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).

In Vitro and In Vivo Metabolism of a Novel Antimitochondrial Cancer Metabolism Agent, CPI-613, in Rat and Human.

CPI-613, an inhibitor of pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) enzymes, is currently in development for the treatment of pancreatic cancer, acute myeloid leukemia, and other cancers. CPI-613 is an analog of lipoic acid, an essential cofactor for both PDH and KGDH. Metabolism and mass balance studies were conducted in rats after intravenous administration of [14C]-CPI-613. CPI-613 was eliminated via oxidative metabolism followed by excretion of the metabolites in feces (59%) and urine (22%). ß-Oxidation was the major pathway of elimination for CPI-613. The most abundant circulating components in rat plasma were those derived from ß-oxidation. In human hepatocytes, CPI-613 mainly underwent ß-oxidation (M1), sulfur oxidation (M2), and glucuronidation (M3). The Michaelis-Menten kinetics (Vmax and Km) of the metabolism of CPI-613 to these three metabolites predicted the fraction metabolized leading to the formation of M1, M2, and M3 to be 38%, 6%, and 56%, respectively. In humans, after intravenous administration of CPI-613, major circulating species in plasma were the parent and the ß-oxidation derived products. Thus, CPI-613 metabolites profiles in rat and human plasma were qualitatively similar. ß-Oxidation characteristics and excretion patterns of CPI-613 are discussed in comparison with those reported for its endogenous counterpart, lipoic acid. SIGNIFICANCE STATEMENT: This work highlights the clearance mechanism of CPI-613 via ß-oxidation, species differences in their ability to carry out ß-oxidation, and subsequent elimination routes. Structural limitations for completion of terminal cycle of ß-oxidation is discussed against the backdrop of its endogenous counterpart lipoic acid.

Safety and tolerability of the first-in-class agent CPI-613 in combination with modified FOLFIRINOX in patients with metastatic pancreatic cancer: a single-centre, open-label, dose-escalation, phase 1 trial.

BACKGROUND: Pancreatic cancer statistics are dismal, with a 5-year survival of less than 10%, and more than 50% of patients presenting with metastatic disease. Metabolic reprogramming is an emerging hallmark of pancreatic adenocarcinoma. CPI-613 is a novel anticancer agent that selectively targets the altered form of mitochondrial energy metabolism in tumour cells, causing changes in mitochondrial enzyme activities and redox status that lead to apoptosis, necrosis, and autophagy of tumour cells. We aimed to establish the maximum tolerated dose of CPI-613 when used in combination with modified FOLFIRINOX chemotherapy (comprising oxaliplatin, leucovorin, irinotecan, and fluorouracil) in patients with metastatic pancreatic cancer. METHODS: In this single-centre, open-label, dose-escalation phase 1 trial, we recruited adult patients (aged ≥18 years) with newly diagnosed metastatic pancreatic adenocarcinoma from the Comprehensive Cancer Center of Wake Forest Baptist Medical Center (Winston-Salem, NC, USA). Patients had good bone marrow, liver and kidney function, and good performance status (Eastern Cooperative Oncology Group [ECOG] performance status 0-1). We studied CPI-613 in combination with modified FOLFIRINOX (oxaliplatin at 65 mg/m2, leucovorin at 400 mg/m2, irinotecan at 140 mg/m2, and fluorouracil 400 mg/m2 bolus followed by 2400 mg/m2 over 46 h). We applied a two-stage dose-escalation scheme (single patient and traditional 3+3 design). In the single-patient stage, one patient was accrued per dose level. The starting dose of CPI-613 was 500 mg/m2 per day; the dose level was then escalated by doubling the previous dose if there were no adverse events worse than grade 2 within 4 weeks attributed as probably or definitely related to CPI-613. The traditional 3+3 dose-escalation stage was triggered if toxic effects attributed as probably or definitely related to CPI-613 were grade 2 or worse. The dose level for CPI-613 for the first cohort in the traditional dose-escalation stage was the same as that used in the last cohort of the single-patient dose-escalation stage. The primary objective was to establish the maximum tolerated dose of CPI-613 (as assessed by dose-limiting toxicities). This trial is registered with ClinicalTrials.gov, number NCT01835041, and is closed to recruitment. FINDINGS: Between April 22, 2013, and Jan 8, 2016, we enrolled 20 patients. The maximum tolerated dose of CPI-613 was 500 mg/m2. The median number of treatment cycles given at the maximum tolerated dose was 11 (IQR 4-19). Median follow-up of the 18 patients treated at the maximum tolerated dose was 378 days (IQR 250-602). Two patients enrolled at a higher dose of 1000 mg/m2, and both had a dose-limiting toxicity. Two unexpected serious adverse events occurred, both for the first patient enrolled. Expected serious adverse events were: thrombocytopenia, anaemia, and lymphopenia (all for patient number 2; anaemia and lymphopenia were dose-limiting toxicities); hyperglycaemia (in patient number 7); hypokalaemia, hypoalbuminaemia, and sepsis (patient number 11); and neutropenia (patient number 20). No deaths due to adverse events were reported. For the 18 patients given the maximum tolerated dose, the most common grade 3-4 non-haematological adverse events were hyperglycaemia (ten [55%] patients), hypokalaemia (six [33%]), peripheral sensory neuropathy (five [28%]), diarrhoea (five [28%]), and abdominal pain (four [22%]). The most common grade 3-4 haematological adverse events were neutropenia (five [28%] of 18 patients), lymphopenia (five [28%]), anaemia (four [22%], and thrombocytopenia in three [17%]). Sensory neuropathy (all grade 1-3) was recorded in 17 (94%) of the 18 patients and was managed with dose de-escalation or discontinuation per standard of care. No patients died while on active treatment; 11 study participants died, with cause of death as terminal pancreatic cancer. Of the 18 patients given the maximum tolerated dose, 11 (61%) achieved an objective (complete or partial) response. INTERPRETATION: A maximum tolerated dose of CPI-613 was established at 500 mg/m2 when used in combination with modified FOLFIRINOX in patients with metastatic pancreatic cancer. The findings of clinical activity will require validation in a phase 2 trial. FUNDING: Comprehensive Cancer Center of Wake Forest Baptist Medical Center.