RESUMO
Acalabrutinib (CALQUENCE; ACB) is a Bruton tyrosine kinase inhibitor (BTKI) used to treat mantle cell lymphoma, small lymphocytic lymphoma (SLL), and chronic lymphocytic leukemia (CLL). On 21 November 2019, ACB was approved by the U.S. FDA for the use as a single therapy for the treatment of CLL/SLL. In silico studies were first done to propose vulnerable sites of metabolism and reactivity pathways by StarDrop software and Xenosite online software; respectively. ACB metabolites and stable adducts were characterized in vitro from rat liver microsomes (RLMs) using Ion Trap LC/MS. Generation of reactive intermediates (RIs) in the in vitro metabolism of ACB was investigated using glutathione, potassium cyanide, and methoxylamine as trapping nucleophiles for the RIs including iminopyridinone, iminium, and aldehyde, respectively, to form stable adducts that can be identified and characterized by Ion Trap LC/MS. Five phase I metabolites, seven 6-iminopyridin-3(6H)-one and five aldehyde RIs of ACB were identified. Based on literature reviews, the generation of RIs of ACB, and the subsequent drug-induced organ toxicity (DIOT) reactions may provide an explanation of ACB ADRs. Additional drug discovery investigations can be performed to facilitate the creation of novel medications with improved safety characteristics.
RESUMO
Spebrutinib is a new Bruton tyrosine kinase inhibitor developed by Avila Therapeutics and Celgene. Spebrutinib (SPB) is currently in phase Ib clinical trials for the treatment of lymphoma in the United States. Preliminary in-silico studies were first performed to predict susceptible sites of metabolism, reactivity pathways and structural alerts for toxicities by StarDrop WhichP450™ module, Xenosite web predictor tool and DEREK software; respectively. SPB metabolites and adducts were characterized in vitro from rat liver microsomes (RLM) using LC-MS/MS. Formation of reactive intermediates was investigated using potassium cyanide (KCN), glutathione (GSH) and methoxylamine as trapping nucleophiles for the unstable and reactive iminium, iminoquinone and aldehyde intermediates, respectively, with the aim to produce stable adducts that can be detected and characterized using mass spectrometry. Fourteen phase I metabolites, four cyanide adducts, six GSH adducts and three methoxylamine adducts of SPB were identified and characterized. The proposed metabolic pathways involved in generation of phase I metabolites of SPB are oxidation, hydroxylation, o-dealkylation, epoxidation, defluorination and reduction. Several in vitro reactive intermediates were identified and characterized, the formation of which can aid in explaining the adverse drug reactions of SPB. Several iminium, 2-iminopyrimidin-5(2H)-one and aldehyde intermediates of SPB were revealed. Acrylamide is identified as a structural alert for toxicity by DEREK report and was found to be involved in the formation of several glycidamide and aldehyde reactive intermediates.
RESUMO
Fenebrutinib is an orally available Bruton tyrosine kinase inhibitor. It is currently in multiple phase III clinical trials for the management of B-cell tumors and autoimmune disorders. Elementary in-silico studies were first performed to predict susceptible sites of metabolism and structural alerts for toxicities by StarDrop WhichP450™ module and DEREK software; respectively. Fenebrutinib metabolites and adducts were characterized in-vitro in rat liver microsomes (RLM) using MS3 method in Ion Trap LC-MS/MS. Formation of reactive and unstable intermediates was explored using potassium cyanide (KCN), glutathione (GSH) and methoxylamine as trapping nucleophiles to capture the transient and unstable iminium, 6-iminopyridin-3(6H)-one and aldehyde intermediates, respectively, to generate a stable adducts that can be investigated and analyzed using mass spectrometry. Ten phase I metabolites, four cyanide adducts, five GSH adducts and six methoxylamine adducts of fenebrutinib were identified. The proposed metabolic reactions involved in formation of these metabolites are hydroxylation, oxidation of primary alcohol to aldehyde, n-oxidation, and n-dealkylation. The mechanism of reactive intermediate formation of fenebrutinib can provide a justification of the cause of its adverse effects. Formation of iminium, iminoquinone and aldehyde intermediates of fenebrutinib was characterized. N-dealkylation followed by hydroxylation of the piperazine ring is proposed to cause the bioactivation to iminium intermediates captured by cyanide. Oxidation of the hydroxymethyl group on the pyridine moiety is proposed to cause the generation of reactive aldehyde intermediates captures by methoxylamine. N-dealkylation and hydroxylation of the pyridine ring is proposed to cause formation of iminoquinone reactive intermediates captured by glutathione. FBB and several phase I metabolites are bioactivated to fifteen reactive intermediates which might be the cause of adverse effects. In the future, drug discovery experiments utilizing this information could be performed, permitting the synthesis of new drugs with better safety profile. Overall, in silico software and in vitro metabolic incubation experiments were able to characterize the FBB metabolites and reactive intermediates using the multistep fragmentation capability of ion trap mass spectrometry.