High Affinity For Glucocorticoid Receptor Research Articles

682 Characterization of a novel selective non-steroidal glucocorticoid receptor agonist with low systemic exposure and reduced potential for skin atrophy

Topical glucocorticoids (GCs) are widely used drugs in dermatology but are associated with both local and systemic adverse effects. With the aim to provide an effective but safer topical treatment, we here describe LEO 134310, a novel non-steroidal glucocorticoid receptor (GR) agonist with low systemic exposure and reduced potential for induction of skin atrophy. LEO 134310 is a full GR agonist with high affinity for GR and nanomolar potency in human PBMCs. Furthermore, selectivity for GR was demonstrated within a panel of nuclear hormone receptors, including the mineralocorticoid receptor, which has been associated with induction of skin atrophy. Topical treatment with LEO 134310 in human skin explants resulted in full agonism, measured by GR target engagement, with maximal effects comparable to BMV and CP, although at higher dosage strength. The lower potency of LEO 134310 in human skin explants could be explained by a combination of lower skin penetration, metabolism in the skin and in vitro potency. LEO 134310 was rapidly deactivated in the blood and other compartments by enzymatic hydrolysis, leading to low systemic exposure of active parent compound. Consequently, a 10-fold higher therapeutic index was achieved with LEO 134310 compared to BMV and CP in the TPA-induced skin inflammation mouse model. Induction of skin atrophy after topical treatment with LEO 134310 in minipigs was minimal in contrast to significant epidermal thinning induced by treatment with CP and BMV. In conclusion, LEO 134310 may potentially offer an improved topical treatment solution for patients with inflammatory skin diseases.

Anti-inflammatory duration of action of fluticasone furoate/vilanterol trifenatate in asthma: a cross-over randomised controlled trial

BackgroundFluticasone furoate/Vilanterol trifenatate (FF/VI) is an inhaled corticosteroid/long-acting beta-agonist combination with a prolonged bronchodilator duration of action. We characterised the time-course of onset and offset of airway anti-inflammatory action of FF/VI, as assessed by fraction of exhaled nitric oxide (FeNO), and compared this to the bronchodilator duration of action.MethodsA single-centre, randomised, double-blind, placebo-controlled, two-period, crossover study was undertaken in 28 steroid-naïve adults with asthma. Participants with an FEV1 ≥ 60% predicted, reversible airway disease, and FeNO > 40 ppb received FF/VI 100/25 mcg or placebo once daily for 14 days. FeNO and peak expiratory flow were measured twice-daily during treatment and during a 21-day washout period. FEV1 was measured for five days from treatment cessation. The primary outcome measure was FeNO change from baseline ratio for 21 days following treatment cessation.ResultsIn the 27 subjects who completed the study, median (range) baseline FeNO was 87 ppb (42–212). FF/VI 100/25 mcg reduced FeNO by day 3, ratio FF/VI versus placebo 0.72 (95% confidence interval 0.61–0.86) with the maximum reduction occurring at day 14, 0.32 (0.27–0.37). Following cessation of treatment FeNO remained suppressed for 18 days, ratio on day 18 0.77 (0.59–1.00), whereas improvements in FEV1 and peak flow were maintained for 3 to 4 days post-treatment.ConclusionsThe anti-inflammatory duration of action of FF/VI is consistent with the high glucocorticoid receptor affinity and long lung retention of fluticasone furoate. The anti-inflammatory effect of FF/VI was of greater duration than its bronchodilator effect in adults with mild asthma.Funding GlaxoSmithKline (201499).Trial registrationProspectively registered on ClinicalTrials.gov registry number NCT02712047.

430 Preclinical evaluation of a novel non-steroidal glucocorticoid receptor agonist with low systemic exposure and reduced potential for skin atrophy

Topical corticosteroids are widely used efficacious drugs but are associated with both local and systemic adverse effects. With the aim to provide an effective but safer topical treatment, we here describe a novel non-steroidal glucocorticoid receptor (GR) agonist with low systemic exposure and reduced potential for skin atrophy induction. LEO 134310 was shown to be a full GR agonist with high affinity for GR and low nanomolar potency in an inflammatory functional assay in human PBMCs. GR was the only nuclear hormone receptor affected by LEO 134310 in a panel of nuclear receptors, including the mineralocorticoid receptor, a receptor which has been associated with induction of skin atrophy. In blood and liver LEO 134310 is rapidly inactivated at a labile site, leading to high systemic clearance. In accordance with this profile, topical treatment with LEO 134310 in a TPA-induced dermatitis mouse model resulted in comparable efficacy to betamethasone valerate (BMV), with no systemic adverse effects at maximally effective doses. In contrast, BMV and clobetasol propionate (CP) markedly reduced body weight at maximally effective doses after topical treatment. Furthermore, skin atrophy after 4-week application to minipigs was minimal after treatment with LEO 134310 compared with significant skin atrophy induced by treatment with CP and BMV. LEO 134310 showed similar levels of target engagement as BMV, measured by FKBP5 mRNA, but lower than CP. Finally, topical treatment with LEO 134310 in a human skin explant model resulted in full agonism, measured by GR target engagement, albeit at higher dosage strength than required for BMV and CP. In conclusion, LEO 134310A shows an improved therapeutic index in preclinical studies compared to traditional corticosteroids and may potentially offer an improved topical treatment solution for patients with atopic dermatitis and psoriasis.

A Mixed Glucocorticoid/Mineralocorticoid Selective Modulator With Dominant Antagonism in the Male Rat Brain.

Adrenal glucocorticoid hormones are potent modulators of brain function in the context of acute and chronic stress. Both mineralocorticoid (MRs) and glucocorticoid receptors (GRs) can mediate these effects. We studied the brain effects of a novel ligand, C118335, with high affinity for GRs and modest affinity for MRs. In vitro profiling of receptor-coregulator interactions suggested that the compound is a "selective modulator" type compound for GRs that can have both agonistic and antagonistic effects. Its molecular profile for MRs was highly similar to those of the full antagonists spironolactone and eplerenone. C118335 showed predominantly antagonistic effects on hippocampal mRNA regulation of known glucocorticoid target genes. Likewise, systemic administration of C118335 blocked the GR-mediated posttraining corticosterone-induced enhancement of memory consolidation in an inhibitory avoidance task. Posttraining administration of C118335, however, gave a strong and dose-dependent impairment of memory consolidation that, surprisingly, reflected involvement of MRs and not GRs. Finally, C118335 treatment acutely suppressed the hypothalamus-pituitary-adrenal axis as measured by plasma corticosterone levels. Mixed GR/MR ligands, such as C118335, can be used to unravel the mechanisms of glucocorticoid signaling. The compound is also a prototype of mixed GR/MR ligands that might alleviate the harmful effects of chronic overexposure to endogenous glucocorticoids.

Health outcomes, education, healthcare delivery and quality – 3057. Randomized, double blind comparative study to assess safety, efficacy with mometasone & formoterol versus fluticasone & formoterol dry powder inhaler (DPI) in the treatment of mild to moderate persistent asthma

This article was originally published online on 23 April 2013 Asthma is a problem worldwide, with an estimated 300 million affected individuals. Mometasone being newer drug has low systemic bioavailability, high glucocorticoid receptor affinity and modifies inflammatory mediators involved in the pathogenesis of asthma. Mometasone significantly improves PFT and symptom control in patients with asthma when used in combination with Formoterol. There are very few studies conducted to assess and compare the safety and efficacy of Mometasone group (Mometasone & Formoterol) versus Fluticasone group (Fluticasone & Formoterol). Hence the present study was undertaken to assess the safety and efficacy of Mometasone group versus Fluticasone group using DPI in patients with mild to moderate persistent asthma and also its effect on symptom control and frequency of rescue medication use. The present study was conducted in PMU, Bangalore during March 2011-2012. 60 patients were recruited in each arm based on inclusion and exclusion criteria. PFT was done pre and post bronchodilator with Salbutamol nebulization with Spirometry. Study medications were randomized, double blinded and were given for 12weeks & comparison was done to know safety, efficacy and frequency of rescue medication use before and after treatment in asthmatics. Statistical test -descriptive statistics, repeated ANOVA, Z- test, t- test. Out of 60 patients in Fluticasone group, 11 developed adverse reactions- 4 developed recurrent URTI whereas in Mometasone group- 7 developed adverse reactions- 2 each developed recurrent URTI & hoarseness of voice. There was an overall improvement in lung function test (FVC, FEV1, FEV1/FVC, FEF25-75, PEF) between Mometasone and a Fluticasone group which was statistically significant when compared within the group (P=0.001), but was not significant when compared between the groups suggesting both are equally efficacious. There was a significant reduction in symptoms before and after treatment within the group, but the reduction was not statistically significant when compared between the groups suggesting both are equally efficacious. There was a significant reduction in dosage of rescue medication used from baseline to the end of 12weeks in Mometasone group compared to Fluticasone group (t value= 6.96; P= 0.001). Both Mometasone +Formoterol and Fluticasone+ Formoterol combinations were safe and equally efficacious in treating asthmatics.

Ligand structural motifs can decouple glucocorticoid receptor transcriptional activation from target promoter occupancy

Glucocorticoid (GC) induction of the tyrosine aminotransferase (TAT) gene by the glucocorticoid receptor (GR) is a classic model used to investigate steroid-regulated gene expression. Classic studies analyzing GC-induction of the TAT gene demonstrated that despite having very high affinity for GR, some steroids cannot induce maximal TAT enzyme activity, but the molecular basis for this phenomenon is unknown. Here, we used RT-PCR and chromatin immunoprecipitation to determine TAT mRNA accumulation and GR recruitment to the TAT promoter (TAT–GRE) in rat hepatoma cells induced by seven GR ligands: dexamethasone (DEX), cortisol (CRT), corticosterone (CCS), 11-deoxycorticosterone (DOC), aldosterone (ALD), progesterone (PRG) and 17-hydroxyprogesterone (17P). As expected, DEX, CRT, CCS and ALD all induced both TAT mRNA and GR recruitment to the TAT–GRE, while PRG and 17P did not. However, while DOC could not induce significant TAT mRNA, it did induce robust GR occupancy of the TAT–GRE. DOC also induced recruitment of the histone acetyltransferase p300 to the TAT–GRE as efficiently as DEX. These DOC-induced effects recapitulated at another GR target gene (sulfonyltransferase 1A1), and DOC also failed to promote the multiple changes in gene expression required for glucocorticoid-dependent 3T3-L1 adipocyte differentiation. Structural simulations and protease sensitivity assays suggest that DOC and DEX induce different conformations in GR. Thus, although steroids that bind GR with high affinity can induce GR and p300 occupancy of target promoters, they may not induce a conformation of GR capable of activating transcription.

Mometasone furoate dry-powder inhaler for the control of persistent asthma

Mometasone furoate dry-powder inhaler (MF-DPI) is an inhaled corticosteroid (ICS) used for the treatment of persistent asthma in patients aged ≥ 12 years. MF-DPI has low systemic bioavailability and high glucocorticoid receptor affinity compared with most other ICSs and modifies inflammatory mediators involved in the pathogenesis of asthma. MF-DPI, unlike other available ICSs, is approved for initiation as a once-daily in the afternoon (q.d. PM) regimen. Studies show that MF-DPI 200 or 400 μg q.d. PM treatment significantly improves lung function and symptom control in patients with mild, moderate or severe asthma. MF-DPI 400 μg q.d. PM is reported to be equivalent to fluticasone propionate 250 μg b.i.d. and beclometasone dipropionate 168 μg b.i.d. and more efficacious than budesonide 400 μg, b.i.d. or q.d. MF-DPI is generally well tolerated, with minimal effects on the hypothalamic-pituitary-adrenal axis.

Lower oropharyngeal deposition of inhaled ciclesonide via hydrofluoroalkane metered-dose inhaler compared with budesonide via chlorofluorocarbon metered-dose inhaler in healthy subjects

Inhaled corticosteroids may cause oropharyngeal side effects if deposited in the oropharynx in active form. Ciclesonide, an inhaled corticosteroid with low glucocorticoid receptor affinity, is activated primarily in the lung by esterases to an active metabolite, desisobutyryl-ciclesonide (des-CIC), with high glucocorticoid receptor affinity. We studied oropharyngeal deposition of ciclesonide, des-CIC, and budesonide. In an open-label, randomized, two-treatment (administered in sequence), five-period study, 18 healthy subjects received 800 microg (ex-valve) inhaled ciclesonide via a hydrofluoroalkane-pressurized, metered-dose inhaler followed by 800 microg budesonide (Pulmicort) by a chlorofluorocarbon-pressurized, metered-dose inhaler (four puffs of 200 microg each, ex-valve) or vice versa. Oropharyngeal cavity rinsing was performed immediately, or 15, 30, 45, or 60 min after inhalation (one rinsing per study period), and the solutions were analyzed using liquid chromatography with tandem mass spectrometric detection. Ciclesonide and budesonide were detected in most oropharyngeal wash samples. Maximal concentration of each inhaled corticosteroid was reached immediately post-inhalation; maximal concentrations of ciclesonide and des-CIC were 30% and 0.67%, respectively, of budesonide. Oropharyngeal deposition of ciclesonide and budesonide decreased rapidly within 15 min post-inhalation, and less rapidly thereafter. Less than 10% of the residual ciclesonide in the oropharynx was converted to des-CIC. The molar dose-adjusted amount of des-CIC was 4% of budesonide (P < 0.0001). There were no significant adverse events. Oropharyngeal deposition of des-CIC was more than one order of magnitude lower than that of budesonide when administered by the respective metered-dose inhalers. This may explain the low frequency of oropharyngeal side effects of ciclesonide in clinical studies.

Ciclesonide, administered once daily, has a low incidence of oropharyngeal adverse events in pediatric asthma patients

RATIONALE: Use of inhaled corticosteroids (ICS) is associated with local oropharyngeal adverse events that can negatively impact treatment initiation and adherence. Ciclesonide (CIC) delivered via hydrofluoroalkane metered-dose inhaler is a new, effective ICS with low oral deposition (<1%) and high lung deposition (~52%). CIC is primarily converted in the lung to its active metabolite, desisobutyryl-ciclesonide (des-CIC). Des-CIC has high serum protein binding and high glucocorticoid receptor affinity.

Trans-Activation and repression properties of the novel nonsteroid glucocorticoid receptor ligand 2,5-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5-(1-methylcyclohexen-3-y1)-1H-[1]benzopyrano[3,4-f]quinoline (A276575) and its four stereoisomers.

Glucocorticoids are potent anti-inflammatory and immunosuppressant agents. However, they also produce serious side effects that limit their usage. It has been proposed that anti-inflammatory properties of glucocorticoids are caused mostly by repression of activator protein 1- and nuclear factor kappabeta-stimulated synthesis of inflammatory mediators, whereas most of their adverse effects are associated with trans-activation of genes involved with metabolic processes. Our laboratories have sought to discover novel glucocorticoid receptor (GR) ligands that have high repression but low trans-activation activities. We describe here cellular properties of 2,5-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5-(1-methylcyclohexen-3-y1)-1H-[1]benzopyrano[3,4-f]quinoline (A276575) and its four enantiomers. Similar to dexamethasone, A276575 exhibited high affinity for GR and potently repressed interleukin (IL) 1beta-stimulated IL-6 production in human skin fibroblasts, prostaglandin (PG) E(2) production in A549 human lung epithelial cells, and concanavalin A-induced monocyte proliferation. In contrast to dexamethasone, A276575 caused smaller induction of aromatase activity in human skin fibroblasts and antagonized dexamethasone-induced activation of an mouse mammary tumor virus-glucocorticoid-response element (GRE) reporter gene construct. Among the four enantiomers of A276575, the two (-)-enantiomers showed 10- to 30-fold higher affinities for GR than their respective (+)-enantiomers. Both (-)-Syn and (-)-Anti enantiomers of A276575 were potent inhibitors of IL-1beta-stimulated PGE2 production in A549 lung epithelial cells; unexpectedly, however, only the (-)-Anti enantiomer inhibited regulated on T-cell activation, normal T-cell expressed and secreted (RANTES) production in A549 cells. In summary, A276575 is a novel, nonsteroidal GR ligand that possesses high repression activities against inflammatory mediator production but has lower GRE trans-activation activities than traditional steroids. Differential repression of RANTES and PGE2 production in a cell by the two (-)-enantiomers of A276575 illustrates the complexity of repression by GR.

Synthesis and characterization of non-steroidal ligands for the glucocorticoid receptor: selective quinoline derivatives with prednisolone-equivalent functional activity.

A novel class of functional ligands for the human glucocorticoid receptor is described. Substituents in the C-10 position of the tetracyclic core are essential for glucocorticoid receptor (GR) selectivity versus other steroid receptors. The C-5 position is derivatized with meta-substituted aromatic groups, resulting in analogues with a high affinity for GR (K(i) = 2.4-9.3 nM) and functional activity comparable to prednisolone in reporter gene assays of glucocorticoid-mediated gene transcription. The biological activity of these novel quinolines was also prednisolone-equivalent in whole cell assays of glucocorticoid function, and compound 13 was similar to prednisolone (po ED(50) = 2.8 mpk for 13 vs ED(50) = 1.2 mpk for prednisolone) in a rodent model of asthma (sephadex-induced eosinophil influx).

Novel glucocorticoid receptor complex with DNA element of the hormone-repressed POMC gene.

Previous studies defined a DNA element necessary for glucocorticoid repression of the pro-opiomelanocortin (POMC) gene. The glucocorticoid receptor (GR) binds this negative glucocorticoid response element (nGRE) with an in vitro affinity similar to that of GR for positive GREs. However, whereas GR binds GREs as homodimers, a novel GR complex which forms with nGRE appears to contain three GR molecules. Biochemical characterization of this complex as well as equilibrium binding studies suggest that it is formed by sequential binding of a GR homodimer followed by binding of a GR monomer on the opposite side of the double helix. The DNA-binding domain (DBD) of GR is sufficient for differential binding of GRE and nGRE, as bacterially-expressed DBD formed unique nGRE complexes that contain three GR polypeptides. Thus, the POMC nGRE provides the first example of an interaction between GR and DNA in which GR binds otherwise than as a homodimer. Despite its high affinity for GR, the nGRE differs significantly from GREs in that it does not activate transcription in any context. As the nGRE appears insufficient on its own to confer hormone responsiveness, other POMC promoter elements are likely to be required to mediate glucocorticoid repression.

Glucocorticoid binding in mouse liver cytosol

Previously, aldosterone has been shown to be a predominant or complete agonist in glucocorticoid effector systems, and to have a moderately high affinity for glucocorticoid receptors. Recently, in two mouse tissues (AtT-20 pituitary cells, liver cytosol) Miras and Harrison reported aldosterone to be without affinity for [ 3H]-triamcinolone binding sites [1]. In view of the possible implications for glucocorticoid receptor ontogeny, and mineralocorticoid specificity, we have repeated these studies on mouse liver, and extended them to kidney. In our hands, aldosterone has a moderately high affinity for glucocorticoid receptors in mouse liver and kidney, indistinguishable from that found in parallel studies in the rat.