Drug Metabolism and Disposition最新文献

Previous studies revealed that pregnane X receptor (PXR) was involved in the "accelerated blood clearance (ABC)" phenomenon induced by repeated injections of PEGylated liposomes, and observed the quickly reduced hepatic accumulation of the second dose in rats at 12 hours after the last injection, compared with the increased hepatic accumulation characteristic of the ABC phenomenon. Among the downstream target genes of PXR, organic anion transporting polypeptide 1a/b (Oatp1a/b), P-glycoprotein (P-gp), and multidrug resistance protein 2 (Mrp2) are responsible for regulating the absorption, distribution, and efflux processes of drugs. This study aimed to investigate the molecular mechanisms behind the accelerated blood and liver clearance of the second dose of PEGylated liposomes from the perspective of PXR-regulated transporters. This study demonstrated that PXR activation promoted the blood clearance and liver clearance of the second dose of PEGylated liposomes in rats via the upregulation of Oatp1a4/1b2 and P-gp/Mrp2, respectively. The long-circulation characteristic and intrahepatic disposition fate of PEGylated liposomes could be reconstructed by inhibition of the PXR-Oatp1a/b/P-gp/Mrp2 axis. Additionally, we found that the repeated injections of PEGylated liposomes could cause significant transporter-mediated drug-drug interactions in combination administration regimens due to the activation of Oatp1a/b and P-gp/Mrp2. This study identifies the PXR-Oatp1a/b/P-gp/Mrp2 axis as a critical molecular regulator of systemic clearance of PEGylated liposomes and provides a therapeutic strategy to mitigate the ABC phenomenon through targeted inhibition of the PXR-Oatp1a/b/P-gp/Mrp2 axis, which is instructive for promoting the development and translation of PEGylated nanoformulations and optimizing their dosing regimens. SIGNIFICANCE STATEMENT: This study reveals that pregnane X receptor activation mediates the accelerated blood and liver clearance of the second dose of PEGylated liposomes in rats via upregulating the organic anion transporting polypeptide 1a4/1b2 and P-glycoprotein/multidrug resistance protein 2, and that PEGylated liposomes can induce transporter-mediated drug-drug interactions in combination administration regimens. These findings advance the understanding for the mechanisms underlying the in vivo disposition fate of repeated injections of PEGylated liposomes, providing guidance and new clues for inhibiting accelerated blood clearance phenomenon and optimizing the dosing regimens of PEGylated formulations.

Caco-2 cells are widely used in drug discovery but have limited drug metabolism, causing discrepancies with human physiology. This study evaluates human intestinal epithelial cells derived from pluripotent stem cell organoids, assessing morphology, gene expression, barrier function, transporter activity, drug metabolism, and cytotoxicity. These cells can be efficiently expanded in 2D culture, ensuring a stable supply of homogeneous, high-performance cells. Importantly, they retain small intestine-specific functions, such as high CYP3A4 activity and efficient peptide transport, over multiple passages. Their barrier integrity, transporter activity, and drug metabolism remain stable, making them a promising alternative to Caco-2 cells. Our findings support their use in microphysiological systems and organ-on-chip technologies, providing a physiologically relevant model for drug absorption, metabolism, and toxicity studies. These findings will aid in developing advanced in vitro models for the small intestine, improving drug absorption, metabolism, and toxicity studies. SIGNIFICANCE STATEMENT: This study shows that human pluripotent stem cell-derived intestinal epithelial cells retain key small intestine functions-drug metabolism, barrier integrity, and transporter activity-even after repeated division, offering a scalable and physiologically relevant alternative to Caco-2 cells.

Human radiolabeled mass balance studies are typically conducted using a single-dose approach to simplify dose recovery calculations and clearance pathway elucidations. A unique aspect of this clinical study is the need for subject confinement of usually exceeding 10 days in the clinical research unit, which adds up to subject burden and may raise ethical concerns especially in patient populations. Additionally, this method has a major inherent limitation, because single-dose measurements may not accurately represent steady-state conditions, particularly for molecules having time-dependent pharmacokinetics or absorption, distribution, metabolism, and excretion characteristics. To address these challenges, a novel study design implementing multiple fractional [¹⁴C]-microtracer doses was proposed and evaluated in a rat proof-of-concept study using [¹⁴C]GDC-0334. The results demonstrated that the multiple-dose approach provides robust assessment of circulating metabolite profile and clearance pathways from the analysis of steady-state samples alone. This approach further eliminates the need for sample pooling, thereby simplifying sample preparation and enhancing sample analysis through the use of undiluted samples. Building on the proof-of-concept study, the proposed design for human mass balance studies can also minimize subject confinement time, substantially reducing participant burden, simplifying study logistics, and reducing overall cost. Furthermore, the reduced confinement makes this approach more feasible for implementation in the target patient population. Accordingly, this study supports the adoption of the proposed multiple dose strategy in human mass balance studies to minimize subject confinement, and enable steady-state assessment of circulating drug-related materials and clearance mechanisms, ultimately improving translatability and robustness of study findings. SIGNIFICANCE STATEMENT: This study validates a novel multiple-dose mass balance design that overcomes the limitations of traditional single-dose methods by enabling evaluation at true steady state. This new approach offers a more reliable and efficient framework for human mass balance studies, with significant benefits including reduced subject confinement, simpler logistics, and more robust sample analysis and data interpretation, ultimately improving the assessment of drug metabolism and clearance.

Parkinson disease (PD) is a complex neurodegenerative condition marked by progressive motor and nonmotor symptoms. Cytochrome P450 (P450) enzymes, notably those from the CYP1 and CYP2 families, are increasingly recognized as significant factors in the development of PD. This review examines the role of P450 enzymes in PD, covering genetic variations, copy number variations, and single nucleotide polymorphisms linked to PD pathogenicity. It also explores the regulatory mechanisms controlling P450 expression in PD and the influence of the gut microbiome and metabolites on P450 activity. Additionally, the review discusses how P450 enzymes metabolically activate drugs used to treat PD and investigates the intricate relationship between P450s and mitochondrial dysfunction. Finally, it underscores the therapeutic potential of targeting P450 enzymes for PD treatment. Understanding the diverse roles of P450 enzymes in PD may lead to innovative treatment approaches and personalized interventions for this challenging neurological disorder. SIGNIFICANCE STATEMENT: Cytochrome P450 (P450) enzymes significantly influence Parkinson disease (PD) development through their roles in drug metabolism and detoxification. Single nucleotide polymorphisms in P450 genes can alter enzyme activity, affecting PD susceptibility and progression. Gut microbiota modulates P450 function, impacting detoxification of PD-related toxins and influencing gut and blood-brain barrier integrity. Additionally, P450-mitochondrial interactions contribute to energy deficits and oxidative stress, exacerbating neurodegeneration in PD. Understanding these pathways may uncover novel therapeutic targets and personalized treatment strategies.

Nucleotidases are enzymes that play vital roles in nucleotide pool balance and purine and pyrimidine metabolism across various tissues. Two major forms of nucleotidases, 5'-nucleotidases (5'-NTs) and nucleoside triphosphate diphosphohydrolases, dephosphorylate nucleoside monophosphates and triphosphates, respectively. Recently, our laboratory reported the dephosphorylation action of these nucleotidases toward the metabolites of clinically used nucleoside analog drugs, including gemcitabine, emtricitabine, tenofovir, and acyclovir. Here, we extended investigating the role of 5'-NTs in disposition of fludarabine, a drug used to treat B-cell chronic lymphocytic leukemia. In vitro incubations carried out using 5 human recombinant 5'-NTs, including cytosolic 5'-nucleotidase 1A (NT5C1A), NT5C2, NT5C3, NT5C, and mitochondrial 5' (3')-deoxyribonucleotidase revealed that NT5C3 catalyzed the dephosphorylation of fludarabine. Although nucleotidases have critical roles in metabolism of endogenous nucleotides and xenobiotics, their spatial localization in tissues is not fully elucidated yet. In the present work, we employed matrix-assisted laser desorption/ionization mass spectrometry imaging to ascertain localizations of tryptic peptides corresponding to major nucleotidases in mouse kidney, colon, and spleen tissues. First, in silico trypsin digestions were performed to determine the trypsin digestion patterns of the above proteins. Then, recombinant nucleotidases were used to characterize tryptic peptides of major nucleotidases. Following this, matrix-assisted laser desorption/ionization mass spectrometry imaging analyses were carried out to localize tryptic peptides corresponding to major nucleotidases in mouse colon, kidney, and spleen tissues. From tissue imaging experiments, we observed localizations of NT5C3 peptides in distinct regions such as the kidney cortex and colonic mucosa. SIGNIFICANCE STATEMENT: Nucleotidases, including cytosolic 5'-nucleotidase (NT5C) 3, have important roles in the endogenous nucleotide metabolism. Additionally, they may catalyze the dephosphorylation reactions of nucleoside analog drugs and their metabolites due to the structural similarities. Using in vitro incubations and enzyme kinetics, we demonstrate the involvement of NT5C3 in the dephosphorylation of an important antineoplastic agent, fludarabine. Furthermore, we employed mass spectrometry imaging to visualize peptides corresponding to NT5C3 and other major nucleotidases in the kidney cortex and colonic mucosa.

Drug monitoring is an essential component of precision therapeutics, yet existing data bases to support therapeutic monitoring are limited to data curated from the scientific literature or predicted in silico. We used human liver S9 fraction to generate metabolites from 1114 therapeutic drugs spanning diverse drug classes. Metabolites were analyzed by liquid chromatography-high-resolution mass spectrometry, annotated through differential analysis of preincubation and postincubation samples, curated by comparison to predicted metabolites from BioTransformer 3.0, and compiled into a human liver pharmaceutical metabolite resource, named "Pharmaceutical Metabolite Data Base (PharmMet DB)." Liquid chromatography-high-resolution mass spectrometry showed heterogeneity in product generation, with some drugs mostly being converted to predicted metabolites, while others were converted to hundreds of unpredicted products characterized by mass-to-charge ratio and chromatographic retention time. Phase I metabolism was dominant, with 30,752 oxidized drug metabolites. Glucuronidation was dominant for phase II metabolism, with 6311 drug metabolites. Notably, 89% of tested drugs produced at least 1 metabolite that was not predicted on BioTransformer 3.0, and these novel metabolites were most frequently detected for anti-inflammatory, central nervous system and antimicrobial drug classes. PharmMet DB provides experimental metabolite profiles to detect therapeutic drug exposures in human biospecimens without a requirement for prescription history. PharmMet DB usage with human epidemiology will advance pharmacometabolomics to improve understanding of drug efficacy, adverse reactions, and interactions in precision medicine. SIGNIFICANCE STATEMENT: Pharmaceutical Metabolite Data Base is a new data base of therapeutic drug metabolites suitable for use with liquid chromatography-high-resolution mass spectrometry to monitor patient adherence, detect unreported drug use, for example, in clinical trials, and enhance pharmacoexposomics and pharmacogenomics research. The data base was generated by incubation of therapeutic agents with human liver S9 fraction and curated relative to in silico predicted metabolites. Associated metadata for metabolic processes and drug classes enhance utility for clinical use, especially with untargeted metabolomics analyses of human samples.

Licorice-induced pseudoaldosteronism is attributed to the inhibition of 11β-hydroxysteroid dehydrogenase 2 in renal tubular cells by glycyrrhizic acid metabolites; however, the marked interindividual variability observed in toxic risk remains unclear. In this study, we established stereoselective toxicokinetic profiles for a recently identified metabolite, 3-epi-18β-glycyrrhetinic acid (3-epi-GA), which were compared with the parent compound, 18β-glycyrrhetinic acid (GA). Following a single intravenous administration of these 2 compounds in rats, 3-epi-GA exhibited a 7-fold longer half-life of the elimination phase and a 22-fold higher area under the curve compared with that of GA, based on a noncompartmental analysis. Two-compartment modeling indicated a 13-fold prolongation in the half-life of the elimination phase and an 18-fold increase in area under the curve for 3-epi-GA. The biliary excretion profiles in rats showed distinct differences between the 2 compounds. In rats administered with GA, 18β-glycyrrhetinyl-30-O-glucuronide, GA-3-O-sulfate-30-O-glucuronide (GA3S30G), and 18β-glycyrrhetinyl-3-O-sulfate (GA3S) were detected in the bile. In contrast, rats administered with 3-epi-GA predominantly excreted 3-epi-18β-glycyrrhetinyl-30-O-glucuronide in the bile, whereas 3-epi-GA3S30G and 3-epi-GA3S were present at trace levels. In vitro studies demonstrated that 3-epi-GA was a poor substrate for human sulfotransferase 2A1. Uptake studies revealed that 18β-glycyrrhetinyl-30-O-glucuronide and 3-epi-18β-glycyrrhetinyl-30-O-glucuronide, but not GA or 3-epi-GA, were actively transported into cells by organic anion transporter 3. Both metabolites exhibited strong binding to serum albumin; however, under hypoalbuminemic conditions, the unbound GA fraction was increased, facilitating passive diffusion into renal tubular cells. Collectively, C-3 epimerization of GA significantly attenuated phase II metabolism and biliary excretion, which resulted in prolonged systemic exposure and potential accumulation of 3-epi-GA and its glucuronide compared with GA. These stereochemical differences provide a mechanistic explanation for the marked interindividual variability observed in licorice-induced pseudoaldosteronism and highlight the importance of monitoring 3-epi-GA-derived compounds as potential biomarkers of licorice-related toxicity. SIGNIFICANCE STATEMENT: C-3 epimerization of 18β-glycyrrhetinic acid (GA) by enterobacteria attenuates phase II metabolism and biliary excretion, resulting in prolonged systemic and renal exposure to 3-epi-GA and its glucuronides compared with GA. This provides interindividual variability in GA-related toxicity.

Tolbutamide is metabolized by cytochrome P450 2C9 into 4-hydroxytolbutamide (4-OH TB), which retains pharmacological activity. 4-OH TB is further oxidized to the inactive metabolite 4-carboxytolbutamide, likely via the intermediate tolbutamide aldehyde (4-CHO TB). Because the conversion of 4-OH TB to 4-CHO TB is considered the rate-limiting step, the enzyme(s) catalyzing this reaction may play a crucial role in determining drug efficacy. We aimed to identify the enzyme(s) responsible for this process in the human liver. 4-CHO TB was formed from 4-OH TB in human liver cytosol (HLC) in the presence of nicotinamide-adenine dinucleotide (NAD⁺). The relative activity factor approach revealed that this reaction was primarily attributed to alcohol dehydrogenase 4 (ADH4). Interestingly, 4-CHO TB was also formed in HLC in the presence of nicotinamide-adenine dinucleotide phosphate (NADP⁺), with a 1.6-fold higher intrinsic clearance than that of NAD⁺. Untargeted proteomic analysis revealed a significant correlation between aldo-keto reductase 1A1 (AKR1A1) protein levels and NADP⁺-dependent 4-CHO TB formation in 15 HLC samples (r = 0.627, P < .05). Recombinant AKR1A1 effectively catalyzed this reaction, contributing 92% of NADP⁺-dependent 4-CHO TB formation in HLC. Based on hepatic NAD⁺ and NADP⁺ concentrations and the expression levels of ADH4 and AKR1A1, AKR1A1 was estimated to contribute one-third of ADH4 to 4-CHO TB formation in the human liver. In conclusion, we demonstrated that ADH4 and AKR1A1 jointly mediate the oxidation of 4-OH TB to 4-CHO TB in the human liver, highlighting the novel role of AKR1A1 as an oxidase in drug metabolism. SIGNIFICANCE STATEMENT: This study identifies aldo-keto reductase 1A1 as a novel enzyme involved in the oxidation of 4-hydroxytolbutamide in the human liver. Alongside alcohol dehydrogenase 4, aldo-keto reductase 1A1 contributes to NADP⁺-dependent aldehyde formation, suggesting a previously unrecognized role in drug metabolism and variability in tolbutamide clearance.

Sterol 12α-hydroxylase (CYP8B1) is a key regulator of bile acid (BA) homeostasis and an emerging therapeutic target for metabolic disorders. To address the challenge of cellular CYP8B1 inhibition characterization, this work developed a pharmacologically optimized HepG2 cells model using triiodothyronine-dexamethasone-bezafibrate (TDB) induction, which significantly enhances the 12α-hydroxylation activity along the acidic pathway of BA biosynthesis in HepG2 cells. Employing stable isotope tracing with apolipoprotein A1-solubilized 2,3,4-¹³C₃-cholesterol, we established a liquid chromatography-mass spectrometry-based flux analysis platform to track de novo BA synthesis. Combined with a recombinant CYP8B1 assay, flux analysis revealed that CYP8B1 participates in cholic acid synthesis in HepG2 cells, typically via 12α-hydroxylation of 7α-hydroxy-3-oxo-4-cholestenoic acid and dihydroxycholestanoic acid. In TDB-HepG2 cells, azole antifungals exhibited differentiated inhibition of 12α-hydroxylation activity, generally mirroring the enzymatic data. Econazole acted as a relatively selective CYP8B1 inhibitor with a cellular half-maximal inhibitory concentration of 0.31-0.45 μM, tioconazole and posaconazole dually inhibited CYP8B1 and sterol 27-hydroxylase (CYP27A1), itraconazole and voriconazole primarily inhibited CYP27A1, and fluconazole showed no activity toward either enzyme. This study provides the first direct evidence that CYP8B1 participates in cholic acid synthesis via the acidic pathway and establishes a high-throughput cellular platform for screening CYP8B1 inhibitors, revealing azoles as effective modulators of this pathway. SIGNIFICANCE STATEMENT: Optimized HepG2 model using a ¹³C₃-cholesterol flux assay provides direct evidence that CYP8B1 participates in cholic acid biosynthesis via the acidic pathway and establishes a high-throughput cellular platform for screening CYP8B1 inhibitors, revealing azoles as effective modulators of this pathway.

The cytochrome P450 (P450) 4F family (CYP4F) are fatty acid ⍵-hydroxylases that catalyze the insertion of a hydroxyl group at the terminal carbon. The enzymes CYP4F3A and CYP4F3B are special cases among all other human P450 enzymes because they are derived from the same gene. The CYP4F3 gene undergoes alternative splicing, resulting in the 2 distinct enzymes. CYP4F3A is exclusively expressed in monocytes and deactivates leukotriene B4 as part of the anti-inflammatory response. Conversely, CYP4F3B is expressed in the liver and kidney where its major function is the production of the potent lipid mediator 20-hydroxyeicosatetraenoic acid from arachidonic acid. Despite these differences, they share a 93% amino acid sequence identity because of their shared gene locus. Both CYP4F3A and CYP4F3B are potential therapeutic targets for autoimmune disorders, cardiovascular diseases, and cancer. Because there is a significant gap in understanding enzyme function, their use as therapeutic targets has not been realized yet. To our knowledge, we present the first protocol for the generation of functional recombinant CYP4F3A and CYP4F3B to high purity. Catalytic assays with arachidonic acid and leukotriene B4 reveal a distinct substrate preference of both enzymes, which confirm their distinct body functions. Spectral analysis confirmed a different binding mode of arachidonic acid to the splice variants with a differential interaction with the respective active site. In addition, we tested the inhibitory effect of the CYP4 pan inhibitor HET0016 on both variants. In conclusion, we successfully implemented a robust protocol for the production of recombinant CYP4F3A and CYP4F3B, which paves the way for more in-depth mechanistic and structural studies and future directed drug design. SIGNIFICANCE STATEMENT: The splice variants CYP4F3A and CYP4F3B originate from the same gene but assume different functions in the human body. However, in-depth structural and functional studies are missing owing to the lack of robust protein expression protocols. In this study, we achieved the first generation of recombinant enzyme and conducted functional studies with fatty acid substrates and drugs, paving a way to a deeper understanding of these fascinating enzymes.