Hyperbilirubinemia may arise due to inadequate clearance of bilirubin from the 
body. Bilirubin elimination is a multifaceted process consisting of uptake of 
bilirubin into the hepatocytes facilitated by OATP1B1 and OATP1B3. Once in the 
hepatocytes, it is extensively glucuronidated by UGT1A1. Eventually, the 
glucuronide metabolite is excreted into the bile via MRP2. UGT1A1 inhibition has 
been previously shown to be linked with hyperbilirubinemia. However, because 
drug transporters also contribute to bilirubin elimination, the purpose of this 
work was to investigate the in vitro inhibition of OATP1B1, OATP1B3, MRP2, and 
BSEP of select test drugs known to elicit hyperbilirubinemia. Test drugs 
investigated in this study were atazanavir and indinavir, which are associated 
with hyperbilirubinemia and elevations in serum transaminase; ritonavir and 
nelfinavir, which are not associated with hyperbilirubinemia; and bromfenac, 
troglitazone, and trovafloxacin, which are associated with severe idiosyncratic 
hepatotoxicity exhibiting elevations in serum bilirubin and transaminase. Due to 
limited solubility and poor ionization of bilirubin and its glucuronide, the 
formation of estradiol 3-glucuronide was used as a surrogate to assess UGT1A1 
activity, while the transport of pitavastatin, CDCF, and taurocholate were used 
as surrogate probe substrates to monitor the function of OATP1B1/OATP1B3, MRP2, 
and BSEP, respectively. It was assumed that any inhibition of the surrogate 
probe substrates by test drugs is indicative of the potential impact of test 
drugs to modulate the function of proteins involved in bilirubin disposition. In 
vitro inhibition was determined by calculating IC50. Moreover, Cmax and 
Cmax,free were integrated with IC50 values to calculate R and Rfree, 
respectively, which represents the ratio of probe drug glucuronidation/transport 
in the absence and presence of test drugs. Analysis of the data showed that 
Rfree demonstrated the best correlation to hyperbilirubinemia. Specifically, 
Rfree was above the 1.1 target threshold against UGT1A1, OATP1B1, and BSEP for 
atazanavir and indinavir. In contrast, Rfree was below this threshold for 
ritonavir and nelfinavir as well as for bromfenac, troglitazone, and 
trovafloxacin. For all test drugs examined, only minor inhibition against 
OATP1B3 and MRP2 were observed. These data suggest that the proposed surrogate 
probe substrates to evaluate the in vitro inhibition of UGT1A1, OATP1B1, and 
BSEP may be suitable to assess bilirubin disposition. For protease inhibitors, 
inclusion of OATP1B1 and BSEP inhibition may improve the predictability of 
hyperbilirubinemia.