This was an open-label, non-randomized, dose escalation PKs study conducted to determine the MTD and dose-limiting toxicities (DLTs) and investigate the PKs, safety, tolerability and preliminary efficacy profiles of antroquinonol. An accelerated titration design was used for this study. For the 6 dose levels (daily dose of 50, 100, 200, 300, 450 and 600 mg), a maximum of 36 patients were scheduled based on the criteria of a maximum of 6 patients per cohort: 1 (lowest level) to 6 (highest level) patients were scheduled for each dose group in the accelerated titration phase, and 3–6 patients for each dose group in the standard titration phase (6 dose cohorts) (Fig. 1). Antroquinonol was administered per os daily, within 15 min after breakfast at the assigned dose levels for 4 weeks. The study protocol was approved by the Institutional Review Boards of Taipei Veterans General Hospital and Tri-Service General Hospital (VGHIRB 201010007MB, TSGHIRB 099-01-007, clinicaltrials.gov protocol registration system NCT011–340-16).

The primary endpoint was MTD. DLT was defined as any ≥grade 3 toxicity according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), version 4.03 (http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf), as determined by the investigator, possibly related in terms of causality to the administration of the study drug, and observed during the first 4 weeks of treatment for patients in the accelerated or standard titration phase. Grade ≥3 nausea, vomiting and diarrhea were considered as DLTs only if they remained at grade 3 for >3 days despite adequate treatment. The secondary endpoints included three parameters: PKs, preliminary efficacy evaluation of changes in measurable tumor size, and safety profile.

No other cytotoxic agents, targeted therapy, investigational drugs, immunotherapy, or radiotherapy was permitted during the study period. The following medications were also prohibited: Levonorgestrel (internal standard in bioanalysis), and drugs known to inhibit or induce cytochrome P (CYP)2C19, CYP3A4, CYP2C8 and CYP2E1.

Patients aged ≥20 years with metastatic NSCLC of the lung who had developed progressive disease after two lines of chemotherapy (including one platinum-based) and one line of epidermal growth factor receptor (EGFR)-targeted therapy (if the patient harboured an EGFR mutation or if the EGFR mutation status was unknown) were enrolled in this study, after providing written informed consent. Other eligibility criteria included: A histological or cytological diagnosis of adenocarcinoma of the lung (or mixed-cell type tumor with an adenocarcinomatous component); life expectancy of ≥3 months; Eastern Cooperative Oncology Group performance status score of ≤2; recovery from toxicities of previous anticancer treatments to ≤grade 1 NCI CTCAE, except for alopecia; clinically measurable disease; no previous radiotherapy directed at the measurable lesion(s); adequate bone marrow reserve, with a white blood cell count of ≥3,500/mm³, hemoglobin concentration of ≥9.0 g/dl, and platelet count of ≥100,000 cells/mm³; adequate liver function; and adequate renal function.

The patients were categorized into an intent-to-treat (ITT) population (patients who received at least one dose of antroquinonol), a per-protocol (PP) population [patients who completed at least 3 cycles of treatment with proper imaging assessment according to Response Evaluation Criteria in Solid Tumors (RECIST) (6)] and a PK population (patients who received at least one dose of antroquinonol with sufficient post-dose bio-samples collected for PKs profile characterization). Baseline and demographic characteristics and safety analyses were performed in the ITT population. The PK population was used for PKs analyses.

For each dose cohort, samples were obtained from all patients treated in the accelerated and standard titration phases of the study. Blood sampling was performed on days 1, 14, 27 and 28 of the first treatment cycle only. Serial blood samples (5 ml per sample) were collected for 24 h at the following time points on study days 1 and 28: Day 1, within 30 min prior to and at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 14 and 24 h after dose administration; and day 28, immediately prior to and at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 14 and 24 h after dose administration. A blood sample for trough plasma concentration was collected immediately prior to dose administration on days 14 and 27.

Determining the plasma PKs of antroquinonol involved investigating the following parameters during a maximum of 4 weeks of dose administration in the accelerated and standard titration phases: C_{max day1}, observed maximum plasma concentration after dosing on day 1; T_{max day1}, time to reach C_{max day1} on day 1; AUC_{0-t day1}, truncated area under the plasma concentration-time curve from the beginning of dosing to the last measurable concentration on day 1; T_{1/2 day1}, half-life on day 1; C_{max day28}, observed maximum plasma concentration after dosing on day 28; T_{max day28}, time to reach C_{max day28} on day 28; AUC_{0-t day28}, truncated area under the plasma concentration-time curve from the beginning of dosing to the last measurable concentration on day 28; T_{1/2 day28}, half-life on day 28; T_{1/2 effective}, half-life derived from the observed antroquinonol accumulation; C_{trough day14}, trough plasma concentration on day 14; C_{trough day27}, trough plasma concentration on day 27; C_{trough day28}, trough plasma concentration on day 28.

The tumor burden was measured according to RECIST, version 1.1, at baseline and at treatment discontinuation. Safety evaluation included complete blood cell counts, biochemistry laboratory data, C-reactive protein level, urinalysis, vital signs, electrocardiogram (ECG) examination and adverse events (AEs).

Patients in the accelerated titration phase were treated for 4 weeks at their first dose level, and then continued with treatment in the extension program for 3 cycles if found to be eligible. Moderate toxicity (MT) and DLTs were observed during the initial 4-week treatment period. The accelerated phase ended when either a DLT or MT was observed. A patient who experienced a DLT at any other level besides the lowest level was returned to the previous dose level. A patient who experienced a DLT at the lowest dose level was discontinued from the study. Patients in the standard titration phase were treated with antroquinonol for 4 weeks. DLTs were evaluated during the 4-week treatment period.

Prior to each 4-week cycle and at mid-cycle (i.e., day 14), a blood sample was drawn for complete blood testing in order to determine whether it was safe to continue with the study drug. If there was a ≥grade 3 toxicity, the patient's dose was reduced to the next lower dose. Dose escalation was allowed in the study and each patient was allowed two dose escalations. Patients who required a dose reduction due to toxicity were not allowed dose escalation at any point during the study. The patients were observed for AEs until 6 weeks after their last treatment.

The extension program included patients who had not withdrawn from the study and wished to participate in the accelerated or standard titration phase following their treatment cycle. The patients were treated for up to three 4-week cycles for as long as they were willing to continue, or until development of intolerable toxicity or progressive disease.

The PKs variables were calculated from the serum concentration data using standard, non-compartmental methods as implemented in WinNonlin, version 5.2 or higher (Pharsight Corp., St. Louis, MO, USA). PKs parameters were presented with descriptive statistics by dose level. Dose proportionality was to be evaluated if sufficient data were available in dose groups.

All statistical analyses were performed using SAS software, version 8.2 or higher (SAS Institute Inc. Cary, NC, USA). Categorical data were summarized using counts and percentages. For continuous variables, descriptive statistics were tabulated, such as number of available observations, mean, median, standard deviation, minimum and maximum. All available data and the tabulation of the results were displayed by initial dose level and with all levels pooled as a whole if applicable. All statistical tests were two-sided, with a P-value of <0.05 indicating statistically significant differences.

A total of 13 patients (7 men and 6 women) were enrolled in this study. All the patients were ethnic Chinese and their mean age was 61 years (range, 43–86 years). Two patients harboured EGFR-activating mutations, 4 patients had EGFR wild-type and 7 patients had unknown EGFR status.

A total of 5 patients were enrolled in the accelerated titration phase (1 patient each in the 50-, 100-, 200-, 300- and 450-mg dose groups) and 8 patients in the standard titration phase (3 patients in the 450- and 5 patients in the 600-mg dose group). All 13 patients were included in the ITT and the PK populations. The PP population consisted of 3 patients, with 1 patient in the 200- and 2 patients in the 600-mg dose group.

There were no DLTs reported in any patient for any of the dose levels in the ITT population in the accelerated or standard titration phase. An MTD was not determined in this study, and was ≥600 mg daily.

Four patients continued to the fourth treatment cycle, 1 patient each in the 200- and 450-mg dose groups and 2 patients in the 600-mg dose group. The best overall response was stable disease in 3 patients (1 patient in the 200-mg dose group and 2 patients in the 600-mg dose group), progressive disease in 7 patients, and not evaluated in 3 patients. Two patients were identified with EGFR-activating mutations, including 1 patient in the 450-mg dose group who discontinued the study drug prior to the completion of the first cycle and 1 patient in the 600-mg dose group who completed two cycles of treatment with the minor response of the sum of the longest diameters of the target lesions being reduced from 41 mm at screening to 31 mm at the end of the study visits.

Overall, antroquinonol at all dose levels exhibited a mild toxicity profile. A total of 4 patients reported 4 serious AEs (2 patients each in the 450- and 600-mg dose groups, including 1 patient from each dose group who succumbed to progressive disease). None of the mortalities, serious AEs or AEs leading to discontinuation was related to the study drug. No grade 4 treatment-emergent AEs (TEAEs) were reported. Grade 3 TEAEs were reported in 2 patients (1 patient in the 200-mg dose group experienced vertigo and 1 patient in the 450-mg dose group experienced decreased appetite). The most commonly occurring TEAEs reported in >2 patients were diarrhea [10 patients, 76.9% (grade 1, 9 patients; and grade 2, 1 patient)], vomiting [9 patients, 69.2% (grade 1, 6 patients; and grade 2, 3 patients)], and nausea [7 patients, 53.8% (grade 1, 6 patients; and grade 2, 1 patient)]. No patient exhibited any laboratory abnormalities recorded as TEAEs, apart from 1 case of grade 1 hematuria in the 450-mg dose group. There were mild increases or decreases in the hematological and biochemical parameters, but none were considered as abnormal results. None of the patients suffered from clinically significant changes in any of the vital signs, ECG readings, or physical examination parameters.

Due to the limited number of patients per dose group (1 patient each received 50, 100, 200 and 300 mg, 4 patients received 450 mg, and 5 patients received 600 mg), the PK results should be interpreted with caution. The power model approach was used to assess the dose proportionality of several antroquinonol doses, ranging from 50 to 600 mg. The values for slope (95% confidence interval) for area under the curve (AUC)_0-t and C_max for day 1 were 0.641 (0.16–1.12) and 0.504 (0.17–0.83), respectively, and for day 28 they were 0.957 (0.59–1.32) and 0.682 (0.08–1.28), respectively. These results indicate a dose-proportional increase over the dose range for AUC_0-t but not for C_max under single-dose conditions, and a dose-proportional increase over the dose range for both AUC_0-t and C_max under multiple-dose conditions.

Under single-dose conditions, the maximum antroquinonol concentration was generally observed between 1.00 and 3.70 h, with the exception of the 200-mg dose group, where a median T_max of 10.00 h was observed. Under multiple-dose conditions, the results were similar with the median T_max ranging from 1.92 to 4.05 h. Antroquinonol was rapidly eliminated following single and multiple administrations, with a mean T_1/2 ranging from 1.30 to 4.33 h, independently of the treatment dose. The trough plasma concentrations were similar on days 14, 27 and 28 within each dose group, and increased with the treatment dose. These results suggest that the steady state was probably attained on day 14. In terms of accumulation, no clear trend was observed when comparing the rate and extent of absorption on day 1 to those on day 28. The PKs mean plasma concentrations vs. time by treatment dose on days 1 and 28 are presented in Figs. 2 and 3, respectively.