Strategies and limitations associated with in vitro characterization of vitamin D receptor activators

Abstract In vitro cell-based assays are common screening tools used for the identification of new VDR ligands. For 25-hydroxyvitamin D 3 [25(OH)D 3 ] and 1α-hydroxyvitamin D 3 [1α(OH)D 3 ], protein expressions of CYP2R1 and CYP27B1, respectively, that form the active 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] ligand were detected in human embryonic kidney (HEK293) cells expressing the GAL4-hVDR, the human brain microvessel endothelial (hCMEC/D3) and adenocarcinoma colonic (Caco-2) cells. The impact of bioactivation enzymes was shown upon the addition of ketoconazole (10 μM KTZ), a pan-CYP inhibitor, which reduced the apparent potency of 25(OH)D 3 and increased the EC 50 from 272 to 608 nM in HEK293 cells. EIA assays verified that 1,25(OH) 2 D 3 was formed and contributed to VDR activity independently of its precursors. In hCMEC/D3 cells where enzyme protein levels were lowest, changes in MDR1/P-gp expression with KTZ were minimal. In Caco-2 cells, the induction of TRPV6 (calcium channel), CYP24A1, CYP3A4, OATP1A2 and MDR1 mRNA expression was 1,25(OH) 2 D 3  > 1α(OH)D 3  > 25(OH)D 3 , with the magnitude of change being blunted by KTZ. Upon inclusion of KTZ in the cell-based assays, high transcriptional activities were observed for synthetic VDR activators from Teijin Pharma. Cyclopentanone derivatives: TPD-003, TPD-005, TPD-006, TPD-008 and TPD-009 (EC 50 s 0.06 to 67 nM, unchanged with KTZ) were found more potent over straight chain and lactone derivatives (antagonists). Most TPD compounds activated OATP1A2, CYP24A1, CYP3A4, and MDR1 (28–67%) and TRPV6 transcriptionally in Caco-2 cells. The results identified that cell-based assays with added KTZ could accurately identify new VDR activators, although these may be hypercalcemic with strong TRPV6 inducing properties.