Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators

Abstract

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.

Figure 1. LY2108491 and LY2109866 are nonsecosteroidal VDR ligands.

(A) Nonsecosteroidal compounds are potent agonists in a VDR-based ligand-sensing assay. SaOS-2 cells were cotransfected with expression vectors encoding Gal4-RXRα-LBD and VP16-VDR-LBD along with a Gal4-responsive luciferase reporter. After transfection, cells were treated with vehicle or various concentrations of 1,25-(OH)₂D₃, LY2108491, or LY2109866, and the reporter activity was expressed as light units α SEM. A schematic of the ligand-sensing assay and the chemical structures of 1,25-(OH)₂D₃, LY2108491, and LY2109866 are also presented. tk-LUC, thymidine kinase–LUC. (B) Nonsecosteroidal VDR ligands induce VDRE-dependent expression of the rat osteocalcin promoter in osteoblasts. ROS17/2.8 cells stably transfected with rat osteocalcin reporter (OCN-LUC) were treated with various concentrations of 1,25-(OH)₂D₃, LY2108491, or LY2109866. Results are shown in percentage of the luciferase activity obtained by treating the cells with 1 μM 1,25-(OH)₂D₃. All the transfections were performed in triplicat

Figure 2. Binding of nonsecosteroidal VDR ligands to VDR-LBD in a classical biochemical ligand-binding assay.

Inhibition of [³H]-1,25-(OH)₂D₃ binding to VDR by cold 1,25-(OH)₂D₃ and its nonsecosteroidal analogs is shown. VDR ligand-binding activity was determined by using purified His-tagged VDR-LBD along with [³H]-1,25-(OH)₂D₃ (1 nM). Concentration of each of the cold competitor ligands is shown.

Figure 3. Nonsecosteroidal VDR ligands are potent agonists in keratinocytes and PBMCs.

(A) LY2108491 and LY2109866 are potent inhibitors of keratinocyte proliferation. KerTr cells plated in 96-well plates were dosed with various concentrations of 1,25-(OH)₂D₃, LY2108491, or LY2109866 for 72 hours at 37°C before BrdU incorporation into DNA was analyzed as a measure of cell proliferation. Results (mean α SEM) of experiments performed in triplicate are shown. (B) Nonsecosteroidal VDR ligands are potent inducers of CYP24 gene expression in keratinocytes. TaqMan quantitative RT-PCR (Q-PCR) was performed on total RNA prepared from KerTr cells treated with various concentrations of 1,25-(OH)₂D₃, LY2108491, or LY2109866 for 24 hours. Levels of GAPDH mRNA were measured in all the samples, and the results were normalized and presented as fold induction (α SEM) compared with normalized CYP24 levels in vehicle-treated cells. (C) Nonsecosteroidal VDR ligands are efficacious in TPA- and PHA-activated PBMCs. Primary cells isolated from donors were stimulated with TPA (100 ng/ml) and PHA (25 μl/ml) and treated with vehicle or 100 nM each of 1,25-(OH)₂D₃, LY2108491, or LY2109866 for 24 hours. TaqMan Q-PCR was performed on RNA obtained from vehicle-treated or VDR ligand–treated samples, using primer pairs and probes for IL-2, IL-4, IL-10, GATA3, and GAPDH. The amount of IL-2, IL-4, IL-10, and GATA3 transcripts relative to GAPDH transcripts is shown as mean α SEM of quadruplicate experimes.

Figure 4. LY2108491 and LY2109866 decrease Th1 cytokine response in vivo.

Immunized mice were treated with LY2108491 (10 and 100 μg/kg), LY2109866 (10 and 100 μg/kg), or vehicle for 10 days. Splenocytes were stimulated ex vivo with 10 μg/ml MOG_35–55 peptide. Supernatants were collected 72 hours after stimulation, and IFN-γ (A) and IL-2 (B) levels were determined by Luminex. The values shown represent the mean of triplicate determinations, and error bars represent SEM. *P < 0.0

Figure 5. LY2108491 and LY2109866 are less potent in inducing VDR-dependent gene expression in intestinal cells.

Luciferase activity (α SEM) of Caco-2 cells transfected with Gal4-VDR-LBD in a mammalian 1-hybrid setting in the presence of vehicle or various concentrations of 1,25-(OH)₂D₃, LY2108491, or LY2109866 are shown. Results are in arbitrary light units obtained from experiments performed in triplicate.

Figure 6. LY2108491 and LY2109866 are less potent and efficacious in inducing the expression of vitamin D–responsive genes in intestinal cells.

(A) Nonsecosteroidal VDR ligands show weak potencies in inducing the expression of endogenous CaT1 genes in differentiated Caco-2 cells. TaqMan Q-PCR was performed on total RNA prepared from differentiated Caco-2 cells treated with various concentrations of 1,25-(OH)₂D₃, LY2108491, or LY2109866 for 24 hours. Levels of GAPDH mRNA were measured in all the samples, and the results were normalized and presented (α SEM) as RLU after normalization with the GAPDH transcript levels. (B) LY2108491 and LY2109866 are poor inducers of endogenous vitamin D–responsive genes in intestinal cells. TaqMan Q-PCR was performed on total RNA prepared from differentiated Caco-2 or rat duodenal IEC-6 cells treated with vehicle or 100 nM each of 1,25-(OH)₂D₃, LY2108491, or LY2109866 for 24 hours. The amount of CYP24 and calbindin-9k transcripts relative to GAPDH transcripts is shown as mean α SEM of quadruplicate experiment

Figure 7. Nonsecosteroidal VDRMs are less potent than 1,25-(OH)₂ D₃ in recruiting p160 family members to VDR.

Luciferase activity (α SEM) of Caco-2 cells transfected with Gal4–SRC-1 (A), Gal4-TIF2 (B), or Gal4-AIB1 (C) and VP16-VDR-LBD in a mammalian 2-hybrid setting in the presence of vehicle or various concentrations of 1,25-(OH)₂D₃, LY2108491, or LY2109866 is shown. Luciferase activity is shown as arbitrary light units obtained from experiments performed in triplicate.

Figure 8. Differential interaction of PGC-1α–receptor interaction with VDR ligands.

Luciferase activity (α SEM) of HEK 293 cells transfected with Gal4-VDR-LBD, Gal4-VDR-LBD^ΔAF-2, or Gal4-VDR-LBD^E420A with or without PGC-1α expression vector in the presence of vehicle or 1 μM each of 1,25-(OH)₂D₃, LY2108491, or LY2109866 is shown. Luciferase activity is shown as arbitrary light units obtained from experiments performed in triplicate.

Figure 9. VDRMs are less calcemic in vivo and show efficacy in a surrogate animal model of psoriasis.

(A) Histological evaluation of hairless mouse skin after topical treatment with 1,25-(OH)₂D₃ at indicated doses. (B) Effect of various concentrations of topical 1,25-(OH)₂D₃ treatment on blood ionized calcium levels in hairless mice. (C) Histological evaluation of hairless mouse skin after topical treatment with LY2108491 at indicated doses. (D) Effect of various concentrations of topical LY2108491 treatment on blood ionized calcium levels in hairless mice. Error bars represent SEM of the mean. *P < 0.05. ^#Hypercalcemia above the normal blood ionized calcium level of 1.34 m

Figure 10. VDRMs do not induce epidermal proliferation in VDR-knockout mice.

Histological evaluation of VDR^+/+ or VDR^–/– mouse skin after topical treatment with vehicle, 1,25-(OH)₂D₃ (10 nmol), LY2108491 (50 nmol), or LY2109866 (50 nmol) is presented. Mice were sacrificed at 72 hours after dosing, and skin samples were collected. Epidermal thickness was examined by H&E staining of paraffin sections. Epidermis is shown with green arrows.

Figure 11. LY2108491 and LY2109866 are noncalcemic in vivo when administered orally in a short-term mouse model of hypercalcemia.

1,25-(OH)₂D₃, LY2108491, or LY2109866 were administered in sesame seed oil at indicated doses to mice by gavage for 6 consecutive days, and blood ionized calcium was measured 6 hours after the last dose. Error bars represent SEM. *P < 0.0