Sulfonation of raloxifene in HEK293 cells overexpressing SULT1A3: Involvement of breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated protein 4 (MRP4/ABCC4) in excretion of sulfate metabolites.

Excretion of sulfate metabolites is an essential process in disposition of raloxifene via the sulfonation pathway. However, the transporters responsible for excretion of raloxifene sulfates remain undefined. Here, sulfonation of raloxifene and excretion of its sulfate metabolites were investigated using SULT1A3-overexpressing HEK293 cells (or SULT293 cells) with significant expression of BCRP and MRP4. SULT293 cell lysate catalyzed the sulfonation of raloxifene at both 6-OH and 4'-OH groups, generating raloxifene-6-sulfate (R-6-S) and raloxifene-4'-sulfate (R-4'-S), respectively. Sulfate formation followed the Michaelis-Menten kinetics (Km = 0.49 μM and Vmax = 5.79 pmol/min/mg for R-6-S; Km = 0.33 μM and Vmax = 1.25 pmol/min/mg for R-4'-S). As expected, the recombinant SULT1A3 enzyme showed a high similarity in raloxifene sulfonation profiles with the lysate preparation. Ko143, a selective inhibitor of BCRP, significantly decreased the excretion rates of raloxifene sulfates (maximal 66.1%) while increasing the intracellular sulfates (maximal 282%). As a result, the apparent efflux clearance (CLef,app, representing the efflux efficiency of raloxifene sulfates) was substantially reduced (maximal 85.6%). Likewise, the pan-MRP inhibitor MK-571 significantly deceased the excretion rates (maximal 69.6%) and CLef,app values (maximal 96.0%) of raloxifene sulfates while increasing the intracellular sulfates (maximal 667%). Further, the short-hairpin RNA (shRNA) targeting BCRP significantly reduced (maximal 35.0%) sulfate excretion. Use of BCRP shRNA also caused significant decreases (maximal 52.5%) in the CLef,app values. Silencing of MRP4 by shRNA led to a substantial alteration in sulfate disposition (i.e., 28.6-37.8% reductions in sulfate excretion, 30.5-59.3% elevations in intracellular sulfates, and 44.8-47.7% deceases in CLef,app values). In conclusion, two sulfate metabolites R-6-S and R-4'-S were generated from raloxifene in SULT293 cells. Cellular excretion of the raloxifene sulfates was mainly mediated by BCRP and MRP4.

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