Drug Metabolism and Disposition最新文献

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.

CYP2B6 is an important enzyme in the phase 1 metabolism of key pharmaceuticals, and inhibition of this enzyme can lead to adverse drug events. Machine learning models can potentially predict interactions with CYP2B6; however, there is limited data with which to train these models in the public domain. We proposed enhancing the applicability domain and improving the predictive capability of our CYP2B6 inhibition model by selecting a small, diverse set of compounds to test in vitro and adding the results to our model training set. We used a distance-based approach to define the applicability domain of the model and then measured the chemical diversity by creating t-distributed stochastic neighbor embedding plots to represent the chemical space of our model. After comparing this chemical space with a 49-plate drug-repurposing library, we were able to identify a plate with the highest average minimum Euclidean distance from the model training set. We then performed in vitro testing of this plate for CYP2B6 inhibition activity at 10 μM and added this new data to our machine learning model. A one-class classification approach was used to evaluate the efficacy of our applicability domain-expansion technique. The results showed that this method did not appreciably increase the performance of the model or the applicability domain, but we did increase the diversity of the training set. Additionally, the in vitro experiments identified vilanterol and allylestrenol as inhibitors of CYP2B6 with IC₅₀ values in the sub to low micromolar range. SIGNIFICANCE STATEMENT: CYP2B6 inhibition can affect the metabolism of important drugs, like methadone and propofol, and result in variability that can lead to adverse events. Machine learning models can help uncover new molecules with inhibitory potential against CYP2B6, but only if predictions of these models are reliable. This study illustrates how the intentional expansion of a machine learning model's applicability domain is neither a simple nor straightforward task, but even a conservative effort can reveal new molecules with CYP2B6 inhibition activity.

Bosutinib monohydrate, a second-generation tyrosine kinase inhibitor, is primarily used to treat Philadelphia chromosome-positive chronic myelogenous leukemia. Pharmacokinetic studies in humans identified 3 metabolites of bosutinib: oxidative dechlorinated bosutinib, N-desmethylated bosutinib, and bosutinib N-oxide. Although a few metabolites have been reported clinically, a comprehensive understanding of bosutinib's metabolic fate is essential for optimizing its therapeutic use and minimizing risks. Therefore, the present study aimed to investigate the detailed metabolism of bosutinib using a combination of in vitro, in vivo, and in silico methods. In vitro experiments were conducted using liver microsomes and S9 fractions in the presence of suitable cofactors, whereas in vivo studies employed Sprague-Dawley rats in which bosutinib was administered as an oral suspension, followed by the collection of blood, urine, and feces at respective time points. The biological samples were analyzed using liquid chromatography-quadrupole-Orbitrap mass spectrometer. A total of 10 metabolites were identified, including 8 novel ones. The diverse metabolic reactions included oxidative O-dealkylation (B-M1, B-M2, B-M4, and B-M7), N-oxidation (B-M5), oxidative dechlorination (B-M2 and B-M3), N-dealkylation (B-M8 and B-M9), hydroxylation (B-M8), and glycine conjugation (B-M10). Interestingly, no metabolites were detected in the plasma, and the major metabolites, B-M3 (13.91%) and B-M9 (10.58%), were found predominantly in the feces. In silico predictions using Meteor Nexus matched with 6 of the experimentally observed metabolites. Toxicity and mutagenicity were further assessed using Deductive Estimation of Risk from Existing Knowledge Nexus and Structure Activity Relationship Analysis using Hypotheses Nexus, which indicated a potential mutagenic concern for B-M7. The integration of experimental and computational approaches in this work contributes significantly to understanding bosutinib's metabolic profile and can guide future strategies for its safe and effective clinical application. SIGNIFICANCE STATEMENT: This study provides an in-depth exploration of bosutinib's metabolic pathways using in vitro models and in vivo analysis of plasma, urine, and fecal samples. Prominently, in silico toxicity assessments indicated that B-M7 may pose mutagenic risks, emphasizing the need for further investigation.

Conventional preclinical in vitro approaches inaccurately predicted clinical trial outcomes of drug-drug interactions involving the peptide NN1177, a glucagon and glucagon-like peptide 1 receptor coagonist. To further study the mechanisms behind this discrepancy, we have exploited a mouse model (8HUM) humanized for the major cytochrome P450 (P450) enzymes involved in drug disposition in humans. We show that NN1177 administration to 8HUM mice suppressed hepatic in vivo expression of CYP3A4 (82% compared to vehicle) and CYP1A2 (58% compared to vehicle). This was consistent with in vitro sandwich culture hepatocyte data reported previously. However, reduction in CYP3A4 and CYP1A2 levels in vivo appeared to resolve over time, despite daily NN1177 administration. These findings suggest an adaptive response to the metabolic effects of NN1177. In vivo pharmacokinetic studies in 8HUM closely matched the findings observed in the clinical trial, because there was no relevant increase in the exposure of the CYP3A4 and CYP1A2 probe drugs. Furthermore, no suppression effects were observed when the mice had been pretreated with the inducing agents, St. John's wort or phenobarbital, respectively, suggesting that the mechanism of P450 reduction does not involve the transcription factors constitutive androgen receptor or pregnane X receptor. These data highlight the complexities associated with therapeutic peptide drug-drug interactions and the remaining challenges for accurate predictions of P450 suppression and potential clinical implications. The humanized 8HUM model provides a promising and informative preclinical tool that can add high value during drug development by providing further insights into the effects on P450 expression, together with the subsequent impact of coadministered probe drugs in an in vivo model. SIGNIFICANCE STATEMENT: The current work describes the application of a humanized cytochrome P450 mouse model that provides further insight into the potential mechanisms and outperforms conventional in vitro approaches for preclinical predictions of peptide drug-drug interaction risk. The results showed no significant effects on the Cooperstown 5 + 1 cocktail, in line with clinical findings, and thereby represent an exciting model to further explore future therapeutic peptide projects during drug development.