effective RXR agonist across cell, rodent and ‘Oncopig’ models. Interestingly, Nano-RXR was not only able to modulate metabolic and transporter genes related to orphan nuclear receptors but also played a major role in modulating programmed cell death in sarcomas, developed in Oncopigs.
Article
Hepatotoxicity remains a major challenge in drug development despite preclinical toxicity screening using hepatocytes of human origin. To overcome some limitations of reproducing the hepatic phenotype, more structurally and functionally authentic cultures in vitro can be introduced by growing cells in 3D spheroid cultures. Characterisation and reproducibility of HepG2 spheroid cultures using a high-throughput hanging drop technique was performed and features contributing to potential phenotypic variation highlighted. Cultured HepG2 cells were seeded into Perfecta 3D® 96-well hanging drop plates and assessed over time for morphology, viability, cell cycle distribution, protein content and protein-mass profiles. Divergent aspects which were assessed included cell stocks, seeding density, volume of culture medium and use of extracellular matrix additives. Hanging drops are advantageous due to no complex culture matrix being present, enabling background free extractions for downstream experimentation. Varying characteristics were observed across cell stocks and batches, seeding density, culture medium volume and extracellular matrix when using immortalized HepG2 cells. These factors contribute to wide-ranging cellular responses and highlights concerns with respect to generating reproducible characteristics in hanging drop spheroids.
Article
Primycin-sulphate is a highly effective compound against Gram (G) positive bacteria. It has a potentially synergistic effect with vancomycin and statins which makes primycin-sulphate a potentially very effective preparation. Primycin-sulphate is currently used exclusively in topical preparations. In vitro animal hepatocyte and neuromuscular junction studies (in mice, rats, snakes, frogs) as well as in in vitro human red blood cell experiments were used to test toxicity. During these studies, the use of primycin-sulphate resulted in reduced cellular membrane integrity and modified ion channel activity. Additionally, parenteral administration of primycin-sulphate to mice, dogs, cats, rabbits and guinea pigs indicated high level of acute toxicity. The objective of this study was to reveal the cytotoxic and gene expression modifying effects of primycin-sulphate in a human system using an in vitro, three dimensional (3D) human hepatic model system. Within the 3D model, primycin-sulphate presented no acute cytotoxicity at concentrations 1 μg/ml and below. However, even at low concentrations, primycin-sulphate affected gene expressions by up-regulating inflammatory cytokines (e.g., IL6), chemokines (e.g., CXCL5) and by down-regulating molecules of the lipid metabolism (e.g., peroxisome proliferator receptor (PPAR) alpha, gamma, etc). Down-regulation of PPAR alpha cannot just disrupt lipid production but can also affect cytochrome P450 metabolic enzyme (CYP) 3A4 expression, highlighting the need for extensive drug–drug interaction (DDI) studies before human oral or parenteral preparations can be developed.
Article
The establishment of a reliable in vitro liver model for drug screening remains challenging with respect to tethering the growth of hepatocyte spheroids and adapting to the current high-throughput system. In the current study, short fibers are utilized as scaffolds for the generation of size-controlled hepatocyte spheroids that recapitulate in vivo hepatic phenotypes and functions. The spheroid formation is modulated by the length and galactose/RGD grafts of short fibers, and short 50 μm long fibers motivate the spheroid formation with optimal hepatic function. Short fibers distribute throughout the entire spheroid for tethering hepatocyte growth to form compact spheroids. Compared with scaffold-free spheroid culture on agarose-coated plates, the spheroid culture with short fibers achieves higher clearance rates of model drugs and provides a better prediction of the in vivo drug clearance rate with a correlation value of 0.886. In addition, the drug metabolism capability is highly sensitive to the inducers and inhibitors of metabolizing enzymes, and the responsiveness is maintained during 20 days of culture, exhibiting an efficient in vitro model for determining drug-drug interactions. Therefore, the spheroid culture with short fibers provides an easily manipulated strategy to maintain hepatocyte functions for a prolonged period and enable ready deployment in conventional multiwell plates and diverse organ-on-a-chip devices for high-throughput drug screening.
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In previous papers we demonstrated that cyclosporin A (CsA) was specifically oxidized in rabbit and human liver by cytochrome P-450IIIA. We therefore anticipated that any drug that is an inducer or an inhibitor of this cytochrome should lead to interaction with CsA when given in association with it. In order to confirm this hypothesis, primary cultures of human hepatocytes and human liver microsomes were used to "reproduce" in vitro clinically significant interactions observed between CsA and drugs known either as specific inducers (i.e., rifampicin) or as specific inhibitors (i.e., erythromycin) of P-450IIIA. Our results were in close agreement with the clinical reports. Human hepatocytes maintained in primary cultures for 72 hr in the presence of 50 microM rifampicin exhibited increased levels of P-450IIIA, determined by Western blot using specific antibodies, and concomitant increase in CsA oxidase activity, determined by HPLC analysis of extra and intracellular media. Conversely, these cultures exhibited erythromycin concentration-dependent decreases in CsA oxidase activity when incubated in the presence of 5, 20, and 100 microM erythromycin. In addition, a Lineweaver-Burk analysis of the erythromycin-mediated inhibition of CsA oxidase activity in human liver microsomes revealed competitive inhibition (with Ki of 75 microM) as expected, this macrolide being a specific substrate of P-450IIIA. Using this experimental approach, 59 molecules representative of 17 different therapeutic classes were screened for inducers and inhibitors of CsA oxidase activity. Our results allowed us to elucidate the molecular mechanism of previously observed, but unexplained, drug interactions involving CsA, and to detect drugs that should interfere with CsA metabolism as inducers or inhibitors. Drugs detected as potential inducers of CsA oxidase included: rifampicin, sulfadimidine, phenobarbital, phenytoin, phenylbutazone, dexamethasone, sulfinpyrazone, and carbamazepine. Drugs detected as potential competitive inhibitors included: triacetyloleandomycin, erythromycin, josamycin, midecamycin, ketoconazole, miconazole, midazolam, nifedipin, diltiazem, verapamil, nicardipine, ergotamine, dihydroergotamine, glibenclamide, bromocriptine, ethynylestradiol, progesterone, cortisol, prednisone, prednisolone, and methylprednisolone. Finally, cefoperazone, cefotaxime, ceftazidime, isoniazide, doxycycline, spiramycin, sulfamethoxazole, norfloxacin, pefloxacin, vancocin, trimethoprim, amphotericin B, valproic acid, quinidine, cimetidine, ranitidine, omeprazole, diclofenac, aspirin, paracetamol, debrisoquine, guanoxan, captopril, furosemide, acetazolamide, sparteine, gliclazide, and imipramine were found not to interfere with the hepatic metabolism of CsA.
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A growing number of human genetic polymorphisms in drug-metabolizing enzymes (DMEs) are being characterized. Some of these have been shown, quite convincingly, to be correlated with risk of toxicity or cancer, whereas others presently remain equivocal. There is good evidence that the correlation is stronger in populations exposed to a variety of environmental procarcinogens; perhaps 30% of DME substrates are able to be metabolically potentiated. Phase I DMEs, most of which represent cytochromes P450, metabolically activate procarcinogens to genotoxic electrophilic intermediates, and Phase II DMEs conjugate the intermediates to water-soluble derivatives, completing the detoxification cycle. It follows that genetic differences in the regulation, expression and activity of genes coding for Phase I and Phase II DMEs would be crucial factors in defining cancer susceptibility and the toxic or carcinogenic power of environmental chemicals. Not all Phase I and Phase II DMEs are implicated in detoxification; previous work from this and from other laboratories has identified candidate Phase I and Phase II genes in which certain alleles are more likely to be associated with cancer susceptibility. In some cases, the allelic frequencies vary dramatically between ethnic groups. In this review, our current knowledge about polymorphisms in the following genes are updated: the aromatic hydrocarbon receptor (AHR), the CYP1A1 structural gene (which encodes aryl hydrocarbon hydroxylase activity), the CYP1A2 structural gene (arylamine oxidations), the CYP2C19 gene (S-mephenytoin 4'-hydroxylase), the CYP2D6 gene (debrisoquine hydroxylase), the CYP2E1 gene (N,N-dimethylnitrosamine N-demethylase), the null mutant for the GSTM1 gene (glutathione transferase mu), and the NAT2 gene (arylamine N-acetyltransferase). If unequivocal biomarkers of genetic susceptibility to cancer and toxicity can be developed successfully, then identification of individuals at increased risk would be very helpful in the fields of public health and preventive medicine.
Article
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The importance of cytochrome P450 isoforms to species differences in the metabolism of foreign compounds and activation of procarcinogens has been identified. The possible range of P450 isozymes in significant variations in toxicity exhibited by experimental rodent species may have a relevance to chemical risk assessment, especially as human P450s are likely to show changes in the way they metabolize xenobiotics. Consequently, in the safety evaluation of chemicals, we should be cautious in extrapolating results from experimental animal models to humans. This paper focuses on examples in which species differences in P450s lead to significant alterations in carcinogenic response, and includes a discussion of the current procedures for toxicity screening, with an emphasis on short-term tests. Images Figure 1 Figure 2 Figure 3 Figure 4
Article
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1. Fully automated inhibition screens for the major human hepatic cytochrome P450s have been developed and validated. Probe assays were the fluorometric-based ethoxyresorufin O-deethylation for CYP1A2 and radiometric analysis of erythromycin N-demethylation for CYP3A4, dextromethorphan O-demethylation for CYP2D6, naproxen O-demethylation for CYP2C9 and diazepam N-demethylation for CYP2C19. For the radiometric assays > 99.7% of 14C-labelled substrate was routinely extracted from incubations by solid-phase extraction. 2. Furafylline, sulphaphenazole, omeprazole, quinidine and ketoconazole were identified as specific markers for the respective CYP1A2 (IC50 = 6 microM), CYP2C9 (0.7 microM), CYP2C19 (6 microM), CYP2D6 (0.02 microM) and CYP3A4 (0.2 microM) inhibition screens. 3. For the radiometric methods, a two-point IC50 estimate was validated by correlating the IC50 obtained with a full (seven-point) assay (r2 = 0.98, p < 0.001). The two-point IC50 estimate is useful for initial screening, while the full IC50 method provides more definitive quantitation, where required. 4. IC50 determined for a series of test compounds in human liver microsomes and cytochrome P450 cDNA-expressed enzymes were similar (r2 = 0.89, p < 0.001). In particular, the CYP1A2, CYP2D6 and CYP3A4 screens demonstrated the flexibility to accept either enzyme source. As a result of incomplete substrate selectivity, expressed enzymes were utilized for analysis of CYP2C9 and CYP2C19 inhibition. Good agreement was demonstrated between IC50 determined in these assays to IC50 published by other laboratories using a wide range of analytical techniques, which provided confidence in the universality of these inhibition screens. 5. These automated screens for initial assessment of P450 inhibition potential allow rapid determination of IC50. The radiometric assays are flexible, sensitive, robust and free from analytical interference, and they should permit the identification and eradication of inhibitory structural motifs within a series of potential drug candidates.
Conference Paper
One of the most challenging research areas in pharmacology in the new millennium is to understand why individuals respond differently to drug therapy and to what extent that individual variability in disposition is responsible for the observed differences in therapeutic efficacy and adverse reactions. To answer these complex questions, drug-metabolism research will rely on multidisciplinary approaches more than ever to investigate the many components involved in drug metabolism and disposition. Major research challenges include the following: (1) the genetic variation of drug targets (receptors, enzymes, etc.), drug transporters (multispecific organic anion transporter, P-glycoprotein, alpha-1-acid glycoprotein, etc.), and drug-metabolizing enzymes (cytochrome P450s and other enzymes); (2) the structure and function of all genetic variants of drug receptors, transporters, and metabolizing enzymes; (3) the induction, repression, and inhibition of ail components involved in drug disposition; (4) the development of noninvasive in vivo methods to determine the physiological significance of various components in the handling of specific therapeutic agents in humans; (5) the mechanism of idiosyncratic adverse drug reactions; and (6) the pharmacokinetic and pharmacodynamic relationships to explain the individual differences in therapeutic efficacy and drug safety, Thus successful drug-metabolism research in the new millennium must integrate receptor biology, enzymology, recombinant DNA technology, biochemical toxicology, and drug disposition into study design and conduct balanced in vitro and in vivo experiments to allow a full understanding of the mechanisms of individual variability in drug therapy and drug safety.
Chapter
The liver is the key organ for biotransformation of xenobiotics that enter the human body, either intentionally (e.g. pharmaceuticals), or unintentionally (e.g. environmental pollutants). All blood-borne xenobiotics are first metabolized by the liver, a process known as first-pass metabolism. The major consequence of the metabolic transformation is the formation of water-soluble metabolites that are removed from the body. In general, the xenobiotic is firstly oxidized (phase I metabolism) and then conjugated to highly polar molecules such as glucose, sulphate, cysteine or glutathione (phase II metabolism). The highly polar metabolites are then transported directly from the hepatocytes into the biliary canaliculi (phase III metabolism) to be excreted in the bile, or are released back into the blood stream to be excreted into the urine via the kidneys. In the intestine, the metabolites may be deconjugated by gut bacterial flora and reabsorbed, leading to a repeat of the metabolic processes (enterohepatic recirculation). The sequential oxidation-conjugation of xenobiotics is commonly known as metabolic detoxification, a process responsible for ridding the body of xenobiotic toxicants. However, it is now also known that many innocuous chemicals can be metabolically “activated” into highly reactive metabolites with toxicological consequences. As the organ where this metabolic “activation” occurs, the liver is therefore often the target organ of chemical toxicants (see Chapter 27).
Article
The nonsteroidal antiinflammatory drug diclofenac causes rare but significant cases of serious hepatotoxicity, typically with a delayed onset (>1-3 months). Because there is no simple dose relationship and because liver injury cannot be reproduced in current animal models, individual patient-specific susceptibility factors have been evoked to account for the increased risk. While these patient factors have remained undefined, a number of molecular hazards have been characterized. Among these are metabolic factors (bioactivation by hCYP2C9 or hCYP3A4 to thiol-reactive quinone imines, activation by hUGT2B7 to protein-reactive acyl glucuronides and iso-glucuronides, and 4'-hydroxylation secondary to diclofenac glucuronidation), as well as kinetic factors (Mrp2-mediated concentrative transport of diclofenac metabolites into bile). From the toxicodynamic view, both oxidative stress (caused by putative diclofenac cation radicals or nitroxide and quinone imine-related redox cycling) and mitochondrial injury (protonophoretic activity and opening of the permeability transition pore) alone or in combination have been implicated in diclofenac toxicity. In some cases, immune-mediated liver injury is involved, inferred from inadvertent rechallenge data and from a number of experiments demonstrating T cell sensitization. Why certain underlying diseases (e.g., osteoarthritis) also increase the susceptibility to diclofenac hepatotoxicity is not clear. To date, cumulative damage to mitochondrial targets seems a plausible putative mechanism to explain the delayed onset of liver failure, perhaps even superimposed on an underlying silent mitochondrial abnormality. Increased efforts to identify both patient-specific risk factors and disease-related factors will help to define patient subsets at risk as well as increase the predictability of unexpected hepatotoxicity in drug development.
Article
Cultured human hepatocytes are a valuable in vitro system for evaluating new molecular entities as inducers of cytochrome P450 (P450) enzymes. The present study summarizes data obtained from 62 preparations of cultured human hepatocytes that were treated with vehicles (saline or dimethylsulfoxide, 0.1%), β-naphthoflavone (33 μM), phenobarbital (100 or 250 μM), isoniazid (100 μM) and/or rifampin (20 or 50 μM), and examined for the expression of P450 enzymes based on microsomal activity toward marker substrates, or in the case of CYP2C8, the level of immunoreactive protein. The results show that CYP1A2 activity was markedly induced by β-naphthoflavone (on average 13-fold, n = 28 preparations), and weakly induced by phenobarbital (1.9-fold, n = 25) and rifampin (2.3-fold, n = 22); CYP2A6 activity tended to be increased with phenobarbital ( n = 7) and rifampin ( n = 3) treatments, but the effects were not statistically significant; CYP2B6 was induced by phenobarbital (6.5-fold, n = 13) and rifampin (13-fold, n = 14); CYP2C8 was induced by phenobarbital (4.0-fold, n = 4) and rifampin (5.2-fold, n = 4); CYP2C9 was induced by phenobarbital (1.8-fold, n = 14) and rifampin (3.5-fold, n = 10); CYP2C19 was markedly induced by rifampin (37-fold, n = 10), but relatively modestly by phenobarbital (7-fold, n = 9); CYP2D6 was not significantly induced by phenobarbital ( n = 5) or rifampin ( n = 5); CYP2E1 was induced by phenobarbital (1.7-fold, n = 5), rifampin (2.2-fold, n = 5), and isoniazid (2.3-fold, n = 5); and, CYP3A4 was induced by phenobarbital (3.3-fold, n = 42) and rifampin (10-fold, n = 61), but not by β-naphthoflavone. Based on these observations, we generalize that β-naphthoflavone induces CYP1A2 and isoniazid induces CYP2E1, whereas rifampin and, to a lesser extent phenobarbital, tend to significantly and consistently induce enzymes of the CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A subfamilies but not the 2D subfamily.
Article
Drug metabolism is a major determinant of drug clearance, interindividual pharmacokinetic differences and, indirectly, of the clinical efficacy and toxicity of drugs. Altered pharmacokinetics can result in inadequate concentration of the drug at the site of action and/or great variations in clinical response. Therefore, the development of a new drug requires not only an exhaustive characterisation of its pharmacological activity, but also knowledge of major enzymes involved in metabolite formation, and the potential enzyme inhibiting or enzyme inducing properties of the drug. Multi-drug therapy is not uncommon in clinical practice. Simultaneous administration of several drugs may result in metabolic drug-drug interactions having pharmacological and/or toxicological implications. As drugs are metabolised by a limited number of enzymes, they can compete each other as substrates for the same enzyme. Thus, competitive/non-competitive/irreversible inhibition of drug-metabolising enzymes by one of the therapeutic agents will result in elevations in plasma/tissue concentrations of the other drugs. For compounds with a narrow therapeutic index, this can lead to overdosage symptoms and/or toxicity. Cytochrome P450 (P450) enzymes are major players in the oxidative metabolism of therapeutic agents and, consequently, the most common mechanism underlying drug-drug interactions is the inhibition of P450 activities. Several drugs in common use cause large increases in exposure to other drugs. As this is an undesirable feature for a drug candidate, information about P450 inhibition by the compound should be obtained before a drug candidate is considered for the clinical stages of development. A combination of biochemical advances in the understanding of the function and regulation of drug-metabolising enzymes, in particular P450s, and automated analytical technologies are revolutionising drug metabolism research.
Article
Hepatocytes entrapped in collagen gel and cultured in serum-free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c-fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin-stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 ± 0.37 and 9 ± 2.7 nmol glucose/h/μg DNA). Collagen-cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 ± 4 nmol urea/h/μg DNA). The rate of albumin synthesis in collagen-entrapped cells was maintained above the day-1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 ± 152 pmol/μg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen-cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and gluthatione-transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione-transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP- and -β, and HNF-1 and -4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate-synthetase I). J Cell Physiol 177:553–562, 1998. © 1998 Wiley-Liss, Inc.
Article
Among the predominantly purple- and red-flowered species of the genusCuphea, only seven yellow floral tube and/or yellow-petaled members in three sections were known. We add here two new species and one new variety.Cuphea congesta from Venezuela andC. splendida var.viridiflava from Bolivia are new to sect.Melvilla.Cuphea xanthopetala from Brazil is described in sect.Euandra and expands the presence of this feature to a fourth section of the genus. A key is provided to allCuphea with yellow floral tubes and/or yellow petals to expedite determinations when these exceptional character states are present.
Article
This report summarizes the results of a multinational pharmaceutical company survey and the outcome of an International Life Sciences Institute (ILSI) Workshop (April 1999), which served to better understand concordance of the toxicity of pharmaceuticals observed in humans with that observed in experimental animals. The Workshop included representatives from academia, the multinational pharmaceutical industry, and international regulatory scientists. The main aim of this project was to examine the strengths and weaknesses of animal studies to predict human toxicity (HT). The database was developed from a survey which covered only those compounds where HTs were identified during clinical development of new pharmaceuticals, determining whether animal toxicity studies identified concordant target organ toxicities in humans. Data collected included codified compounds, therapeutic category, the HT organ system affected, and the species and duration of studies in which the corresponding HT was either first identified or not observed. This survey includes input from 12 pharmaceutical companies with data compiled from 150 compounds with 221 HT events reported. Multiple HTs were reported in 47 cases. The results showed the true positive HT concordance rate of 71% for rodent and nonrodent species, with nonrodents alone being predictive for 63% of HTs and rodents alone for 43%. The highest incidence of overall concordance was seen in hematological, gastrointestinal, and cardiovascular HTs, and the least was seen in cutaneous HT. Where animal models, in one or more species, identified concordant HT, 94% were first observed in studies of 1 month or less in duration. These survey results support the value of in vivo toxicology studies to predict for many significant HTs associated with pharmaceuticals and have helped to identify HT categories that may benefit from improved methods.
Article
Human and animal hepatocytes are now being used as an in vitro technique to aid drug discovery by predicting the in vivo metabolic pathways of drugs or new chemical entities (NCEs), identifying drug-metabolizing enzymes and predicting their in vivo induction. Because of the difficulty of establishing whether the cytotoxic susceptibility of human hepatocytes to xenobiotics/drugs in vitro could be used to predict in vivo human hepatotoxicity, a comparison of the susceptibility of the hepatocytes of human and animal models to six chemical classes of drugs/xenobiotics in vitro have been related to their in vivo hepatotoxicity and the corresponding activity of their metabolizing enzymes. This study showed that the cytotoxic effectiveness of 16 halobenzenes towards rat hepatocytes in vitro using higher doses and short incubation times correlated well with rat hepatotoxic effectiveness in vivo with lower doses/longer times. The hepatic/hepatocyte xenobiotic metabolizing enzyme activities of various animal species and human have been reviewed for use by veterinarians and research scientists. Where possible, recommendations have been made regarding which animal hepatocyte model is most applicable for modeling the susceptibility to xenobiotic induced hepatotoxicity of those humans with slow versus rapid metabolizing enzyme polymorphisms. These recommendations are based on the best human fit for animal drug/xenobiotic metabolizing enzymes in terms of activity, kinetics and substrate/inhibitor specificity. The use of human hepatocytes from slow versus rapid metabolizing individuals for drug metabolism/cytotoxicity studies; and the research use of freshly isolated rat hepatocytes and “Accelerated Cytotoxicity Mechanism Screening” (ACMS) techniques for identifying drug/xenobiotic reactive metabolites are also described. Using these techniques the molecular hepatocytotoxic mechanisms found in vitro for seven classes of xenobiotics/drugs were found to be similar to the rat hepatotoxic mechanisms reported in vivo.
Article
Fatty liver has been associated with an increased risk of primary graft non-function and drug toxicity. However, these effects have been observed mainly in fatty liver with inflammation, a situation characterized by an overall reduction in cytochrome P-450 (CYP)-dependent activities as well as a contrasting increase in CYP2E1 activity. Our aim was to examine the impact of liver-fat accumulation on CYP in two animal models of fatty liver without necroinflammation. Ducks were force-fed with a high-glucidic diet and male Wistar rats, after 48 h fasting, were refed a high-glucidic, fat-free diet for 48 h. Total CYP, aminopyrine- (AND), erythromycin-N-demethylase (END) and chlorzoxazone hydroxylase (CZOHase) activities as well as CYP2E1 and CYP3A proteins were quantified on microsomal proteins. Livers from force-fed ducks exhibited significant decreases in total CYP, AND, END and CZOHase activities, inversely correlated with fat-liver content. Refeeding male Wistar rats a high-glucidic, fat-free diet after 48 h fasting, resulting in a 235% increased liver fat content, was associated with a decrease in total CYP (55%), AND (78%), END (55%) and CZOHase (62%) activities as well as in CYP3A (70%) and CYP2E1 (80%) protein content. A significant inverse correlation was observed between CYP and total lipid content. In these models of steatosis induced by nutritional manipulations, fat liver accumulation was associated with a significant decrease in CYP activities and in CYP protein expression. Furthermore, the decreases in both CYP content and related activities were correlated with the degree of liver fat content.
Article
A certain number of case reports of adverse hepatic reactions to diclofenac are known, suggesting that diclofenac-associated hepatitis may be more common than previously recognized. In order to discriminate among possible molecular mechanisms of toxicity, the following were investigated: (a) cytotoxicity of diclofenac on metabolizing (rat hepatocytes) and non-metabolizing hepatic cells (HepG2, FaO); (b) changes in calcium homoeostasis, glutathione (GSH), lipid peroxidation and ATP levels, and (c) diclofenac metabolism in relation to cytotoxicity. The results indicate that toxicity is associated with the oxidative metabolism of the drug, and correlated with the formation of a minor oxidation metabolite. Inhibitors of diclofenac metabolism concomitantly reduced the toxicity of the drug. Hepatocyte injury was preceded by a decrease in ATP levels. No oxidative stress (no changes in GSH, no lipid peroxidation) could be demonstrated at this early stage. Cytotoxicity was prevented when cells were incubated with fructose, suggesting that the inability of mitochondria to produce ATP is the probable cause of diclofenac hepatotoxicity.
Article
Steroid hydroxylation specificities were determined for 11 forms of human cytochrome P450, representing four gene families and eight subfamilies, that were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. Microsomes isolated from the P450-expressing Hep G2 cells were isolated and then assayed for their regioselectivity of hydroxylation toward testosterone, androstenedione, and progesterone. Four of the eleven P450s exhibited high steroid hydroxylase activity (150-900 pmol hydroxysteroid/min/mg Hep G2 microsomal protein), one was moderately active (30-50 pmol/min/mg) and six were inactive. In contrast, 10 of the P450s effectively catalyzed O-deethylation of 7-ethoxycoumarin, a model drug substrate, while only one (P450 2A6) catalyzed significant coumarin 7-hydroxylation. Human P450 4B1, which is expressed in lung but not liver, catalyzed the 6 beta-hydroxylation of all three steroids at similar rates and with only minor formation of other hydroxylated products. Three members of human P450 family 3A, which are expressed in liver and other tissues, also catalyzed steroid 6 beta-hydroxylation as their major activity but, additionally, formed several minor products that include 2 beta-hydroxy and 15 beta-hydroxy derivatives in the case of testosterone. These patterns are similar to those exhibited by rat family 3A P450s. Although several rodent P450s belonging to subfamilies 2A, 2B, 2C, 2D are active steroid hydroxylases, four of five human P450s belonging to these subfamilies exhibited very low activity or were inactive, as were the human 1A and 2E P450s examined in the present study. These studies demonstrate that individual human cytochrome P450 enzymes can hydroxylate endogenous steroid hormones with a high degree of stereospecificity and regioselectivity, and that some, but not all of the human cytochromes exhibit metabolite profiles similar to their rodent counterparts.
Article
To improve long-term expression of drug biotransformation activities in hepatocytes, we have examined the suitability of several epithelial-like cell lines (MDCK, MS and L-132) for supporting functional co-cultures with rat hepatocytes. Cells were selected on the basis of their compatibility with hepatocytes, formation of stable monolayers in the absence of serum and lack of drug biotransformation activities. The expression of individual elements of the biotransformation system was evaluated in these co-cultures. Co-cultured hepatocytes remained viable and showed a characteristic polygonal shape for more than a week. Depending on the cell line used, levels of aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase activities of co-cultured hepatocytes oscillated between 24-47% of their initial value after 4 days in culture. The highest levels of monooxygenase activity were found in hepatocytes co-cultured with MS cells (41-47%). In contrast, these activities decreased to 6% when hepatocytes were maintained in pure culture for the same period. The activities of the conjugating enzymes UDP-glucuronyltransferase and glutathione S-transferase were maintained at nearly the initial levels during the complete period of study, both in pure and mixed-cultures, regardless of the cell line used. MS cells adapted themselves much better to serum-free culture conditions, and the co-culture with rat hepatocyte was technically easier. After one week, total cytochrome P450 and reduced glutathione in rat hepatocytes/MS co-cultures were 31% and 127% respectively of the day O values, whereas they were undetectable in pure culture. A clear induction of monooxygenase activities by methylcholanthrene, phenobarbital and ethanol could be observed by the 5th day in MS cells/hepatocyte co-cultures. The fact that the results of our work show the suitability of MS cells, an epithelial-derived cell line, for improving the expression of biotransformation enzymes of cultured hepatocytes opens new possibilities of simplifying co-cultures for their use in drug-metabolism studies.
Article
The activity of human cytochrome P450 monooxygenases, aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase can be increased by 3-methylcholanthrene, phenobarbital and ethanol in human hepatocytes maintained in primary culture. Total cytochrome P450 content increased two-fold after 48 hr of incubation with methylcholanthrene or phenobarbital and 1.5-fold after incubation with ethanol. The three chemicals elicited different effects on cytochrome P450 dependent activities. Addition of 3-methylcholanthrene caused a time- and concentration-dependent increase in both monooxygenase activities, aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase, while phenobarbital and ethanol increased 7-ethoxycoumarin O-deethylase activity but had no effect on aryl hydrocarbon hydroxylase. Dexamethasone per se had little or no effect on either monooxygenase activities, but potentiated the effect of the three chemicals on 7-ethoxycoumarin O-deethylase.
Article
The relative importance of drug-induced liver disease assumes much significance in certain groups of patients such as the elderly. The majority of cases occur as unexpected reactions to a therapeutic dose of a drug. Factors affecting susceptibility to drug-induced liver disease are diverse and are discussed in this article.
Article
This article has no abstract; the first 100 words appear below. Drug-induced liver injury is a potential complication of nearly every medication that is prescribed, because the liver is central to the metabolic disposition of virtually all drugs and foreign substances.¹–³ Although drugs are usually metabolized without injury to the liver, many fatal and near-fatal drug reactions occur each year. A few compounds produce metabolites that cause liver injury in a uniform, dose-dependent fashion.⁴–⁶ Most agents form a toxic byproduct only in rare persons. Injury to hepatocytes results either directly from the disruption of intracellular function or membrane integrity or indirectly from immune-mediated membrane damage. Factors promoting the accumulation . . . Supported by the Houghton Foundation and the estate of Alison B. Harwood. I am indebted to Drs. Burton Combes and Ronald W. Estabrook for their critical insights and to Dr. Steve Foster for assistance with the photomicrographs. Source Information From the Liver Unit, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8887, where reprint requests should be addressed to Dr. Lee.
Article
The dealkylations of 7-ethoxy- and 7-pentoxyresorufin,p-nitrophenol hydroxylation, and regio- and stereoselective hydroxylation of testosterone were measured to study the stability and inducibility of cytochrome P450 activities in cultured human hepatocytes. The results showed that human hepatocytes in primary culture retain the ability to increase specific cytochrome P450 activities upon incubation with inducers. 3-Methylcholanthrene produced a strong increase (6- to 21-fold over control) in 7-ethoxyresorufin O-deethylase activity and a small enhancement (1.5- to 2.5-fold) of the p-nitrophenol hydroxylation rate. Incubation of cells with phenobarbital resulted in moderate increases in 7-pentoxyresorufin O-depentylation (1.5- to 2-fold) and in testosterone hydroxylation at 16 alpha (1.5- to 4.5-fold) and 16 beta (1.3- to 4-fold) positions. Ethanol specifically increased p-nitrophenol hydroxylase activity (1.5- to 3.5-fold) and reduced 15 beta- and 6 beta-hydroxylations of testosterone. Treatment of hepatocytes with dexamethasone produced an increase of almost all the activities studied, with 6 beta- (2- to 3-fold) and 16 beta-hydroxytestosterone (1.4- to 2.4-fold) formation showing the greatest enhancement. Clofibric acid exposure resulted in 1.5- to 3-fold increases in 7-pentoxyresorufin O-depentylase and in testosterone 6 beta- and 2 beta-hydroxylase activities. Isosafrol selectively increased 7-ethoxyresorufin O-deethylase activity (2- to 3-fold), and it moderately reduced the other activities studied.
Article
Diclofenac is a nonsteroidal anti-inflammatory drug approved in the United States in 1988 for the treatment of patients with osteoarthritis, rheumatoid arthritis, or ankylosing spondylitis. To characterize the clinical, biochemical, and histological features and possible mechanisms of hepatic injury associated with its use, a retrospective analysis was undertaken of 180 patients whose cases were reported to the Food and Drug Administration from November 1988 through June 1991, as having had possible adverse reactions to diclofenac. Of the reported 180 cases, 79% were female, 71% were 60 years of age or older, and 77% had osteoarthritis. Sixty-seven percent of the cases were detected by symptoms and the remainder by abnormal laboratory tests. Seventy-five percent of the symptomatic patients (90 of 120) were jaundiced. Seven of the 90 icteric patients died. The biochemical pattern of injury was hepatocellular or mixed hepatocellular in 66% of cases. Only 8% had a pattern of cholestatic injury. The remainder, with modestly increased values of both transaminases and alkaline phosphatase, were considered "indeterminate," i.e., either mild hepatocellular or anicteric "cholestatic" injury. Sections of liver from 21 cases were available for study. Hepatic injury was apparent by 1 month after starting the drug in 24%, by 3 months in 63%, and by 6 months in 85% of cases. The latent period in 12% was 6 to 12 months, whereas in 3% it was greater than 12 months.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Serious idiosyncratic hepatic injury has been associated with the use of many nonsteroidal antiinflammatory drugs, including the widely prescribed agent diclofenac. In order to investigate the possibility that covalent protein adducts of reactive metabolites of diclofenac might be responsible for the hepatotoxicity produced by this drug, we have developed a polyclonal antibody that can recognize such adducts in tissues. Immunoblotting revealed that protein adducts of reactive metabolites of diclofenac of 50, 70, 110, and 140 kDa were formed in the livers of mice treated with diclofenac. In the future, it will be determined whether these adducts can cause hepatotoxicity by either a hypersensitivity or metabolic mechanism. Similar approaches may be used to study the protein adducts and mechanisms of hepatotoxicity of other nonsteroidal antiinflammatory drugs.
Article
In a few patients diclofenac produces mild increases in serum aminotransferase activity and in rare cases may be associated with the occurrence of fulminant hepatic necrosis. Both direct toxic effects of a diclofenac metabolite and hypersensitivity reactions have been suggested as possible molecular mechanisms of liver injury. We investigated the pathways of bioactivation and cytotoxicity of diclofenac, which undergoes both aromatic hydroxylation and acyl glucuronidation, in short-term cultured rat hepatocytes. LDH release was first evident after 4 hr of incubation with diclofenac (> 500 microM). In addition, time- and concentration-dependent covalent binding of [14C]diclofenac to hepatocellular proteins occurred, indicating the presence of a reactive intermediate. To specifically explore the role of the acyl glucuronidation pathway in the induction of cytotoxicity and covalent drug-protein adducts, we used two inhibitors of the UDP-glucuronosyltransferase (UDPGT), borneol and 7,7,7-triphenylheptyl-UDP. LDH release was markedly increased in the presence of either UDPGT inhibitor. Alternatively, covalent binding to hepatocellular proteins was greatly reduced when the glucuronide formation was selectively blocked. Furthermore, in vitro inhibition of P450-dependent oxidative biotransformation with the selective inhibitor of the CYP2C subfamily sulfaphenazole or with cimetidine markedly reduced the extent of cytotoxicity, whereas the degree of covalent adduct formation remained unchanged. Similarly, pretreatment of the rats with phenobarbital (80 mg/kg/day for 4 days) delayed the onset and reduced the extent of diclofenac-induced LDH release. Collectively, these results indicate that the formation of a toxic diclofenac metabolite(s) catalyzed by P4502C in hepatocytes leads to acute lethal cell injury, whereas diclofenac acyl glucuronide formation is associated with covalent binding of a reactive metabolite to hepatocellular proteins that is not related to the acute cytotoxicity. The protein adduct formation and its modulation by UDPGT may, however, be toxicologically relevant for the expression of diclofenac hepatitis.
Article
Human hepatocytes stimulated with human recombinant hepatocyte growth factor (h-rHGF) (10 ng/mL) displayed a characteristic lag period before entering into the S phase. The duration of this delay was dependent on the timing of h-rHGF addition to cultures. The highest peak of DNA synthesis was observed at 120 hours of culture when hepatocytes were stimulated with h-rHGF at 72 hours of culture. This was accompanied by an early peak of c-jun and c-fos synthesis (3 hours after addition of h-rHGF) followed by c-myc (6 hours) and increased expression of cyclins A, B, D, and E (12 hours after h-rHGF). A significant dose-dependent increase in inositol 1,4,5-P3 was observed within 45 seconds after stimulation with the factor. This was followed by an immediate increase in the cytosolic-free calcium. Cyclic adenosine monophosphate (cAMP) levels did not change after stimulation with the factor. Tyrosine phosphorylation seems to be an early event in the course of the stimulatory effect of h-rHGF on DNA synthesis of hepatocytes; genistein, a tyrosine kinase inhibitor, impaired the stimulatory effect of h-rHGF on DNA synthesis dose dependently. On the other hand, the action of the factor was negatively regulated by protein kinase C activation, as shown by the increased stimulatory effect of h-rHGF on DNA synthesis upon inhibition of protein kinase C by H7.
Article
The stability of cytochrome P450 enzymes, cytochrome b5, and NADPH-cytochrome c reductase was examined in (A) human liver samples frozen in liquid nitrogen and stored at -80 degrees C, (B) human liver microsomes suspended in 250 mM sucrose and stored at -80 degrees C, and (C) human liver microsomes subjected to as many as 10 cycles of thawing and freezing. In study A, microsomes from five human livers were prepared from fresh (unfrozen) tissue and from tissue that was stored frozen at -80 degrees C for 1, 2, 4, or 6 months. The apparent concentration of cytochromes P450 and b5 and the activity of NADPH-cytochrome c reductase decreased 20-40% as a result of freezing the liver, regardless of whether the liver was stored for 1 or 6 months. Similar decreases were observed in the activities of cytochrome P450 enzymes belonging to several gene families, namely CYP1A2 (7-ethoxyresorufin O-dealkylation and caffeine N3-demethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide methylhydroxylation), CYP2C19 (S-mephenytoin 4'- hydroxylation), CYP2D6 (dextromethorphan O-de-methylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4solidus5 (testosterone 6beta-hydroxylation), and CYP4A9solidus11 (lauric acid 12-hydroxylation). Freezing human liver did not convert cytochrome P450 to its inactive form, cytochrome P420, but it increased the contamination of liver microsomes with hemoglobin or other heme-containing proteins, which resulted in a uniform decrease in the specific activity of cytochromes P450 and b5 and in the specific activity of all P450 enzymes. In study B, the concentration of cytochromes P450 and b5, the activity of NADPH-cytochrome c reductase, and the activity of individual cytochrome P450 enzymes were determined in 10 samples of human liver microsomes stored at -80 degrees C for approximately 0, 1, or 2 years. The sample-to-sample variation in the concentration and activity of cytochrome P450, cytochrome b5, and NADPH-cytochrome c reductase was nominally affected by long-term storage of human liver microsomes at -80 degrees C, indicating there was no differential loss of cytochrome P450 activity, cytochrome b5 concentration, or NADPH-cytochrome c reductase activity. In study C, microsomes from a pool of human livers were subjected to 1, 2, 3, 5, 7, or 10 cycles of freezing at -80 degrees C followed by thawing at room temperature. Freezing/thawing liver microsomes for up to 10 cycles did not convert cytochrome P450 to P420, nor did it cause significant loss of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5, or CYP4A9/11 activity. Overall, these results suggest that our current methods for storing and processing human liver are well suited to preserving microsomal P450 enzyme activity.
Article
Metabolism of the new nonsteroidal antiinflammatory drug aceclofenac ([2-(2',6'-dichlorophenylamino)phenyl]acetoxyacetic acid) was investigated both in the in vitro hepatic human models and in vivo. Aceclofenac is metabolized in human hepatocytes and human microsomes to form [2-(2',6'-dichloro-4'-hydroxy- phenylamino)phenyl]acetoxyacetic acid as the major metabolite, which is then further conjugated. Minor metabolites were [2-(2',6'-dichlorophenylamino)-5-hydroxyphenyl]acetoxyacetic acid and [2-(2',6'-dichlorophenylamino)phenyl]acetic acid, as well as the hydroxylated derivatives [2-(2',6'-dichloro-4'- hydroxyphenylamino)phenyl]acetic acid and [2-(2',6'-dichlorophenylamino)- 5-hydroxyphenyl]acetic acid. After oral administration to human volunteers (100 mg, single dose), aceclofenac reached a Cmax value of 7.6 +/- 1.3 micrograms/ml and a tmax of 2.6 +/- 1.8. The same metabolites as those detected in cell culture or microsome incubations were found in 12-hr urine after an oral administration of 100 mg aceclofenac to human volunteers. Cytochrome 2C9 is the enzyme responsible for the hydroxylation at position 4'. This could be demonstrated by: 1) selective inhibition by sulfaphenazole; 2) correlation between the formation of the hydroxylated metabolite and tolbutamide hydroxylase activity; and 3) formation of this metabolite only when incubated with microsomes obtained from cells expressing human cytochrome 2C9. However, no conclusive information could be obtained concerning the cytochrome catalyzing the hydroxylation at position 5. The comparison between human microsomes and human hepatocytes metabolism on one hand, and human in vivo metabolism on the other, supports human hepatocytes in primary culture as the model that best anticipated the metabolism of the drug in vivo.
Article
Aceclofenac ([2-(2',6'-dichlorophenylamino)phenyl]acetoxyacetic acid) is a novel nonsteroidal antiinflammatory drug, the pharmacokinetics and drug metabolism of which show species differences. After oral administration to the rat, circulating aceclofenac rapidly disappears yielding [2-(2',6'-dichlorophenylamino)phenyl]acetic acid (diclofenac), which is then further oxidized to [2-(2',6'-dichloro-4'-hydroxyphenylamino)phenyl[acetic acid (4'-hydroxydiclofenac) and [2-(2',6'-dichloro-4'-hydroxyphenylamino)phenyl]acetic acid (4'-hydroxydiclofenac) and [2-(2',6'-dichlorophenylamino)-5-hydroxyphenyl]acetic acid (5-hydroxydiclofenac). This is a minor route in humans, wherein aceclofenac is hydroxylated to [2-(2',6'-dichloro-4'-hydroxyphenylamino)phenyl]acetoxyacetic acid (4'-hydroxyaceclofenac), which becomes the major metabolite. In the monkey, the conversion of aceclofenac to diclofenac takes place, but to a much lesser extent than in the rat, and the 4'-hydroxylated metabolites from both compounds are found in monkeys' urine. The mechanistic basis for this species-dependent variations seems to be the different stability of the drug toward liver esterases. In the rat, the most efficient aceclofenac-hydrolyzing activity is found in hepatic microsomes (Vmax = 2113 +/- 177 pmol/min/mg protein and KM = 191 +/- 40 microM) and cytosol (Vmax = 479 +/- 37 pmol/min/mg protein and KM = 75 +/- 22 microM). Consequently, incubation of aceclofenac with cultured rat hepatocytes or in the rat in vivo results in a rapid hydrolysis of the drug, followed by oxidative metabolism of the resulting diclofenac, yielding 4'- and 5-hydroxylated derivatives as the major metabolites. In contrast, the aceclofenac ester bond is much more stable toward human hepatic microsomal (Vmax = 27 +/- 10 pmol/min/mg protein and KM = 792 +/- 498 microM) and cytosolic (Vmax = 87 +/- 5 pmol/min/mg protein and KM 218 +/- 30 microM) esterases, and 4'-hydroxyaceclofenac becomes the major metabolite in cultured human hepatocytes, as well as in human urine. The research presented herein also illustrates the suitability of cultured human hepatocytes for predicting aceclofenac metabolism in humans.
Article