Org 214007-0: a novel non-steroidal selective glucocorticoid receptor modulator with full anti-inflammatory properties and improved therapeutic index

Contains fulltext : 103595.pdf (publisher's version ) (Open Access)Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects

Org 214007-0 has a relatively lower impact on induction than on respression of genes.

<p>Fold changes for the top 25 genes either induced (A) or repressed (B) by 1 μM prednisolone and 1 μM Org 214007-0 in THP-1 cells. NB. Scales are in 2log. Example of an induced gene, FK506 binding protein 51 (FKBP51) (C) and a repressed gene, interleukin 6 (IL-6) (D) in comparison to the vehicle control under either non-stimulated or stimulated (IFNγ/TNFα) condition. Fold changes for the top 25 genes either induced (E) or repressed (F) by 1.5 mg/kg prednisolone and 0.3 mg/kg Org 214007-0 in muscle tissue from arthritic mice. NB. Scales are in 2log. Example of an induced gene (Per-2) (G) and a repressed gene (Ccl8) (H) in comparison to vehicle treated arthritic mice and vehicle treated healthy mice.</p

Org 214007-0 does not effect rates of hepatic enzyme fluxes.

<p>Mass Isotopomer Distribution Analysis (MIDA), as described in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048385#s4" target="_blank">Materials and Methods</a>, was performed in mice treated p.o., once daily, for 7 days with either vehicle, prednisolone (10 mg/kg) or Org 214007-0 (1.5 mg/kg). These doses of each compound are equi-efficacious in suppression of CIA. Neither the glucose-6-phosphatase flux (A) nor the glycogen phosphorylase flux (B) were affected by treatment with prednisolone or Org 214007-0. The glucokinase flux rate (C) was not changed by Org 214007-0, but significantly differed from the effect by prednisolone (##: p = 0.01 <i>vs</i> prednisolone). The glycogen synthase flux rate (D) was significantly decreased by prednisolone (**: p = 0.005 <i>vs</i> vehicle), whereas Org 214007-0 had no significant effect on this flux, but differed significantly from prednisolone (##: p = 0.002 <i>vs</i> prednisolone). Neither Org 214007-0 nor prednisolone, at equi-efficacious dosages, effects the gluconeogenic flux (<i>de novo</i> synthesis of glucose-6-phophate) (E).</p

Org 214007-0 is equally effective as prednisolone in the mouse CIA model.

<p>A) Inhibition of arthritis in the CIA model. Mean clinical score of each group (n = 12), shown as area under the curve (AUC) of the arthritis score monitored every other day during 3 weeks, corrected for baseline, is indicated (± SEM). **  =  significantly different from placebo (p<0.01; ANOVA-test). B) Reduction of bone destruction in the CIA model as measured by X-ray. Mean radiological score (sum of the X-ray scores of left and right hindpaws and knees) of each group of mice (n = 12) at the end of the CIA experiment is indicated (± SEM). **  =  significantly different from placebo (p<0.01; ANOVA-test).</p

Structure and predicted binding mode of Org 214007-0.

<p>A) The structure of ORG 214007-0. This compound, [(-)-N-(2S,10S,14bS)]-N-(8-cyano-1,2,3,4,10,14b-hexahydro-10-methyl dibenzo<i>[c,f]</i>pyrido[1,2-<i>a</i>]azepin-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide] has a molecular weight of 430 g/mole (C₂₄H₂₃N₅OS) B) The predicted binding mode of Org 214007-0 modeled in complex with the glucocorticoid receptor and demonstrating conservation of interactions typical to steroidal glucocorticoids (Gln564, Asn570, Arg611 and Gln642).</p

Org 214007-0 behaves as a partial agonist in vitro.

<p>A) In a co-factor recruitment assay, Org 214007-0, in comparison to prednisolone, shows potent but partial recruitment of a 0.1 μM peptide presenting TIF2 -3. On the Y-axis average fluorescence counts (+/− SD) are shown. EC50 values (%CV) and percentages maximal efficacy (%CV) for Org 214007-0 versus prednisolone were 10 (7.4) nM versus 48 (3.2) nM and 67 (7.1) % vs 100% respectively. B) In THP1 cells Org 214007-0 shows partial induction of FKBP51 protein expression and C) under inflammatory conditions represses the IL-6 protein expression almost as good as prednisolone does.</p

Summary of studies to define the therapeutic index (TI) of Org 214007-0.

<p>A) <i>THP-1</i>: Microarray (<i>m.a</i>.) analysis of mRNA isolated from THP-1 cells incubated for 6 hours with either 1 μM prednisolone or 1 μM Org 214007-0 without (<i>ind</i>) or with IFNγ (220 ng/ml)/TNFα (374 ng/ml) ( = <i>I/T</i>), (<i>rep</i>) The mean percentage repression or induction of genes compared to that by prednisolone (set at 100%) is indicated. B) <i>THP-1 – rep:</i> Repression of gene expression in THP-1 cells. <i>I/T – MCP-1, IL-6, IL-8</i>  =  TNFα (60 ng/ml)/IFNγ (40 ng/ml) induced MCP-1, IL-6 or IL-8 release. C) <i>THP-1 – ind</i>: Induction of FK506 binding protein 51 (<i>FKBP51</i>), glucocorticoid induced leucine zipper (<i>GILZ</i>) and dual specificity phosphatase 1 (<i>DUSP1</i>) in THP-1 cells. D) <i>hWB</i> – <i>rep:</i> Inhibition of LPS-induced (<i>LPS</i>) TNFα release or PMA/anti-CD28 (<i>P/28</i>) induced IL-5 release by primary human whole blood cells and <i>hWB – ind</i>: enhancement of PMA/anti-CD28/compound (<i>P/28</i>) induced G-CSF release by primary human whole blood cells. E) <i>CASM3C – rep</i>: Inhibition of MCP-1 release of coronary artery smooth muscle cells ( = <i>CASM3C</i>) stimulated with a cytokine mixture of IL-1β (1 ng/ml), IFNγ (100 ng/ml) and TFNα (5 ng/ml) ( = <i>1/I/T</i>) by 1 μM prednisolone or 1 μM Org 214007-0. <i>CASM3C – ind</i>: Induction of serum amyloid A (<i>SAA</i>) of the cells mentioned above by 1 μM prednisolone or 1 M Org 214007-0. F) <i>HDF3CGF – rep</i>: inhibition of matrix metalloproteinase (<i>MMP-1</i>) release of human neonatal foreskin fibroblasts stimulated with the cytokine mixture mentioned above plus required growth factors ( = <i>HDF3CGF</i>). <i>HDF3CGF – ind</i>: Activation of plasminogen activator inhibitor-1 (<i>PAI-1</i>) by cells mentioned above by 1 μM prednisolone or 1 μM Org 214007-0. G) <i>CIA mus</i>.: Microarray (<i>m.a</i>.) analysis of mRNA isolated from muscle cells isolated at day 21, 2.5 hours after the final oral administration of either 1.5 mg/kg prednisolone or 0.3 mg/kg Org 214007-0 in the mouse CIA experiment. The mean percentage repression (<i>rep</i>) or induction (<i>ind</i>) of genes compared to that by prednisolone (set at 100%) is indicated.</p><p>IC50 or EC50 values represent the mean concentration of compound (±SD) required to resp. inhibit or effect the response to 50%. Maximal efficacy (Max. eff.) is expressed as the mean relative maximal effect (±SD) compared to the maximal effect by prednisolone (set at 100%). A relative therapeutic index (TI_rel) is calculated by the ratio of (the mean) % maximal efficacy in repression and (the mean) % maximal efficacy in induction of genes by Org 214007, where that of prednisolone is set at 1 (100%/100%). All assays (except for the microarray experiments) are performed at least two times.</p

Org 214007-0 leads to relatively less GR occupancy.

<p>A) Ratio of the read count for clusters induced by 1 μM prednisolone or 1 μM Org 214007-0 in a ChIP-Seq analysis. If Org 214007-0 and prednisolone would induce an equal number of reads, the histogram would be centered on Log0, indicated by the dotted line. There is a clear shift to the right from this line (P-value (mean  = 0) <0.000001), indicating that prednisolone leads to more GR occupancy than Org 214007-0. B) Example profile of the tag clusters in the GR response gene FKBP51. Multiple binding sites are found within this gene, each showing denser clusters after treatment with 1 μM prednisolone than after treatment with 1 μM Org 214007-0. The inset highlights an intronic region at 87 kb downstream of the transcription start site. This region that was identified by Paakinaho et al. (2010) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048385#pone.0048385-Paakinaho1" target="_blank">[54]</a>, as a major intronic enhancer in human A459 lung cancer cells and was shown to be occupied by GR after treatment with the GR ligand dexamethasone.</p

Summary of <i>in vitro</i> studies.

<p>Prednisolone (Pred) and Org 214007-0 (Org) were tested in different <i>in vitro</i> studies. A) <i>GR binding</i>: binding to recombinant human glucocorticoid receptor (GR) assessed by a fluorescence polarization competitor binding assay. Ki  =  inhibition constant or concentration of compound in the competitive binding assay which would occupy 50% of GR if no ligand was present. B) <i>CHO – ind</i>: Induction of gene expression measured in CHO cells stably co-transfected with human GR and a MMTV promoter – luciferase construct. C) <i>HepG2 – ind</i>: Induction of gene expression measured by microarray analysis of mRNA isolated from HepG2 cells incubated with either 1 μM prednisolone or 1 μM Org 214007-0. D) <i>U2OS – rep</i>: Repression of gene expression in U2OS cells overexpressing human GR. INFγ/TNFα – MCP-1  =  IFNγ (100 ng/ml)/TNFα (50 ng/ml) induced MCP-1 release.</p><p>IC50 or EC50 values represent the mean concentration of compound (±SD) required to resp. inhibit or effect the response to 50%. Maximal efficacy (Max. eff.) is expressed as the mean relative maximal effect (±SD) compared to the maximal effect by prednisolone (set at 100%). All assays (except for the micro array experiments) are performed at least two times.</p

Org 214007-0 induces less elevated fasting glucose levels.

<p>Mice (n = 8 per group) are not treated (Control) or treated po, once daily, for 28 days with either vehicle only, prednisolone 0.5 mg/kg/day or 10 mg/kg/day or Org 214007-0 0.17 mg/kg/day or 1.5 mg/kg/day. Both the two lowest doses of each compound are equi-efficacious in suppression of CIA as well as the two highest doses. Blood glucose levels (mean ± SEM) were measured at day 8, 15, 22 and 29, after 9 hours of fasting. * p<0.05, ** p<0.01, *** p<0.001: significantly different (Student's t-test) from vehicle treated group.</p