Mechanisms of Interplay between UGTs and Efflux Transporters in Flavonoid Disposition

Objective: The overall objective is to investigate the mechanism(s) of interplay between UDP-glucuronosyltransferases (UGTs) and efflux transporters in flavonoid disposition. The goals of this research project were to 1) develop and characterize a cell model suitable to study the simplest interplay between one UGT (i.e., UGT1A9) and an efflux transporter (i.e., BCRP); 2) study the interplay between UGT1A9 and BCRP in flavonoid disposition using the established and well-characterized HeLa cell model; 3) compare the interplay between UGTs and efflux transporters in the flavonoid disposition among different cellular and animal models; 4) determine the kinetic parameters of efflux of flavonoid glucuronides that are transported by the specific efflux transporter in the established HeLa cell model. Methods: For objective 1), HeLa cells were stably transfected with UGT1A9 gene which was cloned in the pcDNA3.1 (+/-) vector. The developed “engineered” HeLa cells were characterized at expression levels by the Western blotting and RT-PCR, and at functional levels by metabolism and excretion assay. For objective 2), the engineered HeLa cells overexpressing UGT1A9 were treated with several flavonoids in the presence or absence of efflux transporter inhibitors or siRNA of UGT1A9. For objective 3), Caco-2 cells, the engineered MDCKII-BCRP or MDCKII-MRP2 cells (the MDCKII series cells transiently transfected with UGT1A9) was treated with flavonoids with or without Ko143, a potent chemical inhibitor of BCRP. In addition, FVB and Bcrp (-/-) mice were orally given the selected flavonoid at dose of 20mg/kg. For objective 4), the engineered HeLa cells grown on the 6-well plates were treated with several commercially available mono-hydroxyflavones and other selected compounds using concentrations sufficient to describe kinetics of glucuronide efflux. Results: 1) In the engineered HeLa cells, which were shown to be functional stable for at least five generations, UGT1A9 and BCRP were predominantly expressed. The glucuronidation of model flavonoids (e.g. genistein and apigenin) catalyzed by UGT1A9 overexpressed in the engineered HeLa cells was following the same kinetic mechanism as that by UGT1A9 SupersomeTM, although the UGT1A9 isoform from the HeLa cells usually had a larger Km and smaller Vmax than those from the SupersomeTM. In addition, the glucuronide excretion in the engineered HeLa cells was mainly attributed to the predominant expressed BCRP, because neither the broad specific MRP inhibitor LTC4, nor the potent siRNAs against MRP2 and MRP3 was effective in inhibiting the glucuronide efflux. 2) To study the mechanism of kinetic interplay between UGTs and efflux transporters in flavonoid disposition, we found that in the engineered HeLa cells, the siRNA-mediated UGT1A9 silencing could result in substantial decreases in glucuronide excretion (40-86%, p<0.01), cellular glucuronide levels (45-98%, p<0.01), and cellular glucuronidation activity or fmet (40-87%, p<0.01). However, the BCRP function (measured using glucuronide clearance or CL) was not or marginally affected by siRNA silencing. On the other hand, dose-dependent inhibition of BCRP by Ko143 could lead to the dramatic increase in the intracellular glucuronide levels (4-8 folds), moderate decrease in glucuronide excretion (<60%), and substantial inhibition of CL (>75%). The effect of Ko143 (5µM) on fmet was compound-dependent. 3) To compare the engineered HeLa cells with other cell or animal models, we found that inhibition of BCRP by Ko143 (5µM) did not affect the phase II metabolism of flavonoids but affected the distribution of flavonoid sulfates in Caco-2 cells. In the engineered MDCKII-BCRP or –MRP2 cells, which were transiently transfected with UGT1A9, the excretion rates of wogonin glucuronide, intracellular levels of glucuronides and fmet were all significantly increased. In Bcrp knockout mice, the AUC0-480min values of wogonin and its conjugates were markedly improved (10 folds, P<0.05), compared with FVB mice. 4) In determining the kinetics parameters of glucuronides transported by BCRP, we found that the correlation of Km’ between vesicles and engineered HeLa cells appeared to be better than other kinetics parameters such as Jmax (or maximal efflux rate constant). Conclusion: Engineered HeLa cells which are stably transfected with UGT1A9 are an appropriate tool to study the mechanisms of kinetic interplay between UGTs and efflux transporters and to rapidly identify the glucuronide substrate of BCRP. The bioavailability of flavonoid such as wogonin could be improved experimentally by disrupting the function of BCRP in a mouse pharmacokinetic model in vivo.