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Microsomal Incubations (microsomal + incubation)
Selected AbstractsExtrapolating in vitro metabolic interactions to isolated perfused liver: Predictions of metabolic interactions between R -bufuralol, bunitrolol, and debrisoquineJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2010Sami Haddad Abstract Drug,drug interactions (DDIs) are a great concern to the selection of new drug candidates. While in vitro screening assays for DDI are a routine procedure in preclinical research, their interpretation and relevance for the in vivo situation still represent a major challenge. The objective of the present study was to develop a novel mechanistic modeling approach to quantitatively predict DDI solely based upon in vitro data. The overall strategy consisted of developing a model of the liver with physiological details on three subcompartments: the sinusoidal space, the space of Disse, and the cellular matrix. The substrate and inhibitor concentrations available to the metabolizing enzyme were modeled with respect to time and were used to relate the in vitro inhibition constant (Ki) to the in vivo situation. The development of the liver model was supported by experimental studies in a stepwise fashion: (i) characterizing the interactions between the three selected drugs (R -bufuralol (BUF), bunitrolol (BUN), and debrisoquine (DBQ)) in microsomal incubations, (ii) modeling DDI based on binary mixtures model for all the possible pairs of interactions (BUF,BUN, BUF,DBQ, BUN,DBQ) describing a mutual competitive inhibition between the compounds, (iii) incorporating in the binary mixtures model the related constants determined in vitro for the inhibition, metabolism, transport, and partition coefficients of each compound, and (iv) validating the overall liver model for the prediction of the perfusate kinetics of each drug determined in isolated perfused rat liver (IPRL) for the single and paired compounds. Results from microsomal coincubations showed that competitive inhibition was the mechanism of interactions between all three compounds, as expected since those compounds are all substrates of rat CYP2D2. For each drug, the Ki values estimated were similar to their Km values for CYP2D2 indicative of a competition for the same substrate-binding site. Comparison of the performance between the novel liver physiologically based pharmacokinetic (PBPK) model and published empirical models in simulating the perfusate concentration,time profile was based on the area under the curve (AUC) and the shape of the curve of the perfusate time course. The present liver PBPK model was able to quantitatively predict the metabolic interactions determined during the perfusions of mixtures of BUF,DBQ and BUN,DBQ. However, a lower degree of accuracy was obtained for the mixtures of BUF,BUN, potentially due to some interindividual variability in the relative proportion of CYP2D1 and CYP2D2 isoenzymes, both involved in BUF metabolism. Overall, in this metabolic interaction prediction exercise, the PBPK model clearly showed to be the best predictor of perfusate kinetics compared to more empirical models. The present study demonstrated the potential of the mechanistic liver model to enable predictions of metabolic DDI under in vivo condition solely from in vitro information. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:4406,4426, 2010 [source] In vitro biotransformation of anabolic steroids in caninesJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 2 2000Williams Forensic drug testing of anabolic steroids in racing animals is required because of the potential for steroid abuse. Often when the metabolic products of an administered compound have not been identified, the analysis and verification of the administered compound is difficult. The objective of this study was to qualitatively identify the in vitro phase I biotransformation products of anabolic steroids that have a high potential for abuse in canines. The investigated steroids included testosterone, methyltestosterone, mibolerone and boldenone. Steroid biotransformation products were generated using beagle liver microsomes and analysed by high performance liquid chromatography (HPLC)/mass spectrometry (MS) with an electrospray ionization source. Characterization of steroid metabolites was based on HPLC retention, UV and mass spectra. The major testosterone metabolites were identified as androstenedione and 6,- and 16,-hydroxytestosterone. 6,-Hydroxymethyltestosterone was identified as a major metabolite in the methyltestosterone microsomal incubations. Several mibolerone metabolites were identified as monohydroxylated mibolerones as well as an oxidized mibolerone metabolite. Boldenone metabolites were identified as monohydroxylated boldenones, oxidized boldenone, and testosterone. This information should assist in the determination of anabolic steroid use in canines through the correlation of the urinary metabolites to the administered drug. [source] P450-catalyzed vs. electrochemical oxidation of haloperidol studied by ultra-performance liquid chromatography/electrospray ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2010Tove Johansson Mali'n The metabolites formed via the major metabolic pathways of haloperidol in liver microsomes, N -dealkylation and ring oxidation to the pyridinium species, were produced by electrochemical oxidation and characterized by ultra-performance liquid chromatography/electrospray ionization mass spectrometry (UPLC/ESI-MS). Liver microsomal incubations and electrochemical oxidation in the presence of potassium cyanide (KCN) resulted in two diastereomeric cyano adducts, proposed to be generated from trapping of the endocyclic iminium species of haloperidol. Electrochemical oxidation of haloperidol in the presence of KCN gave a third isomeric cyano adduct, resulting from trapping of the exocyclic iminium species of haloperidol. In the electrochemical experiments, addition of KCN almost completely blocked the formation of the major oxidation products, namely the N -dealkylated products, the pyridinium species and a putative lactam. This major shift in product formation by electrochemical oxidation was not observed for the liver microsomal incubations where the N -dealkylation and the pyridinium species were the major metabolites also in the presence of KCN. The previously not observed dihydropyridinium species of haloperidol was detected in the samples, both from electrochemical oxidation and the liver microsomal incubations, in the presence of KCN. The presence of the dihydropyridinium species and the absence of the corresponding cyano adduct lead to the speculation that an unstable cyano adduct was formed, but that cyanide was eliminated to regenerate the stable conjugated system. The formation of the exocyclic cyano adduct in the electrochemical experiments but not in the liver microsomal incubations suggests that the exocyclic iminium intermediate, obligatory in the electrochemically mediated N -dealkylation, may not be formed in the P450-catalyzed reaction. Copyright © 2010 John Wiley & Sons, Ltd. [source] Improved detection of reactive metabolites with a bromine-containing glutathione analog using mass defect and isotope pattern matchingRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2010André LeBlanc Drug bioactivation leading to the formation of reactive species capable of covalent binding to proteins represents an important cause of drug-induced toxicity. Reactive metabolite detection using invitro microsomal incubations is a crucial step in assessing potential toxicity of pharmaceutical compounds. The most common method for screening the formation of these unstable, electrophilic species is by trapping them with glutathione (GSH) followed by liquid chromatography/mass spectrometry (LC/MS) analysis. The present work describes the use of a brominated analog of glutathione, N -(2-bromocarbobenzyloxy)-GSH (GSH-Br), for the invitro screening of reactive metabolites by LC/MS. This novel trapping agent was tested with four drug compounds known to form reactive metabolites, acetaminophen, fipexide, trimethoprim and clozapine. Invitro rat microsomal incubations were performed with GSH and GSH-Br for each drug with subsequent analysis by liquid chromatography/high-resolution mass spectrometry on an electrospray time-of-flight (ESI-TOF) instrument. A generic LC/MS method was used for data acquisition, followed by drug-specific processing of accurate mass data based on mass defect filtering and isotope pattern matching. GSH and GSH-Br incubations were compared to control samples using differential analysis (Mass Profiler) software to identify adducts formed via the formation of reactive metabolites. In all four cases, GSH-Br yielded improved results, with a decreased false positive rate, increased sensitivity and new adducts being identified in contrast to GSH alone. The combination of using this novel trapping agent with powerful processing routines for filtering accurate mass data and differential analysis represents a very reliable method for the identification of reactive metabolites formed in microsomal incubations. Copyright © 2010 John Wiley & Sons, Ltd. [source] 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 compoundsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 7 2010Timo Rousu 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. Copyright © 2010 John Wiley & Sons, Ltd. [source] Atmospheric pressure desorption/ionization on silicon ion trap mass spectrometry applied to the quantitation of midazolam in rat plasma and determination of midazolam 1,-hydroxylation kinetics in human liver microsomesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2006Rick C. Steenwyk The application of atmospheric pressure desorption/ionization on silicon (AP-DIOS) coupled with ion trap mass spectrometry (ITMS) was investigated for the quantification of midazolam in rat plasma, and determination of midazolam 1,-hydroxylation kinetics in pooled human liver microsomes. Results indicate good sensitivity with absolute detection limits for midazolam in rat plasma of approximately 300 femtograms. A linear dynamic range from approximately 10,5000,ng/mL was obtained in rat plasma with analysis times of 1,min per sample. Kinetic constants for midazolam 1,-hydroxylation in human liver microsomes yielded an apparent Km of 10.0,µM and Vmax of 6.4,nmol/min/mg. Studies investigating the inhibition of 1,-hydroxymidazolam formation by the cytochrome P450 3A4 model inhibitor ketoconazole yielded an IC50 of 0.03,µM. Quantitative precision for replicate analysis of rat plasma and human liver microsomal samples was variable with relative standard deviation (RSD) values ranging from a low of approximately 3% to over 50%, with the highest variability observed in data from human liver microsomal incubations. While preliminary studies investigating the application of AP-DIOS-ITMS suggested feasibility of this technique to typical pharmacokinetic applications, further work is required to understand the underlying causes for the high variability observed in these investigations. Copyright © 2006 John Wiley & Sons, Ltd. [source] In vitro,in vivo correlations for drugs eliminated by glucuronidation: Investigations with the model substrate zidovudineBRITISH JOURNAL OF CLINICAL PHARMACOLOGY, Issue 5 2002Sam Boase Aims, To investigate the effects of incubation conditions on the kinetic constants for zidovudine (AZT) glucuronidation by human liver microsomes, and whether microsomal intrinsic clearance (CLint) derived for the various conditions predicted hepatic AZT clearance by glucuronidation (CLH) in vivo. Methods, The effects of incubation constituents, particularly buffer type (phosphate, Tris) and activators (Brij58, alamethacin, UDP-N-acetylglucosamine (UDP-NAcG)), on the kinetics of AZT glucuronidation by human liver microsomes was investigated. AZT glucuronide (AZTG) formation by microsomal incubations was quantified by h.p.l.c. Microsomal CLint values determined for the various experimental conditions were extrapolated to a whole organ CLint and these data were used to calculate in vivo CLH using the well-stirred, parallel tube and dispersion models. Results, Mean CLint values for Brij58 activated microsomes in both phosphate (3.66 ± 1.40 µl min,1 mg,1, 95% CI 1.92, 5.39) and Tris (3.79 ± 0.74 µl min,1 mg,1, 95% CI 2.87, 4.71) buffers were higher (P < 0.05) than the respective values for native microsomes (1.04 ± 0.42, 95% CI 0.53, 1.56 and 1.37 ± 0.30 µl min,1 mg,1, 95% CI 1.00, 1.73). Extrapolation of the microsomal data to a whole organ CLint and substitution of these values in the expressions for the well-stirred, parallel tube and dispersion models underestimated the known in vivo blood AZT clearance by glucuronidation by 6.5- to 23-fold (3.61,12.71 l h,1vs 82 l h,1). There was no significant difference in the CLH predicted by each of the models for each set of conditions. A wide range of incubation constituents and conditions were subsequently investigated to assess their effects on GAZT formation, including alamethacin, UDP-NAcG, MgCl2, d -saccharic acid 1,4-lactone, ATP, GTP, and buffer pH and ionic strength. Of these, only decreasing the phosphate buffer concentration from 0.1 m to 0.02 m for Brij58 activated microsomes substantially increased the rate of GAZT formation, but the extrapolated CLH determined for this condition still underestimated known AZT glucuronidation clearance by more than 4-fold. AZT was shown not to bind nonspecifically to microsomes. Analysis of published data for other glucuronidated drugs confirmed a trend for microsomal CLint to underestimate in vivo CLH. Conclusions, AZT glucuronidation kinetics by human liver microsomes are markedly dependent on incubation conditions, and there is a need for interlaboratory standardization. Extrapolation of in vitro CLint underestimates in vivo hepatic clearance of drugs eliminated by glucuronidation. [source] | |||||||||||||