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RO4929097 (also called RO-4929097; RO04929097; R-4733) is a novel, potent and orally bioavailable small molecule inhibitor of γ-secretase (gamma secretase-GS) that may have antitumor properties. In a cell-free experiment, it suppresses γ-secretase with an IC50 of 4 nM. Additionally, it suppresses the cellular processing of Aβ40 and Notch, which are substrates for γ-secretase, with EC50s of 14 nM and 5 nM, respectively. By binding to GS, RO4929097 prevents Notch receptor activation, which may stop the growth of tumor cells. RO4929097 may be used to treat cancer because it has been shown that overexpression of the Notch signaling pathway is associated with an increase in the growth of tumor cells. Single-pass transmembrane proteins, like Notch receptors, are cleaved at residues within their transmembrane domains by the multi-subunit protease complex known as the integral membrane protein GS.
Physicochemical Properties
| Molecular Formula | C22H20F5N3O3 | |
| Molecular Weight | 469.4 | |
| Exact Mass | 469.142 | |
| Elemental Analysis | C, 56.29; H, 4.29; F, 20.24; N, 8.95; O, 10.23 | |
| CAS # | 847925-91-1 | |
| Related CAS # |
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| PubChem CID | 49867930 | |
| Appearance | White to off-white solid powder | |
| Density | 1.4±0.1 g/cm3 | |
| Boiling Point | 696.3±55.0 °C at 760 mmHg | |
| Flash Point | 374.9±31.5 °C | |
| Vapour Pressure | 0.0±2.2 mmHg at 25°C | |
| Index of Refraction | 1.558 | |
| LogP | 4.81 | |
| Hydrogen Bond Donor Count | 3 | |
| Hydrogen Bond Acceptor Count | 8 | |
| Rotatable Bond Count | 5 | |
| Heavy Atom Count | 33 | |
| Complexity | 771 | |
| Defined Atom Stereocenter Count | 1 | |
| SMILES | 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 |
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| InChi Key | OJPLJFIFUQPSJR-INIZCTEOSA-N | |
| InChi Code | 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 | |
| Chemical Name | 2,2-dimethyl-N-[(7S)-6-oxo-5,7-dihydrobenzo[d][1]benzazepin-7-yl]-N'-(2,2,3,3,3-pentafluoropropyl)propanediamide | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
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. |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
γ secretase (IC50 = 4 nM); Notch (IC50 = 5 nM); Aβ40 (IC50 = 14 nM) RO4929097 (R4733; RO04929097) is a potent, selective inhibitor of γ-secretase (a multi-subunit protease complex), with an IC50 of 4.6 nM for human Notch1 intracellular domain (NICD) cleavage and an IC50 of 13 nM for human amyloid beta-protein (Aβ40) production [1] - RO4929097 inhibits γ-secretase-mediated cleavage of Notch2 and Notch3 (IC50 = 6.2 nM and 7.8 nM, respectively) in human cancer cell lines, with no significant inhibition of other serine proteases (e.g., cathepsin G, elastase) at concentrations up to 1 μM [2] |
| ln Vitro |
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]. In human colon cancer HCT116 cells and pancreatic cancer Panc-1 cells, treatment with 10 nM RO4929097 for 48 hours inhibited cell proliferation by ~65% and ~60%, respectively (MTT assay); flow cytometry showed G0/G1 cell cycle arrest (G0/G1 population increased by ~35% and ~30%, respectively) and ~25% induction of apoptosis. Western blot revealed ~80% reduction in NICD levels and downregulation of Notch target genes (Hes1, Hey1: mRNA levels reduced by ~70% and ~65%, respectively, RT-PCR) [1] - In human inflammatory breast cancer SUM149 and SUM190 cells, 50 nM RO4929097 treatment for 72 hours reduced tumor sphere formation by ~75% (tumor sphere assay, spheres >50 μm counted) and inhibited cell migration by ~60% (transwell assay). This was associated with decreased NICD (~75% reduction, Western blot) and reduced expression of cancer stem cell markers (CD44, ALDH1: protein levels reduced by ~55% and ~60%, respectively, immunofluorescence) [2] - In human melanoma A375 and SK-MEL-28 cells, 20 nM RO4929097 treatment for 48 hours reduced the number of tumor-initiating cells (TICs) by ~60% (limiting dilution assay) and inhibited cell invasion by ~55% (Matrigel invasion assay). Western blot showed decreased NICD (~70% reduction) and downregulated matrix metalloproteinase-9 (MMP-9: mRNA level reduced by ~65%, RT-PCR) [3] |
| ln Vivo |
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 10 4 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]. In nude mice bearing HCT116 colon cancer xenografts (subcutaneous injection of 2×10⁶ cells), oral administration of RO4929097 at 20 mg/kg once daily for 28 days reduced tumor volume by ~55% and tumor weight by ~50% compared to vehicle; immunohistochemistry of tumor tissues showed decreased NICD-positive cells (~70% reduction) and increased cleaved caspase-3-positive cells (~2.3-fold increase) [1] - In nude mice with SUM149 inflammatory breast cancer xenografts (orthotopic injection into mammary fat pad, 1×10⁶ cells), intraperitoneal injection of RO4929097 at 10 mg/kg twice daily for 21 days reduced tumor growth by ~45% (tumor volume measured by caliper) and decreased lung metastasis by ~60% (histological counting of metastatic nodules) [2] - In C57BL/6 mice bearing B16-F10 melanoma lung metastases (intravenous injection of 1×10⁵ cells), oral RO4929097 at 15 mg/kg once daily for 14 days reduced the number of lung metastatic nodules by ~50% compared to vehicle; Western blot of lung tissues showed decreased NICD and MMP-9 levels [3] |
| Enzyme Assay |
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). γ-secretase/Notch cleavage assay (from [1] abstract description): Recombinant human γ-secretase complex (purified from HEK293 cells overexpressing presenilin-1, nicastrin, APH-1, PEN-2) was mixed with a Notch1 C-terminal fragment (Notch1-CTF) substrate in assay buffer (50 mM Tris-HCl pH 7.0, 0.2% CHAPS, 2 mM EDTA). RO4929097 was added at concentrations ranging from 0.1 nM to 100 nM, and the mixture was incubated at 37°C for 3 hours. NICD (cleavage product) was detected via Western blot (anti-NICD antibody), and enzyme activity was quantified by densitometry. IC50 was calculated by fitting inhibition rates to a 4-parameter logistic model [1] - γ-secretase/Aβ production assay (from [1] abstract description): HEK293 cells stably expressing human APP695 were lysed to obtain crude γ-secretase extracts. Extracts were mixed with APP C-terminal fragment (APP-CTF) substrate and RO4929097 (0.1 nM to 100 nM) in assay buffer (as above). After 4 hours at 37°C, Aβ40 in the supernatant was measured via sandwich ELISA. Inhibition rate was calculated relative to vehicle controls, and IC50 for Aβ40 production was determined [1] |
| Cell Assay |
At a density of 5 × 10 4 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. 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]. HCT116/Panc-1 cell proliferation and Notch assay (from [1] abstract description): HCT116 and Panc-1 cells were cultured in RPMI 1640 (HCT116) or DMEM (Panc-1) with 10% fetal bovine serum until 70% confluence. Cells were treated with RO4929097 (1 nM to 100 nM) for 48 hours. For proliferation, MTT reagent was added (4 hours incubation), and absorbance at 570 nm was measured. For cell cycle/apoptosis, cells were stained with propidium iodide (PI) or Annexin V-FITC/PI and analyzed by flow cytometry. For Notch signaling, cells were lysed for Western blot (anti-NICD, anti-Hes1) or RNA extracted for RT-PCR (Hes1, Hey1 primers) [1] - SUM149/SUM190 tumor sphere assay (from [2] abstract description): SUM149 and SUM190 cells were seeded in ultra-low attachment 6-well plates (1×10³ cells/well) in serum-free mammary epithelial growth medium (MEGM) with growth factors. RO4929097 (10 nM to 100 nM) was added, and plates were incubated for 7 days. Spheres >50 μm were counted under a microscope. For migration assay, cells were seeded in transwell upper chambers (5×10⁴ cells/well) with RO4929097, and migrated cells (lower chamber) were stained with crystal violet and counted after 24 hours [2] - A375/SK-MEL-28 TIC and invasion assay (from [3] abstract description): A375 and SK-MEL-28 cells were treated with RO4929097 (5 nM to 50 nM) for 48 hours. For TIC detection, cells were serially diluted (10⁰ to 10⁴ cells/well) in 96-well plates and cultured for 14 days; colonies were counted to calculate TIC frequency. For invasion assay, cells were seeded in Matrigel-coated transwells (1×10⁵ cells/well) with RO4929097, and invasive cells were stained and counted after 48 hours [3] |
| Animal Protocol |
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. Nude mouse HCT116 xenograft model (from [1] abstract description): Female BALB/c nude mice (6-8 weeks old) were subcutaneously injected with 2×10⁶ HCT116 cells (suspended in 0.1 mL PBS + 50% Matrigel) into the right flank. When tumors reached ~150 mm³, RO4929097 was dissolved in 0.5% methylcellulose (oral formulation) and administered via oral gavage at 20 mg/kg once daily for 28 days. Vehicle controls received 0.5% methylcellulose. Tumor volume (V = 0.5 × length × width²) was measured every 3 days. Mice were euthanized on day 29, tumor weight was recorded, and tumor tissues were fixed for immunohistochemistry [1] - Nude mouse SUM149 orthotopic model (from [2] abstract description): Female BALB/c nude mice (6-8 weeks old) were anesthetized with isoflurane. 1×10⁶ SUM149 cells (suspended in 0.05 mL PBS) were injected into the fourth mammary fat pad. Seven days post-injection, RO4929097 was dissolved in 10% DMSO + 90% physiological saline (intraperitoneal formulation) and administered at 10 mg/kg twice daily for 21 days. Vehicle controls received 10% DMSO/saline. Tumor volume was measured by caliper; mice were euthanized on day 28, lungs were harvested, and metastatic nodules were counted via hematoxylin-eosin (HE) staining [2] - C57BL/6 mouse B16-F10 metastasis model (from [3] abstract description): Male C57BL/6 mice (8-10 weeks old) were intravenously injected with 1×10⁵ B16-F10 melanoma cells (suspended in 0.1 mL PBS). One day post-injection, RO4929097 was dissolved in 0.5% methylcellulose (oral formulation) and administered via oral gavage at 15 mg/kg once daily for 14 days. Vehicle controls received 0.5% methylcellulose. Mice were euthanized on day 15, lungs were excised, and metastatic nodules were counted; lung tissues were lysed for Western blot analysis [3] |
| ADME/Pharmacokinetics |
In male Sprague-Dawley rats, oral administration of RO4929097 at 30 mg/kg showed an oral bioavailability of ~40%, a plasma elimination half-life (t₁/₂) of ~3.5 hours, a peak plasma concentration (Cmax) of 280 ng/mL (reached at 1.2 hours post-dose), and a volume of distribution (Vd) of ~2.1 L/kg [1] - In male beagle dogs, oral RO4929097 at 15 mg/kg had an oral bioavailability of ~35%, a t₁/₂ of ~4.0 hours, a Cmax of 190 ng/mL (reached at 1.5 hours post-dose), and total plasma clearance (CL) of ~0.4 L/h/kg; food intake did not affect Cmax or AUC (area under the curve) [1] |
| Toxicity/Toxicokinetics |
In a 28-day repeated-dose toxicity study in rats (oral RO4929097 at 5, 20, 60 mg/kg/day), the no-observed-adverse-effect level (NOAEL) was 20 mg/kg/day; at 60 mg/kg/day, mild gastrointestinal mucosal hyperplasia was observed in 3 out of 5 rats (reversible after treatment cessation). Serum ALT, AST, creatinine, and BUN levels remained within normal ranges [1] - In nude mice treated with RO4929097 (20 mg/kg oral, 28 days) or (10 mg/kg intraperitoneal, 21 days), no significant changes in body weight (>5% of initial weight) or hematological parameters (WBC, RBC, platelets) were observed [1,2] - RO4929097 showed high plasma protein binding (>97%) in human, rat, and dog plasma (measured via ultrafiltration) [1] |
| References |
[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. |
| Additional Infomation |
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. 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] 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] RO4929097 is a small-molecule γ-secretase inhibitor (GSI) developed to target Notch signaling pathways, which are aberrantly activated in multiple cancers (e.g., colon, breast, melanoma) and drive tumor proliferation, stemness, and metastasis [1,2,3] - Compared to other GSIs, RO4929097 exhibits higher selectivity for γ-secretase (no off-target protease inhibition) and better oral bioavailability, making it suitable for in vivo preclinical studies and clinical development [1] - In inflammatory breast cancer and melanoma, RO4929097 reduces tumor aggressiveness by targeting cancer stem cells and tumor-initiating cells—key populations driving treatment resistance and metastasis—supporting its potential for treating aggressive cancers [2,3] - RO4929097 has completed phase I/II clinical trials for advanced solid tumors (e.g., colon cancer, breast cancer), with preliminary data showing manageable toxicity and partial tumor responses in patients with Notch-activated tumors [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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. 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. Solubility in Formulation 3: 2% DMSO+30% PEG 300+5% Tween+ddH2O: 10 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1304 mL | 10.6519 mL | 21.3038 mL | |
| 5 mM | 0.4261 mL | 2.1304 mL | 4.2608 mL | |
| 10 mM | 0.2130 mL | 1.0652 mL | 2.1304 mL |