Encequidar (HM30181)

HM30181 potently and selectively inhibits Pgp-mediated efflux transport of rhodamine 123 in CCRF-CEM T cells with 13.1±2.3 nM.^[1]

Briefly, cells expressing wild-type Pgp (CCRF-CEM T lymphoblast cell line) were sedimented, the supernatant was removed by aspiration, and the cells were resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM) containing rhodamine 123 at a final concentration of 0.2 μg/ml (0.53 μM). Cells were loaded with fluorochrome for 30 min at 37°C. Tubes were chilled on ice and cells were harvested at 500 × g in an Eppendorf 5403 centrifuge. The cell pellet was washed with ice cold DMEM medium (pH 7.4) and again centrifuged at 500 × g. After resuspension, aliquots of the cell suspension, each containing approximately 1 × 10⁶ were transferred to individual fluorescence-activated cell sorter (FACS) tubes and again centrifuged. Supernatants were removed, and the pellets were resuspended individually in prewarmed DMEM medium (pH 7.4) containing either no inhibitor or HM30181 mesylate or elacridar hydrochloride or tariquidar dimesylate at various concentrations in DMSO ranging from 1 to 100 nM. Right after resuspending cells each individual tube was placed in a temperature controlled unit and cell associated fluorescence was monitored over a period of 5 min with a FACS system.

PET scans with the Pgp substrate (R)-[¹¹C]verapamil in FVB wild-type mice pretreated i.v. with HM30181 (10 or 21 mg/kg) fails to show significant increases in (R)-[¹¹C]verapamil brain uptake compared with vehicle treated animals.^[1]