Journal: Chemical research in toxicology

Article Title: Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib

doi: 10.1021/acs.chemrestox.8b00005

Figure Lengend Snippet: LC–MS/MS analysis of sunitinib GSH conjugates. Sunitinib (50 μM) was incubated with pooled human liver microsomes (0.5 mg/mL) in the presence of an NADPH-regenerating system and supplemented with GSH (5 mM) for 30 min. LC–MS/MS analysis using electrospray ionization was performed in positive ion mode. Shown are representative product ion spectra from UPLC–MS/MS analysis of (A) the putative quinoneimine–GSH conjugate of sunitinib (M5) with precursor ion m/z 702.29 and (B) the direct sunitinib–GSH conjugate (Sun–SG) with precursor ion m/z 706.30. Higher-energy collisional dissociation MS/MS analysis was performed using an LTQ Oribitrap XL (Thermo) in ESI positive ion mode (collision energy 25 V). The predicted structures of (A) M5 and (B) Sun–SG are shown.

Article Snippet: Deuterium-labeled sunitinib ( N -[2-diethylamino(ethyl- d 4 )]-5-[( Z )-(5-fluoro-1,2-dihydro-2-oxo-3 H -indo-3-yldiene)methyl]-2,4-di-methyl-1 H -pyrrole-3-carboxamide, sunitinib- d 4 , S820003), N -desethyl-sunitinib (D289650), sunitinib N -oxide (S820005), furafylline, (+)- N -3-benzylnirvanol, 2-phenyl-2-(1-piperidinyl)propane (PPP), and CY-P3cide were purchased from Toronto Research Chemicals (Toronto, Canada).

Techniques: Liquid Chromatography with Mass Spectroscopy, Incubation, Tandem Mass Spectroscopy

Journal: Chemical research in toxicology

Article Title: Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib

doi: 10.1021/acs.chemrestox.8b00005

Figure Lengend Snippet: LC–MS/MS chromatograms of selected sunitinib metabolites. Sunitinib metabolites were analyzed by LC–MS/MS utilizing MRM. Shown are representative LC–MRM chromatograms of M1 (m/z 371 > 283), M3 (m/z 397 > 281), M5 (m/z 702 > 586), and sunitinib-d4 (internal standard). Metabolites were generated from incubation of sunitinib (10 μM) with recombinant P450 1A2.

Article Snippet: Deuterium-labeled sunitinib ( N -[2-diethylamino(ethyl- d 4 )]-5-[( Z )-(5-fluoro-1,2-dihydro-2-oxo-3 H -indo-3-yldiene)methyl]-2,4-di-methyl-1 H -pyrrole-3-carboxamide, sunitinib- d 4 , S820003), N -desethyl-sunitinib (D289650), sunitinib N -oxide (S820005), furafylline, (+)- N -3-benzylnirvanol, 2-phenyl-2-(1-piperidinyl)propane (PPP), and CY-P3cide were purchased from Toronto Research Chemicals (Toronto, Canada).

Techniques: Liquid Chromatography with Mass Spectroscopy, Generated, Incubation, Recombinant

Journal: Chemical research in toxicology

Article Title: Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib

doi: 10.1021/acs.chemrestox.8b00005

Figure Lengend Snippet: Relative P450 enzyme contribution to sunitinib metabolism. (A) M1, (B) M3, and (C) M5. Sunitinib (10 μM) was incubated with a panel of recombinant human P450 Supersomes (20 nM) in the presence of an NADPH regenerating system and supplemented with GSH (5 mM) for 10 min. Sunitinib metabolites were analyzed by LC–MS/MS analysis utilizing MRM. The MRM transitions for the metabolites indicated were as follows: M1(m/z 371 > 283), M3 (m/z 397 > 281), M5 (m/z 702 > 586). The MRM peak areas are shown for each metabolite. The results are shown as the means ± SD from four independent experiments (n = 4) performed in triplicate each.

Article Snippet: Deuterium-labeled sunitinib ( N -[2-diethylamino(ethyl- d 4 )]-5-[( Z )-(5-fluoro-1,2-dihydro-2-oxo-3 H -indo-3-yldiene)methyl]-2,4-di-methyl-1 H -pyrrole-3-carboxamide, sunitinib- d 4 , S820003), N -desethyl-sunitinib (D289650), sunitinib N -oxide (S820005), furafylline, (+)- N -3-benzylnirvanol, 2-phenyl-2-(1-piperidinyl)propane (PPP), and CY-P3cide were purchased from Toronto Research Chemicals (Toronto, Canada).

Techniques: Incubation, Recombinant, Liquid Chromatography with Mass Spectroscopy

Journal: Chemical research in toxicology

Article Title: Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib

doi: 10.1021/acs.chemrestox.8b00005

Figure Lengend Snippet: Effect of P450 inhibitors on sunitinib metabolite formation: (A) M1, (B) M3, and (C) M5. Sunitinib (10 μM) was incubated with pooled human liver microsomes (0.1 mg/mL) supplemented with 5 mM GSH in the presence or absence of P450-selective chemical inhibitors. The following chemical inhibitors were used to block the respective P450: α-naphthoflavone (1 μM, P540 1A2); furafylline (25 μM, P450 1A2); PPP, phenyl-piperidinyl propane (15 μM, P450 2B6); ticlopidine (5 μM, P450 2B6/2C19); sulfaphenazole (5 μM, P450 2C9); (+)-N-3-benzylnirvanol (5 μM, P450 2C19); quinidine (2 μM, P450 2D6); 4-methylpyrazole (100 μM, P450 2E1); ketoconazole (1 μM, P450 3A); and CYP3cide (2 μM, P450 3A4). Control incubations were carried out with the appropriate vehicle solvent control in the absence of inhibitor. Relative levels of metabolite formed were measured by LC–MS/MS. Metabolite levels (expressed as peak area ratio) under control conditions were: (A) 6.6 ± 1.4 for M1, (B) 0.04 ± 0.02 for M3, and (C) 0.01 ± 0.008 for M5. Percent (%) metabolite formation was based on comparison to vehicle control. The results are shown as the means ± SD from three independent experiments (n = 3) performed in triplicate each. Comparisons of inhibitor vs vehicle control were performed by unpaired two-tailed t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Deuterium-labeled sunitinib ( N -[2-diethylamino(ethyl- d 4 )]-5-[( Z )-(5-fluoro-1,2-dihydro-2-oxo-3 H -indo-3-yldiene)methyl]-2,4-di-methyl-1 H -pyrrole-3-carboxamide, sunitinib- d 4 , S820003), N -desethyl-sunitinib (D289650), sunitinib N -oxide (S820005), furafylline, (+)- N -3-benzylnirvanol, 2-phenyl-2-(1-piperidinyl)propane (PPP), and CY-P3cide were purchased from Toronto Research Chemicals (Toronto, Canada).

Techniques: Incubation, Blocking Assay, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: Chemical research in toxicology

Article Title: Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib

doi: 10.1021/acs.chemrestox.8b00005

Figure Lengend Snippet: Kinetic Parameters of Sunitinib Metabolite Formation by Recombinant P450 1A2 and P450 3A4 a

Article Snippet: Deuterium-labeled sunitinib ( N -[2-diethylamino(ethyl- d 4 )]-5-[( Z )-(5-fluoro-1,2-dihydro-2-oxo-3 H -indo-3-yldiene)methyl]-2,4-di-methyl-1 H -pyrrole-3-carboxamide, sunitinib- d 4 , S820003), N -desethyl-sunitinib (D289650), sunitinib N -oxide (S820005), furafylline, (+)- N -3-benzylnirvanol, 2-phenyl-2-(1-piperidinyl)propane (PPP), and CY-P3cide were purchased from Toronto Research Chemicals (Toronto, Canada).

Techniques: Recombinant

Journal: Chemical research in toxicology

Article Title: Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib

doi: 10.1021/acs.chemrestox.8b00005

Figure Lengend Snippet: Kinetic Parameters of Sunitinib Metabolite Formation in Single Donor Human Liver Microsomes (HH581 and HH741) a

Article Snippet: Deuterium-labeled sunitinib ( N -[2-diethylamino(ethyl- d 4 )]-5-[( Z )-(5-fluoro-1,2-dihydro-2-oxo-3 H -indo-3-yldiene)methyl]-2,4-di-methyl-1 H -pyrrole-3-carboxamide, sunitinib- d 4 , S820003), N -desethyl-sunitinib (D289650), sunitinib N -oxide (S820005), furafylline, (+)- N -3-benzylnirvanol, 2-phenyl-2-(1-piperidinyl)propane (PPP), and CY-P3cide were purchased from Toronto Research Chemicals (Toronto, Canada).

Techniques:

Journal: Chemical research in toxicology

Article Title: Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib

doi: 10.1021/acs.chemrestox.8b00005

Figure Lengend Snippet: LC–MS/MS analysis of the putative sunitinib quinoneimine dansyl–GSH conjugate. Sunitinib (25 μM) was incubated with recombinant P450 1A2 Supersomes (20 nM) in the presence of an NADPH-regenerating system and supplemented with dansyl–GSH (1 mM) for 30 min. Shown is a representative product ion spectrum from LC–MS/MS analysis of the precursor ion m/z 935, corresponding to the quinoneimine dansyl–GSH conjugate. The predicted structure of the sunitinib quinoneimine dansyl–GSH conjugate is shown above.

Article Snippet: Deuterium-labeled sunitinib ( N -[2-diethylamino(ethyl- d 4 )]-5-[( Z )-(5-fluoro-1,2-dihydro-2-oxo-3 H -indo-3-yldiene)methyl]-2,4-di-methyl-1 H -pyrrole-3-carboxamide, sunitinib- d 4 , S820003), N -desethyl-sunitinib (D289650), sunitinib N -oxide (S820005), furafylline, (+)- N -3-benzylnirvanol, 2-phenyl-2-(1-piperidinyl)propane (PPP), and CY-P3cide were purchased from Toronto Research Chemicals (Toronto, Canada).

Techniques: Liquid Chromatography with Mass Spectroscopy, Incubation, Recombinant

Journal: Chemical research in toxicology

Article Title: Cytochromes P450 1A2 and 3A4 Catalyze the Metabolic Activation of Sunitinib

doi: 10.1021/acs.chemrestox.8b00005

Figure Lengend Snippet: Analysis of dansyl–GSH Conjugates and M1 by LC–MS/MS and UPLC–UV Detection. Sunitinib (25 μM) was incubated with pooled human liver microsomes (1 mg/mL) in the presence of dGSH (1 mM) and an NADPH-regenerating system for 30 min. Control incubations were without NADPH, without dGSH, or without substrate (blank sample). Each reaction condition was performed in triplicate. Samples were analyzed by LC–MS/MS utilizing MRM for the (A) quinoneimine–dGSH conjugate of sunitinib: m/z 935 > 862, (B) M1–dGSH conjugate: m/z 911 > 371, and sunitinib–dGSH conjugate(s): m/z 939 > 399. (D) Relative levels of M1 measured by UPLC–UV absorbance at 430 nm. Levels of M1 were compared from incubations with and without dGSH by unpaired two-tailed t test (n = 2-3).

Article Snippet: Deuterium-labeled sunitinib ( N -[2-diethylamino(ethyl- d 4 )]-5-[( Z )-(5-fluoro-1,2-dihydro-2-oxo-3 H -indo-3-yldiene)methyl]-2,4-di-methyl-1 H -pyrrole-3-carboxamide, sunitinib- d 4 , S820003), N -desethyl-sunitinib (D289650), sunitinib N -oxide (S820005), furafylline, (+)- N -3-benzylnirvanol, 2-phenyl-2-(1-piperidinyl)propane (PPP), and CY-P3cide were purchased from Toronto Research Chemicals (Toronto, Canada).

Techniques: Liquid Chromatography with Mass Spectroscopy, Incubation, Two Tailed Test