Current Drug Metabolism - Volume 9, Issue 1, 2008

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.

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.

Cytochrome P450 (P450 or CYP) 3A is one of the most important P450 subfamilies in terms of its broad substrate specificity and relatively high abundance in humans. The substrate specificities of CYP3A4 and CYP3A5 are generally overlapped, but sometimes could differ from each other. It is still important to understand drug interactions more precisely in individual subjects. However, there are few review articles regarding comparative drug oxidation rates catalyzed by CYP3A4 and CYP3A5 and/or substrate inhibition potential towards CYP3A4 and CYP3A5. In this article, we summarize 1) Michaelis-Menten constants (Km), maximal velocities (Vmax), and intrinsic clearance (Vmax/Km) values for 63 substrates (94 reactions) mediated by CYP3A4 and/or CYP3A5, 2) inhibition constants (Ki) and 50% inhibitory concentrations (IC50) of 18 substrates, and 3) maximum inactivation rate constants (kinact) of 14 inhibitors from the literature. The relative contribution of polymorphic CYP3A5 compared with inducible CYP3A4 varies with the substrates and the reaction positions of the substrates. Inhibitory effects of azole antifungal agents and macrolide antibiotics, with low Ki and/or IC50 values for CYP3A4, are likely to be determinant factors for predominant drug interactions in humans, although Asian subjects with relatively high frequency of genetic CYP3A5 expressers should be carefully treated with CYP3A substrates. The collective findings in our present survey provide fundamental and useful information for drug oxidations catalyzed by CYP3A4 and CYP3A5, in spite of some contradictive kinetic parameters for the same reactions reported from many laboratories in different conditions. To understand causal factor(s) and mechanism(s) for such different reports summarized here is still one of the hot research topics to be solved in current drug metabolism.

Drug metabolizing enzymes and transporters are increasingly recognized as key determinants of the inter-individual variability in pharmacokinetic (PK) and pharmacodynamic (PD) outcomes of clinically important drugs. To date, most studies investigating this variability have focused on polymorphisms (e.g. SNPs) in the genes encoding metabolic enzymes and transporters; however, it has recently been reported that the expression of some of these genes is under the control of epigenetic mechanisms. The most common epigenetic mechanism of mammalian genome regulation is DNA methylation, which does not change the genetic code but affects gene expression. Owing to its maintenance of the genomic sequence, DNA methylation is expected to offer an explanation for the controversial phenotypes of certain genetic polymorphisms. It has been recognized that DNA methylation plays a role in the transcriptional regulation of some PK/PD genes. In this review, we describe the impact of various epigenetic mechanisms, especially DNA methylation, on the expression (or activity) of drug metabolizing enzymes and transporter genes.

The in vivo hepatic clearance of tanshinone IIA in the rat was predicted using microsome, cytosol and S9 fractions combined with two different cofactor systems, NADPH-regenerating and UDPGA system. Two different models, the well stirred model and the parallel-tube model, were used in predicting the in vivo clearance in the rat. The in vivo clearance of tanshinone IIA was acquired from a pharmacokinetic study in rat. The results show that the prediction accuracy acquired from the microsome combined with the NADPH is poor. The in vivo clearance in the rat is almost 32 fold higher than the clearance predicted in microsome. The predicted clearance of the S9 model combined with both NADPH and UDPGA system is about 4 fold lower than the in vivo clearance. The predicted clearance of the cytosol combined with the two cofactor system is about 7 fold lower than the in vivo clearance. Although the prediction accuracy acquired from the S9 and cytosol system is not perfect, the prediction accuracy is improved in these two incubation systems. Using S9 combined with both the phase I and phase II metabolism can improve the prediction accuracy.

For drugs that directly act on targets in the central nervous system (CNS), sufficient drug delivery into the brain is a prerequisite for drug action. Systemically administered drugs can reach CNS by passage across the endothelium of capillary vasculatures, the socalled blood-brain barrier (BBB). Literature data suggest that most marketed CNS drugs have good membrane permeability and relatively high plasma unbound fraction, but are not good P-glycoprotein (P-gp) substrates. Therefore, it is important to use the in vitro parameters of P-gp function activity, membrane permeability and plasma unbound fraction as key criteria for lead optimization during the early stage of drug discovery. Evidence from preclinical and clinical studies suggests that drug concentration in cerebrospinal fluid (CSF) appears to be reasonably accurate in predicting unbound drug concentration in the brain. Therefore, CSF can be used as a useful surrogate for in vivo assessment of CNS exposure and provides an important basis for the selection of drug candidates for entry into development. However, it is important to point out that CSF drug concentration is not always an accurate surrogate for predicting unbound drug concentration in the brain. Depending on the physicochemical properties of drugs and the site/timing of CSF sampling, the unbound drug concentration at the biophase within the brain could differ significantly from the corresponding CSF drug concentration.

UDP glucurononosyltransferases (UGT) are a superfamily of enzymes that catalyse the conjugation of a range of structurally diverse drugs, environmental and endogenous chemicals with glucuronic acid. This process plays a significant role in the clearance and detoxification of many chemicals. Over the last decade the regulation and substrate profiles of UGT isoforms have been increasingly characterised. The resulting data has facilitated the prototyping of ligand based in silico models capable of predicting, and gaining insights into, binding affinity and the substrate- and regio- selectivity of glucuronidation by UGT isoforms. Pharmacophore modelling has produced particularly insightful models and quantitative structure-activity relationships based on machine learning algorithms result in accurate predictions. Simple structural chemical descriptors were found to capture much of the chemical information relevant to UGT metabolism. However, quantum chemical properties of molecules and the nucleophilic atoms in the molecule can enhance both the predictivity and chemical intuitiveness of structure-activity models. Chemical diversity analysis of known substrates has shown some bias towards chemicals with aromatic and aliphatic hydroxyl groups. Future progress in in silico development will depend on larger and more diverse high quality metabolic datasets. Furthermore, improved protein structure data on UGTs will enable the application of structural modelling techniques likely leading to greater insight into the binding and reactive processes of UGT catalysed glucuronidation.

The UDP-glucuronosyltransferases (UGTs) are integral membrane proteins of the endoplasmic reticulum that play important roles in the defense against potentially hazardous xenobiotics. The UGTs also participate in the metabolism and homeostasis of many endogenous compounds, including bilirubin and steroid hormones. Most human UGTs can glucuronidate several substrates the chemical structures of which may vary significantly. Understanding the structural basis for the complex substrate specificity of the UGTs is a major challenge that is hampered by the lack of sufficient structural information on these enzymes. Nevertheless, there is currently a broad interest in the structure and function of the UGTs and here we have focused on their oligomeric state. The question whether or not the UGTs are oligomeric enzymes, either dimeric or tetrameric, was frequently addressed in the past, as well as in recent studies. The current knowledge of protein-protein interactions among the UGTs is limited, however, primarily due to considerable difficulties in purifying individual recombinant UGTs as fully active and mono-dispersed proteins. Such hurdles in studying the oligomeric state of the UGTs prompted researchers to develop less direct approaches for examining the quaternary structure of the UGTs and its functional significance. In this article we have reviewed, sometimes critically, most of the available studies about the oligomeric state of the UGTs. Concluding that the UGTs are oligomeric enzymes, we discuss hetero-oligomerization among UGTs and its possible implications for the structure, function and substrate specificity of the enzymes.

Among the naturally-occurring trichothecenes found in food and feed, T-2 toxin is the most potent and toxic mycotoxin. After ingestion of T-2 toxin into the organism, it is processed and eliminated. Some metabolites of this trichothecene are equally toxic or slightly more toxic than T-2 itself, and therefore, the metabolic fate of T-2 toxin has been of great concern. The main reactions in trichothecene metabolism are hydrolysis, hydroxylation and deep oxidation. Typical metabolites of T-2 toxin in an organism are HT-2 toxin, T- 2-triol, T-2-tetraol, 3'-hydroxy-T-2, and 3'-hydroxy-HT-2 toxin. There are significant differences in the metabolic pathways of T-2 toxin between ruminants and non-ruminants. Ruminants have been more resistant to the adverse effects of T-2 toxin due to microbial degradation within rumen microorganisms. Some plant species are resistant to T-2 toxin, while others are capable of its intake and metabolisation.

Excitatory amino acids (EAA) and particularly glutamate toxicity have been implicated in the pathogenesis of neuronal injury occurring in bacterial meningitis by activating the N-methyl-d aspartate (NMDA) receptor complex. Here, we evaluated the effect of adjuvant treatment with the antitussive drug dextromethorphan (DM), a non-competitive NMDA receptor antagonist with neuroprotective potential, in an infant rat model of pneumococcal meningitis. The experiments were carried out in postnatal day 6 (P6) and 11 (P11) animals. Pharmacokinetics of DM and its major metabolite dextrorphan (DO) were performed for dose finding. In our study, DM did not alter clinical parameters (clinical score, motor activity, incidence of seizures, spontaneous mortality) and cortical neuronal injury but increased the occurrence of ataxia (P<0.0001). When DM treatment was started at the time of infection (DM i.p. 15 mg/kg at 0, 4, 8 and 16 hours (h) post infection) in P11 animals, an aggravation of apoptotic neuronal death in the hippocampal dentate gyrus was found (P<0.05). When treatment was initiated during acute pneumococcal meningitis (DM i.p. 15 mg/kg at 12 and 15 h and 7.5 mg/kg at 18 and 21 h after infection), DM had no effect on the extent of brain injury but reduced the occurrence of seizures (P<0.03). We conclude that in this infant rat model of pneumococcal meningitis interference of the EEA and NMDA pathway using DM causes ataxia, attenuates epileptic seizures and increases hippocampal apoptosis, but is not effective in protecting the brain from injury.

Metabolomics is based on the simultaneous analysis of multiple low-molecular-weight metabolites from a given sample. The goals of metabolomics are to catalog and quantify the myriad small molecules found in biological fluids under different conditions. The metabolomics represents the collection of all metabolites in a biological organism, and metabolic profiling can give an instantaneous ‘snapshot’ of the physiology of that cell. Together with the other more established omics technologies, metabolomics will strengthen its claim to contribute to the detailed understanding of the in vivo function of gene products, biochemical analysis, regulatory networks and more ambitious, the mathematical description and simulation of the whole cell in the systems biology approach. This phenomenon will allow the construction of designer organisms for process application using biotransformation and fermentative approaches making effective use of single enzymes, whole microbial and even higher cells and allows the connection of data from genomics, proteomics to enables coordinating the timing of the analysis to physiologically important windows.