Drug Metabolism and Disposition最新文献

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.

Vicadrostat, an aldosterone synthase inhibitor in development in combination with empagliflozin for chronic kidney disease, heart failure, and cardiovascular risk reduction, undergoes extensive hepatic glucuronidation primarily by UDP-glucuronosyltransferase (UGT)2B7 to form BI 689875, an ether glucuronide metabolite. Despite its hepatic formation, BI 689875 is predominantly excreted in urine, as determined in a human ADME study of vicadrostat. This study elucidated mechanisms underlying BI 689875 disposition in humans. BI 689875 was evaluated as a substrate of various drug transporters using transporter-expressing membrane vesicles and HEK293 cells. BI 689875 was identified as a substrate of MRP2, MRP3, MRP4, BCRP, OAT3, OATP1B1, and OATP1B3, but not of P-gp, OAT1, OAT2, OAT4, MATE1, or MATE2-K. The affinity of BI 689875 for MRP3 (K_m = 39 μM) and OAT3 (K_m = 46 μM) was substantially greater than that for other uptake/efflux transporters (not saturable up to 300 μM). In vitro-in vivo extrapolation using a proteomics-informed approach correcting for in vitro versus in vivo transporter expressions revealed that MRP3- and OAT3-mediated intrinsic clearance values for BI 689875 were substantially higher than those of other transporters. These findings suggest that basolateral efflux via MRP3 is the dominant hepatic elimination pathway for BI 689875, explaining its minimal fecal excretion observed in the human ADME study. They also indicate that OAT3-mediated uptake is the primary renal elimination route, with renal basolateral uptake substantially higher than hepatic uptake, consistent with the preferential urinary elimination of BI 689875. Transporter interplay between hepatic MRP3 and renal OAT3 determines the primary route of BI 689875 disposition. SIGNIFICANCE STATEMENT: BI 689875, a glucuronide metabolite, is formed in the liver but eliminated in urine. Through proteomics-informed in vitro-in vivo extrapolation, hepatic MRP3 and renal OAT3 were identified as key contributors to its predominant urinary elimination, highlighting interorgan transporter interplay.

Proteolysis targeting chimeras (PROTACs), a class of targeted protein degraders, are advancing in clinical development, necessitating the accurate prediction of human pharmacokinetics (PK). This study developed a physiologically based pharmacokinetic (PBPK) modeling approach informed by in vitro to in vivo extrapolation to predict the human PK of 2 PROTACs: vepdegestrant (ARV-471) and bavdegalutamide (ARV-110). Bottom-up PBPK models were built in mouse (ARV-471), and in mouse, rat, and dog (ARV-110) using physicochemical and in vitro absorption, distribution, metabolism, and excretion data, including solubility, permeability from a modified Genentech Madin-Darby canine kidney cells assay with 4% bovine serum albumin, and liver microsomal intrinsic clearance (CL). In vitro to in vivo extrapolation gaps were identified and addressed using empirical scalars, including additional systemic CL and tissue partition coefficient scalars, to capture observed intravenous PK. Oral absorption and exposure in preclinical species were predicted using a mechanistic absorption model, assuming passive diffusion driven by total drug concentration. Based on the preclinical PBPK strategy, predicted human apparent CL after oral administration and apparent volume of distribution after oral dosing values for ARV-110 at 35 mg aligned within 2-fold of clinical observations. For ARV-471 at 30 mg oral dose, apparent volume of distribution after oral dosing predictions were within range, but apparent CL after oral administration was overpredicted. To improve alignment with the observed clinical PK, model refinement was limited to adjusting the additional systemic CL scalar, whereas absorption and distribution parameters remained unchanged. The refined PBPK models successfully simulated human oral PK within 2-fold of observed values across multiple doses (60-360 mg for ARV-471 and 70-140 mg for ARV-110). This PBPK modeling framework may support human PK prediction of PROTACs during late-stage drug discovery and development. SIGNIFICANCE STATEMENT: This study highlights that a physiologically based pharmacokinetic (PK)-in vitro to in vivo extrapolation strategy can reliably predict the human PK of proteolysis targeting chimeras, an emerging therapeutic class with complex absorption, distribution, metabolism, and excretion properties. Incorporating mechanistic absorption modeling and permeability data from modified in vitro assays (Genentech Madin-Darby canine kidney cells with 4% bovine serum albumin) improved oral absorption predictions, whereas the integration of multispecies preclinical PK data enhanced the translational accuracy of human PK predictions. Together, these findings establish a translational physiologically based PK framework for estimating oral exposure in first-in-human studies and supporting model-informed development of proteolysis targeting chimeras drug candidates.

The severe acute respiratory syndrome coronavirus-2 main protease inhibitor PF-07321332 (nirmatrelvir), in combination with ritonavir (Paxlovid), has been approved by the US Food and Drug Administration as an oral treatment option for coronavirus disease 2019 patients. In this perspective, we share the expediated absorption, distribution, metabolism, and excretion strategies, which were incorporated as part of discovery efforts, to design orally active severe acute respiratory syndrome coronavirus-2 main protease inhibitors. PF-07321332 (nirmatrelvir) emerged as a potential oral clinical candidate within ∼ 6 months from the time discovery efforts were first initiated. The review also delves into a discussion around the successful use of quantitative fluorine-19 nuclear magnetic resonance spectroscopy in the characterization of the human mass balance and excretion pathways of nirmatrelvir. Human absorption, distribution, metabolism, and excretion data that emerged from the fluorine-19 nuclear magnetic resonance study were used to support the Emergency Use Authorization and new drug application filing, which was accepted by regulatory agencies worldwide. Efficient operational and technical strategies, incorporating the elements of speed without sacrificing data quality, which were crucial to the success of the program, are highlighted. SIGNIFICANCE STATEMENT: This perspective discusses the expedited absorption, distribution, metabolism, and excretion efforts utilized in the discovery and development of the orally active severe acute respiratory syndrome coronavirus-2 main protease inhibitor nirmatrelvir, which in combination with the cytochrome P450 3A inhibitor ritonavir (Paxlovid), is used in the oral treatment of COVID-19. Paxlovid was granted an Emergency Use Authorization by global regulatory agencies in less than 2 years from the initiation of the discovery program and has since been fully approved by the US Food and Drug Administration.

The pregnane X receptor (PXR), a ligand-activated transcription factor, regulates the expression of genes involved in endobiotic and xenobiotic metabolism, inflammation, and fibrosis. Disruption of PXR functions can affect processes critical to metabolic dysfunction-associated steatohepatitis (MASH) progression. Although ligand-dependent PXR functions are well studied, its regulation by post-translational modification, particularly phosphorylation, remains unclear. PXR has a conserved phosphorylation motif within its ligand binding domain (Ser347 in mice; Ser350 in humans). In vitro studies showed that this site mutation impairs human PXR transcriptional activity; however, the mechanism remains elusive. To investigate this phosphorylation site role in MASH development, wild-type and PXR Ser347Ala knock-in mutation (PXR-KI) mice were fed either a high-fat diet or a control chow diet for 16 weeks. On control chow diet, PXR-KI mice exhibited decreased expression of alternative bile acid (BA) synthesis genes compared with wild-type mice. On a high-fat diet, PXR-KI mice manifested more severe hepatic steatosis, revealed by elevated serum total cholesterol, and increased expression of genes involved in lipid metabolism. In addition, changes in BA metabolism and transporter genes suggested a cholestatic pattern in this group of mice. BA profiling showed higher levels of conjugated, hydrophilic, primary BA in the serum and liver, and increased unconjugated BA in the intestine. The data suggest that PXR Ser347 phosphorylation motif is essential for regulating PXR functions to maintain endobiotic metabolism and alleviate hepatotoxicity during MASH progression. SIGNIFICANT STATEMENT: The ligand-independent role of pregnane X receptor (PXR) is unclear. In phosphodeficient PXR knock-in mice, loss of Ser347 phosphorylation worsened hepatic steatosis and altered bile acid homeostasis under high-fat diet feeding, uncovering a novel role and therapeutic potential of PXR phosphorylation in fatty liver diseases.

In our previous study, long-term cocultured hepatocytes were used to estimate the fraction of a drug metabolized by CYP3A4 (f_m,CYP3A4). Metabolic turnover was measured with and without a CYP3A4 selective inhibitor, and the results were verified against in vivo reference data. The current study followed a similar approach using direct or time-dependent inhibitors to evaluate f_m,CYP1A2, f_m,CYP2C8, f_m,CYP2C9, f_m,CYP2C19, and f_m,CYP2D6 for a set of marketed drugs. The used inhibitors were for CYP1A2 (20 μM furafylline), CYP2C8 (40 μM montelukast), CYP2C9 (40 μM sulfaphenazole), CYP2C19 (3 μM (-)N-3-benzyl-phenobarbital), and CYP2D6 (5 μM quinidine). We found that in vitro f_m values above 0.5 were comparable to in vivo values, falling within a 0.5 to 2-fold error in 9 of 11 CYP1A2 substrates, 5 of 8 CYP2C8 substrates, 5 of 8 CYP2C9 substrates, 2 of 3 CYP2C19 substrates, and 11 of 20 CYP2D6 substrates. The study also showed how uncertainty in measured metabolic turnover affects the estimated f_m,CYPs, revealing that when estimated f_m errors are <25%, 89% of predictions are within 2-fold of in vivo f_m, but this drops to 40% when there is higher uncertainty in measured turnover. Although some f_m values were poorly predicted and clinical studies revealed off-target inhibition by certain inhibitors, the chemical inhibition approach using human long-term cocultured hepatocytes showed useful prediction performance for early drug discovery enabling moderate-to-sensitive drug-drug interaction risk assessments, when metabolic turnover is adequate, and inhibitor selectivity is well defined. SIGNIFICANCE STATEMENT: Calculating in vitro fraction metabolized by cytochrome P450 enzymes in liver is vital in drug discovery for assessing the object drug-drug interaction risk of new chemical entities metabolized by cytochrome P450 enzymes before clinical data are available. Despite some limitations, the current study demonstrated that using long-term cocultured hepatocytes with chemical inhibitors is a reliable method for estimating fraction metabolized by cytochrome P450 enzymes in liver, complementing the drug interaction risk assessment.

Organic cation transporter 1 (OCT1, SLC22A1) is a key determinant in the hepatic disposition of cationic drugs, primarily supported by pharmacogenomic studies. However, evidence for OCT1-mediated drug-drug interactions (DDIs) remains limited. This study aimed to elucidate the role of OCT1 in DDIs using cynomolgus monkeys through comprehensive in vitro and in vivo experiments. Cynomolgus monkey OCT1 (cOCT1) shares 94.2% amino acid identity with human OCT1 (hOCT1). Transport assays in transfected human embryonic kidney 293 cells showed that sumatriptan, fenoterol, metformin, quinidine, and 1-methyl-4-phenylpyridinium were transported by cOCT1 at rates comparable to hOCT1 (less than 2-fold difference). The K_m and V_max values for cOCT1-mediated transport of sumatriptan and fenoterol were similar or within 2-fold to those of hOCT1 (K_m: 188 ± 56 vs 178 ± 25 and 1.6 ± 0.48 vs 0.73 ± 0.47 μM, respectively, V_max: 49.4 ± 8.3 vs 83.9 ± 5.2 and 124 ± 8.9 vs 158 ± 22 pmol/min per mg, respectively). Inhibition studies demonstrated that quinidine, rifamycin SV, and ketoprofen inhibited sumatriptan uptake in monkey hepatocytes to a similar extent as in human hepatocytes, with IC₅₀ values within a 2- to 3-fold range. In addition, axitinib, nintedanib, and erlotinib were identified as inhibitors of both cOCT1 and hOCT1. In vivo, coadministration of axitinib (15 mg/kg), nintedanib (40 mg/kg), and erlotinib (15 mg/kg) increased sumatriptan area under the plasma concentration-time curve from zero to 24 hours by 1.3, 2.0, and 1.9-fold, respectively, compared with sumatriptan alone (2 mg/kg). These findings underscore the crucial role of OCT1 in the hepatic disposition and DDIs of cationic drugs, and indicate that cynomolgus monkeys may serve as a valuable model for studying OCT1-mediated drug disposition and interactions. SIGNIFICANCE STATEMENT: This study provides the first evidence that cynomolgus monkey organic cation transporter 1 (OCT1) transport and inhibition characteristics closely align with its human ortholog. Consistent with our in vitro findings, coadministration of OCT1 inhibitors (axitinib, nintedanib, and erlotinib) significantly increased the systemic exposure of sumatriptan in monkeys. These findings offer valuable insights into the role of OCT1 in drug-drug interactions and highlight the potential of cynomolgus monkeys as a useful and potentially translational model for OCT1-mediated disposition and interactions.