Characterization of THLE-cytochrome P450 (P450) cell lines: gene expression background and relationship to P450-enzyme activity.

The hepatic SV40 large T-antigen immortalized human liver epithelial (THLE) cell line and sublines transfected with cytochromes P450 (P450s) are increasingly being used for evaluation of potential drug-induced liver injury. So far, the available information on transporter and enzyme expression in these transfected cell systems is scattered. The purpose of this study was to characterize THLE cell lines with respect to transporter and enzyme expression. The mRNA expression of 96 typical drug absorption, distribution, metabolism and excretion genes, which encode a selection of transporters, phase I and II drug-metabolizing enzymes, and nuclear hormone receptors, was investigated in five THLE cell lines transfected with individual human P450s and in mock-transfected THLE-null cells using real-time polymerase chain reaction. The majority of the analyzed genes was either absent or expressed at low levels in the THLE-null and THLE-P450 cells, apart from housekeeping genes and the individual transfected P450s. Enzyme activity measurements provided confirmatory functional data for CYP2C9 and CYP3A4. Comparison with gene expression in human liver revealed an overall much lower gene expression in the THLE cell lines. The low levels of expression of a broad range of P450 genes in the THLE cell lines highlight the value of studies undertaken with P450-expressing cell lines for investigation of mechanisms of P450 metabolite-mediated hepatotoxicity. However, when attempting to translate between data obtained in THLE cell lines in vitro and functional consequences in vivo, it is important to take account of their limited expression of genes encoding many other drug-metabolizing enzymes and hepatic transporters.

Characterization of type II ligands in CYP2C9 and CYP3A4.

Type II cytochrome P450 (CYP) ligands cause inhibition by direct coordination to the heme iron atom. This interaction usually leads to high inhibitory potential, which can cause drug-drug interaction. The approach to design compounds with diminished CYP inhibition is different depending on whether the compound binds (type II ligand) or not (type I ligand) to the iron atom of the heme group. In this study, the structural characteristics of nitrogen-containing compounds, which bind to the iron atom in two CYP isoforms (CYP2C9 and CYP3A4), were investigated. The in vitro assays applied were fluorescence inhibition assay, difference spectra measurements, and K s determination. Computational modeling as an alternative method to difference spectra measurements to distinguish between type I and type II ligands was also explored. Since two CYP isoforms were used, information about the isoform specificity of type II ligands was also analyzed. The in silico method developed in this study applied together with the information gained from the experimental measurements may result in better decisions during the drug discovery process.

Adjuvant chemotherapy and postoperative radiotherapy in high-risk soft tissue sarcoma patients defined by biological risk factors-A Scandinavian Sarcoma Group study (SSG XX).

PURPOSE: To investigate the outcome following adjuvant doxorubicin and ifosfamide in a prospective non-randomised study based on a soft tissue sarcoma (STS) patient subgroup defined by specific morphological characteristics previously shown to be at a high-risk of metastatic relapse. The expected 5-year cumulative incidence of metastases in patients with this risk profile has previously been reported to be about 50% without adjuvant chemotherapy. METHODS: High-risk STS was defined as high-grade morphology (according to the Fédération Nationale des Centres de Lutte Contre le Cancer [FNCLCC] grade II-III) and either vascular invasion or at least two of the following criteria: tumour size ≥8.0 cm, infiltrative growth and necrosis. Six cycles of doxorubicin (60 mg/m2) and ifosfamide (6 g/m2) were given. Postoperative accelerated radiotherapy was applied and scheduled between cycles 3 and 4. RESULTS: For the 150 eligible patients, median follow-up time for metastases-free survival was 3.9 years (range 0.2-8.7). Five-year metastases-free survival (MFS) was 70.4% (95% confidence interval [CI]: 63.1-78.4) with a local recurrence rate of 14.0% (95% CI: 7.8-20.2). For overall survival (OS), the median follow-up time was 4.4 years (range: 0.2-8.7). The five-year OS was 76.1% (95% CI: 68.8-84.2). Tumour size, deep location and reduced dose intensity (<80%) had a negative impact on survival. Toxicity was moderate with no treatment-related death. CONCLUSIONS: A benefit of adjuvant chemotherapy, compared to similar historical control groups, was demonstrated in STS patients with defined poor prognostic factors. Vascular invasion, tumour size, growth pattern and necrosis may identify patients in need of adjuvant chemotherapy.

CYP2C9 structure-metabolism relationships: substrates, inhibitors, and metabolites.

The cytochrome P450 (CYP) family is composed of monooxygenases, which mediate the metabolism of xenobiotics and endogenous compounds. The characterization of the interactions between these enzymes and candidate drugs is an important part of the drug discovery process. CYP2C9, one isoform of the CYPs, mediates the oxidation of several important drugs. The aim of this work is to investigate the possibility to study inhibition and substrate interactions with CYP2C9, using docking and the site of metabolism predictions. The model compounds used for the study were the COX-2 inhibitor celecoxib and a series of 13 analogues known to be metabolized by CYP2C9. The results obtained using the two methods gave valuable information about important interactions of inhibitors and substrates with CYP2C9. The two methods could be used to predict the site of metabolism and to determine the productive docking pose for each compound. These predictions were verified by metabolite identification using LC/MS/MS after incubation with recombinant CYP2C9.

CYP2C9 structure-metabolism relationships: optimizing the metabolic stability of COX-2 inhibitors.

The cytochrome P450 (CYP) family is composed of a large group of monooxygenases that mediate the metabolism of xenobiotics and endogenous compounds. CYP2C9, one of the major isoforms of the CYP family, is responsible for the phase I metabolism of a variety of drugs. The aim of the present investigation is to use rational design together with MetaSite, a metabolism site prediction program, to synthesize compounds that retain their pharmacological effects but that are metabolically more stable in the presence of CYP2C9. The model compound for the study is the nonsteroidal anti-inflammatory drug celecoxib, a COX-2 selective inhibitor and known CYP2C9 substrate. Thirteen analogs of celecoxib were designed, synthesized, and evaluated with regard to their metabolic properties and pharmacologic effects. The docking solutions and the predictions from MetaSite gave useful information leading to the design of new compounds with improved metabolic properties.

Virtual screening and scaffold hopping based on GRID molecular interaction fields.

In this study, a set of strategies for structure-based design using GRID molecular interaction fields (MIFs) to derive a pharmacophoric representation of a protein is reported. Thrombin, one of the key enzymes involved in the blood coagulation cascade, was chosen as the model system since abundant published experimental data are available related to both crystal structures and structurally diverse sets of inhibitors. First, a virtual screening methodology was developed either using a pharmacophore representation of the protein based on GRID MIFs or using GRID MIFs from the 3D structure of a set of chosen thrombin inhibitors. The search was done in a 3D multiconformation version of the Available Chemical Directory (ACD) database, which had been spiked with 262 known thrombin inhibitors (multiple conformers available per compound). The model managed to find 80% of the known thrombin inhibitors among the 74,291 conformers in the ACD by only searching 5% of the database; hence, a 15-fold enrichment of the library was achieved. Second, a scaffold hopping methodology was developed using GRID MIFs, giving the scaffold interaction pattern and the shape of the scaffold, together with the distance between the anchor points. The scaffolds reported by Dolle in the Journal of Combinatorial Chemistry summaries (2000 and 2001) and scaffolds built or derived from ligands cocomplexed with the thrombin enzyme were parameterized using a new set of descriptors and saved into a searchable database. The scaffold representation from the database was then compared to a template scaffold (from a thrombin crystal structure), and the thrombin-derived scaffolds included in the database were found among the top solutions. To validate the usefulness of the methodology to replace the template scaffold, the entire molecule was built (scaffold and side chains) and the resulting compounds were docked into the active site of thrombin. The docking solutions showed the same binding pattern as the cocomplexed compound, hence, showing that this method can be a valuable tool for medicinal chemists to select interchangeable core structures (scaffolds) in an easy manner and retaining the binding properties from the original ligand.

Comparison of inhibitory effects of the proton pump-inhibiting drugs omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole on human cytochrome P450 activities.

The human clearance of proton pump inhibitors (PPIs) of the substituted benzimidazole class is conducted primarily by the hepatic cytochrome P450 (P450) system. To compare the potency and specificity of the currently used PPIs (i.e., omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole) as inhibitors of four cytochrome P450 enzymes (CYP2C9, 2C19, 2D6, and 3A4), we performed in vitro studies using human liver microsomal preparations and recombinant CYP2C19. Sample analysis was done using selected reaction monitoring liquid chromatography/tandem mass spectometry. With several systems for CYP2C19 activity (two marker reactions, S-mephenytoin 4'-hydroxylation and R-omeprazole 5-hydroxylation, tested in either human liver microsomes or recombinant CYP2C19), the five PPIs showed competitive inhibition of CYP2C19 activity with K(i) of 0.4 to 1.5 microM for lansoprazole, 2 to 6 microM for omeprazole, approximately 8 microM for esomeprazole, 14 to 69 microM for pantoprazole, and 17 to 21 microM for rabeprazole. Pantoprazole was a competitive inhibitor of both CYP2C9-catalyzed diclofenac 4'-hydroxylation and CYP3A4-catalyzed midazolam 1'-hydroxylation (K(i) of 6 and 22 microM, respectively), which were at least 2 times more potent than the other PPIs. All PPIs were poor inhibitors of CYP2D6-mediated bufuralol 1'-hydroxylation with IC(50) > 200 microM. The inhibitory potency of a nonenzymatically formed product of rabeprazole, rabeprazole thioether, was also investigated and showed potent, competitive inhibition with K(i) values of 6 microM for CYP2C9, 2 to 8 microM for CYP2C19, 12 microM for CYP2D6, and 15 microM for CYP3A4. The inhibitory potency of R-omeprazole on the four studied P450 enzymes was also studied and showed higher inhibitory potency than its S-isomer on CYP2C9 and 2C19 activities. Our data suggest that, although the inhibitory profiles of the five studied PPIs were similar, lansoprazole and pantoprazole are the most potent in vitro inhibitors of CYP2C19 and CYP2C9, respectively. Esomeprazole showed less inhibitory potency compared with omeprazole and its R-enantiomer. The inhibitory potency of rabeprazole was relatively lower than the other PPIs, but its thioether analog showed potent inhibition on the P450 enzymes investigated, which may be clinically significant.