Drug Metabolism and Disposition最新文献

A novel workflow for quantification of a drug and its metabolites in in vivo studies has been developed in the context of a radiolabeled human mass balance study. Samples are analyzed with ultra-high-performance liquid chromatography, and fractions are collected in a 384-well plate, which is subjected to offline counting, providing improved detection limits over online radioactivity detection. We discuss an advanced strategy to account for signal suppression or quenching, which significantly affected results in the offline counting of feces and urine samples in the selected case example, to provide more accurate quantification. The new quench model fits 2 data sets from 384-well plates with the actual matrices present to perform counting efficiency correction. Improved results were obtained over the existing approach, where a generic quench curve is defined by only a limited number of points made from a dilution series of a quenching agent. To account for outliers, a robust quartic model was applied. The new model effectively describes matrix-induced quenching and corrects for this, resulting in correct profiles with improved overall recovery as corroborated by comparison with online radioactivity detection and liquid scintillation counting, and can generically be applied postacquisition. The strategy was applied to all 36 fecal extracts from a human absorption, distribution, metabolism, and excretion study, where half of the samples present less than 20,000 disintegrations per min/mL, increasing the average column recovery (sum of individually quantified peaks relative to the total injected radioactivity) to >85%. SIGNIFICANCE STATEMENT: To improve interpretation in radiolabeled absorption, distribution, metabolism, and excretion studies, a matrix-based quench correction model is developed. It compensates for matrix-induced signal suppression when analyzing in vivo samples via offline radioactivity counting. It greatly improves data quality and enables accurate assessment of the true significance of detected metabolites.

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.

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.

Equilibrative nucleoside transporters (ENTs) 1 and 2 are considered critical to the cellular uptake of purine and pyrimidine analogs used to treat cancer and viral infections. However, a detailed understanding of the discrete and overlapping roles of these ENT subtypes in drug activity remains limited. A significant barrier to progress has been the absence of model systems that enable functional characterization of individual nucleoside transporters in the context of their native environment. To address this, we developed and characterized a panel of CRISPR/cas9-engineered human embryonic kidney 293 cell lines with selective deletion of ENT subtypes: ENT1 knockout, ENT2 knockout, and dual knockout. These models were used to dissect subtype-specific roles of ENT1 and ENT2 in nucleoside/nucleobase analog uptake and cytotoxicity. Our data show that ENT1 and ENT2 in their endogenous environment have a similar affinity for a range of both endogenous and chemotherapeutic nucleoside and nucleobase analogs. Deletion of ENT1 generally enhanced the sensitivity of cells to these drugs, particularly the nucleobase analogs, likely due to reduced nucleoside salvage by the cells via ENT1. Deletion of ENT2, on the other hand, dramatically reduced the ability of a number of the tested drugs to impact cell viability, by mechanisms beyond those related to reduced cellular uptake of the drugs. This study highlights distinctive roles of ENT1 and ENT2 in the actions of nucleoside/nucleobase analog drugs. SIGNIFICANCE STATEMENT: A panel of genetically modified human embryonic kidney 293 cells has been created as a model to screen novel nucleoside transporter inhibitors and substrates. Using these cell lines, it was revealed that ENT2 may play a more functionally significant role in nucleoside analog chemotherapeutic drug activity than previously appreciated.

The pharmacokinetic characterization of drug candidates is an essential step in drug development. To date, primary suspension hepatocytes have been widely used for this purpose; however, their poor stability has limited the application of in vitro systems for compounds with low metabolic turnover rates. Highly functional HepaSH cells, prepared from chimeric mice with humanized livers, maintain a cobblestone-like morphology and cytochrome P450-dependent drug-metabolizing activity for up to 168 hours in monolayer culture without medium change using a commercially available long-term hepatocyte culture medium. In this study, we attempted to investigate the utility of long-term culture systems and predict the hepatic clearance of 12 drugs with 9 low and 3 moderate-to-high CL_int in humans using multiple HepaSH monolayers. This culture system successfully monitored the depletion of low (such as diazepam and quinidine) and moderate-to-high CL_int drugs (midazolam). Two low-clearance drugs, disopyramide and warfarin, showed no depletion over 168 hours, indicating limitations in the application of this method for clearance evaluation. Hepatic clearance values obtained from incubation with HepaSH monolayers were predicted for 6-8 of 12 compounds tested with deviations within 3-fold, with an average fold error of 1.14- to 1.19-fold and an absolute average fold error of 1.52- to 1.97-fold, roughly correlating with the clinical reference data. In conclusion, a functionally stable culture method for HepaSH monolayers is highly effective for evaluating low-clearance compounds by greatly extending the metabolic reaction time and will be a valuable tool for determining the pharmacokinetic properties of new drug candidates. SIGNIFICANCE STATEMENT: This study demonstrated that combining highly functional HepaSH monolayers with extended drug incubation enables accurate monitoring of low-turnover compound clearance, an outcome that has been difficult to achieve with traditional assays.

Arachidonic acid (AA) is a polyunsaturated essential fatty acid and a precursor for eicosanoids. It is metabolized by cyclooxygenases, lipoxygenases, and cytochrome P450 (P450) enzymes, which convert AA into hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), chiral eicosanoids with distinct biological activities. Although racemic HETEs and EETs have been studied in cardiovascular diseases, the enantiospecific roles of their enantiomers and the enantioselectivity of P450 enzymes remain largely unexplored. This study aimed to investigate the enantioselective metabolism of AA by human recombinant P450 enzymes, focusing on the formation of R/S-HETEs and (R, S)/(S,R)-EETs. Metabolites were analyzed using liquid chromatography electrospray ionization mass spectrometry. CYP1A2 exhibited the highest activity in forming R-midchain HETEs, followed by CYP3A4. CYP2C19 was the most active enzyme in producing R-subterminal HETEs, with CYP1A2 and CYP1A1, CYP4F3B, and CYP2E1 ranking second. Similarly, CYP2C19 showed the highest activity in generating S-midchain and S-subterminal HETEs, with CYP3A4, CYP2C8, CYP1A1, and CYP1A2 contributing to varying degrees. For EETs, CYP2C19 and CYP1A2 primarily catalyzed the formation of both (R, S)/(S, R)-EETs. These findings emphasize the significant roles of CYP2C19 and CYP1A2 in the regio- and stereoselective metabolism of HETEs and EETs, highlighting their contributions to lipid signaling and potential physiological implications. SIGNIFICANT STATEMENT: This work highlights the importance of profiling P450 with respect to their enantioselectivity in arachidonic acid metabolism. The findings indicate that major P450 differ in the magnitude of their hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acid formation rates, which is a significant for studying diseases that is known to be influenced by alterations in these pathways. Altered enantioselectivity could have implications in diseases such as hypertension, cancer, inflammation, and cardiovascular disorders.

Flux dialysis, a superior method for plasma protein binding (PPB) measurement of compounds with challenging properties, has limitations in early-stage drug discovery due to multi-timepoint sampling and prolonged testing cycles. This study combines flux dialysis with acoustic ejection mass spectrometry (AEMS) to develop an innovative method that accelerates analytical throughput in PPB assays during drug discovery and demonstrates its application for the rapid and precise determination of the unbound fraction (f_u) in plasma. Herein, we validated this approach using 10 commercially available compounds with known f_u values-imipramine, indomethacin, itraconazole, lapatinib, nicardipine, warfarin, chlorpromazine, rivastigmine, zonisamide, and ritonavir-with a wide f_u range covering from very high binding (f_u ≤ 0.01) to low binding (f_u > 0.10) in human plasma. By leveraging the advantages of chromatography-free analysis and nanoliter droplet ejection mode, AEMS achieves a speed of 3 seconds per sample using only 30 nL of sample volume. Our results showed that the f_u values measured correlate strongly (R² > 0.96) with those measured by liquid chromatography-tandem mass spectrometry. Additionally, f_u values by AEMS correlate highly (R² > 0.95) with those reported in the literature. In conclusion, this method presents a high-throughput, accurate, and efficient solution for PPB assays, improving speed by 25-fold compared to the liquid chromatography-tandem mass spectrometry method. SIGNIFICANCE STATEMENT: This study bridges the gap between flux dialysis and acoustic ejection mass spectrometry by creating a synergistic analytical framework for plasma protein binding assays, addressing limitations of both methods and enabling high-throughput applications with improved accuracy and efficiency. The combination of flux dialysis and acoustic ejection mass spectrometry will make a positive contribution to the development of high-throughput in vitro absorption, distribution, metabolism and excretion assays in drug discovery.

Esophageal squamous cell carcinoma (ESCC) is a major global health threat characterized by high incidence and mortality rates. The aberrant suppression of CYP3A5 is frequently observed in ESCC. However, its precise function and the epigenetic mechanism mediating its transcriptional repression remain poorly elucidated. Herein, we found that CYP3A5 expression is significantly reduced in ESCC tumor tissues compared to normal tissues. Crucially, high CYP3A5 expression was associated with a favorable prognosis and reduced tumor metastasis in ESCC. Intriguingly, administration of the histone deacetylase inhibitor trichostatin A resulted in the upregulation of CYP3A5 expression. Further mechanistic experiments revealed that histone deacetylase 4 is the key deacetylase responsible for reducing H3K18/K27 acetylation levels at the CYP3A5 promoter, mediated by P300/CREB binding protein. Functionally, CYP3A5 overexpression effectively inhibited ESCC cell migration and invasion both in vitro and in vivo. In conclusion, CYP3A5 was crucial in ESCC and may serve as a promising therapeutic target for the prevention of tumor metastasis in ESCC. SIGNIFICANCE STATEMENT: CYP3A5 expression was downregulated in esophageal squamous cell carcinoma (ESCC) due to histone hypoacetylation at CYP3A5 promoter region. Because ESCC develops, CYP3A5 suppression promotes tumor metastasis and invasion. CYP3A5 is a potential biomarker and therapeutic target for ESCC.

Although exenatide is approved for patients with type 2 diabetes mellitus (T2DM) with mild to moderate renal impairment, specific dosing guidelines for this population remain undefined. To address this gap, we developed a physiologically based pharmacokinetic model using PK-Sim & MoBi software, integrating target-mediated drug disposition to simulate exenatide's nonlinear pharmacokinetics in normal renal function. The model was extrapolated to renal impairment populations by adjusting physiological parameters and validated against clinical data. The plasma concentrations of exenatide predicted by the established physiologically based pharmacokinetic models for populations with normal renal function and those with renal impairment were in high concordance with the observed values, with fold errors of major pharmacokinetic parameters falling within the 0.5- to 2-fold range. After reducing simulated doses for the renal impairment population to 75%, 50%, and 25% of the 10 μg standard dose, area under the concentration-time curve and C_max were re-predicted to identify optimal doses that bring this population's pharmacokinetic parameters within the normal ranges. On the basis of our findings, we recommend a model-guided dosing strategy for patients with T2DM with renal impairment, consisting of an initial dose of 2.5 μg twice daily, followed by 5-7.5 μg (mild impairment) or 5 μg (moderate impairment) twice daily for maintenance dose. This study suggests that, compared with patients with T2DM with normal renal function, patients with T2DM with renal impairment should begin at half the initial dose and also receive a reduced maintenance dose. SIGNIFICANCE STATEMENT: Exenatide is approved by the US Food and Drug Administration for patients with type 2 diabetes mellitus with mild to moderate renal impairment, but dosing guidelines are still lacking. This study developed and validated physiologically based pharmacokinetic models of exenatide in renal impairment. These new models close the evidence gap for optimal dosing in this population.