Evaluation of the endothelin receptor antagonists ambrisentan, bosentan, macitentan, and sitaxsentan as hepatobiliary transporter inhibitors and substrates in sandwich-cultured human hepatocytes.
BACKGROUND
Inhibition of the transporter-mediated hepatobiliary elimination of bile salts is a putative mechanism for liver toxicity observed with some endothelin receptor antagonists (ERAs).


METHODS
Sandwich-cultured human hepatocytes were used to study the hepatobiliary distribution and accumulation of exogenous taurocholate, ERAs and endogenous bile acids. The molecular mechanisms for findings in hepatocytes or clinical observations were further explored using either vesicular assays (efflux transporters) or transfected cell-lines (uptake transporters). Inhibition constants (IC50) were measured for the human hepatobiliary transporters bile salt export pump (BSEP), sodium taurocholate cotransporting polypeptide (NTCP), multidrug resistance protein 2 (MRP2), P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3.


RESULTS
The ERAs showed dose-dependent reductions in exogenous taurocholate cellular accumulation in human hepatocytes, with macitentan having the greatest effect. Consistent with their effects on bile acids, the ERAs inhibited bile transporters. IC50 values for OATP1B1 and OATP1B3 ranged from 2 µM for macitentan to 47 µM for ambrisentan. Macitentan and bosentan also inhibited NTCP with IC50 values of 10 and 36 µM, respectively. Similar to previously reported findings with sitaxsentan, BSEP inhibition was observed for bosentan and macitentan with IC50 values of 42 and 12 µM, respectively. In contrast, ambrisentan showed little or no inhibition of these transporters. Other transporters tested were weakly inhibited by the ERAs. Accumulation in hepatocytes was also a factor in the effects on bile transport. Macitentan demonstrated the greatest accumulation in human hepatocytes (∼100x) followed by sitaxsentan (∼40x), bosentan (∼20x) and ambrisentan (∼2x).


CONCLUSIONS
Significant differences in the inhibition of hepatic transporters were observed between the evaluated ERAs in vitro. Macitentan had the highest level of cellular accumulation and caused the greatest effects on bile acid distribution in human hepatocytes followed by sitaxsentan and bosentan. Ambrisentan showed a low potential to affect bile acids.
Gilead Sciences, Inc., Foster City, California, United States of America.
Eve-Irene
EI
Lepist
Gilead Sciences, Inc., Foster City, California, United States of America.
Hunter
H
Gillies
Qualyst Transporter Solutions, LLC, Durham, North Carolina, United States of America.
William
W
Smith
Gilead Sciences, Inc., Foster City, California, United States of America.
Jia
J
Hao
Qualyst Transporter Solutions, LLC, Durham, North Carolina, United States of America.
Cassandra
C
Hubert
Qualyst Transporter Solutions, LLC, Durham, North Carolina, United States of America.
Robert L
RL
St Claire
Qualyst Transporter Solutions, LLC, Durham, North Carolina, United States of America.
Kenneth R
KR
Brouwer
Gilead Sciences, Inc., Foster City, California, United States of America.
Adrian S
AS
Ray
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000328:Adult; D001646:Bile / Q000187:drug effects* / Q000378:metabolism; D001647:Bile Acids and Salts / Q000378:metabolism; D000077300:Bosentan; D065128:Endothelin Receptor Antagonists; D005260:Female; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007555:Isoxazoles / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D027381:Liver-Specific Organic Anion Transporter 1; D008297:Male; D026901:Membrane Transport Proteins / Q000378:metabolism; D008875:Middle Aged; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D027361:Organic Anion Transporters / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent / Q000378:metabolism; D029362:Organic Anion Transporters, Sodium-Independent / Q000378:metabolism; D010666:Phenylpropionates / Q000494:pharmacology*; D011724:Pyridazines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D017466:Receptors, Endothelin / Q000378:metabolism; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D013449:Sulfonamides / Q000494:pharmacology*; D027981:Symporters / Q000378:metabolism; D013656:Taurocholic Acid / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D001647:Bile Acids and Salts; D000077300:Bosentan; D065128:Endothelin Receptor Antagonists; D007555:Isoxazoles; D027381:Liver-Specific Organic Anion Transporter 1; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D027361:Organic Anion Transporters; D029363:Organic Anion Transporters, Sodium-Dependent; D029362:Organic Anion Transporters, Sodium-Independent; D010666:Phenylpropionates; D011724:Pyridazines; D011743:Pyrimidines; D017466:Receptors, Endothelin; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D013449:Sulfonamides; D027981:Symporters; D013656:Taurocholic Acid; D013876:Thiophenes; C116614:ABCG2 protein, human; C503999:SLCO1B1 protein, human; C421209:SLCO1B3 protein, human; C071908:sodium-bile acid cotransporter; C467894:ambrisentan; C106276:sitaxsentan; C533860:macitentan