Selectivity of the multidrug resistance modulator, LY335979, for P-glycoprotein and effect on cytochrome P-450 activities.
Overexpression of ATP-dependent drug efflux pumps, P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP), confers multidrug resistance to tumor cells. Modulators of multidrug resistance block the action of these pumps, thereby sensitizing cells to oncolytics. A potent Pgp modulator is LY335979, which fully sensitizes Pgp-expressing cells at 0.1 microM in cytotoxicity assays and for which Pgp has an affinity of 59 nM. The present study examines its effect on MRP1-mediated drug resistance and cytochrome P-450 (CYP) activity and its ability to serve as a Pgp substrate. Drug resistance was examined with HL60/ADR and MRP1-transfected HeLa-T5 cells. Drug cytotoxicity was unaffected by 1 microM LY335979; leukotriene C4 uptake into HeLa-T5 membrane vesicles was unaffected. Because the substrate specificity of Pgp and CYP3A overlap, the effect of LY335979 on the 1'-hydroxylation of midazolam by CYP3A in human liver microsomes was examined. The apparent K(i) was 3.8 microM, approximately 60-fold higher than the affinity of Pgp for LY335979. The modulator's effect on Pgp was evaluated with Pgp-overexpressing CEM/vinblastine (VLB)(100) and parental CCRF-CEM cells. Both cell lines accumulated [(3)H]LY335979 equally well and did not efflux [(3)H]LY335979 during a 3-h incubation, indicating that it is not a substrate of Pgp. Equilibrium-binding studies with CEM/VLB(100) plasma membranes and [(3)H]LY335979 showed that Pgp had a K(d) of 73 nM, which is in good agreement with the previously determined K(i) value. Thus, LY335979 is an extremely potent Pgp, and not MRP1 or MRP2, modulator and has a significantly lower affinity for CYP3A than for Pgp.
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana, USA. Dantzig_Anne_H@lilly.com
A H
AH
Dantzig
R L
RL
Shepard
K L
KL
Law
L
L
Tabas
S
S
Pratt
J S
JS
Gillespie
S N
SN
Binkley
M T
MT
Kuhfeld
J J
JJ
Starling
S A
SA
Wrighton
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000187:drug effects* / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000187:drug effects* / Q000378:metabolism; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects* / Q000378:metabolism; D003986:Dibenzocycloheptenes / Q000378:metabolism / Q000494:pharmacology*; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000187:drug effects; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011804:Quinolines / Q000378:metabolism / Q000494:pharmacology*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System; D003986:Dibenzocycloheptenes; D007527:Isoenzymes; D017997:Leukotriene C4; D006899:Mixed Function Oxygenases; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011804:Quinolines; C095179:zosuquidar trihydrochloride; C104464:CYP3A protein, human