Simultaneous quantification of arctigenin and its glucuronide conjugate in mouse plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry.

Arctigenin is a natural lignin and a main active component of Fructus arctii, the dried fruit of Arctium lappa. This compound was reported to have some biological activities such as anti-inflammatory, antioxidant, antiviral, renoprotective, and antitumor effects. Arctigenin is mainly metabolized to arctigenin-4'-O-glucuronide by UDP-glucuronosyltransferase. In this study, a simultaneous quantification method was established and validated for measuring arctigenin and arctigenin-4'-O-glucuronide in mouse plasma using ultra-high performance liquid chromatography with tandem mass spectrometry. The assay fulfilled the requirements of the United States Food and Drug Administration guideline for assay validation, with a lower limit of quantification of 2.00 ng/mL for arctigenin and 50.0 ng/mL for arctigenin-4'-O-glucuronide. The recovery rate and matrix effect ranged from 78.4 to 102.8% and 92.5 to 106.3%, respectively, for arctigenin, and 74.3 to 109.2% and 94.9 to 110.2% for arctigenin-4'-O-glucuronide. The method was applied to the measurement of plasma concentrations of arctigenin and arctigenin-4'-O-glucuronide in the plasma of mice after administration of arctigenin. All measured concentrations were within the calibration ranges. Our novel method may be useful to measure plasma arctigenin and arctigenin-4'-O-glucuronide concentrations, and contribute to evaluate the pharmacokinetics of arctigenin and arctigenin-4'-O-glucuronide in mice.

TAS6417/CLN-081 Is a Pan-Mutation-Selective EGFR Tyrosine Kinase Inhibitor with a Broad Spectrum of Preclinical Activity against Clinically Relevant EGFR Mutations.

Despite the worldwide approval of three generations of EGFR tyrosine kinase inhibitors (TKI) for advanced non-small cell lung cancers with EGFR mutations, no TKI with a broad spectrum of activity against all clinically relevant mutations is currently available. In this study, we sought to evaluate a covalent mutation-specific EGFR TKI, TAS6417 (also named CLN-081), with the broadest level of activity against EGFR mutations with a prevalence of ≥1%. Lung cancer and genetically engineered cell lines, as well as murine xenograft models were used to evaluate the efficacy of TAS6417 and other approved/in-development EGFR TKIs (erlotinib, afatinib, osimertinib, and poziotinib). We demonstrate that TAS6417 is a robust inhibitor against the most common EGFR mutations (exon 19 deletions and L858R) and the most potent against cells harboring EGFR-T790M (first/second-generation TKI resistance mutation). In addition, TAS6417 has activity in cells driven by less common EGFR-G719X, L861Q, and S768I mutations. For recalcitrant EGFR exon 20 insertion mutations, selectivity indexes (wild-type EGFR/mutant EGFR ratio of inhibition) favored TAS6417 in comparison with poziotinib and osimertinib, indicating a wider therapeutic window. Taken together, we demonstrate that TAS6417 is a potent EGFR TKI with a broad spectrum of activity and a wider therapeutic window than most approved/in-development generations of EGFR inhibitors. IMPLICATIONS: TAS6417/CLN-081 is a potent EGFR TKI with a wide therapeutic window and may be effective in lung cancer patients with clinically relevant EGFR mutations.

Change in plasma lactate concentration during arctigenin administration in a phase I clinical trial in patients with gemcitabine-refractory pancreatic cancer.

Arctigenin is evaluated for antitumor efficacy in patients with pancreatic cancer. It has an inhibitory activity on mitochondrial complex I.Therefore, plasma lactate level of patients after arctigenin administration was evaluated for biomarker of clinical response and/or adverse effect. Plasma lactate level in 15 patients enrolled in a Phase I clinical trial of GBS-01 rich in arctigenin was analyzed by colorimetric assay. Statistical analyses for association of plasma lactate and clinical responses, pharmacokinetics of arctigenin, and background factors of each patient by multivariate and univariate analyses.In about half of the patients, transient increase of lactate was observed. Correlation between plasma lactate level and pharmacokinetic parameters of arctigenin and its glucuronide conjugate, and clinical outcome was not detected. Regarding to the determinant of lactate level, only slight association with liver function test was detected. Plasma lactate level is primary determined by reutilization rather than production for antitumor effect and dose not serve as a biomarker. Arctigenin, inhibition of mitochondrial complex I, plasma lactate concentration, phase I clinical trial of GBS-01, Cori cycle.

Phase I trial of GBS-01 for advanced pancreatic cancer refractory to gemcitabine.

GBS-01, an extract from the fruit of Arctium lappa L. is an orally administered drug rich in arctigenin, which has been reported to exert antitumor activity by attenuating the tolerance of cancer cells to glucose deprivation. We investigated the maximum tolerated dose of GBS-01 based on the frequency of the dose-limiting toxicities (DLTs) and pharmacokinetics in patients with advanced pancreatic cancer refractory to gemcitabine. GBS-01 was given orally at escalating doses from 3.0 g (containing 1.0 g burdock fruit extract) to 12.0 g q.d. A DLT was defined as a grade 4 hematological toxicity and grade 3 or 4 non-hematological toxicity appearing during the first 28 days of treatment. Fifteen patients (GBS-01 dose level 1 [3.0 g], three patients; dose level 2 [7.5 g], three patients; and dose level 3 [12.0 g], nine patients) were enrolled. None of the patients at any of the three dose levels showed any sign of DLTs. The main adverse events were increased serum γ-glutamyl transpeptidase, hyperglycemia, and increased serum total bilirubin; however, all the toxicities were mild. Of the 15 patients, 1 showed confirmed partial response and 4 patients had stable disease. The median progression-free and overall survival of the patients were 1.1 and 5.7 months, respectively. The pharmacokinetic study revealed a high bioavailability of arctigenin and rapid conjugation of the drug with glucuronic acid. The recommended dose of GBS-01 was 12.0 g q.d, and favorable clinical responses were obtained. This trial was registered at UMIN-CTR (http://www.umin.ac.jp/ctr/index-j.htm), identification number UMIN000005787.

Provision of a voluntary exercise environment enhances running activity and prevents obesity in Snark-deficient mice.

The present study was performed to investigate the involvement of SNARK in physical activity levels in mice. To examine the acute effect of SNARK deficiency on voluntary running, Snark-deficient mice (Snark(+/-): n = 16) and their wild-type counterparts (Snark(+/+): n = 16) were assigned to sedentary or exercise (1 wk voluntary wheel running) groups. In addition, to clarify the differences in voluntary running activity and its effect between genotypes, mice (Snark(+/+): n = 16; Snark(+/-): n = 16) were also kept in individual cages with/without a running wheel for 5 mo. Unexpectedly, in both voluntary running experiments, running distances were increased in Snark(+/-) mice compared with Snark(+/+) mice. Under sedentary conditions, body and white adipose tissue weights were increased significantly in Snark(+/-) mice. However, no significant differences were observed between the two genotypes under exercise conditions, and the values were significantly less than those under sedentary conditions in the long-term experiment. In the short-term experiment, serum interleukin-6 level in exercised Snark(+/+) mice was the same as that in sedentary Snark(+/+) mice, whereas that in sedentary Snark(+/-) mice was significantly lower than in the other groups. In contrast, serum leptin level was reduced significantly in exercised Snark(+/-) mice compared with sedentary Snark(+/-) mice. The results of this study demonstrated that exposure to an environment that allows voluntary exercise promotes increased running activity and prevents obesity in Snark-deficient mice.

Susceptibility of Snark-deficient mice to azoxymethane-induced colorectal tumorigenesis and the formation of aberrant crypt foci.

SNF-1/5'-AMP-activated kinase (AMPK)-related kinase (SNARK) is a member of the AMPK-related kinases. Snark(+/-) mice exhibited mature-onset obesity and related metabolic disorders. Obesity is regarded as a risk factor for colorectal cancer. To investigate whether Snark deficiency is involved in tumorigenesis in the large intestine, obese Snark(+/-) mice were treated with a chemical carcinogen, azoxymethane (AOM). The incidences of both adenomas and aberrant crypt foci (ACF) were significantly higher in Snark(+/-) mice than in their wild-type counterparts 28 weeks after the completion of AOM treatment (10 mg/kg/week for 8 weeks). Furthermore, ACF formation was enhanced in Snark(+/-) mice treated with AOM for 2 weeks, suggesting that Snark deficiency contributed to the early phase of tumorigenesis. The total number of ACF was correlated with bodyweight in Snark(+/-) and Snark(+/+) mice, suggesting that obesity was a risk factor for colorectal tumorigenesis in this model. However, the correlation coefficient was higher in Snark(+/-) mice. Moreover, AOM-induced ACF formation was also enhanced in preobese Snark(+/-) mice. Together, these findings suggest that AOM-induced tumorigenesis in Snark(+/-) mice was enhanced via obesity-dependent and -independent mechanisms.