Transport of 5,5-diphenylbarbituric acid and its precursors and their effect on P-gp, MRP2 and CYP3A4 in Caco-2 and LS180 cells.
OBJECTIVE
To examine the transport of 5,5-diphenylbarbituric acid sodium (T2007) and its mono- (MMMDPB) and di- (T2000) methoxymethylated precursors and their inducibility potential in Caco-2 and LS180 cells.


METHODS
Transport studies of T2000, MMMDPB and T2007 in Caco-2 cells were performed in Transwells. P-gp and CYP3A4 activities were assayed by [(3)H]digoxin and rhodamine 123 cellular retention and testosterone 6β-hydroxylation, respectively. Expressions of PXR, VDR and CAR mRNA and CYP3A4, MDR1/P-gp and MRP2 mRNA and protein were determined by qPCR and Western blotting, respectively. PXR siRNA was used to assess the involvement of PXR.


RESULTS
The P(app(A→B))s and P(app(B→A))s of T2000, MMMDPB and T2007 were similar (30-35×10(-6)cm/s) in Caco-2 cells. Treatment for 3 days with T2000 (15μM), MMMDPB (70μM) and T2007 (300μM) generally furnished a greater induction in LS180 cells over the Caco-2 cells due to the higher, natural abundance of PXR. Changes in expression were confined mostly to MDR1 and CYP3A4: in LS180 cells, treatment for 3 days increased MDR1 and CYP3A4 but not MRP2 mRNA, and elevated P-gp and CYP3A4 protein expression that led to decreased cellular accumulation of [(3)H]digoxin and rhodamine 123, and enhanced testosterone 6β-hydroxylase activity towards T2007, respectively. The silencing of PXR by PXR siRNA in LS180 cells significantly attenuated the induction of MDR1 and CYP3A4.


CONCLUSIONS
T2000, MMMDPB, and T2007 exhibited high permeabilities but are not P-gp substrates. T2007 and its analogs upregulated CYP3A4 and MDR1 modestly via the PXR.
Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario, Canada.
J
J
Fan
H-J
HJ
Maeng
Y
Y
Du
D
D
Kwan
K S
KS
Pang
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D001692:Biological Transport; D001711:Biotransformation; D015153:Blotting, Western; D018938:Caco-2 Cells; D018929:Cell Culture Techniques; D002463:Cell Membrane Permeability; D002851:Chromatography, High Pressure Liquid; D000090702:Constitutive Androstane Receptor; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D006801:Humans; D007408:Intestinal Absorption; D007413:Intestinal Mucosa / Q000378:metabolism; D015394:Molecular Structure; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D010634:Phenobarbital / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D000077297:Pregnane X Receptor; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics*; D034741:RNA, Small Interfering / Q000235:genetics; D018167:Receptors, Calcitriol / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D011987:Receptors, Steroid / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000090702:Constitutive Androstane Receptor; D051544:Cytochrome P-450 CYP3A; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010634:Phenobarbital; D000077297:Pregnane X Receptor; D011355:Prodrugs; D034741:RNA, Small Interfering; D018167:Receptors, Calcitriol; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; C000715087:ABCC2 protein, human; C044127:5,5-diphenylbarbituric acid; C510163:CYP3A4 protein, human