Mass balance and pharmacokinetics of an oral dose of 14C‐napabucasin in healthy adult male subjects

Abstract This phase 1, open‐label study assessed14C‐napabucasin absorption, metabolism, and excretion, napabucasin pharmacokinetics, and napabucasin metabolites (primary objectives); safety/tolerability were also evaluated. Eight healthy males (18–45 years) received a single oral 240‐mg napabucasin dose containing ~100 μCi14C‐napabucasin. Napabucasin was absorbed and metabolized to dihydro‐napabucasin (M1; an active metabolite [12.57‐fold less activity than napabucasin]), the sole major circulating metabolite (median time to peak concentration: 2.75 and 2.25 h, respectively). M1 plasma concentration versus time profiles generally mirrored napabucasin; similar arithmetic mean half‐lives (7.14 and 7.92 h, respectively) suggest M1 formation was rate limiting. Napabucasin systemic exposure (per Cmax and AUC) was higher than M1. The total radioactivity (TRA) whole blood:plasma ratio (AUClast: 0.376; Cmax: 0.525) indicated circulating drug‐related compounds were essentially confined to plasma. Mean TRA recovery was 81.1% (feces, 57.2%; urine, 23.8%; expired air, negligible). Unlabeled napabucasin and M1 recovered in urine accounted for 13.9% and 11.0% of the dose (sum similar to urine TRA recovered); apparent renal clearance was 8.24 and 7.98 L/h. No uniquely human or disproportionate metabolite was quantified. Secondary glucuronide and sulfate conjugates were common urinary metabolites, suggesting napabucasin was mainly cleared by reductive metabolism. All subjects experienced mild treatment‐emergent adverse events (TEAEs), the majority related to napabucasin. The most commonly reported TEAEs were gastrointestinal disorders. There were no clinically significant laboratory, vital sign, electrocardiogram, or physical examination changes. Napabucasin was absorbed, metabolized to M1 as the sole major circulating metabolite, and primarily excreted via feces. A single oral 240‐mg dose was generally well tolerated.


| INTRODUC TI ON
Napabucasin is an orally administered reactive oxygen species (ROS) generator bioactivated by the intracellular antioxidant NAD(P) H:quinone oxidoreductase 1 (NQO1). 1,2 Napabucasin exerts its antitumor activity by increasing levels of ROS beyond a cytotoxic threshold, causing cancer cell death. [1][2][3] Treatment of several cancers with napabucasin alone or in combination with other cancer therapies has been assessed in a number of ongoing clinical trials (congress abstracts, [4][5][6][7][8][9][10][11][12][13][14][15][16] and completed clinical trials 17,18 ). An ongoing phase 3 study is assessing napabucasin in combination with 5-fluorouracil, leucovorin, and irinotecan (i.e., FOLFIRI) in patients with previously treated metastatic colorectal cancer (Clinicaltrials. gov identifier: NCT02753127). 19 Preclinical studies of napabucasin suggest it is excreted via the renal as well as biliary/fecal routes in rats and mainly via the bili- The primary objectives of the current study were to characterize the absorption, metabolism, and excretion of 14 C-napabucasin, and to determine the pharmacokinetics (PK) of napabucasin and relevant metabolites in plasma, urine, and feces. Secondary objectives were to assess the safety and tolerability of napabucasin in healthy male subjects following a single oral dose of 240-mg napabucasin.

| Study design
This was a phase 1, open-label, single-dose study (NCT03525405) conducted in healthy male subjects who received a single oral dose of 240-mg napabucasin containing approximately 100 μCi of 14 C-napabucasin ( Figure 1). This study was conducted in compliance with the ethical principles of the International Conference on Harmonization, Good Clinical Practice guidelines, all federal, state, and local laws, and in accordance with the Declaration of Helsinki.
The protocol was approved by the Institutional Review Board at the study site, and all subjects provided written informed consent prior to participation in this study.

| Subjects
Healthy male subjects between the ages of 18

| Analytical methods
For TRA measurement, whole blood and fecal samples were com-  (Table S1). for applied scientific research as previously described. 21 Individual AUC pools were analyzed by LC (Shimadzu Prominence/Nexera)-MS (Thermo Fisher Scientific Q Exactive). The limit of quantitation for radioactivity was set at 1% of each chromatographic analysis (run) and 10 cpm peak height. The cut-off for identification of metabolites was 1% of the radioactive dose for urine and feces. If collective excretion of the metabolites was greater than 10% of the TRA, then structural elucidation of metabolites was performed by comparison with known standards and LC-MS. Detailed methods for measurement of metabolites are described in the supplemental data.

| PK parameters
The PK parameters for 14

| Safety assessments
Adverse events (AEs) were monitored and recorded throughout the study (treatment phase through follow-up call

| Statistical analyses
Data analysis was performed using SAS ® Version 9.4. For PK analysis, TRA in whole blood, plasma, urine, feces, and expired air, and napabucasin and M1 concentrations in plasma and urine were listed and summarized using descriptive statistics. Individual and mean plasma and whole blood concentration versus time profiles were presented graphically on both linear and semi-logarithmic scales. Cumulative radioactivity excreted in urine and feces and total recovery versus collection interval were summarized and presented graphically utilizing the endpoint of the collection interval. Due to a lack of detectable radioactive material, expired air results were not summarized. For safety analysis, AEs were summarized using descriptive statistics. No inferential statistical analyses were performed.

| Demographics and baseline characteristics
Overall, 10 subjects were screened and eight were enrolled and dosed (Table 1) all eight were analyzed for PK and safety. Two of the 10 subjects were screened as backups and were therefore not dosed.

| Excretion
The mean TRA recovered in urine and feces was 81.1%.   There was moderate to high intersubject variability for napabucasin and M1, and high intersubject variability for TRA, reflected by the moderate to high SDs for C max and AUC inf (Table 3).
Drug-related material was essentially confined to the plasma compartment with arithmetic mean TRA whole blood:plasma ratio  Abbreviations: A ef , amount excreted in feces from time 0 to last measurable concentration; A eu , amount excreted in urine from time 0 to last measurable concentration; CL R , renal clearance; CV, coefficient of variation; F ef , percent of dose excreted in feces from time 0 to last measurable concentration; F eu , percent of dose excreted in urine from time 0 to last measurable concentration; mg eq, milligram equivalents; NA, not applicable; SD, standard deviation; TRA, total radioactivity. a Presented in mg eq.

TA B L E 2 Urinary excretion of napabucasin and TRA recovered in urine and feces
plasma M1 to TRA were 0.844, 0.594, and 0.382, for C max , AUC last , and AUC inf , respectively. For plasma napabucasin and M1, the ratio of parent or M1 concentration to TRA based on C max and AUC last was >1 for some individuals, which is also attributed to the heterogeneity of the labeled and nonlabeled material (  TRA could represent labeled napabucasin, labeled M1, or other labeled napabucasin metabolites, which could vary over time, especially at late time points. Abbreviations: AUC, area under the curve; AUC inf , AUC from time 0 to infinity; AUC last , AUC from time 0 to time of last measurable concentration; CL/F, apparent systemic clearance; C max , peak concentration observed; CV, coefficient of variation; eq, equivalent; M/P-AUC, metabolite to parent AUC ratio; M/P-C max , metabolite to parent C max ratio; NA, not applicable; NC, not calculated; R-AUC, ratio of analyte to TRA based on AUC; R-C max , ratio of analyte to TRA based on C max ; SD, standard deviation; t 1/2 , apparent terminal elimination half-life; t lag , time prior to first quantifiable concentration; T max , time to peak concentration; TRA, total radioactivity; V z /F, apparent volume of distribution. a Median (range) data are presented.
b It was not possible to calculate the full summary statistics because a t 1/2 estimate was not always possible (see footnote c) and was more than 30% extrapolated for 2 of 4 subjects. c Half-life estimates were possible for 4 subjects, but for 3 subjects the estimates were determined over a period <2 half-lives.
d Terminal phase could not be defined for any profile in whole blood, so only observed parameters were calculable. the sole fecal metabolite, accounting for a mean of <1% of the initial dose; it was undetected in two subjects and accounted for <2% of the initial dose in the remaining subjects (Table 4). Napabucasin and M1 cumulatively accounted for a mean of approximately 56% of the initial dose (range: 17%-104%).
The most commonly reported TEAEs were gastrointestinal disorders, with seven subjects (87.5%) reporting a total of 21 events. The most frequently reported AEs related to napabucasin were chromaturia (n = 7, 87.5%), diarrhea (n = 4, 50%), and infrequent bowel movements (n = 4, 50%) ( Table 5). Four subjects reported diarrhea (a total of 12 episodes) that started at ~4.5-5.0 h post-dose and lasted up to 11 days. Eight instances were considered definitely related to napabucasin and four were considered possibly or unlikely related.
All episodes were mild in severity and resolved without treatment.
There were no severe AEs, serious AEs, or deaths during the study, and no subject discontinued this study due to an AE. There were no clinically significant laboratory, vital sign, electrocardiogram, or physical examination changes.

| D ISCUSS I ON AND CON CLUS I ON S
The objective of this study was to characterize the absorption, me-  TA B L E 4 Napabucasin and its proposed metabolites (≥1% TRA) identified in plasma, urine, and feces

Subjects, n (%) Events
All F I G U R E 5 Proposed biotransformation pathways of napabucasin in humans. The metabolite profiling results showed that napabucasin was extensively metabolized to produce 30 metabolites, all of which were found in urine, five in plasma, and one in feces. Based on these results, we propose a biotransformation pathway of napabucasin in humans: reduction of the acetyl side-chain and/or the naphthalene dione moiety is the exclusive primary biotransformation pathway, with glucuronidation, sulfonation, and, to a lesser extent, transamination as abundant secondary routes of metabolism. f, feces; p, plasma; u, urine The results of this study show that napabucasin was extensively metabolized to produce 30 metabolites, all of which were found in urine, five in plasma, and one in feces. Based on these findings, we propose the biotransformation pathway of napabucasin in humans illustrated in Figure 5. Reduction in the acetyl side-chain and/or the naphthalene dione moiety is the exclusive primary biotransformation pathway, with glucuronidation, sulfonation, and, to a lesser extent, transamination as abundant secondary routes of metabolism.
A greater, detailed description of this proposed pathway is provided in Figure 5. Reduction of napabucasin on the acetyl side chain pro-  16,23 A limitation of this study was the inability to homogeneously mix the radiolabeled compound with the nonlabeled semisolid suspension due to limited solubility. This lack of homogeneous drug formulation likely resulted in the observed differences in absorption between the radioactive and unlabeled material among subjects, and in the ratio of plasma napabucasin or M1 (based on cold assay) to TRA being >1 in some subjects (based on C max and AUC last ). It was, therefore, not possible to conclude whether the circulating radioactive material was predominantly parent and M1 or other circulating metabolites by comparing the radioactive and nonradioactive profiles. Additionally, the mean TRA recovery was 81.1%, although the discharge criteria specified >90% recovery unless 12 days had elapsed. However, although commonly used as a discharge criterion, 90% is arbitrary, as was the maximum stay required in this study (12 days) if discharge criteria were not met.
This study showed that napabucasin was absorbed following oral dosing and underwent extensive metabolism to yield M1 as the sole major circulating metabolite. Most of the administered radioactive dose was eliminated via the fecal route and to a lesser extent via urine. A single oral 240-mg dose of napabucasin was generally well tolerated in healthy male subjects.

DATA S H A R I N G A N D DATA ACCE SS I B I LIT Y S TAT E M E N T
Certain of the underlying data remains confidential, non-public information and is proprietary to the Company. Access to such confidential information upon request remains at the sole discretion of the Company, and will require the requestor enter into an acceptable confidentiality agreement.

ACK N OWLED G M ENTS
The authors acknowledge the contribution of Jamie Schneider of