Drug Metabolism and Disposition最新文献

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.

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.

Tigecycline (TIG) exhibits poor oral bioavailability and brain distribution. Because bacterial efflux pumps that expel tetracyclines are homologous to mammalian P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), we investigated whether these transporters, along with TIG metabolism, affect TIG pharmacokinetics in mice. BALB/c and wild-type mice received TIG (10 mg/kg) intraperitoneally or orally alone, or in combination with the selective P-gp/BCRP inhibitor elacridar (ELA, 100 mg/kg p.o.) and/or the metabolic inhibitor voriconazole (VORI, 40 mg/kg i.p. or p.o.). One group also received a double TIG dose (20 mg/kg i.p.). Plasma and brain TIG concentrations were quantified via ultraperformance liquid chromatography coupled with tandem mass spectrometry. Differences in area under the curve (AUC) and C_max were analyzed using Bayesian hierarchical models. In both mouse strains, oral ELA substantially increased the plasma AUC_tlast (>1.8-fold; lower 95% credible limit [LCL], >1.6-fold) and C_max (>1.8-fold; LCL, >1.2-fold) of intraperitoneal TIG. The TIG double dose produced somewhat larger increases. With oral TIG, oral ELA markedly increased plasma AUC_tlast (>1.8-fold; LCL, >1.6-fold) in both strains, though C_max increases were more modest (LCLs, ∼ 1). Coadministration with VORI transiently (0.25-0.5 hours) raised plasma TIG, with oral VORI producing greater effects than intraperitoneal VORI. Oral ELA markedly increases systemic exposure to TIG, suggesting that P-gp/BCRP efflux restricts gastrointestinal absorption and increases systemic elimination. Results with VORI suggest that intestinal metabolism further limits oral uptake. Thus, efflux pump inhibition may be a viable strategy to improve TIG therapy. SIGNIFICANCE STATEMENT: Blocking efflux pumps by elacridar in the gut, liver, and brain increases tigecycline absorption and systemic retention. Coadministration of voriconazole, an inhibitor of metabolism, also suggests a significant role of intestinal metabolism in restricting tigecycline's oral bioavailability.

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.

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.

Cytochrome P450 2E1 (CYP2E1) plays an important role in inflammatory disease, but the role of CYP2E1 in gout remains elusive. The pharmacokinetics in rats of Q11, a CYP2E1 inhibitor synthesized by our group, were measured. The anti-inflammatory and uric acid (UA)-lowering levels mechanism of Q11 was investigated in hyperuricemic (HUA) mice and acute gouty arthritis (AGA) rats, and in macrophage cells stimulated by monosodium urate. The half-life (t_1/2) of Q11 in the ankle joint and kidney was approximately 9 hours, which was more than 3 times longer than that in plasma. The expression of CYP2E1 was increased in the kidneys of HUA mice and in the ankles of AGA rats. Q11 reduced plasma UA levels in HUA mice by decreasing xanthine oxidase activity. In addition, Q11 also decreased renal inflammatory injury in HUA mice. Moreover, Q11 decreased the levels of interleukin-1β and tumor necrosis factor-α in AGA rats, increased the levels of catalase and glutathione, and blocked the activation of the Nod-like receptor protein 3 inflammasome. A similar phenomenon was observed in J774A.1 and THP-1 cells treated with sodium urate.CYP2E1 may be a new therapeutic target for the treatment of gout and Q11 has the dual effect of being anti-inflammatory and lowering UA. SIGNIFICANCE STATEMENT: The specific CYP2E1 inhibitor Q11 reduced plasma uric acid levels by decreasing xanthine oxidase activity in a gout animal model, while attenuating joint and renal inflammatory injury through the inhibition of oxidative stress and Nod-like receptor protein 3 inflammasome activation.

Plasma levels of Coproporphyrin I (CP-I), an endogenous biomarker used to gauge hepatic organic anion transporting polypeptide (OATP)1B1 and OATP1B3 activities, are linked to covariates, namely ethnicity, sex, and hemoglobin level. We developed and verified a mechanistic physiologically based pharmacokinetic model for CP-I considering these covariates in basal conditions and with a range of OATP1B perpetrators in virtual healthy subjects of various ethnicities. Simulations recovered the observed steady-state baseline levels and concentrations with interaction (C_max and area under the curve ratios, n = 12 studies) within 2-fold. Published CP-I plasma data in hepatic impairment (HI) indicated a progressive reduction in OATP1B activity in vivo. We applied our verified CP-I model to simulate CP-I plasma levels reported in individuals with increasing severity of HI as classified based on the Child-Pugh classes (A, B, and C) to assess the hepatic OATP1B transporter activity in cirrhotic virtual populations. A biomarker-informed physiologically based pharmacokinetic (BI-PBPK) approach was applied to close the gap between known expression differences for human hepatic OATP1B and multidrug resistance-associated protein 2 (MRP2) in HI and observed activity differences in HI relative to healthy individuals. HI-associated relative activity factor scalars derived from BI-PBPK simulations were developed and verified using 9 OATP1B substrates (n = 7 studies), with an average fold error and absolute average fold error of 0.93 and 1.74 for C_max, and 1.29 and 1.47 for area under the plasma concentration-time curve ratios between HI and healthy. The BI-PBPK approach offers a powerful means to establish model system parameters to improve predictive performance, particularly in disease populations and to explore the mechanisms behind the changes in plasma level. SIGNIFICANCE STATEMENT: Biomarker-informed physiologically based pharmacokinetic approach was used to bridge abundance differences in transporter expression and observed activity differences between healthy volunteer and hepatically impaired patients. A PBPK model for the endogenous biomarker, Coproporphyrin I, was developed where its synthesis rate is linked to body weight, sex, ethnicity, and hemoglobin levels. The model was verified with an extensive set of weak-to-strong OATP1B perpetrator drugs and applied to recover plasma concentrations for multiple OATP1B substrates using associated drug-drug interactions.

(+)-Dihydromethysticin (DHM) is a major kavalactone isolated from kava plants. Previous studies have identified (+)-DHM as a CYP2B6 inhibitor, with intriguing structural specificity. It is the most potent CYP2B6 inhibitor identified to date, and CYP2B6 is the most sensitive of any CYP isoform to inhibition by DHM. This investigation evaluated the stereochemistry of DHM inhibition of CYP2B6 and the role of methylenedioxyphenyl group bioactivation in CYP2B6 inhibition, using expressed CYP2B6 and the probe substrates 7-ethoxy-4-trifluoromethyIcoumarin and S-ketamine. The unnatural enantiomer (-)-DHM and racemic (±)-DHM exhibited similar inhibitory activities. Both DHM enantiomers were noncompetitive inhibitors of CYP2B6, with Ki values of 0.2 μM. DHM analogs lacking a methylenedioxy group were devoid of inhibitory effects in both CYP2B6 metabolism assays. Difluoro substitution of the methylene hydrogens on DHM abolished DHM inhibitory activity, whereas dideuterio substitution had no effect on CYP2B6 inhibition. Both DHM enantiomers and the dideuterio analog, but not the difluorinated analog or methysticin, generated a difference spectrum consistent with a metabolite-inhibitor complex. Results suggest CYP2B6-catalyzed methylenedioxyphenyl bioactivation of DHM to a metabolite-inhibitor complex with subsequent enzyme inhibition. DHM may have potential clinical implications or application as a selective CYP2B6 index inhibitor probe. SIGNIFICANCE STATEMENT: Enantiomers of the kavalactone dihydromethysticin are among the most potent CYP2B6 inhibitors identified to date, undergo metabolite-inhibitor complex formation, and exhibit substrate-dependent competitive and noncompetitive inhibition, which may have potential clinical implications or application. Because there is presently no inhibitor of CYP2B6 recommended for in vitro studies and no strong index inhibitor available for CYP2B6 for clinical studies, due in part to specificity considerations, dihydromethysticin may be a candidate for this purpose.