Cytochromes P450 2C8 and 3A Catalyze the Metabolic Activation of the Tyrosine Kinase Inhibitor Masitinib

Abstract

Masitinib is a small molecule tyrosine kinase inhibitor under investigation for the treatment of amyotrophic lateral sclerosis, mastocytosis, and COVID-19. Hepatotoxicity has been reported in some patients while taking masitinib. The liver injury is thought to involve hepatic metabolism of masitinib by cytochrome P450 (P450) enzymes to form chemically reactive, potentially toxic metabolites. The goal of the current investigation was to determine the P450 enzymes involved in the metabolic activation of masitinib in vitro. In initial studies, masitinib (30 μM) was incubated with pooled human liver microsomes in the presence of NADPH and potassium cyanide to trap reactive iminium ion metabolites as cyano adducts. Masitinib metabolites and cyano adducts were analyzed using reversed-phase liquid chromatography–tandem mass spectrometry. The primary active metabolite, N-desmethyl masitinib (M485), and several oxygenated metabolites were detected along with four reactive metabolite cyano adducts (MCN510, MCN524, MCN526, and MCN538). To determine which P450 enzymes were involved in metabolite formation, reaction phenotyping experiments were conducted by incubation of masitinib (2 μM) with a panel of recombinant human P450 enzymes and by incubation of masitinib with human liver microsomes in the presence of P450-selective chemical inhibitors. In addition, enzyme kinetic assays were conducted to determine the relative kinetic parameters (apparent K m and V max) of masitinib metabolism and cyano adduct formation. Integrated analysis of the results from these experiments indicates that masitinib metabolic activation is catalyzed primarily by P450 3A4 and 2C8, with minor contributions from P450 3A5 and 2D6. These findings provide further insight into the pathways involved in the generation of reactive, potentially toxic metabolites of masitinib. Future studies are needed to evaluate the impact of masitinib metabolism on the toxicity of the drug in vivo.

DeepP450: Predicting Human P450 Activities of Small Molecules by Integrating Pretrained Protein Language Model and Molecular Representation

“…HumanP450Test1 comprised experimentally verified 65 CYP-substrate pairs and 15 CYP-nonsubstrate pairs described in the latest literature published between 2018 and 2023, and the CYPs in this data set refer to the nine categories of CYPs in the TrainSet. − The distribution of Tanimoto Similarity coefficients for small molecules in HumanP450Test1 and the TrainSet is shown in Figure A. Both the t-SNE plot (Figure D) and the similarity distribution indicated that 58% molecules in HumanP450Test1 exhibited similarity values lower than 0.5, with no instances of similarity reaching 0.9 or higher.…”

Cyanide Trapping of Iminium Ion Reactive Metabolites: Implications for Clinical Hepatotoxicity

“…A review of drugs approved by the FDA between 2015 and 2020 showed that 88% of the 164 small molecule drugs contained N-heterocycles, with piperidine and piperazine moieties ranking in the top 3 within this group. 41 Thus, the risk of iminium ion formation from these moieties is highly relevant, as illustrated by the cyanide adduct formation reported for the hepatotoxic compounds listed in Table 1 (5 most-DILI concern and 5 less-DILI concern), as well as reports associating cyanide adduct formation with covalent binding to protein and/or irreversible enzyme inhibition (phencyclidine, 42−44 nicotine, 45−47 rimonabant, 48 pemigatinib, 48,49 masitinib 50 ) and to hepatotoxicity associated with mianserin. 40,51−53 There has been concern that all compounds containing these heterocycles would simply test positive in such assays, regardless of whether they would result in clinical hepatotox-icity�i.e., there was uncertainty as to whether the in vitro data would translate to a positive in vivo outcome�and therefore whether such an assay would have high enough specificity for hepatotoxic compounds.…”

“…A review of drugs approved by the FDA between 2015 and 2020 showed that 88% of the 164 small molecule drugs contained N-heterocycles, with piperidine and piperazine moieties ranking in the top 3 within this group . Thus, the risk of iminium ion formation from these moieties is highly relevant, as illustrated by the cyanide adduct formation reported for the hepatotoxic compounds listed in Table (5 most-DILI concern and 5 less-DILI concern), as well as reports associating cyanide adduct formation with covalent binding to protein and/or irreversible enzyme inhibition (phencyclidine, − nicotine, − rimonabant, pemigatinib, , masitinib) and to hepatotoxicity associated with mianserin. ,− …”

Targeting Sigma Receptors for the Treatment of Neurodegenerative and Neurodevelopmental Disorders

“…Masitinib, a tyrosine kinase inhibitor, has successfully passed a randomized controlled phase II/III trial in ALS patients as an additional drug for riluzole by showing a significant improvement in ALS progression, quality of life, respiratory function and time delay to death [ 107 ]. However, further trials were halted in 2021 when observational studies indicated an increased risk of ischaemic heart disease [ 108 ].…”