Drug Metabolism and Disposition最新文献

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.

Rheumatoid arthritis (RA) patients often develop comorbid RA-associated interstitial lung disease, which is a severe extra-articular manifestation associated with high incidence and mortality rates. Nintedanib, an oral small-molecule tyrosine kinase inhibitor, has shown promise in clinical studies by mitigating disease progression and enhancing lung function among RA-associated interstitial lung disease patients. Adverse reactions to nintedanib are closely linked to its blood concentration, potentially leading to discontinuation due to gastrointestinal side effects and elevated liver enzymes. However, existing research has yet to delve into the pharmacokinetic impact of RA on nintedanib. Therefore, we established a collagen-induced arthritis rat model to examine how RA influences the pharmacokinetics of nintedanib. Additionally, we conducted Caco-2 transport experiments to explore potential factors contributing to these pharmacokinetic alterations. Our findings reveal that nintedanib's pharmacokinetics change in collagen-induced arthritic rats at different disease stages compared to the control group. This alteration results in a notable increase in blood drug concentration and significant changes in pharmacokinetic parameters such as maximum serum concentration (C_max), AUC_0-t, AUC_0→∞, and time to reach peak concentration (T_max). The outcomes from the Caco-2 transport experiments indicate that interleukin 6 stimulation impedes nintedanib efflux, aligning with the observed pharmacokinetic alterations. Further Western blot experiments suggest that the changes in nintedanib's pharmacokinetics are associated with reduced expression of P-glycoprotein. Moreover, our findings suggest that RA may reduce intestinal P-glycoprotein expression by activating the C-Jun N-terminal kinase signaling pathway. SIGNIFICANCE STATEMENT: Rheumatoid arthritis significantly increases the drug concentration of nintedanib. These data suggest that rheumatoid arthritis may be caused by reducing the expression of P-glycoprotein by affecting the C-Jun N-terminal kinase signaling pathway.

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.

Organic anion transporting polypeptide 1B1 (OATP1B1), the hepatic-specific uptake transporter, plays key roles in the absorption, distribution, and excretion of a broad range of endogenous and exogenous compounds. Altered expression and function of OATP1B1 affect the bioavailability and pharmacokinetics of various clinically important drugs. In this study, OATP1B1 uptake function was found to be significantly suppressed by SRC proto-oncogene, non-receptor tyrosine kinase family kinase inhibitors, with SU6656 demonstrating the most potent inhibitory effect. Knockdown and overexpression experiments revealed that YES1 is the specific SRC proto-oncogene, non-receptor tyrosine kinase family kinase responsible for regulating OATP1B1. Further, YES1 was found to interact with OATP1B1, and the tyrosine phosphorylation status of the transporter was suppressed by both the SU6656 treatment and the knockdown of the tyrosine kinase. Moreover, Caveolin 1 (CAV-1), the oligomeric scaffolding protein, was found to interact with OATP1B1. CAV-1 knockdown significantly suppressed the uptake function of OATP1B1. Although the reduction of CAV-1 did not affect the interaction between YES-1 and the transporter, it affected the phosphorylation level of OATP1B1. Immunofluorescence analysis indicated that CAV-1 colocalized with OATP1B1, and the disruption of lipid rafts significantly reduced the association of CAV-1 with OATP1B1, suggesting that the integrity of lipid rafts is essential for the effect of CAV-1 on OATP1B1. In conclusion, YES1 was identified as a regulator for OATP1B1. The tyrosine kinase physically interacts with OATP1B1, and the conformation that facilitates the phosphorylation of OATP1B1 by YES1 is likely maintained by CAV-1. SIGNIFICANCE STATEMENT: The present study found that YES-1 regulates the function of organic anion transporting polypeptide 1B1 (OATP1B1) by interacting with the transporter and influencing its tyrosine phosphorylation status. Caveolin-1 was shown to interact with OATP1B1 as well. Abrogation of Caveolin-1 exhibited no effect on the interaction between YES-1 and OATP1B1 but reduced the phosphorylation level of the transporter. Taken together, inhibitors of YES-1 may alter the uptake function of OATP1B1, potentially leading to drug-drug interactions related to post-translational modification.