Investigation of Fenebrutinib Metabolism and Bioactivation Using MS<sup>3</sup> Methodology in Ion Trap LC/MS
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...
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MDPI AG
2023-05-01
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| Online Access: | https://www.mdpi.com/1420-3049/28/10/4225 |
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| author | Aishah M. Alsibaee Haya I. Aljohar Mohamed W. Attwa Ali S. Abdelhameed Adnan A. Kadi |
| author_facet | Aishah M. Alsibaee Haya I. Aljohar Mohamed W. Attwa Ali S. Abdelhameed Adnan A. Kadi |
| author_sort | Aishah M. Alsibaee |
| collection | DOAJ |
| description | 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(<i>6H</i>)-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. |
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| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
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| spelling | doaj.art-89e56f63b2aa4f13b9ab39b31de340e92023-11-18T02:41:10ZengMDPI AGMolecules1420-30492023-05-012810422510.3390/molecules28104225Investigation of Fenebrutinib Metabolism and Bioactivation Using MS<sup>3</sup> Methodology in Ion Trap LC/MSAishah M. Alsibaee0Haya I. Aljohar1Mohamed W. Attwa2Ali S. Abdelhameed3Adnan A. Kadi4Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi ArabiaDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi ArabiaDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi ArabiaDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi ArabiaDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi ArabiaFenebrutinib 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(<i>6H</i>)-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.https://www.mdpi.com/1420-3049/28/10/4225fenebrutinibBruton tyrosine kinase inhibitormetabolismreactive intermediatestrapping agentsLC-ITMS |
| spellingShingle | Aishah M. Alsibaee Haya I. Aljohar Mohamed W. Attwa Ali S. Abdelhameed Adnan A. Kadi Investigation of Fenebrutinib Metabolism and Bioactivation Using MS<sup>3</sup> Methodology in Ion Trap LC/MS Molecules fenebrutinib Bruton tyrosine kinase inhibitor metabolism reactive intermediates trapping agents LC-ITMS |
| title | Investigation of Fenebrutinib Metabolism and Bioactivation Using MS<sup>3</sup> Methodology in Ion Trap LC/MS |
| title_full | Investigation of Fenebrutinib Metabolism and Bioactivation Using MS<sup>3</sup> Methodology in Ion Trap LC/MS |
| title_fullStr | Investigation of Fenebrutinib Metabolism and Bioactivation Using MS<sup>3</sup> Methodology in Ion Trap LC/MS |
| title_full_unstemmed | Investigation of Fenebrutinib Metabolism and Bioactivation Using MS<sup>3</sup> Methodology in Ion Trap LC/MS |
| title_short | Investigation of Fenebrutinib Metabolism and Bioactivation Using MS<sup>3</sup> Methodology in Ion Trap LC/MS |
| title_sort | investigation of fenebrutinib metabolism and bioactivation using ms sup 3 sup methodology in ion trap lc ms |
| topic | fenebrutinib Bruton tyrosine kinase inhibitor metabolism reactive intermediates trapping agents LC-ITMS |
| url | https://www.mdpi.com/1420-3049/28/10/4225 |
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