DEVELOPMENT AND VALIDATION OF A HIGH-THROUGHPUT RADIOMETRIC CYP3A4/5 INHIBITION ASSAY USING TRITIATED TESTOSTERONE
Drug Metabolism and Disposition, ISSN: 0090-9556, Vol: 33, Issue: 3, Page: 349-358
2005
- 24Citations
- 13Captures
Metric Options: Counts1 Year3 YearSelecting the 1-year or 3-year option will change the metrics count to percentiles, illustrating how an article or review compares to other articles or reviews within the selected time period in the same journal. Selecting the 1-year option compares the metrics against other articles/reviews that were also published in the same calendar year. Selecting the 3-year option compares the metrics against other articles/reviews that were also published in the same calendar year plus the two years prior.
Example: if you select the 1-year option for an article published in 2019 and a metric category shows 90%, that means that the article or review is performing better than 90% of the other articles/reviews published in that journal in 2019. If you select the 3-year option for the same article published in 2019 and the metric category shows 90%, that means that the article or review is performing better than 90% of the other articles/reviews published in that journal in 2019, 2018 and 2017.
Citation Benchmarking is provided by Scopus and SciVal and is different from the metrics context provided by PlumX Metrics.
Example: if you select the 1-year option for an article published in 2019 and a metric category shows 90%, that means that the article or review is performing better than 90% of the other articles/reviews published in that journal in 2019. If you select the 3-year option for the same article published in 2019 and the metric category shows 90%, that means that the article or review is performing better than 90% of the other articles/reviews published in that journal in 2019, 2018 and 2017.
Citation Benchmarking is provided by Scopus and SciVal and is different from the metrics context provided by PlumX Metrics.
Metrics Details
- Citations24
- Citation Indexes24
- 24
- CrossRef20
- Captures13
- Readers13
- 13
Article Description
A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 3A4/5 in human liver microsomes is described. In contrast to the conventional testosterone 6β-hydroxylation assay, the new method does not require high-performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP3A4/5-mediated 6β-hydroxylation of testosterone labeled with tritium in the 6β position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. Using commercially available [1,2,6,7- 3 H]testosterone as substrate, we demonstrated that the reaction is NADPH-dependent, and sensitive to CYP3A4/5/5 inhibitors and a CYP3A4/5/5-specific inhibitory monoclonal antibody, but not to inhibitors of or antibodies against other P450 enzymes. The method was further improved by synthesis of testosterone specifically tritiated in the 6β position, which displayed greatly improved conversion rate with an ensuing increase in assay sensitivity. Competition experiments using tritiated and unlabeled testosterone indicated that CYP3A4/5-mediated 6β-hydroxylation exhibits positive cooperativity and a modest kinetic isotope effect. IC 50 values for more than 40 structurally diverse chemical inhibitors were not significantly different from those determined in the testosterone 6β-hydroxylation assay, using HPLC-tandem mass spectrometry analysis. All the steps of the new assay, namely, incubation, product separation, and radioactivity counting, are performed in 96-well format and can be automated. This assay thus represents a high-throughput version of the classical testosterone 6β-hydroxylation assay, which is the most widely used method to assess the potential for CYP3A4/5 inhibition of new chemical entities.
Bibliographic Details
http://www.sciencedirect.com/science/article/pii/S0090955624031192; http://dx.doi.org/10.1124/dmd.104.002873; http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=14044275075&origin=inward; http://www.ncbi.nlm.nih.gov/pubmed/15608130; https://linkinghub.elsevier.com/retrieve/pii/S0090955624031192; https://dx.doi.org/10.1124/dmd.104.002873; https://dmd.aspetjournals.org/content/33/3/349
Elsevier BV
Provide Feedback
Have ideas for a new metric? Would you like to see something else here?Let us know