Aug 2026· Biomedical chromotography· Vol 40· 0 citations· 17 references
Medicine
TL;DR
It is indicated that a single topical dose of 30 mg does not interfere with SP parameter determinations in routine doping controls, and the metabolism and the systemic exposure of RU 58841 are assessed.
Abstract
ABSTRACT RU 58841 is a nonsteroidal antiandrogen, developed for the treatment of androgen‐dependent conditions. This study aimed to characterize the human metabolism of RU 58841 for the first time and to assess its systemic exposure following topical administration. In an initial in vitro metabolism study, six potential metabolites were identified using liquid chromatography–high resolution (tandem) mass spectrometry (LC‐HRMS/MS). Subsequently, an in vivo administration study was conducted with a RU 58841‐containing hair treatment product, applied once to the scalp of six healthy male volunteers. Dried blood spots (DBS) and urine samples were collected and analyzed by LC‐low resolution MS/MS. Five in vivo‐derived metabolites of RU 58841 were detected in human urine, including products formed via oxidation, N‐dealkylation, and glucuronidation of the parent compound and its phase I metabolites. In DBS, the parent compound and the oxidized metabolite were detected, indicating considerable systemic exposure to topically applied RU 58841. The steroid profile (SP) as part of the Athlete Biological Passport (ABP) was not affected by the administration. These findings provide information about the metabolism and the systemic exposure of RU 58841, and further indicate that a single topical dose of 30 mg does not interfere with SP parameter determinations in routine doping controls.
Cyclophosphamide (CP) is a common alkylating anticancer drug. Artemisinin (ART), an antimalarial agent metabolized by hepatic CYP450s, exhibits promising antitumor activity. This study established a rapid and accurate LC-MS/MS method for the quantitative analysis of CP and its metabolite in mouse plasma, as well as to apply this method in assessing how ART influences the pharmacokinetics of CP. Given the instability of 4-OH-CP, 4-keto-CP was selected as a surrogate metabolite for detection. The effect of ART on the mRNA expression and activity of CYP450 enzymes were assessed by RT-qPCR and substrate cocktail assay. The results indicated that CP and 4-keto-CP were linear in the range of 1-2000 and 2-2000 ng/mL, respectively. The accuracy, precision, extraction recovery, stability, and matrix effect of CP and 4-keto-CP are complied with the Bioanalytical Method Validation Guidelines. ART significantly reduced the AUC0-t of CP by 26.6%, while increasing CL/F by 1.35-fold. Concurrently, the AUC0-t and Cmax of 4-keto-CP increased by 1.42- and 1.45-fold, respectively. Furthermore, ART can markedly upregulate the mRNA expression and activity of CAR/CYP2B6. The results showed that pretreatment with ART could induce CP metabolism to its active metabolite. This study provides implications for the rational use of the ART-CP combination in clinical practice.
Huilong Wang, Wenting Zhang, Haifeng Hu et al.· Biomedical chromotography· 0 citations
Selective androgen receptor modulators (SARMs), such as LGD-4033, are frequently detected in doping control samples and pose significant challenges for result interpretation, particularly at low concentration levels potentially arising from contamination. In this study, a quantitative method for the determination of the carboxylated long-term metabolite (M5-b) of LGD-4033 in human urine was developed and applied to samples obtained from controlled micro-dose administration studies. Urine samples from single- and multiple-dose administration studies (1, 10, and 50 μg) were analyzed following enzymatic hydrolysis and solid-phase extraction using LC-HRMS/MS. Quantification was enabled by the use of a recently synthesized certified reference material, allowing, for the first time, the generation of quantitative excretion data for long-term metabolite under micro-dose conditions. The results demonstrated the rapid formation of long-term metabolite and a clear dose-dependent increase in urinary concentrations. Extended detection windows were observed for all dosing regimens, with long-term metabolite remaining detectable for several days after single-dose administration and for substantially longer periods following consecutive administration (5 days). Accumulation effects during multiple-dose intake resulted in sustained urinary concentrations and prolonged detectability. The quantitative data generated in this study provide an important basis for improved interpretation of adverse analytical findings involving LGD-4033 and support a more evidence-based distinction between different intake scenarios.
Panagiotis Sakellariou, Sebastian Marcel Schröder, Jasmin Thelen et al.· Drug Testing and Analysis· 0 citations
RATIONALE
Duvelisib (DVB), a selective PI3K-δ/γ inhibitor approved for chronic lymphocytic leukemia, requires detailed metabolic characterization to support early drug development and selection of appropriate toxicology species. Because drug metabolism varies across species, an integrated system such as primary hepatocytes containing both Phase I and Phase II enzymes is critical for accurately defining metabolic pathways and identifying potential reactive metabolites.
METHODS
DVB metabolism was investigated using primary hepatocytes from human, monkey, dog, rat, and mouse. Incubated samples were analyzed using high-resolution mass spectrometry (HRMS) to detect and characterize metabolites. Structural elucidation was performed based on MS/MS fragmentation patterns. Mechanistic studies were conducted using a selective aldehyde oxidase (AO) inhibitor to assess the role of AO in DVB metabolism.
RESULTS
DVB underwent diverse metabolic transformations, including oxidation, N-dealkylation, N-glucuronidation and glutathione (GSH) conjugation, leading to the identification of 18 putative metabolites. Thirteen metabolites, including several glucuronide and GSH conjugates were newly identified. AO-mediated oxidation on the purine ring emerged as a major pathway and was significantly reduced in the presence of an AO inhibitor, confirming its involvement. Two previously unreported GSH conjugates were also characterized, suggesting potential sites of reactive metabolite formation and associated toxicity risk. Comparative analysis revealed distinct species-dependent metabolic profiles, with both shared and species-specific metabolites identified among human, mouse, rat, dog, and monkey hepatocytes.
CONCLUSIONS
The present study provides a comprehensive cross-species metabolic profile of DVB, highlighting key pathways and novel metabolites, including those linked to potential toxicity. Mouse hepatocytes are recommended for future toxicological studies.
B. Warkad, Swapnil Sharma, Prakash Niguram et al.· Rapid Communications in Mass...· 0 citations
Background/Objectives: Vonoprazan is a potassium-competitive acid blocker with potential for the treatment of equine gastric ulcer syndrome. Vonoprazan metabolic pathways in horses have not been characterized. This study aimed to identify vonoprazan metabolites following oral administration and to determine the metabolic enzymes responsible for their metabolism using in vitro models. Methods: Six healthy adult Thoroughbred horses received vonoprazan (0.5 and 1 mg/kg PO) in a randomized crossover design. Plasma concentrations of vonoprazan-N-oxide (M-I) and vonoprazan-nitrone (M-III) were quantified using a validated liquid chromatography-tandem mass spectrometry method, and a non-compartmental pharmacokinetic analysis was performed. In vitro metabolism was evaluated using equine liver microsomes (ELMs) and equine recombinant CYP450 (eq-rCYP) enzymes. Enzyme kinetics were characterized using nonlinear regression modeling. Results: Both metabolites were detected in plasma after oral administration. Systemic exposure to M-I was markedly greater than M-III at both doses. At 1 mg/kg, mean ± SD Cmax values were 39.2 ± 28.3 ng/mL for M-I and 1.67 ± 1.66 ng/mL for M-III. AUC0–∞ increased dose-proportionally for both metabolites. In ELMs, M-I formation followed substrate inhibition kinetics, whereas M-III formation followed Michaelis–Menten kinetics. Among recombinant enzymes, CYP2D50 and CYP3A94 were the primary contributors to metabolite formation, exhibiting metabolite-specific kinetic profiles. Conclusions: These findings demonstrate that vonoprazan undergoes hepatic oxidative metabolism in horses, with M-I as the predominant circulating metabolite. Equine recombinants CYP2D50 and CYP3A94 appear to play central roles in equine vonoprazan metabolism, providing foundation for future evaluation of drug–drug interaction potential and clinical use in this species.
Camilo J. Morales, D. McKemie, Jayanti Bhandari Neupane et al.· Metabolites· 0 citations
Metabolic characterization is important for poorly characterized compounds, especially when controlled human administration studies are limited by ethical or safety concerns. Lapisteride is a 4-azasteroid-related compound with 5α-reductase inhibitory activity, but its metabolism and disposition remain unclear. Here, we established an integrated LC-HRMS-based workflow combining human liver microsomal incubation, CYP450 isoform verification, rat administration, multi-matrix sampling, LC-Q-TOF-MS metabolite identification, and urine/bile untargeted metabolomics. Seven lapisteride-related metabolites were tentatively identified. The main biotransformation reactions included mono-oxidation, O-demethylation, di-oxidation, side-chain oxidation to carboxylic acid, and glucuronidation. Diagnostic MS/MS fragments, especially the steroidal core ion at m/z 317 and characteristic C17 aryl side-chain ions, supported the proposed structures and indicated that the C17 aryl-containing side chain was a major metabolic region. CYP450 inhibition results suggested that CYP2C9 and CYP1A2 were the main isoforms involved in lapisteride oxidative metabolism, with CYP2D6 playing a possible secondary role. Multi-matrix concentration-time profiling showed distinct distribution and elimination patterns. Plasma and prostate mainly reflected early exposure, whereas bile and feces were more informative for hepatobiliary excretion and delayed elimination. Untargeted metabolomics identified 79 annotated metabolites in urine and 61 in bile. KEGG analysis showed significant enrichment of unsaturated fatty acid biosynthesis in urine, while primary bile acid biosynthesis showed a non-significant but biologically plausible trend in bile. Overall, this study provides systematic evidence for lapisteride metabolism and offers a practical strategy for metabolic characterization of compounds with limited metabolic information that are difficult to evaluate in controlled human studies.
Zhongquan Li, Bing Liu, Peijie Chen· In Analysis· 0 citations
The pharmacokinetics of the semisynthetic cannabinoid hexahydrocannabinol (HHC) and its 11-nor-carboxy- as well as 11-hydroxy-metabolites has already been the subject of research in various recent publications. However, further metabolism studies showed the presence of other, especially (side-chain-)hydroxylated metabolites in urine and serum. This study aimed to investigate the pharmacokinetics of these metabolites after oral (25 mg fruit gum) and inhalative (three puffs from vape) consumption, using serum and urine samples from a previous study. Serum (up to 48 h) and urine (up to five days) samples of six participants (three per consumption group) were collected at different time points and analyzed by LC-QqTOF and LC-QqLIT. Three metabolites (M1: side-chain-hydroxylated; M2: hydroxylated on alicyclic moiety; M3: 8-OH-HHC) were further investigated. The peak area ratios of analytes and internal standards were plotted against the time after consumption to obtain pharmacokinetic information. Overall, this study proved the presence of these metabolites in biological samples and provided pharmacokinetic insights for the first time. Differences between serum and urine, oral and inhalative consumption as well as inter-individual differences were observed. The investigated metabolites could partially serve as unambiguous consumption markers for HHC, as they are unlikely to be formed in vivo from corresponding Δ9-tetrahydrocannabinol (THC) metabolites.
Lisa Höfert, Willi Schirmer, Isabelle Mösch et al.· Scientific Reports· 0 citations
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