Formation of GSH-trapped reactive metabolites in human liver microsomes, S9 fraction, HepaRG-cells, and human hepatocytes.

The objective was to compare several in vitro human liver-derived subcellular and cellular incubation systems for the formation of GSH-trapped reactive metabolites. Incubations of pooled human liver microsomes, human liver S9 fractions, HepaRG-cells, and human hepatocytes were performed with glutathione as a trapping agent. Experiments with liver S9 were performed under two conditions, using only NADPH and using a full set of cofactors enabling also conjugative metabolism. Ten structurally different compounds were used as a test set, chosen as either "positive" (ciprofloxacin, clozapine, diclofenac, ethinyl estradiol, pulegone, and ticlopidine) or "negative" (caffeine, citalopram, losartan, montelukast) compounds, based on their known adverse reactions on liver or bone marrow. GSH conjugates were observed for seven of the ten compounds; while no conjugates were observed for caffeine, citalopram, or ciprofloxacin. Hepatocyte and HepaRG assays produced a clearly lower number and lower relative abundance of GSH conjugates compared to assays with microsomes and S9 fractions. The major GSH conjugates were different for many compounds in cellular subfractions and cell-based systems. Hepatocytes generally produced a higher number of GSH conjugates than HepaRG cells, although the differences were minor. The results show that the hepatic enzyme system used for screening of GSH-trapped reactive metabolites do have a high impact on the results, and results between different systems are comparable only qualitatively.

Reactive metabolites in early drug development: predictive in vitro tools.

Drug metabolism can result in the formation of highly reactive metabolites that are known to play a role in toxicity resulting in a significant proportion of attrition during drug development and clinical use. Thus, the earlier such reactivity was detected, the better. This review summarizes our multi-year project, together with pertinent literature, to examine a battery of in vitro tests capable of detecting the formation of reactive metabolites. Principal prerequisites for such tests were delineated: chemicals known/not known to cause tissue injury and produce reactive metabolites, activation system (mainly human-derived), small- and large-molecular targets (small-molecular trappers, peptides, proteins), analytical techniques (mass spectrometry), and cellular toxicity biomarkers. The current status of in vitro tools to detect reactive intermediates is the following: 1. Small-molecular trapping agents such glutathione or cyanide detect the production of reactive species with high sensitivity by proper MS technique. However, it seems that also putative "negatives" give rise to corresponding adducts. 2. Results from peptide and dG (DNA targeting) trapper studies are generally in line with those of small-molecular trappers, although also important differences exist. These two trapping platforms do not overlap. 3. It is anticipated that the in vitro adduct studies could be fully interpreted only in conjunction with toxicity biomarker (such as the Nrf2 pathway) information from whole cells or tissues. However, while there are tools to characterize the chemical liability and there are correlation between individual/integrated endpoints and toxicity, there are still severe gaps in understanding the mechanisms behind the link between reactive metabolites and adverse effects.

Comparison of unit resolution SRM and TOF-MS at 12,000 mass resolution for quantitative bioanalysis of 11 steroids from human plasma.

BACKGROUND: The use of high-resolution MS systems for quantitative bioanalysis is a growing field, even though a clear majority of bioanalytical methods are still based on MS/MS with triple quadrupole (QqQ) instrumentation. The recent advances in TOF-MS technology have provided increased linear range and a high selectivity of detection by increased mass resolution and mass accuracy, making these instruments attractive for quantitative analysis due to lack of a need for compound-specific detection reaction optimization and their capability to collect data for a high number of compounds by sensitive wide mass range data acquisition. MATERIALS & METHODS: Here, 11 steroids spiked to human plasma were analyzed by LC-MS using both a QqQ MS system and a TOF instrument operating at 12,000 mass resolution. Sample preparation was performed by hybrid SPE technology. RESULTS: The LOD were 0.5-5 and 0.5-20 ng/ml in plasma for all analytes with QqQ and TOF-MS detection, respectively. CONCLUSION: Although the results show wider linear range and slightly better sensitivity for most of the compounds with QqQ in comparison to TOF, acceptable performance was obtained for most of the compounds within the range of LOD to 2000 ng/ml (in plasma), this was also the case with LC-TOF-MS analysis. The main problem in TOF-MS analysis at 12,000 mass resolution from plasma was selectivity rather than sensitivity or linear range.

Highly variable pH effects on the interaction of diclofenac and indomethacin with human UDP-glucuronosyltransferases.

In vitro glucuronidation assays of diclofenac and indomethacin at pH 7.4 are biased by the instability of the glucuronides due to acyl migration. The extent of this acyl migration may be reduced significantly by performing the glucuronidation reaction at pH 6.0. Testing the human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A and 2B at pH 7.4 revealed that UGT1A10, UGT2B7 and UGT2B17 are the most active enzymes in diclofenac glucuronidation, while the highest indomethacin glucuronidation rates (corrected for relative expression levels) were exhibited by UGT2A1, UGT1A10 and UGT2B7. Interestingly, lowering the reaction pH to 6.0 increased the activity of many UGTs, particularly UGT1A10, toward both drugs, even if the rate of 4-methylumbelliferone glucuronidation by UGT1A10 at pH 6.0 was significantly lower than at pH 7.4. On the other hand, UGT2B15 lost activity upon lowering the reaction pH to 6.0. UGT1A6 does not glucuronidate diclofenac and indomethacin. Nevertheless, both drugs inhibit the 1-naphthol glucuronidation activity of UGT1A6 and their inhibition was stimulated by lowering the reaction pH, yielding significantly lower IC(50) values at pH 6.0 than at pH 7.4. In conclusion, glucuronidation reactions pH affects their outcome in variable ways and could increase the toxicity of drugs that carry a carboxylic acid.

Use of comprehensive screening methods to detect selective human CAR activators.

The so-called human xenosensors, constitutive androstane receptor (hCAR), pregnane X receptor (hPXR) and aryl hydrocarbon receptor (hAhR), participate in drug metabolism and transport as well as in several endogenous processes by regulating the expression of their target genes. While the ligand specificities for hPXR and hAhR are relatively well described, this property of hCAR still remains fairly unclear. Identifying hCAR agonists for drug development and for studying hCAR biology are hindered mainly by the unique properties of the receptor, such as the high constitutive activity and complex signaling network but also by the lack of robust and reliable assays and cellular models. Here, validated reporter assays for these three xenosensors are presented and thereafter used to screen a large set of chemicals in order to find novel selective hCAR ligands. We introduce a novel selective hCAR agonist, FL81, which can be used as a stable positive control in hCAR activity assays. Our established receptor-selective ligand identification methods consisting of supporting biological assays and molecular modeling techniques are then used to study FL81 as well as other discovered ligands, such as diethylstilbestrol, o,p'-DDT, methoxychlor and permethrin, for their ability to specifically activate hCAR and to regulate the CYP enzyme expression and function.

Characterization of cyanide-trapped methylated metabonates formed during reactive drug metabolite screening in vitro.

Reactive metabolites are estimated to be one of the main reasons behind unexpected drug-induced toxicity, by binding covalently to cell proteins or DNA. Due to their high reactivity and short lifespan, reactive metabolites are analyzed after chemical trapping with nucleophilic agents such as glutathione or cyanide. Recently, unexplained and uncharacterized methylated reaction products were reported in a human liver microsome based reactive metabolite trapping assay utilizing potassium cyanide as a trapping agent. Here, a similar assay was utilized to produce mono- or dimethylated and further cyanide-trapped reaction products from propranolol, amlodipine and ciprofloxacin, followed by ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOF-MS) and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) experiments for their more detailed structural elucidation. Formation of all observed cyanide-trapped products was clearly NADPH-dependent and thus metabolism-mediated. The suggested reaction pathways included N-methylation leading to iminium formation in primary and/or secondary amines preceded by cytochrome P450 (CYP)-mediated reactions. As the methylation reaction was suggested to be involved in formation of the actual reactive iminium ion, the observed cyanide-trapped products were experimental artifacts rather than trapped reactive metabolites. The results stress that to avoid overestimating the formation of reactive metabolites in vitro, this methylation phenomenon should be taken into account when interpreting the results of cyanide-utilizing reactive metabolite trapping assays. This in turn emphasizes the importance of identification of the observed cyano conjugates during such studies. Yet, metabolite identification has a high importance to avoid overestimation of in vitro metabolic clearance in the cases where this kind of metabonate formation has a high impact in the disappearance rate of the compound.

Effects of cell differentiation and assay conditions on the UDP-glucuronosyltransferase activity in Caco-2 cells.

Cell differentiation increases UDP-glucuronosyltransferase (UGT) gene expression in Caco-2 cells. Glucuronidation of 13 UGT substrates, 1-naphthol, diclofenac, epitestosterone, estradiol, ethinylestradiol, indomethacin, oxazepam, R- and S-propranolol, propofol, testosterone, trifluoperazine, and zidovudine, were studied to derive a broad view on the effect of cell differentiation on the glucuronidation activities of different human UGTs. In parallel, the glucuronidation of these compounds in human liver microsomes (HLM) and human intestinal microsomes (HIM) was analyzed. Because many of the substrates are highly lipophilic, the effects of dimethyl sulfoxide (DMSO) concentrations in the reaction mixture on glucuronidation rates were tested, as well as the effect of alamethicin, a pore-forming peptide. Large differences were observed in the effects of DMSO and alamethicin between recombinant UGTs and Caco-2 cells and HLM and HIM, and, therefore, the activity assays were performed under multiple conditions. Regardless of the assay conditions, however, the results clearly indicated that although differentiation increases glucuronidation activity, the rates in Caco-2 cells are mostly very low, much lower than those in either HLM or HIM. One clear exception was observed: substrates of UGT1A6, such as 1-naphthol, were glucuronidated at very high rates in both undifferentiated and differentiated Caco-2 cells. It may thus be concluded that Caco-2 cells, even differentiated ones, do not provide a good model system to assess first-pass drug glucuronidation in the intestine.

Comparison of triple quadrupole, hybrid linear ion trap triple quadrupole, time-of-flight and LTQ-Orbitrap mass spectrometers in drug discovery phase metabolite screening and identification in vitro--amitriptyline and verapamil as model compounds.

Liquid chromatography in combination with mass spectrometry (LC/MS) is a superior analytical technique for metabolite profiling and identification studies performed in drug discovery and development laboratories. In the early phase of drug discovery the analytical approach should be both time- and cost-effective, thus providing as much data as possible with only one visit to the laboratory, without the need for further experiments. Recent developments in mass spectrometers have created a situation where many different mass spectrometers are available for the task, each with their specific strengths and drawbacks. We compared the metabolite screening properties of four main types of mass spectrometers used in analytical laboratories, considering both the ability to detect the metabolites and provide structural information, as well as the issues related to time consumption in laboratory and thereafter in data processing. Human liver microsomal incubations with amitriptyline and verapamil were used as test samples, and early-phase 'one lab visit only' approaches were used with all instruments. In total, 28 amitriptyline and 69 verapamil metabolites were found and tentatively identified. Time-of-flight mass spectrometry (TOFMS) was the only approach detecting all of them, shown to be the most suitable instrument for elucidating as comprehensive metabolite profile as possible leading also to lowest overall time consumption together with the LTQ-Orbitrap approach. The latter however suffered from lower detection sensitivity and false negatives, and due to slow data acquisition rate required slower chromatography. Approaches with triple quadrupole mass spectrometry (QqQ) and hybrid linear ion trap triple quadrupole mass spectrometry (Q-Trap) provided the highest amount of fragment ion data for structural elucidation, but, in addition to being unable to produce very high-important accurate mass data, they suffered from many false negatives, and especially with the QqQ, from very high overall time consumption.

Applicability of Generic Assays Based on Liquid Chromatography-Electrospray Mass Spectrometry to Study in vitro Metabolism of 55 Structurally Diverse Compounds.

Liquid chromatography-mass spectrometry (LC-MS) with generic gradient elution for a large number of chemically different compounds is a common approach in drug development, used to acquire a large amount of data in a short time frame for drug candidates. The analysis with non-optimized parameters however may lead to a poor method performance for many compounds, and contains a risk of losing important information. Here, generic electrospray time of flight (ESI-TOF) MS methods in various pH conditions were tested for 55 chemically diverse compounds (10 acids, 25 bases, 17 neutrals, and 3 amphoterics), aiming to find best analytical conditions for each compound, for studies of in vitro metabolic properties in liver preparations. The effect of eluent pH and elution gradient strength on chromatographic performance and electrospray MS ionization efficiency were examined for each compound. The data are evaluated how well the best generic approach could cover the analysis of test compounds and how many compounds would still need completely different analytical conditions after that. Aqueous mobile phase consisting of 0.05% acetic acid and 5 mM ammonium acetate (pH 4.4) showed the best general suitability for the analyses, showing adequate performance for metabolite profiling for 41 out of 55 compounds either in positive or negative ion mode. In positive ion mode, the main limitation of performance in various pH conditions was generally not the lack of ionization, but rather the poor chromatographic performance (inadequate retention or poor peak shape), suggesting that more emphasis should be put in finding conditions providing best chromatographic performance, rather than highest ionization properties. However, a single generic approach for a large number of different compounds is not likely to produce good results for all compounds. Preferably, at least two or three different conditions are needed for the coverage of a larger number of structurally diverse compounds.

Comparison of metabolic stability and metabolite identification of 55 ECVAM/ICCVAM validation compounds between human and rat liver homogenates and microsomes - a preliminary analysis.

In vitro methods to produce metabolic information have increasingly been applied in toxicity risk assessment. In the current contract project of JRC/ECVAM In vitro-Toxicology Unit, 55 organic chemicals, mostly drugs and pesticides, most belonging to ECVAM/ICCVAM validation compounds, expected to be analyzable by LC-MS technique, were subjected to a feasibility study. The simple experimental setup consisted of one concentration of a chemical (25 muM), enzyme preparation (human or rat liver homogenate or microsomes), a set of cofactors (NADPH, UDPGA, PAPS, GSH), 4 time points (0, 15, 30, 60 min, including cofactor-less tubes). Metabolites produced were analyzed and tentatively identified by LC-MS techniques. Most of the chemicals were metabolized and metabolites were tentatively identified by TOF-MS analysis. For some chemicals, about 10 or even more metabolites were detectable (e.g. thioridazine, verapamil, amitriptyline). Altogether 11 out of 55 did not display any metabolites under the experimental conditions of this study. Regarding the metabolites formed, there were mostly quantitative differences, but about 20 substances displayed also species-dependent qualitative differences, i.e. a major metabolite was formed in one species, but not in the other. For most chemicals, differences between microsomes and homogenates were relatively modest at least in the initial analysis. The results demonstrate that LC-MS approach is feasible and rather efficient in providing useful metabolic data from a simple experimental setup. More complex analyses, e.g. quantitative assessment of differences between species or biological preparations, or in vitro-in vivo extrapolations, require more complex approaches and a collection of appropriate, preferably curated, data bases of in vivo characteristics of the studied chemicals.

Rapid detection and characterization of reactive drug metabolites in vitro using several isotope-labeled trapping agents and ultra-performance liquid chromatography/time-of-flight mass spectrometry.

Reactive metabolites are believed to be one of the main reasons for unexpected drug-induced toxicity issues, by forming covalent adducts with cell proteins or DNA. Due to their high reactivity and short lifespan they are not directly detected by traditional analytical methods, but are most traditionally analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) after chemical trapping with nucleophilic agents such as glutathione. Here, a simple but very efficient assay was built up for screening reactive drug metabolites, utilizing stable isotope labeled glutathione, potassium cyanide and semicarbazide as trapping agents and highly sensitive ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOFMS) as an analytical tool. A group of twelve structurally different compounds was used as a test set, and a large number of trapped metabolites were detected for most of them, including many conjugates not reported previously. Glutathione-trapped metabolites were detected for nine of the twelve test compounds, whereas cyanide-trapped metabolites were found for eight and semicarbazide-trapped for three test compounds. The high mass accuracy of TOFMS provided unambiguous identification of change in molecular formula by formation of a reactive metabolite. In addition, use of a mass defect filter was found to be a usable tool when mining the trapped conjugates from the acquired data. The approach was shown to provide superior detection sensitivity in comparison to traditional methods based on neutral loss or precursor ion scanning with a triple quadrupole mass spectrometer, and clearly more efficient detection and characterization of reactive drug metabolites with a simpler test setup.