HE3286, an oral synthetic steroid, treats lung inflammation in mice without immune suppression

Abstract

Background: 17α-Ethynyl-5-androsten-3β, 7β, 17β-triol (HE3286) is a synthetic derivative of an endogenous steroid androstenetriol (β-AET), a metabolite of the abundant adrenal steroid deyhdroepiandrosterone (DHEA), with broad anti-inflammatory activities. We tested the ability of this novel synthetic steroid with improved pharmacological properties to limit non-productive lung inflammation in rodents and attempted to gauge its immunological impact.

Methods and results: In mice, oral treatment with HE3286 (40 mg/kg) significantly (p < 0.05) decreased neutrophil counts and exudate volumes (~50%) in carrageenan-induced pleurisy, and myeloperoxidase in lipopolysaccharide-induced lung injury. HE3286 (40 mg/kg) was not found to be profoundly immune suppressive in any of the classical animal models of immune function, including those used to evaluate antigen specific immune responses in vivo (ovalbumin immunization). When mice treated for two weeks with HE3286 were challenged with K. pneumoniae, nearly identical survival kinetics were observed in vehicle-treated, HE3286-treated and untreated groups.

Conclusions: HE3286 represents a novel, first-in-class anti-inflammatory agent that may translate certain benefits of β-AET observed in rodents into treatments for chronic inflammatory pulmonary disease.

Figure 1

Effect of HE3286 treatment on carrageenan-induced pleurisy. Mice CD1 mice (10 per group) were anesthetized and saline (0.1 mL) alone (sham) or saline containing 2% carrageenan (CAR) was injected into the pleural cavity. Mice were treated (sc) with HE3286 (4 or 40 mg/kg) or vehicle HERF405 alone (0.1 mL) 24 h before and 1 h before CAR. At 4 h after CAR, the animals were killed, the chest opened, and the pleural cavity rinsed with 1 mL of saline solution. The leukocytes in the exudate were suspended in phosphate-buffer saline (PBS) and counted. Data are expressed as mL exudate volume (A) or millions of neutrophils (B) per mouse +/- standard deviation on the Y-axis. Treatment groups are identified on the X-axis. *p < 0.05.

Figure 2

Effect of HE3286 treatment on MPO levels in LPS induced Lung Injury. On day-1, male C57 black/6 mice were pre-treated (gavage) with HE3286 or 0.1 mL vehicle (HERF405). The next day, mice were challenged with 50 μg of E-coli LPS under direct visualization of trachea under light anesthesia. Sixty minutes after the LPS challenge, mice were treated with a second dose of HE3286, or vehicle. Forty-eight hours after LPS challenge, mice were sacrificed and myeloperoxidase (MPO) activity in lungs determined as previously described [45]. Results are from two identical experiments. Data are expressed as O.D at 460 nM.

Figure 3

Effect of HE3286 on OVA-specific immunoglobulin production. Female BALB/c mice (5 per group) were sensitized by intraperitoneal injection (total volume 200 μL) on days 1 and 8 with 100 μg OVA precipitated with alum (25 mg/mL) in saline. Animals were treated (gavage) with HE3286 (40 mg/kg) or with HERF202 vehicle (100 μL) alone once daily for twenty days. On day twenty, animals were sacrificed and OVA-specific immunolglobulin (IgG) levels in serum were measured at various dilutions by ELISA. Data are expressed as optical density +/- standard deviation on the Y-axis versus dilution on the X-axis.

Figure 4

Effect of HE3286 on bacterial infection. Female BALB/c mice (n = 8-10 per group) received daily 0.1 mL administrations (gavage) of HE3286 at 80 mg/kg in vehicle (HERF405), equal volumes of vehicle alone, daily IP administrations of dexamethasone (dex; 0.4 mg/kg) in 0.1 mL saline or sham treated. After 14 days of treatment, infection was induced by SC inoculation of 10⁷ colony-forming units (cfu) of K. pneumoniae. Daily HE3286 or dex treatments continued and all animals monitored twice-daily until the end of the study for health status.