Received for publication April 7, 2006.
Revised June 7, 2006.
Accepted for publication June 16, 2006.

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

Abstract

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 receptor modulators (SERMs) generated by cytochromes P450 (CYPs). We demonstrated this novel concept by investigating the metabolic activation of tamoxifen (TAM) and raloxifene (RAL) by CYP-containing pig and rat liver microsomes. The high resolution screening (HRS) system is based on the coupling of a CYP-bioreactor to an HPLC-based estrogen receptor alpha (ER) affinity assay. CYP-derived metabolites of the SERMs were generated in the bioreactor, subsequently on-line trapped with solid phase extraction (SPE) and finally separated with gradient HPLC. Upon elution the metabolites were screened on affinity for ER with an on-line HRS assay. With this HRS-system, we were able to follow time-dependently the formation of ER-binding metabolites of tamoxifen and raloxifene. By analyzing the bio-affinity chromatograms with LC-MS/MS structural information of the pharmacologically active metabolites was obtained as well. For tamoxifen, 15 active and 6 non-active metabolites were observed of which 5 were of primary, 10 of secondary and 6 of as yet unknown order of metabolism. Raloxifene was biotransformed in 3 primary and 3 secondary metabolites. Tandem MS/MS analysis revealed that three of the observed active metabolites of raloxifene were not described before. This present automated HRS-system on-line coupled to a CYP-containing bioreactor and an ER-affinity detector proved very efficient, sensitive and selective in metabolic profiling of SERMs.

Key words: analytical pharmacology/toxicology, bioactivation, cytochrome P450, drug discovery, drug disposition, high throughput screening, metabolite identification

This article has been cited by other articles:

				C. J. Mark, R. Tatchum-Talom, D. S. Martin, and K. M. Eyster Effects of estrogens and selective estrogen receptor modulators on vascular reactivity in the perfused mesenteric vascular bed Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2007; 293(5): R1969 - R1975. [Abstract] [Full Text] [PDF]