title: Identifying a higher throughput assay for metabolism dependent inhibition (MDI).
abstract: A higher throughput method of screening for the metabolism dependent inhibition of 56 marketed drugs was evaluated and compared data from the PHLM assay (using midazolam as probe) with Cypex assay (using diethoxyfluoresin (DEF) as probe) for CYP3A4 by using 96 well plate. Also 27 marketed drugs were selected to evaluate the reproducibility of Cypex assay using 7-benzyloxyquinoline (7-BQ) as second probe substrate for CYP3A4. Furthermore Cypex CYP2D6 was used to evaluate the reproducibility of this system using 4-methylaminomethyl-7-methoxycoumarin (MMC) as probe substrate with 15 marketed drugs. The fold change was estimated using the fold change obtained from triplicates experiments in same day or different days. All replicates in agreement (i.e. all positive or all negative) for >80% of compounds. The IC50 values for the two assays closely matched only for 13 compounds (23%). Only 5 of the variant 56 compounds had higher IC50 values with the recombinant enzymes, whereas 38 had lower IC50 values with the recombinant cypex CYP3A4 enzyme. The Cypex assay is comparable to PHLM assay in terms of predictivity and reproducibility. The Cypex assay therefore offers a higher throughput, reproducible alternative to PHLM for placement earlier in the lead optimisation process. In conclusion, the results obtained from a fluorescence-based method using Cypex CYP3A4 reflect mostly those obtained from conventional assay using human liver microsomes. This method provides more rapid and reliable detection of MDI inhibitors and may be useful in drug discovery.
authors.affiliation: Neurosciences CEDD DMPK, GlaxoSmithKline, Verona, Italy. mahmud.2.kajbaf@gsk.com.
authors.forename: Mahmud
authors.initials: M
authors.lastname: Kajbaf
authors.forename: Emiliano
authors.initials: E
authors.lastname: Palmieri
authors.forename: Raffaele
authors.initials: R
authors.lastname: Longhi
authors.forename: Stefano
authors.initials: S
authors.lastname: Fontana
mesh_terms: D002851:Chromatography, High Pressure Liquid; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D055808:Drug Discovery; D018757:GABA Modulators / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D008874:Midazolam / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D053719:Tandem Mass Spectrometry
chemical_list: D018757:GABA Modulators; D004364:Pharmaceutical Preparations; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D008874:Midazolam