Discovery of sinomenine/8-Bis(benzylthio)octanoic acid hybrids as potential anti-leukemia drug candidate via mitochondrial pathway.

Traditional Chinese medicine Qingfengteng primarily acquired from the dried canes of Sinomenium acutum (Thunb.) Rehd. et Wils. var. cinereum Rehd. et Wils. and S. acutum (Thunb.) Rehd. et Wils. For the therapeutic treatment of rheumatism, acute arthritis, and rheumatoid arthritis based on Qingfengteng, sinomenine hydrochloride was recently made the principal active ingredient in various dosage forms. 8-Bis(benzylthio)octanoic acid (CPI-613) was an orphan medicine that the FDA and EMA approved orphan for the treatment of certain resistant malignancies. Its unique mode of action and minimal toxicity toward normal tissues made for an apt pharmacophore. In order to expand the field of sinomenine anticancer structures, sinomenine/8-Bis(benzylthio)octanoic acid derivatives were designed and synthesized. Among them, target hybrids e4 stood out for having notable cytotoxic effects against cancer cell lines, especially for K562 cells, with IC50 values of 2.45 µM and high safety. In-depth investigations demonstrated that e4 caused apoptosis by stopping the cell cycle at G1 phase, and doing so by altering the morphology of the nucleus and causing membrane potential of the in mitochondria to collapse. These results indicated e4 exerted an antiproliferative effect through apoptosis induction via mitochondrial pathway.

Comparison of survival for metastatic pancreatic cancer patients treated with CPI-613 versus resected borderline-resectable pancreatic cancer patients.

INTRODUCTION: Treatment of advanced pancreatic adenocarcinoma remains suboptimal. Therapeutic agents with a novel mechanism of action are desperately needed; one such novel agent is CPI-613 targets. We here analyze the outcomes of 20 metastatic pancreatic cancer patients treated with CPI-613 and FOLFIRINOX in our institution and evaluate their outcomes to borderline-resectable patients treated with curative surgery. METHODS: A post hoc analysis was performed of the phase I CPI-613 trial data (NCT03504423) comparing survival outcomes to borderline-resectable cases treated with curative resection at the same institution. Survival was measured by overall survival (OS) for all study cases and disease-free survival (DFS) for resected cases with progression-free survival for CPI-613 cases. RESULTS: There were 20 patients in the CPI-613 cohort and 60 patients in the surgical cohort. Median follow-up times were 441 and 517 days for CPI-613 and resected cases, respectively. There was no difference in survival times between CPI-613 and resected cases with a mean OS of 1.8 versus 1.9 year (p = 0.779) and mean PFS/DFS of 1.4 versus 1.7 years (p = 0.512). There was also no difference in 3-year survival rates for OS (hazard ratio [HR] = 1.063, 95% confidence interval [CI] 0.302-3.744, p = 0.925) or DFS/PFS (HR = 1.462, 95% CI 0.285-7.505, p = 0.648). CONCLUSION: The first study to evaluate the survival between metastatic patients treated with CPI-613 versus borderline-resectable cases undergoing curative resection. Analysis revealed no significant differences in survival outcomes between the cohorts. Study results are suggestive that there may be potential utility with the addition of CPI-613 to potentially resectable pancreatic adenocarcinoma, although additional research with more comparable study groups are required.

Toward a Unifying Hypothesis for Redesigned Lipid Catabolism as a Clinical Target in Advanced, Treatment-Resistant Carcinomas.

We review extensive progress from the cancer metabolism community in understanding the specific properties of lipid metabolism as it is redesigned in advanced carcinomas. This redesigned lipid metabolism allows affected carcinomas to make enhanced catabolic use of lipids in ways that are regulated by oxygen availability and is implicated as a primary source of resistance to diverse treatment approaches. This oxygen control permits lipid catabolism to be an effective energy/reducing potential source under the relatively hypoxic conditions of the carcinoma microenvironment and to do so without intolerable redox side effects. The resulting robust access to energy and reduced potential apparently allow carcinoma cells to better survive and recover from therapeutic trauma. We surveyed the essential features of this advanced carcinoma-specific lipid catabolism in the context of treatment resistance and explored a provisional unifying hypothesis. This hypothesis is robustly supported by substantial preclinical and clinical evidence. This approach identifies plausible routes to the clinical targeting of many or most sources of carcinoma treatment resistance, including the application of existing FDA-approved agents.

Identification of Dihydrolipoamide Dehydrogenase as Potential Target of Vemurafenib-Resistant Melanoma Cells.

BACKGROUND: Despite recent improvements in therapy, the five-year survival rate for patients with advanced melanoma is poor, mainly due to the development of drug resistance. The aim of the present study was to investigate the mechanisms underlying this phenomenon, applying proteomics and structural approaches to models of melanoma cells. METHODS: Sublines from two human (A375 and SK-MEL-28) cells with acquired vemurafenib resistance were established, and their proteomic profiles when exposed to denaturation were identified through LC-MS/MS analysis. The pathways derived from bioinformatics analyses were validated by in silico and functional studies. RESULTS: The proteomic profiles of resistant melanoma cells were compared to parental counterparts by taking into account protein folding/unfolding behaviors. Several proteins were found to be involved, with dihydrolipoamide dehydrogenase (DLD) being the only one similarly affected by denaturation in all resistant cell sublines compared to parental ones. DLD expression was observed to be increased in resistant cells by Western blot analysis. Protein modeling analyses of DLD's catalytic site coupled to in vitro assays with CPI-613, a specific DLD inhibitor, highlighted the role of DLD enzymatic functions in the molecular mechanisms of BRAFi resistance. CONCLUSIONS: Our proteomic and structural investigations on resistant sublines indicate that DLD may represent a novel and potent target for overcoming vemurafenib resistance in melanoma cells.

Identification of PDHX as a metabolic target for esophageal squamous cell carcinoma.

The metabolism in tumors is reprogrammed to meet its energetic and substrate demands. However, this metabolic reprogramming creates metabolic vulnerabilities, providing new opportunities for cancer therapy. Metabolic vulnerability as a therapeutic target in esophageal squamous cell carcinoma (ESCC) has not been adequately clarified. Here, we identified pyruvate dehydrogenase (PDH) component X (PDHX) as a metabolically essential gene for the cell growth of ESCC. PDHX expression was required for the maintenance of PDH activity and the production of ATP, and its knockdown inhibited the proliferation of cancer stem cells (CSCs) and in vivo tumor growth. PDHX was concurrently upregulated with the CD44 gene, a marker of CSCs, by co-amplification at 11p13 in ESCC tumors and these genes coordinately functioned in cancer stemness. Furthermore, CPI-613, a PDH inhibitor, inhibited the proliferation of CSCs in vitro and the growth of ESCC xenograft tumors in vivo. Thus, our study provides new insights related to the development of novel therapeutic strategies for ESCC by targeting the PDH complex-associated metabolic vulnerability.

Inhibition of Mitochondrial Metabolism Leads to Selective Eradication of Cells Adapted to Acidic Microenvironment.

Metabolic transformation of cancer cells leads to the accumulation of lactate and significant acidification in the tumor microenvironment. Both lactate and acidosis have a well-documented impact on cancer progression and negative patient prognosis. Here, we report that cancer cells adapted to acidosis are significantly more sensitive to oxidative damage induced by hydrogen peroxide, high-dose ascorbate, and photodynamic therapy. Higher lactate concentrations abrogate the sensitization. Mechanistically, acidosis leads to a drop in antioxidant capacity caused by a compromised supply of nicotinamide adenine dinucleotide phosphate (NADPH) derived from glucose metabolism. However, lactate metabolism in the Krebs cycle restores NADPH supply and antioxidant capacity. CPI-613 (devimistat), an anticancer drug candidate, selectively eradicates the cells adapted to acidosis through inhibition of the Krebs cycle and induction of oxidative stress while completely abrogating the protective effect of lactate. Simultaneous cell treatment with tetracycline, an inhibitor of the mitochondrial proteosynthesis, further enhances the cytotoxic effect of CPI-613 under acidosis and in tumor spheroids. While there have been numerous attempts to treat cancer by neutralizing the pH of the tumor microenvironment, we alternatively suggest considering tumor acidosis as the Achilles' heel of cancer as it enables selective therapeutic induction of lethal oxidative stress.

A Phase III open-label trial to evaluate efficacy and safety of CPI-613 plus modified FOLFIRINOX (mFFX) versus FOLFIRINOX (FFX) in patients with metastatic adenocarcinoma of the pancreas.

Devimistat (CPI-613®) is a novel lipoate analog that inhibits the tricarboxcylic acid cycle at two key carbon entry points. Through its inhibition of pyruvate dehydrogenase and a-ketoglutarate dehydrogenase complexes, devimistat inhibits the entry of glucose and glutamine derived carbons, respectively. Pancreatic cancer is dependent on mitochondrial function for enhanced survival and aggressiveness. In a Phase I study of modified FOLFIRINOX, in combination with devimistat for metastatic pancreatic cancer patients, there was a 61% objective response rate including a 17% complete response rate. This report outlines the rationale and design of the AVENGER 500 study, a Phase III clinical trial of devimistat in combination with modified FOLFIRINOX compared with FOLFIRINOX alone for patients with previously untreated metastatic adenocarcinoma of the pancreas. Clinical trial registration: NCT03504423.

Devimistat in combination with high dose cytarabine and mitoxantrone compared with high dose cytarabine and mitoxantrone in older patients with relapsed/refractory acute myeloid leukemia: ARMADA 2000 Phase III study.

Devimistat (CPI-613®) is an intravenously administered, novel lipoate analog that inhibits two key tricarboxcylic acid (TCA) cycle enzymes, pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase complexes (KGDH). These complexes control TCA cycle entry of glucose and glutamine-derived carbons, respectively. Acute myeloid leukemia (AML) cells upregulate the TCA cycle in response to DNA damaging agents and treatment with devimistat increases sensitivity to them. A Phase I study of devimistat in combination with cytarabine and mitoxantrone produced a complete remission rate of 50% in patients with relapsed or refractory AML. In the combined Phase I/II experience, older patients with R/R AML treated with 2000 mg/m2 of devimistat had a 52% complete remission/complete remission with incomplete hematologic recovery rate and a median survival of 12.4 months. This report outlines the rationale and design of the ARMADA 2000 study, a Phase III clinical trial of devimistat in combination with high dose cytarabine and mitoxantrone compared with high dose cytarabine and mitoxantrone alone for older patients (≥60 years of age) with relapsed or refractory AML. Clinical trial registration: NCT#03504410.

Inducing Melanoma Cell Apoptosis by ERp57/PDIA3 Antibody in the Presence of CPI-613 and Hydroxychloroquine.

The combination of the cancer mitochondrial metabolic inhibitor CPI-613 and hydroxychloroquine has tumor-suppressive effects on clear cell sarcoma, which shares pathobiological properties with melanoma. Therefore, we intended to examine the effects of a combination of CPI-613 and hydroxychloroquine on the growth of melanoma cells in the present study. However, cell death was not induced in melanoma cells. Therefore, a monoclonal antibody, ICT, that induced apoptosis in melanoma cells in combination with CPI-613 and hydroxychloroquine was developed. Immunoprecipitation, mass spectrometry, and small interfering RNA (siRNA)-mediated gene silencing demonstrated that ICT targeted Endoplasmic Reticulum Resident Protein 57/ Protein Disulfide Isomerase Family A Member 3 (ERp57/PDIA3), which was first identified as being upregulated by metabolic depletion stress and is localized on the cell surface during immunogenic cell death. The combination of CPI-613 and hydroxychloroquine enhanced the localization of ERp57/PDIA3 to the surface of melanoma cells. siRNA-mediated downregulation of ERp57/PDIA3 did not significantly induce ICT-mediated apoptosis in melanoma cells in the presence of CPI-613 and hydroxychloroquine. Therefore, the ICT antibody acts as a tumor suppressor in melanoma cells by targeting the cell membrane ERp57/PDIA3, expression of which was enhanced by the combination of CPI-613 and hydroxychloroquine.

Development and validation of an LC-MS/MS method for determination of a novel anticancer agent (CPI-613) in human plasma.

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.

To achieve the pharmacokinetic objectives of clinical trials, it was necessary to determine the concentrations of CPI-613 and its metabolites in human plasma samples. In this article, the authors describe the development and validation of a highly sensitive and selective LC-MS/MS assay method for the simultaneous quantification of CPI-613 and its major metabolites, CPI-2850 and CPI-1810, in human plasma. Concentration ranges were selected based on previous data where CPI-613 was detected using the HPLC method (rather than LC-MS/MS).

Blockade of glutamine-dependent cell survival augments antitumor efficacy of CPI-613 in head and neck cancer.

BACKGROUND: Alterations in metabolism are one of the emerging hallmarks of cancer cells and targeting dysregulated cancer metabolism provides a new approach to developing more selective therapeutics. However, insufficient blockade critical metabolic dependencies of cancer allows the development of metabolic bypasses, thus limiting therapeutic benefits. METHODS: A series of head and neck squamous cell carcinoma (HNSCC) cell lines and animal models were used to determine the efficacy of CPI-613 and CB-839 when given alone or in combination. Glutaminase 1 (GLS1) depletion was achieved by lentiviral shRNAs. Cell viability and apoptosis were determined in HNSCC cells cultured in 2D culture dish and SeedEZ™ 3D scaffold. Molecular alterations were examined by Western blotting and immunohistochemistry. Metabolic changes were assessed by glucose uptake, lactate production, glutathione levels, and oxygen consumption rate. RESULTS: We show here that HNSCC cells display strong addiction to glutamine. CPI-613, a novel lipoate analog, redirects cellular activity towards tumor-promoting glutaminolysis, leading to low anticancer efficacy in HNSCC cells. Mechanistically, CPI-613 inhibits the tricarboxylic acid cycle by blocking the enzyme activities of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, which upregulates GLS1 and eventually promotes the compensatory role of glutaminolysis in cancer cell survival. Most importantly, the addition of a GLS1 inhibitor CB-839 to CPI-613 treatment abrogates the metabolic dependency of HNSCC cells on glutamine, achieving a synergistic anticancer effect in glutamine-addicted HNSCC. CONCLUSIONS: These findings uncover the critical role of GLS1-mediated glutaminolysis in CPI-613 treatment and suggest that the CB-839 and CPI-613 combination may potentiate synergistic anticancer activity for HNSCC therapeutic gain.

Smart Design of Mitochondria-Targeted and ROS-Responsive CPI-613 Delivery Nanoplatform for Bioenergetic Pancreatic Cancer Therapy.

Mitochondria, as the powerhouse of most cells, are not only responsible for the generation of adenosine triphosphate (ATP) but also play a decisive role in the regulation of apoptotic cell death, especially of cancer cells. Safe potential delivery systems which can achieve organelle-targeted therapy are urgently required. In this study, for effective pancreatic cancer therapy, a novel mitochondria-targeted and ROS-triggered drug delivery nanoplatform was developed from the TPP-TK-CPI-613 (TTCI) prodrug, in which the ROS-cleave thioketal functions as a linker connecting mitochondrial targeting ligand TPP and anti-mitochondrial metabolism agent CPI-613. DSPE-PEG2000 was added as an assistant component to increase accumulation in the tumor via the EPR effect. This new nanoplatform showed effective mitochondrial targeting, ROS-cleaving capability, and robust therapeutic performances. With active mitochondrial targeting, the formulated nanoparticles (TTCI NPs) demonstrate much higher accumulation in mitochondria, facilitating the targeted delivery of CPI-613 to its acting site. The results of in vitro antitumor activity and cell apoptosis revealed that the IC50 values of TTCI NPs in three types of pancreatic cancer cells were around 20~30 µM, which was far lower than those of CPI-613 (200 µM); 50 µM TTCI NPs showed an increase in apoptosis of up to 97.3% in BxPC3 cells. Therefore, this mitochondria-targeted prodrug nanoparticle platform provides a potential strategy for developing safe, targeting and efficient drug delivery systems for pancreatic cancer therapy.

FRI-1 Is an Anti-Cancer Isoquinolinequinone That Inhibits the Mitochondrial Bioenergetics and Blocks Metabolic Shifts by Redox Disruption in Breast Cancer Cells.

Since breast cancer (BC) cells are dependent on mitochondrial bioenergetics for promoting proliferation, survival, and metastasis, mitochondria highlight as an important target for anticancer drug discovery. FRI-1, methyl 1, 3-dimethyl-5, 8-dioxo-5, 8-dihydro-4-isoquinolinecarboxylate, was previously described as a selective cytotoxic compound on cancer cell lines, however, details on the mechanism of action remain unknown. In this work, we describe that FRI-1 inhibits mitochondrial bioenergetics, producing apoptosis in MCF7 and MDA-MB-231 BC cell lines. FRI-1 decreases the maximal oxygen consumption rate (OCR), Δψm, NADH, and ATP levels, with a notable increase of mitochondrial reactive oxygen species (ROS) production, promoting AMPK activation with pro-survival effects. Moreover, FRI-1 inhibits the metabolic remodeling to glycolysis induced by oligomycin. In isolated tumoral mitochondria, FRI-1 increases Complex I and III-dependent OCR state 2, and this is sensitive to rotenone and antimycin A inhibitor additions, suggesting a redox cycling event. Remarkably, α-ketoglutarate and lipoic acid supplementation reversed and promoted, respectively, the FRI-1-induced apoptosis, suggesting that mitochondrial redox disruption affects 2-oxoglutarate dehydrogenase (OGDH) activity, and this is involved in their anticancer mechanism. Consistent with this, the combination of FRI-1 and CPI-613, a dual inhibitor of redox-sensible tricarboxylic acid (TCA) cycle enzymes PDH and OGDH, produced extensive BC cell death. Taken together, our results suggest that FRI-1 exhibits anticancer effects through inhibition of mitochondrial bioenergetics by redox disruption in BC cells.

CPI-613 rewires lipid metabolism to enhance pancreatic cancer apoptosis via the AMPK-ACC signaling.

BACKGROUND: Pancreatic cancer remains one of the most rapidly progressive and deadly malignancies worldwide. Current treatment regimens only result in small improvements in overall survival for patients with this cancer type. CPI-613 (Devimistat), a novel lipoate analog inhibiting mitochondrial metabolism, shows the new hope for pancreatic cancer treatment as an efficient and well-tolerated therapeutic option treated alone or in combination with chemotherapy. METHODS: Pancreatic cancer cells growing in planar 2D cultures and 3D scaffold were used as research platforms. Cell viability was measured by MTT and alamarBlue, and apoptosis was assessed by JC-1 staining and flow cytometry with Annexin V-FITC/PI staining. The mechanism behind CPI-613 action was analyzed by western blot, transmission electron microscopy, and lipolysis assay kits, in the presence or absence of additional signaling pathway inhibitors or gene modifications. RESULTS: CPI-613 exhibits anticancer activity in pancreatic cancer cells by triggering ROS-associated apoptosis, which is accompanied by increased autophagy and repressed lipid metabolism through activating the AMPK signaling. Intriguingly, ACC, the key enzyme modulating lipid metabolism, is identified as a vital target of CPI-613, which is inactivated in an AMPK-dependent manner and influences apoptotic process upon CPI-613. Blockade or enhancement of autophagic process does not increase or blunt apoptosis to CPI-613, but inhibition of the AMPK-ACC signaling significantly attenuates apoptosis induced by CPI-613, suggesting CPI-613-mediated lipid metabolism reduction contributes to its cytotoxicity in pancreatic cancer cells. CONCLUSIONS: These findings explore the critical role of lipid metabolism in apoptosis, providing new insights into the AMPK-ACC signaling axis in crosstalk between lipid metabolism and apoptosis in CPI-613 treatment.

Targeting Altered Energy Metabolism in Colorectal Cancer: Oncogenic Reprogramming, the Central Role of the TCA Cycle and Therapeutic Opportunities.

Colorectal cancer (CRC) is among the most frequent cancer entities worldwide. Multiple factors are causally associated with CRC development, such as genetic and epigenetic alterations, inflammatory bowel disease, lifestyle and dietary factors. During malignant transformation, the cellular energy metabolism is reprogrammed in order to promote cancer cell growth and proliferation. In this review, we first describe the main alterations of the energy metabolism found in CRC, revealing the critical impact of oncogenic signaling and driver mutations in key metabolic enzymes. Then, the central role of mitochondria and the tricarboxylic acid (TCA) cycle in this process is highlighted, also considering the metabolic crosstalk between tumor and stromal cells in the tumor microenvironment. The identified cancer-specific metabolic transformations provided new therapeutic targets for the development of small molecule inhibitors. Promising agents are in clinical trials and are directed against enzymes of the TCA cycle, including isocitrate dehydrogenase, pyruvate dehydrogenase kinase, pyruvate dehydrogenase complex (PDC) and α-ketoglutarate dehydrogenase (KGDH). Finally, we focus on the α-lipoic acid derivative CPI-613, an inhibitor of both PDC and KGDH, and delineate its anti-tumor effects for targeted therapy.

A Single-Center Retrospective Study of Acute Kidney Injury Incidence in Patients With Advanced Malignancies Treated With Antimitochondrial Targeted Drug.

INTRODUCTION: Mitochondrial dysfunction plays an important role in the pathophysiology of kidney disease. Inhibitors of mitochondrial metabolism are being developed for the treatment of solid organ and hematologic malignancies. We describe the incidence and clinical features of acute kidney injury (AKI) in patients treated with the antimitochondrial drug CPI-613. METHODS: We identified 33 patients with relapsed or refractory malignancy, previously enrolled in 3 open-label phase II studies, who received single-agent CPI-613 chemotherapy. AKI was defined by the Kidney Disease Improving Global Outcomes serum creatinine criteria. Participants were followed for a median (25th-75th percentile) of 120.0 (74.0-301.0) days. Risk factors for AKI were assessed by proportional hazards regression using univariate and multivariate analyses. RESULTS: Participants had baseline mean (SD) age of 63.8 (11.6) years and serum creatinine 0.9 (0.3) mg/dl. AKI developed in 9 (27%) patients; chart review failed to identify a potential cause of AKI other than CPI-613 administration in 5 (15%) patients, of whom 1 had AKI stage 1, 1 had AKI stage 2, and 3 experienced AKI stage 3. Time from initiation of CPI-613 treatment to AKI was 51.0 (16.0-58.0) days. Age, per 5-year increase, was associated with higher risk of AKI (adjusted hazard ratio 2.01, 95% confidence interval 1.06-3.79, P = 0.03). Follow-up serum creatinine was available in 4 participants 174.8 (139.6) days after the episode of AKI; 3 patients had complete recovery in kidney function and 1 had partial recovery. CONCLUSION: AKI is a possible complication during treatment with mitochondria-targeted chemotherapy.

Translational assessment of mitochondrial dysfunction of pancreatic cancer from in vitro gene microarray and animal efficacy studies, to early clinical studies, via the novel tumor-specific anti-mitochondrial agent, CPI-613.

STUDY RATIONALE AND OBJECTIVES: Via genetic alterations, malignant transformation and proliferation are associated with extensive alterations of mitochondrial energy metabolism of tumor cells. Thus, inhibition of the altered form of mitochondrial energy metabolism of tumor cells may be an effective therapy for cancers. This study performed translational assessment of mitochondrial dysfunction of pancreatic cancer from in vitro gene microarray and animal efficacy studies, to early clinical studies, via the novel tumor-specific anti-mitochondrial agent, CPI-613. METHODS: The gene profiles of BxPC-3 human pancreatic tumor cells and non-transformed NIH-3T3 mouse fibroblast cells (negative control), after CPI-613 or sham treatment, were assessed and compared using microarray technique. The anti-cancer efficacies of CPI-613 and Gemcitabine were assessed and compared in mice with xenograft from inoculation of BxPC-3 human pancreatic tumor cells, based on the degree of tumor growth inhibition and prolongation of survival when compared to vehicle treatment. The anti-cancer activities, according to overall survival (OS), of CPI-613 alone and in combination with Gemcitabine were assessed in patients with Stage IV pancreatic cancer. RESULTS: Microarray studies indicated that CPI-613 down-regulated the expression of Cyclin D3, E1, E2, F, A2, B1 and CDK2 genes of BxPC-3 pancreatic cancer cells but not non-transformed NIH-3T3 mouse fibroblast cells (negative control). In mice with pancreatic carcinoma xenografts, four weekly intraperitoneal injections of either CPI-613 (25 mg/kg/administration) or Gemcitabine (50 mg/kg/administration) inhibited tumor growth and prolonged survival when compared to vehicle treatment. The degree of tumor growth inhibition was ~2×, and prolongation of survival was ~4×, greater with CPI-613 treatment than with Gemcitabine treatment. In patients with Stage IV advanced pancreatic cancer, CPI-613 at 420-1,300 mg/m(2), given twice weekly for three weeks followed by a week of rest (i.e., 3-week-on-1-week-off) as monotherapy, provided median OS of 15 months in three patients. CPI-613 at 150-320 mg/m(2) given twice weekly on the 3-week-on-1-week-off dosing schedule, coinciding with Gemcitabine (1,000 mg/m(2)) given once weekly on the 3-week-on-1-week-off dosing schedule, provided median OS of 17.8 months in four patients. These median OS values from CPI-613 monotherapy and CPI-613 + Gemcitabine treatment tend to be longer than those in patients treated with Abraxane + Gemcitabine combination or FOLFININOX (median OS ~12 months). CONCLUSIONS: The dysfunctional mitochondria of pancreatic cancer cells was translationable from in vitro gene alteration and animal tumor model studies to patients with advanced Stage IV pancreatic cancer, as reflected by the anti-cancer activities of the tumor-specific anti-mitochondrial agent, CPI-613, in these studies.