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- Lutz, Mareike, 1967-, et al.
(author)
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Monolithic silica rod liquid chromatography with ultraviolet or fluorescence detection for metabolite analysis of cytochrome P450 marker reactions
- 2002
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In: Journal of chromatography. B. - Amsterdam : Elsevier. - 1570-0232 .- 1873-376X. ; 780:2, s. 205-215
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Journal article (peer-reviewed)abstract
- In vitro cytochrome P450 assays are used in metabolism studies in support of early phases of drug discovery to investigate, e.g., metabolic stability, enzyme inhibition and induction by new chemical entities. LC-UV and LC-fluorescence are traditional analytical tools in support of such studies. However, these tools typically comprise different methods of relatively low throughput for the various metabolites of probe reactions. In recent years, LC-MS methods have been developed to increase throughput. Increased throughput can also be achieved by means of modern chromatographic tools in combination with UV and fluorescence detection. This approach is especially suitable when cytochrome P450 isoforms are investigated by means of single probe incubations. Here, an LC-UV/fluorescence system based on a monolithic porous silica column is described for the analysis of metabolites of nine cytochrome P450 marker reactions [phenacetin to paracetamol (CYP1A2), coumarin to 7-hydroxycoumarin (CYP2A6), paclitaxel to 6alpha-hydroxypaclitaxel (CYP2C8), diclofenac to 4-hydroxydiclofenac (CYP2C9), mephenytoin to 4-hydroxymephenytoin (CYP2C19), bufuralol to 1-hydroxybufuralol (CYP2D6), chlorzoxazone to 6-hydroxychlorzoxazone (CYP2E1), midazolam to 1-hydroxymidazolam (CYP3A4), and testosteron to 6beta-hydroxytestosteron (CYP3A4)]. While offering sensitivities and linear ranges comparable to previously reported methods, the set-up described here provides ease of use and increased throughput with maximum cycle times of 4.5 min. © 2002 Elsevier Science B.V. All rights reserved.
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- Persson, Kajsa P., et al.
(author)
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Evaluation of Human Liver Slices and Reporter Gene Assays as Systems for Predicting the Cytochrome P450 Induction Potential of Drugs in Vivo in Humans
- 2006
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In: Pharmaceutical research. - New York : Springer. - 0724-8741 .- 1573-904X. ; 23:1, s. 56-69
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Journal article (peer-reviewed)abstract
- PurposeThe aim of the study was to investigate the feasibility of predicting human in vivo cytochrome P450 (CYP) induction properties of drugs using in vitro methods.MethodsThe CYP induction potential of compounds was tested in human liver slices and in reporter gene assays for the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR).ResultsIn human liver slices, CYP activities decreased dramatically over the experimental period, whereas mRNA levels could reliably be used to investigate CYP1A, 2C9, and 3A4 induction. However, the interindividual variations and demanding experimentation limit the use of liver slices in screening programs. Reporter gene assays are robust and reliable assays, amenable to high throughput screening. Several compounds activated AhR. The relevance of this activation, however, needs to be further investigated since there are no clear reports on drugs inducing CYP1A in vivo. The results from the PXR assay could be used to correctly classify compounds with known CYP3A induction properties when relating in vivo AUCtot to PXR EC50 values.ConclusionsLiver slices are a valuable model to study the regulation of a larger number of enzymes by single compounds. The PXR reporter gene assay could be used as a reliable screening method to predict CYP3A induction in vivo. © 2006 Springer Science + Business Media, Inc.
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