Peripheral glucocorticoid receptor antagonism by relacorilant with modest HPA axis disinhibition

Glucocorticoid stress hormones are produced in response to hypothalamic–pituitary–adrenal (HPA) axis activation. Glucocorticoids are essential for physiology and exert numerous actions via binding to the glucocorticoid receptor (GR). Relacorilant is a highly selective GR antagonist currently undergoing a phase 3 clinical evaluation for the treatment of endogenous Cushing’s syndrome. It was found that increases in serum adrenocorticotropic hormone (ACTH) and cortisol concentrations after relacorilant treatment were substantially less than the increases typically observed with mifepristone, but it is unclear what underlies these differences. In this study, we set out to further preclinically characterize relacorilant in comparison to the classical but non-selective GR antagonist mifepristone. In human HEK-293 cells, relacorilant potently antagonized dexamethasone- and cortisol-induced GR signaling, and in human peripheral blood mononuclear cells, relacorilant largely prevented the anti-inflammatory effects of dexamethasone. In mice, relacorilant treatment prevented hyperinsulinemia and immunosuppression caused by increased corticosterone exposure. Relacorilant treatment reduced the expression of classical GR target genes in peripheral tissues but not in the brain. In mice, relacorilant induced a modest disinhibition of the HPA axis as compared to mifepristone. In line with this, in mouse pituitary cells, relacorilant was generally less potent than mifepristone in regulating Pomc mRNA and ACTH release. This contrast between relacorilant and mifepristone is possibly due to the distinct transcriptional coregulator recruitment by the GR. In conclusion, relacorilant is thus an efficacious peripheral GR antagonist in mice with only modest disinhibition of the HPA axis, and the distinct properties of relacorilant endorse the potential of selective GR antagonist treatment for endogenous Cushing’s syndrome.


Introduction
Glucocorticoid (GC) stress hormones are essential for physiology and exert numerous functions via binding to the glucocorticoid receptor (GR). The predominant active GC hormone is cortisol in humans, while rats and mice adrenals exclusively secrete corticosterone. The GR is a ligand-dependent transcription factor that is expressed in a wide variety of tissues and cell types (Weikum et al. 2017) and is activated during the circadian GC peak, upon stress, or due to hypercortisolism , Joëls 2018). The maintenance of homeostasis by GCs is tightly regulated by hypothalamus-pituitary-adrenal (HPA) axis activity (Lightman et al. 2020). The hypothalamic neurons from the paraventricular nucleus release the corticotropinreleasing hormone which reaches the corticotropic cells of the anterior pituitary gland and stimulates the release of adrenocorticotropic hormone (ACTH) into the circulation. ACTH in turn induces the production and secretion of GCs from the adrenal gland (Spencer & Deak 2017). Dysregulation of GC secretion is responsible for several pathologies, the most obvious being Cushing's syndrome (CS). Endogenous CS is a condition characterized by cortisol overproduction that can be ACTH-dependent, caused by either a pituitary adenoma or an ectopic source, or ACTH-independent when caused by an adrenal gland tumor or hyperplasia (Barbot et al. 2020). Hypercortisolism in CS is associated with multisystem morbidity including all features of the metabolic syndrome, i.e. visceral fat accumulation, insulin resistance, hypertension and dyslipidemia (Kalsbeek et al. 2015), and with a range of neuropsychiatric symptoms such as depression, impaired memory and sleep disturbances (Pivonello et al. 2015). Similar symptoms are observed in some patients who use exogenous corticosteroid medication (Barbot et al. 2020).
Under normal physiological conditions, GCs exert negative feedback on the HPA axis to maintain homeostasis. This feedback mechanism is mediated by the GR via the hypothalamus and the hippocampus in the central nervous system (CNS), the pituitary and possibly the adrenal gland (Ratka et al. 1989, Walker et al. 2015. As such, GR agonists such as dexamethasone (DEX) suppress HPA axis activity and thereby diminish endogenous cortisol/corticosterone secretion (Andrews et al. 2012, Asirvatham et al. 2020. Vice versa, GR antagonists like mifepristone disinhibit HPA axis activity, resulting in increased ACTH production with concomitant elevated secretion of cortisol/corticosterone (Gaillard et al. 1984). GR antagonists counteract GC-driven metabolic disruption, but the disinhibition of the HPA axis can be an issue in such treatment regimens. The resulting elevated endogenous GCs compete for GR occupancy and can thereby interfere with effective GR antagonism. In addition, high GC levels can override the capacity of 11β-hydroxysteroid dehydrogenase type 2, that is expressed in the kidney and converts cortisol into inactive cortisone. This can lead to overactivation of mineralocorticoid receptors affecting the sodium/potassium balance (Ferrari 2010, Shearer et al. 2019. Unlike mifepristone, relacorilant selectively antagonizes GR activity while not affecting the progesterone receptor (PR) and the androgen receptor (AR) (Hunt et al. 2017). In phase 2 clinical trial, relacorilant was shown to be efficacious in reducing hyperglycemia, hypertension and other manifestations of hypercortisolism and was generally well-tolerated in patients with CS. Increases in ACTH and cortisol were substantially less than typically observed with mifepristone, which will have clinical advantages for patients with CS (Pivonello et al. 2021). Relacorilant is currently undergoing phase 3 clinical evaluation in this patient population (https:// clinicaltrials.gov/ct2/show/NCT03697109). In the current preclinical study, we compared the effects of relacorilant and mifepristone in vitro and in male mice, with a focus on HPA axis (re-)activity.

Luciferase reporter assay in HEK-293 cells
HEK-293 cells were seeded as 80,000 cells/well in 24-well plates in culture medium DMEM (1X) + GlutaMAX™ with 10% charcoal-stripped FBS, supplemented with penicillin/ streptomycin. The cells were transfected after 24 h using the FuGENE®HD Transfection Reagent (Promega Corporation) according to the manufacturer's instructions. Per well, 10 ng human GR plasmid, 25 ng tyrosine aminotransferase (TAT1)-firefly-luciferase (reporter gene), 1 ng CMVrenilla-luciferase plasmid (control reporter gene) and 264 ng pcDNA were transfected. The TAT1 reporter construct is composed of a GR-responsive element of the Tat gene coupled to the firefly-luciferase and is transcribed upon GR activation (Liu et al. 1996, Kroon et al. 2021. One day later, we tested GR agonism using 0.1% dimethyl sulfoxide (DMSO) as vehicle, 1 µM relacorilant, 3 nM and 100 nM dexamethasone (DEX), or 100 nM and 1 µM cortisol. Both concentrations of DEX and cortisol corresponded to the EC 80 and EC max in HEK-293 cells. To test GR antagonism, cells were pre-treated for 1 h with 0.1-1000 nM relacorilant or mifepristone before treatment with either 3 nM DEX or 100 nM cortisol. After 24 h, the medium was removed, and the wells were washed once with PBS before the cold acetonitrile, vortexed and centrifuged at 16,200 g for 5 min at 4°C. The supernatant was diluted in water (200 µL in 800 µL) in a HPLC vial. A total of 10 µL of the resulting samples were added tn a 50 × 4.6 mm C18 Ascentis Express 2.7 µm column with a mobile phase A (0.1% formic acid in water) and a mobile phase B (0.1% formic acid in acetonitrile) with an average flow rate of 1 mL/min. Data were then analyzed at Saretius Ltd. using Phoenix64/ WinNonlin 6.3 and presented as mean. The plasma bioavailability (F) was calculated using the equation: ) and expressed as a percentage (100 × F), where units are consistent for both AUCs (e.g. h × ng/mL) and for both doses (e.g. mg/kg). Brain concentrations of relacorilant in mice were assessed by administering 90 mg/kg relacorilant diluted in 10% DMSO, 0.1% Tween in hydroxypropyl methylcellulose (HPMC 5%) via oral gavage to six CD-1 male mice. Mice were killed by CO 2 infixation 1 h (n = 3) or 3 h (n = 3) postdosing. Brain samples were collected and homogenised in acetonitrile:water (1:1), with 300 µL for 100 mg. The resulting samples were added on a Acquity UPLC HSS T3 1.8 µm 2.1 × 50 mm column in the Xevo TQ-D UHPLC-MS/ MS, using multiple reaction monitoring. The mobile phase A (0.1% formic acid) and mobile phase B (methanol) had an average flow rate of 0.6 mL/min.

Relacorilant treatment and continuous corticosterone exposure
The animal study was approved by the ethics committee of Leiden University Medical Center. Male 8-week-old C57BL/6J mice were housed in conventional cages with a 12 h light:12 h darkness cycle and were given ad libitum access to food and water. To evaluate relacorilant in a disease model of continuous glucocorticoid exposure, mice were subcutaneously implanted with 12.5 mg of corticosterone-release pellets (Koorneef et al. 2020, Kroon et al. 2021. Mice were treated via daily oral gavage (09:00 h) with 60 mg/kg relacorilant or vehicle (10% DMSO, 0.1% Tween-80, 0.5% hydroxypropylmethylcellulose in PBS). After 5 days, mice fasted for 6 h, and blood was collected for immune cell analysis (Sysmex XT-2000iV) and to analyze plasma biochemistry. Mice were killed by CO 2 infixation, perfused with ice-cold PBS for 5 min and tissues were collected, snap-frozen in liquid nitrogen and stored at −80˚C until further processing.

Relacorilant treatment and acute dexamethasone exposure
The animal study was approved by the ethics committee of Leiden University Medical Center. Male 8-week-old 256:2 Journal of Endocrinology e220263 C57BL/6J mice were housed in conventional cages with a 12 h light:12 h darkness cycle and were given ad libitum access to food and water. To test HPA axis responsiveness, a 3-day experiment was performed with daily administration of 60 mg/kg relacorilant, 60 mg/kg mifepristone or vehicle (10% DMSO, 0.1% Tween-80, 0.5% hydroxypropylmethylcellulose in PBS) via oral gavage (09:00 h). On day 1, a novelty stress test was performed 1 h after oral gavage treatment. Blood was collected at T = 0 min and animals were placed in a new cage without bedding for 10 min. Mice were then placed back in their home cages, and blood was collected at T = 30, 60, 120 and 180 min. On day 3, animals were subcutaneously injected with solvent (PBS) or 5 mg/kg of dexamethasone-phosphate for 1 h after the last oral gavage with vehicle, relacorilant or mifepristone. After 6 h, animals were killed by CO 2 infixation, trunk blood was collected by heart puncture before the animals were perfused with ice-cold PBS for 5 min and tissues were collected, snap-frozen in liquid nitrogen and stored at −80˚C until further processing. Corticosterone levels were determined in all blood samples by using an HS EIA kit (Immunodiagnosticsystems, Boldon Business Park, UK).

Plasma biochemistry
Plasma samples were analyzed for glucose using the Glucose liquiUV mono Multipurpose reagent (10786, Diagnostics Worldwide) for ELISA. The coefficient of variation (CV) for the glucose assay was 5.23% in commercial control serum (Autocal -IMTTC 13160, Diagnostics Worldwide) across 25 assays. Insulin was measured in plasma samples using the Ultra-Sensitive Mouse Insulin ELISA Kit Crystal Chem Inc.). According to the manufacturer's instructions, the average CV was 25.82% in commercial control serum across 19 assays. The homeostatic model assessment for insulin resistance (HOMA-IR) was calculated by multiplying the fasted glucose concentration (mmol/L) by the fasted insulin concentration (mmol/L).

RNA isolation, cDNA synthesis and RT-PCR analysis
Total RNA was isolated from liver, adrenal gland, pituitary gland, hippocampus, interscapular brown adipose tissue (iBAT) and gonadal white adipose tissue (gWAT) samples using TriPure Isolation reagent (Roche). We used 1 µg RNA for cDNA synthesis using the Recombinant RNasin® Ribonuclease Inhibitor kit (Promega Corporation) and BioRad T100™ Thermal Cycler. Gene expression was assessed by real-time quantitative PCR using GoTaq® RT-qPCR system (Promega Corporation) and thermal cycler Bio-Rad CFX96.

Mouse AtT20 cells
Mouse corticotroph AtT20/D16v-F2 cells (ATCC number: CRL-1795) were routinely cultured in 75 cm 2 flasks in DMEM containing 10% FCS, l-glutamine (2 mmol/L) and penicillin (100 U/mL) at 37°C in a humidified incubator at 5% CO 2 , as previously described (Creemers et al. 2021). Once every week, cells were harvested using trypsin (0.05%) -EDTA (0.48 mM) solution. For experiments, cells were seeded at a density of 40,000 cells/well in 24-well culture plates in 1 mL of culture medium. After 6 h, cells were treated with vehicle (0.1% EtOH or 0.1% DMSO), dexamethasone, relacorilant or mifepristone at indicated concentrations and combinations. After 72 h, supernatant was collected, protease inhibitor trasylol (5 IU/mL) was added to prevent ACTH degradation and samples were stored at −20°C. ACTH was measured using the Immulite 2000 XPi immunoassay analyzer (Siemens Medical Solutions). Cells were used for RNA isolation using a High Pure RNA kit (Roche, 11828665001) according to manufacturer's protocol. All experiments were performed twice, and experimental groups were in quadruplicate. The relative expression of genes was normalized using the geometric mean of the expression of three reference genes (RG) Hprt, GusB and Gapdh, as previously described (Blažević et al. 2022).

Microarray assay for real-time coregulator-nuclear receptor interaction
GR-coregulator interactions were assessed in the presence of 1 µM cortisol, mifepristone or relacorilant as compared to vehicle (DMSO), as previously described (Desmet et al. 2014). A total of 154 short leucine-rich binding motifs representing 64 known nuclear receptor coregulators were attached to the solid support of the PamChip® array. The coregulator-derived motifs were incubated with HEK-293 cell lysates containing a GR LGD-GST (Invitrogen), the ligand (DMSO, cortisol, mifepristone or relacorilant) and a tag-specific antibody coupled to a fluorophore. The interaction between GR and the coregulator motifs was assessed by detection of the immunofluorescent signal. For all coregulator motifs, each treatment condition had four replicates from which the mean and the standard error of mean (s.e.m) of the immunofluorescent signal were calculated and expressed as relative interaction. For cortisol, mifepristone and relacorilant treatments, the modulation 256:2 Journal of Endocrinology e220263 index (MI) was calculated as the log 10 -transformed normalized interaction using DMSO as baseline.

Statistical analysis
All data are expressed as mean (±s.e.m). Statistical significance was calculated using the one-way ANOVA or two-way ANOVA in GraphPad Prism 8. All normalized data were corrected for outliers using GraphPad outlier calculator. The IC50 values for relacorilant and mifepristone in HEK-293 and AtT20 cells, and DEX EC50 in AtT20 cells were determined using GraphPad Prism 8 non-linear fitting. The MI statistical significance was calculated using the t.test in R v.4.0.

Relacorilant antagonizes GR signaling in HEK-293 cells
HEK-293 cells were treated with increasing doses of relacorilant or mifepristone to evaluate antagonism of DEX-or cortisol-induced human GR signaling. Against 3 nM DEX, relacorilant showed a potency (IC50 = 2 nM) that was five-fold lower than mifepristone (IC50 = 0.4 nM) but reached the same efficacy at 1 µM (Fig. 1A). In agonism mode, both relacorilant and mifepristone were not significantly different as compared to the vehicle group (Fig. 1B). Against 100 nM cortisol, relacorilant effectively antagonized GR activity (IC50 = 5.6 nM), with a four-fold lower potency as compared to mifepristone (IC50 = 1.3 nM) (Fig. 1C). Consistent with previous findings, no significant GR agonism was observed for either relacorilant or mifepristone (Fig. 1D). The results suggest that relacorilant antagonizes GR activity in HEK-293 cells with a lower potency but a similar efficacy as mifepristone. Relacorilant binding does not result in any prominent GR agonism.
The maximum detected concentration of relacorilant after i.v. dosing was 623.9 ng/mL at 0.083 h (5 min), with a plasma half-life of 0.69 h (41 min) following i.v. dosing. Upon oral dosing, the maximum concentration was 130 ng/mL at time point 0.5 h (30 min), with a plasma halflife of 1.62 h (97 min) following oral dosing. Based on these  Journal of Endocrinology e220263 data, the bioavailability (F) of relacorilant was estimated at 20.5% (Table 1).

Relacorilant treatment prevents glucocorticoidinduced hyperinsulinemia and immunosuppression
We next tested the effects of relacorilant in mice, in a model of continuous corticosterone exposure (Koorneef et al. 2020, Kroon et al. 2021. Daily administration of relacorilant partially prevented corticosterone-induced hyperinsulinemia (P < 0.001, Supplementary Fig. 1A, see section on supplementary materials given at the end of this article) without influencing plasma glucose levels ( Supplementary Fig. 1B), which resulted in a lower HOMA-IR (P < 0.01, Supplementary Fig. 1C). These beneficial effects of relacorilant on metabolic features were accompanied by effective GR antagonism on expression of its target genes Fkbp5 and Gilz in metabolic tissues including liver, brown adipose tissue and white adipose tissue ( Supplementary Fig. 1D, E and F). In addition to metabolic symptoms, continuous corticosterone exposure resulted in lower levels of total white blood cells (P < 0.001, Fig. 2A), lymphocytes (P < 0.001, Fig. 2B), eosinophils (P < 0.05, Fig. 2C) and monocytes (Fig. 2D), which were all normalized by relacorilant treatment (P < 0.001, Fig. 2A and B -P < 0.01, Fig. 2C -P < 0.05, Fig. 2D). To further investigate the effects of relacorilant on human immune cells, we utilized PBMCs isolated from four healthy donors in an ex vivo model of LPS-induced inflammation. DEX markedly reduced LPS-induced IL-6 production in all donors. Co-administration with mifepristone fully (donor 1-3) or partially (donor 4) prevented DEX-induced reduction of IL-6 levels ( Fig. 2E, F, G and H). Co-administration with relacorilant partially prevented the effect of DEX in donor 1 (Fig. 2E), fully prevented the effect of DEX in donors 2 and 3 ( Fig. 2F and G) and showed no effect in donor 4 (Fig.  2H). In conclusion, relacorilant treatment successfully counteracted corticosterone-induced hyperinsulinemia and immunosuppression in mice, as well as DEX-induced anti-inflammatory effects in human PBMCs for three out of four donors.

Relacorilant treatment shows modest disinhibition of the HPA axis compared to mifepristone
To evaluate the effects on HPA axis reactivity, a novelty stress test was performed in male mice 1 h after treatment with vehicle, mifepristone or relacorilant. In vehicletreated mice, the novelty stressor significantly elevated plasma corticosterone levels at T = 30 min (P < 0.01 vs T = 0), and these gradually declined to reach pre-novelty stress levels at T = 120 min and T = 180 min (Fig. 3).
Mifepristone pretreatment significantly increased plasma corticosterone before the novelty stressor (P < 0.001 vs vehicle). Corticosterone levels increased further due to the novelty stressor at T = 60 min (P < 0.01 vs T = 0) and remained high throughout the test (P < 0.001 vs vehicle at T = 30, 60, 120 and 180, Fig. 3). Relacorilant treatment significantly elevated corticosterone levels at T = 60 (P < 0.05) and T = 120 (P < 0.01) as compared to vehicle, demonstrating a relatively modest disinhibition of the HPA axis as compared to mifepristone (P < 0.001 between relacorilant and mifepristone at all time points). Novelty stress-induced corticosterone levels remained high at T = 180 after mifepristone but not relacorilant, suggesting rapid recovery of basal corticosterone levels upon relacorilant treatment (Fig. 3). In conclusion, relacorilant only modestly and transiently disinhibited the HPA axis, while mifepristone strongly disinhibited HPA axis activity, reflecting differences between the compounds with respect to interference with GR-mediated negative feedback.

Relacorilant treatment antagonizes DEX-induced gene expression in the adrenal gland and the pituitary but not in the CNS
Next, we more directly assessed GR antagonism in vivo.
After 3 consecutive days of treatment with either vehicle, mifepristone or relacorilant, male mice were injected with 5 mg/kg DEX 1 h after the last treatment, and tissues were harvested 6 h later. DEX treatment completely suppressed HPA axis activity as apparent from diminished endogenous corticosterone levels ( Supplementary Fig. 2A). This effect was neither influenced by relacorilant nor mifepristone pre-treatment (Supplementary Fig. 2A). Negative feedback on the HPA axis is regulated at multiple levels i.e. in the adrenal gland, the pituitary, the hypothalamus and the hippocampus. To evaluate GR antagonism at different levels of the HPA axis, we performed RT-qPCR analysis of well-known GR-responsive genes Fkbp5, Gilz and Mt2a. As a positive control for peripheral antagonism of GR signaling, we included gene expression analysis in the liver. In this tissue, relacorilant and mifepristone both effectively

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Journal of Endocrinology e220263 reduced DEX-induced Fkbp5 and Mt2a expression (P < 0.01 and P < 0.05 respectively, Fig. 4A and 4C), while Gilz expression was lowered by relacorilant but not by mifepristone treatment (P < 0.01, Fig. 4B). In the adrenal gland, DEX-induced upregulation of Fkbp5 and Gilz was prevented by both relacorilant (P < 0.05) and mifepristone (P < 0.001) ( Fig. 4D and E), while DEX-induced increase of Mt2a expression was only significantly reduced by mifepristone (P < 0.05, Fig. 4F). In the pituitary, relacorilant and mifepristone did not lower Fkbp5 expression (Fig. 4G) but significantly lowered DEX-induced Gilz (P < 0.05, Fig.  4H) and Mt2a expression (P < 0.05 and P < 0.01 respectively, Fig. 4I). As a proxy for GR signaling in the CNS, we analyzed gene expression in the hippocampus, where mifepristone significantly reduced DEX-induced expression of Fkbp5 (P < 0.05) and Mt2a (P < 0.05) but not Gilz (Fig. 4J, K and L). Relacorilant, on the other hand, did not influence any DEX-induced GR target gene expression in the hippocampus (Fig. 4J, K and L). We next investigated the expression of genes with tissue-specific function. Hepatic Mttp and adrenal Cyp11b1 were not influenced by DEX or GR antagonist pre-treatment (Supplementary Fig. 2B and C). In the pituitary, expression of Pomc (precursor for ACTH) was downregulated by DEX but not influenced by relacorilant nor mifepristone ( Supplementary Fig. 2D).
Hippocampal Sgk1 was upregulated after DEX injection but neither influenced by relacorilant nor mifepristone treatment ( Supplementary Fig. 2E). Finally, Nr3c1 (Gr) expression was altered by DEX treatment in the pituitary but not in the other tissues of interest (P < 0.001, Supplementary Fig. 2F, G, H and I), and this effect was not further influenced by either relacorilant or mifepristone pre-treatment ( Supplementary Fig. 2H). In the adrenal gland, Nr3c1 expression was higher after relacorilant treatment as compared to mifepristone (P < 0.01, Supplementary Fig. 2G). Altogether, the results suggest that relacorilant exerts GR antagonism in peripheral tissues but not in the CNS.

Relacorilant is less potent than mifepristone in AtT20 mouse pituitary cells
Slow feedback on the HPA axis by synthetic glucocorticoids takes place at the level of GR-mediated gene expression in the anterior pituitary. In addition, pituitary tumors constitute a common cause of endogenous CS (Barbot et al. 2020). We therefore evaluated the effects of relacorilant on DEX-induced gene expression in the mouse AtT20 pituitary cell line, in comparison with mifepristone. AtT20 cells were treated with vehicle or 1 nM -1 µM relacorilant or mifepristone in the presence of 5 nM DEX (corresponding to the EC80 for the measured genes, Supplementary Fig. 3A). DEX induced the expression of Fkbp5 and Gilz, which was dose-dependently prevented by relacorilant and mifepristone treatment, and IC50 values of relacorilant were approximately eight times higher as compared to mifepristone ( Fig. 5A and B). Both relacorilant and mifepristone only partially prevented DEX-induced downregulation of Nr3c1 expression (Fig. 5C). Finally, DEX treatment suppressed Pomc expression and ACTH release, which was prevented by both GR antagonists. Relacorilant showed a substantially lower potency as compared to mifepristone with a 26-fold difference in IC50 for Pomc expression and a 38-fold difference for ACTH release ( Fig.  5D and E). Altogether, these results suggested that both relacorilant and mifepristone antagonized GR signaling in mouse AtT20 pituitary cells, but relacorilant showed lower potency as compared to mifepristone, in particular with respect to ACTH production and release. These results demonstrate that the difference in potency between mifepristone and relacorilant depends on a GR-dependent process and was most substantial for the suppression of Pomc transcription and ACTH release.

Relacorilant induces a unique GR interactome that lacks two key transcriptional corepressors
The proteins that form the transcriptional complexes that mediate GR-dependent gene transcription differ per cell type and per gene. In view of the gene-dependent potency of relacorilant and mifepristone, we next characterized the GR interactome in the presence of 1 µM cortisol, mifepristone or relacorilant. We assessed the interaction between the ligand-binding domain of GR and 154 short leucine-rich binding motifs representing 64 known nuclear receptor coregulators. GR antagonists typically only stimulate a limited subset of GR interactions with transcriptional coregulators (Zalachoras et al. 2013, Viho et al. 2019. In the presence of cortisol, GR showed a significant interaction with a total of 55 motifs, while mifepristone and relacorilant significantly induced GR interaction with 15 and 8 motifs, respectively (P < 0.05, Fig.  6A). As anticipated, we also observed similar interaction scores for many GR coactivators for both GR antagonists (Fig. 6B). Several GR interactions were substantially weaker in the presence of GR antagonists as compared to cortisol for example, HAIR, NR0B2, PRGC1 and PRGC2 (P < 0.001, Fig. 6C, Supplementary Fig. 3B). When focusing further on the differences in the GR recruitment profile of both GR antagonists, we observed that relacorilant did not result in GR recruitment of corepressor motifs NCOR1 and NCOR2, while these were readily recruited by GR in the presence of mifepristone (P < 0.001, Fig. 6B and C, Supplementary Fig. 3B). In fact, NCOR1 and NCOR2 were specific to the mifepristone-bound GR complex as these interactions were also lacking in the presence of cortisol (P < 0.001, Fig. 6C, Supplementary Fig. 3B). Our results suggested that both mifepristone and relacorilant induced an antagonist-like GR transcriptional interactome, but the antagonistic properties of mifepristone seemed to involve GR interaction with corepressors such as NCOR1 and NCOR2, while relacorilant antagonism seemed to depend exclusively on the absence of coactivators in the GR interactome.

Discussion
In this study, we set out to further characterize the selective GR antagonist relacorilant preclinically, in particular with respect to HPA axis disinhibition (Fig.  7). There is a great need for improved GR antagonists, as the only marketed GR antagonist, mifepristone, exhibits cross-reactivity for the PR and is clinically associated with numerous side effects such as irregular vaginal bleeding and endometrial thickening (Sun et al. 2014). The use of mifepristone is also associated with mineralocorticoid effects (hypokalemia, hypertension and edema) (Yuen et al. 2015, Sai et al. 2019, Brown et al. 2020. Effective GR antagonism with relacorilant was previously shown in healthy human subjects, in which single or multiple daily dosing of relacorilant was reported to reduce prednisone-induced Fkbp5 expression in blood (Bali et al. 2016) and reversed the decrease in eosinophils caused by prednisone treatment (Hunt et al. 2018). In mice, we observed additional beneficial effects as relacorilant co-administration prevented corticosterone-induced hyperinsulinemia and immunosuppression. Due to the short length of this experiment, we were unable to evaluate the full range of metabolic effects of relacorilant treatment in this cohort. Our current results also confirmed GR antagonism in human HEK-293 cells and mouse AtT20 cells and showed that relacorilant treatment prevented the anti-inflammatory effect of DEX in human PBMCs exposed to LPS. Mifepristone treatment causes disinhibition of the HPA axis by inhibiting negative feedback, and this results in excessive levels of circulating ACTH and cortisol (Morgan & Laufgraben 2013). Previous results in patients with endogenous CS due to pituitary corticotroph adenomas showed that long-term treatment with mifepristone resulted in a continuous elevation of circulating ACTH (Fleseriu et al. 2014), driving subsequent cortisol elevations. In the same patient population, treatment with The IC50 values were determined using GraphPad Prism 8 non-linear fitting. The relative expression of genes was normalized using the geometric mean of the expression of three reference genes (RG) Hprt, GusB and Gapdh. DEX, dexamethasone; Mif., mifepristone; Rela., relacorilant; Fkbp5, FK506-binding protein 5; Gilz, glucocorticoidinduced leucine zipper protein; Nr3c1, nuclear receptor subfamily 3 group C member 1; Pomc, proopiomelanocortin; ACTH, adrenocorticotropic hormone; Hprt, hypoxanthine phosphoribosyltransferase 1; GusB, glucuronidase beta; Gapdh, glyceraldehyde-3-phosphate dehydrogenase.
relacorilant was not associated with a significant increase in serum ACTH and cortisol concentrations (Pivonello et al. 2021). In a novelty stress test in mice, we confirmed the strong disinhibition of the HPA axis by mifepristone treatment (Gaillard et al. 1984, Ratka et al. 1989. In contrast, relacorilant treatment only modestly and transiently disinhibited the HPA axis in mice in vivo. This was in line with our in vitro observations in mouse corticotroph cells in which relacorilant less potently counteracted DEXmediated suppression of Pomc mRNA and ACTH release compared to mifepristone. Interestingly, the fold difference between relacorilant and mifepristone for suppression of Pomc mRNA and ACTH release was four times larger in comparison to the effect on classical GR target genes Fkbp5 and Gilz, and Nr3c1 (GR). Thus, there seems to be an intrinsically low potency of relacorilant in the regulation of components of the HPA axis. It is important to note that AtT20 cells differ from normal corticotroph cells. For example, they have dysfunctional arginine-vasopressin (AVP) receptors, as AVP treatment fails to alter ACTH secretion (Lutz-Bucher et al. 1987). Therefore, differential modulation of ACTH secretion by the two GR antagonists may not reflect the in vivo situation.
Many GC effects are genomic and mediated by intracellular GRs; however, rapid effects on ACTH secretion can also be mediated by non-genomic pathways (Uchoa et al. 2014). It was previously shown in AtT20 cells and rat primary pituitary cells that corticosterone-mediated Relacorilant induces a unique GR interactome. (A) Interaction score of GR with 154 short leucine-rich binding motifs of 64 known nuclear receptor coregulators as measured by the modulation index (MI) in the presence of 1 µM cortisol, mifepristone or relacorilant. (B) Fold change-fold change plot of the interaction score of GR with nuclear receptor coregulator motifs as measured by the modulation index (MI) in the presence of 1 µM relacorilant compared to 1 µM mifepristone. (C) Interaction score of GR with a subset of nuclear receptor coregulator motifs as measured by relative binding in the presence of 1 µM cortisol, mifepristone or relacorilant. The relative interaction is expressed as mean ± s.e.m. and statistical significance was calculated using a one-way ANOVA in GraphPad Prism 8. * Significant difference with cortisol; $ Significant difference between mifepristone and relacorilant. The MI is expressed as the log 10 -transformed normalized relative interaction, and statistical significance was calculated with t-test using R v.4.0. e220263 inhibition of ACTH secretion involved membraneassociated GRs (Deng et al. 2015). Although mifepristone is unable to inhibit rapid GC effects in rat hypothalamus (Di et al. 2003), it is possible that mifepristone acts via membrane-associated GRs in corticotroph cells to a different extent than relacorilant. It is also clear from the study of GR dim/dim mice that ACTH production and release exhibit different sensitivity to GR-mediated inhibition (Reichardt et al. 1998). In these mice with impaired genomic GR signaling, Pomc mRNA levels were clearly altered via a genomic mechanism, but ACTH blood levels were not strongly affected. In the current study, the difference in potency of the two GR antagonists may therefore reflect differences in both genomic and nongenomic regulatory mechanisms.
Differences between relacorilant and mifepristone treatment could be the result of different pharmacokinetic properties. The pharmacokinetics of mifepristone have been assessed in rats, monkeys and humans but not in mice. It has been reported to be metabolized within 1-4 h following oral administration in rats with a bioavailability (F) of 39% (Deraedt et al. 1985, Sitruk-Ware & Spitz 2003, and within 20-30 h in humans (Heikinheimo 1997, Sarkar 2002. In mice, relacorilant had a plasma half-life of 1.62 h (97 min) following oral dosing with a bioavailability (F) of 20.5% (Table 1). Based on these data, we estimate that the half-life of mifepristone is slightly longer than relacorilant. There are major differences in half-life between rodents and humans, as in humans, the half-life of relacorilant is between 11 and 19 h (Hunt et al. 2018).

Figure 7
Graphical overview of the effects of relacorilant and mifepristone on HPA axis activity. Under normal physiological conditions in mice (central panel), CRH is released from the hypothalamus and stimulates the release of ACTH from the pituitary gland. ACTH induces the secretion of corticosterone from the adrenal gland into the bloodstream. Corticosterone exerts -via the GR -a negative feedback on the HPA axis to maintain homeostasis. Mifepristone treatment antagonizes the GR in the CNS, the pituitary and the adrenal gland which results in a strong disinhibition of the HPA axis and a significant increase of endogenous ACTH and corticosterone (right panel). Relacorilant treatment exhibits GR antagonism in the pituitary and the adrenal gland but to a much lesser extent in the CNS. Therefore, GR antagonism by relacorilant only results in a modest and transient disinhibition of the HPA axis which limits the increase of endogenous ACTH and corticosterone (left panel). CRH, corticotropin-releasing hormone; ACTH, adrenocorticotropic hormone;