Discovery of a highly potent glucocorticoid for asthma treatment

Abstract

Glucocorticoids are the most effective treatment for asthma. However, their clinical applications are limited by low efficacy in severe asthma and by undesired side effects associated with high dose or prolonged use. The most successful approach to overcome these limitations has been the development of highly potent glucocorticoids that can be delivered to the lungs by inhalation to achieve local efficacy with minimal systemic effects. On the basis of our previous structural studies, we designed and developed a highly potent glucocorticoid, VSGC12, which showed an improved anti-inflammation activity in both cell-based reporter assays and cytokine inhibition experiments, as well as in a gene expression profiling of mouse macrophage RAW264.7 cells. In a mouse asthma model, VSGC12 delivered a higher efficacy than fluticasone furoate, a leading clinical compound, in many categories including histology and the number of differentiated immune cells. VSGC12 also showed a higher potency than fluticasone furoate in repressing most asthma symptoms. Finally, VSGC12 showed a better side effect profile than fluticasone furoate at their respective effective doses, including better insulin response and less bone loss in an animal model. The excellent therapeutic and side effect properties of VSGC12 provide a promising perspective for developing this potent glucocorticoid as a new effective drug for asthma.

Keywords: Glucocorticoids; VSGC12; asthma; glucocorticoid receptor; potency.

Figure 1

VSGC12 is a highly potent glucocorticoid. (a) Chemical structure of VSGC12. (b) VSGC12 has potency higher than VSG22, but lower than FF, in reporter assays in AD293 cells. Left panel, MMTV induction reporter assay; right panel, AP-1 repression reporter assay. RLU, relative luciferase units. (c) An examination of off-target effects in MR and PR reporter assays. Steroid concentration: 1 μM. Data were plotted as percent of that at 1 μM native ligands (corticosterone for MR, progesterone for PR). *P<0.05; **P<0.01; ***P<0.001, n.s., not significant; n=3. (d). An in vitro H³-Dex competition binding assay for examining ligand-binding affinity to GR. Radioactive ligand H³-DEX: 15 nM. The IC₅₀ for DEX, VSG22, VSGC12 and FF are 13.4, 4.1, 6.1 and 2.7  nM, respectively. (e) A cytokine inhibition assay for testing the ability of candidate ligands to suppress cytokine IL-6 release from A549 cells. The IC₅₀ for DEX, VSG22, VSGC12 and FF are 0.80, 0.07, 0.05 and 0.03 nM, respectively.

Figure 2

Gene expression profile of VSGC12. (a) A microarray gene expression analysis of VSGC12, VSG22, FF and DEX in mouse macrophage RAW264.7 cells on the induction of an inflammation response. Inflammation was induced by 1 μg ml^–1 LPS at a steroid hormone concentration of 100 nM. Data were plotted as relative expression level to vehicle (DMSO), and aligned to the gene expression pattern seen upon DEX treatment, from most downregulated to most upregulated genes. (b) Venn diagrams of genes induced or repressed more than twofold in RAW264.7 cells. (c) Gene expression profile of pro-inflammatory cytokines in RAW264.7 cells. Data were normalized to DEX as 0. (d) A validation of gene expression in RAW264.7 cells by quantitative PCR. Error bars indicate s.d., n=3, statistics analysis is based on ΔΔCt, *P<0.05; **P<0.01, n.s., not significant.

Figure 3

Examination of the efficacy of VSGC12 in a mouse OVA-induced asthma model. (a) Lung function (AHR) of BALB/c mice with different treatment. RL, resistance of lung, cm H₂O.s/ml. ACH, acetylcholine. (b) Differentiated cells count. Macro, macrophage; Eos, eosinophils; LC, lymphocytes; PMN, phagocytic monocytes. (c) OVA-specific IgE. OD, arbitrary unit=(OD₄₀₅–OD₅₄₀)×dilution factor. (d) Histology score, left panel, hematoxylin and eosin (HE) staining; right panel, PAS staining. Error bars indicate s.e.m., each group n=10. *P<0.05; **P<0.01; ***P<0.001; n.s., not significant.

Figure 4

Examination of the potency of VSGC12 in a mouse OVA-induced asthma model. (a) Lung function of BALB/c mice treated with 0.25 mg kg^–1 VSGC12, or FF. (b) Differential cells count in the lungs of mice treated with 0.25 mg kg^–1 VSGC12 or FF. (c) Lung function of BALB/c mice treated with the indicated amounts of VSGC12 or FF. (d) Differential cell count in the lungs of mice treated with the indicated amounts of VSGC12 or FF. (e) A side-by-side comparison of the repression activities of various doses of VSGC12 and FF on AHR. Data were plotted as percent of repression at the maximal ACH challenge of 4 mg kg^–1, with vehicle control (OVA+DMSO) as 0 and saline as 100. (f) A side-by-side comparison of the repression activity of VSGC12 and FF at various doses on total differential cell count. Data were plotted as percent of repression between saline (100) and OVA+DMSO (0). Error bars indicate s.e.m., each group n=8. *P<0.05; **P<0.01; ***P<0.001.

Figure 5

Examination of side effects of VSGC12 and FF at the effective dose. (a) Body weight of DBA/1 mice treated with the indicated daily dose of steroids for 2 weeks. Mice body weight was measured at day 0, 7 and 14. (b) Insulin-tolerance test of mice treated with the indicated daily doses of steroids for 7 days. Glucose level was measured at day 7 at various time points before and after insulin injection. Data were plotted as percent of the glucose level of time 0. (c) Plasma insulin levels of mice treated with the indicated daily doses of steroid for 7 days. (d) Spleen size and weight for mice treated with the indicated daily doses of steroid for 2 weeks. (e) Cortical bone thickness of femurs of mice treated with the indicated daily doses of steroid for 2 weeks. (f) Bone stiffness of femurs of mice treated with the indicated daily doses of steroid for 2 weeks. Mouse strain: DBA/1. Each treatment n=5, error bars indicate s.e.m., *P<0.05; **P<0.01; ***P<0.001.