Characterization of Novel Mechanisms of Efflux Transport of the Glucuronides of Testosterone and its Metabolites: a Quantitative Proteomics‐based IVIVE Study and Relevance to Enterohepatic Recirculation of Androgens

Abstract

Testosterone (T) is primarily eliminated via glucuronidation upon oral administration. As a result, testosterone glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG) and dihydrotestosterone glucuronide (DHTG) are produced as the key circulating metabolites, which are excreted in urine and bile. The goal of this study was to characterize the in vivo fractional contribution of individual hepatic transporters (ft) in the elimination of the glucuronides of T and its metabolites. We first performed vesicular transport screening assay with recombinant human MRP2, MRP3, MRP4, MDR1, and BCRP to identify the transporters that are involved in the efflux of TG, AG, EtioG and DHTG. The transport kinetic analyses were then carried out for the transporters that exhibited activity in the initial screening. The inside‐out vesicle ratio was determined by 5′‐nucleotidase activity assay. We next quantified the protein abundances of these efflux transporters in vesicles, human liver, and intestine by quantitative LC‐MS/MS proteomics using a validated surrogate peptide‐based approach. The ft for each transporter was estimated by proteomics‐based physiological scaling factors, i.e., transporter abundance in whole tissue versus vesicles, and by correction for the inside‐out vesicles. The transport screening assays revealed that TG, AG, EtioG, and DHTG were primarily effluxed by MRP2 and MRP3 with differential contribution of individual transporters. EtioG and DHTG were transported by MDR1 and BCRP, respectively, but at much lower rates than by MRPs. MRP2 and MRP3 exhibited higher intrinsic clearance for TG, followed by DHTG, AG, and EtioG (Figure 1). The Km value for EtioG transport by MRP3 was about 3 to 20‐fold lower than for the other glucuronides. After integrating the transporter abundance data from human tissues, the ft of MRP2 and MRP3 in efflux of TG, EtioG, and DHTG in liver was calculated to be around 0.6 and 0.4, respectively. For AG, MRP3 played a major role (68%) to its efflux in liver [corroborates with our in vivo clinical data (Basit et al., CTS, 11, 2018)]. In conclusion, our results for the first‐time demonstrated that MRP2 and MRP3 are the major transporters that are involved in the efflux of T glucuronide conjugates. Important role of MRP2 in transport of TG, EtioG, and DHTG indicates the potential importance of enterohepatic recirculation in regulating the circulating T, Etio, and DHT after deconjugation in the gut. With this respect, our unpublished data suggest efficient deconjugation of these glucuronides by human relevant β‐glucuronidases. Characterization of the efflux mechanisms of glucuronides of T and DHT is important for predicting the androgen disposition and interindividual variability, including drug‐androgen interaction in humans. The mechanistic transport data can be extrapolated (using quantitative proteomics) to other androgen relevant tissues, such as kidney, prostate, testis and placenta.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.