Drug Metabolism and Disposition最新文献

Corydalis Decumbentis Rhizoma (CDR) is a traditional Chinese herbal medicine, which has been clinically used for treating migraine, relieving the symptoms of rheumatoid arthritis, and reversing osteoporosis. Bicuculline (BIC) is one of the main bioactive ingredients present in CDR. The purpose of this study was to comprehensively investigate the interaction of BIC with cytochrome P450 enzymes and the alteration of the pharmacokinetic properties of the active thiol metabolite 4 of clopidogrel when coadministered. BIC showed time-, concentration-, and NADPH-dependent inhibitory effects on CYP2C19. BIC (2.0 μM) inhibited CYP2C19 activity by approximately 64% after 9 minutes of incubation at 30 °C, and K_I and k_inact were 0.09 μM and 0.051 min^-1, respectively. Ticlopidine showed a significant protective effect against BIC-induced CYP2C19 inactivation. Dialysis did not restore the inhibited enzyme activity induced by BIC. Moreover, the addition of catalase/superoxide dismutase or glutathione did not show protective effects against BIC-induced enzyme inactivation. It is speculated that carbene intermediates were involved in BIC-induced inactivation of CYP2C19 because K₃Fe (CN)₆ restored the enzymatic activity. In addition, BIC and CDR extract pretreatment resulted in a significant decrease in the C_max and area under the curve of the plasma thiol metabolite in rats given clopidogrel. SIGNIFICANCE STATEMENT: This study identifies bicuculline, a major component of Corydalis Decumbentis Rhizoma, as a mechanism-based inhibitor of CYP2C19 through carbene-mediated coordination with heme iron. Such inhibition reduces clopidogrel activation and indicates clinically relevant herb-drug interactions.

Targeted covalent inhibitors, such as acrylamide covalent drugs (ACDs), offer advantages in potency, selectivity, and duration of effect compared with traditional small-molecule inhibitors. However, ACDs undergo unique biotransformation pathways in humans, including CYP-mediated metabolism, protein covalent binding, and nonenzymatic glutathione (GSH) adduction, which make standard in vitro metabolism assays for small molecules unsuitable for characterizing ACDs. This study aimed to develop a specialized panel of in vitro metabolism experiments for characterizing ACDs. The approach included metabolism stability assays in human liver microsomes with or without NADPH, covalent binding to human serum albumin with or without GSH, and metabolite profiling in human liver microsomes with or without GSH. In vitro metabolic data were generated for 5 ACDs, abivertinib, afatinib, osimertinib, ibrutinib, and pyrotinib, and compared with reported human metabolism and disposition data. In general, in vitro biotransformation pathways determined in this study are consistent with major metabolic clearance pathways observed in humans. For example, osimertinib showed the highest nonspecific protein covalent binding, a high oxidation-to-GSH adduct ratio, and moderate NADPH-dependent metabolic rates, supporting protein covalent binding as the major metabolic pathway in humans. In contrast, afatinib exhibited minimal CYP-mediated metabolism after accounting for covalent binding to microsomal proteins, low serum protein binding, and a very low oxidation-to-GSH adduct ratio, consistent with GSH adduction being the predominant biotransformation pathway in humans. The results demonstrate that the newly developed in vitro metabolism workflow enables more accurate predictions of CYP-mediated clearance rates and clarifies the relative contributions of CYP metabolism, nonspecific protein covalent binding, and GSH adduction to overall metabolic clearance in humans. SIGNIFICANT STATEMENT: This study established a novel in vitro metabolism approach for characterizing acrylamide covalent drugs. By comparing in vitro metabolic data for abivertinib, afatinib, osimertinib, ibrutinib, and pyrotinib with reported human metabolism and disposition data, we demonstrated that this method improves the accuracy of predicting CYP-mediated metabolic rates. Furthermore, it provides clearer insights into the relative contributions of CYP metabolism, nonspecific protein covalent binding, and glutathione adduction to the overall metabolic clearance of acrylamide covalent drugs in humans.

Encequidar is a gut-restricted P-glycoprotein inhibitor that is a useful tool molecule to boost the oral bioavailability of P-glycoprotein substrates. In this article, we demonstrate that encequidar has moderate to high clearance and volume of distribution, and low oral bioavailabilities (<10%) in rat, dog, and monkey. We show, in vivo, the ability of encequidar to inhibit gut P-glycoprotein and boost the oral exposures of numerous P-glycoprotein probe substrates by 5- to 20-fold in rat, dog, and monkey. In addition, we show low portal vein levels of encequidar, suggesting that it is an efficient gut P-glycoprotein inhibitor but unlikely to inhibit bile canicular P-glycoprotein. We leverage this gut-restricted nature of encequidar to differentiate between intestinal excretion/secretion mediated by gut P-glycoprotein and biliary elimination mediated by canicular P-glycoprotein without the need for bile duct-cannulated animal studies. We show that encequidar can inhibit intestinal secretion of known P-glycoprotein substrates (paclitaxel, apixaban, and talinolol) in rat and dog. The reduction in the amount of parent in feces, post-intravenous dosing, by encequidar reflects intestinal secretion, whereas the remaining amount of parent in feces in the presence of encequidar reflects biliary elimination. In all cases, renal elimination was unaffected by encequidar. In summary, we demonstrate that encequidar can differentiate between the various disposition pathways-renal, biliary, and intestinal-in animals and provides a quick qualitative estimate of the human disposition pathways. SIGNIFICANCE STATEMENT: Encequidar is a potent, gut-restricted P-glycoprotein (P-gp) inhibitor that boosts oral bioavailability of P-gp substrates in the commonly used nonclinical species of rat, dog, and monkey. Encequidar is a suitable in vivo P-gp inhibitor to determine the main routes of elimination and differentiate between intestinal secretion and biliary elimination of P-gp substrates using intact animal models.

Tobacco use remains the leading cause of preventable death worldwide. The major metabolic pathway for nicotine, the addictive component in tobacco, is via cytochrome P450 (CYP) 2A6-mediated metabolism to cotinine. Cannabidiol has been shown to reduce cigarette consumption in vivo and inhibit CYP2A6-mediated nicotine metabolism in vitro. In the present study, Δ-8-tetrahydrocannabinol (Δ8-THC), an isomer of Δ-9-tetrahydrocannabinol, was examined as a potential inhibitor of CYP2A6-mediated nicotine metabolism. While Δ-9-tetrahydrocannabinol showed no significant inhibition of nicotine metabolism to cotinine, Δ8-THC demonstrated unbound IC₅₀ values of 0.57 ± 0.04 μM in microsomes from recombinant wild-type CYP2A6 overexpressing human embryonic kidney 293 cells and 0.70 ± 0.16 μM in human liver microsomes (HLMs). A similar unbound IC₅₀ value was observed for recombinant CYP2A6∗5 microsomes (0.52 ± 0.17 μM) and was modestly elevated in recombinant CYP2A6∗2 microsomes (1.00 ± 0.12 μM). IC₅₀ shift experiments were consistent across pooled HLM (5.3-fold) and microsomes from liver specimens exhibiting the CYP2A6 (∗2/∗2) and (∗9/∗9) genotypes (6.1- and 4.0-fold, respectively) but were reduced in CYP2A6 (∗35/∗35) microsomes (1.0-fold). Irreversible inhibition kinetics in pooled HLMs by Δ8-THC yielded a k_inact value of 0.022 ± 0.001 min^-1 and an unbound K_I value of 0.232 ± 0.062 μM. Static modeling predicted that oral dosing with 10 mg Δ8-THC increased the nicotine plasma area under the curve by 189%, with further increases observed at 20 mg and 40 mg; interactions were also observed with inhalation doses ≥70 mg. These findings suggest that, based on CYP2A6 genotype, Δ8-THC could be a candidate for smoking cessation therapy. SIGNIFICANCE STATEMENT: This study is the first, to the best of our knowledge, to identify Δ-8-tetrahydrocannabinol as a potent and irreversible inhibitor of nicotine metabolism to cotinine. The extent of inhibition is modulated by genetic variation in cytochrome P450 2A6. These findings suggest that further investigations focusing on Δ-8-tetrahydrocannabinol and its potential as a candidate for smoking cessation therapy are warranted.

Effective drug delivery to the inner ear is severely limited by the restrictive nature of the round window membrane (RWM). In this study, coumarin-6-labeled chitosan nanoparticles were administered via intratympanic injection in guinea pigs to investigate their spatiotemporal transport across the RWM and entry into the inner ear. The nanoparticles exhibited prolonged residence on the RWM and efficient presence in the perilymph in a time- and concentration-dependent manner. Mechanistic analyses demonstrated that nanoparticle transport occurred through coordinated paracellular and transcellular pathways. Transient modulation of tight junctions facilitated paracellular diffusion, whereas active transcellular transport involved multiple endocytic routes. After cellular uptake, nanoparticles underwent intracellular trafficking and were released into the perilymph via Golgi-mediated exocytosis. Collectively, these findings reveal an in vivo, RWM-specific transport cascade characterized by coordinated paracellular tight junction modulation, multiroute endocytic uptake, and Golgi-mediated exocytotic release into the perilymph, providing mechanistic insight into nanoparticle disposition in the inner ear and supporting their potential for local drug delivery. SIGNIFICANCE STATEMENT: This study defines an in vivo, round window membrane-specific transport cascade governing nanoparticle disposition in the inner ear. It demonstrates coordinated paracellular and transcellular transport enabling nanoparticle entry into the perilymph, advancing mechanistic insight into physiological barrier regulation of inner ear drug delivery and supporting rational design of nanocarrier-based otologic therapies.

Clinical trials have demonstrated that venlafaxine affects brain functions in healthy subjects, but its underlying mechanisms remain unclear. The objective of this study was to systematically evaluate the effects of venlafaxine on the expression and activity of catechol-O-methyltransferase (COMT) in cortex of nondepressed rats and mice. Chronic in vivo exposure to venlafaxine for 8 days led to increases in cerebral COMT expression and activity, decreases in the methyl donor S-adenosylmethionine (SAM) levels, downregulation of H3K4me3 and H3K27me3 expression, and alterations in locomotor and exploration activities. Data from U251 cells and primary astrocytes showed that venlafaxine significantly upregulated COMT, p-AKT, p-P70S6K, and p-4EBP1 expression and decreased cellular SAM levels. Phosphatidylinositol 3-kinase inhibitor LY294002, mammalian target of rapamycin inhibitor rapamycin, silencing P70S6K, or silencing 4EBP1 remarkably attenuated venlafaxine-induced upregulation of COMT. Rapamycin or silencing P70S6K and 4EBP1 reversed venlafaxine-mediated deficiency of cellular SAM levels. In mice, rapamycin significantly attenuated venlafaxine-induced increases in cortical expression of COMT, p-P70S6K, and p-4EBP1, decreases in cortical SAM levels and locomotor and exploration activities, and downregulations of H3K4me3 and H3K27me3 expression. Furthermore, COMT inhibitor tolcapone reversed venlafaxine-induced decreases in SAM levels, H3K4me3 and H3K27me3 expression, and locomotor and exploration activities. Supplementing SAM also remarkably attenuated venlafaxine-induced decreases in H3K4me3 and H3K27me3 expression and behavioral alterations. These observations were further confirmed in U251 cells and primary astrocytes. These results indicate that venlafaxine induces cortical COMT via phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin pathway to decreasing SAM levels. Depletion of cortical SAM levels partly contributes to the decreases in activities of locomotor and exploration and expression of H3K4me3 and H3K27me3. SIGNIFICANCE STATEMENT: This study revealed that venlafaxine upregulated cortical catechol-O-methyltransferase expression and activity via activating phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin pathway. The induction of catechol-O-methyltransferase led to depletion of cortical S-adenosylmethionine, which may partly contribute to the decreases in locomotor and exploration activities and downregulations of H3K4me3 and H3K27me3 expression in cortex of rats and mice.

Altered drug pharmacokinetics during inflammation or infection have been linked to elevated plasma concentrations of proinflammatory cytokines. Data on how these cytokines affect the expression and activity of intestinal drug transporters and, therefore, bioavailability of transported drugs, remain limited. Here, we used a novel human enteroid in vitro model to investigate the effects of key proinflammatory cytokines (ie, interleukin [IL]-1β, IL-6, tumor necrosis factor-α, and interferon-gamma) on the mRNA expression of major intestinal transporters and activity of intestinal breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp). Differentiated enteroid monolayers (in 96-well plates) were treated for 48 hours with each cytokine individually or in combination (cocktail) at 0.1, 1, or 10 ng/mL, encompassing their pathophysiological plasma concentrations in various inflammatory conditions. In a concentration-dependent manner, the cytokine cocktail significantly reduced the mRNA expression of BCRP, P-gp, multidrug resistance proteins 2/3, organic solute transporter α/β, serotonin transporter, and organic anion transporter polypeptide 2B1, while increasing multidrug resistance protein4 mRNA expression. Among individual cytokines, IL-1β elicited the most pronounced effects. To quantify the effect of cytokines on mRNA expression and activity of BCRP and P-gp, these treatments, at 1 ng/mL of individual cytokines or the cocktail, were repeated in the Transwell format. The efflux ratio of nitrofurantoin (a selective BCRP substrate), after exposure to 1 ng/mL of each cytokine or the cytokine cocktail for 48 hours, was significantly reduced, whereas the efflux ratio of digoxin (a P-gp substrate) remained unchanged. SIGNIFICANCE STATEMENT: Proinflammatory cytokines significantly downregulate major intestinal drug transporter expression and breast cancer resistance protein activity in human enteroid monolayers, highlighting the potential impact of inflammation on oral drug bioavailability. These results can be used to populate physiologically-based pharmacokinetic models to predict transporter-mediated drug absorption under inflammatory conditions, guiding safer and more effective dosing regimens.

Lung cancer is the leading cause of cancer deaths worldwide with non-small cell lung cancer (NSCLC) as the predominant subtype. Drug resistance in patients with NSCLC often limits treatment effectiveness, underscoring the need for novel therapeutic targets. We have previously demonstrated that a knockdown of CYP4F11 attenuates the proliferation and migration of NCI-H460 cells. CYP4F11 is a fatty acid ω-hydroxylase and metabolizes arachidonic acid to the important lipid mediator 20-hydroxyeicosatetraenoic acid. However, the underlying mechanism of how CYP4F11 promotes cancer progression is unknown. Here, we first confirmed that a genetic ablation of CYP4F11 reduces cell proliferation and migration in an additional NSCLC cell line. Conversely, CYP4F11 overexpression markedly enhanced proliferation and migration in both cell models, underlining the relevance of CYP4F11 as a putative drug target. To further examine the impact of CYP4F11, transcriptomic profiling was conducted comparing CYP4F11 knockdown and control cells. Most intriguingly, fatty acid desaturase 2 (FADS2), a key enzyme in arachidonic acid biosynthesis, was one of the most significantly downregulated genes. Further validation confirmed a significant downregulation of FADS2 at both mRNA and protein levels in CYP4F11 knockdown cells, while a CYP4F11 overexpression triggered its expression. This suggests a regulatory mechanism between CYP4F11 and FADS2 through the joint metabolite arachidonic acid. Collectively, our studies identify CYP4F11 as a promoter of NSCLC cell proliferation and migration and establish a crosstalk between CYP4F11 and FADS2. This work provides new mechanistic insights into lipid metabolism-driven oncogenesis and highlights CYP4F11 as a promising therapeutic target for NSCLC. SIGNIFICANCE STATEMENT: CYP4F11 promotes non-small cell lung cancer progression by driving cell proliferation and migration, as evidenced by both loss-of-function and gain-of-function assays. Importantly, we for the first time identified a positive association between CYP4F11 and fatty acid desaturase 2, uncovering a previously unrecognized tumorigenic mechanism at the cancer-lipid metabolism interface that provides new opportunities for targeted intervention.

A dual prodrug linking clopidogrel and indobufen-an established dual antiplatelet therapy combination-was designed to enhance the bioactivation of clopidogrel while enabling coordinated inhibition of the ADP and thromboxane A₂ pathways of platelet activation. Because these 2 agents differ markedly in mechanism and duration of action, conventional combination therapy necessitates asymmetrical dosing. The fixed 1:1 molar ratio imposed by covalent conjugation introduces an inherent constraint on achieving balanced dual-pathway inhibition, a key consideration for defining the conjugate's therapeutic positioning. Three conjugates-deuterated clopidogrel-indobufen (1a), clopidogrel-indobufen (1b), and clopidogrel-(S)-indobufen (1c)-were synthesized and evaluated in rats. A single dose of these conjugates produced a delayed time to maximum plasma concentration and a sustained-release profile for both active metabolites. Covalent conjugation enhanced systemic exposure to the clopidogrel active metabolite while reducing exposure to released indobufen. Because conjugates 1b and 1c exhibited pharmacokinetic profiles more comparable to equimolar coadministration, they were selected for pharmacodynamic assessment. ADP receptor P2Y₁₂ occupancy and plasma thromboxane B₂ served as pathway-specific biomarkers, each bridging the pharmacokinetics and pharmacodynamics of the irreversible inhibition by clopidogrel and the reversible inhibition by indobufen, respectively. Both biomarkers showed strong correlations with inhibition of the corresponding platelet activation pathways. A single dose of 1b or 1c yielded synchronized maximal inhibition of both pathways at 8 hours-4 hours later than conventional coadministration-while retaining comparable peak efficacy. In the repeated dosing study, assessments aligned with the maximal-effect time point of the coadministration reference demonstrated that both conjugates-when supplemented with an interdose of indobufen-achieved pathway inhibition equivalent to the clinical regimen. These findings support conjugates 1b and 1c as promising alternatives to standard clopidogrel therapy and as potential tools for controlled de-escalation of antiplatelet therapy. SIGNIFICANCE STATEMENT: The clopidogrel-indobufen dual prodrugs enable synchronous, sustained release of both antiplatelet species in rats. P2Y₁₂ receptor occupancy and plasma thromboxane B₂ effectively capture the pharmacokinetic-pharmacodynamic relationships of this irreversible/reversible dual-antagonist combination.