BOL-303242-X, a novel selective glucocorticoid receptor agonist, with full anti-inflammatory properties in human ocular cells

Abstract

Purpose: BOL-303242-X is a novel selective glucocorticoid receptor agonist under clinical evaluation for the treatment of inflammatory skin and eye diseases. Data from in vitro and in vivo studies suggest an improved side-effect profile of this compound compared to classical glucocorticoids. The aim of this study was to determine the anti-inflammatory effect of BOL-303242-X in ocular cells.

Methods: Four primary human ocular cell cultures, including human conjunctival fibroblasts (HConFs), human corneal epithelial cells (HCEpiCs), human optic nerve astrocytes (HONAs), and human retinal endothelial cells (HRECs), as well as a human monocytic cell line, THP-1, were challenged with either lipopolysacharide (LPS) or interleukin-1ss (IL-1ss). Luminex technology was used to determine the effect of BOL-303242-X on LPS- or IL-1ss-induced cytokine release and intercellular adhesion molecule-1 (ICAM-1) levels. Effects of BOL-303242-X on nuclear factor kappa B (NFkappaB) and mitogen-activated protein kinase (MAPK) in HCEpiCs were also assessed by measuring inhibitory kappa B protein-alpha (IkappaB-alpha), phosphorylated p65 NFkappaB, and MAPK levels by western blotting. Dexamethasone (DEX) or triamcinolone acetonide (TA) was used as the control.

Results: LPS or IL-1ss induced multiple cytokine release in all cell types studied. BOL-303242-X significantly reduced LPS- or IL-1ss-induced inflammatory cytokine release in a dose-dependent manner, including granulocyte colony-stimulating factor (G-CSF), IL-1ss, IL-6, IL-8, IL-12p40, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-alpha). BOL-303242-X showed activity and potency comparable to that observed for DEX or TA. A statistically significant inhibitory effect of BOL-303242-X was observed at doses ranging from 1 to 100 nM in HConFs, HCEpiCs, HONAs, and THP-1. The IC(50) values for these effects were in the low nM range. BOL-303242-X also significantly reduced LPS-induced IL-1ss release and ICAM-1 levels in HRECs. Furthermore, BOL-303242-X inhibited IL-1ss-induced decreases in IkappaB-alpha levels, as well as IL-1ss-induced phosphorylation of NFkappaB, p38, and c-Jun-N-terminal kinase (JNK) MAPKs in HCEpiCs.

Conclusions: BOL-303242-X acts as a potent anti-inflammatory agent in various primary human ocular cells with similar activity and potency compared to classical steroids. Results also suggest that MAPK (p38 and JNK) and NFkappaB signaling pathways are involved in the anti-inflammatory properties of BOL-303242-X in HCEpiCs. An improved side effect profile of this novel SEGRA compound has been reported recently. Thus, BOL-303242-X may provide a new option for the treatment of ophthalmic conditions with an inflammatory component.

Figure 1

BOL-303242-X demonstrates similar activity as dexamethasone (DEX) in reducing IL-1ß-induced cytokine release from human conjunctival fibroblasts. Cells were pretreated with BOL-303242-X or DEX for 2 h, and then further treated for 18 h with vehicle (0.1% DMSO), IL-1ß, BOL-303242-X, DEX, or combinations of them. Cytokine content in the conditioned media was determined by Luminex. Data are means±SEM or geometric means±SE estimated by the Taylor Series expansion for the MCP-1 data; n=3. The asterisk indicates a p≤0.05 versus that for IL-1ß. The dagger indicates a p≤0.05 versus BOL-303242-X at same concentration. Statistical analysis was performed using two-way ANOVA followed by the Tukey-Kramer test on raw data for G-CSF and IL-8. Data were elevated to the power of 0.4 for IL-6 and by taking the logarithm of MCP-1 data. A Student’s t-test was used to determine whether IL-1ß was effective at increasing cytokine release. The solid bar is statistically significant versus the gray bar at increasing cytokine release.

Figure 2

BOL-303242-X demonstrates similar activity as dexamethasone (DEX) in reducing IL-1ß-induced cytokine release from human corneal epithelial cells. Cells were pretreated with BOL-303242-X or DEX for 2 h, and then further treated with vehicle (0.1% DMSO), IL-1ß, BOL-303242-X, DEX, or combinations of them for 18 h. Cytokine content in the conditioned media was determined by Luminex. Data are means±SEM; n=3. The asterisk indicates a p≤0.05 versus IL-1ß, and the dagger indicates a p≤0.05 versus BOL-303242-X at the same concentration. Statistical analysis was performed using two-way ANOVA followed by the Tukey-Kramer test on raw data. A Student’s t-test was used to determine whether IL-1ß was effective. The solid bar is statistically significant versus the gray bar at increasing cytokine release.

Figure 3

BOL-303242-X demonstrates similar activity as triamcinolone acetonide (TA) in reducing LPS-induced cytokine release in human optic nerve astrocytes. Cells were pretreated with BOL-303242-X or TA for 2 h, and then further treated with vehicle (0.1% DMSO), LPS, BOL-303242-X, TA, or combinations of them at the indicated doses for 18 h. Cytokine content in the conditioned media was determined by Luminex. Data are means±SEM; n=3. The asterisk indicates a p≤0.05 versus LPS. The dagger indicates a p≤0.05 versus TA at the same concentration. Statistical analysis was performed using two-way ANOVA followed by the Tukey-Kramer test on raw data. A Student’s t-test was used to determine whether LPS was effective at increasing cytokine release. The solid bar is statistically significant versus the gray bar.

Figure 4

BOL-303242-X demonstrates similar activity as triamcinolone acetonide (TA) in reducing LPS-induced IL-1ß and ICAM-1 levels in human retinal endothelial cell lysates. Cells were pretreated with BOL-303242-X or TA for 2 h, and then further treated with vehicle (0.1% DMSO), LPS, BOL-303242-X, TA, or combinations of them at the indicated doses for 18 h. Cytokine and ICAM-1 content in the cell lysates was determined by Luminex, and protein content in the lysates by the Micro BCA assay. Data are means±SEM; n=3. The asterisk indicates a p≤0.05 versus the control, and the dagger indicates a p≤0.05 versus LPS. Statistical analysis was performed using one-way ANOVA followed by the Tukey-Kramer test on raw data.

Figure 5

BOL-303242-X reduces LPS-induced cytokine release with similar activity as dexamethasone (DEX) from THP-1 cells. Cells were pretreated with BOL-303242-X or DEX for 2 h, and then further treated with vehicle (0.1% DMSO), LPS, BOL-303242-X, DEX, or their combinations at the indicated doses for 18 h. Cytokine content in the conditioned media was determined by Luminex. Data are means±SEM or geometric means±SE estimated by the Taylor series expansion for the MCP-1 data; n=3. The asterisk indicates a p≤0.05 versus LPS. The dagger indicates a p≤0.05 versus DEX at the same concentration. Statistical analysis was performed using two-way ANOVA followed by the Tukey-Kramer test, and data elevated to the power of 0.4 for IL-6, 0.8 for IL-12p40, and by taking the logarithm of MCP-1 data. A Student’s t-test was used to determine whether LPS was effective at increasing cytokine release. The solid bar is statistically significant versus the gray bar.

Figure 6

BOL-303242-X and dexamethasone (DEX) reduce IL-1ß-induced JNK and p38 MAPK phosphorylation in human corneal epithelial cells. Cells were pretreated with vehicle, BOL-303242-X, or DEX for 2 h, and then further treated with vehicle, IL-1ß, IL-1ß plus BOL303242-X, or DEX in basic EpiLife medium for 30 min. Cell lysates were prepared, resolved by SDS-PAGE, and western blotting was performed with phospho and total p38 or JNK antibodies. The top images show representative western blots for phosphorylated JNK (pJNK), total JNK, phosphorylated p38 (pp38), and total p38. At the bottom, the results of the quantification by densitometry and phosphorylated:total JNK or p38 ratios are shown for the pJNK (left panel) and pp38 (right panel) blots. The data are expressed as % of IL-β. Data are means±SEM from three to four independent experiments. The asterisk indicates a p≤0.05 versus IL-1ß. One-way ANOVA followed by the Dunnett’s test on data elevated to the power of 0.8 for pJNK and on raw data for p38. In the figure, BOL represents BOL-303242-X.

Figure 7

Effects of BOL-303242-X and dexamethasone (DEX) on p65 NFκB phosphorylation and IκB-α protein levels in human corneal epithelial cells. Cells were pretreated with vehicle, BOL-303242-X, or DEX for 2 h, and then further treated with vehicle, IL1ß, IL-1ß plus BOL-303242-X, or DEX in basic EpiLife medium for 10 min. Cell lysates were prepared and resolved by SDS-PAGE. Western blotting was performed with phospho-p65 NFκB followed by p65 NFκB antibodies, or with IκB-α followed by keratin antibodies. The top images show representative blots for phosphorylated p65 NFκB (pp65 NFκB), total p65 NFκB, IκB-α, and keratin. At the bottom, the results of the densitometric quantification of the blots for pp65NFκB (left panel) and IκB-α (right panel) phosphorylated:total p65 NFκB or IκB-α:keratin ratios are shown. The data are expressed as % of IL-β. Data are means±SEM from three to four independent experiments. The asterisk indicates a p≤0.05 versus IL-1ß. One-way ANOVA followed by the Dunnett’s test on raw data for pNFκB and IκB-α. In the figure, BOL represents BOL-303242-X.