On-line Formation, Separation, and Estrogen Receptor Affinity Screening of Cytochrome P450-Derived Metabolites of Selective Estrogen Receptor Modulators

• DOI: 10.1124/dmd.106.010355
• Publication Date: 2006-06-22T01:02:00Z
• Authors: S.M. van Liempd; J. Kool; W.M.A. Niessen; D.E. van Elswijk; H. Irth; N.P.E. Vermeulen
• Keywords: Raloxifene; Antiestrogen

We have developed a fully automated bioreactor coupled to an on-line receptor affinity detection system. This analytical system provides detailed information on pharmacologically active metabolites of selective estrogen modulators (SERMs) generated by cytochromes P450 (P450s). demonstrated this novel concept investigating the metabolic activation tamoxifen and raloxifene P450-containing pig rat liver microsomes. The high resolution screening (HRS) is based coupling P450-bioreactor HPLC-based alpha (ERα) assay. P450-derived SERMs were in bioreactor, subsequently trapped with solid phase extraction, finally separated gradient HPLC. Upon elution, screened for ERα HRS With system, we able follow, time-dependent manner, formation ERα-binding raloxifene. By analyzing bioaffinity chromatograms liquid chromatography-tandem mass spectrometry, structural was obtained as well. For tamoxifen, 15 6 nonactive observed, which 5 primary, 10 secondary, yet unknown order metabolism. Raloxifene biotransformed three primary secondary metabolites. MS/MS analysis revealed that observed not described before. present ERα-affinity detector proved very efficient, sensitive, profiling SERMs.