VBP15, a glucocorticoid analogue, is effective at reducing allergic lung inflammation in mice

Abstract

Asthma is a chronic inflammatory condition of the lower respiratory tract associated with airway hyperreactivity and mucus obstruction in which a majority of cases are due to an allergic response to environmental allergens. Glucocorticoids such as prednisone have been standard treatment for many inflammatory diseases for the past 60 years. However, despite their effectiveness, long-term treatment is often limited by adverse side effects believed to be caused by glucocorticoid receptor-mediated gene transcription. This has led to the pursuit of compounds that retain the anti-inflammatory properties yet lack the adverse side effects associated with traditional glucocorticoids. We have developed a novel series of steroidal analogues (VBP compounds) that have been previously shown to maintain anti-inflammatory properties such as NFκB-inhibition without inducing glucocorticoid receptor-mediated gene transcription. This study was undertaken to determine the effectiveness of the lead compound, VBP15, in a mouse model of allergic lung inflammation. We show that VBP15 is as effective as the traditional glucocorticoid, prednisolone, at reducing three major hallmarks of lung inflammation--NFκB activity, leukocyte degranulation, and pro-inflammatory cytokine release from human bronchial epithelial cells obtained from patients with asthma. Moreover, we found that VBP15 is capable of reducing inflammation of the lung in vivo to an extent similar to that of prednisone. We found that prednisolone--but not VBP15 shortens the tibia in mice upon a 5 week treatment regimen suggesting effective dissociation of side effects from efficacy. These findings suggest that VBP15 may represent a potent and safer alternative to traditional glucocorticoids in the treatment of asthma and other inflammatory diseases.

Conflict of interest statement

Competing Interests: Jesse M. Damsker is an employee of ReveraGen BioPharma and has stock options. Blythe C. Dillingham was an employee of ReveraGen BioPharma during the time the work was done on this manuscript. Erica K. M. Reeves is an employee of ReveraGen BioPharma and has stock options. John M. McCall is an employee of PharMac LLC and has founder shares and a board membership with ReveraGen BioPharma. Eric P. Hoffman has founder shares and a board membership with ReveraGen BioPharma. Finally, Kanneboyina Nagaraju has founder shares and a board membership with ReveraGen BioPharma. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Structure of VBP15 and schematic of the OVA-induced model of acute allergic lung inflammation.

(A) Chemical structures of prednisone (left panel), prednisolone (mid panel), and VBP15 (right panel). VBP compounds include a delta-9,11 double bond and tail group modifications. (B) Diagram of OVA-induced mouse model of allergic lung inflammation.

Figure 2. VBP15 reduces leukocyte infiltration in the OVA-induced model of acute allergic lung inflammation.

OVA-challenged mice were either left untreated or treated with oral doses of prednisone, VBP15 (20 mg/kg), or cherry syrup alone daily for 6 days. A group of non-challenged mice (naïve) was included in order to assess basal inflammatory parameters. Lung tissue and BAL cells underwent FACS analysis to determine the number of infiltrating eosinophils (A and C) and effector/memory CD4+T cells (B and D). Bar graphs represent mean (±SE) cell numbers. Percentages indicate percentage reduction compared to vehicle control. *p<0.05; **p<0.01; ***p<.001 compared to syrup group with n = 5 mice per group.

Figure 3. VBP15 reduces acute allergic lung inflammation.

OVA-challenged mice were either left untreated or treated with oral doses of prednisone, VBP15 (20 mg/kg), or cherry syrup alone daily for 6 days. A group of non-challenged mice (naïve) was included in order to assess basal inflammatory parameters. Perfused whole lungs were processed for histological analysis and stained with H&E (A) or PAS (B). Images (10× magnification) represent areas of tissue surrounding bronchioles. Arrows on H&E sections indicate inflammatory foci. Percentage of PAS positive airways were counted via bright field microscopy (C). Bar graph represents mean (±SE)% PAS positive airways. *p<.05; **p<0.01 compared to the vehicle control group with n = 5 mice per group. IL-13 (D) and RANTES (E) were measured in BAL fluid by flow cytometric bead array. Bar graphs represent mean (±SE) cytokine concentration values. *p<.05; **p<0.01 compared to syrup group with n = 5 mice per group.

Figure 4. VBP15 inhibits NFκB activity.

A549 cells stably-transfected with a luciferase NFκB construct were exposed to increasing concentrations of VBP15 or prednisolone (3, 30, 300, 3000 nM) followed by TNFα stimulation before measuring luciferase activity. Bar graph represents mean (±SE) luciferase units. *p<.012 (due to the Bonferroni adjustment for multiple comparisons) compared to treatment with vehicle control. Data represents 4 biological replicates with assay performed in triplicate.

Figure 5. VBP15 reduces leukocyte degranulation.

Anti-DNP-sensitized RBL-2H3 cells were treated with prednisolone (50 µM), VBP-15 (50 µM), or vehicle control (DMSO) for 7 minutes followed by addition of DNP to induce degranulation. The reaction was allowed to proceed for an additional 20 minutes before supernatant was removed and tested for β-hexosaminidase content. A well of untreated cells was lysed to gauge total β-hexosamindase content. Release percentage was determined using a formula described in Materials and Methods. Bar graph represents mean (±SE) release percentage. **p

Figure 6. VBP15 reduces basolateral cytokine secretion from human bronchial epithelial cells obtained from asthmatic patients.

HBE cells from 3 separate human donors were pulse-treated with VBP15 (10 µM) or vehicle control (DMSO). Basolateral surface supernatant was tested for the presence of TGFβ1 (left panel) and IL-13 (right panel) by flow cytometric bead array. Bar graphs represent mean (±SE) concentration values. **, p<0.01 compared to vehicle control with n = 3 donors. N.D. = Not Detectible (lower limit of detection = 4.5 pg/ml).

Figure 7. VBP15 does not induce tibia length shortening.

Wildtype outbred CD1 mice were treated daily for 5 weeks with VBP15 (30 and 45 mg/kg), prednisolone (10 mg/kg) or vehicle control starting at 12 days of age. At the end of the treatment, tibias were harvested and measured. Bar graph represents mean (±SE) tibia length values. *p