Figure 1.
Structure and predicted binding mode of Org 214007-0.
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[c,f]pyrido[1,2-a]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).
Figure 2.
Org 214007-0 behaves as a partial agonist in vitro.
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.
Table 1.
Summary of in vitro studies.
Figure 3.
Org 214007-0 has a relatively lower impact on induction than on respression of genes.
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.
Table 2.
Summary of studies to define the therapeutic index (TI) of Org 214007-0.
Figure 4.
Org 214007-0 leads to relatively less GR occupancy.
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) [54], 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.
Figure 5.
Org 214007-0 is equally effective as prednisolone in acute inflammation mouse models.
A) Inhibition of acute inflammation in the LPS-induced TNFα mouse model. Mean percentage inhibition (of each group of n = 8) (± SEM) of the LPS-induced TNFα release by the vehicle treated group is shown (no TNFα is detectable without LPS injection). ** = significantly different from placebo (p<0.01; ANOVA-test). B) Inhibition of acute T cell-driven inflammation in the anti-CD3-induced IL-2 mouse model. Org 214007-0 shows a more potent activity than prednisolone in this model. Mean percentage inhibition (of each group of n = 2) (± SEM) of the anti-CD3-induced IL-2 release relative to a positive control group (treated with 10 mg/kg A420983, a pan-SRC inhibitor) (set at 100%) is shown (no IL-2 is detectable without anti-CD3 injection). ** = significantly different from placebo (p<0.01; ANOVA-test).
Figure 6.
Org 214007-0 is equally effective as prednisolone in the mouse CIA model.
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).
Figure 7.
Org 214007-0 induces less elevated fasting glucose levels.
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.
Figure 8.
Org 214007-0 does not effect rates of hepatic enzyme fluxes.
Mass Isotopomer Distribution Analysis (MIDA), as described in detail in Materials and Methods, 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 vs prednisolone). The glycogen synthase flux rate (D) was significantly decreased by prednisolone (**: p = 0.005 vs vehicle), whereas Org 214007-0 had no significant effect on this flux, but differed significantly from prednisolone (##: p = 0.002 vs prednisolone). Neither Org 214007-0 nor prednisolone, at equi-efficacious dosages, effects the gluconeogenic flux (de novo synthesis of glucose-6-phophate) (E).
Figure 9.
Org 214007-0 does not cause a shift in the liver glucose/glycogen balance.
As determined in MIDA (details in legend Figure 8), the glucose balance (A), which represents the difference between the glucose-6-phophatase and glucokinase flux, is not affected by Org 214007-0 treatment but is significantly changed by the prednisolone treated group (*: p = 0.046 vs vehicle.; ##: p = 0.004 vs prednisolone). The glycogen balance (B), representing the difference between the glycogen synthase and glycogen phophorylase flux, is not effected by Org 214007-0 treatment but is significantly changed by the prednisolone treated group (*: p = 0.024 vs vehicle; ##: p = 0.005 vs prednisolone). Neither Org 214007-0 nor prednisolone, at equi-efficacious dosages, effects the metabolic clearance rate of blood glucose (C).