Early enterovirus translation deficits extend viral RNA replication and elicit sustained MDA5-directed innate signaling.

IMPORTANCE: Multiple pattern recognition receptors sense vRNAs and initiate downstream innate signaling: endosomal Toll-like receptors (TLRs) 3, 7, and 8 and cytoplasmic RIG-I-like receptors (RLRs) RIG-I, and MDA5. They engage distinct signaling scaffolds: mitochondrial antiviral signaling protein (RLR), MyD88, and TLR-adaptor interacting with SLC15A4 on the lysosome (TLR7 and TLR8) and toll/IL-1R domain-containing adaptor inducing IFN (TLR3). By virtue of their unusual vRNA structure and direct host cell entry path, the innate response to EVs uniquely is orchestrated by MDA5. We reported that PVSRIPO's profound attenuation and loss of cytopathogenicity triggers MDA5-directed polar TBK1-IRF3 signaling that generates priming of polyfunctional antitumor CD8+ T-cell responses and durable antitumor surveillance in vivo. Here we unraveled EV-host relations that control suppression of host type-I IFN responses and show that PVSRIPO's deficient immediate host eIF4G cleavage generates unopposed MDA5-directed downstream signaling cascades resulting in sustained type-I IFN release.

Interindividual Variability in Cytochrome P450 3A and 1A Activity Influences Sunitinib Metabolism and Bioactivation.

Sunitinib is an orally administered tyrosine kinase inhibitor associated with idiosyncratic hepatotoxicity; however, the mechanisms of this toxicity remain unclear. We have previously shown that cytochromes P450 1A2 and 3A4 catalyze sunitinib metabolic activation via oxidative defluorination leading to a chemically reactive, potentially toxic quinoneimine, trapped as a glutathione (GSH) conjugate (M5). The goals of this study were to determine the impact of interindividual variability in P450 1A and 3A activity on sunitinib bioactivation to the reactive quinoneimine and sunitinib N-dealkylation to the primary active metabolite N-desethylsunitinib (M1). Experiments were conducted in vitro using single-donor human liver microsomes and human hepatocytes. Relative sunitinib metabolite levels were measured by liquid chromatography-tandem mass spectrometry. In human liver microsomes, the P450 3A inhibitor ketoconazole significantly reduced M1 formation compared to the control. The P450 1A2 inhibitor furafylline significantly reduced defluorosunitinib (M3) and M5 formation compared to the control but had minimal effect on M1. In CYP3A5-genotyped human liver microsomes from 12 individual donors, M1 formation was highly correlated with P450 3A activity measured by midazolam 1'-hydroxylation, and M3 and M5 formation was correlated with P450 1A2 activity estimated by phenacetin O-deethylation. M3 and M5 formation was also associated with P450 3A5-selective activity. In sandwich-cultured human hepatocytes, the P450 3A inducer rifampicin significantly increased M1 levels. P450 1A induction by omeprazole markedly increased M3 formation and the generation of a quinoneimine-cysteine conjugate (M6) identified as a downstream metabolite of M5. The nonselective P450 inhibitor 1-aminobenzotriazole reduced each of these metabolites (M1, M3, and M6). Collectively, these findings indicate that P450 3A activity is a key determinant of sunitinib N-dealkylation to the active metabolite M1, and P450 1A (and potentially 3A5) activity influences sunitinib bioactivation to the reactive quinoneimine metabolite. Accordingly, modulation of P450 activity due to genetic and/or nongenetic factors may impact the risk of sunitinib-associated toxicities.

Detoxication versus Bioactivation Pathways of Lapatinib In Vitro: UGT1A1 Catalyzes the Hepatic Glucuronidation of Debenzylated Lapatinib.

O-Dealkylation of the tyrosine kinase inhibitor lapatinib by cytochrome P450 3A enzymes is implicated in the development of lapatinib-induced hepatotoxicity. Conjugative metabolism of debenzylated lapatinib (M1) via glucuronidation and sulfation is thought to be a major detoxication pathway for lapatinib in preclinical species (rat and dog), limiting formation of the quinoneimine reactive metabolite. Glucuronidation of M1 by human recombinant UDP-glucuronosyltransferases (UGTs) has been reported in vitro; however, the relative UGT enzyme contributions are unknown, and the interspecies differences in the conjugation versus bioactivation pathways of M1 have not been fully elucidated. In the present study, reaction phenotyping experiments using human recombinant UGT enzymes and enzyme-selective chemical inhibitors demonstrated that UGT1A1 was the major hepatic UGT enzyme involved in lapatinib M1 glucuronidation. Formation of the M1-glucuronide by human liver microsomes from UGT1A1-genotyped donors was significantly correlated with UGT1A1 activity as measured by 17ß-estradiol 3-glucuronidation (R 2 = 0.90). Interspecies differences were found in the biotransformation of M1 in human, rat, and dog liver microsomal and 9000g supernatant (S9) fractions via glucuronidation, sulfation, aldehyde oxidase-mediated oxidation, and bioactivation to the quinoneimine trapped as a glutathione (GSH) conjugate. Moreover, we demonstrated the sequential metabolism of lapatinib in primary human hepatocytes to the M1-glucuronide, M1-sulfate, and quinoneimine-GSH conjugate. M1 glucuronidation was highly correlated with the rates of M1 formation, suggesting that O-dealkylation may be the rate-limiting step in lapatinib biotransformation. Interindividual variability in the formation and clearance pathways of lapatinib M1 likely influences the hepatic exposure to reactive metabolites and may affect the risk for hepatotoxicity. SIGNIFICANCE STATEMENT: We used an integrated approach to examine the interindividual and interspecies differences in detoxication versus bioactivation pathways of lapatinib, which is associated with idiosyncratic hepatotoxicity. In addition to cytochrome P450 (P450)-mediated bioactivation, we report that multiple non-P450 pathways are involved in the biotransformation of the primary phenolic metabolite of lapatinib in vitro, including glucuronidation, sulfation, and aldehyde oxidase mediated oxidation. UGT1A1 was identified as the major hepatic enzyme involved in debenzylated lapatinib glucuronidation, which may limit hepatic exposure to the potentially toxic quinoneimine.