The toxicokinetics of β-amanitin, a toxic bicyclic octapeptide present abundantly in Amanitaceae mushrooms, was evaluated in mice after intravenous (iv) and oral administration. The area under plasma concentration curves (AUC) following iv injection increased in proportion to doses of 0.2, 0.4, and 0.8 mg/kg. β-amanitin disappeared rapidly from plasma with a half-life of 18.3–33.6 min, and 52.3% of the iv dose was recovered as a parent form. After oral administration, the AUC again increased in proportion with doses of 2, 5, and 10 mg/kg. Absolute bioavailability was 7.3–9.4%, which resulted in 72.4% of fecal recovery from orally administered β-amanitin. Tissue-to-plasma AUC ratios of orally administered β-amanitin were the highest in the intestine and stomach. It also readily distributed to kidney > spleen > lung > liver ≈ heart. Distribution to intestines, kidneys, and the liver is in agreement with previously reported target organs after acute amatoxin poisoning. In addition, β-amanitin weakly or negligibly inhibited major cytochrome P450 and 5′-diphospho-glucuronosyltransferase activities in human liver microsomes and suppressed drug transport functions in mammalian cells that overexpress transporters, suggesting the remote drug interaction potentials caused by β-amanitin exposure.

Zastaprazan (JP-1366), a novel potassium-competitive acid blocker, is a new drug for the treatment of erosive esophagitis. JP-1366 is highly metabolized in human, mouse, and dog hepatocytes but moderately metabolized in rat and monkey hepatocytes when estimated from the metabolic stability of this compound in hepatocyte suspension and when 18 phase I metabolites and 5 phase II metabolites [i.e., N-dearylation (M6), hydroxylation (M1, M19, M21), dihydroxylation (M7, M8, M14, M22), trihydroxylation (M13, M18), hydroxylation and reduction (M20), dihydroxylation and reduction (M9, M16), hydrolysis (M23), hydroxylation and glucuronidation (M11, M15), hydroxylation and sulfation (M17), dihydroxylation and sulfation (M10, M12), N-dearylation and hydroxylation (M3, M4), N-dearylation and dihydroxylation (M5), and N-dearylation and trihydroxylation (M2)] were identified from JP-1366 incubation with the hepatocytes from humans, mice, rats, dogs, and monkeys. Based on the cytochrome P450 (CYP) screening test and immune-inhibition analysis with CYP antibodies, CYP3A4 and CYP3A5 played major roles in the metabolism of JP-1366 to M1, M3, M4, M6, M8, M9, M13, M14, M16, M18, M19, M21, and M22. CYP1A2, 2C8, 2C9, 2C19, and 2D6 played minor roles in the metabolism of JP-1366. UDP-glucuronosyltransferase (UGT) 2B7 and UGT2B17 were responsible for the glucuronidation of M1 to M15. However, JP-1366 and active metabolite M1 were not substrates for drug transporters such as organic cation transporter (OCT) 1/2, organic anion transporter (OAT) 1/3, organic anion transporting polypeptide (OATP)1B1/1B3, multidrug and toxic compound extrusion (MATE)1/2K, P-glycoprotein (P-gp), and breast cancer-resistant protein (BCRP). Only M1 showed substrate specificity for P-gp. The findings indicated that drug-metabolizing enzymes, particularly CYP3A4/3A5, may have a significant role in determining the pharmacokinetics of zastaprazan while drug transporters may only have a small impact on the absorption, distribution, and excretion of this compound.

The well-known hepatotoxicity mechanism resulting from alpha-amanitin (α-AMA) exposure arises from RNA polymerase II (RNAP II) inhibition. RNAP Ⅱ inhibition occurs through the dysregulation of mRNA synthesis. However, the signaling pathways in hepatocytes that arise from α-AMA have not yet been fully elucidated. Here, we identified that the RAS/RAF/ERK signaling pathway was activated through quantitative phosphoproteomic and molecular biological analyses in Huh-7 cells. Bioinformatics analysis showed that α-AMA exposure increased protein phosphorylation in a time-dependent α-AMA exposure. In addition, phosphorylation increased not only the components of the ERK signaling pathway but also U2AF65 and SPF45, known splicing factors. Therefore, we propose a novel mechanism of α-AMA as follows. The RAS/RAF/ERK signaling pathway involved in aberrant splicing events is activated by α-AMA exposure followed by aberrant splicing events leading to cell death in Huh-7 cells.

We aimed to investigate the metabolic pathways of kobusin, a tetrahydrofurofuran lignan with benzo[1,3]dioxole group, which exhibits antioxidant, anti-inflammatory, and anticancer activities. The metabolic profiles of kobusin, following incubation with hepatocytes and human liver microsomes (HLMs), were categorized into three clusters using ion identity molecular networking, with each cluster subsequently linked by glucuronidation and sulfation. These clusters were predicted to correspond to demethylenation, demethylation, and methylation with demethylenation. The structures of the metabolites and the responsible metabolizing enzymes were further characterized using hepatocytes, HLMs, S9 fractions, cDNA-expressed cytochrome P450 isozymes (CYPs), UDP-glucuronosyltransferase isozymes (UGTs), and sulfotransferases (SULTs). Kobusin yielded five phase 1 metabolites, including kobusin catechol (M1) formed via CYP2C8, CYP2C9, CYP2C19, and CYP3A4/5; O-desmethylkobusin (M2 and M3) via CYP2C9 and CYP2C19; and hydroxykobusin (M4 and M5) via CYP3A4/5. The major phase 1 metabolites (M1, M2, and M3) were subsequently converted into 14 phase 2 metabolites, including O-methyl-M1 (M6-M8) via catechol O-methyltransferase, as well as six glucuronide and sulfate conjugates of kobusin metabolites (M1-G, M2-G, M3-G, M6-G, M7-G, and M8-G; M1-S, M2-S, M3-S, M6-S, and M7-S) formed by UGTs and SULTs, respectively. Two glutathione (GSH) conjugates of the reactive intermediate derived from M1 (kobusin catechol) were identified after incubation of kobusin with HLMs in the presence of NADPH and GSH; however, these conjugates were not detected in hepatocyte incubations. In conclusion, kobusin undergoes extensive metabolism, yielding a total of 19 metabolites in human hepatocytes. These findings provide insight into the potential contributions of metabolizing enzymes to kobusin's pharmacokinetics and drug interactions associated with the reactive intermediate of M1.