Drug Metabolism and Disposition最新文献

(+)-Dihydromethysticin (DHM) is a major kavalactone isolated from kava plants. Previous studies have identified (+)-DHM as a CYP2B6 inhibitor, with intriguing structural specificity. It is the most potent CYP2B6 inhibitor identified to date, and CYP2B6 is the most sensitive of any CYP isoform to inhibition by DHM. This investigation evaluated the stereochemistry of DHM inhibition of CYP2B6 and the role of methylenedioxyphenyl group bioactivation in CYP2B6 inhibition, using expressed CYP2B6 and the probe substrates 7-ethoxy-4-trifluoromethyIcoumarin and S-ketamine. The unnatural enantiomer (-)-DHM and racemic (±)-DHM exhibited similar inhibitory activities. Both DHM enantiomers were noncompetitive inhibitors of CYP2B6, with Ki values of 0.2 μM. DHM analogs lacking a methylenedioxy group were devoid of inhibitory effects in both CYP2B6 metabolism assays. Difluoro substitution of the methylene hydrogens on DHM abolished DHM inhibitory activity, whereas dideuterio substitution had no effect on CYP2B6 inhibition. Both DHM enantiomers and the dideuterio analog, but not the difluorinated analog or methysticin, generated a difference spectrum consistent with a metabolite-inhibitor complex. Results suggest CYP2B6-catalyzed methylenedioxyphenyl bioactivation of DHM to a metabolite-inhibitor complex with subsequent enzyme inhibition. DHM may have potential clinical implications or application as a selective CYP2B6 index inhibitor probe. SIGNIFICANCE STATEMENT: Enantiomers of the kavalactone dihydromethysticin are among the most potent CYP2B6 inhibitors identified to date, undergo metabolite-inhibitor complex formation, and exhibit substrate-dependent competitive and noncompetitive inhibition, which may have potential clinical implications or application. Because there is presently no inhibitor of CYP2B6 recommended for in vitro studies and no strong index inhibitor available for CYP2B6 for clinical studies, due in part to specificity considerations, dihydromethysticin may be a candidate for this purpose.

Drug-induced kidney injury, often resulting from the intracellular accumulation of drugs in renal proximal tubule cells via uptake transporters such as organic anion transporters 1 and 3 (OAT1/3), remains a major obstacle in drug development. Conventional 2-dimensional cultures of human renal proximal tubule epithelial cells (RPTECs) hardly express OAT1/3, limiting their utility for toxicity assessment. In contrast, 3-dimensional (3D) cultures of RPTEC have been shown to markedly upregulate OAT1/3 expression, offering a more physiologically relevant in vitro model for evaluating the toxicity of anionic compounds. In this study, we investigated the mechanism underlying OAT1/3 upregulation in 3D-RPTEC and explored a strategy to mitigate transporter-mediated toxicity. We found that hepatocyte nuclear factor (HNF) 4α expression is also increased in 3D-RPTEC. Motif analysis and cleavage under targets and release using nuclease-quantitative polymerase chain reaction revealed that HNF4α directly binds to the promoters of SLC22A6 and SLC22A8, identifying it as a key transcriptional regulator of OAT1/3 expression. Activation of the farnesoid X receptor (FXR), which represses the binding of HNF4α to promoters through the upregulation of small heterodimer partner (SHP), decreased OAT1/3 expression. Treatment with FXR ligands reduced cellular uptake of OAT1/3 substrates (eg, tenofovir and adefovir) and decreased their cytotoxic effects in 3D-RPTEC. These findings elucidate a transcriptional mechanism by which HNF4α regulates OAT1/3 expression in 3D-RPTEC and demonstrate that FXR agonists can downregulate OAT1/3 expression via the HNF4α-SHP axis. The present study highlights the utility of 3D-RPTEC as a valuable platform for mechanistic studies of transporter regulation. SIGNIFICANCE STATEMENT: This study shows that farnesoid X receptor agonists suppress hepatocyte nuclear factor 4α-mediated OAT1/3 activity in 3-dimensional-cultured renal proximal tubular cells and reduce nucleotide analog-induced toxicity. These findings provide mechanistic insight into transporter regulation and suggest a potential strategy to prevent nephrotoxicity.

A novel workflow for quantification of a drug and its metabolites in in vivo studies has been developed in the context of a radiolabeled human mass balance study. Samples are analyzed with ultra-high-performance liquid chromatography, and fractions are collected in a 384-well plate, which is subjected to offline counting, providing improved detection limits over online radioactivity detection. We discuss an advanced strategy to account for signal suppression or quenching, which significantly affected results in the offline counting of feces and urine samples in the selected case example, to provide more accurate quantification. The new quench model fits 2 data sets from 384-well plates with the actual matrices present to perform counting efficiency correction. Improved results were obtained over the existing approach, where a generic quench curve is defined by only a limited number of points made from a dilution series of a quenching agent. To account for outliers, a robust quartic model was applied. The new model effectively describes matrix-induced quenching and corrects for this, resulting in correct profiles with improved overall recovery as corroborated by comparison with online radioactivity detection and liquid scintillation counting, and can generically be applied postacquisition. The strategy was applied to all 36 fecal extracts from a human absorption, distribution, metabolism, and excretion study, where half of the samples present less than 20,000 disintegrations per min/mL, increasing the average column recovery (sum of individually quantified peaks relative to the total injected radioactivity) to >85%. SIGNIFICANCE STATEMENT: To improve interpretation in radiolabeled absorption, distribution, metabolism, and excretion studies, a matrix-based quench correction model is developed. It compensates for matrix-induced signal suppression when analyzing in vivo samples via offline radioactivity counting. It greatly improves data quality and enables accurate assessment of the true significance of detected metabolites.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a significant public health concern. Accumulating evidence suggests that long noncoding RNAs are dysregulated in MASLD. However, the roles and underlying mechanism of long noncoding RNAs in MASLD progression have not been fully elucidated. Here, we investigated the liver-specific functions of hepatocyte nuclear factor 4 α antisense 1 (HNF4A-AS1) and its mouse homolog, HNF4A opposite strand (Hnf4aos), in the pathogenesis of MASLD. HNF4A-AS1 and Hnf4aos were significantly upregulated in MASLD and diet-induced obese mice, respectively. Functionally, liver-specific knockdown of Hnf4aos reduced blood glucose levels and improved insulin sensitivity in the MASLD mouse model. Similarly, HNF4A-AS1 knockdown suppressed lipid droplet formation, intracellular triglyceride accumulation, and total cholesterol production in free fatty acid-induced HepG2 cells. Conversely, HNF4A-AS1 overexpression produced the opposite effects. Mechanistically, HNF4A-AS1 bound to the HNF4A protein and recruited heterogeneous nuclear ribonucleoprotein C (HNRNPC), thereby promoting HNF4A protein degradation. Taken together, our findings highlight the critical role of HNF4A-AS1 in MASLD progression and indicate that repressing HNF4A-AS1/HNF4A axis might be a potential therapeutic strategy for MASLD. SIGNIFICANCE STATEMENT: Long noncoding RNA HNF4A-AS1 and its mouse homolog, Hnf4aos, are upregulated in metabolic dysfunction-associated steatotic liver disease (MASLD) progression. Knockdown of HNF4A-AS1 or Hnf4aos alleviates MASLD progression in vitro or in vivo. HNF4A-AS1 interacts with HNF4A and promotes its protein degradation via HNRNPC, therefore aggravating MASLD progression.

The bioavailability of a drug is influenced by its physicochemical properties, dissolution rate, solubility, gastrointestinal tract permeability, and its absorption, distribution, and metabolism. Zileuton, a poorly soluble biopharmaceutical classification system class II drug prescribed for the treatment of asthma, was selected as a candidate for nanocrystal drug formulation. Our previous study demonstrated that zileuton nanocrystal formulation (NfZ) efficiently suppressed proinflammatory cytokines associated with asthma pathogenesis. Hence, this study aims to evaluate whether sex-dependent differences in the expression of genes related to drug metabolism and intestinal permeability influence the activity of the nanocrystal-formulated drug. In this study, we used the same animal samples as those reported in our earlier publications. To the best of our knowledge, this is the first study to conduct a comparative analysis of the effects of pure active pharmaceutical ingredient (API) and its nanocrystal formulation on metabolic enzyme genes and intestinal permeability-related genes. Our results demonstrated a significant increase in several genes involved in the metabolism of API, physical mixture (PM), and NfZ in males compared with females. PM and NfZ-treated animals showed increased expression of metabolic enzyme-related genes (phase I and phase II) and reduced alanine aminotransaminase activity at an equivalent dose compared with API-treated animals. However, genes related to oxidative metabolism and detoxification pathways were highly expressed in PM-treated animals; comparatively, NfZ treatment showed modest induction. Genes related to intestinal permeability were most significantly altered in API-treated animals compared to those dosed with NfZ. SIGNIFICANCE STATEMENT: The significance of this study is the sex-dependent difference in the expression of phase I and phase II metabolic enzymes and intestinal permeability-related genes upon treatment with different formulations of zileuton. Approximately 5% of asthmatic patients develop hepatocytotoxicity due to zileuton treatment and show several fold elevated levels of ALT in plasma. In the present study, differences in the decrease of ALT levels between the formulations highlight the advantages of the nanocrystal formulation.

Paracetamol is one of the most frequently used medications worldwide because of its analgesic and antipyretic properties. Despite its widespread use, its effects on metabolic changes in human plasma are unclear. This study aimed to assess the impact of a single therapeutic dose of paracetamol on the plasma metabolites of fasting participants. This cross-sectional study involved 28 age-matched fasting participants, comprising 14 controls and 14 individuals treated with paracetamol. Demographic analysis, clinical characteristics, and laboratory test results were evaluated. Blood samples were collected for metabolite extraction using a liquid chromatography mass spectrometry untargeted metabolomic approach, followed by a series of metabolomic analyses investigating alterations in metabolomic plasma profiles. There were significant metabolite differences between the control and paracetamol-treated groups. A total of 51 metabolites were significantly altered by paracetamol treatment, with 28 downregulated and 23 upregulated. Analysis of the differential metabolic pathways demonstrated metabolite enrichment in various pathways, including purine metabolism, nicotinamide metabolism, fatty acid biosynthesis, and the oxidation of branched-chain fatty acids. These findings enhance the understanding of the metabolic targets that may contribute to paracetamol's therapeutic effects and potential toxicity. SIGNIFICANT STATEMENT: Paracetamol is extensively used for its analgesic and antipyretic effects, but its impact on metabolic alterations in human plasma remains inadequately elucidated. In our study, we identified significant dysregulation of specific metabolites and disrupted biological pathways in fasting plasma samples following paracetamol administration. These findings provide valuable insights into the pharmacological efficacy of paracetamol while also highlighting potential toxicological implications.

Pregnane X receptor (PXR) is a key transcriptional regulator of drug-metabolizing enzymes and transporters, notably CYP3A4, which metabolizes a significant proportion of clinically used drugs. PXR activation can induce CYP3A4 expression, potentially leading to drug-drug interactions (DDIs) by altering the pharmacokinetics of CYP3A4 substrates, particularly for narrow therapeutic index drugs. Conventional induction assays rely on measuring CYP3A4 mRNA and enzyme activity, but mRNA levels often do not correlate with enzyme activity, which can lead to mispredictions of DDIs. To address this gap, we incorporated our newly established Fast and Surfactant-Treated proteomic workflow into the current in vitro induction assay to enable simultaneous quantification of CYP3A4 mRNA, protein, and enzyme activity induction from a single experiment. Using rifampicin as a PXR agonist, we demonstrated that the unified All-in-One assay provided consistent induction parameters with discrete assays, offering a robust method for assessing CYP3A4 induction. We also applied this approach to the tyrosine kinase inhibitors pazopanib and crizotinib, revealing nonuniformities in their induction profiles across mRNA, protein, and enzyme activity endpoints. Specifically, although both tyrosine kinase inhibitors induced CYP3A4 mRNA expression in a dose-dependent manner, they do not lead to protein induction, suggesting that the in vitro induction observed at the mRNA level may not translate to clinical induction. Collectively, these preliminary findings suggest that protein measurements may provide a more holistic representation of CYP3A4 induction and can potentially improve the predictability of clinical DDIs in drug development. SIGNIFICANCE STATEMENT: We described and validated a unified assay that can simultaneously measure CYP3A4 mRNA, protein, and enzyme activity induction from a single human hepatocyte experiment. This unified All-in-One approach has the potential to improve in vitro-in vivo correlation and translation of CYP3A4-mediated induction drug-drug interactions for new chemical entities. However, further work, including the integration of static or dynamic physiologically based pharmacokinetic modeling with protein induction data, will be required to fully confirm these insights.

NADPH cytochrome P450 reductase is the required redox partner for the majority of human cytochrome P450 enzymes, which are critically important for phase I drug metabolism of a wide variety of substrates. It is well understood that cytochrome P450 reductase supports P450 catalysis when its flavin mononucleotide (FMN)-containing domain (FMND) binds to the proximal side of P450 enzymes to deliver electrons to the P450 heme. Herein, we describe mass spectrometry-based footprinting approaches to compare the surface labeling of CYP2A6 and that of an artificial fusion protein composed of the reductase FMND linked to the N-terminus of CYP2A6 (FMND/CYP2A6). Three complementary footprinting approaches were used: hydrogen-deuterium exchange, benzoyl fluoride labeling, and fast photochemical oxidation of proteins (FPOP). Although the different labeling approaches target different amino acids and occur over varying reaction timescales, their outcomes generally agree. These experiments did not detect differential protection on the proximal P450 face where FMND is expected to bind. Instead, they consistently demonstrated increased exposure of CYP2A6 surface residues, indicative of structural changes in CYP2A6 in the presence of the FMND. Overall, the reduced protection is consistent with the FMN domain causing long-range allosteric modulation of the CYP2A6 structure. This structural evidence is consistent with increasing functional evidence that the reductase is an allosteric modulator of P450 enzymes in addition to its role in electron transfer. SIGNIFICANCE STATEMENT: Both established and new mass-spectrometry footprinting methods support structural changes in the CYP2A6 structure upon interaction with the FMN-containing domain of its reductase. This evidence supports the idea that the reductase is an allosteric modulator of P450 enzymes, in addition to its established role in electron transfer.

Accurately predicting human exposure to food-related compounds is crucial for evaluating their health effects without conducting animal testing. We previously reported an in vitro method using human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) for predicting human maximum plasma concentrations (C_max) of food-related compounds. However, the C_max predictivity of flavonoids and their glycosides is relatively low, potentially due to complex gastrointestinal absorption processes. This study aimed to assess whether hiSIECs cultured using with a modified culture method (mod-hiSIECs) can mimic human intestinal absorption and enhance C_max predictivity. Consistent with observations in the human intestine, the mod-hiSIECs formed a column-like morphology. The expression levels of key saccharide-related genes, such as sucrase-isomaltase, lactase, and cytosolic β-glucosidase, and disaccharidase and glycosidase activities similar to those of human primary enterocytes were obtained using mod-hiSIECs when compared with hiSIECs cultured using the conventional hiSIEC culture method and Caco-2 cells. In addition, the mod-hiSIECs accurately mimicked the intestinal glucosidase, glucuronidase, and sulfatase activities against daidzein, genistein, and their corresponding glucosides. The C_max predictability for flavonoid aglycones and their glucosides was significantly improved with mod-hiSIECs compared with conventional hiSIEC culture method, reducing the fold-difference from 2.3-9.0 to 0.46-5.5. The mod-hiSIECs could be a useful tool for predicting gastrointestinal absorption of flavonoids in humans. SIGNIFICANT STATEMENT: Human induced pluripotent stem cell-derived small intestinal epithelial cells via a modified method, exhibited gene expression and metabolic profiles akin to primary enterocytes, and their permeability data predicted human plasma concentrations of isoflavones and glucosides more accurately than Caco-2 cells or conventional human induced pluripotent stem cell-derived small intestinal epithelial cells.