Drug Metabolism and Disposition最新文献

Based on literature reports of the increasing importance of non-cytochrome P450 (non-P450) metabolism, an initial evaluation of the prevalence of non-P450 metabolism was undertaken and published in 2016. This initial evaluation covered 10 years and included 125 intravenously and orally administered small-molecule drugs approved by the US Food and Drug Administration. However, the current evaluation has been expanded to cover 20 years (2005-2024) of US Food and Drug Administration-approved drugs and includes 316 small-molecule intravenously and orally administered drugs for which adequate data from human radiolabeled absorption, distribution, metabolism, and excretion are available. Contributions of P450 and non-P450 enzymes to the formation of major metabolites (≥10% of dose) were assessed and tabulated. The involvement of P450 versus non-P450 enzymes in the formation of major metabolites is compared, and the individual non-P450 enzymes responsible are described. This second analysis indicates that non-P450 enzymes contribute significantly to the metabolism of the 316 drugs analyzed with approximately 30% of the metabolism of these drugs carried out by non-P450 enzymes, with the predominant non-P450 enzymes identified being glucuronosyltransferases (10.2%), hydrolases (8.2%), gut microbes (2.7%), and carbonyl reductases (2.6%). As with the previous assessment, the relative contribution of non-P450 enzymes to drug metabolism does not appear to have increased dramatically over the last 20 years of drugs analyzed. Metabolism by uridine 5'-diphospho-glucuronosyltransferase, hydrolase, and carbonyl-reducing enzymes, as well as by gut microbes, is significant and occurs frequently through the 20 years being evaluated. Therefore, further efforts toward characterizing non-P450 metabolism will be needed because it is anticipated that metabolism by non-P450 enzymes will continue to be prevalent in new drug approvals and especially in early drug discovery and development. SIGNIFICANCE STATEMENT: Understanding the metabolic fate of drugs and the enzymes involved in their metabolism is a major focus in both drug discovery and development and provides insights into the possible impact of polymorphic enzymes and drug-drug interaction potential. The current evaluation indicates that although there has not been a temporal increase in non-cytochrome P450 metabolism, metabolism by non-cytochrome P450 enzymes such as uridine 5'-diphospho-glucuronosyltransferase, hydrolases, carbonyl reductase, and gut microbes continues to be prevalent in drug approvals.

Dictamni Cortex (DC) has been associated with herb-induced liver injury, and fraxinellone (FRA), a furan-containing constituent, is implicated as a major hepatotoxic component. However, direct evidence linking FRA bioactivation to hepatic protein adduction in vivo remains limited. Here, we synthesized pyrroline- and pyrrole-type conjugates derived from the reactive FRA-cis-enedial intermediate and established liquid chromatography-tandem mass spectrometry methods to quantify corresponding lysine and cysteine/lysine adduct markers in the mouse liver after oral dosing with FRA or DC extract. Protein adduct levels increased in a dose- and time-dependent manner, peaked at 12 hours, and remained detectable up to 120 hours. Pretreatment with the CYP3A inhibitor ketoconazole markedly reduced adduct formation, supporting a CYP3A-dependent bioactivation pathway. To enable immunochemical detection, oxidized FRA was coupled to keyhole limpet hemocyanin to generate a polyclonal antiserum that selectively recognized FRA-derived pyrroline and pyrrole motifs. Western blotting and immunofluorescence revealed increased immunoreactive bands and a pericentral (central vein > portal triad) gradient in liver lobules, with prominent nuclear staining in hepatocytes after FRA or DC extract exposure; these signals were attenuated by ketoconazole. Collectively, these data demonstrate that CYP3A-mediated formation of an electrophilic cis-enedial intermediate drives covalent modification of hepatic proteins during FRA/DC exposure. The combined liquid chromatography-tandem mass spectrometry and antibody-based assays provide complementary tools for mechanistic studies and biomarker development for furan-containing herbal constituents. SIGNIFICANCE STATEMENT: This work provides direct in vivo evidence that CYP3A-dependent bioactivation of fraxinellone generates a reactive cis-enedial that covalently modifies hepatic proteins. The liquid chromatography-tandem mass spectrometry adduct markers and a selective antiadduct antibody enable semi‑quantitative detection and spatial mapping of protein adduction, supporting mechanistic investigations and biomarker development for furan-containing herbal constituents associated with liver injury.

Drug-induced liver injury can occur when the canalicular phospholipid floppase multidrug resistance protein 3 (Mdr2 in rodents) is inhibited, but there is still a lack of early biomarkers to detect this risk. In this study, bile duct-cannulated rats were dosed with multidrug resistance protein 3/Mdr2 inhibitor itraconazole (ITZ; 100 mg/kg/d for 3 days) to assess phospholipid changes via an untargeted-to-targeted lipidomics workflow. Untargeted profiling of bile and liver samples identified 1347 and 2475 tentative lipids, of which 221 and 404 were phosphatidylcholines (PCs) in bile and the liver, respectively. Unsupervised principal component analysis revealed strong treatment effects on bile PCs. A volcano plot indicated a selective, but not global, reduction in biliary PCs after ITZ treatment. Among these, PC 38:4 stood out as the most consistently decreased bile species. Structural elucidation using multistage collision-induced dissociation/mass spectrometry³ fragmentations confirmed its identity as arachidonyl PC 18:0/20:4. Subsequent absolute quantitation showed that bile PC 38:4 remained stable in controls (10.5 ± 1.02 μM; 5.6% CV) but declined rapidly after the first dose of ITZ (6.89 ± 1.50 μM at 0-4 hours) and continued to decrease to 4.22 ± 0.958 μM by day 3, a 2.7-fold decrease. Conversely, hepatic PC 38:4 showed a modest, yet significant increase (∼1.2-fold). Plasma bile acids remained unaffected, supporting a mechanism involving Mdr2 rather than the bile salt export pump. These findings identify PC 38:4 (18:0/20:4) as a sensitive and mechanistically relevant marker of Mdr2 inhibition. Monitoring PC 38:4 in nonclinical species may enable early, transporter-specific drug-induced liver injury risk assessment during drug development. SIGNIFICANCE STATEMENT: Untargeted-to-targeted lipidomics workflows identified phosphatidylcholine 38:4 as a sensitive, specific, and mechanistically linked biomarker of Mdr2 inhibition in rats. Multistage collision-induced dissociation/mass spectrometry³ fragmentation further confirmed the identity as arachidonyl phosphatidylcholine 18:0/20:4. Its rapid and specific decline in the presence of the Mdr2 inhibitor itraconazole offers a potential new tool for early detection of human multidrug resistance protein 3-related liver injury risk during drug development.

The therapeutic efficacy of many anticancer drugs is frequently compromised by multidrug resistance, a process often driven by elevated activity of ATP-binding cassette (ABC) efflux pumps in tumor cells. These membrane transporters actively expel chemotherapeutic agents in an ATP-dependent fashion, thereby lowering intracellular drug exposure and diminishing treatment responses. The shortage of clinically approved agents capable of overcoming ABC transporter-mediated resistance highlights the urgency of identifying alternative approaches, including the repurposing of small-molecule targeted therapies to inhibit drug efflux. Here, we examine lirafugratinib, an orally available and highly selective fibroblast growth factor receptor 2 inhibitor currently undergoing clinical evaluation for intrahepatic cholangiocarcinoma and other solid tumors, as a potential modulator of ABCG2-mediated drug resistance. Our findings reveal that lirafugratinib, at concentrations that do not impair cell viability, restores sensitivity to ABCG2-substrate chemotherapeutic drugs and enhances apoptosis in ABCG2-overexpressing nonsmall cell lung cancer cells. Mechanistically, lirafugratinib impedes the efflux capability of ABCG2 without altering its protein expression. ATPase experiments and molecular docking analysis further indicate that lirafugratinib engages the drug-binding region of ABCG2 and modulates its ATP hydrolysis cycle. Collectively, these results suggest that lirafugratinib may be utilized as a chemosensitizing agent to counteract multidrug resistance in nonsmall cell lung cancer with high ABCG2 expression, supporting its evaluation in combination therapies. Further in vivo studies and clinical trials are required to substantiate its clinical applicability. SIGNIFICANCE STATEMENT: This work identifies lirafugratinib, a highly selective fibroblast growth factor receptor 2 inhibitor, as a previously unrecognized suppressor of ABCG2-dependent MDR. By limiting efflux-mediated depletion of anticancer drugs while maintaining its own activity, lirafugratinib resensitizes resistant nonsmall cell lung cancer cells to cytotoxic agents, supporting its potential utility in combination regimens for tumors with elevated ABCG2 expression.

Doxorubicin (Dox) cardiotoxicity can worsen due to increased cardiac accumulation. P-glycoprotein downregulation, common in irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), may influence Dox pharmacokinetics (PK) and cardiotoxicity, but its impact remains unexplored. This study investigated the mechanistic effects of IBS/IBD on Dox-induced cardiotoxicity. An IBS/IBD rat model assessed Dox PK after intravenous bolus (2.5 mg/kg) and oral (10 mg/kg) dosing. A whole-body physiologically based pharmacokinetic model was developed to predict Dox cardiac concentration in rats and humans. In rats, intravenous Dox clearance decreased by 70% in IBS, tripling plasma concentration, while IBD had a milder effect. Oral Dox bioavailability dropped by 85% in IBS and 50% in IBD, likely due to delayed gastric emptying. The whole-body physiologically based pharmacokinetic model predicted elevated cardiac interstitial fluid (ISFHeart) Dox concentrations surpassing cardiotoxicity thresholds in IBS/IBD rats. Human simulations showed 4- to 5-fold higher plasma concentration in IBS/IBD patients receiving intravenous Dox (60 mg/m²), nearing toxic levels. IBS/IBD prolongs Dox ISFHeart retention, increasing apoptosis-mediated cardiotoxicity risk, especially with bolus dosing. These findings highlight the critical impact of IBS/IBD on Dox PK and toxicity, advocating for personalized chemotherapy approaches. SIGNIFICANCE STATEMENT: This study shows that irritable bowel syndrome and inflammatory bowel disease alter doxorubicin disposition via impaired P-glycoprotein activity, reducing clearance, elevating systemic exposure, and increasing cardiac concentration of doxorubicin. Using rat models with whole-body physiologically based pharmacokinetic simulations, this study reveals how transporter dysfunction and gastrointestinal changes influence pharmacokinetics and toxicity. Findings extend knowledge beyond dose-dependent cardiotoxicity, highlighting comorbidities in drug disposition and underscoring disease-drug interactions for precision dosing in cancer patients.

The direct effects of drug metabolites on efficacy and safety have been evaluated carefully according to the regulatory guidelines by major authorities such as the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, US Food and Drug Administration, and Chinese National Medical Product Administration. In recent years, there has been increasing interest in and concern about the effects of drug metabolites on drug-drug interactions (DDIs) by inhibiting or inducing the activities of drug metabolism enzyme and/or drug transporters, termed metabolite-mediated DDIs (Met DDIs). Actually, the clinical risks of Met DDIs had been identified as gemfibrozil and mibefradil; mibefradil was withdrawn from the market because of serious adverse reactions. This review will focus on (1) the recommendations of various regulatory agencies regarding Met DDIs, (2) strategies and methodologies for evaluating Met DDIs for supporting the registrations of investigational new drug applications and new drug applications, and (3) the prediction of Met DDIs with the application of in vitro/nonclinical/clinical data by using relevant silicon models such as physiologically based pharmacokinetic and population pharmacokinetic models. SIGNIFICANCE STATEMENT: Metabolite-mediated drug-drug interactions (Met DDIs) can significantly affect clinical safety and efficacy. This review comprehensively analyzes regulatory guidelines by major authorities (National Medical Product Administration, US Food and Drug Administration, Pharmaceuticals and Medical Devices Agency, European Medicines Agency, and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use), summarizing their consensus on proactive Met DDI risk assessment while highlighting key discrepancies in scope, thresholds, and implementation requirements. Furthermore, it presents a translational evaluation strategy-from early in vitro characterization to advanced modeling approaches (physiologically based pharmacokinetic/population pharmacokinetic simulations) and optimized clinical study designs-with critical support from multiple case studies that illustrate practical applications, challenges, and solutions in Met DDI risk mitigation.

This study evaluated the properties of fatty acid amide hydrolase 2 (FAAH2) as a drug-metabolizing enzyme, specifically its substrate specificity and functionality. We used expression systems and various microsomes, with DS-8500a and its analogs as model substrates for xenobiotic hydrolysis. Among FAAH1, FAAH2, carboxylesterase 1, and carboxylesterase 2, FAAH2 exhibited significant hydrolytic activity, producing M20, a metabolite of DS-8500a. FAAH2 showed the highest affinity for the substrate DS-8500a with a Michaelis constant, which agreed with that observed in human liver microsomes. Furthermore, the FAAH2 protein levels in human liver microsomes showed a positive correlation with the amounts of M20 production. These findings suggested that FAAH2 is the primary enzyme responsible for metabolizing DS-8500a. We also found that human liver microsomes showed interindividual variability in FAAH2 activity with approximately a 15-fold maximal difference and a different extent of inhibitory effect by an FAAH2 inhibitor on M20 formation, possibly affecting the pharmacokinetic profile of FAAH2 substrates. Experiments using DS-8500 analogs suggested that structural modifications in the ethanolamide moiety influence the hydrolytic activity of FAAH2. Modifications to this moiety led to alterations not only in the hydrolytic activity of FAAH2 but also in the substrate specificity for FAAH1. In conclusion, we have demonstrated for the first time that FAAH2 catalyzes the hydrolysis of xenobiotics. This hydrolytic enzyme may affect the pharmacokinetics of FAAH2 substrates. SIGNIFICANCE STATEMENT: This study focuses on fatty acid amide hydrolase 2 (FAAH2) as a hydrolytic enzyme capable of metabolizing not only fatty acid amides but also xenobiotics, using DS-8500a as a model substrate. This work highlights the property of FAAH2 as a drug-metabolizing enzyme, emphasizing the need for evaluations with inhibitors such as cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester when xenobiotics are hydrolyzed. In addition, the quantification of FAAH2 protein levels and the assessment of individual variability provide insights into the influence of FAAH2 on drug metabolism across different individuals.

UVB radiation, a major environmental carcinogen, induces cyclobutane pyrimidine dimers and contributes to the development of squamous cell carcinoma. The RNA N⁶-methyladenosine modification (m⁶A) plays a critical role in regulating the DNA damage response. This study demonstrates that notoginsenoside R1 (NGR1), a bioactive ginsenoside derived from Panax notoginseng, protects against UVB induced skin sunburn injury. ABCG2 was identified as a key epidermal transporter responsible for the efflux of NGR1 from keratinocytes, revealing a previously unrecognized function of this efflux pump: the capacity to mediate the nonspecific import of NGR1. Mechanistically, NGR1 significantly upregulated WTAP expression, enhanced global m⁶A levels, and activated the m⁶A/DDB2 axis, resulting in a substantial reduction in cyclobutane pyrimidine dimers. These findings elucidate a molecular pathway through which NGR1, via ABCG2 mediated transport, mitigates UVB induced DNA damage responses by promoting m⁶A dependent DNA repair, positioning it as a promising candidate for topical therapeutic intervention. SIGNIFICANCE STATEMENT: Mechanisms by which NGR1 alleviates UVB induced skin sunburn injury via the WTAP/m⁶A axis and ABCG2 mediated trafficking offer a promising avenue for developing improved epidermal therapeutics for the related skin disorders.

SR12813 is an experimental cholesterol-lowering drug that reduces intracellular cholesterol through accelerated proteasomal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and is also recognized as a prototypical activator of the pregnane X receptor (PXR, NR1I2). Rifampicin, a clinically used antibiotic, likewise functions as a human PXR agonist. Although PXR-mediated induction of drug metabolism genes has been extensively characterized in hepatocytes and humanized mouse liver, comparatively little is known about the transcriptional effects of these ligands in intestinal and colon cancer cells. Here, we used RNA-sequencing in LS180 colon adenocarcinoma cells to compare transcriptional responses elicited by SR12813 and rifampicin. Both compounds induced canonical PXR targets, including CYP3A4, UGT1A1, and MDR1 (P-glycoprotein), whereas SR12813 preferentially upregulated genes associated with ketone body metabolism, lipid storage, and glycolysis. Complementary nuclear receptor reporter assays demonstrated that, in addition to robust PXR activation, SR12813 also functions as a partial agonist of peroxisome proliferator-activated receptor gamma, a receptor with critical roles in lipid metabolism and colon cancer biology. These findings demonstrate that SR12813 elicits overlapping, yet distinct transcriptional profiles relative to rifampicin, extending beyond xenobiotic metabolism to include metabolic pathways relevant to tumor progression. Collectively, our results highlight SR12813 as a dual-acting modulator of PXR and peroxisome proliferator-activated receptor gamma, and underscore its utility as a pharmacological tool for investigating nuclear receptor crosstalk in intestinal models. SIGNIFICANCE STATEMENT: SR12813 activates both pregnane X receptor and peroxisome proliferator-activated receptor gamma, demonstrating dual nuclear receptor modulation in colon cancer cells. By linking xenobiotic metabolism with lipid and mitochondrial pathways, this work uncovers previously unreported receptor crosstalk and provides a mechanistic framework for how diverse ligands can differentially shape transcriptional programs relevant to drug metabolism and tumor biology.