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

Abstract

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

Effects of estrogens and selective estrogen receptor modulators on vascular reactivity in the perfused mesenteric vascular bed

“…Moreover, TAM and RAL also undergo metabolism to form active metabolites (3,40). Thus the effects of TAM and RAL may be due to direct effects of the SERMs on estrogen receptors in the mesenteric vasculature, or to effects of their metabolites.…”

Chromatography in Bioactivity Analysis of Compounds

“…acetylcholinesterase, phosphodiesterase, glutathione-S-transferase (EAD -Enzyme Activity/Affinity Detection), affinity of bioactive substances receptors, e.g. estrogen receptor (RAD -Receptor Affinity Detection) [76,77,78,79,80,81,82]. Conjunction of the chromatographic methods of component separation with methods of analysis of biological properties provides great opportunities in their analysis.…”

Development of On-Line High Performance Liquid Chromatography (HPLC)-Biochemical Detection Methods as Tools in the Identification of Bioactives

“…EAD and RAD assays can further be classified as homogeneous assays (Figure 2b) [11], where there is a marked difference in the signal intensity obtained from bound and unbound ligands, or heterogeneous assays (Figure 2c) [12], where a further separation step is required to distinguish between bound and unbound ligands. The metabolite profiling assays use a configuration where a metabolism system is inserted between the automatic sample injector and the HPLC gradient pump (Figure 2d) [13]. This system, which can be further combined with BCD systems, consists of pumps for addition of the reagents and a reaction coil.…”

“…However, the rather broad description of EAD or RAD does not take into account that some hyphenated techniques are based on the metabolism of compounds and not their affinity for the enzymes responsible for the metabolism. These include systems using cytochromes P450 and liver slices [13,29–31]. Furthermore, there are also assays where HPLC is combined with bio-affinity compounds which may be based on orphan receptors and others based on immunological assays with both types not employing “classical” receptors [32–37].…”

“…Due to an 80 s assay time with subsequent band broadening, highly concentrated extracts were analyzed and ELISA blocking reagent was used to prevent non-specific binding. Van Liempd et al [13,30] combined metabolism assays and ER screening which is described in more detail in section 3.3. Kool et al [54] used an analogous method to that of Oosterkamp et al [53] to investigate pig and rat liver microsomal metabolites of tamoxifen and ( Z )-4-hydroxytamoxifen for ERα affinity.…”