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Ponatinib (Iclusig®) is a multi-targeted tyrosine kinase inhibitor (TKIs). It is active against T315I and other BCR-ABL mutants. Investigation of in vivo metabolism of ponatinib was done using Sprague Dawley rats by giving one oral dose of PNT (4.7 mg/kg) to each rat and urine samples were gathered at several time intervals from dosing. Filteration of urine samples was done through 0.45 μm syringe filters. Phase separation using ACN was applied for extraction of ponatinib related metabolites. Characterization and identification of one in vivo phase II metabolite and thirteen in vivo phase I of PNT were done using LC-MS/MS. Phase I metabolic reactions were reduction, N-demethylation, hydroxylation, N-oxidation, oxidation and amide hydrolysis. Phase II metabolic reaction was glucuronidation of hydroxyl benzyl metabolites of ponatinib. The major in vivo metabolic reactions were α hydroxylation and α oxidation at piperazine ring. Literature review revealed no articles that have been published on in vivo metabolism of ponatinib in Sprague Dawley rats or ponatinib in vivo phase I and phase II metabolites structural characterization and identification., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Entrectinib (ENC) is a potent orally available anaplastic lymphoma kinase (ALK) inhibitor. In 10 July 2017, biotechnology company (Ignyta) announced that granted orphan drug designation approval was given by the FDA to ENC for "treatment of NTRK fusion-positive solid tumors". A validated LC-MS/MS methodology was developed for ENC quantification in human plasma matrix. The supposed method characterized by high speed, specificity and sensitivity. This established method was applied for metabolic degradation assessment of ENC. Reversed stationary phase (C 18 column) and elution mobile phase (48% 10 mM ammonium formate in H 2 O (pH: 4.2 adjusted by adding few drops of formic acid): 52% ACN) were utilized for chromatographic resolution of ENC and lapatinib as internal standard (IS). Total elution time, flow rate and injection volume were 4 min., 0.25 mL/min., and 5 μL, respectively. Electrospray ionization source was used for ions generation, while positive multiple reactions monitoring (MRM) mode was used for ion analysis. The data of calibration curve of ENC in human plasma was linear in the range of 5-500 ng/mL with correlation coefficient (r 2 ) >0.999. LOQ and LOD for ENC were 2.17 ng/mL and 0.71 ng/mL, respectively. Inter-day and intra-day precision and accuracy were 97.52 to 101.83%, and 0.38 to 1.32%, respectively. Intrinsic clearance (Clint) and in vitro half-life (t 1/2 ) were equal to 15.67 mL/min/kg and 9.1 min, respectively. To our knowledge, this is considered the first method for ENC quantification in human plasma and its metabolic degradation assessment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Saracatinib (AZD-0530) is a drug under clinical trials that developed by AstraZeneca. It is considered a dual kinase inhibitor, with selective actions as a Src inhibitor and a Bcr-Abl tyrosine-kinase inhibitor. Saracatinib chemical structure contains N-methyl piperazine group and 1,3 benzodioxole group. N-methyl piperazine group that can be bioactivated to form iminium intermediates which can be captured by KCN. 1,3-Benzodioxole group can be bioactivated to form ortho-quinone intermediate that can be conjugated with GSH. The formed conjugates are stable and can be identified using LC-MS/MS. In our current work, we are trying to give insight into the reasons that may be responsible for saracatinib side effects. Using LC-MS/MS, in vitro metabolic pathways were investigated for saracatinib in rat liver microsomes. Ten saracatinib phase I metabolites were characterized and the metabolic pathways were found to be hydroxylation, oxidation, reduction, dealkylation, N-oxidation and ether cleavage. Also, four potential reactive intermediates (three cyanide adducts and one GSH conjugate) were identified and the bioactivation mechanisms were explained. The existence of these four reactive metabolites may be the main reason for observed saracatinib side effects in clinical trials. Literature review showed no previous articles have been proposed the detailed structural identification of the formed reactive metabolites., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Brigatinib (BGB) belongs to a class of drugs called ALK inhibitor. On April 28, 2017, BGB has been approved by U.S. FDA for use in metastatic ALK-positive NSCLC. A fast, specific, sensitive and validated LC-MS/MS method was developed for the quantification of BGB in human plasma matrix. This method was applied successfully to study metabolic stability of BGB. Reversed phase (C18 column) and isocratic binary mobile phase (55% 0.1% formic acid: 45% ACN) were used for chromatographic separation of BGB and ponatinib (IS). The flow rate, total run time and injection volume were fixed at 0.2 mL/min, 4 min, 5 μL respectively. ESI source was utilized for ions formation, while multiple reaction monitoring (MRM) mode was used for ion analysis. In human plasma matrix, the Linearity range of the calibration curve was 5-500 ng/mL (r 2  ≥ 0.9982). LOQ and LOD were found to be 1.89 and 5.72 ng/mL. The precision and accuracy for the intra-day and inter-day were 0.45 to 1.85% and 97.37 to 104.85%. In vitro half-life (t 1/2 ) and intrinsic clearance (CL int ) were equal to 12.0 min and 13.1 ± 0.15 mL/min/kg respectively. The quantification of BGB in human plasma or its metabolic stability has not been studied as seen in literature review., (Copyright © 2018 Elsevier B.V. All rights reserved.)