Drug Metabolism and Disposition最新文献

Pregnane X receptor (PXR) is a key transcriptional regulator of drug-metabolizing enzymes and transporters, notably CYP3A4, which metabolizes a significant proportion of clinically used drugs. PXR activation can induce CYP3A4 expression, potentially leading to drug-drug interactions (DDIs) by altering the pharmacokinetics of CYP3A4 substrates, particularly for narrow therapeutic index drugs. Conventional induction assays rely on measuring CYP3A4 mRNA and enzyme activity, but mRNA levels often do not correlate with enzyme activity, which can lead to mispredictions of DDIs. To address this gap, we incorporated our newly established Fast and Surfactant-Treated proteomic workflow into the current in vitro induction assay to enable simultaneous quantification of CYP3A4 mRNA, protein, and enzyme activity induction from a single experiment. Using rifampicin as a PXR agonist, we demonstrated that the unified All-in-One assay provided consistent induction parameters with discrete assays, offering a robust method for assessing CYP3A4 induction. We also applied this approach to the tyrosine kinase inhibitors pazopanib and crizotinib, revealing nonuniformities in their induction profiles across mRNA, protein, and enzyme activity endpoints. Specifically, although both tyrosine kinase inhibitors induced CYP3A4 mRNA expression in a dose-dependent manner, they do not lead to protein induction, suggesting that the in vitro induction observed at the mRNA level may not translate to clinical induction. Collectively, these preliminary findings suggest that protein measurements may provide a more holistic representation of CYP3A4 induction and can potentially improve the predictability of clinical DDIs in drug development. SIGNIFICANCE STATEMENT: We described and validated a unified assay that can simultaneously measure CYP3A4 mRNA, protein, and enzyme activity induction from a single human hepatocyte experiment. This unified All-in-One approach has the potential to improve in vitro-in vivo correlation and translation of CYP3A4-mediated induction drug-drug interactions for new chemical entities. However, further work, including the integration of static or dynamic physiologically based pharmacokinetic modeling with protein induction data, will be required to fully confirm these insights.

NADPH cytochrome P450 reductase is the required redox partner for the majority of human cytochrome P450 enzymes, which are critically important for phase I drug metabolism of a wide variety of substrates. It is well understood that cytochrome P450 reductase supports P450 catalysis when its flavin mononucleotide (FMN)-containing domain (FMND) binds to the proximal side of P450 enzymes to deliver electrons to the P450 heme. Herein, we describe mass spectrometry-based footprinting approaches to compare the surface labeling of CYP2A6 and that of an artificial fusion protein composed of the reductase FMND linked to the N-terminus of CYP2A6 (FMND/CYP2A6). Three complementary footprinting approaches were used: hydrogen-deuterium exchange, benzoyl fluoride labeling, and fast photochemical oxidation of proteins (FPOP). Although the different labeling approaches target different amino acids and occur over varying reaction timescales, their outcomes generally agree. These experiments did not detect differential protection on the proximal P450 face where FMND is expected to bind. Instead, they consistently demonstrated increased exposure of CYP2A6 surface residues, indicative of structural changes in CYP2A6 in the presence of the FMND. Overall, the reduced protection is consistent with the FMN domain causing long-range allosteric modulation of the CYP2A6 structure. This structural evidence is consistent with increasing functional evidence that the reductase is an allosteric modulator of P450 enzymes in addition to its role in electron transfer. SIGNIFICANCE STATEMENT: Both established and new mass-spectrometry footprinting methods support structural changes in the CYP2A6 structure upon interaction with the FMN-containing domain of its reductase. This evidence supports the idea that the reductase is an allosteric modulator of P450 enzymes, in addition to its established role in electron transfer.

Prediction of human intestinal metabolism within physiologically based pharmacokinetic models is now well established within drug development. Extrapolation of in vitro kinetic parameters accounts for differences in abundance between different in vitro systems and tissues. The existing data assume that the activity of CYP3A4 is consistent between the intestine and liver once adjusted for its tissue-specific expression level. However, the validity of this assumption for other enzymes and other tissues remains uncertain. In the current study, indicators of "activity per unit of enzyme," namely, turnover number (k_cat) or specificity constant (k_sp), were measured for 7 enzymes (CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT1A6, UGT2B7, and UGT2B17) in microsomes prepared from 4 paired (same donor) intestine and liver tissue samples. After excluding 1 donor with low intestinal activity, the intestinal k_cat and k_sp for the studied CYPs were within 2-fold of the liver values, with the exception of 1 donor with 4-fold lower CYP2D6 k_cat in the intestine compared with the liver. Conversely, the UGT1A1 k_sp and UGT2B7 k_cat were 5-fold and 7-fold higher in intestinal microsomes compared with liver microsomes, respectively. Trends in interdonor variability in k_cat were noted and require further evaluation in a larger set of donors. The current paradigm of extrapolation of hepatic metabolism data to predict in vivo first-pass metabolism in the intestine using tissue abundances appears to be valid for CYPs but should be approached with caution when predicting intestinal glucuronidation. SIGNIFICANCE STATEMENT: This study assessed whether hepatic metabolism data can predict intestinal metabolism in physiologically based pharmacokinetic models by comparing enzyme abundance and activity in matched liver and intestine microsomes from 4 donors. Seven key drug-metabolizing enzymes were quantified. While CYP-mediated intestinal metabolism could generally be predicted from liver data after adjusting for tissue abundance, caution is warranted for enzymes involved in intestinal glucuronidation, where assumptions of equivalent activity across tissues may not hold.

Mycobacterium abscessus (Mab, MAB) poses a rising health threat worldwide. Infections occur in hospital settings, affecting immunocompromised and immunocompetent patients alike. Individuals with underlying lung diseases, such as cystic fibrosis and bronchiectasis, are particularly at risk. Mab is intrinsically multidrug resistant to most antibiotics, has high treatment failure to current treatment regimens, and lacks a vaccine. Targeting bacterial metabolism has historically resulted in successful therapies. We discovered that the cytochrome P450 isoform CYP123 encoded by MAB_1216c is required for host infection. To determine the role of CYP123 during host infection, we generated highly active recombinant CYP123 and found CYP123 interactions with steroid hormones, which are key players in host immune response. All tested steroids induced a reverse type I shift when titrated to the enzyme. Their binding affinity was dictated by the presence of hydroxyl groups at certain positions in the steroid scaffold. Metabolism assays with a surrogate redox system revealed that CYP123 is a steroid hydroxylase and can convert 11-deoxycorticosterone and progesterone to a single monohydroxylated product, respectively. Mab infection has been associated with fungal coinfection, and cytochrome P450 enzymes have been shown to interact with azoles. We found that CYP123 binds to various triazole and azole drugs in the low micromolar range. Our results indicate that Mab CYP123 can interfere with host endobiotics with a potential implication in host cell reprogramming and can bind antifungal therapeutics possibly leading to worse polymicrobial infections. CYP123 could emerge as a potential drug target for an orthogonal approach to treating Mab infections. SIGNIFICANCE STATEMENT: Infections with the pathogen Mycobacterium abscessus are on the rise with limited treatment options. The M. abscessus cytochrome P450 CYP123 was identified to play an essential role for host infection. Steroids do not only bind to CYP123 but are also metabolized to monohydroxylated products implicating the potential to interfere with steroidogenesis and immune antagonism by this bacterium.

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.

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.

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.

The cytochromes P450 (P450s) are essential drug-metabolizing enzymes. In humans, P450 genetic variants partly account for interindividual variability in drug metabolism. However, in dogs, a species often used in drug metabolism studies, the genetic variants of P450s remain to be fully investigated. In this study, the sequencing of 6344 dog genomes identified 8 variants in CYP2B6 (formerly CYP2B11), including 7 nonsynonymous variants (R74C, A83T, V103I, R145Q, Q151H, Q233L, and F389Y). Of these variants, V103I is located in a substrate recognition site, a domain crucial for enzyme function. Notably, the R74C variant exhibited a highly breed-specific distribution across the 119 dog breeds analyzed. The eighth variant, c.823-2_823delAGG, was located at the boundary of intron 5 and exon 6 and, in transcripts generated by minigene assay in human embryonic kidney 293 cells, led to the deletion of 3 bases of exon 6. This resulted in the deletion of 1 amino acid residue (p.E275del). To perform metabolic assays, recombinant proteins of all 8 variants were prepared in Escherichia coli. The metabolic activities of some variants were different from that of the reference CYP2B6 protein. These results suggest the possible contribution of genetic variants to the variability of CYP2B-dependent drug metabolism in dog liver. SIGNIFICANCE STATEMENT: Seven nonsynonymous dog cytochrome P450 2B6 variants were identified. Eighth variant, c.823-2_823delAGG, shifted the splice acceptor site, resulting in a 3-nucleotide deletion. Potential importance of CYP2B6 variants in the variability exists in metabolic activities among individual animals.

Changes in the expression of drug-metabolizing enzymes and transporters can alter the pharmacokinetics of drugs, potentially affecting their efficacy and safety. In this study, we investigated the effects of decreased multidrug resistance-associated protein (MRP) 2 expression on the gene expression of other drug-metabolizing enzymes and transporters. Variations in the mRNA expression of drug-metabolizing enzymes and transporters were observed in MRP2-knockdown human hepatocellular carcinoma HepG2 cells and the liver of MRP2-deficient Eisai hyperbilirubinemic rats (EHBR). Both models showed decreased mRNA and protein expression of sulfotransferase (SULT) 1E1, a phase II drug-metabolizing enzyme, suggesting a relationship between the transcriptional regulation of MRP2 and SULT1E1. The plasma levels of bilirubin, bile acids, and cholesterol were higher in EHBR than in control Sprague-Dawley rats. Treatment with chenodeoxycholic acid (CDCA), a primary bile acid, reduced SULT1E1 mRNA expression in HepG2 cells and suppressed human SULT1E1 promoter activity in a luciferase reporter assay using HepG2 cells. CDCA is a known agonist of the farnesoid X receptor (FXR), and transcriptome analysis of the EHBR liver also suggested FXR activation, as inferred from changes in its target gene expression. These findings suggest that decreased MRP2 expression causes coordinated changes in the SULT1E1 gene expression via FXR activation by endogenous substances. These indirect changes in the expression of drug-metabolizing enzymes or transporters should be considered during drug development and in clinical practice. SIGNIFICANCE STATEMENT: This study investigated compensatory or coordinated changes in gene expression of drug-metabolizing enzymes and transporters in multidrug resistance-associated protein (MRP) 2-knockdown HepG2 cells and in the liver of MRP2-deficient rats. Decreased expression of MRP2 affects the gene expression of drug-metabolizing enzymes and transporters, including a decrease in SULT1E1, likely through nuclear receptor activation by endogenous molecules.