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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.

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.

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).

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.

Drug-induced photoallergy is an immune adverse reaction to the combined effect of drugs and light. From the mechanistic point of view, it first involves covalent binding of drug to protein resulting in the formation of a photoantigen. Hence, determination of the structures of drug−protein photoadducts is of great relevance to understand the molecular basis of photoallergy and cross-immunoreactivity among drugs. Looking for new strategies to investigate the covalent photobinding of drugs to proteins, we generated highly specific antibodies to drug chemical substructures. The availability of such antibodies has allowed us to discriminate between the different modes by which tiaprofenic acid (TPA), suprofen (SUP), and ketoprofen (KTP) photobind to proteins. The finding that the vast majority of the TPA photoadduct can be accounted for by means of antibody anti-benzoyl strongly supports the view that the drug binds preferentially via the thiophene ring, leaving the benzene ring more accessible. By contrast, selective recognition of SUP-protein photoadducts by antibody anti-thenoyl evidences a preferential coupling via the benzene ring leaving the thiophene moiety more distant from the protein matrix. In the case of KTP, photoadducts are exclusively recognized by antibody anti-benzoyl, indicating that the benzene ring is again more accessible. As a result of this research, we have been able to identify a common substructure that is present in TPA−albumin and KTP−albumin photoadducts. This is remarkable since, at a first sight, the greatest structural similarities can be found between TPA and SUP as they share the same benzoylthiophene chromophore. These findings can explain the previously reported observations of cross-reactivity to KTP (or TPA) in patients photosensitized to TPA (or KTP).