A novel strategy for development of glucocorticoids through non-genomic mechanism

Abstract

Glucocorticoids (GCs) are routinely believed to take effect through genomic mechanisms, which are also largely responsible for GCs' side effects. Beneficial non-genomic effects of GCs have been reported as being independent of the genomic pathway. Here, we synthesized a new type of GCs, which took effect mainly via non-genomic mechanisms. Hydrocortisone was conjugated with glycine, lysine and phenylalanine to get a bigger molecular structure, which could hardly go through the cell membrane. Evaluation of the anti-inflammatory efficacy showed that hydrocortisone-conjugated glycine (HG) and lysine could inhibit neutrophil degranulation within 15 min. HG could inhibit IgE-mediated histamine release from mast cells via a non-genomic pathway, and rapidly alleviate allergic reaction. Luciferase reporter assay showed that HG would not activate the glucocorticoid response element within 30 min, which verified the rapid effects independent of the genomic pathway. The work proposes a novel insight into the development of novel GCs, and provides new tools for experimental study on non-genomic mechanisms.

Fig. 1

Schematic presentation of the synthesis of HG. 1 Glycine, 2 N-Boc-glycine, 3 N-Boc-glycine-21-hydrocortisone-ate, and 4 Glycine-21-hydrocortisone–ate

Fig. 2

Structural formula of HG (a) and phenylalanine (b)

Fig. 3

Rapid effects of the new type of GCs on human neutrophil degranulation. HH (P < 0.05), HL (P < 0.05) and HG (P < 0.01) could inhibit fMLP-stimulated MPO release from human neutrophils within 15 min. Data are expressed as means ± SE of four independent experiments performed in duplicate, *P < 0.05, **P < 0.01 in comparison with control group

Fig. 4

Non-genomic effects of HG on histamine release from mast cells. a Dose–response curve of HG’s rapid inhibition on histamine release of mast cells. HG could inhibit histamine release in dose-dependent manner within 15 min. b Summary of histamine release percentage of Control, HG (10⁻⁷ M), HH (10⁻⁷ M), and Gly (10⁻⁷ M). Glycine could not inhibit the histamine release itself. c Summary of histamine release percentage of Control, HG (10⁻⁷ M), RU (RU486, 10⁻⁶ M), and HG (10⁻⁷ M) + RU (10⁻⁶ M). RU486 could not block the rapid inhibitory effects of HG. d Summary of histamine release percentage of Control, ATI (Actidione, 10⁻⁴ M), HG (10⁻⁷ M), and HG (10⁻⁷ M) + ATI (10⁻⁴ M). Actidione could not block the rapid inhibitory effects of HG. e Time-course curve of HG’s inhibition on histamine release. Data are expressed as means ± SD of histamine release percentage. *P < 0.05, **P < 0.01 in comparison with control group

Fig. 5

Rapid effect of HG and HH on activation of GRE. RBL-2H3 cells (1 × 10⁵) were transiently co-transfected with 1 μg MMTV-Luc, 1 ng of pRL-TK Renilla reniformis luciferase plasmid as control. Transfected cells were treated with or without 100 nM HH, HG or solvent control for 7, 15 and 30 min. The ratios of firefly to R. reniformis Luc activities were calculated to correct for differences in transfection efficiency. The average ratio of the untreated control group was used to calculate the percentage of change in the reporter activity of treated groups, and results are expressed as the mean ± SD (n = 3 each). HH **P < 0.01 in comparison with control group

Fig. 6

Influence of HG on intracellular cAMP levels in mast cells. The sensitized mast cells were incubated with 10⁻⁴–10⁻⁷ M HG or solvent control for 7 min at 37°C, and then the reaction was terminated by ice-bath. The cAMP levels were measured by using a cAMP [³H] assay system. Data are expressed as means ± SD (n = 5 each)

Fig. 7

Acute toxicity studies of HG on mice by i.v. and i.m. Groups of 10 mice each were administrated with different concentrations of HG by i.v. and i.m. The calculated median lethal dose (LD₅₀) were 567.8 mg/kg and 776.5 mg/kg, respectively