γ secretase (IC50 = 4 nM); Notch (IC50 = 5 nM); Aβ40 (IC50 = 14 nM)

RO4929097 inhibits the synthesis of ICN, which lowers the expression of Hes1, the downstream Notch target, and causes A549 cells to have a less altered morphology. In human tumor-derived cells, RO4929097 inhibits Notch processing[1]. Breast cancer cells are inhibited by RO4929097 (1 µM) to a 20% extent for SUM149 cells and 10% for SUM190 cells. The invasiveness of SUM149 cells is not significantly affected by RO4929097. Both cell lines experience a significant reduction in colony formation when exposed to RO4929097, with SUM149 cells showing the greatest effect[2]. In vitro, RO4929097 suppresses primary melanoma cell proliferation, anchorage independent growth, and sphere formation[3].

RO4929097 (3-60 mg/kg, p.o.) has a substantial inhibitory effect on tumor growth in nude mice with A549 NSCLC xenografts when compared to animals receiving vehicle treatment. Initially, treatment causes established A549 tumors to regress when mice are given 60 mg/kg RO4929097 twice daily according to the 7+/14-schedule[1]. Primary melanoma cell growth is inhibited in vivo by RO4929097. After mice were injected with 10⁴ cells in vivo, it was found that the RO4929097-treated cells significantly delayed the formation of tumors compared to the vehicle-treated ones. The percentage of secondary tumors formed by RO4929097-treated cells is lower, and the secondary tumors formed by RO4929097-treated cells are smaller[3].

The Aβ peptides are quantified by ECL assays employing an Origen 1.5 Analyzer and a range of anti-Aβ antibodies following the use of RO4929097. The α-secretase cleavage site is immediately distal to an epitope in the Aβ peptide (within amino acids 18–21) that is bound by the 4G8 murine mAb. The C terminus, which is exposed following γ-secretase-mediated cleavage to produce amino acid 40 of the Aβ40 peptide, is bound by the G2–10 murine mAb. The C terminus of the Aβ42 peptide, which is generated by γ-secretase-mediated cleavage to produce amino acid 42, is bound by the rabbit antibody FCA3542. Biotin-LC-sulfo-N-hydroxysuccinimide-ester is used to biotinylate the 4G8 mAb. Using TAG-N-hydroxysuccinimide ester, the G2–10 and FCA3542 antibodies are ruthenylated. Biotinylated 4G8 and ruthenylated G2–10 are used to detect Aβ(x–40). Biotinylated 4G8 and ruthenylated FCA3542 are used to detect Aβ(x-42).

At a density of 5 × 10⁴ cells, the IBC cell lines SUM149 and SUM190 are seeded. 0.1 nM to 10 μM of RO4929097 are administered as a vehicle or escalating doses the following day. Trypsinization of the cells is done after 72 hours, and a hemocytometer is used to count the viable cells.

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Basal breast cancer, common among patients presenting with inflammatory breast cancer (IBC), has been shown to be resistant to radiation and enriched in cancer stem cells. The Notch pathway plays an important role in self-renewal of breast cancer stem cells and contributes to inflammatory signaling which promotes the breast cancer stem cell phenotype. Herein, we inhibited Notch signaling using a gamma secretase inhibitor, RO4929097, in an in vitro model that enriches for cancer initiating cells (3D clonogenic assay) and conventional 2D clonogenic assay to compare the effect on radiosensitization of the SUM149 and SUM190 IBC cell lines. RO4929097 downregulated the Notch target genes Hes1, Hey1, and HeyL, and showed a significant reduction in anchorage independent growth in SUM190 and SUM149. However, the putative self-renewal assay mammosphere formation efficiency was increased with the drug. To assess radiosensitization of putative cancer stem cells, cells were exposed to increasing doses of radiation with or without 1 μM RO4929097 in their standard (2D) and self-renewal enriching (3D) culture conditions. In the conventional 2D clonogenic assay, RO4929097 significantly sensitized SUM190 cells to ionizing radiation and has a modest radiosensitization effect in SUM149 cells. In the 3D clonogenic assays, however, a radioprotective effect was seen in both SUM149 and SUM190 cells at higher doses. Both cell lines express IL-6 and IL-8 cytokines known to mediate the efficacy of Notch inhibition and to promote self-renewal of stem cells. We further showed that RO429097 inhibits normal T-cell synthesis of some inflammatory cytokines, including TNF-α, a potential mediator of IL-6 and IL-8 production in the microenvironment. These data suggest that additional targeting agents may be required to selectively target IBC stem cells through Notch inhibition, and that evaluation of microenvironmental influences may shed further light on the potential effects of this inhibitor[2].

Mice: Mice treated with RO4929097 are given oral doses of suspensions ranging from 3 to 60 mg/kg RO4929097 in accordance with the prescribed regimens. RO4929097 is dosed at 60 mg/kg/d every other week for 4 weeks (7+/7- × 2 cycles) in the Calu-6 xenograft model. RO4929097 is dosed once daily at 10 mg/kg for 21 days for all other xenograft models. One-way ANOVA, the post hoc Bonferroni t test, and the Mann-Whitney rank-sum test are used to determine statistical analysis. When P ≤ 0.05, differences between groups are deemed significant. A549 tumors from both the vehicle-treated and specific RO4929097-treated groups are taken, processed, and embedded in paraffin for an overnight fix. They are then sectioned at 5 μM and stained with H&E for histopathology evaluation. The histology images were obtained with an Olympus BX51 microscope (×40 objective) mounted on a Nikon DS-Fi1 equipped with the NIS-Elements F2.20 software. Three A549 tumours are flash-frozen for Western blot analysis, one for each of the two groups (7 (60 mg/kg) or 21 days (3 and 30 mg/kg). H-200 antibody is used at a dilution of 1:1,000 to detect collagen type V, and at a dilution of 1:5,000 to detect MFAP5.

[1]. Luistro L, et al. Preclinical profile of a potent gamma-secretase inhibitor targeting notch signaling with in vivo efficacy and pharmacodynamic properties. Cancer Res. 2009, 69(19), 7672-7680.

[2]. Debeb BG, et al. Pre-clinical studies of Notch signaling inhibitor RO4929097 in inflammatory breast cancer cells. Breast Cancer Res Treat. 2012 Jul;134(2):495-510.

[3]. Huynh C, et al. The novel gamma secretase inhibitor RO4929097 reduces the tumor initiating potential of melanoma. PLoS One. 2011, 6(9), e25264.

RO4929097 is a member of the class of dibenzoazepines that is the amide formed from formal condensation of the carboxy group of 2,2-dimethyl-3-oxo-3-[(2,2,3,3,3-pentafluoropropyl)amino]propanoic acid with the amino group of (7S)-7-amino-5,7-dihydrodibenzo[b,d]azepin-6-one. It has a role as an EC 3.4.23.46 (memapsin 2) inhibitor. It is a dibenzoazepine, a lactam, an organofluorine compound and a dicarboxylic acid diamide.
Ro4929097 has been used in trials studying the treatment of Sarcoma, LYMPHOMA, Neoplasms, Wilm's Tumor, and OSTEOSARCOMA, among others.
Gamma-Secretase Inhibitor RO4929097 is an orally bioavailable, small-molecule gamma secretase (GS) inhibitor with potential antitumor activity. Gamma secretase inhibitor RO4929097 binds to GS and blocks activation of Notch receptors, which may inhibit tumor cell proliferation. The integral membrane protein GS is a multi-subunit protease complex that cleaves single-pass transmembrane proteins, such as Notch receptors, at residues within their transmembrane domains. Overexpression of the Notch signaling pathway has been correlated with increased tumor cell growth.

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Basal breast cancer, common among patients presenting with inflammatory breast cancer (IBC), has been shown to be resistant to radiation and enriched in cancer stem cells. The Notch pathway plays an important role in self-renewal of breast cancer stem cells and contributes to inflammatory signaling which promotes the breast cancer stem cell phenotype. Herein, we inhibited Notch signaling using a gamma secretase inhibitor, RO4929097, in an in vitro model that enriches for cancer initiating cells (3D clonogenic assay) and conventional 2D clonogenic assay to compare the effect on radiosensitization of the SUM149 and SUM190 IBC cell lines. RO4929097 downregulated the Notch target genes Hes1, Hey1, and HeyL, and showed a significant reduction in anchorage independent growth in SUM190 and SUM149. However, the putative self-renewal assay mammosphere formation efficiency was increased with the drug. To assess radiosensitization of putative cancer stem cells, cells were exposed to increasing doses of radiation with or without 1 μM RO4929097 in their standard (2D) and self-renewal enriching (3D) culture conditions. In the conventional 2D clonogenic assay, RO4929097 significantly sensitized SUM190 cells to ionizing radiation and has a modest radiosensitization effect in SUM149 cells. In the 3D clonogenic assays, however, a radioprotective effect was seen in both SUM149 and SUM190 cells at higher doses. Both cell lines express IL-6 and IL-8 cytokines known to mediate the efficacy of Notch inhibition and to promote self-renewal of stem cells. We further showed that RO429097 inhibits normal T-cell synthesis of some inflammatory cytokines, including TNF-α, a potential mediator of IL-6 and IL-8 production in the microenvironment. These data suggest that additional targeting agents may be required to selectively target IBC stem cells through Notch inhibition, and that evaluation of microenvironmental influences may shed further light on the potential effects of this inhibitor.[2]

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Several reports have demonstrated a role for aberrant NOTCH signaling in melanoma genesis and progression, prompting us to explore if targeting this pathway is a valid therapeutic approach against melanoma. We targeted NOTCH signaling using RO4929097, a novel inhibitor of gamma secretase, which is a key component of the enzymatic complex that cleaves and activates NOTCH. The effects of RO4929097 on the oncogenic and stem cell properties of a panel of melanoma cell lines were tested both in vitro and in vivo, using xenograft models. In human primary melanoma cell lines, RO4929097 decreased the levels of NOTCH transcriptional target HES1. This was accompanied by reduced proliferation and impaired ability to form colonies in soft agar and to organize in tridimensional spheres. Moreover, RO4929097 affected the growth of human primary melanoma xenograft in NOD/SCID/IL2gammaR-/- mice and inhibited subsequent tumor formation in a serial xenotransplantation model, suggesting that inhibition of NOTCH signaling suppresses the tumor initiating potential of melanoma cells. In addition, RO4929097 decreased tumor volume and blocked the invasive growth pattern of metastatic melanoma cell lines in vivo. Finally, increased gene expression of NOTCH signaling components correlated with shorter post recurrence survival in metastatic melanoma cases. Our data support NOTCH inhibition as a promising therapeutic strategy against melanoma.[3]

C22H20F5N3O3

469.4

469.142

C, 56.29; H, 4.29; F, 20.24; N, 8.95; O, 10.23

847925-91-1

49867930

White to off-white solid powder

1.4±0.1 g/cm3

696.3±55.0 °C at 760 mmHg

374.9±31.5 °C

0.0±2.2 mmHg at 25°C

1.558

4.81

3

8

5

33

771

1

N([C@@H]1C(=O)NC2C=CC=CC=2C2C=CC=CC1=2)C(=O)C(C)(C)C(=O)NCC(F)(F)C(F)(F)F

OJPLJFIFUQPSJR-INIZCTEOSA-N

InChI=1S/C22H20F5N3O3/c1-20(2,18(32)28-11-21(23,24)22(25,26)27)19(33)30-16-14-9-4-3-7-12(14)13-8-5-6-10-15(13)29-17(16)31/h3-10,16H,11H2,1-2H3,(H,28,32)(H,29,31)(H,30,33)/t16-/m0/s1

2,2-dimethyl-N-[(7S)-6-oxo-5,7-dihydrobenzo[d][1]benzazepin-7-yl]-N'-(2,2,3,3,3-pentafluoropropyl)propanediamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.33 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.33 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 2% DMSO+30% PEG 300+5% Tween+ddH2O: 10 mg/mL

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