Cell-based models to predict human hepatotoxicity of drugs / Modelos celulares para predecir la hepatotoxicidad humana de fármacos

Drug-induced liver injury is a significant leading cause of liver disease and post-market attrition of approved drugs. Several hepatic cell-based models have been used for early safety risk assessment during drug development. Their capacity to predict hepatotoxicity depends on cells' functional performance. Cultured hepatocytes have contributed to increase knowledge of the metabolic patterns and mechanisms involved in drug toxicity. A major limitation of monolayer hepatocytes is that they undergo rapid loss of hepatic functionality over time, particularly drug metabolising capability. The sandwich culture model promotes polarised cell surface and stabilises hepatocyte functionality, particularly transport systems, better than monolayer cultures. As 3D spatial organisation and complex heterotypic cell interactions are essential for the functional homeostasis of the liver, hepatocyte models (3D cultures, co-cultures with NPCs and microfluidic systems) that mimic cell-cell, cell- matrix interactions and nutrient flow characteristic of the liver microenvironment have been shown to improve the metabolic competency of hepatocytes and have been proposed for better in vitro predictions of drug hepatotoxicity. In addition to hepatocytes, other cell-based models have been proposed for hepatotoxicity studies. Hepatoma cell lines are metabolically poor compared to hepatocytes, but offer key advantages, such as unlimited life span, reproducibility, high availability and easy handling, which make them useful for screening purposes. Alternatively, hepatic cell lines engineered for stable or transient expression of key drug-metabolising enzymes have also been used. Finally, stem cell-derived hepatocytes are emerging in vitro systems that would provide a stable source of hepatocytes from individuals with highly valuable particular polymorphic characteristics for preclinical drug metabolism and toxicity prediction of new drugs (AU)

La lesión del hígado por fármacos es una de las causas principales de enfermedad hepática y de retirada del mercado de fármacos autorizados. Son varios los modelos de células hepáticas utilizados durante el desarrollo de fármacos para la valoración temprana de su seguridad. Los estudios basados en hepatocitos cultivados han contribuido al conocimiento de los mecanismos implicados en la toxicidad por fármacos. Una limitación fundamental de los hepatocitos cultivados en monocapa es la pérdida temprana de funciones hepáticas, en particular la capacidad para metabolizar fármacos. El cultivo tipo sándwich mantiene la polaridad de los hepatocitos y los sistemas de transporte y estabiliza su funcionalidad mejor que el cultivo en monocapa. Puesto que la organización espacial 3D y las interacciones celulares heterotípicas son esenciales para la homeostasis funcional del hígado, los hepatocitos cultivados en sistemas que reproducen las interacciones entre células, célula-biomatriz y el flujo de nutrientes característicos del microambiente hepático (cultivos 3D, co-cultivos con células no parenquimales, sistemas microfluidicos) presentan mayor capacidad metabólica y han sido propuestos para mejorar la predicción in vitro de la hepatotoxicidad. Otras células hepáticas han sido propuestas como alternativa a los hepatocitos para evaluar la hepatotoxicidad. Si bien las líneas celulares de hepatoma tienen menor capacidad metabólica que los hepatocitos, presentan ventajas clave para el cribado de fármacos (vida ilimitada, reproducibilidad, gran disponibilidad, fácil manejo). También se utilizan células manipuladas para la expresión estable o transitoria de enzimas de biotransformación. Por último, los hepatocitos procedentes de células madre son sistemas in vitro emergentes que proporcionarían una fuente estable de hepatocitos, a partir de individuos con características polimórficas especiales, sumamente valiosa para la predicción preclínica de la toxicidad de nuevos fármacos (AU)

The thromboembolic risk in giant cell arteritis: a critical review of the literature.

Giant cell arteritis is a systemic vasculitis characterized by granulomatous inflammation of the aorta and its main vessels. Cardiovascular risk, both for arterial and venous thromboembolism, is increased in these patients, but the role of thromboprophylaxis is still debated. It should be suspected in elderly patients suffering from sudden onset severe headaches, jaw claudication, and visual disease. Early diagnosis is necessary because prognosis depends on the timeliness of treatment: this kind of arteritis can be complicated by vision loss and cerebrovascular strokes. Corticosteroids remain the cornerstone of the pharmacological treatment of GCA. Aspirin seems to be effective in cardiovascular prevention, while the use of anticoagulant therapy is controversial. Association with other rheumatological disease, particularly with polymyalgia rheumatica is well known, while possible association with antiphospholipid syndrome is not established. Large future trials may provide information about the optimal therapy. Other approaches with new drugs, such as TNF-alpha blockades, Il-6 and IL-1 blockade agents, need to be tested in larger trials.

In vitro evaluation of potential hepatotoxicity induced by drugs.

The liver is the most important target for toxicity caused by drugs. This vulnerability is a consequence of the functional features of the liver and their role in the metabolic elimination of most drugs. Therefore, evaluation of potential hepatotoxicity represents a critical step in the development of new drugs. The liver is very active in metabolising foreign compounds and, although biotransformation reactions generally parallel detoxification processes, the formation of reactive metabolites is relatively frequent. Thus, drug-induced hepatotoxicity can be due to the administered compound itself or to metabolites formed by hepatic metabolism. The most important systems to study hepatotoxicity and metabolic activity in vitro are liver slices, isolated liver cells in suspensions or in primary cultures including co-culture methods and special 3D techniques, various subcellular fractions and hepatic cell lines. These models can be used for cytotoxicity and genotoxicity screening, and also to identify the mechanisms involved in drug-induced hepatotoxicity. Assessment of current cytotoxicity and hepatic-specific biochemical effects are limited by the inability to measure a wide spectrum of potential mechanistic changes involved in the drug-induced toxic injury. A convenient selection of end-points allows a multiparametric evaluation of drug toxicity. In this regard, omic (cytomic, metabonomic, proteomic and toxicogemic) approaches help defining patterns of hepatotoxicity for early identification of potential adverse effects of the drug to the liver. The development of robust in vitro-based multiparametric screening assays covering a wider spectrum of key effects will heighten the predictive capacity for human hepatotoxicity, and accelerate the drug development process.

Functional characterization of hepatocytes for cell transplantation: customized cell preparation for each receptor.

The first indication of hepatocyte transplantation is inborn liver-based metabolic disorders. Among these, urea cycle disorders leading to the impairment to detoxify ammonia and Crigler-Najjar Syndrome type I, a deficiency in the hepatic UDP-glucuronosyltransferase 1A1 present the highest incidence. Metabolically qualified human hepatocytes are required for clinical infusion. We proposed fast and sensitive procedures to determine their suitability for transplantation. For this purpose, viability, attachment efficiency, and metabolic functionality (ureogenic capability, cytochrome P450, and phase II activities) are assayed prior to clinical cell infusion to determine the quality of hepatocytes. Moreover, the evaluation of urea synthesis from ammonia and UDP-glucuronosyltransferase 1A1 activity, a newly developed assay using beta-estradiol as substrate, allows the possibility of customizing cell preparation for receptors with urea cycle disorders or Crigler-Najjar Syndrome type I. Sources of human liver and factors derived from the procurement of the liver sample (warm and cold ischemia) have also been investigated. The results show that grafts with a cold ischemia time exceeding 15 h and steatosis should not be accepted for hepatocyte transplantation. Finally, livers from non-heart-beating donors are apparently a potential suitable source of hepatocytes, which could enlarge the liver donor pool.

Potential hepatoprotective effects of new Cuban natural products in rat hepatocytes culture.

The protective effects of five Cuban natural products (Mangifera indica L. (MSBE), Erythroxylum minutifolium, Erythroxylum confusum, Thalassia testudinum and Dictyota pinnatifida extracts and mangiferin) on the oxidative damage induced by model toxicants in rat hepatocyte cultures were studied. Cells were pre-incubated with the natural products (5-200 microg/mL) for 24 h. Then hepatotoxins (tert-butyl hydroperoxide, ethanol, carbon tetrachloride and lipopolysaccharide) were individually added and post-incubated for another 24 h. After treatments, cell viability was determined using the MTT assay. Mangiferin and MSBE exhibited the highest cytoprotective potential (EC50 between 50 and 125 microg/mL), followed by T. testudinum and Erythroxylum extracts, whereas no significant protective effects was produced by Dictyota extract treatment. Antioxidant properties of the natural products against lipid peroxidation and GSH depletion induced by tert-butyl hydroperoxide were then investigated. The results show that at 36 h pre-treatment of cells with mangiferin or MSBE, concentrations of T. testudinum and Erythroxylum extracts ranging from 25 to 100 microg/mL significantly inhibited lipid peroxidation induced by tert-butyl hydroperoxide (100 and 250 microM) and increased the GSH levels reduced by the toxicant. D. pinnatifida inhibited lipid peroxidation, but did not preserve GSH levels. In conclusion, MSBE, E. minutifolium, E. confusum and T. testudinum extracts and mangiferin showed hepatoprotective activity against induced damage in all the experimental series, where mangiferin and the extracts of MSBE and T. testudinum were the best candidates to inhibit "in vitro" damage to rat hepatocytes. This hepatoprotective effect found could be associated with the antioxidant properties observed for the products.

Desarrollo, análisis y optimización de modelos celulares hepáticos para estudios de fármacotoxicología y terapia celular / Development and optimization of hepatic cell models for pharmaco-toxicology and cell therapy research

El hígado juega un papel fundamental en el metabolismo de medicamentos y enel mantenimiento de la homeostasis del organismo y, por tanto los modelos celulareshepáticos desempeñan un papel clave para estudios fármaco-toxicológicos ymás recientemente en el campo de la terapia celular. Sin embargo, la limitada disponibilidadde hepatocitos viables y funcionales, debido a la falta de tejido hepático,es la principal limitación para utilizar estos recursos celulares. El objetivo delpresente trabajo se ha basado en el desarrollo y caracterización de modelos celulareshepáticos que puedan constituir una alternativa a los hepatocitos para estetipo de aplicaciones. Para ello se han abordado tres estrategias diferentes: 1) optimizacióndel proceso de obtención de hepatocitos a partir de hígados enterosdescartados para transplante, determinando las condiciones adecuadas para elaislamiento y cultivo de hepatocitos; 2) caracterización funcional de las células delhepatoblastoma HepG2, y 3) desarrollo de un protocolo para inducir la diferenciaciónhepatogénica de células madre mesenquimales adultas derivadas de tejidoadiposo (ADSC). Para conseguir un buen aprovechamiento de hígados descartadospara transplante resulta necesario optimizar los protocolos de aislamiento y criopreservaciónde hepatocitos. El estudio con células madre adultas se presenta comouna alternativa muy válida para la obtención de hepatocitos-like viables y funcionalmenteactivos, útiles a corto plazo en estudios de fármaco-toxicología y en unfuturo para terapia celular hepática. El uso de células madre abre un gran abanicode posibilidades, facilitando el establecimiento de un modelo celular diferenciadoadulto con características que otros modelos celulares, como son el hepatomahumano HepG2, no presentan. No obstante, es necesario adquirir un mayor conocimientode los mecanismos celulares y moleculares que controlan la transdiferenciacióna hepatocitos

Given the importance of the liver in the metabolism and maintenance of thehomeostasis of the organism, many studies have been conducted in the area oftoxicology and, more recently, in hepatic cellular therapy. However, the maindrawback is the limited availability of viable and functional hepatocytes due tothe scarcity of liver tissue. The purpose of this work was based on the developmentand characterization of hepatic cellular models to become an alternative tohepatocytes in toxicology studies and cellular therapy. To this end, three mainobjectives have been investigated: 1) to adopt a procedure of hepatocyte isolationfrom discarded organs for transplantation which determines the optimal conditions for the isolation and culture of hepatocytes, 2) to characterize the cells from thehepatoblastome HepG2, and 3) to develop a hepatogenic differentiation protocolto induce the hepatic differentiation in adipose-derived stem cells (ADSC). Inparticular, the hepatogenic differentiation of stem cells opens a wide range ofpossibilities to facilitate the establishment of an adult differentiated cellular modeluseful for pharmaco-toxicological studies and for hepatic cellular therapy. The useof adult stem cells may allow the establishment of an adult cellular model withproperties that others cellular models, like HepG2, do not show. However, it isnecessary to optimise the isolation and cryopreservation procedures, as well as thedifferentiation protocols from adult stem cells and try to acquire a wide knowledgeof the cellular and molecular mechanisms that control the transdifferentiation tohepatocytes

Interactions of polyphenols with the P450 system: possible implications on human therapeutics.

Polyphenols are a family of natural compounds with many biological properties. This review focuses on their potential interaction on the cytochrome P450 system. Effects of phenolic acids, anthocyanins, stilbenes, catechins and other flavonoids on the drug metabolising function are revised. Their daily intake and presence in herbal medicines justify the study of potential drug-interaction to prevent undesirable clinical consequences.

Cell lines: a tool for in vitro drug metabolism studies.

Primary cultured hepatocytes are a valuable in vitro model for drug metabolism studies. However, their widespread use is greatly hindered by the scarcity of suitable human liver samples. Moreover, the well-known in vitro phenotypic instability of hepatocytes, the irregular availability of fresh human liver for cell harvesting purposes, and the high batch-to-batch functional variability of hepatocyte preparations obtained from different human liver donors, seriously complicate their use in routine testing. To overcome these limitations, different cell line models have been proposed for drug metabolism screening. Human liver-derived cell lines would be ideal models for this purpose given their availability, unlimited life-span, stable phenotype, and the fact that they are easy to handle. However, the human hepatoma cells currently used (i.e. HepG2, Mz-Hep-1) show negligible levels of drug-metabolizing and do not constitute a real alternative to primary hepatocytes. Different strategies have been proposed to generate metabolically competent immortalized hepatocytes (transformation of human hepatocytes with plasmids encoding immortalizing genes, hepatocyte-like cells derived from stem cells, cell lines generated from transgenic animals, hepatocyte/hepatoma hydrid cells). Moreover, recombinant models heterologously expressing P450 enzymes in different host cells have been developed and successfully used in drug metabolism testing. In addition, new strategies have recently been explored to upregulate the expression of drug-metabolizing enzymes in cell lines of a human origin (i.e. transfection with expression vectors encoding key hepatic transcription factors). Among metabolic-based drug-drug interactions, P450 inhibition seems to be the most important. A major application of recombinant models expressing a single P450 is the screening of potential enzyme inhibitors. Therefore, pharmaceutical companies increasingly make use of cell lines to speed up the selection of new drugs with favourable pharmacokinetic and metabolic properties.

Strategies to in vitro assessment of major human CYP enzyme activities by using liquid chromatography tandem mass spectrometry.

At the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single candidate can be identified for development. Determining the role of CYP enzymes in the metabolism of a compound and evaluating the effect of NCEs on human CYP activities are key issues in pharmaceutical development as they may explain inter-subject variability, drug-drug interactions, non-linear pharmacokinetics and toxic effects. Reliable methods for determining enzyme activities are needed to characterize an individual CYP enzyme and to obtain a tool for the evaluation of its role in drug metabolism in humans. Different liquid chromatography tandem mass spectrometry methodologies have been developed for the fast and routine analysis of major in vivo and in vitro CYPs enzyme activities. The high sensitivity and selectivity of mass spectrometry allow traditional assays to be minimized, thus saving time, efforts and money. Therefore this technology has become the method of choice for the fast assessment of CYP enzyme activities in early drug discovery development. Our intention herein is to review the most recent approaches that have been developed to quickly assess CYPs activities using in vitro models and liquid chromatography coupled with mass spectrometry, as well as their application in early drug discovery.

Modulation of P450 enzymes by Cuban natural products rich in polyphenolic compounds in rat hepatocytes.

This paper reports cytotoxic effects and changes in the P450 system after exposing rat hepatocytes to four polyphenol-rich products widely used in Cuban traditional medicine (Mangifera indica L. (MSBE), Thalassia testudinum (Tt), Erythroxylum minutifolium and confusum extracts). Effects of mangiferin, the main polyphenol in MSBE, were also evaluated. Cytotoxicity was assayed by the MTT test after exposure of cells to the products (50-1000 microg/mL) for 24 or 72 h. The results showed that 500 microg/mL MSBE was moderately cytotoxic after 72 h, while mangiferin was not. Marked reductions in cell viability were produced by Erythroxylum extracts at concentrations > or = 200 microg/mL, whereas only moderate effects were induced by 1000 microg/mL Tt. Seven specific P450 activities were evaluated after 48 h exposure of cells to the products. MSBE reduced phenacetin O-deethylation (POD; CYP1A2) activity in a concentration-dependent manner (IC(50)=190 microg/mL). No decreases were observed in other activities. In contrast, mangiferin produced reductions in five P450 activities: IC(50) values of 132, 194, >200, 151 and 137 microg/ml for POD (CYP1A2), midazolam 1'-hydroxylation (M1OH; CYP3A1), diclofenac 4'-hydroxylation (D4OH; CYP2C6), S-mephenytoin 4'-hydroxylation (SM4OH), and chlorzoxazone 6-hydroxyaltion (C6OH; CYP2E1), respectively. E. minutifolium, E. confusum and Tt extracts produced small reductions in SM4OH and C6OH activities, but no significant changes were noted in the other P450 activities. On the other hand, all the products increased the benzyloxyresorufin O-debenzylation (BROD; CYP2B1) activity, with MSBE, mangiferin or E. minutifolium showing the highest effects (about 2-fold over control). Our results showed in vitro effects of these natural products on P450 systems, possibly leading to potential metabolic-based interactions.

Effects of Mangifera indica L. aqueous extract (Vimang) on primary culture of rat hepatocytes.

Vimang is an aqueous extract from stem bark of Mangifera indica L. (Mango) with pharmacological properties. It is a mixture of polyphenols (as main components), terpenoids, steroids, fatty acids and microelements. In the present work we studied the cytotoxic effects of Vimang on rat hepatocytes, possible interactions of the extract with drug-metabolizing enzymes and its effects on GSH levels and lipid peroxidation. No cytotoxic effects were observed after 24 h exposure to Vimang of up to 1000 microg/mL, while a moderate cytotoxicity was observed after 48 and 72 h of exposure at higher concentrations (500 and 1000 microg/mL). The effect of the extract (50-400 microg/mL) on several P450 isozymes was evaluated. Exposure of hepatocytes to Vimang at concentrations of up to 100 microg/mL produced a significant reduction (60%) in 7-methoxyresorufin-O-demethylase (MROD; CYP1A2) activity, an increase (50%) in 7-penthoxyresorufin-O-depentylase (PROD; CYP2B1) activity, while no significant effect was observed with other isozymes. To our knowledge, this is the first report regarding the modulation of the activity of the P450 system by an extract of Mangifera indica L. The antioxidant properties of Vimang were also evaluated in t-butyl-hydroperoxide-treated hepatocytes. A 36-h pre-treatment of cells with Vimang (25-200 microg/mL) strongly inhibited the decrease of GSH levels and lipid peroxidation induced by t-butyl-hydroperoxide dose- and time-dependently.

Determination of major human cytochrome P450s activities in 96-well plates using liquid chromatography tandem mass spectrometry.

At the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single candidate can be identified for development. Evaluation of the effect of NCEs on human CYP450 enzyme activities is a key issue in pharmaceutical development as it may explain inter-subject variability, drug-drug interactions, non-linear pharmacokinetics and toxic effects. A liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method has been developed for the fast and routine analysis of major human CYP450s enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in primary hepatocyte cell cultures. The high sensitivity and selectivity of mass spectrometry has allowed traditional assays to be minimized, thus enabling the use of 96-well plate format which markedly reduced the number of hepatocytes needed for each cytochrome CYP450 activity measurement, a fact that is particularly critical concerning human hepatocytes.

Effects of steatosis on drug-metabolizing capability of primary human hepatocytes.

The suitability of liver grafts discarded for transplantation because of macrosteatosis for preparing human hepatocyte cultures for in vitro drug metabolism studies has been examined. Lower cell viability and yield of isolation procedure were obtained from fatty livers (>40% steatosis) with respect to normal tissue. Significant reductions in 7-ethoxycoumarin O-deethylation (ECOD) and testosterone oxidations were found in hepatocytes prepared from steatotic livers. The potential impact of lipid accumulation on P450 enzymes was studied in vitro by incubation of cultured hepatocytes with long chain free fatty acids (FFA). Treatment of cells with 0.25-3mM FFA induced dose-dependent accumulation of lipids in the cytosol. Decreased ECOD and testosterone oxidation were found after 14h of exposure to 1mM or 2mM FFA (about 60-70% and 30-60% of control, respectively). The effects of fat-overloading on individual P450s were analyzed both at activity and mRNA level. CYP1A2, CYP2C9, CYP2E1 and CYP3A4 activities were reduced after hepatocyte incubation with 1mM (to 45-65% of control) or 2mM (to 20-50%) FFA for 14h. Reductions in P450 transcripts were also found in hepatocytes treated with 1mM FFA. Our findings showed a general down-regulation of P450s involved in drug metabolism in fat-overloaded hepatocytes. The results suggest that, despite their reduced P450 function, human hepatocytes obtained from donors with steatosis are metabolically competent and could be used for drug metabolism studies.

In vitro ADME medium/high-throughput screening in drug preclinical development.

The study of the ADME features of the huge number of new chemical entities (NCEs) produced mainly by combinatorial chemistry has become a bottleneck in the drug development process. In response the pharmaceutical industry is involved in the development of new medium/high-throughput screening capabilities. The aim of this paper is to review some of the available in vitro ADME systems adapted to screening requirements together with the technological approaches which can be linked to medium/high-throughput molecular screening.

Cryopreservation of rat, dog and human hepatocytes: influence of preculture and cryoprotectants on recovery, cytochrome P450 activities and induction upon thawing.

Several cryopreservation protocols for hepatocytes have been proposed over the past few years, but their effectiveness varies greatly as a function of the characteristics of the method used. One factor in the success of cryopreservation is the quality of cells before freezing. The results suggest that the cryopreservation of hepatocytes in a medium containing polyvinylpyrrolidone (PVP), in addition to DMSO, constitutes a convenient means of long-term storage of hepatocytes for preparing primary cultures to be used in drug metabolism studies. The combined use of the two cryoprotectants is particularly critical for low-viability cell suspensions. An interesting alternative to increase cell viability is the preculture of hepatocytes before cryopreservation. By the use of this procedure, high-quality cells, estimated in terms of post-thaw recovery, viability, adaptation of hepatocytes to culture, drug-metabolizing capability and cytochrome P450 induction, are obtained. Therefore, cryopreserved hepatocytes can provide a regular source of metabolically competent cells for in vitro investigations of the metabolic profile of new drugs and drug-drug interactions in pharmaco-toxicological research.

[Risk management project: reactive or proactive approach?]. / Progetto Risk Management: metodologia reattiva o proattiva?

Risk management in healthcare refers to the process of developing strategies aimed at preventing and controlling the risk of occurrence of errors and harmful events. The final objective is primarily that of increasing patient safety and secondarily, that of reducing the financial burden of adverse events. The implementation of a risk management system is therefore of vital strategic importance. Nevertheless, a fundamental question that needs to be answered in the operational phase is: should a proactive or reactive approach to risk management be taken? In our view, proactive risk management has many advantages over a reactive approach and is therefore preferable. The reactive approach should be taken exclusively to obtain information regarding risk and errors, in the preliminary, as well as monitoring and follow-up phases of the project.

The immunosuppressant drug FK506 prevents Fas-induced apoptosis in human hepatocytes.

FK506 is a potent immunosuppressive drug used for the prevention of graft rejection in organ transplantation. Experimental and clinical studies have shown correlations between apoptosis and graft rejection, and apoptosis also plays a role in cell death after ischemia-reperfusion injury in the rat liver. Fas-mediated apoptosis is very likely involved in allograft rejection and experimental evidence has shown a decrease of FasR expression in mouse hepatocytes produced by the drugs. On the basis of these findings we have investigated the protective effect of FK506 in comparison with cyclosporine A (CsA) on Fas-induced apoptosis, by analysing the activation of downstream effector caspases in human hepatocytes. Apoptosis was induced by treatment with agonistic antibodies against FasR, which resulted in a significant activation of caspase-3 after 12 h. Prevention of the downstream activation of the caspase cascade and apoptosis was observed when hepatocytes were pre-treated for 3 h with immunosuppressant drugs. A significant reduction (ca. 30-40%) of caspase-3 activation by 5 microM FK506 and CsA was observed. Along with less activation of caspase-3 a decrease of apoptotic DNA fragmentation was found. In addition, FK506 significantly reduced not only caspase-8 but also caspase-9 activation, to a similar extent as CsA, thus suggesting a protective effect at the mitochondrial level of this drug, as has already been reported for CsA. These effects of FK506 help to explain its strong anti-rejection properties and suggest promising benefits of pharmacological preconditioning on ischemia-reperfusion injury following liver transplantation.

Human hepatocytes in primary culture: the choice to investigate drug metabolism in man.

Different types of hepatic tissue, including whole or split livers from organ donors or waste liver from therapeutic liver resections, are used to prepare human hepatocyte cultures. Characteristics of liver samples from different origins (gender, age, healthy/pathological status, xenobiotic treatment) as sources of human hepatocytes are key factors which notably determine viability and functionality of hepatocytes. The characterisation of the CYP system can be assessed in terms of activity (using specific substrates/inhibitors), protein (antibody analysis) and molecular biology-based mRNA amplification techniques (PCR technology and DNA microarrays). It could reasonably be considered that human hepatocytes reflect the heterogeneity of CYP expression in human liver and is a suitable model for drug metabolism studies. Several key issues need to be addressed at the early stages of drug development to better select drug candidates (metabolic profile and rate, identification of CYPs involved, drug-drug interactions due to enzyme induction/inhibition). The metabolic stability and metabolite profile of new chemicals can be easily investigated by incubating the drugs with fully competent metabolic models like hepatocyte suspensions or 24 h-cultured hepatocytes. CYP inhibitory effects are usually screened in recombinant CYP enzymes or microsomes, however, the actual concentration of substrate and inhibitor available to the CYP enzyme depends on processes missing in subcellular models (transport mechanisms, cytosolic enzymes, binding to intracellular proteins). Since intact cells more closely reflect the environment to which drugs are exposed in the liver, cultured hepatocytes constitute a more predictive model for drug-drug interactions. Screening of CYP inducers cannot be done in microsomes as it requires a cellular system fully capable of expressing CYP genes. Primary hepatocytes are still the unique in vitro model for global examination of inductive potential of drugs (monitored as increases in mRNA content or activity).

Functionality of cultured human hepatocytes from elective samples, cadaveric grafts and hepatectomies.

The major possible sources of human liver for hepatocyte isolation are elective liver biopsies, cadaveric liver grafts and therapeutic liver resections. The suitability in terms of metabolic-competent hepatocyte cultures and risk/benefit of these resources has been comparatively studied. To this end, viability of isolated hepatocytes, yield of isolation procedure, hepatocyte survival during culture and CYP activities were the parameters analysed. The best results were found in hepatocytes prepared from elective biopsies, whereas a marked reduction in viability and functional competence was seen in hepatocytes from hepatectomy samples. Metabolic differences were observed in total CYP oxidative metabolism (7-ethoxycoumarin O-deethylation, total testosterone hydroxylation), as well as in CYP3A4, CYP2C9 or CYP2C19 activities (testosterone oxidations at 6beta-, 16beta- and 17-positions, respectively). Vascular control during the hepatectomy procedure influenced hepatocyte functionality: higher CYP activities were found in hepatocytes isolated from samples obtained under non-ischemic conditions or continuous vascular clamping than in those obtained under intermittent vascular clamping. In addition to cellular functionality, other criteria such as sample availability or ethical aspects should be considered. Elective biopsies have low, but not absent, surgical risk. However, the better functionality and the higher accessibility of elective liver samples in comparison to the other groups suggest this source of liver tissue as the most appropriate for cell harvesting purposes.

Human hepatocytes as a tool for studying toxicity and drug metabolism.

Drugs are usually biotransformed into new chemical species that may have either toxic or therapeutic effects. Drug metabolism studies are routinely performed in laboratory animals but, due to metabolic interspecies differences when compared to man, they are not accurate enough to anticipate the metabolic profile of a drug in humans. Human hepatocytes in primary culture provide the closest in vitro model to human liver and the only model that can produce a metabolic profile of a given drug that is very similar to that found in vivo. However their availability is limited due to the restricted access to suitable tissue samples. The scarcity of human liver has led to optimising the cryopreservation of adult hepatocytes for long-term storage and regular supply. Human hepatocytes in primary culture express typical hepatic functions and express drug metabolising enzymes. Moreover, qualitative and quantitative similarities between in vitro and in vivo metabolism of drugs were observed. Different strategies have been envisaged to prolong cell survival and delay the spontaneous decay of the differentiated phenotype during culture. Thus, hepatocytes represent the most appropriate model for the evaluation of integrated drug metabolism, toxicity/metabolism correlations, mechanisms of hepatotoxicity, and the interactions (inhibition and induction) of xenobiotics and drug-metabolising enzymes. However, in view of limitations of primary hepatocytes, efforts are made to develop alternative cellular models (i.e. metabolic competent CYP-engineered cells stably expressing individual CYPs and transient expression of CYPs by transduction of hepatoma cells with recombinant adenoviruses). In summary, several cellular tools are available to address key issues at the earliest stages of drug development for a better candidate selection and hepatotoxicity risk assessment.