Drug Metabolism and Disposition最新文献

{"title":"Lysosomal membrane localization of organic anion transporting polypeptide 2B1 in human hepatocytes.","authors":"Yeseul Ahn, Jacqueline B Tiley, Bhagwat Prasad, Kim L R Brouwer","doi":"10.1016/j.dmd.2026.100386","DOIUrl":"https://doi.org/10.1016/j.dmd.2026.100386","url":null,"abstract":"<p><p>Organic anion transporting polypeptide 2B1 (OATP2B1) is a basolateral plasma membrane uptake transporter expressed in hepatocytes. However, the cellular internalization and degradation pathways of OATP2B1 remain poorly characterized. Filling this knowledge gap is critical for improving predictions of OATP2B1 substrate disposition and OATP2B1-mediated drug interactions. This study investigated the localization and primary degradation pathway of OATP2B1 in human hepatocytes and examined whether OATP2B1 localization was affected by metabolic dysfunction-associated steatohepatitis in human liver tissue. Sandwich-cultured human hepatocytes (SCHHs) were treated with either 25 μM chloroquine (CQ) or 10 μM MG-132 to inhibit lysosomal or proteasomal degradation, respectively. Following treatment, SCHHs were fixed and stained for OATP2B1 and lysosome-associated membrane protein (LAMP) 1, followed by confocal imaging and subsequent object-based quantitative analysis. Lysosomal inhibition by CQ in SCHHs resulted in significant accumulation of intracellular OATP2B1, which displayed major colocalization with the lysosomal marker, LAMP1 (20% ± 9.38% vs 9.5% ± 5.25%; CQ vs control, respectively; P < .0001). In contrast, the OATP2B1 total object volume and intracellular localization remained unchanged after MG-132 treatment. OATP2B1 colocalization with LAMP1 was significantly higher in liver tissue from patients with metabolic dysfunction-associated steatohepatitis (10.47% ± 4.95%) compared with control liver tissue (6.00% ± 2.06%; P < .0001). Super-resolution imaging using stimulated emission depletion microscopy revealed that OATP2B1 was localized on lysosomal membranes. In conclusion, these findings demonstrate that OATP2B1 degrades primarily via lysosomes in human hepatocytes. Lysosomal dysfunction associated with some xenobiotics or disease may increase OATP2B1 localization on lysosomal membranes. SIGNIFICANCE STATEMENT: This study confirms that lysosomes are the predominant degradation pathway for the basolateral membrane protein organic anion transporting polypeptide 2B1 (OATP2B1) in human hepatocytes. Super-resolution imaging revealed that OATP2B1 is localized on lysosomal membranes. Both lysosomal inhibition by chloroquine in vitro and lysosomal dysfunction associated with metabolic dysfunction-associated steatohepatitis in human liver tissue increased OATP2B1 colocalization with the lysosomal membrane marker.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"54 10","pages":"100386"},"PeriodicalIF":3.6,"publicationDate":"2026-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149015891","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteomic characterization of human kinases across tissue types: Implications for kinase-activated drugs.","authors":"Griffin D Shaffer, Dilip Kumar Singh, Sandhya Subash, Bhagwat Prasad","doi":"10.1016/j.dmd.2026.100385","DOIUrl":"https://doi.org/10.1016/j.dmd.2026.100385","url":null,"abstract":"<p><p>Kinases catalyze the activation of certain nucleoside and nucleotide analog drugs used in the treatment of various cancers and viral infections. Because these drugs require intracellular phosphorylation to their active form, the abundance of kinases within tissues is a key factor in determining both variability in response and off-target effects. In this study, we quantified and compared protein abundances of kinases in commercially available pooled S9 fractions of adult human liver (n = 50), kidney (n = 12), lung (n = 5), intestine (n = 4), and individual adult primary hepatocytes (n = 8). These data were compared with the proteomic abundance profile of red blood cells (n = 4) from healthy donors and bone marrow samples (n = 44) from patients with acute myeloid leukemia, in addition to kinase abundance in human plasma from the publicly available data base Human Protein Atlas. Kinase protein abundance was estimated using the total protein approach in pmol/mg total protein. We detected 364 kinases, including 23 drug-metabolizing kinases, with varying abundances across tissue and biofluid types. Four drug-metabolizing kinases, adenosine kinase, cytidine/uridine monophosphate kinase 1, and nucleoside diphosphate kinases 1 and 3, were detected in all sample types. In addition, by comparing tissue-to-plasma abundance ratios of kinases with those of known secreted versus nonsecreted marker proteins, we showed that kinases are primarily present in plasma due to tissue shedding, with few (phosphoglycerate kinase 1, pyruvate kinase M, adenylate kinase 1, and nucleoside diphosphate kinase 1) that may be present as part of extracellular vesicles. Lastly, we identified the presence of 24 kinases commonly targeted by inhibitors across sample types. Overall, this study advances the understanding of the presence and variability of kinases in different tissues. SIGNIFICANCE STATEMENT: This study quantified and compared the presence and abundance of kinases, including drug-metabolizing kinases, across various human sample types. In total, 364 kinases, including 23 known drug-metabolizing kinases, were detected with varying abundances across tissue and biofluid types. This study provides important quantitative data about drug-metabolizing kinases that can be used to understand variability in response and the off-target effects of nucleoside analog drugs.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"54 10","pages":"100385"},"PeriodicalIF":3.6,"publicationDate":"2026-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148943854","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Systemic inflammation reprograms human hepatic phenotype and suppresses absorption, distribution, metabolism, and excretion gene expression: Validation in a 3-dimensional human liver spheroid system.","authors":"Inger Johansson, Allan Zhao, Lili Milani, Qiaolin Deng, Magnus Ingelman-Sundberg, Sander van Riet","doi":"10.1016/j.dmd.2026.100384","DOIUrl":"https://doi.org/10.1016/j.dmd.2026.100384","url":null,"abstract":"<p><p>Inflammation profoundly alters hepatic drug metabolism and transport, requiring careful consideration in pharmacotherapy to minimize adverse outcomes. In this study, we compared global transcriptomic profiles of macroscopically and histologically normal liver tissue from patients undergoing tumor metastasis resection (n = 37) and critically ill patients requiring intensive care (n = 50), alongside an advanced 3-dimensional primary human liver spheroid model. Transcriptomic analysis revealed clear segregation between cohorts, identifying 6250 differentially expressed genes and a pronounced inflammatory signature in livers from critically ill patients, marked by elevated acute-phase proteins, including C-reactive protein, serum amyloid A1, and serum amyloid A2. Three-dimensional primary human liver spheroids were analyzed with increasing cellular complexity: hepatocyte monocultures, hepatocyte-nonparenchymal cell cocultures, and triple cultures incorporating liver sinusoidal endothelial cells, with or without free fatty acid supplementation to mimic metabolic stress. Incorporation of nonparenchymal cells and liver sinusoidal endothelial cells markedly upregulated C-reactive protein expression and induced transcriptomic shifts closely mirroring inflamed human liver tissue. Pathway enrichment and targeted analysis of absorption, distribution, metabolism, and excretion genes demonstrated consistent inflammation-driven downregulation of cytochrome P450 enzymes, phase II enzymes, transporters, and nuclear receptors critical for drug metabolism and biliary efflux-patterns nearly identical to those observed between inflamed liver tissue and tissue obtained by tumor resection. These findings highlight substantial phenotype changes in macroscopically and histologically normal livers and validate advanced multicellular 3-dimensional human liver spheroids as physiologically relevant models for recapitulating inflammation-induced perturbations in hepatic pharmacokinetics, offering predictive tools for improved preclinical safety assessment and personalized dosing in patients with systemic inflammation. SIGNIFICANCE STATEMENT: Inflammation profoundly alters hepatic drug metabolism and transport, requiring careful consideration in pharmacotherapy to minimize adverse outcomes. This study highlights substantial phenotype changes caused by inflammation in macroscopically and histologically normal livers and validates advanced multicellular 3-dimensional human liver spheroids as physiologically relevant models for recapitulating inflammation-induced perturbations in hepatic pharmacokinetics.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"54 10","pages":"100384"},"PeriodicalIF":3.6,"publicationDate":"2026-08-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149001500","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A framework for optimized reaction phenotyping of the seven major hepatic P450 isoforms in human liver microsomes using six mechanism-based inactivators and one reversible inhibitor.","authors":"Ting Wang, Kevin Jacob, Klairynne Raymond, Mitchell E Taub","doi":"10.1016/j.dmd.2026.100349","DOIUrl":"10.1016/j.dmd.2026.100349","url":null,"abstract":"<p><p>Cytochrome P450 (CYP) enzymes play a central role in drug metabolism. Understanding the contribution of individual CYP isoforms, known as CYP reaction phenotyping, is essential for elucidating metabolic pathways and predicting drug-drug interactions. Reversible inhibitors are commonly used in human liver microsome (HLM)-based CYP reaction phenotyping. However, selecting appropriate inhibitor concentrations is challenging as experimental results will vary depending on the HLM concentration, substrate concentration, and incubation duration selected. These factors collectively influence the observed inhibitory potency, often complicating data interpretation. The use of mechanism-based inactivators (MBIs) of P450 enzyme can potentially overcome these issues. However, the lack of standardized rigorously evaluated protocols and concerns about off-target inhibition have limited the broader application of MBIs for reaction phenotyping. This study systematically evaluated assay conditions for the use of P450 isoform-selective MBIs across the 7 major hepatic P450 isoforms (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) to establish an HLM concentration-independent, universal protocol for CYP reaction phenotyping. Inactivation kinetics were determined, and potential off-target P450 inhibition of each MBI across all 7 P450 isoforms was assessed. Optimized assay conditions were established for each P450 isoform-MBI pair, including HLM concentration, MBI concentration, and incubation time. Under these optimized assay conditions, all selected MBI demonstrated potent and selective inhibition of their target P450 isoforms, demonstrating ≥90% activity loss and minimal off-target inhibition for most MBIs (none >16%). Overall, the established protocol provides a robust and reproducible framework for HLM-based P450 reaction phenotyping using MBIs. This approach enhances the specificity of P450 isoform contribution assessments and supports improved drug-drug interaction prediction in early drug development. SIGNIFICANT STATEMENT: Mechanism-based inactivators offer a powerful approach for defining individual P450 isoform contributions. This study systematically optimized mechanism-based inactivator assays across all major hepatic P450 isoforms in human liver microsomes (HLMs), establishing a unified protocol that balances inactivator concentration, HLM level, and incubation duration to achieve ≥90% target inhibition with minimal off-target effects. The resulting framework improves assay specificity, enhances reliability of P450 reaction phenotyping in HLM, and supports more accurate prediction of metabolic pathways and drug-drug interaction risk.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"54 8","pages":"100349"},"PeriodicalIF":3.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148600383","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nucleolar and coiled-body phosphoprotein 1 enhances the induction of CYP3A4 by promoting nuclear translocation of pregnane X receptor.","authors":"Kyoka Nagaoka, Masataka Nakano, Kiamu Kurosawa, Yuichiro Higuchi, Shotaro Uehara, Nao Yoneda, Hiroshi Suemizu, Tatsuki Fukami, Miki Nakajima","doi":"10.1016/j.dmd.2026.100350","DOIUrl":"10.1016/j.dmd.2026.100350","url":null,"abstract":"<p><p>Pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor that plays a central role in the regulation of drug-metabolizing enzymes and transporters. Under basal conditions, PXR is predominantly localized in the cytoplasm. Upon activation by ligands such as rifampicin, PXR translocates into the nucleus and induces the transcription of its target genes, including CYP3A4, a major drug-metabolizing enzyme in the human liver. From our previous proteomic analysis of coimmunoprecipitated proteins obtained from lysates of rifampicin-treated ShP51 cells (a HepG2 cell line stably expressing human PXR) using an anti-PXR antibody, we identified nucleolar and coiled-body phosphoprotein 1 (NOLC1), a protein known to shuttle among the cytoplasm, nucleus, and nucleolus, as a potential PXR-interacting factor. This study aimed to clarify the role of NOLC1 in PXR nuclear translocation. Knockdown of NOLC1 in ShP51 cells or HepaSH cells (primary hepatocytes from humanized-liver mice) attenuated the rifampicin-induced expression and activity of CYP3A4, suggesting that NOLC1 positively regulates PXR-mediated CYP3A4 induction. Rifampicin significantly increased luciferase activity driven by the promoter and distal enhancer regions of the CYP3A4 gene; however, this induction was diminished by NOLC1 knockdown in ShP51 cells. Furthermore, immunofluorescence staining of HepaSH cells revealed that rifampicin promoted PXR nuclear translocation, whereas this effect was suppressed by NOLC1 knockdown. Taken together, these findings demonstrate that NOLC1 facilitates the ligand-dependent nuclear translocation and transcriptional activity of PXR, thereby functioning as a regulatory factor for drug metabolism in the human liver. SIGNIFICANCE STATEMENT: This study demonstrates that nucleolar and coiled-body phosphoprotein 1 promotes ligand-dependent pregnane X receptor (PXR) nuclear translocation, enhances PXR recruitment to chromatin, and increases PXR-mediated transactivation, thereby enhancing the induction of CYP3A4 expression. These findings provide mechanistic insight into potential strategies for mitigating PXR-mediated adverse effects.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"54 8","pages":"100350"},"PeriodicalIF":3.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148618684","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modeling cortisol and cortisone binding to corticosteroid-binding globulin and albumin based on clinical protein binding data.","authors":"Aurora K Authement, Abhinav Nath, Katya B Rubinow, John K Amory, Nina Isoherranen","doi":"10.1016/j.dmd.2026.100362","DOIUrl":"10.1016/j.dmd.2026.100362","url":null,"abstract":"<p><p>Cortisol is a major endogenous glucocorticoid that regulates numerous physiological processes. In plasma, cortisol and its inactive metabolite cortisone bind to corticosteroid-binding globulin (CBG) and albumin, leaving only the unbound fraction available for receptor activation and metabolism. Changes in ligand or protein concentrations alter unbound fractions. Existing binding equations are difficult to extend to multiligand, multiprotein systems and do not readily capture competitive endogenous binding interactions. The goal of this study was to develop a plasma protein binding model that quantitatively describes binding species and predicts unbound concentrations across physiological states. Total and unbound cortisol and cortisone, CBG, and albumin were measured in plasma from healthy premenopausal women (n = 13) at baseline and after 7 days of 30 mg hydrocortisone treatment. Reversible 1:1 binding models were implemented in COPASI and MATLAB/Simulink, and dissociation constants (K_d) were estimated by fitting equilibrium binding models to observed unbound concentrations. A model describing simultaneous binding of cortisol and cortisone to CBG and albumin yielded in vivo K_d values for cortisol:CBG, cortisone:CBG, cortisol:albumin, and cortisone:albumin of 0.0130 μM, 0.169 μM, 172 μM, and 519 μM, respectively. Model predictions agreed with observed unbound cortisol and cortisone concentrations, and bootstrap resampling confirmed stable K_d estimates. This work provides a quantitative framework for predicting unbound cortisol and cortisone concentrations across physiological and disease states by accounting for changes in ligand and protein concentrations. This enables extrapolation to conditions such as pregnancy, adrenal insufficiency, and liver disease, informing interpretation of altered cortisol concentrations in these populations. SIGNIFICANCE STATEMENT: This work establishes a framework to predict in vivo cortisol and cortisone binding. The developed model was applied to predict unbound cortisol and cortisone concentrations in physiological and pathophysiological states and can be integrated into pharmacokinetic models. Our analysis demonstrates that cortisol and cortisone binding affinities estimated in the native plasma environment differ from those measured using purified proteins. These differences have important implications for predicting and analyzing unbound cortisol concentrations.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"54 8","pages":"100362"},"PeriodicalIF":3.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148653306","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CYP1A2 and ADH1C contribute to abacavir aldehyde intermediate formation in the human liver.","authors":"Rei Sato, Masataka Nakano, Yuichiro Higuchi, Shotaro Uehara, Nao Yoneda, Hiroshi Suemizu, Miki Nakajima, Tatsuki Fukami","doi":"10.1016/j.dmd.2026.100348","DOIUrl":"10.1016/j.dmd.2026.100348","url":null,"abstract":"<p><p>Abacavir, an anti-human immunodeficiency virus agent, is metabolized to the pharmacologically inactive abacavir carboxylate via an aldehyde intermediate, abacavir aldehyde (abacavir-CHO). Because aldehyde formation is generally rate-limiting, the enzymes responsible for abacavir-CHO formation may significantly influence drug efficacy and toxicity. In this study, we aimed to identify the enzymes involved in abacavir-CHO formation in the human liver. Using 5 recombinant human NAD⁺-dependent alcohol dehydrogenase (ADH) isoforms, which are well known to catalyze aldehyde formation from alcohols, ADH1C was identified as the primary catalyst of abacavir-CHO formation. In human liver S9 fractions, abacavir-CHO formation was observed not only in the presence of NAD⁺ but also in the presence of NADPH, with intrinsic clearance comparable between the 2 conditions. Notably, NADPH-dependent activity was higher in human liver microsomes than in cytosolic fractions. This activity was markedly inhibited by 1-aminobenzotriazole, a general cytochrome P450 (P450) inhibitor, indicating the involvement of P450 enzymes. Among recombinant human P450 isoforms, CYP1A2 exhibited the highest catalytic activity. Quantitative analysis using the relative activity factor approach revealed that CYP1A2 accounted for 89.8% of abacavir-CHO formation under NADPH-dependent conditions in human liver microsomes. In HepaSH cells, hepatocytes from chimeric mice with humanized livers, abacavir carboxylate formation was decreased by 50% by α-naphthoflavone, a CYP1A2 inhibitor, and increased approximately 7-fold by omeprazole, a CYP1A2 inducer. Collectively, these findings demonstrate that CYP1A2 plays a significant role in abacavir-CHO formation in the human liver, comparable to ADH1C, and may have implications for variability in abacavir response. SIGNIFICANCE STATEMENT: This study demonstrates that CYP1A2, in addition to ADH1C, significantly contributes to the formation of the reactive aldehyde metabolite abacavir-CHO in the human liver. This finding suggests a need to reconsider the conventional view that alcohol dehydrogenases are the primary enzymes responsible for this pathway and indicates that CYP1A2-mediated metabolism may influence interindividual variability, drug-drug interactions, and the risk of abacavir-associated adverse reactions.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"54 8","pages":"100348"},"PeriodicalIF":3.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148411033","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Diagnostic exogenous probe strategy using ezetimibe-glucuronide effectively identifies metabolic dysfunction-associated steatohepatitis.","authors":"Dominique O Farrera, Kayla L Frost, Joseph L Jilek, Paxton A Sample, Jailyn M Izu, Mina M Alaaldin, Paige Lindberg, Max M Maloney, David Rodrigues, Emi Yamaguchi, Sangwoo Ryu, Yi-An Bi, Mark West, Sumathy Mathialagan, Sarah Lazzaro, Brooke Rabe, Aiden-Hung P Nguyen, Katherine Ho, Susie Messenger, Ahmad Omid Rahimi, Ryan Akhbari, Carolina Säll, Walter Klimecki, Mary F Paine, Stephen H Wright, Dean Billheimer, Geoffrey Block, Iman Ghaderi, Nathan J Cherrington","doi":"10.1016/j.dmd.2026.100351","DOIUrl":"10.1016/j.dmd.2026.100351","url":null,"abstract":"<p><p>Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease with an urgent need for reliable, noninvasive biomarkers that can serve as reasonably likely surrogate endpoints, as diagnosis remains limited by the invasiveness of liver biopsy and the inaccuracy of existing noninvasive tests. MASH disrupts hepatic and renal transporter expression and function, leading to increased systemic retention of drug substrates. Because ezetimibe-glucuronide (EZEG) is a substrate of transporters altered in MASH, we evaluated whether EZEG could serve as a noninvasive, mechanism-based exogenous diagnostic function test. Plasma and urine EZEG concentrations were quantified by liquid chromatography-tandem mass spectrometry in biopsy-confirmed patients with nonalcoholic fatty liver disease activity scores (NAS) of 0-5 after administration of a 1 mg subtherapeutic dose of ezetimibe. Patients with MASH (NAS = 5, n = 8) exhibited approximately 4.5-fold higher plasma EZEG concentrations than non-MASH individuals (NAS 0-4, n = 45), demonstrating excellent discrimination with an area under the receiver operating characteristic curve of 0.92 (95% CI, 0.79-1.00) using the 60-minute postdose concentration. Plasma EZEG was strongly associated with hepatocyte ballooning, the U.S. Food and Drug Administration-defining and most difficult-to-detect histologic feature of MASH, supporting mechanistic specificity. In contrast, urinary EZEG excretion showed only modest increases and poor diagnostic discrimination. These findings indicate that MASH-associated transporter dysfunction leads to systemic EZEG accumulation, supporting its use as a mechanism-based diagnostic function test and a promising noninvasive strategy for early detection and disease stratification. SIGNIFICANCE STATEMENT: Metabolic dysfunction-associated steatohepatitis (MASH) lacks reliable noninvasive tests for diagnosis and therapeutic development. This study demonstrates that plasma ezetimibe-glucuronide, measured after a subtherapeutic ezetimibe dose, sensitively detects MASH-associated transporter dysfunction and strongly correlates with hepatocyte ballooning, the defining histologic feature of MASH. This mechanism-based probe-drug approach provides a highly discriminative, noninvasive strategy to support early disease detection and patient stratification.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"54 8","pages":"100351"},"PeriodicalIF":3.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148629697","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PSMD2 mediates the degradation of CYP1A2 and promotes the progression of metabolic dysfunction-associated steatohepatitis.","authors":"Min Liu, Tianhao Liu, Dongsheng Liu, Jiaying He, Qin Shao, Jiye Aa, Guangji Wang, Yuan Xie","doi":"10.1016/j.dmd.2026.100363","DOIUrl":"10.1016/j.dmd.2026.100363","url":null,"abstract":"<p><p>Despite the well recognized downregulation of cytochrome P450 enzymes in metabolic dysfunction-associated steatohepatitis (MASH), the posttranscriptional mechanisms underlying this phenomenon are still not well understood. This study investigates the role of proteasomal degradation in the regulation of CYP1A2, a major hepatic P450 enzyme, during MASH progression. Using a high-fat diet-induced MASH mouse model and palmitic acid-treated hepatocytes model, we found that CYP1A2 protein levels were significantly decreased without corresponding changes in mRNA expression, indicating predominant posttranslational modification regulation. Mechanistically, we first identified 26S proteasome non-ATPase regulatory subunit 2 (PSMD2) as a novel and crucial regulatory factor that dominates the posttranslational degradation of CYP1A2 during MASH development, and confirmed it functions as a key interacting protein mediating the accelerated proteasomal degradation of CYP1A2. Inhibition of proteasome activity with bortezomib or genetic silencing of PSMD2 effectively blocked CYP1A2 degradation and ameliorated hepatocyte lipid accumulation. Importantly, hepatocyte-specific knockdown of PSMD2 in vivo restored CYP1A2 expression and significantly alleviated MASH-associated pathologies, including hepatic steatosis, inflammation, and liver injury. Collectively, these findings establish PSMD2 as a novel and essential modulator that governs the posttranslational degradation of CYP1A2. Moreover, PSMD2 plays a critical role in the progression of MASH, highlighting its potential as a therapeutic target for this disease. SIGNIFICANCE STATEMENT: This study provides the first evidence that hepatocyte proteasome non-ATPase regulatory subunit 2 (PSMD2) drives metabolic dysfunction-associated steatohepatitis pathology by promoting CYP1A2 degradation and lipid accumulation. PSMD2 deficiency alleviates hepatic steatosis, reduces triglyceride and free fatty acid levels, and restores liver function. These findings establish PSMD2 as a critical regulator of lipid metabolism and hepatocyte integrity, positioning it as a promising therapeutic target for metabolic dysfunction-associated steatohepatitis intervention.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"54 8","pages":"100363"},"PeriodicalIF":3.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148648509","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"3D hepatic spheroid arrays for clearance assessment of small and bRo5 molecules.","authors":"Viraj Mehta, Rupa Yellanki, Parusharamulu Molgara, Venkatesham Uthukam, Prabhakar Reddy Kolli, Tarun Sharma, Vamsi Madgula, Guruswamy Karnam","doi":"10.1016/j.dmd.2026.100365","DOIUrl":"10.1016/j.dmd.2026.100365","url":null,"abstract":"<p><p>Accurately predicting hepatic clearance remains a critical hurdle in preclinical pharmacokinetics, especially for metabolically stable compounds and emerging modalities such as proteolysis targeting chimeras (PROTACs) that fall in beyond-rule-of-five chemical space. In this study, we developed and evaluated a 3-dimensional hepatic spheroid-array system that improves clearance prediction for both small molecules and PROTACs. Using a panel of 15 small molecules and 9 PROTACs, we compared in vitro clearance predictions from the spheroid-array model with conventional suspension hepatocytes. The spheroid-array predicted human hepatic clearance within a 2-fold range for 9 of 15 and within a 3-fold range for 12 of 15 small molecules. The average absolute fold error was lower for the spheroid system than the suspension system (1.74 vs 3.52). Notably, unlike the suspension system, the spheroid-array quantified clearance for several low-clearance compounds such as warfarin and theophylline. Regression correction improved prediction accuracy for both, with a significant improvement for the suspension model. For several PROTACs, metabolic turnover could not be measured in the suspension system, highlighting its limited suitability for highly stable compounds. In contrast, the spheroid system demonstrated significant metabolic activity, enabling characterization of hepatic clearance for these otherwise stable PROTACs. Together, these findings establish the spheroid-array system as a sensitive and physiologically relevant tool for preclinical clearance assessment across traditional and emerging therapeutic modalities. SIGNIFICANCE STATEMENT: We developed a 3-dimensional hepatic spheroid-array that shows excellent viability and functional activity for 3 weeks. The system showed better hepatic clearance predictivity with a lower average absolute fold error than the suspension system. Our work also first time validates the spheroid-array system for beyond-rule-of-five molecules showing the possibility for evaluating clearance of proteolysis targeting chimeras. This work establishes spheroid as a reliable tool for preclinical hepatic clearance assessment.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"54 8","pages":"100365"},"PeriodicalIF":3.6,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148683918","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}