In vitro enantioselective inhibition of the main human CYP450 enzymes involved in drug metabolism by the chiral pesticide tebuconazole.

Tebuconazole (TEB) is a chiral triazole fungicide worldwide employed to control plant pathogens and preserve wood. People can be exposed to TEB either through diet and occupational contamination. This work investigates the in vitro inhibitory potential of rac-TEB, S-(+)-TEB, and R-(-)-TEB over the main cytochrome P450 enzymes (CYP450) using human liver microsomes to predict TEB in vivo inhibition potential. The IC50 values showed that in vitro inhibition was enantioselective for CYP2C9, CYP2C19, and CYP2D6, but not for CYP3A4/5. Despite enantioselectivity, rac-TEB and its single enantiomers were always classified in the same category. The inhibition mechanisms and constants were determined for rac-TEB and it has shown to be a mixed inhibitor of CYP3A4/5 (Ki = 1.3 ± 0.3 µM, αKi = 3.2 ± 0.5 µM; Ki = 0.6 ± 0.3 µM, αKi = 1.3 ± 0.3 µM) and CYP2C9 (Ki = 0.7 ± 0.1 µM, αKi = 2.7 ± 0.5 µM), and a competitive inhibitor of CYP2D6 (Ki = 11.9 ± 0.7 µM) and CYP2C19 (Ki = 0.23 ± 0.02 µM), respectively, suggesting that in some cases, rac-TEB has a higher or comparable inhibitory potential than well-known strong inhibitors of CYP450 enzymes, especially for CYP2C9 and CYP2C19. In vitro-in vivo extrapolations (IVIVE) were conducted based on the results and data available in the literature about TEB absorption and metabolism. R1 values were estimated based on the Food and Drug Administration guideline and suggested that in a chronic oral exposure scenario considering the acceptable daily intake dose proposed by the European Food and Safety Authority, the hypothesis of rac-TEB to inhibit the activities of CYP3A4/5, CYP2C9, and CYP2C19 in vivo and cause pesticide-drug interactions cannot be disregarded.

Risk assessment of the chiral pesticide fenamiphos in a human model: Cytochrome P450 phenotyping and inhibition studies.

Fenamiphos (FS) is a chiral organophosphate pesticide that is used to control nematodes in several crops. Enantioselective differences may be observed in FS activity, bioaccumulation, metabolism, and toxicity. Humans may be exposed to FS through occupational and chronic (food, water, and environmental) exposure. FS may cause undesirable CYP450 pesticide-drug interactions, which may impact human health. Here, the CYP450 isoforms involved in enantioselective FS metabolism were identified, and CYP450 inhibition by rac-FS, (+)-FS, and (-)-FS was evaluated to obtain reliable information on enantioselective FS risk assessment in humans. CYP3A4 and CYP2E1 metabolized FS enantiomers, and CYP2B6 may participate in rac-FS metabolism. In addition, rac-FS, (+)-FS, and (-)-FS were reversible competitive CYP1A2, CYP2C19, and CYP3A4/5 inhibitors. High stereoselective inhibition potential was verified; rac-FS and (-)-FS strongly inhibited and (+)-FS moderately inhibited CYP1A2. Stereoselective differences were also detected for CYP2C19 and CYP3A4/5, which were strongly inhibited by rac-FS, (+)-FS, and (-)-FS. Our results indicated a high potential for CYP450 drug-pesticide interactions, which may affect human health. The lack of stereoselective research on the effect of chiral pesticides on the activity of CYP450 isoforms highlights the importance of assessing the risks of such pesticides in humans.

Prediction of seriniquinone-drug interactions by in vitro inhibition of human cytochrome P450 enzymes.

Seriniquinone is a secondary metabolite isolated from a rare marine bacterium of the genus Serinicoccus. This natural quinone is highlighted for its selective cytotoxic activity toward melanoma cancer cells, in which rapid metastatic properties are still a challenge for clinical treatment of malignant melanoma. The progress of seriniquinone as a promising bioactive molecule for drug development requires the assessment of its clinical interaction potential with other drugs. This study aimed to investigate the in vitro inhibitory effects of seriniquinone on the main human CYP450 isoforms involved in drug metabolism. The results showed strong inhibition of CYP1A2, CYP2E1 and CYP3A, with IC50 values up to 1.4 µM, and moderate inhibition of CYP2C19, with IC50 value >15 µM. Detailed experiments performed with human liver microsomes showed that the inhibition of CYP450 isoforms can be explained by competitive and non-competitive inhibition mechanisms. In addition, seriniquinone demonstrated to be an irreversible and time-dependent inhibitor of CYP1A2 and CYP3A. The low inhibition constants values obtained experimentally suggest that concomitant intake of seriniquinone with drug metabolized by these isoforms should be carefully monitored for adverse effects or therapeutic failure.

In vitro enantioselective study of the toxicokinetic effects of chiral fungicide tebuconazole in human liver microsomes.

Tebuconazole (TEB) is a chiral triazole fungicide that is globally marketed and used as a racemic mixture to control plant pathogens. Due to its use as a racemic mixture, TEB may exhibit enantioselective toxicokinetics toward nontarget organisms, including humans. Therefore, the in vitro enantioselective metabolism of TEB by cytochrome P450 enzymes (CYP450) was studied using human liver microsomes, and the in vivo toxicokinetic parameters were predicted. A new enantioselective, reversed-phase LC-MS/MS method was developed and validated to analyze the enantiomers of TEB and its main metabolite, 1-hydroxytebuconazole (TEBOH). In vitro metabolic parameters were obtained, and in vitro-in vivo extrapolations were performed. Michaelis-Menten and atypical biphasic kinetic profiles were observed with a total intrinsic clearance ranging from 53 to 19 mL min-1 mg-1. The in vitro-in vivo extrapolation results showed that TEB first passage effect by the liver seems to be negligible, with hepatic clearance and extraction ratios ranging from 0.53 to 5.0 mL min-1 kg-1 and 2.7-25%, respectively. Preferential metabolism of (+)-TEB to rac-TEB and (-)-TEB was observed, with preferential production of (+)-TEBOH. Furthermore, reaction phenotyping studies revealed that, despite the low hepatic clearance in the first pass metabolism of TEB, multiple human CYP450 isoforms were involved in TEB metabolism when TEBOH enantiomers were generated, mainly CYP3A4 and CYP2C9, which makes TEB accumulation in the human body more difficult due to multiple metabolic pathways.

Metabolism studies of chiral pesticides: A critical review.

The consumption of pesticides worldwide has been growing in recent decades, and consequently the exposure of humans and other animals to them as well. However, even though it is known that chiral pesticides can behave stereoselectively, the knowledge about the risks to human health and the environment is scarce. Among the pesticides registered to date, approximately 30% have at least one center of asymmetry, and just 7% of them are currently marketed as a pure stereoisomer or as an enriched mixture of the active stereoisomer. There are several in vitro, in vivo, and in silico models available to evaluate the enantioselective metabolism of chiral pesticides aiming ecotoxicological and risk assessment. Therefore, this paper intends to provide a critical view of the metabolism of chiral pesticides in non-target species, including humans, and discuss their implications, as well as, conduct a review of the analytical techniques employed for in vitro and in vivo metabolism studies of chiral pesticides.

In Vitro Inhibition of Human CYP450s 1A2, 2C9, 3A4/5, 2D6 and 2E1 by Grandisin.

Grandisin, a lignan isolated from many species of plants, such as Virola surinamensis, is a potential drug candidate due to its biological properties, highlighted by its antitumor and trypanocidal activities. In this study, the inhibitory effects of grandisin on the activities of human cytochrome P450 enzymes were investigated by using human liver microsomes. Results showed that grandisin is a competitive inhibitor of CYP2C9 and a competitive and mechanism-based inhibitor of CYP3A4/5. The apparent Ki value for CYP2C9 was 50.60 µM and those for CYP3A4/5 were 48.71 µM and 31.25 µM using two different probe substrates, nifedipine and midazolam, respectively. The apparent KI, kinact, and kinact/KI ratio for the mechanism-based inhibition of CYP3A4/5 were 6.40 µM, 0.037 min-1, and 5.78 mL ·â€Šmin-1 µmol-1, respectively, by examining nifedipine oxidation, and 31.53 µM, 0.049 min-1, and 1.55 mL ·â€Šmin-1 µmol-1, respectively, by examining midazolam 1'-hydroxylation. These apparent kinact/KI values were comparable to or even higher than those for several therapeutic drugs that act as mechanism-based inhibitors of CYP3A4/5. CYP1A2 and CYP2D6 activities, in turn, were not substantially inhibited by grandisin (IC50 > 200 µM and 100 µM, respectively). In contrast, from a concentration of 4 µM, grandisin significantly stimulated CYP2E1 activity. These results improve the prediction of grandisin-drug interactions, suggesting that the risk of interactions with drugs metabolized by CYP3A4/5 and CYP2E1 cannot be overlooked.

In vitro metabolism of the lignan (-)-grandisin, an anticancer drug candidate, by human liver microsomes.

(-)-grandisin is a tetrahydrofuran lignan that displays important biological properties, such as trypanocidal, anti-inflammatory, cytotoxic, and antitumor activities, suggesting its utility as a potential drug candidate. One important step in drug development is metabolic characterization and metabolite identification. To perform a biotransformation study of (-)-grandisin and to determine its kinetic properties in humans, a high performance liquid chromatography (HPLC) method was developed and validated. After HPLC method validation, the kinetic properties of (-)-grandisin were determined. (-)-grandisin metabolism obeyed Michaelis-Menten kinetics. The maximal reaction rate (Vmax ) was 3.96 ± 0.18 µmol/mg protein/h, and the Michaelis-Menten constant (Km ) was 8.23 ± 0.99 µM. In addition, the structures of the metabolites derived from (-)-grandisin were characterized via gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analysis. Four metabolites, 4-O-demethylgrandisin, 3-O-demethylgrandisin, 4,4'-di-O-demethylgrandisin, and a metabolite that may correspond to either 3,4-di-O-demethylgrandisin or 3,5-di-O-demethylgrandisin, were detected. CYP2C9 isoform was the main responsible for the formation of the metabolites. These metabolites have not been previously described, demonstrating the necessity of assessing (-)-grandisin metabolism using human-derived materials.