Investigation of Fenebrutinib Metabolism and Bioactivation Using MS3 Methodology in Ion Trap LC/MS

by Selleckchem | Oct 17 2023

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.

Comments:

Fenebrutinib is an orally available Bruton tyrosine kinase inhibitor that is currently being investigated in multiple phase III clinical trials for the treatment of B-cell tumors and autoimmune disorders. Prior to these clinical trials, preliminary studies were conducted using in silico and in vitro methods to predict and characterize the metabolism and potential toxicities of fenebrutinib.

In the in silico studies, the StarDrop WhichP450™ module was used to identify potential sites of metabolism, while the DEREK software was employed to detect structural alerts for potential toxicities. These computational methods helped in predicting how fenebrutinib might be metabolized in the body and identified structural features that could be associated with adverse effects.

In the subsequent in vitro experiments, fenebrutinib metabolites and adducts were characterized using rat liver microsomes (RLM) and a specialized LC-MS/MS technique called MS3 in Ion Trap. This method allowed for the identification and analysis of reactive and unstable intermediates produced during the metabolism of fenebrutinib.

To capture these transient intermediates, nucleophiles such as potassium cyanide (KCN), glutathione (GSH), and methoxylamine were used. These trapping agents reacted with the reactive intermediates, forming stable adducts that could be studied using mass spectrometry.

Through these experiments, ten phase I metabolites, four cyanide adducts, five GSH adducts, and six methoxylamine adducts of fenebrutinib were identified. The proposed metabolic reactions involved hydroxylation, oxidation of a primary alcohol to an aldehyde, N-oxidation, and N-dealkylation. These reactions were responsible for the formation of the identified metabolites and adducts.

The mechanism of reactive intermediate formation was also investigated. It was found that N-dealkylation followed by hydroxylation of the piperazine ring resulted in the bioactivation of fenebrutinib to iminium intermediates, which were captured by cyanide. Oxidation of the hydroxymethyl group on the pyridine moiety led to the generation of reactive aldehyde intermediates, which were captured by methoxylamine. Additionally, N-dealkylation and hydroxylation of the pyridine ring were proposed to cause the formation of iminoquinone reactive intermediates, which were captured by glutathione.

The identification and characterization of these reactive intermediates and adducts provide insights into the potential adverse effects of fenebrutinib. The formation of these reactive species may contribute to the observed adverse effects associated with the drug. Understanding the bioactivation pathways and the reactive intermediates involved can help in further evaluating the safety profile of fenebrutinib.

The information obtained from these studies can also be used in future drug discovery experiments. By utilizing this knowledge, researchers can design and synthesize new drugs with improved safety profiles by modifying the chemical structure to mitigate the formation of reactive intermediates or enhance their detoxification.

Overall, the combination of in silico software and in vitro metabolic incubation experiments using mass spectrometry enabled the characterization of fenebrutinib metabolites and reactive intermediates. These findings contribute to a better understanding of the drug's metabolism and potential adverse effects, facilitating the development of safer and more effective therapies in the future.