Mass balance, metabolic disposition, and pharmacokinetics of a single oral dose of regorafenib in healthy human subjects

Purpose To evaluate the mass balance, metabolic disposition, and pharmacokinetics of a single dose of regorafenib in healthy volunteers. In addition, in vitro metabolism of regorafenib in human hepatocytes was investigated. Methods Four healthy male subjects received one 120 mg oral dose of regorafenib containing approximately 100 µCi (3.7 MBq) [14C]regorafenib. Plasma concentrations of parent drug were derived from HPLC–MS/MS analysis and total radioactivity from liquid scintillation counting (LSC). Radiocarbon analyses used HPLC with fraction collection followed by LSC for all urine samples, plasma, and fecal homogenate extracts. For the in vitro study, [14C]regorafenib was incubated with human hepatocytes and analyzed using HPLC–LSC and HPLC–HRMS/MS. Results Regorafenib was the major component in plasma, while metabolite M-2 (pyridine N-oxide) was the most prominent metabolite. Metabolites M-5 (demethylated pyridine N-oxide) and M-7 (N-glucuronide) were identified as minor plasma components. The mean concentration of total radioactivity in plasma/whole blood appeared to plateau at 1–4 h and again at 6–24 h post-dose. In total, 90.5% of administered radioactivity was recovered in the excreta within a collection interval of 12 days, most of which (71.2%) was eliminated in feces, while excretion via urine accounted for 19.3%. Regorafenib (47.2%) was the most prominent component in feces and was not excreted into urine. Excreted metabolites resulted from oxidative metabolism and glucuronidation. Conclusions Regorafenib was eliminated predominantly in feces as well as by hepatic biotransformation. The multiple biotransformation pathways of regorafenib decrease the risk of pharmacokinetic drug–drug interactions. Electronic supplementary material The online version of this article (10.1007/s00280-017-3480-9) contains supplementary material, which is available to authorized users.


M-1
M-1 was found in trace amounts in incubations with human hepatocytes. The high-resolution mass spectral data showed [M+H]+ at m/z 515.073 (Fig. S2), which is 32 Da (+2O) higher the than the parent drug. The MS2 product ion spectrum showed major fragment ions at m/z 497.063 (loss of H 2 O), m/z 424.047 (loss of N-hydroxymethylformamide CHONHCH 2 OH), m/z 302.057, and m/z 201.986 with the urea amide bonds as sites of fragmentation. Therefore, M-1 was interpreted as a pyridine N-oxide and N-methylhydroxylated product (combination of M-2 and M-3) of regorafenib.

M-2 (BAY 75-7495)
M-2 was found as a major metabolite in incubations with human hepatocytes. The high-resolution mass spectral data showed [M+H]+ at m/z 499.079 (Fig. S3), which is 16 Da (+O) higher than the parent drug. The MS2 product ion spectrum showed major fragment ions at m/z 424.045 (loss of N-methylformamide and oxygen CHONHCH 3 + O), m/z 304.072, m/z 247.051, and m/z 201.986 with the urea amide bonds as sites of fragmentation. Therefore, M-2 is interpreted as a pyridine N-oxide product of regorafenib. Furthermore, all mass spectral data were identical to the synthetic reference material BAY 75-7495.

M-3 (BAY 81-8753)
Only small amounts of M-3 were found in human hepatocytes. The high-resolution mass spectral data showed [M+H]+ at m/z 499.077 (Fig. S4), which is 16 Da (+O) higher than the parent drug. The MS2 product ion spectrum showed major fragment ions at m/z 424.045 (loss of Nhydroxymethylformamide CHONHCH 2 OH), m/z 286.061, and m/z 201.986 with the urea amide bonds as sites of fragmentation. Therefore, M-3 is interpreted as an N-methylhydroxylated product of regorafenib. Furthermore, all mass spectral data were identical to the synthetic reference material BAY 81-8753.

M-4 (BAY 75-1098)
Only small amounts of M-4 were found in human hepatocytes. The high-resolution mass spectral data showed [M+H]+ at m/z 469.067 (Fig. S5), which is a loss of 14 Da (-CH 2 ), compared with the parent drug. The MS2 product ion spectrum showed major fragment ions at m/z 424.045 (loss of formamide CHONH 2 ), m/z 274.061, m/z 247.050, and m/z 201.986 with the urea amide bonds as sites of fragmentation. Therefore, M-4 was interpreted as an N-demethylated product of regorafenib. Furthermore, all mass spectral data were identical to the synthetic reference material BAY 75-1098.

M-5 (BAY 81-8752)
M-5 was found as a metabolite in incubations with human hepatocytes. The high-resolution mass spectral data showed [M+H]+ at m/z 485.061, which is 2 Da (-CH 2 , +O) higher than the parent drug. The MS2 product ion spectrum (Fig. S6) showed fragment ions at m/z 424.045 (loss of formamide and oxygen CHONH 2 and O), m/z 290.056, m/z 247.050, and m/z 201.986 with the urea amide bonds as sites of fragmentation. Therefore, M-5 was interpreted as a pyridine N-oxide and N-demethylated product (combination of M-2 and M-4) of regorafenib. Furthermore, all mass regorafenib 16 spectral data were identical to the synthetic reference material BAY 81-8752.

M-6 (BAY 1005352)
Only small amounts of M-6 were found in human hepatocytes. The high-resolution mass spectral data showed [M+H]+ at m/z 470.051 (Fig. S7), which is a loss of 13 Da (-NH 2 CH 3 , +H 2 O) compared with the parent drug. The MS2 product ion spectrum showed major fragment ions at m/z 424.045 (loss of CO 2 ), m/z 231.055, m/z 247.050, and m/z 201.986 with the urea amide bonds as sites of fragmentation. Therefore, M-8 was interpreted as an N-demethylated and hydrolyzed carboxylic acid derivative of regorafenib. Furthermore, all mass spectral data were identical to the synthetic reference material BAY 1005352.

M-7
M-7 was found as a metabolite in incubations with human hepatocytes. The high-resolution mass spectral data showed [M+H]+ at m/z 659.113 (Fig. S8), which is 176 Da (+C 6 H 8 O 6 ) higher than the parent drug. The MS2 product ion spectrum showed fragment ions at m/z 641.103 (loss of H 2 O), m/z 483.082 (loss of -C 6 H 8 O 6 , anhydro glucuronic acid, 176 Da), which is the pseudomolecular ion of parent drug, m/z 288.077, and m/z 270.067 with the urea amide bonds as sites of fragmentation. Therefore, M-7 was interpreted as a glucuronic acid conjugate of parent drug regorafenib. For further structure elucidation, M-7 was isolated and purified by HPLC from human urine (GCM 2082-01), and subjected to LC-MS and NMR analysis (Fig. S10). From these data it was proposed that M-7 is N-glucuronidated at the urea nitrogen adjacent to the trifluoromethyl-chloro phenyl moiety.

M-8
M-8 was found as a metabolite in incubations with human hepatocytes. The high-resolution mass spectral data showed [M+H]+ at m/z 675.109 (Fig. S9), which is 192 Da (+C 6 H 8 O 6 , +O) higher than the parent drug. The MS2 product ion spectrum showed fragment ions at m/z 657.099 (loss of H 2 O), m/z 499.077 (loss of -C 6 H 8 O 6 , anhydro glucuronic acid, 176 Da), which is the pseudomolecular ion of the hydroxylated parent drug, and m/z 304.072 with the urea amide bonds as sites of fragmentation. The fragment ion m/z 304.072 is characteristic for M-2; therefore, M-8 was interpreted as a glucuronic acid conjugate of M-2. For further structure elucidation, M-8 was isolated and purified by HPLC from human urine (GCM 2082-02) and subjected to LC-MS and NMR-analysis. From these data it was proposed that M-8 is N-glucuronidated at the urea nitrogen adjacent to the trifluoromethylchloro phenyl moiety.