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].

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Relationship between ARID1A and Harakiri in apoptosis of gastric cancer cells [1]
Harakiri (also known as death protein 5, DP5) is well-characterized as promoting apoptosis by interacting with the apoptotic inhibitors Bcl-2 and Bcl-XL via the mitochondrial alteration. Devimistat is a recently developed lipoic acid antagonist that abrogates mitochondrial energy metabolism to induce apoptosis in various cancer cells. Devimistat also induced apoptosis of GPM-2 gastric cancer cells in the present study. Interestingly, siRNA-mediated downregulation of ARID1A conferred resistance to devimistat-induced apoptosis in GPM-2 cells. Notably, exogenous expression of Harakiri significantly restored the sensitivity of GPM-2 gastric cancer cells to devimistat-induced apoptosis even when ARID1A was downregulated. Representative data are shown in Fig. 3. These findings imply a relationship between ARID1A and the Harakiri-mediated apoptosis pathway in gastric cancer cells.

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.

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Devimistat (CPI-613)  Treatment Negatively Impacts CSC-Rich Spheres and Results in a Decrease in Tumorigenicity In Vivo [2]
Many studies provide evidence that sphere-forming conditions enrich CSCs in vitro. To confirm the CPI-613 target effect on the CSC population, we treated UWB1.289 MUT and OVCAR3 spheres after incubating these cells for 14 days in sphere promoting culture conditions in low-adhesion plates (Figure 2A). In contrast to what was observed in the previous monolayer experiments, carboplatin/paclitaxel treatment, used as positive control, had no effect (p-value > 0.05) on CD133+ and CD117+ cell frequency at this concentration of drug under these sphere-forming culture conditions which preferentially enrich for CSCs when compared to vehicle, indirectly confirming CSC resistance to cytotoxics. Interestingly, CPI-613 treatment decreased CD133+ and CD117+ cell frequency (p-value < 0.01) in CSC-rich spheres, corroborating its target effect on CSC population. Combining CPI-613 and carboplatin/paclitaxel on CSC-rich spheres resulted in a decrease in CD133+ and CD117+ cells frequency compared to treatment with vehicle or carboplatin/paclitaxel treatments alone (Figure 2A, p-value < 0.001). The only additive effect of combining CPI-613 with carboplatin/paclitaxel was observed in the CD117 population in the UWB1.289 cells.

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Devimistat (CPI-613)  Treatment In Vivo Induces a Decrease in CD133+ and CD117+ Cell Frequency [2]
The CPI-613 target effect on the CSC population in vitro led us to investigate whether we would observe a similar effect in vivo. OVCAR3 cells were injected s.c. in NOD/SCID (NOD.CB17-Prkdcscid/NCrCrl congenic immunodeficient) mice, and once tumors reached 200 mm3 of volume, the mice were treated weekly i.p. with CPI-613 at a concentration of 12.5 mg/kg. The mice were euthanized 48 h after the second injection, which was day 9 post initiating treatment (Figure 3A). At this time point, flow cytometric analysis of tumor cells revealed a decrease in CD133+ and CD117+ tumor cell frequency in CPI-613-treated mice compared with vehicle-treated mice (p-value < 0.001) (Figure 3B lower panel). This effect on CD133+ and CD117+ cell populations was similar to what was observed in the in vitro analysis, confirming the ability of CPI-613 to reduce CSC frequency in the tumor.

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Combining Devimistat (CPI-613)  and Carboplatin/Paclitaxel Treatment Impacts Tumor Growth Compared to CPI-613 Single Agent [2]
Combination drug therapy has played a particularly prominent role in the treatment of cancers as it targets multiple cancer cell-survival, promoting pathways delaying the onset of treatment resistance. We demonstrated the combination of CPI-613 and carboplatin/paclitaxel in vitro negatively impacted the enrichment of chemoresistant cells in culture (Figure 1B). To assess CPI-613 in combination with carboplatin/paclitaxel in vivo, we injected OVCAR3 cells in NOD/SCID mice to compare antitumor activity of 12.5 mg/kg CPI-613 delivered once weekly either alone or in combination with carboplatin/paclitaxel (respectively 25 mg/kg and 7 mg/kg i.p. once per week). Tumor volume was assessed every 3 days. Though we used a lower dose of CPI-613 compared to other in vivo experimental protocols, CPI-613 single-agent inhibition of tumor growth was evident when compared to the vehicle-treated arm (p-value < 0.01). As expected, the carboplatin/paclitaxel treatment group and the carboplatin/paclitaxel CPI-613 combination treatment group showed reduced tumor burden as compared to the CPI-613 single agent and vehicle groups (p-value < 0.001; Figure 4A). More importantly, flow cytometric analysis of tumors harvested at the end of the treatment period revealed a decreased frequency of CD133+ and CD117+ cells in CPI-613 treated tumors compared with vehicle-treated controls, again implying CPI-613 preferentially targets the CSCs (p-value < 0.01). Of interest, however, was the combination treatment effect on CSC frequency. Despite there being no significant difference in CD133+ and CD117+ cells compared to CPI-613 single-agent treatment, the combination of CPI-613 and carboplatin/paclitaxel negated the carboplatin/paclitaxel-induced enrichment of CD133+ and CD117+ cell frequency (p-value < 0.001) (Figure 4B). Annexin/PI analysis of cells in the harvested tumors confirmed the increase in necrosis in the combination treatment group (Figure S2), suggesting an additive benefit of using CPI-613 in combination with classical cytotoxic.

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 Devimistat (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[2]
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 Devimistat (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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Tumorigenicity In Vivo Assay [2]
To analyze the in vivo tumorigenicity rate after Devimistat (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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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

Background: This article describes the development and validation of a bioanalytical assay to quantify CPI-613 and its major metabolites, CPI-2850 and CPI-1810, in human plasma matrix using LC-MS/MS. Methodology: Sample extraction procedure following protein precipitation with acetonitrile was optimized to extract all three analytes from plasma with maximum recovery. The final extracted supernatants were diluted with water and injected onto an Xbridge C18 (50 × 2.1 mm; 5 μm) column for analysis. The analytes were separated by a gradient elution, and detection was performed on a triple quadrupole mass spectrometer (Sciex API 5000) operating in the negative ion mode. Results: The assay was linear over a range of 50-50,000 ng/ml for CPI-613, 250-250,000 ng/ml for CPI-2850 and 10-10,000 ng/ml for CPI-1810. Benchtop stability was established for 24 h, and four freeze-thaw cycles were evaluated for CPI-613 and its metabolites. Long-term freezer (-60 to -80°C) stability for about 127 days was established in this validation. Mean matrix recovery was more than 80% for all analytes. Conclusion: A robust LC-MS/MS method was developed for the quantification of CPI-613 and its major metabolites. The current assay will be used to support ongoing and future CPI-613 clinical trials. https://pubmed.ncbi.nlm.nih.gov/35172610/

[1]. 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]. The Metabolic Inhibitor CPI-613 Negates Treatment Enrichment of Ovarian Cancer Stem Cells. Cancers (Basel). 2019 Oct 29;11(11):1678.

Devimistat (CPI-613) has been used in trials studying the treatment of Cancer, Lymphoma, Solid Tumors, Advanced Cancer, and Pancreatic Cancer, among others.
Devimistat is a racemic mixture of the enantiomers of a synthetic alpha-lipoic lipoic acid analogue with potential chemopreventive and antineoplastic activities. Although the exact mechanism of action is unknown, devimistat has been shown to inhibit metabolic and regulatory processes required for cell growth in solid tumors. Both enantiomers in the racemic mixture exhibit antineoplastic activity.

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This study was designed to unravel the pathobiological role of impaired ARID1A expression in gastric carcinogenesis. We examined ARID1A expression immunohistochemically in 98 gastric cancer tissue specimens with regard to the clinicopathological features. Based on the proportion and intensity of ARID1A immunoreactivity at the cancer invasion front, we subdivided the specimens into low- and high-expression ARID1A groups. Notably, low ARID1A expression was significantly correlated with overall survival of the patients. Subsequently, we determined the molecular signature that distinguished ARID1A low/poor prognosis from ARID1A high/good prognosis gastric cancers. A comprehensive gene profiling analysis followed by immunoblotting revealed that a mitochondrial apoptosis mediator, Harakiri, was less expressed in ARID1A low/poor prognosis than ARID1A high/good prognosis gastric cancers. siRNA-mediated ARID1A downregulation significantly reduced expression of the Harakiri molecule in cultured gastric cancer cells. Interestingly, downregulation of ARID1A conferred resistance to apoptosis induced by the mitochondrial metabolism inhibitor, devimistat. In contrast, enforced Harakiri expression restored sensitivity to devimistat-induced apoptosis in ARID1A downregulated gastric cancer cells. The present findings indicate that impaired ARID1A expression might lead to gastric carcinogenesis, putatively through gaining resistance to the Harakiri-mediated apoptosis pathway.[1]

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One of the most significant therapeutic challenges in the treatment of ovarian cancer is the development of recurrent platinum-resistant disease. Cancer stem cells (CSCs) are postulated to contribute to recurrent and platinum-resistant ovarian cancer (OvCa). Drugs that selectively target CSCs may augment the standard of care cytotoxics and have the potential to prevent and/or delay recurrence. Increased reliance on metabolic pathway modulation in CSCs relative to non-CSCs offers a possible therapeutic opportunity. We demonstrate that treatment with the metabolic inhibitor CPI-613 (devimistat, an inhibitor of tricarboxylic acid (TCA) cycle) in vitro decreases CD133+ and CD117+ cell frequency relative to untreated OvCa cells, with negligible impact on non-CSC cell viability. Additionally, sphere-forming capacity and tumorigenicity in vivo are reduced in the CPI-613 treated cells. Collectively, these results suggest that treatment with CPI-613 negatively impacts the ovarian CSC population. Furthermore, CPI-613 impeded the unintended enrichment of CSC following olaparib or carboplatin/paclitaxel treatment. Collectively, our results suggest that CPI-613 preferentially targets ovarian CSCs and could be a candidate to augment current treatment strategies to extend either progression-free or overall survival of OvCa.[2]

C22H28O2S2

388.59

388.153

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

95809-78-2

Devimistat-d10;2586055-61-8

24770514

White to off-white solid powder

1.1±0.1 g/cm3

553.0±50.0 °C at 760 mmHg

63-65℃

288.3±30.1 °C

0.0±1.6 mmHg at 25°C

1.595

5.66

1

4

13

26

363

0

O=C(CCCCC(CCSCC1C=CC=CC=1)SCC1C=CC=CC=1)O

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.)