Mitochondrial metabolism

GPM-2 stomach cancer cells undergo apoptosis when exposed to devimistat. Devimistat specifically targets a modified version of mitochondrial energy metabolism that tumor cells use. Devimistat causes alterations in the cellular redox state and mitochondrial enzyme activity, which result in cell death, including apoptosis [1].

CPI-613 (25 mg/kg) has potent anticancer activity in a human tumor xenograft model of of a pancreatic tumor cell (BxPC-3). Similarly, CPI-613 (10 mg/kg) also produces significant tumor growth inhibition of H460 human non-small cell lung carcinoma in mouse model. Besides, CPI-613 produces little or no side-effect toxicity in expected therapeutic dose ranges in large animal models and has the maximum tolerated dose of 100 mg/kg in mice.

JC-1 analysis for mitochondrial membrane potential (MMP)[2]
MMP was measured by the JC-1 fluorescent probe. CPI-613-treated or non-treated cells were incubated with JC-1 (1:1000 dilution) for 20 min at 37 °C. After PBS washing, cells were observed under a fluorescence microscope with the red fluorescence (550 nm excitation/600 nm emission) and green fluorescence channels (485 nm excitation/535 nm emission). Quantitative analysis of Red/Green fluorescence ratio was measured by NIH ImageJ software.

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Measurement of ROS levels[2]
Intracellular ROS production was determined using the oxidant-sensing fluorescent probe DCFH-DA. Briefly, cells were incubated with 10 μM of DCFH-DA for 20 min at 37 °C and images were captured using a fluorescence microscope. Median fluorescence intensity from at least 100 cells in randomly selected fields were quantified by NIH Image J software as we previously described.

Transmission electron microscopy (TEM)[2]
Approximately 1.0 × 107 cells treated with 200 μM CPI-613 or vehicle were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate (NaCAC) buffer (pH 7.4) for 45 min. The samples were post-fixed in 2% osmium tetroxide in NaCAC, stained with 2% uranyl acetate, dehydrated with a graded ethanol series and embedded in Epon-Araldite resin. Thin sections were cut with a Leica EM UC6 ultramicrotome, collected on copper grids, and stained with uranyl acetate and lead citrate. Cells were observed in a Hitachi HT7700 transmission electron microscope and imaged with an UltraScan 4000 CCD camera and First Light Digital Camera Controller.

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Three-dimensional (3D) cell culture[2]
Briefly, 1 × 105 cells were seeded into 48-well SeedEZ scaffold supplied with complete medium. After 3 days of culture, cells growing in the SeedEZ scaffold were treated with 200 μM CPI-613 for 5 days, and cell viability was measured by alamarBlue at 545/590 nm ex/em, followed by phalloidin staining and imaging as we previously described.

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Lipolysis analysis[2]
Lipid droplets and free fatty acids (FFA) released into the culture medium of pancreatic cancer cells were measured to evaluate lipolysis. AsPC-1 and PANC-1 cells were treated with 200 μM CPI-613 for 48 h prior to lipolysis assessment. To determine lipid droplets, cells were fixed with 4% paraformaldehyde and stained with the dye Oil-Red-O for 30 min using the isopropanol method, followed by processed for haematoxylin staining. The released FFA levels were measured by Free Fatty Acid Quantification Kit according to the manufacturer’s instruction. The absorbance at 570 nm was measured immediately afterwards on a microplate reader.

Tumorigenicity In Vivo Assay[3]
To analyze the in vivo tumorigenicity rate after CPI-613 pretreatment in vitro, OVCAR3 cells were treated in vitro with either CPI-613 (75 µM) or vehicle every 72 h. Cells were harvested after 7 d and 1 × 106 cells were injected respectively in 5 mice for each arm: vehicle and CPI-613 pretreated. The tumorigenicity rate was analyzed after 21, 35 and 48 d. All mice were euthanized with CO2 after 48 d.

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In Vivo Experiment[3]
Using an institutionally approved Institutional Care and Use Committee (IACUC) protocol (2017N0000236), twelve-week old NOD/SCID mice were subcutaneously injected with 3 × 106 OVCAR3 cells 1:1L PBS:Matrigel. Measurements of the resulting tumors were determined by calipers every other day, and the bodyweight of each mouse was assessed twice per week. The tumor volume was calculated using the following formula: (width2 × height)/2. When the tumor volume reached 150 to 200 mm3, the mice were randomly divided into four arms. The treatments included vehicle, carboplatin/paclitaxel (25 mg/kg and 7 mg/kg, respectively), and CPI-613 (12.5 mg/kg) as single agents or carboplatin/paclitaxel in combination with CPI-613. A second in vivo 4 arm experiment was conducted only the treatments included vehicle, olaparib (50 mg/kg), and CPI-613 (25 mg/kg) as single agents or olaparib in combination with CPI-613 (25 mg/kg). Both the experiments were 14 days in length, and treatments were administered via intraperitoneal injection (carboplatin/paclitaxel and CPI-613 weekly administration, olaparib daily administration).
Tumor volume was measured every three days. At the completion of the experiment, mice were euthanized in accordance the with IACUC approved protocol, and xenografts were harvested. Portions of each xenograft were snap-frozen as well as formaldehyde-fixed and paraffin-embedded for further analyses. Tumors were processed following a previously described protocol (and H-2Kd+ mouse cells were removed using a fluorescein isothiocyanate (FITC) conjugated antibody and Macs LD columns as per manufacturers’ recommendations. H-2Kd- cells were stained with Live-Dead (Pacific Blue, 1:600), anti-CD133 (CD133/2 clone 293C3, 1:10, PE-conjugated) and anti-CD117 (clone A3C6E2, 1:10, APC-conjugated) and analyzed using FACS LSRII cytofluorimeter. Data were collected from at least 1 × 105 live cells/sample and analyzed with FlowJo 10.1 version.

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Dissolved in DMSO and then diluted in water.; 25 mg/kg; i.p. administration

CD1 nu/nu mice bearing BxPC-3 and H460 cells tumor models

[1]. Sakuratani T, et al. Downregulation of ARID1A in gastric cancer cells: a putative protective molecular mechanism against the Harakiri-mediated apoptosis pathway. Virchows Arch. 2021;478(3):401-411.
[2]. CPI-613 rewires lipid metabolism to enhance pancreatic cancer apoptosis via the AMPK-ACC signaling. J Exp Clin Cancer Res. 2020; 39: 73.
[3]. The Metabolic Inhibitor CPI-613 Negates Treatment Enrichment of Ovarian Cancer Stem Cells.Cancers (Basel). 2019 Nov; 11(11): 1678.

C22H28O2S2

388.59

388.15307

C, 68.00; H, 7.26; O, 8.23; S, 16.50

95809-78-2

Devimistat-d10;2586055-61-8

White to off-white solid powder

5.6

87.9

O=C(O)CCCCC(SCC1=CC=CC=C1)CCSCC2=CC=CC=C2

ZYRLHJIMTROTBO-UHFFFAOYSA-N

InChI=1S/C22H28O2S2/c23-22(24)14-8-7-13-21(26-18-20-11-5-2-6-12-20)15-16-25-17-19-9-3-1-4-10-19/h1-6,9-12,21H,7-8,13-18H2,(H,23,24)

6,8-bis(benzylthio)octanoic acid

CPI613; CPI-613; Devimistat; CPI 613

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.35 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 20.8 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: ≥ 2.08 mg/mL (5.35 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.35 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2 mg/mL (5.15 mM) in 2% DMSO + 40% PEG300 + 5% Tween80 + 53% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 5: ≥ 2 mg/mL (5.15 mM) (saturation unknown) in 2% DMSO 98% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

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Solubility in Formulation 6: 1% DMSO+30% polyethylene glycol+1% Tween 80:30 mg/mL

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