Endoxifen and Other Metabolites of Tamoxifen Inhibit Human Hydroxysteroid Sulfotransferase 2A1 (hSULT2A1)

Abstract

Although tamoxifen is a successful agent for treatment and prevention of estrogen-dependent breast cancer, its use has been limited by the low incidence of endometrial cancer. Human hydroxysteroid sulfotransferase 2A1 (hSULT2A1) catalyzes the formation of an a-sulfooxy metabolite of tamoxifen that is reactive toward DNA, and this has been implicated in its carcinogenicity. Also, hSULT2A1 functions in the metabolism of steroid hormones such as dehydroepiandrosterone (DHEA) and pregnenolone (PREG). These roles of hSULT2A1 in steroid hormone metabolism and in generating a reactive metabolite of tamoxifen led us to examine its interactions with tamoxifen and several of its major metabolites. We hypothesized that metabolites of tamoxifen may regulate the catalytic activity of hSULT2A1, either through direct inhibition or through serving as alternate substrates for the enzyme. We found that 4-hydroxy-N-desmethyltamoxifen (endoxifen) is a potent inhibitor of hSULT2A1-catalyzed sulfation of PREG and DHEA, with K i values of 3.5 and 2.8 mM, respectively. In the hSULT2A1-catalyzed sulfation of PREG, 4-hydroxytamoxifen (4-OHTAM) and N-desmethyltamoxifen (N-desTAM) exhibited K i values of 12.7 and 9.8 mM, respectively, whereas corresponding K i values of 19.4 and 17.2 mM were observed with DHEA as substrate. A K i value of 9.1 mM was observed for tamoxifen-N-oxide with DHEA as substrate, and this increased to 16.9 mM for the hSULT2A1-catalyzed sulfation of PREG. Three metabolites were substrates for hSULT2A1, with relative sulfation rates of 4-OHTAM > N-desTAM > > endoxifen. These results may be useful in interpreting ongoing clinical trials of endoxifen and in improving the design of related molecules.

The Effects of Endoxifen and Other Major Metabolites of Tamoxifen on the Sulfation of Estradiol Catalyzed by Human Cytosolic Sulfotransferases hSULT1E1 and hSULT1A1*1

“…The enzymatic reactions were analyzed by LC-MS, and the negative ion ESI-MS of the products formed by the hSULT1A1*1-catalyzed sulfation of 4-OHTAM, endoxifen, and N-desTAM (Supplemental Figs. 8-10, respectively) were similar to previous ESI-MS data from reactions catalyzed by hSULT2A1 (Squirewell et al, 2014). The retention times of 4-TAM-SO 4 , endoxifensulfate, and N-desTAM-S on LC-MS chromatography (data not shown) were 16.30, 16.07, and 21.81 minutes, respectively.…”

“…Products of sulfation catalyzed by hSULT1E1 and hSULT1A1*1 were identified by liquid chromatography-mass spectrometry (LC-MS) analysis on a Waters Q-TOF Premiere mass spectrometer, as described previously (Squirewell et al, 2014). Briefly, each 50 ml reaction was performed at pH 7.4 and used 50 mM substrate with 50 mM PAPS in the presence of 0.25 M potassium phosphate, 8.3 mM 2-mercaptoethanol, and 2% ethanol (v/v).…”

“…Synthesis of tamoxifen-Noxide (TAM-NO) was performed, as described elsewhere (Foster et al, 1980;Mani and Kupfer, 1991). N-desmethyltamoxifen-sulfamate (N-desTAM-S) and 4-hydroxytamoxifen-sulfate (4-TAM-SO 4 ) were synthesized from sulfuryl imidazolium triflate, according to our previously published method (Squirewell et al, 2014). [ 3 H]-Estradiol (81.0 Ci/mmol) was obtained from Perkin Elmer (Waltham, MA).…”

“…Major metabolites of tamoxifen, including N-desmethyltamoxifen (N-desTAM), tamoxifen-N-oxide (TAM-NO), 4-OHTAM, and endoxifen, were recently shown to inhibit the sulfation of steroid substrates for hSULT2A1 (Squirewell et al, 2014). Thus, we hypothesized that tamoxifen metabolites were also inhibitors of estradiol sulfation catalyzed by hSULT1E1 and hSULT1A1*1.…”

Genetic polymorphisms of CYP2D6*10 and the effectiveness of combined tamoxifen citrate and testosterone undecanoate treatment in infertile men with idiopathic oligozoospermia

“…Tamoxifen citrate, as a first line of treatment for infertile men with idiopathic oligozoospermia, was proposed by the World Health Organization (WHO) Working Committee (Rowe et al, 2000). Tamoxifen undergoes extensive hepatic and gut wall metabolism in humans for several primary and secondary metabolites that exhibit a range of pharmacologic activity (Squirewell et al, 2014). Previous research demonstrated that endoxifen formation proceeds stepwise by oxidation of tamoxifen with N-desmethyltamoxifen (NDM) as the predominant intermediate (Desta et al, 2004), and patients receiving tamoxifen are influenced by cytochrome P4502D6 (CYP2D6) genetic variants (Stearns et al, 2003;Jin et al, 2005).…”