Search Results (3,261)

Short-chain fatty acids (SCFAs) and medium-chain fatty acids (MCFAs) include small organic anions derived from the gut microbiome that interact with organic anion transporters of the SLC22 family, many of which are expressed in the kidney proximal tubule. According to the Remote Sensing and Signaling Theory (RSST), crosstalk between organs (e.g., gut–liver–kidney axis, gut–brain axis) and the gut microbiome is mediated by metabolites and signaling molecules transported by multi-specific “drug” transporters. The renal drug transporter OAT1 (SLC22A6) is also a major transporter of gut-microbiome products and uremic toxins (e.g., indoxyl sulfate); it has been shown to act as part of a regulatory feedback loop involving the gut microbiome. SCFAs, especially propionate and butyrate, have been shown to play a central role in the transcriptional regulation of OAT1 through HDAC inhibition. By fecal metagenomics analyses of Oat1 knockout mice, we now find that propionate synthesis is among the most altered pathways in the gut microbiome. In contrast, these pathways were only minimally altered in the Oat3 (Slc22a8) knockout. Metabolomics analyses indicate that serum propionate derivatives (e.g., propionyl glycine) and 3-hydroxybutyrate are dependent on OAT1 in the knockout mice and in humans treated with probenecid, an OAT1 inhibitor. The gut microbiome of the Oat1 knockout mice also exhibited greater fatty acid synthesis, which generates odd-chain-length fatty acids (e.g. heptanoate) when propionate is available. Overall, the data, especially when considered in light of in vitro experiments of others, indicates the in vivo existence of a feedback loop connecting gut-microbiome-derived SCFAs and MCFAs to kidney proximal tubule uptake via OAT1. This bidirectional feedback loop in turn regulates OAT1 expression through HDAC inhibition. The feedback loop is clearly consistent with the Remote Sensing and Signaling Theory—in particular, the centrality of multi-specific “drug” transporters in organ crosstalk and host–microbiome interactions via small molecules with “high information content.” The key role of OAT1 function in maintaining tubular secretion in CKD supports the importance of this RSST loop in renal pathophysiology. Modulating this RSST loop could have therapeutic value in chronic kidney disease and other contexts. Full article

Background: Diabetes mellitus remains a major public health concern, particularly in sub-Saharan Africa where type 2 diabetes predominates. In West Africa, Uvaria chamae P. Beauv. is traditionally used for diabetes management. This study investigates previously reported metabolites from Uvaria chamae using an integrated in silico approach to explore their potential antidiabetic activity and underlying mechanisms. Methods: A comprehensive literature survey identified 106 phytochemicals from stems, roots, leaves, and seeds. Diabetes-related protein targets were retrieved from the RCSB Protein Data Bank, while ligand structures were obtained from PubChem and the COCONUT database. Molecular docking, MM-GBSA rescoring, induced-fit docking, QSAR, and ADMET analyses were performed to evaluate interaction profiles, predicted activity, and developability. Results: The integrated analysis supports a polypharmacological mixture-based profile with organ-associated trends. Stem- and root-derived flavonoids, particularly isouvaretin and diuvaretin, showed the most consistent profiles for PPARγ-related pathways, while uvarinol was associated with PTP1B. Leaf alkaloids were mainly linked to DPP-4 and digestive enzyme inhibition. These compounds displayed more favorable predicted pharmacokinetic and toxicity profiles compared to acetogenins, which, despite favorable binding energies, were not prioritized as drug-like candidates due to their high lipophilicity, low QED values, and predicted toxicity liabilities, but may contribute to extract-level activity. Conclusion: These findings provide a hypothesis-generating and hierarchical framework for the prioritization of Uvaria chamae metabolites and extracts, supporting further experimental validation through enzymatic, cellular, and gene expression studies. Full article

Background/Objectives: Flavonoids are low-molecular-weight polyphenolic compounds that are universally distributed in plants. They are a chemically varied group of secondary metabolites with a broad range of biological activity. The use of flavonoids is known to decrease the risk of many chronic diseases due to their radical scavenging, antioxidant, anti-inflammatory, anticarcinogenic, and antimutagenic properties. Limitations in the use of flavonoids include their low water solubility and poor stability, and therefore their low bioavailability. The encapsulation of flavonoids in different nanocarriers has helped to overcome this limitation. Taking this into account, in this work, the encapsulation of four flavones with several therapeutic applications—7-hydroxyflavone, 7,8-dihydroxyflavone, baicalein, and luteolin—in poly(lactic-co-glycolic) acid (PLGA)-derived polymeric nanoparticles (NPs) has been investigated. Methods: A physicochemical characterization of the NPs has been carried out using different techniques, including the evaluation of antioxidant and antimicrobial activities. Results: In all cases, the encapsulation efficiency of the four flavones in the prepared NPs was high (>90%), the zeta potential was about −31 mV, and the size was nanometric (~450 nm). The drug release from the nanoparticles was also studied, showing first-order kinetics. Statistical tools were applied to the release rate constants. The antioxidant activity and the in vitro antimicrobial activity of the free and flavone-loaded NPs were investigated, in the case of the latter using Gram-positive and Gram-negative bacteria. Results show that when the flavones are encapsulated, they retain their therapeutic properties. Conclusions: In summary, PLGA-based NPs not only prevent flavone degradation but also significantly boost solubility, ultimately optimizing bioavailability. Our results underscore these NPs as a promising platform for efficient flavone delivery. Full article

In the search for new antischistosomal 3-benzylmenadiones (benzylMDs), the screening of a library developed in our laboratory led to the identification of two regioisomeric analogues, the 2′,5′- and 3′,5′-dimethoxy-benzylMD—designated schistodiones A_2′,5′ and A_3′,5′—which were investigated for their activity against the platyhelminth Schistosoma mansoni and various protozoan parasites, bacteria, and fungi. Reported work has shown that benzylMDs act as prodrugs: their bioactivation undergoes a cascade of redox reactions within the parasite, generating multiple drug metabolites, e.g., the main benzoylmenadione (benzoylMD) intermediates, and reactive oxygen species that interfere with key metabolic pathways. Among the secondary metabolites, benzoxanthones have been identified as potential products generated along this oxidative pathway. The aim of the study was to synthetize methoxylated benzoxanthones, as putative metabolites generated from these antischistosomal benzylMDs. During the synthetic work, several difficulties arose, including the absence of starting reagents, the incompatibility of certain reactions with methoxy groups, the possible formation of several isomers, and the easy re-oxidation of sensitive intermediates. To overcome these obstacles, we developed a new retrosynthetic strategy using modified precursors: replacing methoxy groups with O-methylenemethoxy (OMOM) groups that are more stable in basic media, using aldehydes or aromatic esters as precursors, and replacing certain substituents with groups that are easier and less costly to introduce (chlorine or nitro). Selected metabolites (benzoylMDs, benzoxanthones) were then tested in parasite and cellular assays. Furthermore, benzoylMDs were tested as subversive substrates of S. mansoni thioredoxin-glutathione reductase (SmTGR) and selected drug metabolites were investigated in SmTGR modeling experiments. From a One Health perspective, these benzylMD derivatives pose limited environmental risk because their metabolites lack toxicity when encountered externally, as toxicity requires intracellular metabolic activation and localized formation of reactive intermediates in close proximity to their cellular targets inside parasites. Full article

Background: Toddaliae Asiaticae Radix (TA) boasts a long history of medicinal application. However, origin traceability and quality assessment of the widely distributed original plant Toddalia asiatica have not been fully elucidated. Methods: A hybrid framework integrating targeted metabolomics, network pharmacology (NP), and machine learning (ML) was established. By optimizing key parameters, a high-coverage and rapid method for multiple categories compounds was developed using ultra-high performance liquid chromatography-multiple reaction monitoring tandem mass spectrometry (UPLC-MRM MS/MS). Using samples collected across 16 geographical regions, redundancy analysis (RDA) and pattern recognition techniques were applied to explore environment-sensitive metabolites. Taking into account five types of diseases, NP analysis was employed to obtain the bioactive components and their contribution weight in disease treatment. Subsequently, core Quality Markers (Q-Markers) with dual functions of responsive to geographic variations and biologically relevant to therapeutic efficacy were figured out, and were used to establish origin scoring model and discrimination model. Results: The geographical metabolic characteristics of the TA from broad regions in China were thoroughly analyzed, and 60 geographically sensitive compounds were identified. Through NP analysis, 27 core Q-Markers were locked. The bioactivity-weighted scoring model based on Q-Markers revealed the consistency of regional rankings as well as minor fluctuations across five diseases. ML demonstrated that the Q-Markers preserved regional discrimination performance, and the introducing of practical-oriented weights enhanced overall discriminative confidence. Conclusions: This research decodes the Geographical metabolic characteristics of TA, and highlights the necessity of function-oriented prioritization of drug resources. Full article

Background: Irinotecan hydrochloride (CPT-11) is an important anticancer drug used in a wide range of regimens to treat colorectal and gastric cancers, and one of its severe side effects is delayed diarrhea. Therefore, based on known circadian variations in intestinal function and drug metabolism, we investigated whether CPT-11-induced delayed diarrhea may be attenuated by the time of dosing. Methods: When CPT-11 was administered to rats at 9:00 or 21:00, CPT-11-induced delayed diarrhea was assessed, and concentrations of CPT-11, its active metabolite SN-38, and SN-38 glucuronide (SN-38GL) in blood, intestinal tissues, and intestinal contents were measured. Results: The severity of diarrhea was significantly less in the 21:00 dosing group compared with the 9:00 dosing group. Blood SN-38 concentrations 8 h after the administration of CPT-11 were significantly higher in the 9:00 dosing group than in the 21:00 dosing group. SN-38, which exerts potent cytotoxic effects, circulates enterohepatically. When SN-38 is absorbed from the intestinal mucosa, intestinal tissues may be injured, resulting in delayed diarrhea. CPT-11 and SN-38 concentrations in intestinal tissues and contents 8 h after the administration of CPT-11 were significantly higher in the 9:00 dosing group than in the 21:00 dosing group at all measurement points. This was consistent with more severe CPT-11-induced delayed diarrhea in the 9:00 dosing group. Conclusions: Chronotherapy with CPT-11 may reduce CPT-11-induced delayed diarrhea. These differences in SN-38 concentrations in the intestinal tract at different dosing times may contribute to the time-dependent reduction in CPT-11-induced delayed diarrhea. Full article

Drug exposure during pregnancy and early life is typically considered a short-term clinical intervention rather than a determinant of long-term pharmacological outcomes. Consequently, the developmental context is largely absent from drug discovery and drug development paradigms, where efficacy, safety and target engagement are evaluated predominantly in adult, steady-state systems. This disconnect may contribute to unexplained variability in drug response and toxicity later in life. Pregnancy is accompanied by dynamic remodeling of the maternal gut microbiota and its metabolic output, generating bioactive microbial metabolites that regulate immune tone, metabolic homeostasis and the expression of drug-metabolizing enzymes and transporters. These microbial signals intersect with pharmacological interventions across gestation, shaping maternal pharmacokinetics, placental regulation and fetal drug exposure during developmentally sensitive windows. Importantly, microbiota–drug interactions initiated during pregnancy do not terminate at birth. Instead, they extend into infancy through vertical microbial transmission, breast milk-mediated metabolic signaling, and the immaturity of neonatal drug-handling systems, collectively contributing to developmental programming of drug responsiveness beyond early life. In this review, we propose a microbiota-informed framework that reframes perinatal drug exposure as a developmentally embedded signal operating across a maternal–placental–infant continuum. This perspective introduces a missing developmental dimension into drug discovery and highlights new opportunities to improve translational predictability and precision pharmacotherapy across the life course. Full article

Inflammatory bowel disease (IBD), comprising Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic relapsing–remitting condition characterized by systemic and intestinal inflammation and immune dysregulation. Up to 47% of IBD patients develop extraintestinal manifestations, yet kidney and urological involvement remain underrecognized. Accumulating evidence has linked IBD to nephrolithiasis, glomerular diseases, tubulointerstitial nephritis, acute kidney injury, chronic kidney disease, and, rarely, amyloid A amyloidosis. Population studies have consistently shown elevated risks for various important kidney disorders in IBD, with CD generally posing a greater risk than UC. The pathogenesis of kidney complications in IBD reflects complex gut–kidney interactions, including metabolic and absorptive abnormalities, shared genetic and immune pathways, intestinal dysbiosis with nephrotoxic microbial metabolites, systemic inflammation, and drug-related nephrotoxicity. Strategies for kidney protection in IBD include increased awareness, close monitoring of kidney function, urinary metabolic profiling in high-risk patients, and prompt nephrology referral for early detection and treatment. Management should include an effective and sustained control of intestinal inflammation, discontinuation of potential nephrotoxic drugs when indicated, and timely diagnosis and treatment of kidney manifestations, as well as integrating kidney complications into IBD guidelines to enhance awareness, ultimately optimizing both the kidney and overall outcomes for IBD patients. Future studies are needed to validate the potential predictive biomarkers for kidney complications and to develop targeted interventions to address shared gut–kidney pathogenic mechanisms in IBD. Full article

Background: Daidzein, a secondary metabolite primarily obtained from soybean (Glycine max L.) and other legumes, has significant nutritional and pharmacological value. Chemically, daidzein is an isoflavone and plays a crucial role in the therapeutic amelioration of numerous disorders, including allergies, inflammation, diabetes, cardiovascular, and neurodegenerative diseases. Emerging preclinical evidence suggests potential antineoplastic activity of daidzein against various cancers. This current work aims to perform a critical evaluation of daidzein’s potential as an anticancer molecule with an in-depth understanding of its mechanisms of action. Methods: The data for this review were obtained from various sources, including PubMed, Scopus, and Web of Science. Results: Daidzein, as a pure phytochemical or in combination with other phytochemicals and anticancer drugs, has been reported to induce apoptotic and autophagic cell death, impeding cell growth, viability, proliferation, and angiogenesis, and arresting cell division at various phases in vitro. Various daidzein formulations also exhibited similar anticancer effects by immunomodulation and genetic alteration in the cancer cells. In vivo anticancer studies of daidzein also suggest modulation of several hallmark pathways, such as inhibition of nuclear factor-κB, Janus kinase/signal transducer and activator of transcription, and rat sarcoma virus/rapidly accelerated fibrosarcoma. Conclusions: Irrespective of numerous promising preclinical studies, the absence of clinical studies provides a major challenge to establishing daidzein’s safety and efficacy in human cancers. Therefore, further advancements in clinical research of daidzein are vital for manifesting as an antineoplastic drug. Full article

Alzheimer’s disease (AD) is a common age-related neurodegenerative disorder with extremely low drug development success rates, making nutritional intervention a promising strategy. Cerebral energy metabolism dysfunction is a core pathological feature of AD. Odd-chain fatty acids (OCFAs) can generate propionyl-CoA via β-oxidation to replenish the impaired tricarboxylic acid (TCA) cycle. This study characterized the lipid composition of OCFAs-enriched algal oil by UPC²-Q-TOF-MS, evaluated its neuroprotective effects on Caenorhabditis elegans (C. elegans) models with AD, Parkinson’s disease (PD), and Huntington’s disease (HD), and explored the metabolic mechanism of its key component pentadecanoic acid (C15:0) using untargeted metabolomics. Results showed that triglycerides (TAGs) represented the predominant lipid class, accounting for 97.3% of the total lipid content in the algal oil. Among all the identified TAG molecular species, TAGs containing C15:0/C17:0 accounted for more than 90%. OCFAs-enriched algal oil exhibited disease-selective neuroprotection. It significantly improved locomotor function in AD nematodes, moderately ameliorated PD-related deficits, whereas showed no efficacy in HD nematodes. Metabolomics revealed that C15:0 produced propionyl-CoA to rescue TCA cycle dysfunction and energy deficits, upregulated membrane phospholipids to repair membrane integrity, and reduced abnormal metabolites to restore metabolic homeostasis. KEGG analysis confirmed that C15:0 globally regulated core metabolic pathways including amino acid, cofactor, nucleotide, and carbon metabolism. OCFAs-enriched algal oil exerted selective anti-AD effects by repairing energy metabolism, remodeling membrane phospholipids, and restoring metabolic homeostasis, providing a novel nutritional candidate for AD intervention. Full article

Background: Abemaciclib (ABM) in combination with tamoxifen (TAM) is an extremely significant treatment regimen for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer. It is approved for patients to reduce the risk of cancer recurrence. A bioanalytical method for the simultaneous determination of this new anti-breast cancer combination and its pharmacokinetic application has not yet been reported. Methods: An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed for quantifying ABM, TAM, and its metabolites, including abemaciclib active metabolites M2, M18, and M20 and tamoxifen active metabolite N-desmethyl tamoxifen (NDTAM), in rat plasma using econazole as the internal standard (IS). Chromatographic separation was achieved on a Kinetex C18 column (100 × 2.1 mm ID, 2.6 µm) using gradient elution with 5 mM ammonium formate in water (eluent A) and 5 mM ammonium formate in water/methanol (1:9, v/v, eluent B) at a flow rate of 0.4 mL/min. Detection was performed on a TSQ Fortis Plus mass spectrometer employing multiple reaction monitoring mode under positive electrospray ionization. Results: The developed method was validated according to the guidance of the FDA. Linearity in rat plasma (ng/mL) was achieved from 1 to 1000 for ABM, TAM, and M20; 3 to 1000 for M2; 5 to 500 for M18; and 1 to 500 for NDTAM; with correlation coefficients ranging from 0.9991 to 0.9931 for all analytes using a weighting factor of 1/X². The lower limit of detection (LLOD) ranged between 0.3 and 1.5 ng/mL for all drugs. The accuracy ranged from 96 to 108% and the precision was less than 7.6% RSD for all analytes. For the first time, the newly developed approach was effectively used in a pharmacokinetic study on the simultaneous oral administration of ABM and TAM in rats that received 30.0 mg/kg of ABM and 8.0 mg/kg of TAM. Conclusions: To the best of our knowledge, this is the first reported UPLC–MS/MS method for the assay of ABM, TAM, and its active metabolites in plasma. This method offers a bioanalytical tool for assessing the pharmacokinetics of ABM and TAM. Therefore, this study makes a definite significant contribution to the field of bioanalytical research. Further validation in human plasma is required for future clinical or therapeutic drug monitoring applications, as the approach was developed in an animal model. Full article

Euphorbia clavarioides Boiss. is traditionally used in wound healing and other medicinal applications. Its bioactive compounds and pharmacological potential remain underexplored. This study investigated the phytochemical composition, antioxidant, anti-inflammatory, and anticancer activities of E. clavarioides Boiss. traditionally used in wound healing. Plant extracts were characterized using phytochemical screening, Fourier-transform infrared spectroscopy (FTIR), and liquid chromatography–mass spectrometry (LC-MS). Antioxidant activity was evaluated via DPPH and nitric oxide (NO) scavenging assays, anti-inflammatory effects through nitrite inhibition in LPS-stimulated RAW 264.7 macrophages, and anticancer potential using the MTT assay against DU-145, PC-3, SKU-T, and AGS cell lines. Phytochemical screening confirmed tannins, phlobatannins, saponins, flavonoids, alkaloids, steroids, terpenoids, and cardiac glycosides. FTIR spectra of aqueous extracts revealed peaks at 2990.66 cm⁻¹ (O–H), 1738.68 cm⁻¹ (C=O), 1217.22 cm⁻¹ (C–N), and 527.37 cm⁻¹ (C–Cl). LC-MS profiling identified diverse metabolites, including phenolics (pseudolaroside B, cinnamtannin A2, (−)-medicarpin, butyrolactol A) and terpenoids (zerumbone, sclareol isomer, diterpenoid-like compounds), underpinning the plant’s bioactivity. Methanol extracts exhibited the strongest DPPH scavenging activity (IC₅₀ = 755.71 µg/mL), whereas aqueous and ethanol extracts demonstrated superior NO scavenging. Ethanol extracts showed maximal anti-inflammatory activity, while aqueous extracts induced pro-inflammatory effects. Cytotoxicity assays indicated negligible toxicity. In anticancer assays, ethanol and methanol extracts significantly inhibited the proliferation of all tested cell lines at 100 µg/mL, exceeding drug control, whereas aqueous extracts displayed lower activity. The bioactive compounds in E. clavarioides support its traditional wound-healing use and demonstrate mechanistic antioxidant, anti-inflammatory, and anticancer activities, highlighting its potential as a source of multi-target natural therapeutics. Full article

This article discusses rheumatoid arthritis (RA) as a chronic, systemic autoimmune disease leading to progressive joint damage and multi-organ complications. The complex pathogenesis of the disease is presented, involving the interaction of environmental, genetic, and immunological factors, including the role of autoantibodies and proinflammatory cytokines. Particular attention is paid to leflunomide, a disease-modifying antirheumatic drug (DMARD), which primarily works by inhibiting the DHODH enzyme, leading to reduced T and B cell proliferation. The additional anti-inflammatory properties of the drug’s active metabolite, teriflunomide, and its impact on signaling pathways related to the immune response are also discussed. This article examines the variability in patient responses to leflunomide treatment in terms of both efficacy and toxicity, with particular emphasis on the potential role of pharmacogenetic factors. It was pointed out that polymorphisms in genes related to drug metabolism, transport, and mechanism of action may influence the pharmacokinetics and safety of the therapy. It was also emphasized that the available data are primarily derived from observational studies and small cohorts, and the results are often inconsistent. Although some genetic variants and plasma teriflunomide concentrations show potential as predictors of treatment response, the current level of evidence does not support the routine use of pharmacogenetic testing in clinical practice. The article emphasizes that the pharmacogenetics of leflunomide represents a promising, yet still exploratory, avenue of research in the context of personalized RA therapy. It emphasizes the need for larger, well-designed clinical trials and the development of standardized guidelines, which would be necessary before the potential implementation of such strategies in routine clinical practice. Full article

Intensive aquaculture of rice field eel (Monopterus albus) is constrained by oxidative stress induced by high-density culture resulting in growth inhibition, while prophylactic antibiotics pose escalating risks of drug resistance and food safety hazards. This study addresses the critical need for developing efficient, environmentally friendly functional feed additives as sustainable growth promoters in intensive aquaculture. To investigate the dietary berberine (BBR) effect on promoting growth performance, hepatic antioxidant capacity and metabolism in M. albus, four experimental groups were established: control (CON, 0 mg/kg) and berberine-supplemented groups (BBR25, 25 mg/kg; BBR50, 50 mg/kg; BBR100, 100 mg/kg). Growth performance, serum biochemical parameters, hepatic antioxidant capacity, and liver metabolomics (LC-MS) were evaluated after the 8-week feeding trial. BBR50 and BBR100 had significantly increased final weight, weight gain rate (WG), and survival rate (SR), while reducing feed conversion ratio (FCR) (p < 0.05). Serum glucose (Glc), total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were decreased (p < 0.05), while high-density lipoprotein cholesterol (HDL-C) and phosphofructokinase (PFK) activity were increased (p < 0.05). Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were significantly reduced (p < 0.05). Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were upregulated (p < 0.05), whereas malondialdehyde (MDA) was downregulated (p < 0.05). Metabolomics identified 98 differential metabolites, with significant enrichment of metabolites associated with arachidonic acid metabolism, histidine metabolism, arginine/proline metabolism, tryptophan metabolism, and pathways related to mTOR signaling. Overall, dietary supplementation with 50 mg/kg BBR emerged as a practically favorable dose among the tested concentrations for promoting growth performance and feed utilization efficiency, whereas 100 mg/kg BBR was associated with lipid and amino acid metabolic alterations suggestive of metabolic reprogramming and antioxidant-related shifts, without conferring additional growth benefits. Full article

Interpretation of pharmacometabolomics results, aiming particularly at biomarker (sets) discovery for drug exposure, remains a major challenge. The metabotyping of drug exposure depends on resolution of specific metabolomics techniques and comprises individual metabolic phenotypes (“metabotypes”), disease-, drug- and microbiome-specific patterns, as well as conditional metabolic states (e. g. fasting). In this clinical trial with 16 healthy participants, an exploratory objective was to evaluate the untargeted urinary metabolomics of voriconazole, administered in four single doses, using proton nuclear magnetic resonance (¹H-NMR) spectroscopy. Voriconazole is a second-generation triazole and a potent inhibitor of drug-metabolizing enzymes such as cytochrome P450 (CYP) isozymes CYP3A4 and CYP2C19. Therefore, identification of metabolites reflecting acute CYP3A4 inhibition was of particular interest. On two treatment days without and with voriconazole (with background microdosed midazolam and omeprazole administration for CYP3A4 and CYP2C19 phenotyping, respectively), spot urine was collected after overnight fasting (predose) and 4 h later (postdose fasting). In the postdose versus predose fingerprints, most changes at the annotated metabolite level were attributable to fasting metabolomics or potential confounders. ¹H-NMR spectroscopy identified neither a short-term voriconazole-specific signature nor patterns or metabolites potentially reflecting acute CYP3A4 inhibition. Our study emphasizes crucial significance of strict standardization of fasting time and minimization of confounder influences by clinical trial design as well as selection of adequate baselines and high-resolution analytical techniques in pharmacometabolomics research, especially for biomarker discovery. Full article