GR/glucocorticoid receptor (Ki: 0.15 nM)

Nenocorilant (Relacorilant) (0-1000 nM; 0-72 h; OvCa cells) exhibits pro-apoptotic properties that partially restore the induction of apoptosis in tumor cells by paclitaxel and gemcitabine, while also counteracting the effects of cortisol [1]. The GR target genes SGK1 and MKP1 are not upregulated by dex when applied at a concentration of 100 nM for four hours in MIA PaCa-2 and OVCAR5 cells [2].

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In the MIA PaCa-2 cell line, paclitaxel-driven apoptosis was blunted by cortisol and restored by Relacorilant. In the OVCAR5 cell line, relacorilant improved the efficacy of paclitaxel and the potency of platinum agents. A screen to identify optimal combination partners for relacorilant showed that microtubule-targeted agents consistently benefited from combination with relacorilant.[1]

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Cortisol suppresses and Relacorilant restores apoptotic effects of paclitaxel in vitro [1]
To interpret the effects of relacorilant in combination with cytotoxic therapy, the effects of GC alone first needed to be characterized. Solid-tumor cell lines whose growth was affected by GC were identified (Table 1). GC increased the viability of three cell lines, suggesting that a GR antagonist might decrease the viability of those lines independent of cytotoxic agent activity. Such cell lines were excluded from further study as the potentiation of the cytotoxic agent could not be easily distinguished from direct effects on viability. Cell lines like OVCAR5, in contrast, provide a more interpretable model to investigate the effects of GC on cytotoxic agent activity because GC alone did not significantly affect growth. To avoid interference from GCs in FBS, FBS was diluted to 2.5% v/v or charcoal-dextran stripped FBS (CDS-FBS, in which the GCs are removed) was used. Similar effects of GC were observed across various tissues of origin and in both standard 2.5% FBS and CDS-FBS. These initial experiments were conducted using a viability assay (CellTiter-Glo), and thus effects on growth rate could not be distinguished from effects on apoptosis.

To explore the breadth of these pro-apoptotic effects of relacorilant in combination with paclitaxel, the MIA PaCa-2 cells were next assessed with distinct cytotoxic agents and dexamethasone, a GR agonist. Using 100 nM dexamethasone, MIA PaCa-2 viability was assessed using a viability (CellTiter-Glo) endpoint assay across a range of gemcitabine combinations. Dexamethasone numerically increased the IC50 (i.e., reduced potency) of gemcitabine (Figure 1C), though the effect was non-significant. This modest effect was dose-dependently reversed by adding Relacorilant (Figure 1C), while the effect was again non-significant. Dexamethasone increased the residual viability (i.e., reduced the maximal efficacy) of high doses (> 0.1 μM) of gemcitabine (Figure 1D). Relacorilant alone had no off-target, agonist-independent activity, which is consistent with the mechanism of a competitive antagonist neutralizing the activity of an agonist (dexamethasone or cortisol). Both cortisol, used to model endogenous GR agonism, and dexamethasone, used to assess a more GR-specific agonist were assessed in these experiments. The data in Figure 1 demonstrates that both agonists can promote MIA PaCa-2 cell survival in the presence of distinct cytotoxic agents and relacorilant can reverse these effects.

To confirm the dose dependency of the observed GR agonist and antagonist effects, dexamethasone or relacorilant were titrated while cell viability was assessed. OVCAR5 cells were used to extend findings into cells of distinct tissue of origin, and carboplatin was chosen given its use in ovarian cancer clinical practice. In the presence of 100 nM dexamethasone, carboplatin potency was improved by increasing concentrations of relacorilant (Figure 2A). The IC50 of carboplatin was calculated at each concentration of relacorilant and plotted to demonstrate the dose-dependent effect of relacorilant on carboplatin potency (Figure 2B). Conversely, the carboplatin potency was diminished by increasing concentrations of dexamethasone (Figure 2C). The effect of dexamethasone on carboplatin potency was eliminated by the presence of relacorilant (Figure 2C). These data support on-target, GR-mediated, dose-dependent effects of relacorilant on carboplatin potency.

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Relacorilant improves efficacy of microtubule inhibitors [1]
The observations in Figure 2 and Table 1 raised the possibility that the effects of relacorilant on a cytotoxic agent’s potency or efficacy could be related to the cytotoxic agent’s mechanisms of action. Viability of OVCAR5 cells was initially tested with 19 cytotoxic agents alone to determine baseline potency and efficacy (data not shown). Subsequently, the dose response for each agent was conducted with GR agonist, GR antagonist, both, or neither. Relacorilant improved IC50, residual viability, or both for multiple agents (Figure 3). Microtubule inhibitors showed a consistent benefit from combination with relacorilant in the OVCAR5 cell line.

In xenograft models under physiological cortisol conditions, Nenocorilant (Relacorilant) (30 mg/kg; po; every four days for 25 days; female Balb/c nude mice) enhances the effectiveness of cytotoxic therapy and stimulates apoptotic activity [1].

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Relacorilant increases apoptotic activity of cytotoxic therapy in xenograft models [1]
To assess the effects of relacorilant under physiological GC conditions, xenograft studies were conducted. The mice were treated and handled to avoid altering normal GC levels, which in the mouse are primarily corticosterone levels, by allowing extra time for acclimatization and minimizing handling. Consistent with the in vitro results, Relacorilant improved the efficacy of paclitaxel in the MIA PaCa-2 xenograft (Figure 4A). Because gemcitabine + paclitaxel is an approved standard of care in pancreatic cancer, relacorilant benefit on top of gemcitabine + paclitaxel was also assessed (Figure 4B). Gemcitabine + paclitaxel was more effective than paclitaxel alone, and relacorilant further improved the efficacy of gemcitabine + paclitaxel. To extend these findings, xenografts from distinct tissues of origin were also assessed. Both the HeLa (cervical) xenograft and CC6279 (a patient-derived cholangiocarcinoma xenograft) showed significant improvements in the efficacy of paclitaxel when relacorilant was added (Figure 4C and 4D). As expected from the in vitro results, relacorilant alone had no effects in the CC6279 models (Figure 4D) and was thus not included in other xenograft studies as it would be unethical and unnecessary to repeatedly confirm this. Together, these data demonstrate that relacorilant can improve the efficacy of chemotherapies in vivo.

The initial in vitro observations suggested that an increase in apoptosis, rather than a decrease in proliferation rate, was achieved when Relacorilant was added to a cytotoxic therapy (Figure 1). To determine if this was recapitulated in vivo, apoptosis markers were assessed in relacorilant-treated xenografts. The CC6279 cholangiocarcinoma model was assessed because the relacorilant effect size was greatest. Cleaved caspase 3 activity was qualitatively and quantitatively elevated in mice treated with relacorilant + paclitaxel compared to paclitaxel alone, while no difference was observed for CK18 (a tumor cell marker) and Ki67 (a proliferation marker) (Figure 4E and 4F). Consistent with the initial in vitro observation, relacorilant promoted tumor cell apoptosis in xenograft models.

In vitro viability assays [1]
For viability assays, tumor cells were seeded in 96-well plates in a volume of 180 μL at a density designed to achieve 3–5 doublings during the incubation period. Dexamethasone, cortisol, and relacorilant were added 1 day later, and cytotoxic agents were added an additional day after that. Cells were incubated for an additional 3 days before assaying with CellTiter-Glo®. High (untreated) and low (no plated cells) controls for each plate were used for normalization and to calculate residual viability using the formula: Residual viability = (Max viability of cytotoxic agent at top concentrations)/(High control – low control) × 100. Curve fits were conducted in GraphPad Prism using a 4-parameter fit. Media were removed and 200 μL of CellTiter-Glo reagent were added to each well. The plate was shaken for 2 minutes and then left to equilibrate for 10 minutes prior to reading luminescence on the Biotech Synergy II microplate reader.

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In vitro apoptosis assays [1]
For kinetic image-based assays, tumor cells were seeded in a volume of 180 μL with Incucyte® NucLight Rapid Red and Incucyte Caspase 3/7 reagent. One to three images/day/well were acquired using an Incucyte S3 live cell imaging system. Image acquisition began 1 day after seeding and coincided with the addition of cortisol/relacorilant (day 1; 0 h). Paclitaxel was added on day 2 (24 h). Image analysis was conducted using the Incucyte Analysis Software to determine the number of cells positive for each fluorescence marker. The apoptotic index was calculated by dividing the number of caspase 3/7 positive cells by the total number of NucLight positive cells.

Animal/Disease Models: Female Balb/c nude mice [1]
Doses: 30 mg/kg
Route of Administration: po (po (oral gavage)) once every four days for 25 days
Experimental Results: Inhibited tumor growth and increased the expression level of caspase 3.

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MIA PaCa-2 xenografts [1]
3 × 106 MIA PaCa-2 (pancreatic) cells were implanted in the right flank of female Balb/c nude mice in 100 μL phosphate-buffered saline (PBS) mixed 1:1 with Matrigel®. Treatment began upon randomization with relacorilant 30 mg/kg formulated in 10% DMSO/0.1% Tween® 80/89.9% HPMC (0.5%) and/or paclitaxel in 0.9% sodium chloride solution. Paclitaxel was dosed once every four days (Q4D) via intravenous injection, and relacorilant was dosed the day prior and day of paclitaxel administration by oral gavage.

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HeLa xenograft [1]
5 × 106 HeLa (cervical) cells were implanted in the right flank of female Balb/c nude mice in 100 μL PBS mixed 1:1 with Matrigel. Treatment began upon randomization with relacorilant 30 mg/kg formulated in 10% DMSO/0.1% Tween 80/89.9% HPMC (0.5%) and/or paclitaxel in 0.9% sodium chloride solution. Paclitaxel was dosed Q4D via intravenous injection, and relacorilant was dosed the day prior and day of paclitaxel administration by oral gavage.

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CC6279 xenograft [1]
Fresh tumor tissues from mice bearing established CC6279 (cholangiocarcinoma) patient-derived tumors were harvested and cut into small pieces (approximately 2–3 mm in diameter). Tumor fragments were implanted in the right flank of female Balb/c nude mice in 100 μL PBS mixed 1:1 with Matrigel. Treatment began upon randomization with relacorilant 30 mg/kg formulated in 10% DMSO/0.1% Tween 80/89.9% HPMC (0.5%) and/or paclitaxel in 0.9% sodium chloride solution. Paclitaxel was dosed Q4D via intravenous injection, and relacorilant was dosed daily by oral gavage. Upon termination, tumors were excised and fixed in 10% neutral buffered formalin then paraffin embedded.

[1]. Glucocorticoid receptor antagonism promotes apoptosis in solid tumor cells. Oncotarget. 2021 Jun 22;12(13):1243-1255.

[2]. Glucocorticoid receptor activation inhibits chemotherapy-induced cell death in high-grade serous ovarian carcinoma. Gynecol Oncol. 2015 Sep;138(3):656-62.

[3]. WHO Drug Information. International Nonproprietary Names for Pharmaceutical.

Background: Resistance to antiproliferative chemotherapies remains a significant challenge in the care of patients with solid tumors. Glucocorticoids, including endogenous cortisol, have been shown to induce pro-survival pathways in epithelial tumor cells. While pro-apoptotic effects of glucocorticoid receptor (GR) antagonism have been demonstrated under select conditions, the breadth and nature of these effects have not been fully established.
Materials and Methods: To guide studies in cancer patients, relacorilant, an investigational selective GR modulator (SGRM) that antagonizes cortisol activity, was assessed in various tumor types, with multiple cytotoxic combination partners, and in the presence of physiological cortisol concentrations.
Results: In the MIA PaCa-2 cell line, paclitaxel-driven apoptosis was blunted by cortisol and restored by relacorilant. In the OVCAR5 cell line, relacorilant improved the efficacy of paclitaxel and the potency of platinum agents. A screen to identify optimal combination partners for relacorilant showed that microtubule-targeted agents consistently benefited from combination with relacorilant. These findings were confirmed in xenograft models, including MIA PaCa-2, HeLa, and a cholangiocarcinoma patient-derived xenograft. In vivo, tumor-cell apoptosis was increased when relacorilant was added to paclitaxel in multiple models.
Conclusions: These observations support recently reported findings of clinical benefit when relacorilant is added to paclitaxel-containing therapy in patients with ovarian and pancreatic cancers and provide a new rationale for combining relacorilant with additional cytotoxic agents.[1]

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Objectives: To test the hypothesis that glucocorticoid receptor (GR) activation increases resistance to chemotherapy in high-grade serous ovarian cancer (HGS-OvCa) and that treatment with a GR antagonist will improve sensitivity to chemotherapy.
Methods: GR expression was assessed in OvCa cell lines by qRT-PCR and Western blot analysis and in xenografts and primary human tumors using immunohistochemistry (IHC). We also examined the effect of GR activation versus inhibition on chemotherapy-induced cytotoxicity in OvCa cell lines and in a xenograft model.
Results: With the exception of IGROV-1 cells, all OvCa cell lines tested had detectable GR expression by Western blot and qRT-PCR analysis. Twenty-five out of the 27 human primary HGS-OvCas examined expressed GR by IHC. No cell line expressed detectable progesterone receptor (PR) or androgen receptor (AR) by Western blot analysis. In vitro assays showed that in GR-positive HeyA8 and SKOV3 cells, dexamethasone (100nM) treatment upregulated the pro-survival genes SGK1 and MKP1/DUSP1 and inhibited carboplatin/gemcitabine-induced cell death. Concurrent treatment with two GR antagonists, either mifepristone (100nM) or CORT125134 (100nM), partially reversed these effects. There was no anti-apoptotic effect of dexamethasone on chemotherapy-induced cell death in IGROV-1 cells, which did not have detectable GR protein. Mifepristone treatment alone was not cytotoxic in any cell line. HeyA8 OvCa xenograft studies demonstrated that adding mifepristone to carboplatin/gemcitabine increased tumor shrinkage by 48% compared to carboplatin/gemcitabine treatment alone (P=0.0004).
Conclusions: These results suggest that GR antagonism sensitizes GR+ OvCa to chemotherapy-induced cell death through inhibition of GR-mediated cell survival pathways.[2]

C26H21F4N7O3S

587.548657178879

587.136

C, 53.15; H, 3.60; F, 12.93; N, 16.69; O, 8.17; S, 5.46

1496509-78-4

89954189

White to off-white solid powder

3.2

0

12

5

41

1140

1

C([C@@]12C(=CC3N(C4=CC=C(F)C=C4)N=CC=3C1)CCN(S(C1C=NN(C)N=1)(=O)=O)C2)(C1=NC=CC(C(F)(F)F)=C1)=O

PZIQQHVUAHTSJN-VWLOTQADSA-N

InChI=1S/C26H21F4N7O3S/c1-35-32-14-23(34-35)41(39,40)36-9-7-17-11-22-16(13-33-37(22)20-4-2-19(27)3-5-20)12-25(17,15-36)24(38)21-10-18(6-8-31-21)26(28,29)30/h2-6,8,10-11,13-14H,7,9,12,15H2,1H3/t25-/m0/s1

[(4aR)-1-(4-fluorophenyl)-6-(2-methyltriazol-4-yl)sulfonyl-4,5,7,8-tetrahydropyrazolo[3,4-g]isoquinolin-4a-yl]-[4-(trifluoromethyl)pyridin-2-yl]methanone

Nenocorilant; 1496509-78-4; Nenocorilant [INN]; MV83V7AT2Z; CHEMBL4068844; HY-147293; [(4aR)-1-(4-fluorophenyl)-6-(2-methyltriazol-4-yl)sulfonyl-4,5,7,8-tetrahydropyrazolo[3,4-g]isoquinolin-4a-yl]-[4-(trifluoromethyl)pyridin-2-yl]methanone; ((4aR)-1-(4-Fluorophenyl)-1,4,5,6,7,8-hexahydro-6-((2-methyl-2H-1,2,3-triazol-4-yl)sulfonyl)-4ah-pyrazolo(3,4-g)isoquinolin-4a-yl)(4-(trifluoromethyl)-2-pyridinyl)methanone;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)