Drug Metabolism and Disposition最新文献

{"title":"Alterations of valsartan pharmacokinetics in a rodent model of metabolic dysfunction-associated steatohepatitis.","authors":"Dominique O Farrera, Mina M Alaaldin, Paige Lindberg, Paxton A Sample, Paige Lenzen-Hammerel, Christopher S LaMadrid, Ryan Haymore, Stephen H Wright, Nathan J Cherrington","doi":"10.1016/j.dmd.2025.100043","DOIUrl":"10.1016/j.dmd.2025.100043","url":null,"abstract":"<p><p>Valsartan (VAL) is commonly prescribed for patients with cardiovascular disease (CVD) to lower blood pressure, reduce heart failure risk, and prevent heart attacks or strokes by blocking the effects of angiotensin II. Many patients with CVD also suffer from metabolic dysfunction-associated steatohepatitis (MASH), which disrupts several xenobiotic transporters, affecting the pharmacokinetics of numerous drugs. Medications used in patients to treat comorbidities associated with MASH may be subject to this altered disposition and potential toxicity. This study aimed to assess how MASH alters the pharmacokinetics of VAL using a rodent model that mimics human MASH. MASH was induced in rats via a methionine- and choline-deficient (MCD) diet. Rats received VAL-a substrate of organic anion-transporting polypeptide (OATP) 1B1/1B3 and reported for multidrug resistance-associated protein-2-(2 mg/kg) through intravenous injection to isolate hepatic transport processes, and bile, serum, and liver concentrations measured using liquid chromatography-tandem mass spectrometry. Consistent with MASH progression, MCD rats presented with more gross pathology, including increased liver-to-body weight ratios, along with macrosteatosis, hepatocyte ballooning, and lobular inflammation. In MCD rats, the expression of Oatp1b2 was significantly reduced, and Mrp2 was internalized, resulting in higher systemic exposure to VAL compared with controls. Additionally, cumulative biliary excretion of VAL was lower in MCD rats. To further assess VAL disposition in MASH, transport kinetics were evaluated in human embryonic kidney 293 cells overexpressing OATP1B1 or OATP1B3, revealing similar affinity for VAL between both transporters. These findings suggest that changes in OATP function in MASH may alter VAL pharmacokinetics, which may have implications for personalized treatments. SIGNIFICANCE STATEMENT: Although expression of drug transporters in metabolic dysfunction-associated steatohepatitis (MASH) has been explored, the combined effect between MASH and genetic loss of transporters on the disposition of sartan drugs has not been determined. This study applied liquid chromatography-tandem mass spectrometry analyses and immunohistological staining to assess drug disposition and identify alterations to drug transporters in rodents on a methionine- and choline-deficient diet. The observations made in this study have significant implications regarding its disposition in the context of hepatic dysfunction associated with MASH.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"53 3","pages":"100043"},"PeriodicalIF":4.4,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143585030","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":"Structure-activity relationship of isoprenoid triazole bisphosphonate-based geranylgeranyl diphosphate synthase inhibitors: Effects on pharmacokinetics, biodistribution, and hepatic transporters.","authors":"Staci L Haney, Yashpal S Chhonker, Mamunur Rashid, Daryl J Murry, Sarah A Holstein","doi":"10.1016/j.dmd.2025.100038","DOIUrl":"10.1016/j.dmd.2025.100038","url":null,"abstract":"<p><p>Geranylgeranyl diphosphate synthase produces the isoprenoid geranylgeranyl diphosphate, which is used in protein geranylgeranylation. Our previous work illustrates that geranylgeranyl diphosphate synthase inhibitors (GGSIs) disrupt Rab-mediated protein trafficking in cells, inducing the unfolded protein response pathway and apoptosis. Structure-function studies of our GGSIs, which are isoprenoid triazole bisphosphonates, have revealed a complex relationship between GGSI structure and enzymatic, cellular, and in vivo activities. The dose-limiting toxicity of this family of GGSIs is hepatic, and the mechanisms underlying their hepatic uptake are unexplored. Here, we evaluate the pharmacokinetics (PK) and biodistribution of a pair of potent GGSIs that are olefin isomers (homogeranyl [HG] and homoneryl [HN]). We investigate whether these isomers, as well as their a-methylated analogs (HG-me and HN-me), are substrates for key hepatic transporters and explore the effects of these GGSIs on the expression of a panel of hepatic transporters and cytochrome P450s. The PK/biodistribution studies revealed that both systemic exposure and liver levels of HG were significantly higher than that of HN across multiple time points. Conversely, HN was present at 4-fold higher concentrations in the bile at 2 hours postinjection relative to HG. HG-me and HN-me, but not HG or HN, were determined to be substrates of hepatic transport proteins OATP1B1 and OATP1B3. While the hepatic expression of several transporters and cytochrome P450 were altered by GGSI treatment, no significant differences in expression patterns between pairs of olefin isomers were observed. Collectively, these studies reveal that GGSI structure, including olefin stereochemistry, impacts PK profile, biodistribution, and hepatic transporter affinity. SIGNIFICANCE STATEMENT: Our understanding of the in vivo structure-activity relationship of our novel geranylgeranyl diphosphate synthase inhibitors has expanded, demonstrating that isoprenoid olefin stereochemistry impacts pharmacokinetic and biodistribution patterns and that other modifications impact transporter affinity. These studies reveal the underlying complexity of the mechanisms regulating hepatic exposure to these agents. Future studies will focus on optimizing tumor-directed geranylgeranyl diphosphate synthase inhibitor delivery while minimizing hepatic uptake.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"53 3","pages":"100038"},"PeriodicalIF":4.4,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143536794","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":"Toward improved clearance predictions and distribution profiles employing the isolated perfused rat liver model: Experimental optimization.","authors":"Sofie Heylen, Johan Nicolaï, Stijn Van Asten, An Tuytelaars, Katie De Wagter, Jan Snoeys, Raymond Evers, Stephanie Kourula, Pieter Annaert","doi":"10.1016/j.dmd.2025.100045","DOIUrl":"10.1016/j.dmd.2025.100045","url":null,"abstract":"<p><p>To reduce the drug attrition due to failure in clinical trials, an early accurate hepatic clearance (CL_H) prediction for small molecule drugs is critical. However, the routinely used in vitro to in vivo extrapolation (IVIVE) methods to predict human CL_H have led to significant underpredictions for many drug discovery programs. The ex vivo isolated perfused rat liver (IPRL) can be employed as a model to gain a mechanistic understanding of the root cause(s) of the frequent suboptimal performance of IVIVE methods. Although the IPRL perfusate composition is a critical factor to obtain reliable IPRL data to bridge the in vitro to in vivo clearance gap, no studies have experimentally explored the effect of different perfusate compositions on drug disposition profiles. Initially, in this study, acetaminophen and atazanavir, showing low or high protein binding, respectively, were tested in the IPRL system with 2 reperfusion media. One medium contained 3% bovine serum albumin, whereas the other was 10% rat blood to determine optimal experimental conditions, analogous to best practices in literature. This research provides new insights into the effect of IPRL perfusate composition on accurate CL_H predictions and liver disposition profiles. Specifically, this study demonstrates for the first time that 3% bovine serum albumin and 0.01% bovine α-1-acid glycoprotein are essential in an IPRL experiment to mimic biorelevant conditions and to achieve in vivo-relevant unbound clearance values, alongside 10% blood to maintain liver viability and functionality. The unique mechanistic insights derived from these robust IPRL data will aid in the development of improved IVIVE methods for CL_H. SIGNIFICANCE STATEMENT: This study explored the effect of perfusate composition on compound disposition profiles in an isolated perfused rat liver system. Reliable disposition profiles and improved clearance predictions were obtained, employing biorelevant plasma protein concentrations alongside 10% blood to obtain in vivo-relevant unbound clearance values and to preserve liver viability and functionality. Robust conditions are critical in isolated perfused rat liver experiments to gain mechanistic insights into the in vitro to in vivo clearance gap, enabling optimization of in vitro to in vivo extrapolation methods.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"53 3","pages":"100045"},"PeriodicalIF":4.4,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143630269","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":"Physiologically based pharmacokinetic modeling of midostaurin and metabolites at steady-state to bridge drug interaction scenarios in lieu of clinical trials.","authors":"Helen Gu, Romain Sechaud, Imad Hanna, Ryan Pelis, Heidi J Einolf","doi":"10.1016/j.dmd.2025.100036","DOIUrl":"10.1016/j.dmd.2025.100036","url":null,"abstract":"<p><p>Midostaurin and its active metabolites are substrates, mixed inhibitors/inducers of cytochrome P450 (CYP)3A4. The main objective of this study was to develop/refine a physiologically based pharmacokinetic (PBPK) model that incorporated recent clinical drug-drug interaction (DDI) data with midazolam after multiple dosing, to qualify the pharmacokinetic (PK) model simulations of midostaurin and its metabolites, and to apply it to predict untested clinical DDI scenarios with potential comedications. In this study, Simcyp PBPK model of midostaurin and its 2 metabolites was refined from a previously published model associated with endogenous biomarker 4β-hydroxycholesterol data through further optimization of CYP3A4 inhibition/induction potency and was qualified to simulate midostaurin steady-state PK. The incorporation of these parameters enabled DDI predictions of high midostaurin doses on the PK of midazolam and oral contraceptives containing ethinyl estradiol. Additionally, scaling factors for in vitro breast cancer resistance protein and the organic anion transporting polypeptide (OATP1B) inhibition were applied to account for the observed single-dose DDI with rosuvastatin and further extrapolated to predict steady-state DDI with other OATP1B drug substrates. The overall prediction results showed minimal impact of midostaurin at high doses on CYP3A substrates or an effect on the exposure of OATP1B substrates. In summary, the midostaurin PBPK model was retrospectively refined, requalified, and used to simulate the steady-state perpetrator DDI of midostaurin and its metabolites. This PBPK modeling approach and the resulting model predictions were implemented into the midostaurin product label (up to 100 mg twice a day) without the need for confirmatory clinical studies. SIGNIFICANCE STATEMENT: The manuscript describes how a midostaurin PBPK model was updated, verified, and applied to untested scenarios by a predict-learn-confirm cycle as new clinical data become available. It also provides a learning experience of prospective prediction by utilizing endogenous biomarker 4β-hydroxycholesterol to evaluate a complex CYP3A4-mediated drug interaction.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"53 3","pages":"100036"},"PeriodicalIF":4.4,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143476346","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":"Cross-industry demonstration of the validity of the mixed matrix method for the assessment of cross-species exposure coverage of human circulating drug metabolites.","authors":"Alexander D James, Christian Lanshoeft, Gregory S Steeno, Douglas K Spracklin, Zhiping You, Raman Sharma, Xue-Qing Li, Linda C Andersson, Isabel Piel, Alison Webb, Janine M Doyle, Laurent Laboureur, Georges Da Violante, David Rizzo, Yves Martin Siegrist","doi":"10.1016/j.dmd.2025.100041","DOIUrl":"10.1016/j.dmd.2025.100041","url":null,"abstract":"<p><p>The mixed matrix method (MmM) is a widely used approach by the pharmaceutical industry for early assessment of whether exposures to major human circulating metabolites, of traditional small-molecule drugs, are adequately covered by the species used for toxicology assessment, which is a key requirement of the safety testing of drug metabolites (metabolites in safety testing guidelines). However, questions remain regarding its accuracy and utility in replacing conventional bioanalytical approaches. Furthermore, the available literature on the topic is not fully consistent in terms of how the assay should be conducted. As a result, encouraged by health authority advice on this topic, a cross-industry group under the European Federation of Pharmaceutical Industries and Associations was formed to: (1) further investigate the MmM accuracy, including a robust statistical analysis covering a diverse chemical space of commercially available drugs and drug candidates as well as their metabolites; (2) propose recommendations for best practice including a decision tree that the industry should consider when using the MmM; and (3) discuss whether the MmM could be used to support metabolites in safety testing assessment and could potentially be included into new drug application submissions without the need for additional measurements using the conventional bioanalytical approach. The outcome of this European Federation of Pharmaceutical Industries and Associations assessment shows that MmM measured exposure ratios of 1.9 and 1.4 are statistically sufficient to demonstrate adequate exposure coverage of human metabolites above 50% or between 10% and 50% of drug-related exposure, respectively, by toxicology species. The aim is to encourage both industry and regulatory agencies to consider MmM as an acceptable approach to compare major human circulating metabolite exposures across species. SIGNIFICANCE STATEMENT: The outcome of our mixed matrix method assessment showed that measured exposure ratios of 1.9 and 1.4 are adequate to demonstrate coverage of human metabolites above or below 50% drug-related exposure by toxicology species. Recommendations for best practice and a decision tree for conducting metabolites in safety testing evaluations are proposed. Our investigations show that mixed matrix method data are sufficiently robust for the intended purpose and that the assay provides an opportunity to streamline drug development and reduce the need for resource-intensive bioanalysis and certain animal studies.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"53 3","pages":"100041"},"PeriodicalIF":4.4,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143572460","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":"Application of small interfering RNA technology in cytochrome P450 gene modulation.","authors":"Wenzhao Jiang, Ruoyao Sang, Cai Zhang, Runting Yin, Zhen Ouyang, Yuan Wei","doi":"10.1016/j.dmd.2025.100040","DOIUrl":"10.1016/j.dmd.2025.100040","url":null,"abstract":"<p><p>Cytochrome P450 plays key roles in the biotransformation of endogenous and exogenous chemicals including drugs and environmental pollutants. The inhibition and downregulation of P450s can have therapeutic effects, and/or modulate drug metabolism. P450s are largely inhibited by small molecules; however, this strategy is often hampered by intrinsic toxicity and drug-drug interactions. Furthermore, it is challenging for small molecules to exhibit high selectivity and inhibitory efficiencies. Recently, small interfering RNA (siRNA) technology has demonstrated the potential for P450 modulation. Examples of recent applications of siRNAs in P450 gene modulation, in vitro and in vivo, are highlighted in this review. The necessity of siRNA techniques and their advantages as P450 modulators are discussed, along with a review of current obstacles and a perspective on future advancements. SIGNIFICANCE STATEMENT: This article reviews studies on the application of small interfering RNA technology to cytochrome P450 gene modulation. The necessity of siRNA methods and the benefits of their use as P450 modulators have been suggested by comparison with small-molecule drugs. Additionally, the challenges that presently limit the broader implementation of this topic are examined, and a perspective for future developments is proposed.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"53 3","pages":"100040"},"PeriodicalIF":4.4,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143515056","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":"Identification of selective substrates and inhibitors of the major human renal uptake transporters.","authors":"Yik Pui Tsang, Acilegna G Rodriguez, Mark S Warren, Jashvant D Unadkat","doi":"10.1016/j.dmd.2025.100046","DOIUrl":"10.1016/j.dmd.2025.100046","url":null,"abstract":"<p><p>Renal clearance of drugs mediated by transporters can be affected by diseases (eg, inflammation due to infections), physiological changes (eg, pregnancy), or drug-drug interactions. To elucidate the transporters involved, the magnitude of effect, and the underlying mechanisms, human proximal tubular epithelial cells could be exposed to potential perpetrators (eg, cytokines, pregnancy-related hormones or the interacting drug), and the activity of transporters quantified. A crucial prerequisite for such studies is the identification of selective substrates or substrate-inhibitor pairs for each renal transporter. Using transporter-transfected mammalian cells and membrane vesicles, we systematically evaluated the selectivity of 6 substrates (or substrate-inhibitor pairs) for the major uptake and efflux renal transporters. Cidofovir, levocetirizine, and ergothioneine were found to be selective substrates of the organic anion transporter (OAT) 1, 4, and organic cation/carnitine transporter 1, respectively. Nicotinic acid was transported by OAT2, but also by OAT1 and 3, though to a lesser extent. Probenecid did not selectively inhibit OAT1/3-mediated uptake of nicotinic acid, but quercetin did, allowing selective measurement of OAT2 activity. Interestingly, nicotinic acid was also transported by the endogenous monocarboxylate transporter 1 in HEK293 cells. Glycochenodeoxycholic acid sulfate was transported by OAT3 and multidrug resistance-associated protein 2 (MRP2), with MRP2 selectively inhibited by cyclosporine A, allowing selective measurement of OAT3 activity. Atenolol was transported by organic cation transporter 2 and multidrug and toxin extrusion proteins 1 and 2-K, with multidrug and toxin extrusion proteins activity selectively inhibited by mitoxantrone, allowing selective measurement of organic cation transporter 2 activity. SIGNIFICANCE STATEMENT: These findings provide a framework for measuring the in vitro activity of individual uptake transporters in primary human proximal tubular epithelial cells. By applying our proposed methodology, researchers can quantify how various factors (eg, cytokines, pregnancy-related hormone, drug interactions) modulate individual renal uptake transporter activity in proximal tubular epithelial cells.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"53 3","pages":"100046"},"PeriodicalIF":4.4,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143536777","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":"Organic cation transporters 2: Structure, regulation, functions, and clinical implications.","authors":"Anoud Ailabouni, Bhagwat Prasad","doi":"10.1016/j.dmd.2025.100044","DOIUrl":"10.1016/j.dmd.2025.100044","url":null,"abstract":"<p><p>The SLC22A2 gene encodes organic cation transporter 2 (OCT2), which is predominantly expressed in renal proximal tubule cells. OCT2 is critical for the active renal excretion of various cationic drugs and endogenous metabolites. OCT2 expression varies across species, with higher levels in mice and monkeys compared with humans and rats. The human OCT2 protein consists of 555 amino acids and contains 12 transmembrane domains. OCT2 functions as a uniporter, facilitating the bidirectional transport of organic cations into renal tubular cells, driven by the inside-negative membrane potential. Its expression is regulated by sex hormones, contributing to potential sex differences in Oct2 activity in rodents. OCT2 has been linked to tissue toxicity, such as cisplatin-induced nephrotoxicity. Factors such as genetic variants, age, disease states, and the coadministration of drugs, including tyrosine kinase inhibitors, contribute to interindividual variability in OCT2 activity. This, in turn, impacts the systemic exposure and elimination of drugs and endogenous substances. Regulatory agencies recommend evaluating the potential of a drug to inhibit OCT2 through in vitro and clinical drug-drug interaction (DDI) studies, often using metformin as a probe substrate. Emerging tools like transporter biomarkers and physiologically based pharmacokinetic modeling hold promise in predicting OCT2-mediated DDIs. While several OCT2 biomarkers, such as N1-methylnicotinamide, have been proposed, their reliability in predicting renal DDIs remains uncertain and requires further study. Ultimately, a better understanding of the factors influencing OCT2 activity is essential for achieving precision medicine and minimizing renal and systemic toxicity. SIGNIFICANCE STATEMENT: Organic cation transporter 2 (OCT2) is essential for the active tubular secretion of xenobiotics and endogenous cationic substances in the kidneys. This article offers a comprehensive overview of the tissue distribution, interspecies differences, and factors affecting its activity-critical for evaluating tissue toxicity and systemic exposure to cationic substances. Using OCT2 biomarkers and integrating OCT2 activity and expression data into physiologically based pharmacokinetic models are valuable tools for predicting OCT2 function and its clinical implications.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"53 3","pages":"100044"},"PeriodicalIF":4.4,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143531330","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":"Two-step metabolic activation to ortho-benzoquinone intermediate and its role in 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside-induced liver injury in mice.","authors":"Hong Pan, Guohong Zhai, Qiuyi Jing, Yaya Fan, Chao Fang, Fuguo Shi","doi":"10.1016/j.dmd.2025.100047","DOIUrl":"10.1016/j.dmd.2025.100047","url":null,"abstract":"<p><p>2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) is the most abundant constituent of Polygonum multiflorum and is exclusively found in this herb. This renowned herbal medicine has been documented to lead to liver damage in humans. The present study demonstrated that TSG underwent 2-step metabolic activation to generate a reactive metabolite, involving both intestinal and hepatic metabolisms. TSG was hydrolyzed to its aglycone 2,3,5,4'-tetrahydroxystilbene (TS) in the intestine, and then, the 2,3 catechol of 2,3,5,4'-tetrahydroxystilbene was metabolized to an ortho-benzoquinone intermediate in the liver. The reactive metabolite was characterized as the N-acetyl-cysteine conjugate both in vivo and in vitro. Its structure was verified by a combined isotope-labeling strategy using the ¹⁴N/¹⁵N, H/D, and ⁷⁹Br/⁸¹Br isotope pattern-based mass shifts. Intestinal β-glucosidase and hepatic CYP3A4 and CYP2C9 contributed to the reactive metabolite formation. The reactive intermediate could covalently modify the hepatic proteins through cysteine in mice. Combined with the treatment with β-glucosidase, a single oral administration of 400 mg/kg TSG caused liver centrilobular necrosis and degeneration in mice. Selective CYP3A inhibitor ketoconazole protected TSG-induced liver injury, concurrently attenuating protein adduct formation modified by reactive metabolites. The results indicate that TSG does not exert hepatotoxic effects but that the reactive ortho-benzoquinone metabolite from the oxidation of the 2,3 catechol of aglycone is responsible for TSG-induced liver injury. The study also facilitates a better understanding of the principal hepatotoxic chemicals in Polygonum multiflorum. SIGNIFICANCE STATEMENT: 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) undergoes intestinal deglycosylation to generate its aglycone, and then, the 2,3 catechol of aglycone was metabolized to an ortho-benzoquinone intermediate in the liver. β-glucosidase potentiates TSG-induced liver injury and protein adduction by the reactive metabolite. The results indicate that the reactive metabolite of TSG exerts hepatotoxic effects rather than the parent compound.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"53 3","pages":"100047"},"PeriodicalIF":4.4,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143556133","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":"Efficient protein quantification in drug metabolism and pharmacokinetics with an accelerated proteomic workflow.","authors":"Xiaofeng Wu, Nicholas Ferguson, Lloyd Wei Tat Tang","doi":"10.1016/j.dmd.2025.100048","DOIUrl":"10.1016/j.dmd.2025.100048","url":null,"abstract":"<p><p>Quantifying proteins involved in the absorption, distribution, metabolism, and excretion (ADME) of drugs is essential to improve understanding of their disposition and pharmacokinetics. Proteomics, because of its great versatility, is a widely used approach for protein analysis. However, existing protocols face challenges, such as poor peptide identification in liquid chromatography with tandem mass spectrometry under multiple reaction monitoring mode as well as the time- and labor-intensive nature of detergent-engaged workflows. In this study, we compared and evaluated several targeted ADME proteomic methods, including a novel approach called Fast Surfactant-Treated (FAST). Using FAST in ADME proteome analysis of primary human hepatocytes revealed that most proteins, especially membrane proteins, were efficiently solubilized and digested, with the ionic detergent sodium deoxycholate and rapidly removed during preparation by the incorporation of a centrifugation step following acetonitrile precipitation. Compared with the traditional proteomic workflow involving dithiothreitol reduction and iodoacetamide alkylation, FAST achieved an approximately 4-fold increase in cytochrome P450 and UDP-glucuronosyltransferases quantification and 5-fold increase in transporters, based on endogenous tryptic peptide signals. For specific proteins such as CYP2J2, organic anion transporter, and organic anion transporting polypeptide 1B1, FAST generated peptide quantification peaks with significantly higher signal-to-noise ratios and in a shorter amount of sample processing time. We then further validated the FAST proteomics workflow using the pregnane X receptor agonist rifampicin in human hepatocytes, which revealed that CYP3A4 protein levels were induced to a similar extent as observed in the CYP3A midazolam-1'-hydroxylase activity assay. Altogether, these results suggest that FAST proteomics is a robust, efficient, and versatile method for ADME bioanalysis. SIGNIFICANCE STATEMENT: Quantifying absorption, distribution, metabolism, and excretion (ADME) proteins from in vitro matrices remains a challenge, particularly when speed and efficiency are critical. By incorporating sodium deoxycholate detergent into the ADME proteome sample preparation workflow, we developed a methodology called Fast Surfactant-Treated proteomics. This approach enabled more efficient quantification of drug-metabolizing enzymes and membrane transporters, offering a streamlined protocol with reduced bench time.</p>","PeriodicalId":11309,"journal":{"name":"Drug Metabolism and Disposition","volume":"53 3","pages":"100048"},"PeriodicalIF":4.4,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143603467","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}