LC-ESI-MS/MS reveals the formation of reactive intermediates in brigatinib metabolism: elucidation of bioactivation pathways

Abstract

Brigatinib (BGB) is a newly approved anaplastic lymphoma kinase (ALK) inhibitor. On April 28, 2017, BGB was approved by the U.S. FDA for the treatment of metastatic anaplastic lymphoma kinase-positive non-small cell lung cancer. The toxicity profile of BGB includes nausea, fatigue, diarrhea, elevated lipase, dyspnoea, hypertension, hypoxia, pneumonia, elevated amylase, pulmonary embolism, elevated ALT, hyponatraemia and hypophosphatemia. Using LC-MS/MS, we investigated the in vitro phase I metabolism of for BGB in rat liver microsomes (RLMs). In the in vitro metabolism of BGB, iminium reactive intermediates were trapped by potassium cyanide forming a stable complex that can be characterized by LC-MS/MS. Four BGB in vitro phase I metabolites were identified. In vitro phase I metabolic pathways were N-dealkylation, α hydroxylation and α oxidation. Additionally, three iminium reactive metabolites were found and the bioactivation mechanisms were proposed. A piperidine ring was found to be responsible for BGB bioactivation. The presence of these three reactive metabolites may be the main reason for BGB side effects. A literature review showed no previous article reported the in vitro phase I metabolism study of BGB or structural identification of the formed reactive metabolites.

Fig. 1. Chemical structure of brigatinib.

Fig. 2. PI chromatogram of MIP at m/z 584 showing BGB peak at 26.6 min (A), PI mass spectrum of BGB (B).

Scheme 1. Fragmentation pattern of BGB.

Fig. 3. PI chromatogram of MIP at m/z 570 showing BGB570 peak at 29.5 min (A), PI spectrum of BGB570 (B).

Scheme 2. Fragmentation pattern of BGB570.

Fig. 4. PI chromatogram of MIP at m/z 600 showing BGB600 peak at 32.9 min (A), PI spectrum of BGB600 (B).

Scheme 3. Fragmentation pattern of BGB600.

Fig. 5. PI chromatogram of MIP at m/z 486 showing BGB486 peak at 30.9 min (A), PI mass spectrum of BGB486 (B).

Scheme 4. Fragmentation pattern of BGB486.

Fig. 6. PI chromatogram of MIP at m/z 486 showing BGB598 peak at 27.3 min (A), PI mass spectrum of BGB598 (B).

Scheme 5. Fragmentation pattern of BGB598.

Fig. 7. PI chromatogram of MIP at m/z 609 showing BGB609 peak at 31.4 min (A), PI mass spectrum of BGB609 (B).

Scheme 6. Fragmentation pattern of BGB609.

Fig. 8. PI chromatogram of MIP at m/z 623 showing BGB623 peak at 42.8 min (A), PI mass spectrum of BGB623 (B).

Scheme 7. Fragmentation pattern of BGB623.

Fig. 9. PI chromatogram of MIP at m/z 527 showing BGB527 peak at 43.9 min (A), PI mass spectrum of BGB527 (B).

Scheme 8. Fragmentation pattern of BGB527.

Scheme 9. Proposed mechanism of bioactivation of BGB into reactive intermediates and trapping strategy.

Fig. 10. Chemical structure of BGB showing places of phase I metabolic reaction and bioactivation pathways.